Development of a photoswitchable antagonist of NMDA receptors

Article abstract of DOI:10.1016/j.tet.2017.06.056

N-methyl–Daspartate receptors (NMDARs) are vital for neurological processes such as learning, memory, and synaptic plasticity. As such, small molecules that modulate their function are of interest in the study of numerous neurological diseases. We have synthesized a small library of photoswitches that modulate NMDAR function. The most efficient photoswitch to date is based on a known ligand of the glycine binding site and shows significant subtype selectivity.

Full text of DOI:10.1016/j.tet.2017.06.056

Accepted Manuscript
Development of a photoswitchable antagonist of NMDA receptors
Felix W.W. Hartrampf, David M. Barber, Kevin Gottschling, Philipp Leippe, Michael
Hollmann, Dirk Trauner
PII:
S0040-4020(17)30699-3
10.1016/j.tet.2017.06.056
TET 28821
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 15 May 2017
Revised Date: 26 June 2017
Accepted Date: 27 June 2017
Please cite this article as: Hartrampf FWW, Barber DM, Gottschling K, Leippe P, Hollmann M, Trauner
D, Development of a photoswitchable antagonist of NMDA receptors, Tetrahedron (2017), doi: 10.1016/
j.tet.2017.06.056.
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Development of a photoswitchable antagonist
of NMDA receptors
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Felix W. W. Hartrampf, David M. Barber, Kevin Gottschling, Philipp Leippe, Michael Hollmann and Dirk
Trauner*
Department of Chemistry, New York University, New York, NY 10003

1
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Tetrahedron
journal homepage: www.elsevier.com
Development of a photoswitchable antagonist of NMDA receptors
Felix W. W. Hartrampfa , David M. Barbera, Kevin Gottschlingb, Philipp Leippea, Michael Hollmannb and
Dirk Traunera,c *
a Department of Chemistry and Pharmacology, Ludwig-Maximilians-Universität, München, and Center for Integrated Protein Science, Munich 81377, Germany
bRuhr University Bochum, Department of Biochemistry I – Receptor Biochemistry, Bochum 44780, Germany
cDepartment of Chemistry, New York University, New York, NY 10003
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
N-methyl-D-aspartate receptors (NMDARs) are vital for neurological processes such as
learning, memory, and synaptic plasticity. As such, small molecules that modulate their function
are of interest in the study of numerous neurological diseases. We have synthesized a small
library of photoswitches that modulate NMDAR function. The most efficient photoswitch to
date is based on a known ligand of the glycine binding site and shows significant subtype
selectivity.
Available online
Keywords:
glutamate receptor
antagonist
2017 Elsevier Ltd. All rights reserved
.
azobenzene
photopharmacology
photoswitch
1. Introduction
glutamate itself or well-established (ant-)agonists like MK-801 or
NMDA.7-10 A long-standing goal of our and other groups is the
application of synthetic photoswitches to the optical control of
various targets with light,11-16 with a special focus on reversibly
controlling glutamate receptors using either freely diffusible or
covalently linked ligands.17-21 To date, all three receptor
subclasses have been addressed using freely diffusible
photoswitchable agonists.22-24 Most recently, we developed a
photochromic agonist of NMDA receptors named ATG that
could be used for precise two-photon activation upon irradiation
with 740 nm light.24 While these agonists represent powerful
tools, they create conditions that are not always physiological
(e.g. anomalous overexcitation) and thus may render the analysis
of neuronal networks more complicated. It is therefore
advantageous to develop photoswitchable antagonists of
glutamatergic signaling.25 Herein, we present the design and
Ionotropic glutamate receptors are the workhorses of fast
synaptic transmission and are therefore ubiquitously expressed
throughout the nervous system.1,2 The ionotropic glutamate
receptors are commonly divided into three classes, each named
after a selective agonist mimicking glutamate (1) itself (Figure
1): the kainate (2) receptors, the AMPA receptors (α-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid, 3) and the NMDA
receptors (N-methyl-D-aspartate, 4). Each type is marked by
specific pharmacology: kainate receptors are thought to have
more of a supporting and modulatory role, AMPA receptors are
synthesis of
a small library of photoswitchable NMDA
antagonists using the concepts of azologization26 and azo-
extension as well as their biological evaluation in Xenopus
oocytes, which demonstrated the viability of azo-L-701324 as a
reversible antagonist of NMDA receptors.
Figure 1. Structure of glutamate and selective agonists of different classes
of glutamate channels.
responsible for the majority of excitatory synaptic transmission.
NMDA receptors are both ligand- and voltage-sensitive and play
a major role in coincidence detection related to postsynaptic
depolarization and synaptic glutamate release.3-6 As such, they
are of key importance in synaptic plasticity, the molecular basis
2. Results and discussion
2.1. Chemistry
of memory and learning, as well as in
a range of
neurodegenerative disorders such as Alzheimer’s disease and
Parkinson’s disease. In order to study the molecular
underpinnings of glutamatergic neuronal networks with the high
spatial and temporal precision of light, numerous optical tools
have been developed, most of which are caged variants of either
We first identified NMDA antagonists from the large body of
literature that would be amenable to azologization, i.e. the
replacement of structural units such as diaryl ethers or stilbenes
with azobenzenes, or azo-

2
Reduction of the nitro group with tin in ethanolic HCl gave
ACCEPTED MANUSCRIPT
aniline 17. At this point, simple Mills coupling with
nitrosobenzene gave azobenzene 18, whilst azo coupling with
diethylaniline via the diazonium salt resulted in substituted red-
shifted azobenzene 19.
