Azologization and repurposing of a hetero-stilbene-based kinase inhibitor: Towards the design of photoswitchable sirtuin inhibitors

Article abstract of DOI:10.3762/bjoc.15.214

The use of light as an external trigger to change ligand shape and as a result its bioactivity, allows the probing of pharmacologically relevant systems with spatiotemporal resolution. A hetero-stilbene lead resulting from the screening of a compound that was originally designed as kinase inhibitor served as a starting point for the design of photoswitchable sirtuin inhibitors. Because the original stilbenoid structure exerted unfavourable photochemical characteristics it was remodelled to its heteroarylic diazeno analogue. By this intramolecular azologization, the shape of the molecule was left unaltered, whereas the photoswitching ability was improved. As anticipated, the highly analogous compound showed similar activity in its thermodynamically stable stretched-out (E)-form. Irradiation of this isomer triggers isomerisation to the long-lived (Z)-configuration with a bent geometry causing a considerably shorter end‐to‐end distance. The resulting affinity shifts are intended to enable real‐time photomodulation of sirtuins in vitro.

Full text of DOI:10.3762/bjoc.15.214

Azologization and repurposing of a hetero-stilbene-based
kinase inhibitor: towards the design of photoswitchable
sirtuin inhibitors
Christoph W. Grathwol1, Nathalie Wössner2, Sören Swyter2, Adam C. Smith3,
Enrico Tapavicza3, Robert K. Hofstetter1, Anja Bodtke1, Manfred Jung2
and Andreas Link*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 2170–2183.
1Institute of Pharmacy, University of Greifswald,
Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany, 2Institute
of Pharmaceutical Sciences, University of Freiburg, Albertstr. 25,
79104 Freiburg, Germany and 3Department of Chemistry and
Biochemistry, California State University Long Beach, 1250 Bellflower
Boulevard, Long Beach, CA, 90840 USA
doi:10.3762/bjoc.15.214
Received: 28 June 2019
Accepted: 29 August 2019
Published: 16 September 2019
This article is part of the thematic issue "Molecular switches".
Guest Editor: W. Szymanski
Email:
Andreas Link* - link@uni-greifswald.de
* Corresponding author
© 2019 Grathwol et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
azo compounds; epigenetics; photoswitch; sirtuins; stilbenes
Abstract
The use of light as an external trigger to change ligand shape and as a result its bioactivity, allows the probing of pharmacological-
ly relevant systems with spatiotemporal resolution. A hetero-stilbene lead resulting from the screening of a compound that was
originally designed as kinase inhibitor served as a starting point for the design of photoswitchable sirtuin inhibitors. Because the
original stilbenoid structure exerted unfavourable photochemical characteristics it was remodelled to its heteroarylic diazeno ana-
logue. By this intramolecular azologization, the shape of the molecule was left unaltered, whereas the photoswitching ability was
improved. As anticipated, the highly analogous compound showed similar activity in its thermodynamically stable stretched-out
(E)-form. Irradiation of this isomer triggers isomerisation to the long-lived (Z)-configuration with a bent geometry causing a
considerably shorter end‐to‐end distance. The resulting affinity shifts are intended to enable real‐time photomodulation of sirtuins
in vitro.
Introduction
Sirtuins are protein deacylases that cleave off not only acetyl, isoforms have been identified to date [1]. These can be grouped
but also other acyl groups from the ε-amino group of lysines in into five classes (I, II, III, IV and V) according to their phyloge-
histones and many other substrate proteins. This class of lysine netic relationship [2]. The isoforms Sirt1, Sirt2 and Sirt3 origi-
deacetylases (KDACs) is distinguished from others by their de- nate from the same phylogenetic branch (class I), but differ in
pendence on the cosubstrate NAD+. In mammals, seven sirtuin their subcellular localization. Although Sirt1 and Sirt2 were
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shown to shuttle between nucleus and cytoplasm in a cell-type resented [31]. The approach to new chemotypes for sirtuin inhi-
and cell-cycle dependent manner, Sirt1 is mainly found in the bition via known adenosine mimicking kinase inhibitors has
nucleoplasm and Sirt2 in the cytoplasm [3-7]. Sirt3 primarily already been fruitful in the past [32,33]. Therefore, a focused
resides in the mitochondrion [8]. Facing the multitude of kinase inhibitor library from GlaxoSmithKline was screened for
diseases that are associated with a dysregulation of sirtuin activ- biological activity on human sirtuin isoforms Sirt1–Sirt3. Aza-
ity, they represent a promising target for pharmaceutical inter- stilbene derivative GW435821X (2a, Figure 1), initially
vention. For example, selisistat (EX-527, 1), a nanomolar and published as c-RAF kinase inhibitor, was identified as a moder-
selective Sirt1 inhibitor, passed phase II clinical trials as a ately active Sirt2 inhibitor with low selectivity [34,35]. In this
disease-modifying therapeutic for Huntington’s disease (HD) work, the photoresponsiveness of the hetero-stilbene core struc-
and was acquainted by AOP Orphan Pharmaceuticals AG for ture is examined. Furthermore, an intramolecular azologization
phase III trials in 2017 [9,10]. Its structure comprises a carbox- approach is performed in order to obtain photoswitchable
amide moiety, which mimics the amide group of the endoge- sirtuin inhibitors, which could be useful tools in the further in-
nous pan-sirtuin inhibitor nicotinamide (Figure 1). Likewise vestigation of the biochemistry and pharmacology of sirtuins.
