# Bistable and Water-Operable Hemiindigo Photoswitches
Allow Optical Control of Acetylcholinesterase Activity
with Visible Light

Letícia da Mata Lazinski, Morane Beaumet, Frédérique Loiseau, Cyril Goudet, Martial Boggio-pasqua, Guy Royal, Romain Haudecoeur

► To cite this version:

Letícia da Mata Lazinski, Morane Beaumet, Frédérique Loiseau, Cyril Goudet, Martial Boggio-pasqua, et al. Bistable and Water-Operable Hemiindigo Photoswitches Allow Optical Control of Acetylcholinesterase Activity with Visible Light. ChemistryEurope, 2025, 3 (5), pp.e202500221. 10.1002/ceur.202500221. hal-05339073

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# Bistable and Water-Operable Hemiindigo Photoswitches
Allow Optical Control of Acetylcholinesterase Activity
with Visible Light

Leticia M. Lazinski, Morane Beaumet, Frédérique Loiseau, Cyril Goudet,
Martial Boggio-Pasqua, Guy Royal, and Romain Haudecoeur*

While photopharmacology enables precise spatiotemporal con-
trol over drug activity, its widespread reliance on UV-responsive
molecules often hinders preclinical and clinical advancements.
Thus, alternative scaffolds can offer promising advantages over
the commonly used azobenzene family. In this study, we intro-
duce the first hemiindigo-based photopharmacological agents,
which are capable of isomer-dependent human acetylcholines-
terase inhibition in the nanomolar range. These hemiindigo
photoswitches exhibit highly favorable properties: They are fully
functional in aqueous medium with good photoisomerization

quantum yields, respond exclusively to blue-to-orange visible
light (415–590 nm), and maintain high stability in their
metastable form, even in physiological buffers. Notably, indoxyl
*N*-methylation emerged as a key structural feature for optimizing
photophysical properties, as demonstrated through crystallogra-
phy and a theoretical model describing the photochemical path-
ways governing photoisomerization. Overall, this study paves the
way for broader exploration and future applications of the largely
overlooked hemiindigo scaffold in photopharmacology.

**1. Introduction**

Over the past years, light has emerged as a valuable tool for devel-
oping innovative therapeutic options. Upon integration of optically
responsive units into molecules and biomolecules, light can be used
to deliver a safe, non-invasive, and potentially orthogonal stimulus

for biological systems, providing unparalleled spatiotemporal
precision.[1] This property appears particularly compelling when
employing molecular photoswitches, that undergo fast and revers-
ible isomerization upon light irradiation, thereby altering their inter-
action pattern with biological targets and leading to changes in
pharmacological effects. When only one isomer is biologically
active, both drug activation and deactivation could be precisely con-
trolled, confining the effect to specific area and timeframe.[2,3]
Photopharmacology has developed for 15 years holding this prom-
ise, yielding many inspirational proof-of-concept studies but scarce
clinical output to date. Notably, around 95% of the reported systems
relied on the introduction of an azobenzene moiety incorporated
into a bioactive scaffold.[4] Azobenzene is indeed very convenient
as an easily accessible, compact, and efficient photoswitchable
group with a large geometrical change upon nitrogen–nitrogen
double bond E→Z photoisomerization. However, it comes with
drawbacks that often hamper the transposition of in vitro results
to in vivo or clinical studies, such as a low thermal stability of the
Z isomer in aqueous medium at 37 °C, or the requirement for
low-penetration and potentially harmful UV irradiation in most
cases—although recent design efforts produced promising red-
shifted azobenzene derivatives.[5–8] Therefore, the development of
alternative photoswitchable scaffolds that operate in aqueous
medium, exhibit high thermal stability in both isomeric states,
and allow visible-light-driven isomerization would be highly benefi-
cial in this context. Hemiindigoids, including hemithioindigos (HTIs)
and hemiindigos (HIs), have recently emerged as attractive candi-
dates (Figure 1).[9] These photoswitches show visible-light-induced
photoisomerizations (Z→E and E→Z) and were often associated
with high bistability,[10–14] with some axially chiral analogs achieving
metastable isomer lifetimes exceeding 3,400 years—the longest

