Design, synthesis, and functional evaluation of a novel series of phosphonate-functionalized 1,2,3-triazoles as positive allosteric modulators of α7 nicotinic acetylcholine receptors

Article abstract of DOI:10.1021/acschemneuro.0c00348

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel widely distributed in the central nervous system, mainly in the hippocampus and cortex. The enhancement of its activity by positive allosteric modulators (PAMs) is a promising therapeutic strategy for cognitive deficits and neurodegenerative disorders. With the aim of developing novel scaffolds with PAM activity, we designed and synthesized a series of phosphonate-functionalized 1,4-disubstituted 1,2,3-triazoles using supported copper nanoparticles as the cycloaddition reaction catalyst and evaluated their activity on α7 receptors by single-channel and whole-cell recordings. We identified several triazole derivatives that displayed PAM activity, with the compound functionalized with the methyl phosphonate group being the most efficacious one. At the macroscopic level, α7 potentiation was evidenced as an increase of the maximal currents elicited by acetylcholine with minimal effects on desensitization, recapitulating the actions of type I PAMs. At the single-channel level, the active compounds did not affect channel amplitude but significantly increased the duration of channel openings and activation episodes. By using chimeric and mutant α7 receptors, we demonstrated that the new α7 PAMs share transmembrane structural determinants of potentiation with other chemically nonrelated PAMs. To gain further insight into the chemical basis of potentiation, we applied structure-activity relationship strategies involving modification of the chain length, inversion of substituent positions in the triazole ring, and changes in the aromatic nucleus. Our findings revealed that the phosphonate-functionalized 1,4-disubstituted 1,2,3-triazole is a novel pharmacophore for the development of therapeutic agents for neurological and neurodegenerative disorders associated with cholinergic dysfunction.

Full text of DOI:10.1021/acschemneuro.0c00348

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Article
Design, Synthesis and Functional Evaluation of a Novel Series
of Phosphonate-functionalized 1,2,3-triazoles as Positive
Allosteric Modulators of #7 Nicotinic Acetylcholine Receptors
Beatriz Elizabeth Nielsen, Santiago Stabile, Cristian Vitale, and Cecilia Bouzat
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.0c00348 • Publication Date (Web): 28 Jul 2020
Downloaded from pubs.acs.org on August 1, 2020
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Design, Synthesis and Functional Evaluation of a Novel Series of Phosphonate-
functionalized 1,2,3-triazoles as Positive Allosteric Modulators of α7 Nicotinic
Acetylcholine Receptors
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Beatriz Elizabeth Nielsen1#, Santiago Stabile2#, Cristian Vitale2* and Cecilia Bouzat1*
1Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB), Departamento de
Biología, Bioquímica y Farmacia, Universidad Nacional del Sur-Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET), Bahía Blanca, Argentina.
2Instituto de Química del Sur (INQUISUR), Departamento de Química, Universidad
Nacional del Sur-Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET), Bahía Blanca, Argentina.
#These authors contributed equally to the work.
Correspondence to:
*Dr. Cecilia Bouzat. Instituto de Investigaciones Bioquímicas de Bahía Blanca, UNS-
CONICET, Bahía Blanca 8000, Argentina., E-mail: inbouzat@criba.edu.ar
*Dr. Vitale Cristian. Instituto de Química del Sur, UNS-CONICET, Bahía Blanca 8000,
Argentina., E-mail: cvitale@criba.edu.ar
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ABSTRACT
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α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel widely
distributed in the central nervous system, mainly in hippocampus and cortex. The
enhancement of its activity by positive allosteric modulators (PAMs) is a promising
therapeutic strategy for cognitive deficits and neurodegenerative disorders. With the aim
of developing novel scaffolds with PAM activity, we designed and synthesized a series
of phosphonate-functionalized 1,4-disubstituted 1,2,3-triazoles using supported copper
nanoparticles as cycloaddition reaction catalyst, and evaluated their activity on α7
receptors by single-channel and whole-cell recordings. We identified several triazole
derivatives that displayed PAM activity, the compound functionalized with the methyl
phosphonate group being the most efficacious one. At the macroscopic level, α7
potentiation was evidenced as an increase of the maximal currents elicited by
acetylcholine with minimal effects on desensitization, recapitulating the actions of type I
PAMs. At the single-channel level, the active compounds did not affect channel
amplitude, but significantly increased the duration of channel openings and activation
episodes. By using chimeric and mutant α7 receptors, we demonstrated that the new α7
PAMs share transmembrane structural determinants of potentiation with other
chemically non-related PAMs. To gain further insight into the chemical basis of
potentiation, we applied structure-activity relationship strategies involving modification of
the chain length, inversion of substituent positions in the triazole ring and changes in the
aromatic nucleus. Our findings revealed that the phosphonate-functionalized 1,4-
disubstituted 1,2,3-triazole is a novel pharmacophore for the development of therapeutic
agents for neurological and neurodegenerative disorders associated to cholinergic
dysfunction.
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KEYWORDS
Nicotinic receptors; ligand-gated ion channels; patch-clamp; triazole; phosphonates;
click chemistry.
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INTRODUCTION
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The α7 receptor is one of the major nicotinic acetylcholine receptors (nAChR) in
the central nervous system, particularly present in hippocampus, cortex and subcortical
limbic regions. It is involved in the modulation of neural circuits associated to cognition,
memory, learning, attention and sensory gating information1,2. Also, α7 is expressed in
many non-neuronal cells, where it plays a role in immunity, inflammation and
neuroprotection3,4.
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α7 is a pentameric ligand-gated ion channel (pLGIC) that contains an extracellular
domain (ECD), which carries the agonist binding sites at subunit interfaces, a
transmembrane domain (TMD), that forms the ion pore, and an intracellular domain
(ICD). Between ECD and TMD there is a transition zone, composed by several loops,
that is essential to couple agonist binding to channel gating5. Binding of acetylcholine
(ACh) to the receptor triggers the opening of the ion channel permeable to cations
including calcium, which results in rapid membrane depolarization and calcium influx into
the cell3,6. This fast ionotropic response is further prolonged by signal transduction
pathways and the release of calcium from intracellular stores due to the dual
ionotropic/metabotropic nature of α73,4.
Decline in α7 activity has been associated with several neurological,
neurodegenerative and inflammatory disorders, such as Alzheimer’s disease and
schizophrenia. The enhancement of α7 function has, therefore, emerged as a
therapeutic strategy for these disorders2,3,7. Positive allosteric modulators (PAMs) are
promising therapeutic tools, since they exhibit several advantages over orthosteric
agonists, which include conservation of the temporal and spatial pattern of the
endogenous activation by ACh, higher selectivity, less side effects and an enormous
structural diversity3,8,9. Based on their effects on macroscopic currents evoked by ACh,
PAMs have been classified as type I and type II. Both types of PAMs enhance the
agonist-elicited peak currents, but whereas type I PAMs exert minimal or no changes in
desensitization, type II PAMs decrease the desensitization rate and reactivate receptors
from desensitized states3,8,10
.
Both types of PAMs exhibit a wide chemical and structural diversity, ranging from
small molecules to entire proteins11. Their structures include aromatic rings, nitrogen,
oxygen and sulfur-containing heterocycles, donors and acceptors of hydrogen bonds,
etc. Although this broad spectrum of structures constitutes a disadvantage for identifying
a lead structure, it also brings the possibility to explore a wide range of molecules and
functional groups for improving potency, efficacy and selectivity during the development
of novel therapeutic compounds.
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Among the several nitrogen-containing heterocycles present in α7 PAMs
(pyridines, isoxazoles, thiazoles, indoles, etc.) triazoles have been explored only in a few
studies as ligands for nAChRs12,13. However, they have shown a wide range of
pharmacological activities, including antimicrobial, analgesic, anti-inflammatory, local
anaesthetic, anticonvulsant, antineoplastic, antimalarial, antiviral, antiproliferative,
anticancer, antimicotic and antihistaminic activities14,15. In particular, 1,4-disubstituted
1,2,3-triazoles have not been explored in the design and development of α7 PAMs, even
when the structure has been used as scaffold for several drugs in medicinal chemistry
as it can be obtained in a regiospecific manner through the copper(I)-catalyzed azide-
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alkyne cycloaddition (CuAAC)14,16
.