Sirt2 inhibition was shown to have beneficial effects in animal
Results
and cell models of neurodegenerative diseases like HD and
Parkinson’s disease [11,12]. Sirt3 activity recently was found to Chemistry of azastilbenes
play an important role in cardiovascular diseases and extended All azastilbene derivatives were synthesised by palladium-cata-
ageing in humans [13-16]. Regarding tumorigenesis, the know- lysed cross-coupling reactions using either commercially avail-
ledge on the influence of sirtuins is inconsistent. Sirt1, Sirt2 and able 5-bromonicotinamide (3a) or methyl 5-bromonicotinate
Sirt3 all have been reported to act either as tumor suppressors or (3b). If 3b was used, transformation to the nicotinamide was
promotors, depending on the particular cell type [1,17].
accomplished almost quantitatively by addition of a saturated
solution of ammonia in anhydrous methanol and stirring in a
closed vessel at 40 °C. Compounds 4a and b could easily be ob-
tained through Suzuki coupling with commercially available
naphthalene-2-ylboronic acid or (3,4-dihydronaphthalen-2-
yl)boronic acid (Scheme 1). The latter was synthesized accord-
ing to a literature procedure [36].
Figure 1: Selisistat (1) and hit compound GW435821X (2a).
The ability to externally control the biological activity of small
molecules in vitro or in vivo comprises numerous opportunities
for example in the elucidation of biochemical pathways or the
reduction of systemic side effects in drug therapy. Molecular
photoswitches, i.e., compounds that undergo changes in their
geometry and physicochemical properties upon irradiation with
light, represent one major approach to this. One of the most
common light-driven transformations exploited in molecular
Scheme 1: Reagents and conditions: a) appropriate boronic acid,
Pd(PPh3)4, Na2CO3, DMF, H2O, microwave, 15 min, 150 °C, 43–64%.
photoswitches is the E–Z isomerization of double bonds [18]. In Formation of compounds 2b–h was accomplished through Heck
this context, the photochemistry of stilbenes and the closely coupling of aryl bromides with the appropriate styrenes
related azobenzenes has been studied intensely in the past [19- (Scheme 2) [37].
23]. Due to the multifaceted photoreactivity of unsubstituted
stilbenes, an appropriate modification of the stilbene core is Compounds 2b and 2e were obtained in moderate yield using
necessary to prevent unwanted irreversible side reactions 3a as the aryl halide in the Heck reaction. The use of 3b in the
[24,25]. On the contrary, the photochemical properties of Heck reaction resulted in a substantial improvement of yield in
azobenzenes are more convenient as already proven by their use the synthesis of 2g but not for 2c. Interchanging the roles by
as photoswitches in countless biological applications [26-30]. using 5-vinylnicotinamide (5a) or methyl 5-vinylnicotinate (5b)
However, their heteroaromatic counterparts still seem underrep- as alkene component had detrimental effects on the yields in the
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Scheme 2: Reagents and conditions: a) Pd2(dba)3 or Pd(OAc)2, P(o-tol)3, TEA, DMF, 120–140 °C, 0.7–24 h, 11–75%; b) potassium vinyltrifluorobo-
rate, Cs2CO3, PdCl2(PPh3)2, ACN, H2O, 1.5 h, 120 °C, 78% c) tributylvinyltin, Pd(PPh3)4; toluene, reflux, 3 h, 76%; d) NH3, MeOH, 40 °C, 3 d,
87–95%.
synthesis of 2d, 2f and 2h. Intermediates 5a and 5b were acces- Compared to the lead structure 2a, all compounds except 2e–h
sible from 3a and 3b via Suzuki–Miyaura or Stille coupling show increased inhibitory activity against Sirt2 (Table 1). Com-
[34].
pound 2c represents the most potent inhibitior with an IC50
value of about 7 µM. Moreover, a slight increase in selectivity
for Sirt2 and Sirt3 over Sirt1 could be observed for 2c, 4a and
Biology
The influence on deacetylase activity of three human sirtuin 4b. While none of the modifications provided complete
isoforms (Sirt1–3) was determined in a fluorescence-based isoenzyme specificity, 2c preferentially inhibited Sirt2
assay, using Z-Lys(acetyl)-AMC (ZMAL) as a substrate [38]. (IC50 6.6 0.5) and Sirt3 (IC50 7.5 0.9 µM) compared to
Table 1: Sirt1–3 inhibition for compounds 2a–h, 4a/4b and 8a.
Compound
Sirt1 inhibitiona
Sirt2 inhibitiona
Sirt3 inhibitiona
2a
2b
2c
2d
2e
2f
2g
2h
4a
4b
8a
27% @ 50 µM
71% @ 10 µM
51% @ 100 µM
51% @ 10 µM
61% @ 50 µM
26% @ 10 µM
52% @ 50 µM
n.i.
24.6 2.8 µMb
8.7 0.2 µMb
6.6 0.5 µMb
64% @ 10 µM
69% @ 50 µM
21% @ 10 µM
62% @ 50 µM
9% @ 10 µM
48% @ 10 µM
45% @ 10 µM
n.i.
41.7 2.0 µMb
89% @ 50 µM
7.5 0.9 µMb
90% @ 50 µM
60% @ 50 µM
79% @ 50 µM
87% @ 50 µM
n.i.
n.i.
n.i.
n.i.
38% @ 10 µM
38% @ 10 µM
n.i.
aPercent inhibition relative to controls at the indicated concentration, n.i. = no inhibition detected. bIC50 values (μM) with statistical limits; values are
the mean SD of duplicate experiments.