L. M. Lazinski, M. Beaumet, R. Haudecoeur

Univ. Grenoble Alpes

CNRS

DPM

Grenoble 38000, France

E-mail: Romain.Haudecoeur@univ-grenoble-alpes.fr

L. M. Lazinski, F. Loiseau, G. Royal

Univ. Grenoble Alpes

CNRS

DCM

38000 Grenoble, France

C. Goudet

IGF

Université de Montpellier

CNRS

INSERM

34094 Montpellier, France

M. Boggio-Pasqua

Université de Toulouse

CNRS

Laboratoire de Chimie et Physique Quantiques

31062 Toulouse, France

<figure><img src="image_3.png" /></figure>

Supporting information for this article is available on the WWW under https://
doi.org/10.1002/ceur.202500221

<figure><img src="image_4.png" /></figure>

© 2025 The Author(s). ChemistryEurope published by Chemistry Europe and Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 1.** General structure and numbering of HI/HTI scaffolds; structure of AChE inhibitor donepezil; general structure of the photoisomerizing HI compounds developed in this study; crystal structures of Z-6d and E-6d (ORTEP).</figcaption></figure>

reported to date.[15] However, their properties in aqueous environments remain underexplored, despite a few pioneering studies.[10,16] Although the HTI scaffold has long been exploited for general biological applications,[17–19] its use in photopharmacology stricto sensu remains limited,[20–24] and HIs have been largely overlooked. In response, we present the first prototypes of HI-based photopharmacological tools, along with comprehensive experimental characterizations of their photoswitching performances in water and physiological buffer, supported by theoretical investigations of their photoswitching mechanism. Inspired by the structural resemblance of HIs to the marketed drug donepezil, we designed HI photo-switches as inhibitors of acetylcholinesterase (AChE), the main therapeutic target for Alzheimer's disease (AD) management (Figure 1).[25] AD is a neurodegenerative condition and the leading cause of dementia in people over 65, currently affecting nearly 55 million individuals worldwide, with healthcare costs expected to exceed $1 trillion by 2050.[26] Since AChE catalyzes the inactivating hydrolysis of the neurotransmitter acetylcholine (ACh), inhibiting its activity is a key strategy to elevate synaptic ACh levels and alleviate

early to moderate stage AD-related cognitive impairment. Several photopharmacological studies have investigated cholinesterase kinetics and AChE's role in AD progression, as a precise spatiotemporal control over cholinergic modulation could overcome the limitations of conventional drugs that lack selectivity and rely solely on pharmacokinetics.[27] However, to date, all relied exclusively on UV-light-responsive agents.[28–31] Thus, we introduce the first photoswitchable inhibitors of AChE operating in the visible range.

## 2. Results and Discussion

Given the established structural resemblance between donepezil and hemiindigoids, which has previously inspired medicinal chemistry research on analogous aurones and indanones, we initially designed HI compounds 5a–d by incorporating donepezil-like substitutions at the A-ring—specifically 5- and/or 6-methoxy groups—and 2-(pyrrolidin-1-yl)ethoxy or 2-(piperidin-1-yl)ethoxy groups at the B-ring, known to enhance AChE inhibition

<figure><img src="image_7.png" /><figcaption>**Scheme 1.** Chemical synthesis of compounds 5a–d, 6a, 6d, 7a, and 7d. Reagents and conditions: a) i. $ClCH_2CN$, AlCl$_3$, BCl$_3$, 1,2-dichloroethane, reflux, ii. HCl, $H_2O$, reflux, iii. $Ac_2O$, 85 °C, iv. NaH, dimethoxyethane, rt; b) i. glycine, Cu powder, $K_2CO_3$, KOH, $H_2O$, reflux, ii. $Ac_2O$, NaOAc, reflux, iii. $Na_2SO_3$, EtOH, $H_2O$, 80 °C; c) i. 4-(2-(pyrrolidin-1-yl)ethoxy)benzaldehyde or 4-(2-(piperidin-1-yl)ethoxy)benzaldehyde (1.2 equiv), piperidine (cat.), PhMe, reflux, ii. KOH 50%, MeOH, rt; d) NaH, Mel (2.4 equiv), DMF, rt; e) Mel (1.1 equiv), EtOAc, rt.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Table 1.** Photophysical properties of compounds **5a–d**, **6a**, **6d**, **7a**, and **7d**. Unless otherwise stated, measures were performed in DMSO. Measures in water and PBS buffer (pH 8.0) were carried out with 2% DMSO for solubility.</figcaption></figure>