Figure 2. Diversification sites identified by SAR studies. Red arrows
indicate the azologization or azo-extension site.
extension, the appendage of an azobenzene unit to existing
biologically active compounds. Ideally, these would cover the
various known binding sites of NMDA receptors identified
through classical pharmacology, as a crystal structure of a full
tetrameric NMDA receptor was not published until very
recently.27,28
A survey of NMDA antagonists with well-
established structure-activity relationship (SAR) studies yielded
four scaffolds that featured sites tolerant of substitution with
large hydrophobic residues (Figure 2). Specifically, these were
the α-tertiary amines memantine hydrochloride (5) and
amantadine (6), two channel blockers approved for the treatment
of Alzheimer’s disease, cis-PPDA (7),
a glutamate site
Scheme 2. Synthesis of azo-memantine derivatives 18 and 19.
antagonist,29 and finally L-701324 (8),30 a hydroxyquinolone
targeting the glycine binding site. It was known that the primary
amine of memantine could be coupled to bulky sulfonamides like
dansyl chloride without significantly compromising the binding
ability.31 A range of derivatives of cis-PPDA were investigated,
all of them bearing very bulky aromatic residues such as
phenanthrene, biphenyl or anthracene.29 For L-701324, studies
established that the phenyl ether moiety could be exchanged for
substituted benzyl or aniline groups.30,32,33
For the synthesis of azo-PPDA derivatives, we utilized
precedent from the original paper, in which cis-2,3-
piperazinedicarboxylic acid (21) was coupled under Schotten–
Baumann conditions (Scheme 3).29 When trying to couple this
building block to the azobenzene carboxylic acid chloride
derived from 20, only traces of product 22 could be obtained. We
thus switched to an alkylation approach. To this end, we started
with known cis-piperazinedicarboxylic acid dimethyl ester (23).29
This meso compound could be alkylated with (E)-1-(4-
(bromomethyl)phenyl)-2-phenyldiazene (24)36 to give the
racemic diester 25. This compound could then be saponified with
aqueous LiOH to afford cis-PPDA derivative 26, “azo-PPDA”.
We started the synthesis of the photoswitchable memantine
derivatives from 1-bromo-3,5-dimethyl-adamantane (9), which
could be aminated to formamide 10 and hydrolyzed to
memantine hydrochloride (5) in two steps according to a
literature procedure (Scheme 1).34 A more direct C,H-activation
Ritter-type reaction did not reproducibly yield the desired
product.35
N
O
N
N
H
N
H
(12%)
(61%)
Et3N
R
O
O
S
R=Br
N
H
HCONH2
HCl
R=NHCHO
R=NH3Cl
N
N
N
N
Y
X
N
X=NCO, Y=H
X=SO2Cl, Y=NMe2
Scheme 3. Failed acylation attempts toward azo-PPDA (top)
and successful alkylation approach (bottom) to racemic
bisaminoacid azo-PPDA (26).
In order to access a photoswitchable version of our fourth
azologization template, L-701324, we prepared the amide 30
according to a modified literature procedure. More precisely,
benzylic cyanide 27 was hydrolyzed to 28 in quantitative yield
with sulfuric acid/hydrochloric acid in acetic acid. After
activation of the carboxylic acid to the acid chloride, amide
coupling with methyl 4-chloroanthranilate (29) gave the amide
Scheme 1. Synthesis of azo-amantadine derivatives via
diversification of an aniline building block.
Under standard conditions, it was coupled to p-azobenzene
isocyanate (11) to yield the first NMDA receptor antagonist
candidate 13. A second derivative was obtained by condensation
of 5 to commercially available dabsyl chloride (12), yielding the
corresponding photoswitchable sulfonamide 14. Two more
derivatives were accessed through alkylation of the slightly less
hindered adamantylamine (amantadine, 6) with p-nitrobenzyl
bromide (15) to give the secondary amine 16 (Scheme 2).

3
30 in good overall yield (Scheme 4). Under the reduction
conditions described by Magata, we isolated aniline 31 in low
yield and purity (Table 1).32
With a set of compounds in hand, we looked at their
ACCEPTED MANUSCRIPT
photoswitching properties using UV-Vis spectroscopy. The data
obtained indicated that the azoswitches behaved like regular,
“unbiased” azobenzenes with optimal trans/cis switching
wavelengths between 340 and 360 nm. The exceptions were azo-
memantine 14, whose optimal switching wavelength was
strongly red-shifted (450 nm) due to its push-pull system, and the
amantadine derivative 19, which switched at 440 nm.37 For the
characterization of all other azoswitches, see the supporting
information (Figure S2).
CN
COOH
AcOH
COOMe
O
HCl, H2SO4
(COCl)2
(78%)
(99%)
Cl
COOMe
NH2
N
H
NO2
NO2
NO2
Cl
Scheme 4. Synthesis of nitroaryl 30 through amide coupling
of arylacetic acid 28 with anthranilate 29.
Using sodium sulfide, we only isolated small amounts of
product. We therefore attempted reduction under palladium
catalysis, which resulted in ester hydrolysis and decomposition.
We thus turned to metal reductants such as tin and zinc, which
offered a small improvement. Finally,, we found that zinc in
ethanol with CaCl2 as a mild Lewis acid gave the desired product
in good yield and sufficient purity.
Figure 3. Photoswitching of PNRA in PBS buffer. τ(420 nm)=2.868 ±
0.00674 min; τ(360 nm)=2.868 ± 0.00674 min.
Table 1. Reduction of the nitro group in 30.
2.2. Biological evaluation
Having established the switching wavelengths for our
compounds, we set out to investigate whether they would act as
functional mimics of their parent compounds and, more
importantly, whether they would do so in a light-dependent
manner. As the test system, we chose Xenopus oocytes, as they
had served us well in the biological evaluation of
photoswitchable NMDA receptor agonists as well as AMPA
receptor antagonists due to the high level of heterologous
expression.24,25 Furthermore, we wanted to probe the effect on
different receptor subunit combinations using two-electrode
voltage clamp electrophysiology. Solutions of compounds were
washed in under ambient light (predominantly as the trans-
isomer), then irradiated with 360 or 415 nm light to induce
switching to the cis-state.
reductant
reaction conditions
AcOH, 80 °C
Yield %
Fe
27
Na2S
dioxane, H2O, 80 °C
dioxane, H2O, 100 °C
CH2Cl2, EtOH, rt
25
Na2S
35
Pd/C, N2H4·H2O
decomposition
Sn
10% HCl, EtOH, 80 °C
10% HCl, EtOH, 80 °C
EtOH, H2O, 80 °C
39
50
73
Zn
Zn, CaCl2
We first examined the action of our photoswitchable channel
blockers 13, 14, 18 and 19. The first memantine-derived
antagonist was the unsymmetrical urea 13. Whilst it acted as an
antagonist at 20 µM, the overall block was only about 10% with
a light-dependent current change of only roughly 5%. Dabsyl-
conjugated memantine 14 showed very modest block of GluN1-
1a+GluN2A and hardly any of GluN1-1a+GluN2B at 50 µM,
neither of which were influenced by light. We thus turned to the
amantadine derivatives 18 and 19, each at a concentration of
50 µM. In Xenopus oocytes, 18 showed around 15% block that
was not abridged with light. Essentially the same outcome was
observed with the red-shifted variant 19 that was switched with
410 nm light. Interestingly, while all channel blockers performed
rather poorly overall, some selectivity of GluN1-1a+GluN2A
over GluN1-1a+GluN2B was obtained in all cases. At this stage,
we decided to pursue ligands of other binding sites. The next
compound tested was azo-PPDA (26), whose bisamino acid
portion binds to the glutamate site of NMDA receptors.