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Sirt1 (51% inhibition at 100 µM). Though not photoswitchable, for 3-styrylpyridines, forming two regioisomeric dihydroaza-
compounds 4a and 4b were synthesized to test the influence of phenanthrenes that are oxidized to 2- and 4-azaphenantrene (not
a rigid conformation around the C=C double bond on sirtuin shown), respectively [50].
inhibition. Interestingly, this increased rigidity provokes a com-
plete loss of activity against Sirt1. Despite the fact, that all Photochemistry of compounds 2b and 2f was investigated via
mammalian sirtuins possess profound similarity in their catalyt- UV–vis spectroscopy, LC–HRMS and NMR spectroscopy.
ic core domains, many isotype selective inhibitors have been Compound 2b represents the core structure of the azastilbenes
developed in recent years [39-45]. In the case of Sirt2 it was investigated, whereas in 2f the influence of ortho methylation
shown that appropriate ligand binding can induce conformation- was intended to be examined. For UV–vis spectroscopy 50 µM
al changes of the enzyme, revealing a so-called selectivity solutions in 5% DMSO (v/v) in enzyme assay buffer were used,
pocket, which allows for isotype-specific interactions [46]. A as this reflects the enzyme assay conditions. However, for
recently developed fluorescence polarization (FP)-based assay LC-HRMS and NMR analysis, a higher concentration of
enables mapping of ligand binding to this specific binding site 10 mM in methanol was necessary to receive reliable chro-
[35]. For 2a an interaction with the selectivity pocket was matograms and spectra.
already implied in the same work. Additionally performed
docking studies proposed a binding mode in which 2a mimics Upon exposure of 2b to radiation of 365 nm, changes in the
the nicotinamide residue of NAD+, whereas aromatic amino UV–vis spectra proceeded slowly, due to the low absorbance of
acid residues of the selectivity pocket stabilize the dimethyl- 2b in this wavelength region. However, shorter wavelengths,
phenol ring [35]. As photoisomerization in stilbenes and azo i.e. 254 nm, revealed fast and dramatic changes (Figure 2).
dyes is accompanied by a perpendicular twist of the phenyl ring After an initial decline and blue shift of the absorption
towards the former molecular plane, we assumed that this con- maximum, the UV–vis spectrum of 2b developed a more com-
formational change should provoke a differential binding situa- plex structure with further illumination. The initial spectrum did
tion at least for the dimethylphenol residue in 2a. Unfortu- not restore, neither thermally by standing in the dark nor photo-
nately, binding of 2b and c could not be localised in the vicinity chemically when exposed to daylight. Regarding 2f, 254 nm ra-
of the selectivity pocket of Sirt2, so that the binding pose diation was obligatory to obtain changes in the UV–vis spec-
remains unclear.
trum. However even long-term radiation did not lead to a com-
plex spectrum as with 2b, yet no stationary state was reached in
the examined time. As in the case of 2b, the spectrum of 2f was
Photochemistry of azastilbenes
The photochemical behaviour of stilbenes has been subject to not altered by daylight, nor by standing several days in the dark
intense investigation in the past. It is reported that unsubsti- at room temperature.
tuted stilbene undergoes E→Z photoisomerization [47], as well
as photocyclization to dihydrophenanthrene upon UV irradia- LC–HRMS analysis provided deeper insights and clarified the
tion, which is oxidized to phenantrene in the presence of differential behaviour observed in the UV–vis spectra of 2b and
oxygen [48]. In high concentrations, (E)-stilbene furthermore 2f after UV irradiation. As anticipated, UV irradiation lead to
undergoes photocyclodimerization to cyclobutane derivatives E→Z isomerization of the C=C double bond in both com-
[49]. Photoisomerization and photocyclization are also reported pounds. The (Z)-isomers were found to be slightly more polar
Figure 2: (Left) UV–vis spectrum of 2b 50 µM in 5% DMSO (v/v) in assay buffer after varying durations of irradiation with 254 nm and 365 nm, re-
spectively. (Right) UV–vis spectrum of 2f 50 µM in 5% DMSO (v/v) in assay buffer after varying durations of UV radiation.
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than the respective (E)-isomers and their absorption maxima The degree of photoisomerization could not be enhanced by ex-
appeared blue shifted as demonstrated by the UV–vis spectra tended illumination. Instead, for 2b prolonged irradiation
extracted from the LC runs. Unfortunately, the amount of pho- resulted in the formation of several side products, so that after
toisomerization was only moderate, since after 100 minutes of 10 hours the fractions containing (E)-2b and (Z)-2b had
continuous irradiation still substantial amounts of the (E)- declined significantly. This decrease was primarily accompa-
isomers were present in the mixtures (Figure 3). Proton NMR nied by an increase of the fractions containing the benzoquino-
analysis implied photostationary states comprising a relative line carboxamide isomers 8a and b formed by photocyclization
percentage of 45% (Z)-2b and 57% (Z)-2f, respectively after and successive oxidation (Scheme 3). Furthermore, small
100 minutes of 254 nm irradiation. The NMR spectra can be amounts of cycloaddition products in two fractions were found,
found in Supporting Information File 1.
probably due to the high concentration of 2b in the irradiated
Figure 3: (Left) LC chromatogram of the LC–HRMS analysis of 2b after varying durations of irradiation with 254 nm. Identity of 8a was assigned by
the reference compound synthesized and allowed differentiation of the two fractions containing photocyclized compounds, as indicated by mass spec-
tra. (Right) LC chromatogram of the LC–HRMS analysis of 2f after varying durations of irradiation with 254 nm.
Scheme 3: Photocyclization and oxidation reaction of 2b upon UV irradiation.
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solution. In contrast, 2f was remarkably stable to long-term UV order (ADC(2)) [51-53]. ADC2 calculations have also been
radiation. Even though the ratio of the double bond isomers was carried out with the implicit solvent continuum model COSMO
left unaffected, only small traces of the cycloaddition product using a dielectricity constant and refractive index of a methanol/
and some unidentified compounds were registered. No forma- water mixture, which was used as solvent in the experimental
tion of benzoquinoline carboxamides was registered as in the UV–vis measurements of the LC-HRMS fractions [54,55].
case of 2b. Hence, due to the sterically blocking ortho methyl Geometries for reactants (E)-2b and (Z)-2b were optimized for
groups in 2f, intramolecular photocyclization could be two different rotational isomers ((E)-2b-A and (E)-2b-B;
prevented.