With the aim of developing new synthetic PAMs, we designed and synthetized a
novel series of 1,4-dibustituted 1,2,3-triazoles using supported copper nanoparticles on
activated carbon (CuNPs/C) as cycloaddition reaction catalyst. This kind of catalysts is
readily prepared under mild conditions, and exhibits a high versatility in the
multicomponent click synthesis of 1,2,3-triazoles17,18. We chose an aryl group and a
phosphonate group as substituents in positions 1 and 4, respectively. The phosphonate
group, which is characterized by a stable carbon-phosphorus bond (C-P), was selected
because it is a bioactive functional group poorly exploited and not explored in nAChRs
to date19,20. However, it has a promising therapeutic potential for several human health
disorders and has been included in antibiotics, herbicides, antivirals, glutamate receptors
antagonists, anticancer and antiresorptive agents, acetylcholinesterase (AChE)
inhibitors, metal chelators, etc.19,20
.
The compounds showing functional activity were identified through high-resolution
single-channel and macroscopic current recordings of human α7, which allowed to
determine their potencies and efficacies as well as the mechanisms and structural
determinants underlying potentiation of receptor function. We also applied structure-
activity relationship (SAR) strategies to obtain derivatives by modifying the chain length,
inverting the substituent positions on the triazole ring and varying the aromatic nucleus.
Our findings revealed that the phosphonate-functionalized 1,4-disubstituted 1,2,3-
triazole is a key pharmacophore for the development of new potential therapeutic agents.
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RESULTS AND DISCUSSION
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Design of novel α7 positive allosteric modulators and evaluation of their functional
activities.
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Since potentiation of α7 is emerging as a promising therapeutic strategy for the
treatment of cognitive deficits, schizophrenia, pain and inflammatory processes, the
synthesis of novel α7 ligands is of current relevance. This work combines synthetic
chemistry with electrophysiological recordings of human α7 to generate novel ligands
that modify receptor function, and to reveal their molecular mechanisms of action.
Given that the 1,2,3-triazole ring can act as bioisostere of several nitrogen-
containing heterocycles21, in particular 1,2,4-triazoles22 and isoxazoles23 that are present
in some structures of known α7 PAMs, we evaluated the potential of this scaffold as a
novel PAM for α7. We also included other moieties found in reported α7 PAMs, the
naphthyl group present in TQS10 and the hydroxymethyl group present in JNJ-193094224,
which in turn, allowed us further functionalization into phosphonates (Figure 1).
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Figure 1. Design of 1,4-disubstituted 1,2,3-triazoles as α7 PAMs. The moieties
present in reported α7 PAMs were the naphthyl group of TQS (4-(naphthalen-2-yl)-
3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) and the hydroxymethyl
group of JNJ-1930942 (2-[[4-fluoro-3-(trifluoromethyl)phenyl]amino]-4-(4-pyridinyl)-5-
thiazolemethanol).
Synthesis of 1,4-disubstituted 1,2,3-triazole series I
As shown in Scheme 1, triazole 2 was obtained in high yield (80%) from 1-
(chloromethyl)naphthalene (1) and propargyl alcohol, through the multicomponent
CuAAC in water, using CuNPs/C as catalyst18. Subsequently, compound 2 was treated
with CBr4 and PPh3 in dichloromethane (DCM) to afford bromide 3 (80%). Michaelis-
Becker reaction on compound 3 was performed with two different H-phosphonates, to
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give phosphonates 4a-b in good yields (70-76%). Finally, dimethyl phosphonate 4a was
subjected to hydrolysis with trimethylsilyl bromide (TMSBr) in DCM to give the
corresponding phosphonic acid 5 (85%).
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Scheme 1. Synthesis of 1,4-disubstituted 1,2,3-triazole series I. Reagents and
conditions. i. CuNPs/C (5 mol%), NaN3, H2O, 75°C (82%). ii. CBr4, PPh3, DCM, 0°C - r.t
(80%). iii. (a) NaH, HPO3R2, 0°C (b) 3, 0°C- r.t (70-76%). iv. 4a, TMSBr, DCM, r.t (85%).
Pharmacological activity of triazole derivatives series I on human α7 receptors
To evaluate the potential activity of series I as modulators of human α7, we
performed single-channel recordings in the cell-attached patch configuration from
BOSC23 cells expressing the receptor. In these experiments, the compounds were
applied together with 100 μM ACh, which is close to the EC50 concentration for receptor
activation3. Compounds were evaluated at the micromolar range based on the active
concentrations of α7 PAMs previously described and considering this range as promising
for therapeutic use25.
For α7 receptors, single-channel activity induced by ACh appeared mainly as brief
and isolated openings shown as upward deflections and flanked by long closed periods,
or less frequently as activation episodes named bursts25,26. Bursts consist of a few
opening events in quick succession, which are evoked by a single receptor molecule25,26
(Figure 2). The mean open and burst durations can be obtained from the corresponding
histograms as described in Materials and Methods (Figure 2).
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Figure 2. Effects of the 1,4-disubstituted 1,2,3-triazole series I on α7 receptors at
the single-channel level. Left: Typical traces of α7 channel currents in the presence of
100 M ACh alone or combined with each compound (50 M). Channel openings are
shown as upward deflections. Right: Representative open and burst duration histograms
for each condition. Membrane potential: -70 mV. Filter: 9 kHz.
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α7 PAMs have been classified in two types according to their effects on
macroscopic currents evoked by ACh. However, single-channel recordings are more
sensitive to detecting kinetic changes than macroscopic recordings. At the single-
channel level, potentiation of α7 by both types of PAMs is detected by the increase in
burst and open durations3,27,28. The impact of both types of PAMs on α7 kinetics covers
a wide spectrum of enhanced durations. Type II PAMs produce a more profound
potentiation and, in some cases, they can increase the activation episodes from the sub
millisecond range to the second range. Type I PAMs typically exert more modest effects
on the scale of several milliseconds, which, in turn, make them more suitable and
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promising for therapeutic use3,25,29
.
To identify triazole derivatives with functional α7-PAM activity we measured single-
channel currents in the presence of ACh (100 µM) and each compound (50 µM). Open
and burst duration histograms of α7 in the absence of PAMs were described by the sum
of two exponential components. From the corresponding histograms, we obtained the
durations and relative areas of the components in the absence or presence of the
compounds, and the average values are shown in Table 1. The burst duration
corresponds to the duration of the slowest component of the corresponding histogram
(See Materials and Methods). Potentiation is typically detected as an increase in burst
and open durations. Additionally, an increase in the relative area of the longest duration
open component without changes in its duration may underlie potentiation. However,
relative areas of components are usually more variable among recordings, and the
increase in the proportion of longer opening events leads to a concomitant increase in
burst duration. Thus, in general, the efficacy of α7 PAMs can be more accurately
determined from their capability to increase primarily burst duration and also open
durations3,28,29
.
Compound 2 increased the duration of the brief open component and induced a
small increase in the mean burst duration, indicating slight PAM activity (1.2 times with
respect to the control, Table 1, Figure 2). In contrast, compound 3 did not exert any
statistically significant effect on the single-channel currents elicited by ACh (Table 1).
Open and burst durations histograms were fitted by two exponential components for both
conditions (Figure 2). The fact that compounds 2 and 3 differ only in the presence of a
hydroxyl or a bromide group (Scheme 1) suggests that higher polarity is required for the
potentiating activity. Then, we evaluated the two phosphonates, compounds 4a and 4b,
and the phosphonic acid 5 (Scheme 1).
Compound 4a carrying the methyl phosphonate group exhibited a robust α7 PAM
activity. Open and burst durations histograms were fitted by three exponential
components instead of the two described for the control condition (Figure 2). The open
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time histograms showed that the first open component diminished its area at the expense
of an increase in the area of the second component, and that a novel long duration
component appeared (Table 1). Thus, potentiation was evidenced by a statistically
significant 4-fold increase in the mean open duration (considering the longest duration
component) and by a 7-fold increase in mean burst duration, with respect to the control
condition (Table 1, Figure 2).