<table><thead><tr><th rowspan="2">Cd. λ<sub>abs</sub><br>[nm]</th><th rowspan="2">ε<sup>2</sup><br>[M<sup>-1</sup> cm<sup>-1</sup>]</th><th colspan="2">PSS [%Z/E]<sup>a</sup>[at λ in nm]</th><th rowspan="2">ΔG<sup>*</sup> H<sub>2</sub>O<br>[kcal. mol<sup>-1</sup>]</th><th rowspan="2">t<sub>1/2</sub><sup>E</sup> H<sub>2</sub>O<sup>b</sup>[25 °C]</th><th rowspan="2">ΔG<sup>*</sup> PBS<br>[kcal. mol<sup>-1</sup>]</th><th rowspan="2">t<sub>1/2</sub><sup>E</sup>PBS<sup>b</sup>[25 °C]</th><th colspan="2">Φ<sub>iso</sub></th><th rowspan="2">Φ<sub>iso</sub><br>H<sub>2</sub>OZ<sup>→</sup>E</th></tr><tr><th>Z→E</th><th>E→Z</th><th>Z→E</th><th>E→Z</th></tr></thead><tbody><tr><td><b>5a</b></td><td>455</td><td>18,600</td><td>29/71 (415)</td><td>98/2 (530)</td><td>26.2</td><td>21 d</td><td>23.7</td><td>8 h</td><td>7%</td><td>7%</td><td>3%</td></tr><tr><td><b>5b</b></td><td>468</td><td>8900</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td></tr><tr><td><b>5c</b></td><td>498</td><td>9500</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>n.d.</td></tr><tr><td><b>5d</b></td><td>479</td><td>16900</td><td>31/69 (450)</td><td>99/1 (546)</td><td>26.0</td><td>15 d</td><td>24.4</td><td>26 h</td><td>5%</td><td>3%</td><td>2%</td></tr><tr><td><b>6a</b></td><td>457</td><td>8800</td><td>5/95 (415)</td><td>76/24 (530)</td><td>27.4</td><td>5 mo</td><td>26.1</td><td>17 d</td><td>24%</td><td>5%</td><td>8%</td></tr><tr><td><b>6d</b></td><td>481</td><td>11700</td><td>6/94 (450)</td><td>93/7 (590)</td><td>26.8</td><td>2 mo</td><td>25.3</td><td>4 d</td><td>12%</td><td>25%</td><td>7%</td></tr><tr><td><b>7a</b></td><td>454</td><td>13500</td><td>21/79 (415)</td><td>99/1 (530)</td><td>26.1</td><td>17 d</td><td>24.2</td><td>17 h</td><td>9%</td><td>5%</td><td>2%</td></tr><tr><td><b>7d</b></td><td>478</td><td>17500</td><td>19/81 (450)</td><td>89/11 (530)</td><td>26.0</td><td>15 d</td><td>24.5</td><td>29 h</td><td>6%</td><td>3%</td><td>2%</td></tr></tbody></table>

activity.[32,33] Their synthesis was relatively straightforward, starting with the preparation of appropriately substituted 1-acetylindolin-3-ones **4a–d**. This was achieved either by Sugasawa chloroacetylation of commercially available anilines[34] or through a three-step procedure involving Ullmann cross-coupling between glycine and bromobenzoic acid followed by cyclization (Supporting Information, SI).[35] These intermediates were then condensed with the corresponding aldehydes under basic conditions, followed by saponification of the N-acetyl protecting group using KOH.[36] Second-generation compounds **6a**, **6d**, **7a**, and **7d** were synthesized through methylation of the most promising HIs **5a** and **5d**, using two distinct sets of conditions to obtain both the monomethyl and dimethyl derivatives (**Scheme 1**).