(Z)-2b-A and (Z)-2b-B), defined in Supporting Information
File 1. In the following, we report only the results for (E)-2b-B
To verify the hypothetical structures derived from irradiation of and (Z)-2b-A, since they possess lower ground state energies
2b, we carried out quantum chemical calculations of the double and therefore are expected to be the dominant species at room
bond isomers (E)-2b and (Z)-2b as well as the oxidized com- temperature. Energy differences of the ground state structures
pounds 8a and 8b. We used density functional theory (DFT) to of two pairs of isomers, however, are less than 0.6 kcal/mol,
optimize the ground state equilibrium structures of (E)-2b, (Z)- and computed spectra differ only slightly. Extensive results of
2b, 8a and 8b, and used time-dependent DFT (TDDFT) and all structures and all applied computational methods are sum-
high-level correlated methods to obtain UV–vis absorption marized in the Supporting Information. While TDDFT system-
energies and oscillator strengths. To obtain the simulated atically underestimates the λmax values of the lowest absorption
absorption spectrum and λmax values, oscillator strengths were of all compounds by 0.1–0.75 eV, CC2 and ADC(2) agree with
converted into molar decadic extinction coefficients using a the λmax values of the lowest absorption bands with a maximum
Gaussian line shape with a full-width-at-half-maximum of deviation of 0.15 eV, similar to the previously determined accu-
0.3 eV. The correlated methods used were second-order approx- racy [56]. We notice a good agreement between ADC(2) gas
imated coupled cluster singles and doubles (CC2) and its phase calculations with CC2 gas phase calculations, which
approximation, algebraic diagrammatic construction to second- justifies the usage of the approximate ADC(2) method.
Figure 4: Calculated and experimental absorption spectra of compounds (E)-2b-B (A), (Z)-2b-A (B), and products 8a (C) and 8b (D). Oscillator
strengths (green sticks) correspond to the ADC(2)/COSMO calculation.
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Synthesis and photochemistry of
Comparing the calculated absorption spectra for (E)-2b-B and
(Z)-2b-A to the experimental spectra obtained from LC-HRMS photoswitchable diazeno analogue
(Figure 4A,B), we see that all calculations consistently confirm Even though the photochemical properties of ortho methylated
the experimentally found blue shift of about 15 nm (0.22 eV) azastilbenes like 2f could be improved by preventing photocy-
for the λmax value of the lowest absorption band. Blue shifts clization, they were still unsuitable for the use as photoswitch-
predicted by CC2, ADC(2), ADC(2)/COSMO are 14, 16, and able sirtuin inhibitors in the enzyme assay. The long irradiation
20 nm, respectively. Consistent with the experimental spectra, periods that were necessary to obtain significant amounts of the
all theoretical methods predict the maximum extinction of the (Z)-isomers did not permit switching of the inhibitors in the en-
lowest absorption band of (Z)-2b to approximately one half of zyme assay mixture, as the fluorescent substrate and the en-
the one of (E)-2b. Since the maximum error of the methods zyme would be harmed by long-term UV radiation. We envi-
(0.15 eV) is smaller than the observed blue shift (0.22 eV), we sioned to replace the stilbene motive of selected stilbene 2c by a
conclude that the computed λmax values are meaningful and diazeno group, because photoisomerization of azo dyes was an-
clearly support the successful formation of the Z-isomer. ticipated to proceed fast and reversible by application of UV ir-
Regarding the spectra of the photocyclization and oxidation radiation and visible light, respectively in this analogue.
products 8a and 8b (Figure 4C,D), theoretical methods predict
the λmax value of the lowest absorption bands within 8 nm 5-Diazenylnicotinamide 11 was synthetically accessible in two
(≈0.15 eV) of the value of the experimental spectrum of the steps through conversion of commercially available methyl
LC–HRMS, clearly confirming the experimentally found blue 5-aminonicotinate (9) and 4-fluoroaniline to 10 under Mill’s
shift of 0.75 eV and 0.54 eV compared to compounds (E)-2b reaction conditions and subsequent ammonolysis of the methyl
and (Z)-2b, respectively. Also here, we conclude that the calcu- ester 10 to amide 11 (Scheme 4).
lations clearly support the formation of compounds 8a and/or
8b. However, due to the similarity of the spectra of 8a and 8b, Photoswitching of (E)-11 to a long-lived PSS (t½ = 300 h) con-
calculations do not allow to predict which of the two isomers taining 84% of (Z)-11 was possible by short term UV irradia-
was present in the respective fraction analysed.
tion of 365 nm. The photoisomerization could be reversed by
exposure to visible light, i.e. 452 nm, albeit the PSS at 452 nm
Regarding the high similarity between 8a/8b and selisistat, it still comprised about 25% of (Z)-11 as determined by HPLC
was likely that these cyclized compounds could possess biologi- analysis using UV–vis detection at the isosbestic points
cal activity against sirtuins, too. On the other hand they (Table 2). Light of 500 nm could also reverse photoisomeriza-
resemble a fixed (Z)-configuration of the stilbene double bond. tion, but was not as effective as 452 nm radiation. 630 nm irra-
Therefore, comparison with 2b could provide information con- diation, in contrast, did not lead to an altered PSS composition
cerning differential biological activity of the two photoisomers.