Originally designed by Monaghan and coworkers,29 cis-PPDA (7)
was developed as a subtype-selective antagonist that would show
a different selectivity profile. At the time, most antagonists
preferentially bound to GluN2A>GluN2B>GluN2C≈GluN2D
(high to low affinities) whereas cis-PPDA showed the opposite
When we attempted to cyclize compound 31 using base
(K2CO3 or KHMDS) to the hydroxyquinolone as a platform for
diversification, intractable mixtures of highly polar compounds
were found. We thus changed our approach and first coupled
aniline 31 to nitrosobenzene in a Mills reaction. With azobenzene
32 in hand, the cyclization occurred in moderate yield to give
azo-L-701324 (33), which we termed PNRA (Photoswitchable
NMDA Receptor Antagonist) (Scheme 5).
Scheme 5. Synthesis of PNRA (33) via Mills reaction and
cyclization.

4
4. Experimental section
affinity pattern (GluN2C/GluN2D over GluN2A/GluN2B).
However, our azo-modified version of cis-PPDA showed the
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4.1. Chemical methods and materials
opposite selectivity, with little antagonistic effect observed in the
case of NMDA receptors bearing GluN2A/GluN2B subunits, and
no affinity to GluN2C, each at a concentration of 100 µM. Our
final compound, PNRA (33), was investigated next. To our
delight, it was shown to be a strong antagonist of GluN1-
1a+GluN2A at 2 µM, blocking 80% of the glutamate/glycine-
induced current, which could be restored once perfusion of the
compound was stopped (Figure 3). A possible explanation for the
moderately light-dependent antagonism could be the known
tolerance for modification on the right-hand side of L-701324-
like antagonists, hinting at a large binding pocket able to
accommodate both isomers of PRNA.32
All reactions were performed with standard Schlenk
techniques under an atmosphere of nitrogen in oven-dried
glassware (100 °C oven temperature) that was further dried using
a heatgun (set to 650 °C) for all water-sensitive reactions.
Tetrahydrofuran (THF) and diethyl ether (Et2O) were distilled
prior to use from sodium and benzophenone, triethylamine
(Et3N), and dichloromethane (CH2Cl2) were distilled from
calcium hydride. N,N-dimethylformamide (DMF), acetonitrile
(MeCN), and methanol (MeOH) were purchased from Acros
Organics as 'extra dry' reagents under inert gas atmosphere and
over molecular sieves. All other reagents were purchased from
commercial sources and used without further purification.
Reaction progress was monitored by analytical thin-layer
chromatography (TLC), which was carried out using pre-coated
glass plates (silica gel 60 F254) from Merck. Visualization was
achieved by exposure to ultraviolet light (254 nm) where
applicable followed by staining with potassium permanganate
solution. Flash column chromatography was performed using
Merck silica gel (40–63 ꢀm particle size). Proton nuclear
magnetic resonance (1H NMR) spectra were recorded on a Varian
400, Inova 400, Varian 600 or Varian 800 spectrometer.
Chemical shifts (δ scale) are expressed in parts per million (ppm)
and are calibrated using residual protic solvent as an internal
reference (CHCl3: δ = 7.26 ppm, CD2HOD: δ = 3.31 ppm,
DMSO-d5: δ = 2.50 ppm, HOD: δ = 4.79 ppm). Data for 1H
NMR spectra are reported as follows: chemical shift (ppm)
(multiplicity, coupling constants (Hz), integration). Couplings are
expressed as: a = apparent, s = singlet, d = doublet, t = triplet,
q = quartet, m = multiplet, or combinations thereof. Carbon
nuclear magnetic resonance (13C NMR) spectra were recorded on
the same spectrometers at 100, 150 or 200 MHz (±1 MHz
variance). Carbon chemical shifts (δ scale) are also expressed in
parts per million (ppm) and are referenced to the central carbon
resonances of the solvents (CDCl3: δ = 77.16 ppm, MeOH-d4:
δ = 49.00 ppm). In order to assign the 1H and 13C NMR spectra, a
range of 2D-NMR experiments (COSY, HMQC, HMBC,
NOESY) was used as appropriate. Infrared (IR) spectra were
recorded on a Perkin Elmer Spectrum BX II (FTIR System)
equipped with an attenuated total reflection (ATR) measuring
unit. IR data are reported in frequency of absorption (cm−1). Mass
spectroscopy (MS) experiments were performed on a Thermo
Finnigan MAT 95 (electron ionization, EI) or on a Thermo
Finnigan LTQ FT (electrospray ionization, ESI) instrument.
(2S,3R)-piperazine-2,3-dicarboxylic acid (21), dimethyl (2S,3R)-
GluN1-1a + GluN2A - PNRA (2 µM)
glutamate/glycine
PNRA
360 nm
1 µA
20 s
Figure 4. Two-electrode voltage clamp electrophysiology of Xenopus
ooctyes expressing GluN1-1a+GluN2A channels. Evaluation of PNRA (33)
(2 ꢀM) revealed that it can modulate NMDA receptor currents in a light-
dependent fashion in the presence of glutamate (100 ꢀM) and glycine
(10 ꢀM). Trace representative of n = 5 oocytes.