Then, we performed the complete characterization of important photophysical parameters, which, in the case of photochroms designed for photopharmacology, are typically carried out in DMSO and aqueous solutions. The studied derivatives were investigated in DMSO, 2% DMSO in water and phosphate buffer (pH 8.0) focusing on the determination of molar extinction coefficients (ε<sup>2</sup>), isomer ratios at the photostationary state (PSS), activation free energies (ΔG<sup>*</sup>, E→Z), thermal half-life of the metastable isomer E (t<sub>1/2</sub><sup>E</sup>), and photoisomerization quantum yields (Φ<sub>iso</sub>). Results summarized in **Table 1** indicate that the Z isomers of all derivatives display absorption maxima at the lowest energy absorption band between 450 and 500 nm. Although the impact of B-ring and C-ring modifications on the photochromic properties has been previously documented,[10,37] no information regarding the effects of A-ring functionalization has been reported so far for HIs. By comparing the properties of unsubstituted compounds **5–7d** with their methoxy analogs **5–7a**, **5b**, and **5c**, we observed that the presence of a methoxy group at position 6 induced a blue shift in absorption, whereas the same substituent at position 5 caused a bathochromic effect and a significant decrease in molar extinction coefficients. These effects are consistent with previous observations on HTI compounds and could be explained by Kauffmann's distribution rule for auxochromes.[11] Photoisomerization experiments were conducted by illuminating the compounds with blue light (415–450 nm) for Z→E conversion, and with green-orange light (530–590 nm) for E→Z conversion. The impact of light irradiation on

the chemical stability of the compounds was investigated. While no photodegradation or photobleaching was observed after repeated short irradiations, continuous high-power illumination over longer times eventually led to some degradation through multiple pathways, especially involving dimerization via [2 + 2] photocycloaddition, as already reported,[38] and oxidation of the central double bond, likely due to singlet oxygen generation (SI). However, this observation is not a significant concern, as the time required to reach the PSS at biologically relevant concentrations falls within the range of seconds. Overall, all HIs showed photochromic behavior in DMSO, but the presence of a methoxy group at position 5, as in **5b** and **5c**, had a detrimental effect, resulting in poor photoconversion in DMSO (< 60/40%Z/E) at the tested wavelengths and absence of spectroscopical changes indicative of photochromism when irradiated in aqueous medium under the studied conditions. The methoxy group in the *para* position relative to the indoxyl heteroatom induces a bathochromic shift and reduced absorption, in line with Kauffmann's rule. This may account for the absence of photoswitching in water, potentially due to increased spectral overlap between isomers or altered excited-state dynamics favoring non-isomerizing decay pathways. Confirming these effects would require advanced theoretical modeling beyond the scope of this study. In contrast, unsubstituted and 6-methoxy derivatives **5a**, **5d**, **7a**, and **7d** yielded moderate to excellent photoconversions in the Z→E (31/69 to 19/81% Z/E) and E→Z (89/11 to 99/1%Z/E) directions. In addition, the thermal half-lives of E isomers in water were found to be in the week timescale (t<sub>1/2</sub><sup>E</sup> = 15–21 d), but decreased significantly in phosphate-buffered saline (PBS, pH 8.0), with half-lives ranging from 8 to 29 h, consistent with previously reported trends.[16,39] Indoxyl N-methylation had an overall highly positive impact on photoisomerization, in line with observations from previous reports studying the effect of N-alkylation.[10,37] Indeed, compared to indoxyl N-H derivatives, **6a** and **6d** showed the highest isomeric ratios for the Z→E conversion at the PSS (> 6/94, %Z/E), the highest quantum yields Φ<sub>iso</sub><sup>Z→E</sup> in DMSO (24% and 12%, respectively, vs. 5–9% for N-H counterparts) and water (8% and 7%, respectively, vs. 2–3%). These derivatives also exhibited higher activation barriers for the E→Z thermal backwards reaction both in water

<footnote><sup>a</sup>Calculated from ¹H NMR spectra performed in *d*<sub>6</sub>-DMSO; <sup>b</sup>In some cases, *t*<sub>1/2</sub> values at 25°C were extrapolated from ΔG<sup>*</sup> (SI).</footnote>
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**Figure 2.** a) Crystal structures of Z-6d, E-6d, and Z-7d with associated torsion angles around the rotatable single bond next to the double bond and b) potential energy profile and chemical structures of 8 and 9 model systems. Curved arrows represent the main relaxation pathways. Red dashed curve: S₁ relaxation path. Black dashed curve: unreactive S₀ relaxation path from minimum energy conical intersections (MECI). Green dashed curve: reactive S₀ relaxation path from MECI.</figcaption></figure>

(27.4 and 26.8 kcal mol⁻¹, respectively, vs. 26.0–26.2 kcal mol⁻¹ for N-H derivatives) and in PBS buffer (26.1 and 25.3 kcal mol⁻¹, respectively, vs. 23.7–24.5 kcal mol⁻¹), resulting in significantly longer t₁/₂ E values (months vs. weeks in water, days vs. hours in PBS). These improvements made N-methyl HIs 6a and 6b high-performance photoswitches for physiological applications.