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Compound 4b also exerted PAM activity but was less efficacious than 4a. The
histograms of open durations showed the presence of a new component of longer
duration and no statistically significant differences in the durations and areas of the other
two open components with respect to the control condition (Figure 2). The mean open
duration (considering the longest duration component) and the mean burst duration
increased 2.5 and 4.6 times, respectively (Table 1, Figure 2).
Compound 5, carrying the phosphonic acid group, produced a slight potentiation,
which was evidenced only as an increase in the mean burst duration (Table 1, Figure 2).
In this case, the open and burst duration histograms were fitted by two and three
exponential components, respectively.
Table 1. Single-channel parameters of α7 receptors in the presence of compounds
series I.
o1 (ms)
(area)
o2 (ms)
(area)
o3 (ms)
(area)
Series I
n N

burst (ms)
0.07 ± 0.02
(0.90 ± 0.10)
0.16 ± 0.03###
(0.85 ± 0.06)
0.09 ± 0.02
(0.77 ± 0.15)
0.27 ± 0.03
(0.10 ± 0.05)
0.30 ± 0.04
(0.15 ± 0.06)
0.29 ± 0.07
(0.23 ± 0.15)
0.34 ± 0.07
-
-
-
0.32 ± 0.06
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3
3
3
5
3
2
0.42 ± 0.05**
0.34 ± 0.07
3
-
50 0.09 ± 0.04
1.06 ± 0.04***
4a
4b
5
2.26 ± 0.34***
1.46 ± 0.67***
0.50 ± 0.10**
μM (0.64 ± 0.09)## (0.33 ± 0.09)## (0.03 ± 0.01)
0.08 ± 0.01
(0.82 ± 0.09)
0.07 ± 0.01
(0.87 ± 0.05)
0.30 ± 0.09
(0.17 ± 0.09)
0.25 ± 0.06
(0.13 ± 0.05)
0.68 ± 0.12***
(0.01 ± 0.004)
-
Single-channel currents were recorded from cells expressing human α7 wild-type
activated by 100 μM ACh in the absence or presence of the synthetic compounds shown
in Scheme 1. o1, o2 and o3 correspond to the time constants of the open components
determined from fitting the open time histograms. The relative area of each component is
shown in brackets below its duration. The mean burst duration (burst) corresponds to the
time constant of the slowest exponential component of the burst duration histogram.
Values are mean ± SD. n: number of independent experiments, each from different cell
patches. N: number of cell transfections. Statistical analysis was conducted by One Way
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ANOVA followed by Bonferroni’s post hoc tests for multiple comparisons. The symbol *
indicates statistical significance determined by comparing the mean durations of the
slowest components of open and burst duration histograms in the presence of each
#
compound with respect to those in the absence of compounds. The symbol indicates
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statistically significant differences in duration or area among the same components
between experimental and control conditions. The number of symbols (one, two or three)
indicates different significant p-values independently of the type of symbol (For instance,
p<0.05*, p<0.01**, p<0.001***).
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In order to examine the potentiation by phosphonates and the phosphonic acid in
more detail, we measured single-channel currents elicited by 100 μM ACh together with
the synthetic compounds at a broader concentration range, from 5 to 150 μM (Table 2).
For this analysis, we only considered the burst duration and the duration of the slowest
component of the open time histogram (named as open in Table 2) since, as shown
below, these were the parameters that better revealed the potentiating effect.
We found that compound 4a was the most potent and efficacious, exerting its
potentiating effect from 10 μM to 100 μM (Table 2). A slight potentiating effect was
observed at 5 μM in only two out of six recordings, which showed an additional open and
burst components with increased durations (open  0.62 ms and burst  0.86 ms).
Potentiation by compound 4b was evident at 10 μM as a slight increase (1.8 times) in
the mean burst duration (Table 2). However, its maximal positive modulatory activity was
observed at 50 μM (Table 2, Figure 2, Figure S1). In the presence of compound 5, we
detected in only two out of four recordings a modest increase in the mean burst duration
at 5 µM (0.95 ms). The maximal potentiation was achieved at 10 μM and was mainly
observed as an increase of 3.7 times in the burst duration with respect to the control
condition (Table 2, Figure S1). In three out of eight recordings, an additional third
component showing a 2.8 increased duration with respect to that of the slowest open
component of the control condition was detected (0.76 ± 0.31 ms, p<0.001). However,
increasing compound 5 concentration to 50 μM decreased its potentiating effect as the
burst duration was only 1.5-fold longer than that of the control (Table 2). Therefore,
compounds 4b and 5 only exerted their maximal potentiation at one of the tested
concentrations, 50 μM and 10 μM respectively, thus showing a more limited active
concentration range compared to compound 4a.
The three compounds exhibited a lower potentiation ability at the highest
concentrations of their active ranges (Table 2). Although it is not possible to
unequivocally determine from our recordings the cause of this inhibitory effect, a possible
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explanation is that it may be due to channel blockade. The dual action of compounds
mediating opposite effects on nAChR function, such as agonism or potentiation at low
concentrations and open channel blockade at higher concentrations, has been described
for other ligands and arises from binding to different sites25,30–32. Alternatively, the
compounds may have a dual negative allosteric modulator (NAM)-PAM activity. This
possibility appears to be less probable since such a behavior has not been reported
before for other α7 PAMs, and it has been suggested that NAMs compete for the same
allosteric sites of PAMs12,33. Also, we cannot discard from these experiments that the
inhibitory activity of the compound takes place at the same concentration range as that
of its potentiating activity. This may result in competition between potentiation and
inhibition activities and may lead to the underestimation of its efficacy as a PAM.
By comparing the maximal potentiation evoked by compounds 4a, 4b and 5 using
One Way ANOVA followed by Bonferroni’s post-hoc tests, statistically significant
differences were detected in the mean durations of the slowest open component
(p<0.001) and bursts (p<0.01). The maximal enhancement of the channel open duration
by compound 4b in all the recordings was lower than that exerted by compound 4a
(p<0.001). Conversely, compound 5 prolonged openings in only a few recordings, to a
lesser extent than compound 4a (p<0.05) and without significant differences with respect
to compound 4b (p>0.05). Also, compound 5 increased the mean burst duration in a
similar manner to compound 4b (p>0.05). For both compounds the increase in burst
duration was lower than that induced by compound 4a (p<0.05). Thus, considering the
mean burst and open durations as a measurement of the efficacy, the order for the
maximal PAM activity was compound 4a > 4b > 5.
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In our experimental conditions, the main chemical species of the phosphonic acid
group in compound 5 exhibits only one negative net charge, due to a single hydroxyl
group ionized at physiological pH 7.4. There are no charged molecules previously
reported as PAMs, so it is possible that the presence of ionized groups gives origin to
electrostatic forces which impede or hinder hydrophobic interactions in the allosteric site,
leading to the poor effect of compound 5. On the other hand, given that the phosphonates
are not charged, they may be capable of establishing hydrophobic interactions in the
allosteric site and inducing more significant potentiation. It was also noticeable a
decrease in potentiation of 4b with the increase in the aliphatic chain length of the group
esterifying the phosphonic acid. This effect may be due to a higher steric hindrance since
the ability to form hydrophobic interactions does not change substantially.
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Table 2. Single-channel properties of α7 in the presence of 1,2,3-triazoles
functionalized with phosphonic acid or phosphonate groups.