Furthermore, the block of the NMDA current caused by trans-
PNRA could be reversibly reduced when exposed to 360 nm
light. The same was true for GluN1-1a+GluN2C, albeit with a
lower (60%) initial block (Figure S1c). Interestingly,
heterologously expressed GluN1-1a+GluN2B and GluN1-
1a+GluN2D were blocked with much lower efficiency (30%),
although the reduction in antagonism upon illumination with
360 nm light was rather similar (Figure S1b and S1d). This
selectivity profile is rather unusual, as alluded to before. To the
best of our knowledge, PNRA is the first NMDA antagonist
displaying higher affinity for GluN2A and GluN2C over GluN2B
and GluN2D. As such, it could be used as a starting point for the
development of other, even more subtype-specific NMDAR
antagonists.
piperazine-2,3-dicarboxylate
(23)
and
(E)-1-(3-
(bromomethyl)phenyl)-2-phenyldiazene (24) were prepared
3. Conclusion
according to literature procedures.29,36
4.2. Biological methods
In summary, we have synthesized and characterized a series of
azobenzene photoswitches for the optical control of NMDA
receptors and evaluated them in vitro. From this work, azo-L-
701324 or PNRA (35) emerged as a viable photochromic
antagonist of NMDA receptors that shows subtype selectivity for
GluN2A and GluN2C over GluN2B and GluN2D. It is an active
blocker in its dark-adapted trans-state and can be switched into
its less active cis-state using near-UV light. Future research will
focus on the development of red-shifted and other variants to
increase the light-dependent current change, enabling the study of
NMDA receptors in more advanced biological systems like acute
mouse brain slices and whole-cell patch-clamp electrophysiology
of hippocampal CA1 neurons.
cRNA was synthesized from cDNA clones of GluN1-1a
(GenBank accession number U08261), GluN2A (NM_012573),
GluN2B (U11419), GluN2C (NM_012575), GluN2D
(NM_022797), GluA1(Q)flip (P19490.2), GluA1(Q)flop
(P19490.1), GluA2(R)flip (P19491.2), and GluK2(P42260)
subcloned in the Xenopus laevis oocyte expression vector
pSGEM using the T7 mMESSAGE mMACHINE Kit (Ambion).
Synthesized cRNA was isolated via the Clean & Concentrator 25
kit (Zymo), the quality of the cRNA controlled via denaturating
agarose gel electrophoresis and the concentration, after
photometrical determination, adjusted to 400 ng·µL−1 with
nuclease-free water. All animal procedures were performed in
accordance with the Tierschutzgesetz (TierSchG) and the

5
Tierschutz-Versuchstierverordnung (TierSchVersV). Xenopus
oocytes of stages V or VI were obtained by surgical removal of
the ovaries of a Xenopus laevis frog previously anesthetized with
ethyl 3-aminobenzoate methanesulfonate (2.3 gL−1; Sigma). The
ovaries were cut and under constant shaking incubated with 300
U·mL−1 (10 mg·mL−1) collagenase type I 44 (Worthington
Biochemicals) for 3 h at 21 °C in Ca2+-free Barth's solution (in
and cooled to 0 °C. The second solution was added dropwise to
ACCEPTED MANUSCRIPT
the first containing p-azobenzene isocyanate (11) and the
resulting reaction mixture was stirred for 17 h. The solvent was
removed by rotary evaporation and the residue was redissolved in
EtOAc (40 mL). The organic phase was washed with H2O
(15 mL) and saturated aqueous NaCl (25 mL), dried over MgSO4
and the solvent was removed by rotary evaporation. Two
purifications by column chromatography (pentane:EtOAc 7:1 and
15:1) yielded urea 13 (50 mg, 0.12 mmol, 12%) as an orange
solid. Rf 0.49 (hexanes:EtOAc 5:1). 1H NMR (400 MHz, CDCl3)
δ = 7.98 (d, J = 8.6, 2H), 7.94–7.87 (m, 4H), 6.76 (d, J = 8.9,
2H), 4.52 (s, 1H), 3.12 (s, 5H), 2.10–2.02 (m, 1H), 1.64–1.60 (m,
2H), 1.55–1.38 (m, 4H), 1.30–1.16 (m, 4H), 1.07 (s, 2H), 0.78 (s,
6H). 13C NMR (101 MHz, CDCl3) δ = 153.7, 152.8, 148.2,
142.1, 130.7, 129.2, 124.4, 122.8, 119.4, 53.4, 50.7, 48.3, 42.8,
40.9, 32.6, 30.2. IR (ATR) 3324, 2900, 1746, 1652, 1541, 1454,
1303, 1220, 1142, 1098, 1017, 843, 763, 685. HRMS (ESI)
Calculated for C26H34N4O2S 466.2402, found 466.2393. Mp 184–
185 °C.
mM: 88 NaCl, 1.1 KCl, 2.4 NaHCO3, 0.8 MgSO4, 15 HEPES, pH
adjusted to 7.6 with NaOH). The Ca2+-sensitive collagenase
reaction was stopped by rinsing with Barth's solution with (in
mM: 88 NaCl, 1.1 KCl, 2.4 NaHCO3, 0.8 MgSO4, 0.4 CaCl2, 0.3
Ca(NO3)2, 15 HEPES, pH adjusted to 7.6 with NaOH). Oocytes
were kept in Barth's medium supplemented with 100 µg·mL−1
gentamycin, 40 µg·mL-1 streptomycin, and 63 mg·mL−1
penicillin. Oocytes of stages V or VI were selected and injected
with cRNA within 8 h after surgery using a Nanoliter 2010
injector (WPI). For the expression of the heteromeric NMDA
receptors, 4 ng (10 nL) GluN1-1a cRNA were coinjected with
one of the following: 7 ng (17 nL) GluN2A, or 6 ng (15 nL)
GluN2B, or 5 ng (13 nL) GluN2C, or 5 ng (13 nL) GluN2D.
Electrophysiological recordings were performed on days 4–7.
4.3.3. N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-
yl)-4-((E)-(4-(dimethylamino)phenyl)diazenyl)-
benzenesulfonamide (14)
Two-electrode voltage clamping was performed using
a
TurboTec-10CX amplifier (npi electronic) controlled by Pulse
software (HEKA). Borosilicate glass capillaries (Harvard
Instruments) were pulled to resistances of 0.1–1.0 Mꢁ and filled
Memantine hydrochloride (5) (43 mg, 0.20 mmol, 1.0 eq) was
dissolved in CH2Cl2 (3 mL) and Et3N (84 µL, 0.60 mmol, 3.0 eq)
was added before the reaction mixture was cooled to 0 °C.