The crystal structures of compounds Z-6d, E-6d, and Z-7d provided additional insights into their differing photophysical behaviors (Figure 2a). While the N-H derivative Z-7d exhibited an almost planar conformation (torsion angle around the rotatable single bond next to the double bond of 6.3°), N-methylation of the indoxyl group in Z-6d introduced significant steric hindrance between the N-methyl group and the hydrogen atom at position 2', resulting in a twisted structure (19.5°). In contrast, E-6d maintained a planarity similar to Z-7d (6.7°). This steric effect likely contributes to the balanced relative stability observed between the Z and E isomers in N-methyl compounds and more generally in N-alkyl derivatives,[10,37] compared with N-H counterparts. To bring further support to the observed increased Z→E and E→Z photoisomerization efficiencies upon indoxyl N-methylation, theoretical calculations of the static photochemical path were performed on two isolated model systems 8 and 9 representative of the indoxyl N-H and N-methyl derivatives (Figure 2b and Table 2). The lowest excited-state (S₁) potential energy surface (PES) displays three minima: Z* corresponds to the relaxed (ππ*) excited-state structure for the Z isomer; likewise, E* is the (ππ*) excited-state structure for the E isomer; and DB-Twi* is a twisted intermediate minimum involving strong double-bond torsion (of around 75°) and a non-negligible adjacent single-bond torsion (of 25°). This last structure, which occurs along the relevant Z→E photoisomerization coordinate, is the lowest energy structure on the S₁ PES and features a noticeable charge transfer character from the C ring to the B ring (Figure S80, SI, Supporting Information) accompanied by an increase of the excited-state dipole moment from 5.6 D at Z* to 10.3 D at DB-Twi*. The adiabatic photoisomerization mechanism (Figures S80 and S81, SI, Supporting

<table><caption>**Table 2.** Theoretical values for energetics (in kcal mol⁻¹) relevant to the Z→E photoisomerization of 8 and 9 H1 model systems.</caption><thead><tr><th>Relative energies<sup>a)</sup> [kcal mol<sup>-1</sup>]</th><th>N-H H1 8</th><th>N-methyl H1 9</th></tr></thead><tbody><tr><td>ΔE(Z<sup>*</sup>→Twi*)</td><td>-2.9</td><td>-8.0</td></tr><tr><td>ΔE<sup>+</sup>(Z<sup>*</sup>→Twi*)</td><td>3.5</td><td>0.3</td></tr><tr><td>ΔE(Twi*→E<sup>*</sup>)</td><td>1.9</td><td>3.7</td></tr><tr><td>ΔE<sup>+</sup>(Twi*→E<sup>*</sup>)</td><td>7.2</td><td>8.0</td></tr><tr><td>ΔE(Z<sup>*</sup>→MECI-Twi1)</td><td>6.4</td><td>0.4</td></tr><tr><td>ΔE(Z<sup>*</sup>→MECI-Twi2)</td><td>-0.6</td><td>-6.0</td></tr></tbody></table>

Information) is thus a two-step process Z*→DB-Twi*→E*, favorable energetically with DB-Twi* lower than Z* by 2.9 (8.0) kcal mol⁻¹ and E* lower than Z* by 1.0 (4.3) kcal mol⁻¹ for compound 8 (9). Interestingly, the first step (Z*→DB-Twi*) involves a small barrier of 3.5 kcal mol⁻¹ for 8, whereas it is quasi barrierless for 9. In addition, the second step (DB-Twi*→E*) requires to overcome a much more significant barrier of 7 and 8 kcal mol⁻¹, respectively. The adiabatic photoisomerization pathway involves mainly a double-bond isomerization (DBI) mechanism but possesses also a significant hula-twist (HT) character due to the non-negligible rotation around the adjacent single bond. DBI and HT mechanisms have been reported before for this and are system- and environment-dependent.[40–42]