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Compound (μM)
n N

open (ms)
burst (ms)
-
0.27 ± 0.03
0.22 ± 0.06
1.05 ± 0.02***
1.06 ± 0.04***
0.52 ± 0.01***
0.13 ± 0.02***
0.36 ± 0.20
0.32 ± 0.09
0.68 ± 0.12***
0.20 ± 0.03**
0.14 ± 0.04***
0.23 ± 0.06
0.23 ± 0.04
0.25 ± 0.06
0.26 ± 0.01
0.22 ± 0.11
0.32 ± 0.06
0.27 ± 0.09
2.08 ± 0.13***
2.26 ± 0.34***
0.96 ± 0.04***
0.25 ± 0.05
0.33 ± 0.08
0.57 ± 0.06***
1.46 ± 0.67***
0.23 ± 0.06
0.24 ± 0.09
0.27 ± 0.15
1.19 ± 0.39***
0.50 ± 0.10**
0.28 ± 0.06
0.28 ± 0.09
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5
10
4a
4b
5
50
100
150
5
10
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100
150
5
10
50
100
150
Single-channel currents were recorded from cells expressing human α7 wild-type
activated by 100 μM ACh in the absence or presence of compounds 4a, 4b and 5. open
and burst correspond to the time constants of the slowest exponential component of the
corresponding histograms obtained from single-channel recordings. Values are mean ±
SD. n: number of independent experiments, each from different cell patches. N: number
of cell transfections. Statistical significance was determined by One Way ANOVA followed
by Bonferroni’s post hoc tests for multiple comparisons. The symbol * indicates statistical
significance between the longest duration values (open or burst) in the presence of each
compound with respect to those in the absence of compounds (p<0.05*, p<0.01**,
p<0.001***). The structures of compounds 4a, 4b and 5 are shown in Scheme 1.
As the ability to penetrate the blood-brain barrier (BBB) is a requirement for
neurotherapeutic drugs34,35, we also estimated this ability for the active compounds 4a,
4b, and 5 using two different computational methods. Results from the BOILED-egg
method36 (SwissADME platform: http://www.swissadme.ch/) predicted that 4a and 4b but
not phosphonic acid 5 can cross the BBB. Application of the online BBB prediction server
(http://www.cbligand.org/BBB/) yielded scores of 0.110, 0.092, and 0.075 for 4a, 4b and
5, respectively, which are significantly higher than the minimum required for BBB
permeation (0.02)34. Thus, predictive models indicated that our most active compound,
4a, as well as 4b, meet the requirements to cross the BBB.
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To our knowledge, this is the first work exploring phosphonate groups as nAChR
modulators. Some background reports sustained our selection of phosphonate groups
as substituents for the triazole heterocycle because they are present in some AChE
inhibitors, which in turn, are potential candidates to act directly on nAChRs as allosteric
agents. Some of these compounds potentiate nAChR function at low concentrations, but
exert a negative effect due to open-channel blockade at higher concentrations31,32, in
agreement with our observations for some of the 1,2,3-triazole derivatives.
As compound 4a was the synthetic phosphonate-functionalized 1,2,3-triazole
showing the most potent and efficacious PAM activity, we explored its effects at the
macroscopic level and in further detail at the microscopic level, and also used it to
perform SAR studies.
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Detailed characterization of compound 4a as a positive allosteric modulator for α7
In order to determine whether compound 4a behaves as a type I or a type II PAM
on α7 receptors, we evaluated by whole-cell recordings its effects on macroscopic
currents, which result from the activation of all receptors in a cell.
ACh elicited rapid macroscopic currents from cells expressing human α7, which
reached the peak in about 5-10 ms and decayed in the continuous presence of the
agonist due to desensitization (Figure 3A). The current decays were fitted by two
exponential components with time constants of 30-50 ms (fast) and 500-1200 ms (slow
)
under the present experimental conditions. The desensitization rate of α7 receptors
cannot be accurately determined from the decay rate of macroscopic currents because,
as it is extremely fast, its measurement is limited by the speed of the perfusion system.
Nevertheless, a decrease of the desensitization rate as that exerted by type II PAMs is
translated into a decrease of the decay rate, which can be quantified by the increase of
the decay time constants3,25
.
We recorded macroscopic currents evoked by ACh in the absence and presence
of compound 4a (50 μM) in the same cell and averaged from different cells the relative
changes for peak current and net charge, and the absolute values for the decay time
constants. Compound 4a significantly enhanced the peak current in 1.51 ± 0.26 times
(p<0.001, n=7, N=3, Figure 3A). The decay in the presence of the compound was also
fitted by two components, fast = 29 ± 6 ms and slow = 901 ± 649 ms, which did not
markedly change with respect to the components of the control current (fast = 37 ± 7 ms
and slow = 1294 ± 1034 ms). The fast component showed a very slight increase
compared to the control (p=0.03), but the net charge remained without changes (1.02 ±
0.22, p>0.05), with a net charge/peak ratio of 0.68 ± 0.15. Typically, type I PAMs show
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net charge/peak ratios of about one whereas type II PAMs show higher values because
they also decrease the current decay rate, which leads to a large increase of the net
charge. Therefore, compound 4a recapitulates the macroscopic potentiation pattern of
type I PAMs.
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Figure 3. Positive allosteric modulation of compound 4a on α7 receptors. (A)
Representative whole-cell α7-currents evoked by ACh (black traces) and ACh co-applied
with 50 μM compound 4a (grey traces). The horizontal bars over the currents indicate
the application of agonist alone (black) or together with the compound (grey). Holding
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potential: 50 mV. (B) Typical traces of α7 single-channel currents in the presence of 100
M ACh with and without 5-150 M compound 4a. Membrane potential: -70 mV. Filter:
9 kHz. (C) Mean open (open, black) and burst (burst, grey) durations in presence of
different concentrations of compound 4a. The data correspond to the duration of the
slowest component of each corresponding histogram. Data are plotted as mean ± SD.
The number of independent experiments (n) and the number of cell transfections (N) for
each condition are shown in Table 2. Statistical significance was determined by One
Way ANOVA followed by Bonferroni’s post hoc tests for multiple comparisons. The
symbol * indicates statistical significance between the longest duration values (open or
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
burst) in the presence of each compound with respect to those in the absence of
compounds (p<0.05*, p<0.01**, p<0.001***). The structures of compounds 4a, 4b and 5
are shown in Scheme 1.
Figure 3B-C illustrates and summarizes the actions of compound 4a at the
microscopic level. As mentioned above, potentiation started to be evident at 5 μM and
maintained its maximal level in the 10-50 μM concentration range (Figure 3B-C). At 100
μM, the potentiating effect was reduced but still detectable whereas at 150 μM there was
no potentiation and, instead, a slight reduction in the open duration was observed.
For α7 activated by ACh, a wide range of channel amplitudes are detected because
the brief open-channel lifetime does not allow full amplitude resolution27,28. Hence, to
explore if compound 4a alters channel amplitude, we constructed amplitude histograms
only for events longer than 0.3 ms, which are fully resolved in our system. The maximal
single-channel amplitude did not exhibit significant differences in the presence (9.76 ±
0.37 pA) or absence of compound 4a (9.99 ± 0.41 pA, p>0.05, n=4, N=4, Figure 3B), as
described for other α7 PAMs25,28,29
.
Overall, compound 4a increased the macroscopic peak current elicited by ACh
without significantly decreasing the desensitization rate, which is the hallmark of a type
I PAM behaviour. It did not affect the single-channel conductance but prolonged the
duration of openings (4 times) and induced frequent activation in sustained episodes or
bursts (7-fold longer duration). These properties allowed us to classify compound 4a as
the first synthetic phosphonate-functionalized 1,2,3-triazole with type I α7-PAM activity.
Structural determinants of compound 4a potentiation
The locations of α7-PAM binding sites have not been determined unequivocally
since crystal structures of α7 either alone or in complex with modulators have not been
reported yet. However, the use of chimeric and/or mutant receptors has provided an
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approximation of PAM binding sites and has allowed the discovery of amino acids whose
presence is essential for modulation, either at the binding site or in the transduction
pathway. In order to provide further information about the α7-PAM activity of compound
4a, we explored its activity in the chimeric receptor α7-5HT3A (Figure 4A) and in the
quintuple mutant α7 TSLMF receptor (Figure 4B), which constitute valuable approaches
to identify structural determinants of potentiation.
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The chimeric α7-5HT3A receptor carries human α7 sequence in the extracellular
domain, up to the beginning of the first transmembrane domain, and mouse 5-HT3A
sequence thereafter. It has been used as a model of the extracellular domain of α7 as
well as to define domains involved in drug action5,37 (Figure 4A). The chimeric receptor
activated by 500 μM ACh exhibited a slow open component of 5.39 ± 0.18 ms (area 0.20
± 0.10), and a mean burst duration of 13.33 ± 3.16 ms (n=5, N=4). In the presence of 50
μM compound 4a, there were no statistically significant differences in the area and the
duration of the longest openings (5.01 ± 0.56 ms, area 0.16 ± 0.09) and in the mean
duration of bursts elicited by ACh (10.89 ± 3.06 ms, n=4, N=3, p>0.05 in each case,
determined by two-tailed Student’s t-test). The open time and burst duration histograms
were fitted by three exponential components in the absence and presence of compound
4a (Figure 4C).