By applying Mallory reaction conditions to a solution of 2b in
Table 2: Percentage of E/Z-isomers of 11 at the thermal equilibrium
methanol utilizing oxygen and iodine as oxidants we were able
to isolate a preparative amount of 8a and tested it for its biolog-
ical activity against Sirt1, Sirt2 and Sirt3. Surprisingly, 8a
showed complete inactivity towards all sirtuins tested (Table 1).
Hence it can be assumed that E→Z photoisomerization in simi-
lar compounds lowers inhibitory strength accordingly.
(∆), and photostationary states (PSS) after 365 nm and 452 nm irradi-
ation.
∆
PSS 5 min 365 nm PSS 1 min 452 nm
(E)-11/(Z)-11 99:1 16:84 75:25
Scheme 4: Reagents and conditions: a) 4-fluoroaniline, oxone, HAc, 60 °C, 14 d, 42%; b) NH3, MeOH, rt, 3 d, 98%.
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obtained by UV irradiation of 365 nm. Switching between the switchable element in this application. Even though competing
two PSS could be repeated several times without any observ- azaphenantrene formation could be prevented by implementa-
able fatigue of the compound (Figure 5).
tion of blocking ortho methyl groups in 2f, the degree of photo-
isomerization in the two compounds observed was only moder-
The photoswitchable diazeno compound 11 was subjected to bi- ate and required UV radiation over an extended period of time.
ological evaluation to test the effect of photoisomerization on Furthermore, the irreversibility of photoisomerization remained
the inhibitory activity. The enzyme assay mixture containing 11 a major drawback and made an exchange with a diazeno group
was exposed to 5 minutes of 365 nm radiation and compared mandatory. Typically, it is not clear from the beginning, if the
with the results of a non-irradiated measurement. The applied remodelling of the bioactive compounds will lead to an active
radiation did not perturb the proper enzyme functioning as diazeno derivative or not. The so-called azologization approach,
proved by an unaltered enzyme activity in the blank tests. moulded by Trauner et al., features a rational strategy for the
Unfortunately, 365 nm radiation turned out to have only minor design of photoswitchable compounds from established drug
effects on the IC50 values of 11 (Table 3).
molecules through replacing certain core motives with an bio-
isosteric azobenzene moiety [57-59]. Recent examples have
proven successful for receptor ligands by exchange of a linear
alkinyl spacer for the zigzag shaped (E)-diazeno group [60,61].
In that instance, the geometry of the lead had to be changed
considerably but careful design led to useful photoswitches. In
the case of lead 2a no such alteration of geometry was neces-
sary and thus it seemed highly likely, that biological activity
could be maintained. Indeed, this hypothesis could be proven.
Exchange of the azastilbene double-bond with a diazeno bridge
caused only a slight decrease in inhibitory potency against Sirt2
and Sirt3, and the selectivity profile of diazeno compound 11
equals the profile of its direct stilbene analogue 2c. Concerning
Table 3: Sirt1-3 inhibition for compound 11 at the thermal equilibrium
(∆) and the photostationary state (PSS) after 5 minutes of 365 nm irra-
diation.
Entry
Sirt1 inhibitiona Sirt2 inhibitiona Sirt3 inhibitiona
∆
PSS
35% @ 100 µM 18.9 1.38 µM 27.5 3.42 µM
19% @ 100 µM 24.1 1.69 µM 29.9 2.11 µM
aPercent inhibition relative to controls at the indicated concentration,
n.i. = no inhibition detected.
Discussion
In recent years, photopharmacology has become a reputable photoswitchability, 11 was superior to the stilbenoid structures,
strategy to optically control biochemical processes in the field as it could be toggled reversibly between two states comprising
of enzyme and ion channel modulation and recently 7TM-re- high amounts of (E)-11 and (Z)-11, respectively. The other part
ceptors also called GPCRs. Whereas in most approaches of the hypothesis was, that by this photoinduced isomerization a
towards photoswitchable ligands the structure of the lead has to considerable drop of activity would occur due to the conforma-
be changed considerable in order to incorporate a photoswitch- tional change and the resulting changed geometry and polarity.
able structural element, this was not the case with azastilbene- However, this part of our hypothesis turned out to be wrong.
based lead structure 2a. Unfortunately, due to several disadvan- The over-all conformational changes upon photoisomerization
tages the azastilbene moiety itself was unsuitable as photo- were too small or did not lead to a hindered binding, as antici-
Figure 5: (Left) UV–vis spectrum of 11, 50 µM in 5% DMSO (v/v), in assay buffer at the thermal equilibrium and the photostationary states (PSS) after
365 nm or 452 nm radiation. (Right) Fatigue resistance of 11, 50 µM in 5% DMSO (v/v), in assay buffer over 10 cycles of alternating 365 nm and
452 nm radiation.
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pated. This result is disappointing, because the photoswitchable staining with iodine, DNPH/H2SO4 (2 g 2,4-dinitrophenylhy-
sirtuin inhibitor 11 cannot be switched between active and inac- drazine and 5 mL H2SO4 in 50 mL EtOH and 16 mL water) or
tive state, as envisioned. Possible reasons could be assigned to vanillin/sulfuric acid (3.0 g vanillin and 0.5 mL H2SO4 in
substituent effects as demonstrated by Simeth et al. [62]. As 100 mL EtOH) reagent. Synthesis was additionally monitored
recently reported by Schehr et al., reducing agents like DTT, using high speed SFC/MS runs performed by a Nexera SFE-
used to prevent enzyme oxidation in crystallization mixtures or SFC/UHPLC switching system (Shimadzu Corporation, Kyoto,
in vitro assays, can reduce azo dyes to hydrazine derivatives Japan) consisting of a pumping system (one LC-30ADSF for
very fast and thus disable photoisomerization [63]. However, in liquid CO2 and two LC-20ADXR for modifier and make-up
our enzyme assay no such reducing agents were present, which delivery), an on-line supercritical fluid extraction module (SFE-
is why we assume that the photoswitchable diazeno group 30A auto extractor equipped with 0.2 mL extraction vessels) for
should still be intact in the enzyme assay mixture. Even if the reaction monitoring, an autosampler (SIL-30AC) for purified
change in space orientation does not alter binding after irradia- compounds, a column thermostat (CTO-20AC) equipped with a
tion, we would have predicted, that at least the significant Torus DIOL (Waters) or Phenomenex CSP (Lux Amylose-2,
difference in polarity of (E)-11 and (Z)-11 should lead to i-Amylose-3, i-Cellulose-5), a degasser (DGU-20A5R), a com-
marked differences of sirtuin engagement in vitro. However, munications module (CBM-20A), and two back pressure regu-
recent results from a carefully designed azologization study per- lators BPR A and B (SFC-30A). UV and MS spectra were re-
formed by Rustler et al. led to comparable difficulties [64].