Dabsyl chloride (12) (68 mg, 0.21 mmol, 1.05 eq) was added,
whereupon the reaction mixture turned dark red. After 30 min,
the solution was warmed to room temperature and stirred for
14 h. At this point CH2Cl2 (15 mL) was added and the organic
phase was washed with H2O (15 mL), saturated aqueous
NaHCO3 (15 mL) and saturated aqueous NaCl (15 mL). The
organic phase was dried over MgSO4 and the solvent was
removed by rotary evaporation. Purification by column
chromatography (pentane:EtOAc 5:1) yielded sulfonamide 14 as
with 3
were performed in barium Ringer’s solution (BaR, in m
NaCl, 2.5 KCl, 1.8 BaCl2, 10 HEPES-NaOH, pH 7.2)
supplemented with 250 m niflumic acid (NFA) to prevent the
opening of endogenous calcium-induced chloride channels. For
the recording of the NMDA receptor currents 100 µ glutamate
and 10 µ glycine were used. The photoswitches were irradiated
M
KCl. Oocytes were clamped at 70 mV. All recordings
M
: 115
M
M
M
with high-power fiber-coupled Prizmatix LED (360 and 415 nm)
after wash-in. Before perAll compounds were handled under
ambient light conditions without precautions to exclude light.
a
dark red solid (57 mg, 0.12 mmol, 61%). Rf 0.28
(hexanes:EtOAc 3:1). Dark-red solid. 1H NMR (400 MHz,
CDCl3) δ = 7.98 (d, J = 8.6, 2H), 7.94–7.87 (m, 4H), 6.76 (d,
J = 8.9, 2H), 4.52 (s, 1H), 3.12 (s, 5H), 2.10–2.02 (m, 1H), 1.64–
1.60 (m, 2H), 1.55–1.38 (m, 4H), 1.30–1.16 (m, 4H), 1.07 (s,
2H), 0.78 (s, 6H). 13C NMR (101 MHz, CDCl3) δ = 155.3, 153.2,
143.7, 143.5, 128.0, 125.8, 122.5, 111.6, 56.9, 50.3, 49.4, 42.3,
41.5, 40.5, 32.8, 30.2, 30.0. IR (ATR) 3473 w, 3288 w, 2958 m,
2928 m, 2358 w, 1815 w, 1690 s, 1478 m, 1454 m, 1416 m, 1366
m, 1299 m, 1247 m, 1166 m, 1056 w, 889 w. HRMS (ESI)
Calculated for C26H34N4O2S 466.2402, found 466.2393. Mp 178–
180 °C.
4.3. Synthetic procedures and characterization data
4.3.1. (1r,3R,5S,7r)-3,5-dimethyladamantan-1-
aminium chloride (memantine hydrochloride) (5)
Bromide 9 (4.03 g, 16.6 mmol, 1.00 eq) was dissolved in
formamide (17 mL) and heated to 125 °C for 14 h, at which point
GC-MS indicated full conversion, then cooled to room
temperature. The mixture was diluted with H2O (50 mL), then
extracted with CH2Cl2 (2 × 80 mL). The combined organic
phases were washed with concentrated aqueous NaCl (100 mL),
then dried and concentrated to give a yellow oil that was directly
taken on to the next step. Crude 10 was dissolved in concentrated
aqueous HCl (35 mL) and heated to 100 °C, resulting in the slow
formation of a voluminous precipitate. Ater 5 h, the mixture was
cooled to room temperature and concentrated. The residue was
washed with ice-cold Et2O (60 mL) and dried to give 5 as a
colorless solid (2.28 g, 10.6 mmol, 64% over two steps). 1H
NMR (400 MHz, CDCl3) δ = 2.29–2.19 (m, 1H), 1.72 (d,
J = 3.3, 2H), 1.50 (q, J = 12.0, 11.4, 3H), 1.42 (ad, J = 2.8, 4H),
1.23 (aq, J = 13.5, 12.7, 2H), 0.93 (s, 6H).Analytical data
matched the literature values.34
4.3.4. (3s,5s,7s)-N-(4-nitrobenzyl)adamantan-1-
amine (16)
To adamantylamine (6) (302 mg, 2.00 mmol, 1.00 eq) in
MeCN (2.5 mL) was added K2CO3 (373 mg, 2.70 mmol, 1.35 eq)
and p-nitrobenzyl bromide (15) (389 mg, 1.80 mmol, 0.90 eq).