Recordings of α7-5HT3A were carried out with low calcium solutions to avoid
channel block. This could interfere with potentiation since Ca2+ has been shown to act
as a PAM of α7 and also to enhance potentiation by some modulators38,39. However,
some degree of potentiation could have still been observed in our recording conditions
because, though at lower concentration, Ca2+ was still present in the solution (0.2 mM),
and also because a prototype type I PAM, 5-HI, has been shown to potentiate the same
chimeric receptor under low calcium concentration27,29,40. Thus, despite the fact that other
type I PAMs can modulate positively the chimera29,40, compound 4a was incapable of
potentiating it, suggesting a role of the ECD-TMD interface or the TMD of α7 for
potentiation.
The quintuple mutant α7 TSLMF receptor contains mutations in five residues of
TMD in α7 that inhibit potentiation by type II PAMs, which led to propose the region or
cavity lined by this residues as a PAM binding site41,42 (Figure 4B). However, the
mutations differentially affect potentiation by type I PAMs. Whereas some type I PAMs,
such as NS-1738 and flavonoids, lose their ability to potentiate the quintuple mutant
receptor25,43, others, such as 5-HI, keep such ability29. The open and burst duration
histograms of α7 TSLMF activated by 100 µM ACh were fitted by three exponential
components, which showed no differences in the absence and presence of compound
4a (Figure 4C). In the absence of 4a, the mean duration of the slowest open component
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was 1.17 ± 0.32 ms (area 0.18 ± 0.10), and the mean burst duration was 2.03 ± 0.77 ms
(n=5, N=4). In the presence of 50 μM compound 4a, there were no significant differences
in the open duration and area (1.04 ± 0.20 ms, area 0.17 ± 0.07), and in the burst duration
(1.76 ± 0.05 ms, n=4, N=3, p>0.05 in each case determined by two-tailed Student’s t-
test). In summary, the five transmembrane residues or at least some of them are
essential for potentiation by compound 4a, either by conforming the binding site or by
being involved in the transduction pathway.
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Type II PAMs, and some type I PAMs, such as NS-1738, LY-2087101, ivermectin
and flavonoids, share some of the transmembrane structural determinants25,29,41,43,44. It
has been proposed that PAMs may use overlapping sites at the transmembrane cavity
with type I PAMs binding in an upper position related to type II PAMs13,43,44. For some
PAMs, including type I PAMs as ivermectin44, an important delay in the potentiation has
been detected from macroscopic currents, whereas for compound 4a (Figure 3A) as well
as for other type I PAMs, such as NS-1738 and flavonoids25,29, no significant delays have
been observed. It is therefore possible that the time required for exerting potentiation by
different PAMs may be determined by their locations and dispositions at the overlapping
transmembrane binding sites, by the pathway used to reach their transmembrane sites,
and by their interaction with the lipid membrane, all governed by their chemical
properties.
We showed that compound 4a shares transmembrane structural determinants of
potentiation with other chemically non-related PAMs. This finding suggests a promising
α7 selectivity of compound 4a since the transmembrane residues are not well conserved
among Cys-loop receptors, and most α7 PAMs requiring these residues for their action
do not potentiate or even inhibit other receptors of the family, including 5-HT3A and other
nAChR subtypes10,25,45,46. Nevertheless, future studies are needed to experimentally
determine the selectivity of this novel series of compounds among different
neurotransmitter receptors.
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Figure 4. Structural determinants of potentiation of compound 4a. (A) View of a
chimeric α7-5HT3A subunit5. (B) View of a quintuple mutant α7 TSLMF subunit, showing
the three helices of the TMD (M1, M2 and M3), carrying the five mutations that make α7
insensitive to the prototype type II PAM PNU-12059642 (S223T and A226S in M1, M254L
in M2, and I281M and V288F in M3). (C) Typical traces from single-channel recordings
of α7-5HT3A activated by 500 μM ACh alone or combined with 50 μM compound 4a (top),
and of α7 TSLMF activated by 100 μM ACh alone or combined with 50 μM compound 4a
(bottom). Channel openings are shown as upward deflections. Right: Representative
open and burst duration histograms for each condition. Membrane potential: -70 mV.
Filter: 9 kHz.
Synthesis of 1,4-disubsituted 1,2,3-triazoles series II: SAR studies
On the basis of the biological results obtained for the 1,2,3-triazole series I, we
selected dimethyl phosphonate 4a as lead compound and performed SAR studies in
order to find derivatives with enhanced activity. The strategies employed, shown in
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Scheme 2, can be classified into three categories: i) modification of chain length between
triazole-aryl group and triazole-phosphonate group, i.e. the generation of a homologous
series, ii) inversion of substituent positions in triazole ring, and iii) variation of aryl group
attached to the triazole ring.
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None of these strategies involves the modification of the phosphonate group, which
seemed to be essential for the α7-PAM activity exerted by compound 4a. Synthetic
routes for obtaining these derivatives are shown in Scheme 2, and experimental details
for the synthesis of precursors are described in the Supporting Information.
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Scheme 2. Synthesis of 1,4-disubstituted 1,2,3-triazole series II. (A) Synthesis of
homologues of phosphonate 4a. Reagents and conditions. i. (a) HCl / H2O, (b) NaNO2
(aq.), (c) NaN3, r.t. (90%) ii. CuNPs/C (5 mol%), H2O, NaN3, 70°C. iii. CBr4, PPh3, DCM,
0°C - r.t. iv. P(OMe)3, 110°C. (B) Synthesis of a 4a analogue with inversion of
substituents on triazole ring. Reagents and conditions. i. (H2CO)n , K2CO3, MeOH, 65°C
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(quant.) ii. (a) Tf2O, 2,6-Lut, DCM, -78°C- 0°C (83%). (b) NaN3, DMF, 0°C (83%). iii. (a)
CuI, K2CO3, TBAI, MeCN, 80°C (93%). (b) K2CO3, MeOH, 0°C (quant.). iv. CuNPs/C (10
mol%), TEA, THF, 65°C (54%). (C) Synthesis of benzyl derivative of 4a. Reagents and
conditions. i. CuNPs/C (5 mol%), H2O, NaN3, 70°C (84%). ii. CBr4, PPh3, DCM, 0°C - r.t.
(85%) iii. P(OMe)3, 110°C (67%).
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Generation of a homologous series
To study the effect of the chain length on α7-PAM activity we proposed the
synthesis of two derivatives: phosphonates 10a and 10b from alcohols 8a and 8b,
respectively (Scheme 2A). As previously reported, 8a-b can be obtained through CuAAC
reaction47,48. Compound 10a presents the naphthyl group directly attached to the triazole
ring, modifying both the geometry and electronic distributions of the aromatic ring. On
the other hand, compound 10b presents an elongation of the alkyl chain between the
triazole moiety and phosphonate group.
1-azidonaphthalene (7) was obtained in excellent yield from 1-aminonaphthalene
(6) via diazonium salt (Supporting Information). It was subsequently submitted to click
reaction with propargyl alcohol (2), in presence of CuNPs/C (5 mol%), to give triazole
8a, whereas compound 1 was reacted with 3-butyn-1-ol, in presence of sodium azide
and the same catalyst, to give compound 8b. Both alcohols were transformed in their
corresponding bromides via Appel reaction, and then subjected to Arbuzov reaction with
trimethyl phosphite, affording the desired products 10a and 10b, with higher yields when
compared to Michaelis-Becker reaction.