To determine the nonadiabatic photoisomerization pathways, minimum energy conical intersections (MECIs) between the ground and first excited states, responsible for efficient S₁→S₀ non-radiative decay, were located in the vicinity of the adiabatic photoisomerization path presented above. Figure 2 shows two relevant S₀/S₁ MECIs easily accessible energetically. MECI-Twi1 is found 6.4 kcal mol⁻¹ above the Z* minimum in 8 and only 0.4 kcal mol⁻¹ in 9. It is characterized by a double-bond torsion of about 120°. MECI-Twi2 lies past the twisted minimum DB-Twi* with a double-bond torsion of about 65° at an even lower energy.

<footnote><sup>a)</sup>ΔE: relative energy with respect to the first structure indicated inside the parentheses. ΔE*: energy barrier between the first and second structures indicated inside the parentheses.</footnote>
<figure><img src="image_11.png" /></figure>

It is located 0.6 and 6.0 kcal mol⁻¹ below the Z⁺ minimum, for derivatives **8** and **9**, respectively. Note that in these two MECIs, the HT character is exacerbated, as the torsion around the adjacent single bond increases by a further 15° compared to *DB-Twi*\*. The topology of these MECIs are sloped (Figures S82 and S83, SI, Supporting Information) and the ground-state relaxation paths arising from these MECIs are expected to be the Z isomer ground-state recovery and the *E* isomer photoproduct formation for *MECI-Twi1* and *MECI-Twi2*, respectively (Figure S84 and S85, SI, Supporting Information). The mechanistic picture that we can derive from this simple PES topology for the Z→*E* photoisomerization thus involves two possible nonadiabatic routes: (1) Z⁺→*MECI-Twi1*→*E*, and (2) Z⁺→*DB-Twi*\*→*MECI-Twi2*→*E*, with route (1) possibly less efficient than route (2), as decay from *MECI-Twi1* favors Z reformation in the simple static picture (Figure S84, SI, Supporting Information). For both pathways (1) and (2), the photoisomerization is expected to be more efficient in **9**, as the access to the MECIs and to the intermediate *DB-Twi*\* structure is energetically more favorable. This is due to the fact that these structures involve some amount of charge transfer character from the C ring to the B ring, and the presence of the methyl group on the C ring increases the electron donor character, thus favoring the charge transfer energetically. This result supports the experimental observations of an increased photoisomerization efficiency upon indoxyl *N*-methylation. Importantly, we could not find the presence of any accessible twisted intramolecular charge transfer (TICT) pathway for these two systems, even in polar solvents. This type of unproductive photophysical pathway has been reported in this to account for the decrease of photoisomerization efficiency in polar solvents.[40,43,44] Thus, in our systems, the presently observed decrease of Z→*E* photoisomerization efficiency in **6d** and **7d** going from DMSO to water (Table 1) solvent cannot be accounted for by TICT formation. One possible explanation could be strong direct interactions (e.g., through H-bonding) with polar protic solvent molecules favoring unproductive deactivation pathways, as recently suggested.[45]

After demonstrating that HI photoswitches can function in physiological environments with relevant bistability and modeling their photoswitching paths, the next step involved assessing their ability to inhibit different forms of AChE using Ellman's assay (Table 3). Most of the Z isomers tested exhibited greater activity

against human AChE (*hAChE*) than the electric eel version (*eeAChE*), with compounds **6a** and **7a** achieving *IC*₅₀ values in the hundred-nanomolar range (160 nM and 70 nM, respectively). In contrast, the unsubstituted **6d** and **7d** showed weaker inhibition, in the micromolar range. This suggests the critical importance of the 6-methoxy substitution for *hAChE* inhibition, mirroring the structure of donepezil, although this trend was less pronounced for compounds **5a** and **5d**. The assay was then modified to compare activity under *dark* and *lit* conditions. The compounds were incubated with AChE either in the dark or under pulsed illumination at 445 or 470 nm, followed by the addition of the substrate acetylthiocholine iodide (ATCI) and the subsequent record of enzymatic activity after 5 min. Absorbance spectra of control wells containing only the test compounds were monitored throughout irradiation and the enzymatic reaction to confirm that the compounds remained stable and did not degrade or undergo thermal back-isomerization during the assay.