The resulting suspension was stirred at 80 °C for 24 h before it
was cooled to room temperature and filtered through a plug of
celite. The solution was concentrated and purified by column
chromatography (hexanes:EtOAc 5:1) to yield 16 (363 mg,
1.27 mmol, 70%) as an off-white solid. Rf 0.11 (hexanes:EtOAc
4.3.2. 1-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-
yl)-3-(4-((E)-phenyldiazenyl)phenyl)urea (13)
1
5:1). Off-white solid. H NMR (400 MHz, CDCl3) δ = 8.16 (d,
J = 8.7, 2H), 7.53 (d, J = 8.4, 2H), 3.87 (s, 2H), 2.09 (d, J = 4.4,
3H), 1.74–1.58 (m, 12H). HRMS (ESI): Calculated for
C17H23N2O2 287.1754, found 287.1754. The data matched
literature values.38
4-(phenyldiazenyl)aniline (197 mg, 1.00 mmol, 1.00 eq) was
dissolved in dry CH2Cl2 and the solution was cooled to 0°C
before Et3N (420 µL, 3.00 mmol, 3.00 eq) and triphosgene
(148 mg, 500 µmol, 0.50 eq) were added. The reaction mixture
was stirred for 6 h. In a separate vial, a solution containing
memantine hydrochloride (5) (215 mg, 1.00 mmol, 1.00 eq) and
Et3N (420 µL, 3.00 mmol, 3.00 eq) in dry CH2Cl2 was prepared
4.3.5. (3s,5s,7s)-N-(4-aminobenzyl)adamantan-1-
amine (17)

6
Nitroaryl 16 (0.14 g, 0.50 mmol, 1.0 eq) was dissolved in
EtOH (5 mL) before tin(II) chloride (95 mg, 0.80 mmol, 1.6 eq)
and aqueous HCl (2 M, 4.5 mL) were added, turning the colorless
solution bright yellow. After stirring at 80 °C for 15 h, the
mixture was cooled to room temperature before saturated
aqueous NaHCO3 (10 mL) was added. The solution was
extracted with CH2Cl2 (3 × 15 mL), then washed with saturated
aqueous NaCl (15 mL), dried over MgSO4 and concentrated. The
off-white solid 17 (0.10 g, 0.40 mmol, 80%) thus obtained was
redissolved in EtOAc (40 mL). The organic phase was washed
ACCEPTED MANUSCRIPT
with H2O (2 × 10 mL), HCl (1 M, 20 mL), NaOH (1 M, 20 mL),
H2O (15 mL) and saturated aqueous NaCl (15 mL). The solvent
was removed and the crude product was purified by column
chromatography (CH2Cl2:MeOH 49:1 to 9:1) to give 20 (227 mg,
1.00 mmol, 50%) as an orange solid. Rf 0.44 (hexanes:EtOAc
1
9:1). H NMR (400 MHz, CDCl3) δ = 8.66 (s, 1H), 8.24 (d,
J = 7.6, 1H), 8.18 (d, J = 7.7, 1H), 7.97 (d, J = 7.6, 2H), 7.65 (t,
J = 7.8, 1H), 7.59–7.49 (m, 3H). 13C NMR (151 MHz, CDCl3)
δ = 170.8, 152.8, 152.5, 132.3, 131.7, 130.4, 129.5, 129.3, 127.9,
124.9,123.2. IR (ATR) 2822, 2563, 1889, 1676, 1585, 1421,
1308, 1211, 1160, 1077, 917, 762, 680. HRMS (EI) Calculated
for C13H11N2O2 226.0742, found 226.0729. Mp 169–170 °C. The
data matched literature values.36
1
deemed pure enough for further transformations. H NMR (400
MHz, CDCl3) δ = 7.14 (d, J = 7.9, 2H), 6.64 (d, J = 8.2, 2H),
3.66 (s, 2H), 3.59 (s, 2H), 2.09 (s, 3H), 1.77–1.60 (m, 12H). The
data matched literature values.38
4.3.6. (3s,5s,7s)-N-(4-((E)-henyldiazenyl)benzyl)-
adamantan-1-amine (18)
4.3.9. dimethyl cis-1-(3-((E)-phenyldiazenyl)-
benzyl)piperazine-2,3-dicarboxylate (25)
Amine 17 (51 mg, 0.20 mmol, 1.0 eq) was dissolved in
CH2Cl2 (0.5 mL) in an open flask. Glacial AcOH (0.5 mL) and
nitrosobenzene (23 mg, 0.21 mmol, 1.0 eq) were added. The
flask was sealed with a septum and the solution was heated to 40
°C, whereupon it turned dark brown. After 20 h the reaction was
cooled to room temperature, the solvent was removed by rotary
evaporation and the crude product was purified by column
chromatography (CH2Cl2:MeOH 12:1). Further purification by
preparative TLC (CH2Cl2:MeOH 7:1) yielded azobenzene 18
(17 mg, 50 mmol, 40%) as an orange solid. Rf 0.29
To a solution of dimethyl cis-piperazine-2,3-dicarboxylate
(24) in THF (1.0 mL) was added Et3N (20 µL, 0.15 mmol,
1.0 eq) at 0 °C. (E)-1-(3-(bromomethyl)phenyl)-2-phenyldiazene
in THF (0.50 mL) was subsequently added dropwise and the
mixture was stirred for 30 min at this temperature before the
cooling bath was removed. The mixture was stirred for 16 h
before it was concentrated in vacuo. The residue was redissolved
in EtOAc (25 mL), then washed with H2O (5 mL) and
concentrated aqueous NaCl (5 mL), dried over MgSO4 and
concentrated. The crude product was purified by column
chromatography (pentane:EtOAc 1:1 to 0:1) to yield azobenzene
25 (33 mg, 83 µmol, 55%) as a red oil. Rf 0.50 (CH2Cl2:MeOH
1
(hexanes:EtOAc 5:1). H NMR (400 MHz, CDCl3) δ = 7.94–
7.78 (m, 4H), 7.57–7.41 (m, 5H), 3.86 (s, 2H), 2.10 (t, J = 3.3,
3H), 1.76 (d, J = 2.8, 6H), 1.73–1.58 (m, 7H). 13C NMR (101
MHz, CDCl3) δ = 152.7, 151.7, 130.9, 129.1, 128.8, 123.0, 122.8,
120.9, 120.4, 44.7, 42.3, 36.6, 29.6. IR (ATR) 2900, 2848, 1453,
1356, 1308, 1137, 828, 685. HRMS (ESI) Calculated for
C23H29N3 346.2278, found 346.2275. Mp 110–112 °C.
1
9:1). H NMR (400 MHz, CDCl3) δ = 7.96–7.86 (m, 3H), 7.85–
7.78 (m, 1H), 7.56–7.42 (m, 5H), 4.00–3.83 (m, 4H), 3.73 (s,
3H), 3.73 (s, 3H), 3.12–3.00 (m, 1H), 2.96–2.81 (m, 2H), 2.60–
2.48 (m, 1H), 2.17 (s, 1H). 13C NMR (101 MHz, CDCl3)
δ = 171.1, 171.0, 153.0, 152.7, 139.9, 131.3, 131.1, 129.2, 123.1,
122.9, 121.8, 62.1, 60.8, 60.0, 52.3, 51.5, 46.6, 45.4. IR (ATR)
3349, 2950, 1738, 1600, 1434, 1357, 1200, 1148, 1076, 1034,
1013, 910, 797, 764, 660. HRMS (ESI) Calculated for
C21H25N4O4 397.1870, found 397.1875.
4.3.7. (3s,5s,7s)-N-(4-((E)-(4-(diethylamino)-
phenyl)diazenyl)benzyl)adamantan-1-amine (19)
Amine 17 (52 mg, 0.20 mmol, 1.0 eq) was dissolved in MeOH
(0.5 mL) and H2O (0.5 mL) and cooled to 0 °C before
concentrated HCl (63 µL, 0.60 mmol, 3.0 eq) was added. NaNO2
(15 mg, 0.22 mmol, 1.1 eq) dissolved in H2O (0.1 mL) was then
added dropwise, turning the reaction mixture bright yellow. After
40 min at 0 °C, NaOAc (98 mg, 1.2 mmol, 6.0 eq) was added,
followed by N,N-diethylaniline (25 µL, 0.20 mmol, 1.0 eq). At
this point, the reaction was slowly warmed to room temperature.