Inversion of substituent positions in triazole ring
Dimethyl phosphite was added to paraformaldehyde (PFA) in methanol (MeOH) to
afford quantitatively hydroxymethylphosphonate 11 (Scheme 2B), which was converted
in triflate 12 by treatment with Tf2O in presence of 2,6-lutidine in DCM. Reaction of the
triflate derivative with sodium azide give the desired dimethyl (azidomethyl)phosphonate
13 (quant.). On the other hand, alkyne 14 was obtained through copper-promoted
coupling of chloride 1 and trimethylsilylacetylene, and subsequently cleavage of silyl
group, in the presence of K2CO3 (for experimental details see Supporting Information).
Finally, CuAAC reaction between 13 and 14 was carried out in
tetrahydrofuran/triethylamine (THF/TEA), with CuNPs/C as catalyst, affording the
desired phosphonate 15 (54%).
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Variation of aryl group attached to the triazole ring
In order to evaluate the importance of naphthyl group in α7-PAM activity exerted
by 4a, an analogue bearing a benzyl group was synthesized (Scheme 2C). The synthetic
approach employed was similar to the one used for obtaining phosphonate 4a. The main
difference was that for obtaining compound 19, Arbuzov reaction was used instead of
Michaelis-Becker reaction. Synthesis of this derivative started with the multicomponent
click reaction of benzyl bromide 16 with propargylic alcohol, which afforded triazole
1749,50, followed by Appel reaction, yielding bromide 1851 and then reacted with P(OMe)3
to give the desired phosphonate 19.
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Functional characterization of series II compounds on α7 receptors: SAR studies
To evaluate the α7-PAM activity of the synthetized compounds series II we
performed single-channel recordings in cell-attached configuration in BOSC23 cells
expressing human α7 receptor. The compounds were added to the pipette solution at a
concentration of 50 μM together with 100 μM ACh to compare their effects with those of
the original compound 4a at the same concentrations.
In the majority of the recordings, no potentiation of α7 by compound 10a was
detected (Table 3, Figure 5). The open time and burst duration histograms were fitted by
two exponential components and were similar to those of the control condition (Figure
5A). From a total of ten recordings in the presence of compound 10a, only one showed
a slight increase in the duration of the slowest open component ( 1 ms) and three
recordings showed a slight increase of the mean burst duration (1.28 ± 0.27 ms). The
increase of the mean burst duration was statistically significant with respect to the control
condition in the absence of modulators (p<0.05); however, it was detected in less than
30% of the recordings and was lower compared to that exerted by compound 4a
(p<0.05). Overall, the shortening of the triazole-aryl group length in compound 10a
diminished the α7-PAM activity respect to the lead compound.
Compound 10b did not potentiate α7, since there were no significant differences
in the mean open and burst durations with respect to the control condition (Table 3,
Figure 5). Both histograms were fitted by two exponential components, similar to
histograms of control recordings (Figure 5A). Therefore, the elongation of the triazole-
phosphonate chain revoked the α7-PAM activity of the compound 4a.
Compound 15 carrying the inverted triazole exhibited α7-PAM activity, which was
evidenced by increased open-channel lifetime and prolonged bursts (Table 3, Figure 5).
The open time and burst duration histograms were described by three exponential
components (Figure 5A). The first two open components were similar to those of the
control condition, and the main difference was given by the appearance of a third long-
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duration component with low relative area. The duration of this component was
statistically significantly longer than that of the slowest open component of the control
condition (Figure 5B). Also, the mean burst duration was prolonged in the presence of
compound 15 (Figure 5B). Although potentiation was significant, the degree of
potentiation of compound 15 was slightly lower than that exerted by compound 4a (Table
3, Figure 5).
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The analysis of compound 19 revealed that the variation of the aryl group annulled
the α7-PAM activity of the original triazole derivative. The mean open and burst durations
did not show significant differences with the control condition and were shorter than in
the presence of compound 4a (Table 3, Figure 5). Both histograms were fitted by two
exponential components as those described for α7 in the presence of ACh alone (Figure
5A).
Therefore, the inversion of substituent positions in the triazole ring was the only
SAR strategy that preserved α7-PAM activity of the triazole derivatives, although this
activity was slightly lower than that exerted by the original structure. The retention of
PAM activity may be due to the fact that the steric disposition of the substituents on the
triazole ring of compound 15 is still similar to that of compound 4a.
Table 3. Single-channel parameters of α7 receptors in the presence of compounds
series II.
Compound
n
7
4
10
7
4
N
6
3
5
4
3
3

open (ms)
burst (ms)
0.32 ± 0.06
2.26 ± 0.34***
0.30 ± 0.11###
0.28 ± 0.11###
-
0.27 ± 0.03
4a
1.06 ± 0.04***
0.31 ± 0.16###
0.23 ± 0.08###
10a
10b
μM
15
50
0.45 ± 0.13** ### 1.27 ± 0.24*** ###
19
0.29 ± 0.02###
0.37 ± 0.07###
5
Single-channel currents were recorded from cells expressing human α7 wild-type
activated by 100 μM ACh in the absence or presence of synthetic compounds. open and

burst correspond to the slowest component of the corresponding histograms. Values are
mean ± SD. n: number of independent experiments, each from different cell patches. N:
number of cell transfections. Statistical significance was determined by One Way
ANOVA followed by Bonferroni’s post hoc tests for multiple comparisons. The different
symbols indicate statistically significant differences compared to the control in the
absence of compounds (*) and compared to the condition in the presence of compound
4a (#). The number of symbols (one, two or three) indicates different significant p-values
independently of the type of symbol (For instance, p<0.05*, p<0.01**, p<0.001***). The
structures of compounds are shown in Scheme 2.
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Figure 5. Effects of the 1,4-disubstituted 1,2,3-triazole series II on α7 receptors at
the single-channel level. (A) Left: Typical traces of α7 single-channel currents in the
presence of 100 M ACh alone or combined with 50 M of each series II compound.
Channel openings are shown as upward deflections. Right: Representative open and
burst duration histograms for each condition. Membrane potential: -70 mV. Filter: 9 kHz.
(B) Bar chart showing the mean open (open) and burst (burst) durations in presence of
the different compounds. open corresponds to the longest duration component of the
corresponding open time histogram. Data are plotted as mean ± SD. The number of
independent experiments (n) and the number of cell transfections (N) for each condition
is shown in Table 3. Statistical significance was determined by One Way ANOVA
#
followed by Bonferroni’s post hoc tests for multiple comparisons. The symbols * and
indicate comparison with control without compounds and with compound 4a condition
respectively. The number of symbols (one, two or three) indicates different significant p-
values independently of the type of symbol (For instance, p<0.05*, p<0.01**,
p<0.001***).
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The SAR modifications in this work led to compounds lacking positive modulatory
activity or with the same effect as that of the original structure. These results are in
agreement with previous reports demonstrating that minimal changes in the chemical
structure of a modulator can lead to dramatic differences in the pharmacological profile
of the compound, involving the interconversion to allosteric agonists, silent allosteric
modulators (SAMs) and NAMs8,33. In this case, from compound 4a we obtained
compound 15, which retained α7-PAM activity, and compounds 10a, 10b and 19, which
lost this activity. This lack of PAM activity may result from either the conversion to SAM
activity or the loss of the binding capacity of the compound to the allosteric site.
Our study opens doors for future modifications through SAR studies, which are
almost unlimited and offer the possibility to generate a broad range of structures for the
search of promising therapeutic compounds.
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CONCLUSIONS
Positive allosteric modulation of α7 nAChRs has emerged as a promising
therapeutic strategy for neurological, inflammatory and neurodegenerative disorders with
a dysfunction in cholinergic signaling3,7,9. The development of new synthetic PAMs offers
not only the possibility of providing prototype compounds, but also the chance of
improving pharmacological properties, such as efficacy, potency, selectivity, and
reduced side effects. Here, we synthetized a novel series of 1,4-disubstituted 1,2,3-
triazoles with aryl and phosphonates groups, whose biological activities on α7 receptors
were evaluated for the first time by electrophysiological techniques.