corded via photodiode array detection (SPD-M20A) and elec-
trospray ionization single quadrupole MS (LCMS-2020) con-
trolled by Shimadzu LabSolution software (Version 5.91).
Conclusion
Based on lead structure GW435821X (2a) a small library of Chromatographic purification of products was performed by
analogous azastilbene compounds was designed, synthesized flash chromatography on silica gel (20–45 µm, Carl Roth)
and tested for their inhibitory activity against the human sirtuin applying pressured air up to 0.8 bar. NMR spectra were re-
isoforms Sirt1, Sirt2 and Sirt3. Compared to the lead structure corded on a Bruker Avance III instrument (1H NMR: 400 MHz,
the inhibitory potency could be increased to single digit µM po- 13C NMR: 100.6 MHz). Chemical shifts were referenced to
tency for some compounds, while isoenzyme selectivity still tetramethylsilane (TMS) as internal standard in deuterated sol-
remains an issue. The photochemistry of azastilbene com- vents and reported in parts per million (ppm). Coupling con-
pounds 2b and 2f was studied. For 2b, besides photoisomeriza- stants (J) are reported in Hz using the abbreviations: s = singlet,
tion, formation of benzoquinoline carboxamides by photocy- d = doublet, t = triplet, q = quartet, m = multiplet and combina-
clization and oxidation was indicated by high accuracy mass tions thereof, br = broad. Infrared (IR) spectra were recorded on
spectroscopy. Formation of 4-azaphenantrene derivative 8a a Bruker Alpha FT-IR spectrometer equipped with a diamond
could be proven by isolation and characterization of a prepara- ATR unit and are indicated in terms of absorbance frequency
tive sample. Theoretical UV–vis spectra for (E)-2b, (Z)-2b and [cm−1]. Microwave synthesis was conducted in a Monowave
two isomeric benzoquinoline carboxamides reproduced the ex- 300 microwave synthesis reactor from Anton Paar equipped
perimental data. Compound 2f was unsusceptible to photocy- with appropriate sealed reaction vessels G10 (6 mL) or G30
clization due to sterically blocking ortho methyl groups but (20 mL), applying a maximum initial power of 850 W to reach
could not be toggled between (E)- and (Z)-configuration. This a given temperature (IR sensor) for a given time with stirring at
lead to the synthesis of a first diazenyl derivative of the lead 600 rpm. Melting points were measured in open capillary tubes
structure 2a with promising photochemical characteristics for a using a Melting Point M-565 apparatus from Büchi and are
new class of photoswitchable sirtuin inhibitors, but the activity uncorrected. High accuracy mass spectra were recorded on a
difference for the (E)- and (Z)-isomers needs dramatic improve- Shimadzu LCMS-IT-TOF using ESI ionization. Purity of final
ment before a useful molecular probe can be obtained by this compounds was determined by HPLC with DAD (applying the
approach.
100% method at 220 nm). Preparative and analytical HPLC
were performed using Shimadzu devices CBM-20A, LC-20A P,
SIL-20A, FRC-10A with SPD 20A UV–vis detector and an
ELSD-LTII. In analytical mode a LiChroCART® (250 × 4 mm)
Experimental
General remarks
All solvents and reagents were obtained from commercial and in preparative mode a Hibar® RT (250 × 25 mm) column,
suppliers and were used without purification. Anhydrous sol- both containing LiChrospher® 100 RP-18e (5 µm), were used.
vents were purchased from Acros Organics. Thin-layer chroma- An Elementar Vario MICRO cube was used for the experimen-
tography (TLC) was executed on silica gel 60 F254 aluminium tal determination of elemental configurations of final pure prod-
plates purchased from Merck. Visualization of the compounds ucts. UV–vis spectra were obtained using a Thermo Scientific
was accomplished by UV-light (254 nm and 366 nm) and by Genesys 10S UV–vis spectrophotometer.
2178

Beilstein J. Org. Chem. 2019, 15, 2170–2183.