After 15 h, EtOAc (10 mL) was added and the phases were
separated. The organic phase was washed with H2O (5 mL),
NaOH (2 M, 5 mL) and concentrated aqueous NaCl (5 mL),
dried over MgSO4 and concentrated. The crude product was
purified by column chromatography (pentane:EtOAc 1:1) to
yield azobenzene 19 (17 mg, 50 mmol, 40%) as an orange solid.
4.3.10. cis-1-(3-((E)-phenyldiazenyl)benzyl)-
piperazine-2,3-dicarboxylate (26)
To a solution of 25 in THF (1.0 mL) was added LiOH (41 mg,
0.96 µmol, 12 eq) in H2O (0.5 mL) at room temperature. The
orange solution was stirred for 36 h before it was treated with
aqueous HCl (2 M, 0.5 mL) until pH ≈ 3. The mixture was
concentrated to half volume, diluted with H2O (2 mL), and
concentrated to half volume again. The suspension was filtered
and the orange residue washed with ice-cold water and Et2O to
yield azobenzene 26 (14 mg, 38 µmol, 40%) as an orange-red
solid.
1
Rf 0.39 (CH2Cl2:MeOH 9:1). H NMR (600 MHz, CDCl3)
1H NMR (400 MHz, MeOH-d4) δ = 8.03–7.80 (m, 4H), 7.65–
7.43 (m, 5H), 4.34 (d, J = 3.9, 1H), 4.22 (d, J = 4.0, 1H), 4.19 (d,
J = 13.7, 1H), 3.94 (d, J = 13.7, 1H), 3.37 (d, J = 12.6, 1H), 3.22
(td, J = 12.4, 4.0, 1H), 2.93 (dd, J = 13.4, 3.6, 1H), 2.82 (td,
J = 12.9, 3.0, 1H). 13C NMR (101 MHz, MeOH-d4) δ = 171.5,
168.7, 154.2, 153.9, 140.4, 132.9, 132.4, 130.5, 130.3, 124.2,
123.8, 123.2, 60.3, 60.0, 58.0, 44.6, 44.0. IR (ATR) 3079, 2925,
2339, 1748, 1643, 1459, 1380, 1240, 1203, 1159, 1107, 1056,
998, 905, 829, 771, 694. HRMS (ESI) Calculated for C19H21N4O4
369.1557, found 369.1561. Mp 180–188 °C.
δ = 7.84 (d, J = 9.2, 2H), 7.78 (d, J = 8.4, 2H), 7.47–7.39 (m,
2H), 6.75–6.67 (m, 2H), 3.82 (s, 2H), 3.45 (q, J = 7.1, 4H), 2.10
(q, J = 2.9, 3H), 1.73 (d, J = 2.8, 6H), 1.72–1.61 (m, 7H), 1.23 (t,
J = 7.1, 6H). 13C NMR (151 MHz, CDCl3) δ = 152.2, 150.0,
143.1, 128.7, 125.1, 122.2, 110.9, 44.9, 44.7, 42.9, 36.7, 29.6,
12.6. IR (ATR) 2972, 2899, 2845, 1597, 1513, 1393, 1349, 1137,
1096, 1008, 823, 812. HRMS (ESI) Calculated for C27H36N4
416.2934, found 416.2931. Mp 172–174 °C.
4.3.8. 3-(phenyldiazenyl)benzoic acid (20)
4.3.11. 2-(3-nitrophenyl)acetic acid (28)
3-aminobenzoic acid (274 mg, 2.0 mmol, 1.0 eq) was
dissolved in MeOH (10 mL). AcOH (0.34 mL, 6.0 mmol, 3.0 eq)
and nitrosobenzene (214 mg, 2.0 mmol, 1.0 eq) were added,
turning the reaction mixture dark brown. After heating to 50 °C
for 22 h, the solvent was removed and the residue was
2-(3-nitrophenyl)acetonitrile (27) (1.95 g, 12.0 mmol, 1.00 eq)
was dissolved in H2O (7 mL) before concentrated H2SO4 (7 mL)
and AcOH (7 mL) were added dropwise. The solution was stirred
at 110°C for 5 h. After cooling to room temperature, H2O

7
(40 mL) was added carefully. The solution was extracted with
(hexanes:EtOAc 6:1) to give 32 as an orange solid (0.13 g,
0.32 mmol, 63%).
ACCEPTED MANUSCRIPT
EtOAc (3 × 50 mL), then washed with H2O (90 mL) and
saturated aqueous NaCl (90 mL), dried over MgSO4 and
concentrated. The off-white solid 28 (2.15 g, 11.9 mmol, 99%)
1
Rf 0.51 (hexanes:EtOAc 5:1). H NMR (400 MHz, CDCl3)
δ = 11.22 (s, 1H), 8.84 (d, J = 2.0, 1H), 8.01–7.81 (m, 5H), 7.64–
7.38 (m, 5H), 7.04 (dd, J = 8.7, 2.0, 1H), 3.88 (s, 2H), 3.84 (s,
3H). 13C NMR (101 MHz, CDCl3) δ = 169.8, 168.0, 153.1,
152.7, 142.3, 141.0, 135.2, 132.1, 131.9, 131.2, 129.7, 129.2,
123.8, 123.1, 123.0, 122.5, 120.4, 113.5, 52.6, 45.6. IR (ATR)
3246 w, 3119 w, 2918 w, 1693 s, 1580 s, 1512 m, 1252 s, 1236 s,
1098 m, 759 s, 690 s. HRMS (ESI) Calculated for C22H19ClN3O3
408.1109, found 408.1118. Mp 89–92°C.