In the context of nAChR ligands, there is only one series of PAMs with triazoles in
their structure, in particular the isomer 1,2,4-triazole12,13. In contrast, the isomer 1,2,3-
triazole was explored for the first time in the present work. Triazole features that make
them optimal in medicinal chemistry include the formation of hydrogen bonds, dipole-
dipole and π-π interactions; stability to hydrolysis, metabolic degradation and redox
conditions; and water solubility14. Several triazoles have been reported with a wide range
of biological activities14,15. Another singularity is that substitutions in positions 1 and 4 of
the triazole ring give place, in terms of distance and planarity, to non-classical
bioisosteres of the amide group, one of the most frequent in bioactive drugs14,16. Also,
phosphonates have been unexplored among the nAChR ligands but are present in
several AChE inhibitors20. The unique biological properties of phosphonates lie in their
highly stable C-P bond, which makes them hydrolysis resistant and capable of mimicking
phosphorous-oxygen and carbon-oxygen bonds of endogenous molecules19,20
.
In conclusion, we here designed and synthetized a novel series of 1,4-disubstituted
1,2,3-triazole derivatives with aryl and phosphonates groups that exhibited α7-PAM
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activity, the compound functionalized with the methyl phosphonate group being the most
efficacious one. We deciphered the molecular mechanism of action, classified the most
active compound as a type I PAM, identified transmembrane amino acids required for
potentiation, and applied several SAR strategies from its structure. We propose
phosphonate-functionalized 1,2,3-triazoles not only as a novel pharmacophore with
intrinsic α7-PAM activity, but also as a scaffold for developing new potential therapeutic
agents for neurological diseases.
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MATERIALS AND METHODS
Synthetic procedures
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General. All moisture sensitive reactions were carried out under a nitrogen atmosphere.
Anhydrous tetrahydrofuran was freshly distilled from sodium/benzophenone ketyl and
DCM from CaH2. All starting materials were of the best available grade (Aldrich, Merck)
and were used without further purification. Commercially available copper(II) chloride
dihydrate was dehydrated upon heating in oven (150°C, 60ꢀmin) prior to use for the
preparation of CuNPs. Column chromatography was performed with Merck silica gel 60
(0.040–0.063ꢀμm, 240–400ꢀmesh) and hexane/ethyl acetate (EtOAc), MeOH/DCM, or
MeOH/EtOAc as eluent. Reactions were monitored by thin‐layer chromatography (TLC)
on silica gel plates (60F‐254) visualized under UV light and/or using 5%
phosphomolybdic acid in ethanol. Nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker ARX‐300 spectrometer (300 MHz 1H, 75 MHz 13C, 121 MHz 31P).
Chemical shifts (δ) were reported in parts per million (ppm) from tetramethylsilane (TMS)
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using the residual solvent resonance (CDCl3: 7.26 ppm for 1H NMR, 77.16 ppm for 13
C
NMR). Multiplicities were abbreviated as follows: s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet; brs = broad signal). Coupling constants (J) were reported in Hz.
Melting points (m.p) were uncorrected.
Preparation of the CuNPs/C. Anhydrous copper(II) chloride (135ꢀmg, 1ꢀmmol) was
added to a suspension of lithium (14ꢀmg, 2ꢀmmol) and 4,4′‐di‐tert‐butylbiphenyl (DTBB,
27ꢀmg, 0.1ꢀmmol) in dry THF (2ꢀmL) at room temperature under nitrogen atmosphere.
The reaction mixture, which was initially dark brown, rapidly changed to black, indicating
that the suspension of copper nanoparticles was formed. This suspension was diluted
with THF (10ꢀmL), followed by the addition of activated carbon (0.8ꢀg). The resulting
mixture was stirred for 1ꢀh at room temperature, filtered, and the solid successively
washed with THF (5ꢀmL) and ethanol (10ꢀmL), and then dried under vacuum. The
catalyst prepared in this way did not require dry or inert atmosphere storage and was
used without any pre-treatment in all CuAAC reactions.
General procedure for the 1,3-dipolar cycloaddition catalyzed by CuNPs/C in
water. Sodium azide (72 mg, 1.1 mmol), the organic halide (1 mmol) and the alkyne (1
mmol) were added to a suspension of CuNPs/C (40 mg, 5 mol% Cu) in H2O (2 mL). The
reaction mixture was warmed to 70°C and monitored by TLC until total conversion of the
starting materials. Water (10 mL) was added to the resulting mixture followed by
extraction with EtOAc (3 x 10 mL). The collected organic phases were dried with Na2SO4,
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solvent was removed under vacuum and the product was purified by flash column
chromatography (hexane‐EtOAc) to give the corresponding triazole.
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{1-[(naphthalen-1-yl)methyl]-1H-1,2,3-triazol-4-yl}methanol (2). White solid, (196
mg, 82%). m.p: 132-134 °C. Rf = 0.29 (EtOAc). 1H NMR (CD3CN) δ 8.05 – 7.98 (m, 1H),
7.89 – 7.81 (m, 2H), 7.51 (s, 1H), 7.50 – 7.44 (m, 2H), 7.44 – 7.38 (m, 1H), 7.37 – 7.32
(m, 1H), 5.91 (s, 2H), 4.45 (d, J = 5.1 Hz, 2H), 3.13 (t, J = 5.1 Hz, 1H). 13C NMR (75 MHz,
CD3OD) δ 148.0, 135.4, 132.5, 131.9, 130.8, 129.9, 128.8, 128.00, 127.95, 127.3, 126.5,
124.0, 56.4, 52.9.
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{[1-(naphthalen-1-yl)-1H-1,2,3-triazol-4-yl]methanol (8a). This compound was directly
synthetized from 1-azidonaphthalene. Pale yellow solid (180 mg, 80%), m.p: 109-110
°C. Rf = 0.47 (EtOAc).1H NMR (CDCl3) δ 8.04 – 7.97 (m, 1H), 7.96 – 7.91 (m, 2H), 7.63
– 7.46 (m, 5H), 4.98 (s, 2H), 3.41 (s, 1H). 13C NMR (CDCl3) δ 147.7, 134.1, 133.6, 130.4,
128.5, 128.3, 127.9, 127.1, 125.0, 124.5, 123.6, 122.3, 56.5. Spectroscopic data are in
1
agreement with previously reported H and 13C NMR characterization data for the title
compound47.
2-{1-[(naphthalen-1-yl)methyl]-1H-1,2,3-triazol-4-yl}ethan-1-ol (8b). White solid, (164
mg, 65%). m.p: 145-146.5 °C. Rf = 0.26 (EtOAc).1H NMR (CDCl3) δ 8.01 – 7.93 (m, 1H),
7.93 – 7.86 (m, 2H), 7.57 – 7.49 (m, 2H), 7.49 – 7.40 (m, 2H), 7.17 (s, 1H), 3.87 (c, J =
5.9 Hz, 2H), 2.84 (t, J = 5.9 Hz, 2H), 2.65 (s, 1H). 13C NMR (CDCl3) δ 145.8, 133.9,
131.2, 130.0, 129.9, 128.9, 127.9, 127.3, 126.4, 125.4, 122.9, 121.4, 61.6, 52.3, 28.7.
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Spectroscopic data are in agreement with previously reported H and 13C NMR
characterization data for the title compound48.
(1-benzyl-1H-1,2,3-triazol-4-yl)methanol (17). White solid, (159 mg, 84%). m.p: 74-
75°C. Rf = 0.12 (50% EtOAc/Hexane). 1H NMR (CDCl3) δ 7.45 (s, 1H), 7.36 – 7.29 (m,
3H), 7.26 – 7.21 (m, 2H), 5.45 (s, 2H), 4.70 (s, 2H), 4.17 – 3.69 (s, 1H). 13C NMR (CDCl3)
δ 148.3, 134.6, 129.2, 128.8, 128.2, 121.9, 56.2, 54.2. Spectroscopic data are in
1
agreement with previously reported H and 13C NMR characterization data for the title
compound49,50
.
dimethyl ({4-[(naphthalen-1-yl)methyl]-1H-1,2,3-triazol-1-yl}methyl)phosphonate
(15). In this case, CuAAC was carried out in THF/TEA instead of water. Alkyne 14 (0.05
g, 0.3 mmol) and the organic azide 13 (0.049 g, 0.3 mmol) were added to a suspension
of CuNPs/C (24 mg, 10 mol% Cu) in THF (2 mL) and TEA (42 µL, 0.3 mmol). The reaction
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mixture was warmed to 65°C and monitored by TLC until total conversion of the starting
materials. Water (10 mL) was added to the resulting mixture followed by extraction with
EtOAc (3 x 10 mL). The collected organic phases were dried with Na2SO4, solvent was
removed under vacuum and the product was purified by flash column chromatography
(DCM/MeOH) to give phosphonate 15 as a colourless oil, (54 mg, 54 %). Rf = 0.65 (10%
MeOH/DCM). 1H NMR (CDCl3) δ 8.03 – 7.94 (m, 1H), 7.88 – 7.79 (m, 1H), 7.79 – 7.71
(m, 1H), 7.48 – 7.42 (m, 2H), 7.42 – 7.37 (m, 2H), 7.21 – 7.17 (m, 1H), 4.63 (d, J = 13.0
Hz, 2H), 4.52 (s, 2H), 3.65 (d, J = 10.9 Hz, 6H). 13C NMR (CDCl3) δ 148.2, 134.8, 134.0,
131.7, 128.8, 127.7, 127.0, 126.2, 125.8, 125.7, 124.1, 123.0, 53.65 (d, J = 6.6 Hz),
45.09 (d, J = 156.0 Hz), 30.1. 31P NMR (CDCl3) δ 18.4.