For *eeAChE* inhibition under illuminated and dark conditions, only compound **7d** showed a notable *lit/dark* *IC*₅₀ ratio of 3.9, suggesting that the formation of the *E* isomer through photogeneration led to reduced inhibitory activity. For the other compounds, photoisomerization did not result in significant changes in activity. In contrast, for *hAChE* inhibition, derivatives with 6-methoxy groups on the A-ring (i.e., **5a**, **6a**, and **7a**) exhibited higher *lit/dark* *IC*₅₀ ratios (ranging from 1.3 to 3.7) compared with their nonmethoxylated counterparts (**5d**, **6d**, and **7d**), which had ratios between 0.7 and 1.5. This could be due to the 6-methoxy group disrupting the apparent symmetry between the isomers of the HI compounds. Testing donepezil under *lit* and *dark* conditions against *hAChE* revealed no variation in *IC*₅₀, confirming that the observed differences are specific to the photoswitchable nature of the HI structures. Overall, the best ratio obtained (3.7–3.9) closely align with the median value of 4.5 calculated from reported dark-active azobenzene-based examples in a recent literature survey, highlighting the ability of HIs to enable isomer-dependent activity modulation.[4] This was further validated by evaluating the ability of HIs to function as light-responsive AChE inhibitors in a complex medium. Cholinesterase activity in 1% fetal bovine serum was measured in the presence of HI compounds, either in the dark or after pre-illumination of inhibitor stock solutions at 420 or 450 nm.

<table><caption>**Table 3.** Biological activities of compounds **5a**, **5d**, **6a**, **6d**, **7a**, and **7d** and the reference AChE inhibitor donepezil (dpz) under *dark* and *lit* conditions.</caption><thead><tr><th rowspan="2">Cd.</th><th colspan="3">eeAChE</th><th colspan="3">hAChE</th><th colspan="3">Cholinesterase activity from fetal bovine serum</th></tr><tr><th>IC<sub>50</sub> dark [nM]</th><th>IC<sub>50</sub> lit [nM]</th><th>lit/dark ratio</th><th>IC<sub>50</sub> dark [nM]</th><th>IC<sub>50</sub> lit [nM]</th><th>lit/dark ratio</th><th>EC<sub>50</sub> dark [nM]</th><th>EC<sub>50</sub> lit [nM]</th><th>lit/dark ratio</th></tr></thead><tbody><tr><td>5a</td><td>710 ± 60</td><td>1200 ± 100</td><td>1.7</td><td>210 ± 40</td><td>270 ± 50</td><td>1.3</td><td>250 ± 30</td><td>530 ± 40</td><td>2.1<sup>a)</sup></td></tr><tr><td>5d</td><td>1090 ± 50</td><td>2000 ± 100</td><td>1.8</td><td>440 ± 50</td><td>330 ± 40</td><td>0.7</td><td>860 ± 70</td><td>1050 ± 90</td><td>1.2</td></tr><tr><td>6a</td><td>1050 ± 70</td><td>1200 ± 100</td><td>1.1</td><td>160 ± 10</td><td>590 ± 60</td><td>3.7</td><td>250 ± 60</td><td>640 ± 70</td><td>2.6<sup>a)</sup></td></tr><tr><td>6d</td><td>2800 ± 100</td><td>2200 ± 200</td><td>0.8</td><td>2000 ± 200</td><td>3000 ± 300</td><td>1.5</td><td>>1000</td><td>>1000</td><td>n.d.</td></tr><tr><td>7a</td><td>1800 ± 100</td><td>1800 ± 200</td><td>1.0</td><td>70 ± 10</td><td>230 ± 30</td><td>3.3</td><td>430 ± 40</td><td>530 ± 50</td><td>1.2</td></tr><tr><td>7d</td><td>3100 ± 200</td><td>12000 ± 2000</td><td>3.9</td><td>900 ± 100</td><td>1300 ± 100</td><td>1.4</td><td>>1000</td><td>>1000</td><td>n.d.</td></tr><tr><td>dpz</td><td>n.d.</td><td>n.d.</td><td>n.d.</td><td>4.5 ± 0.2</td><td>5.3 ± 0.4</td><td>1.1</td><td>41 ± 5</td><td>40 ± 6</td><td>1.0</td></tr></tbody></table>