Synthesis
150.1, 147.7, 135.0, 134.0, 135.7, 132.4 (d, J = 3.4 Hz), 132.2,
General procedure for synthesis of nicotinamides from 128.8 (d, J = 8.2 Hz), 126.9, 122.9 (d, J = 2.3 Hz), 116.2 (d, J =
methyl nicotinates: The respective methyl nicotinate was 21.8 Hz), 52.9; IR (ATR) ν (cm−1): 2957, 1718, 1508, 1433,
treated with a saturated solution of ammonia in anhydrous 1299, 1230, 986, 821, 763; HRESIMS: calcd for [C15H12NO2F
MeOH (30 mL) and stirred in a sealed vessel at 40 °C until thin + H]+ 257.0852, found 257.0850.
layer chromatography indicated complete conversion of the
starting material. The solvent was evaporated under reduced (E)-5-(4-Fluorostyryl)nicotinamide (2c): Synthesis was con-
pressure and the residue washed sparingly with cold MeOH. ducted following the general procedure of nicotinamides from
methyl nicotinates, using methyl (E)-5-(4-fluorostyryl)nicoti-
(E)-5-Styrylnicotinamide (2b): In a microwave reaction vessel nate (75 mg, 0.31 mmol, 1.00 equiv). The product was ob-
3a (1.01 g, 5.00 mmol, 1.00 equiv) was mixed with styrene tained as colourless solid (65 mg, 0.27 mmol, 87%): Rf = 0.48
(651 mg, 6.25 mmol, 1.25 equiv), tris(o-tolyl)phosphine (EtOAc/MeOH 95:5); mp: 205.6 °C; 1H NMR, H,H-COSY
(61 mg, 0.20 mmol, 0.04 equiv), Pd2(dba)3 (92 mg, 0.10 mmol, (400 MHz, DMSO-d6) δ (ppm) 8.91 (d, J = 1.9 Hz, 1H), 8.88
0.02 equiv) and NEt3 (863 ΜL, 0.63 g, 6.25 mmol, 1.25 equiv) (d, J = 2.0 Hz, 1H), 8.47 (pseudo-t, J = 2.0 Hz, 1H), 8.22 (s,
and suspended in anhydrous DMF (6 mL). The reaction was 1H), 7.76–7.68 (m, 2H), 7.49 (d, J = 16.6 Hz, 1H), 7.31 (d,
conducted at 120 °C for 40 min in a microwave reactor. After J = 16.6 Hz, 1H), 7.29–7.22 (m, 2H); 13C NMR, DEPT135,
cooling to room temperature the mixture was taken up in HSQC, HMBC (75.5 MHz, DMSO-d6) δ (ppm) 166.4, 161.9,
EtOAc and filtered through a pad of Celite®. The filtrate was 150.3, 147.3, 133.1 (d, J = 3.2 Hz), 132.3, 131.4, 130.1, 129.7,
washed with water (3 × 30 mL) and sat. aq. NaCl solution 128.6 (d, J = 8.2 Hz), 124.1 (d, J = 2.2 Hz), 115.7 (d,
(30 mL), dried over MgSO4 and concentrated under reduced J = 21.6 Hz); IR (ATR) ν (cm–1): 3364, 3172, 1650, 1620,
pressure. The formed precipitate was collected by filtration and 1507, 1397, 1212, 968, 857, 601; HRESIMS: calcd for
recrystallized from EtOAc. The product was obtained as colour- [C14H11N2OF + H]+ 242.0855, found 242.0844; comp. purity
less crystals (0.55 g, 2.45 mmol, 49%): Rf = 0.25 (cyclohexane/ (220 nm): 100%; anal. calcd for C14H11N2OF: N, 11.56; C,
THF 1:1); mp: 196.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 69.41; H, 4.58; found: N, 11.53; C, 69.89; H, 4.51.
(ppm) 8.93 (d, J = 2.0 Hz, 1H), 8.90 (d, J = 2.1 Hz, 1H), 8.50
(pseudo-t, J = 2.0 Hz, 1H), 8.24 (s, br, 1H), 7.71–7.62 (m, 3H), Methyl 5-[(4-fluorophenyl)diazenyl]nicotinate (10):
7.54–7.28 (m, 5H); 13C NMR, DEPT135, HSQC, HMBC 4-Fluoroaniline (444 mg, 4.00 mmol, 1.00 equiv) was dis-
(75.5 MHz, DMSO-d6) δ (ppm) 166.4, 150.4, 147.3, 136.4, solved in DCM (15 mL) and treated with a solution of oxone
132.4, 131.4, 131.3, 129.6, 128.7, 128.2, 126.7, 124.2; IR (4.92 g, 8.00 mmol, 2.00 equiv) in water (50 mL). The biphasic
(ATR) ν (cm−1): 3372, 3168, 1649, 1619, 1492, 1394, 961, 746, mixture was vigorously stirred until thin layer chromatography
691, 568; HRESIMS: calcd for [C14H12N2O + H]+ 224.0950, indicated complete consumption of the starting material. The
found 224.0939; comp. purity (220 nm): 100 %; anal. calcd for watery phase was discarded and the organic phase washed with
C14H12N2O: N, 12.49; C, 74.98; H, 5.39; found: N, 12.38; C, an aq. HCl-solution (1 M, 3 × 10 mL) and water (3 × 10 mL),
74.81; H, 5.15.
then dried over MgSO4. The solution was concentrated to a
volume of 5 mL under reduced pressure and added to a solu-
Methyl (E)-5-(4-fluorostyryl)nicotinate: Synthesis was con- tion of 9 (609 mg, 4.00 mmol, 1.00 equiv) in acetic acid
ducted according to the procedure of 2b using 3b (648 mg, (20 mL). The reaction mixture was stirred at 60 °C for two
3.00 mmol, 1.00 equiv), 1-fluoro-4-vinylbenzene (550 mg, weeks, cooled to room temperature, poured onto ice cooled sat.
4.50 mmol, 1.50 equiv), tris(o-tolyl)phosphine (183 mg, aq. NaHCO3-solution and extracted with EtOAc (3 × 50 mL).