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General procedure for bromination of alcohols (Appel reaction). To a cooled
solution (0°C) of the alcohol (0.5 mmol) and CBr4 (0.6 mmol) in dry DCM (10 mL), PPh3
(0.8 mmol) was slowly added. Then, the reaction mixture was stirred at room temperature
until total conversion of the starting materials (1h, monitored by TLC). The solvent was
removed under vacuum and the residue was purified by flash column chromatography
(hexane‐EtOAc) to give the corresponding bromide.
4-(bromomethyl)-1-[(naphthalen-1-yl)methyl]-1H-1,2,3-triazole (3). Pale yellow solid,
1
(120 mg, 80%). m.p: 108-109°C. Rf = 0.35 (50% EtOAc/Hexane). H NMR (CDCl3) δ
7.98 – 7.86 (m, 3H), 7.56 – 7.50 (m, 2H), 7.50 – 7.46 (m, 1H), 7.46 – 7.42 (m, 1H), 7.34
(s, 1H), 5.96 (s, 2H), 4.47 (s, 2H). 13C NMR (CDCl3) δ 144.9, 134.1, 131.3, 130.3, 129.6,
129.1, 128.2, 127.5, 126.6, 125.5, 122.9, 122.8, 52.6, 21.7.
4-(bromomethyl)-1-(naphthalen-1-yl)-1H-1,2,3-triazole (9a). Orange oil (118 mg,
82%). Rf = 0.71 (50% EtOAc/Hexane). 1H NMR (CDCl3) δ 8.02 (m, 1H), 8.00 – 7.93 (m,
2H), 7.66 – 7.50 (m, 5H), 4.73 (s, 2H). 13C NMR (CDCl3) δ 144.8, 134.3, 133.5, 130.8,
128.41, 128.35, 128.2, 127.3, 125.6, 125.1, 123.7, 122.3, 77.6, 77.2, 76.7, 21.6.
4-(2-bromoethyl)-1-[(naphthalen-1-yl)methyl]-1H-1,2,3-triazole (9b). Pale yellow
solid (115 mg, 73%). m.p: 159-161°C. Rf = 0.61 (50% EtOAc/Hexane). 1H NMR (CDCl3)
δ 7.99 – 7.89 (m, 1H), 7.89 – 7.80 (m, 2H), 7.52 – 7.40 (m, 3H), 7.39 – 7.32 (m, 1H),
7.22 (s, 1H), 5.90 (s, 2H), 3.54 (t, J = 7.0 Hz, 2H), 3.14 (t, J = 7.0 Hz, 2H). 13C NMR
(CDCl3) δ 145.0, 133.8, 131.0, 129.93, 129.87, 128.9, 127.6, 127.2, 126.3, 125.3, 122.8,
52.1, 31.4, 29.4.
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1-benzyl-4-(bromomethyl)-1H-1,2,3-triazole (18). Pale yellow solid (107 mg, 85%).
m.p: 135-136°C. Rf = 0.60 (50% EtOAc/Hexane). 1H NMR (CDCl3) δ 7.49 (s, 1H), 7.40 –
7.35 (m, 3H), 7.31 – 7.25 (m, 2H), 5.51 (s, 2H), 4.54 (s, 2H). 13C NMR (CDCl3) δ 145.1,
134.4, 129.3, 129.0, 128.3, 122.8, 54.4, 21.7. Spectroscopic data are in agreement with
previously reported 1H and 13C NMR characterization data for the title compound51.
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General procedure for the synthesis of phosphonates via Michaelis-Becker
reaction. To a stirred suspension of NaH (2 mmol) in dry dimethylformamide (DMF, 3
mL), H-phosphonate (2 mmol) was slowly added. After 30 min, a solution of the
brominated compound (0.5 mmol) in DMF (2 mL) was added and the mixture was stirred
overnight at room temperature. The reaction was quenched with H2O and extracted with
EtOAc (3 x 10 mL). The organic layers were washed with brine, dried over Na2SO4,
filtered and evaporated. The residue was purified by flash column chromatography
(MeOH/DCM or MeOH/EtOAc) to afford the corresponding phosphonates.
dimethyl ({1-[(naphthalen-1-yl)methyl]-1H-1,2,3-triazol-4-yl}methyl)phosphonate
(4a). Colourless oil (126 mg, 76%). Rf = 0.50 (10% MeOH/DCM). 1H NMR (CDCl3) δ 8.16
– 8.07 (m, 1H), 8.00 – 7.90 (m, 2H), 7.64 (d, J = 2.4 Hz, 1H), 7.61 – 7.52 (m, 2H), 7.52
– 7.47 (m, 1H), 7.47 – 7.40 (m, 1H), 6.00 (s, 2H), 3.63 (d, J = 10.9 Hz, 6H), 3.30 – 3.19
(m, 2H). 13C NMR (CDCl3) δ 139.36 (d, J = 7.8 Hz), 139.3, 134.8, 132.2, 131.9, 130.3,
129.8, 128.5, 127.8, 127.2, 126.5, 124.18 (d, J = 5.0 Hz), 53.39 (d, J = 6.5 Hz), 52.5,
23.35 (d, J = 141.4 Hz). 31P NMR (CDCl3) δ 27.2.
diethyl
({1-[(naphthalen-1-yl)methyl]-1H-1,2,3-triazol-4-yl}methyl)phosphonate
1
(4b). Colourless oil (125 mg, 70%). Rf = 0.64 (10% MeOH/EtOAc). H NMR (CDCl3) δ
7.97 – 7.90 (m, 1H), 7.90 – 7.84 (m, 2H), 7.52 – 7.46 (m, 2H), 7.46 – 7.40 (m, 2H), 7.37
(d, J = 2.3 Hz, 1H), 5.94 (s, 2H), 4.24 – 4.14 (m, 4H), 3.21 (d, J = 20.5 Hz, 2H), 1.14 (t,
J = 7.1 Hz, 6H). 13C NMR (CDCl3) δ 138.7 (d, J = 6.6 Hz), 134.0, 131.2, 130.1, 129.9,
129.0, 128.0, 127.3, 126.4, 125.4, 122.9, 122.7 (d, J = 4.1 Hz), 62.42 (d, J = 6.6 Hz),
52.6, 24.2 (d, J = 142.7 Hz), 16.3 (d, J = 6.0 Hz). 31P NMR (CDCl3) δ 24.72.
({1-[(naphthalen-1-yl)methyl]-1H-1,2,3-triazol-4-yl}methyl)phosphonic acid (5).
TMSBr (0.065 mL, 0.492 mmol) was added to a solution of the phosphonate 4a (0.059
g, 0.164 mmol) in anhydrous DCM (5 mL). After stirring for 5 h at room temperature, the
solution was concentrated under reduced pressure. Then, MeOH (5 mL) was added, and
the mixture was stirred for 30 min at room temperature. The solvent was evaporated,
and the product was dried under vacuum to afford 6 (white solid, 43 mg, 85%). m.p: 150-
29
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