<footnote><sup>a)</sup>Differences between *dark* and *lit* *IC*₅₀ values were determined as statistically significant by unpaired t-test.</footnote>
<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 3.** Compound 6a: a) photoisomerization between Z and E isomers until PSS in water using blue light (450 nm); b) naked eye distinction between isomers (Z and PSS at 450 nm) in aqueous solution (50 µM, 2% DMSO/water); c) photobleaching experiment involving successive cycles of irradiation in water with blue (450 nm) and green light (530 nm); d) portion of ¹H NMR spectra in *d*₂-DMSO measured in the dark and at the PSS (415 nm and 530 nm); e) human AChE inhibition in the dark or under irradiation at 445 nm; and f) comparison of *p*EC₅₀ against cholinesterase activity from fetal bovine serum 1% in *dark* (gray) and *lit* (blue) conditions, with statistical analysis by two-tailed unpaired t-test.</figcaption></figure>

All tested compounds produced inhibition values comparable to those obtained from the hAChE assay, with two compounds (5a, and especially 6a, Figure 3) showing statistically significant differences (2.1–2.6-fold) between dark and illuminated conditions, confirmed by unpaired t-test. It is important to note that the global pharmacokinetic properties of quaternary salts are often suboptimal, which makes compound 5a a more pharmacologically suitable candidate. The experiments involving isolated enzymes and fetal bovine serum utilized different irradiation protocols—applying light either before (on stock solutions) or after (during incubation) the addition of HI compounds in the reaction medium—both yielding comparable results.

### 3. Conclusion

In conclusion, we have developed the first prototypes of HI-based photopharmacological agents. These compounds, which are straightforward to synthesize, exhibit several advantageous properties compared to azobenzenes, including high bistability in both water and physiological buffers, high visible-light-mediated interconversion, and good quantum yields. Notably, N-substitution on the HI C-ring significantly enhances these characteristics, resulting in high-performance photoswitches suitable for biological applications—an option obviously not available in analogous HTIs.

Theoretical analyses and crystallography provided insights to rationalize these findings, laying the foundation for future works on N-substituted HIs. Inhibition studies further revealed that, despite the inherent pseudosymmetric nature of hemiindigoid isomers,⁴⁶⁾ isomer-specific activity differences emerged with appropriate substitution, aligning with standard *lit/dark* ratios observed for azobenzenes in the literature.

### Supporting Information

Material and methods; synthesis; chemical, photochemical/physical, biochemical characterizations; photostability and singlet oxygen generation; crystal structures; computations; additional references[47–59] (PDF). XYZ files for cartesian coordinates of **8** and **9** optimized structures (XYZ). Crystallographic information files for Z-6d, E-6d, and Z-7d structures (CIF).

### Acknowledgements

This work was supported by the ANR ("Agence Nationale de la Recherche") through Labex ARCANE and CBH-EUR-GS (ANR-17-EURE-0003) and through ANR project EPIDERMIS (ANR-19-CE44-0002). The authors thank Labex ARCANE for the fellowship
<figure><img src="image_14.png" /></figure>

grant of L.M.L in the frame of project SHAPESHIFT and the ANR for
the fellowship grant of M.B. in the frame of project EPIDERMIS. This
work is part of the HELIOPHORE project of PEPR LUMA and is sup-
ported by the ANR, as a part of the France 2030 program, under
grant ANR-24-EXLU-0005. This work was granted access to the
HPC resources of CALMIP supercomputing center under the alloca-
tion 2024-[24022]. The authors thank Dr. Christian Philouze from the
DCM, UMR 5250, for the X-ray structures acquisition; ICMG UAR 2607
(PRMN and PSM platforms) for NMR and MS analyses; and Thomas
Curel, Sylvie Claeysen and the staff from the ARPEGE platform
facilities at the Institut de Génomique Fonctionnelle for helpful
discussions.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability Statement

The data that support the findings of this study are available in
the supplementary material of this article.

Keywords: acetylcholinesterase • hemiindigo • hemiindigoids • photopharmacology • photoswitch

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