1
thus obtained was pure enough for further transformations. H
NMR (400 MHz, CDCl3) δ = 8.17 (d, J = 7.0, 2H), 7.63 (d,
J = 7.6, 1H), 7.58–7.48 (m, 1H), 3.79 (s, 2H). The data matched
literature values.39
4.3.12. methyl 4-chloro-2-(2-(3-nitrophenyl)-
acetamido)benzoate (30)
2-(3-nitrophenyl)acetic acid (28) (1.62 g, 9.00 mmol, 2.00 eq)
was dissolved in CH2Cl2 (28 mL) and cooled to 0 °C before
oxalyl chloride solution (2M, 5.90 mL, 11.7 mmol, 2.60 eq) was
added dropwise over 30 min, followed by addition of DMF
(70 µL, 0.90 mmol, 0.20 eq) over 2 min. The solution was
warmed to room temperature and stirred for 5 h. After this time,
the solvent was removed in a nitrogen stream. The residue was
redissolved in 1,2-dichloroethane (30 mL) and methyl 2-amino-
4-chlorobenzoate (29) (835 mg, 4.50 mmol, 1.00 eq) was added
in one portion. The dark-yellow solution was stirred at 80 °C for
15 h, then cooled to room temperature. Saturated aqueous
NaHCO3 (20 mL) was added and the solution was extracted with
CH2Cl2 (3 × 60 mL), then washed with H2O (50 mL) and
saturated aqueous NaCl (90 mL), dried over MgSO4 and
concentrated. The residue was purified by column
chromatography (hexanes:EtOAc 4:1) to yield 30 (1.18 g,
3.38 mmol, 75%) as a white solid. Rf 0.38 (hexanes:EtOAc 3:1)
1H NMR (400 MHz, CDCl3) δ = 11.32 (s, 1H), 8.79 (d, J = 2.1,
1H), 8.25 (d, J = 2.0, 1H), 8.19 (dt, J = 8.2, 1.5, 1H), 7.94 (d,
J = 8.6, 1H), 7.72 (d, J = 7.6, 1H), 7.56 (t, J = 7.9, 1H), 7.07 (dd,
J = 8.6, 2.1, 1H), 3.89 (s, 3H), 3.88 (s, 2H). The data matched
literature values.32,39
4.3.15. (E)-7-chloro-4-hydroxy-3-(3-
(phenyldiazenyl)phenyl)quinolin-2(1H)-one,
PNRA (33)
To a solution of 32 (62 mg, 0.15 mmol, 1.0 eq) in THF
(1.0 mL) was added KHMDS (0.5 M in PhMe, 0.90 mL,
0.45 mmol, 3.0 eq) dropwise at 0 °C, turning the solution dark-
red. The cooling bath was removed and the mixture was stirred at
room temperature. After 2.5 h, MeOH (2 mL) was added,
followed by careful addition of TFA until pH = 8 and EtOAc
(20 mL). The organic phase was washed with concentrated
aqueous NaCl (10 mL), dried and evaporated to give a red oil that
was purified by column chromatography (hexanes:EtOAc 2:1 to
1:2) to give 33 as an orange solid (39 mg, 0.96 µmol, 63%). Rf
0.18 (hexanes:EtOAc 2:1). 1H NMR (800 MHz, DMSO-d6)
δ = 11.65 (s, 1H), 7.99 (d, J = 8.6, 1H), 7.94–7.91 (m, 3H), 7.88
(dt, J = 7.9, 1.6, 1H), 7.64 (d, J = 7.8, 1H), 7.63–7.57 (m, 5H),
7.36 (d, J = 2.1, 1H), 7.25 (dd, J = 8.6, 2.1, 1H). 13C NMR (201
MHz, DMSO-d6) δ = 163.0, 158.0, 152.4, 152.1, 139.5, 135.6,
134.9, 134.8, 131.9, 130.0, 129.3, 125.8, 125.6, 123.0, 122.2,
121.8, 114.9, 114.6, 112.4. IR (ATR) 3025 w, 2928 w, 1642 s,
1599 m, 1500 w, 1365 m, 1270 m, 1149 m, 880 m, 795 s, 688 s.
HRMS (ESI) Calculated for C21H15ClN3O2 376.0847, found
376.0847. Mp >275 °C.
4.3.13. methyl 2-(2-(3-aminophenyl)acetamido)-4-
chlorobenzoate (31)
Methyl
2-(2-(3-aminophenyl)acetamido)-4-chlorobenzoate
Acknowledgments
(30) (736 mg, 2.11 mmol, 1.00 eq) was dissolved in EtOH
(85 mL) and H2O (28 mL) before zinc powder (4.12 g,
63.0 mmol, 30.0 eq) and a CaCl2·6H2O (468 mg, 4.22 mmol,
2.00 eq) were added in one portion. The suspension was stirred at
80 °C for 3 h, then cooled to room temperature and filtered over a
plug of celite with EtOAc (50 mL). The filtrate was diluted with
H2O (50 mL) and the phases were separated. The aqueous phase
was extracted with EtOAc ((3 × 60 mL). The combined organic
phases were washed with saturated aqueous NaCl (90 mL), dried
over MgSO4 and concentrated. The residue was purified by
column chromatography (hexanes:EtOAc 2:1) to yield 31
D. M. B. gratefully acknowledges the European Commission
for a Marie Skłodowska-Curie Intra-European Fellowship (PIEF-
GA-2013-627990). D. T. thanks the Munich Center for
Integrated Protein Science (CIPSM) and the European Research
Council (Advanced Grant 268795).
Appendix A. Supplementary Data
Supplementary data related to this article can be found at
http:// dx.doi.org/10.1016/XXXXXXX and contains H and 13C
1
(1.18 g, 3.38 mmol, 75%) as
a
white solid. Rf 0.37
1
(hexanes:EtOAc 1:1) H NMR (400 MHz, CDCl3) δ = 11.06 (s,
1H), 8.82 (d, J = 2.1, 1H), 7.91 (d, J = 8.6, 1H), 7.15 (t, J = 7.8,
1H), 7.03 (dd, J = 8.6, 2.1, 1H), 6.76 (ddd, J = 7.5, 1.7, 1.0, 1H),
6.71 (t, J = 2.0, 1H), 6.62 (ddd, J = 8.0, 2.4, 0.9, 1H), 3.87 (s,
3H), 3.65 (s, 2H). The data matched literature values.32
NMR spectra for all new compounds as well as additional traces
for the other subunit combinations and photochemical
characterization for compounds 13, 14, 18, 19 and 26.
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