0.60 mmol, 0.20 equiv), Pd2(dba)3 (67 mg, 0.30 mmol, The combined organic extracts were washed with water
0.10 equiv) and NEt3 (1.25 mL, 9.00 mmol, 3.00 equiv) in an- (3 × 50 mL), sat. aq. NaCl solution (30 mL) and dired over
hydrous DMF (4 mL). The reaction was conducted at 140 °C MgSO4. The solvent was evaporated under reduced pressure
for 1.5 h. The raw product was purified by silica gel column and the residue purified by silica gel column chromatography
chromatography (n-hexane/EtOAc 2:1) yielding a colourless (cyclohexane/EtOAc 3:1). The product was obtained as orange
solid (97 mg, 0.38 mmol, 13%): Rf = 0.50 (n-hexane/EtOAc solid (431 mg, 1.67 mmol, 42%): Rf = 0.52 (cyclohexane/
2:1); mp: 108.2 °C; 1H NMR, H,H-COSY (400 MHz, CDCl3) δ EtOAc 3:1); mp: 103.6 °C; 1H NMR, H,H-COSY (400 MHz,
(ppm) 9.09 (d, J = 1.8 Hz, 1H), 8.90 (d, J = 2.1 Hz, 1H), 8.52 DMSO-d6) δ (ppm) 9.34 (d, J = 2.3 Hz, 1H), 9.22 (d,
(pseudo-t, J = 2.0 Hz, 1H), 7.57–7.49 (m, 2H), 7.26 (d, J = J = 2.0 Hz, 1H), 8.50 (pseudo-t, J = 2.2 Hz, 1H), 8.09–8.01 (m,
16.4 Hz, 1H), 7.13–7.06 (m, 2H), 7.03 (d, J = 16.4 Hz, 1H); 2H), 7.52–7.44 (m, 2H), 3.95 (s, 3H); 13C NMR, DEPT135,
4.00 (s, 3H, H-8); 13C NMR, DEPT135, HSQC, HMBC HSQC, HMBC (75.5 MHz, DMSO-d6) δ (ppm) 164.5, 164.4 (d,
(75.5 MHz, CDCl3) δ (ppm) 165.3, 163.2 (d, J = 249.4 Hz), J = 251.8 Hz), 151.8, 150.3, 148.5 (d, J = 2.8 Hz), 146.7, 126.4
2179

Beilstein J. Org. Chem. 2019, 15, 2170–2183.
(d, J = 6.7 Hz), 125.4 (d, J = 9.5 Hz), 116.6 (d, J = 23.2 Hz), 254 nm) or six Vilber-Lourmat T8-L lamps (8W, 365 nm), re-
52.7; IR (ATR) ν (cm−1): 3081, 1713, 1583, 1496, 1286, 1222, spectively. Visible light radiation of 630 nm (red), 500 nm
1092, 1000, 843, 498.
(green) and 452 nm (blue) was derived from a Paulmann
FlexLED 3D strip. All compounds were irradiated in solution,
5-[(4-Fluorophenyl)diazenyl]nicotinamide (11): Synthesis using spectrophotometric grade solvents. Photoisomerization
was conducted following the general procedure of nicoti- and UV–vis spectra measurement was conducted in quartz
namides from methyl nicotinates, using 10 (160 mg, 0.62 mmol, cuvettes at room temperature.
1.00 equiv). The product was obtained as orange solid (149 mg,
0.61 mmol, 98%): Rf = 0.65 (EtOAc/MeOH 95:5); mp: Computational details: All calculations were carried out using
212.3 °C; 1H NMR, H,H-COSY (400 MHz, DMSO-d6) δ (ppm) the TURBOMOLE version 7.2 quantum chemistry package
9.24 (d, J = 2.3 Hz, 1H), 9.20 (d, J = 2.0 Hz, 1H), 8.58 (pseudo- [67]. Geometry optimizations of all compounds in different
t, J = 2.2 Hz, 1H), 8.38 (s, br, 1H), 8.08–8.02 (m, 2H), 7.80 (s, conformers were carried out using DFT with PBE approxima-
br, 1H), 7.53–7.45 (m, 2H); 13C NMR, DEPT135, HSQC, tion to the exchange-correlation (XC) functional and employ-
HMBC (75.5 MHz, DMSO-d6) δ (ppm) 165.6, 164.3 (d, ing the SV(P) basis set [68,69]. The 10 lowest excitation ener-
J = 251.4 Hz), 150.7, 148.6 (d, J = 2.8 Hz), 148.2, 146.7, 130.5, gies and their oscillator strengths were computed using the
125.7, 125.3 (d, J = 9.4 Hz), 116.6 (d, J = 23.2 Hz); IR (ATR) ν SV(P) basis and the larger def2-TZVP basis set [69]. This was
(cm−1): 3359, 3125, 1669, 1628, 1496, 1398, 1136, 838, 808, done using TDDFT with the hybrid approximation to the XC
692; HRESIMS: calcd for [C12H9N4OF + H]+ 224.0760, found functional PBE0, CC2, and ADC(2) [51-53,70-72]. ADC(2) and
224.0753; comp. purity (220 nm): 100%; anal. calcd for CC2 calculations make use of the resolution-of-identity approx-
C12H9N4OF: N, 22.94; C, 59.02; H, 3.71; found: N, 22.95; C, imation [73]. ADC(2) calculations were also done using the
59.46; H, 3.92.
continuum solvent model COSMO as previously described
[54,55,74-76]. A dielectric constant of 62.14 and a refractive
Cloning, expression and purification of recombinant pro- index of 1.3379 were used, which corresponds to a solvent of a
teins: Expression and purification of Sirt1133-747, Sirt256−356, 6/4-mixture of methanol/water, as experimentally determined
and Sirt3118−395 was carried out as described previously. Iden- [77,78]. Broadened absorption spectra were simulated by
tity and purity were verified by SDS-PAGE [65]. Protein con- converting oscillator strengths to decadic extinction coeffi-
centration was determined by the Bradford assay [66]. Deacy- cients using a Gaussian line shape with a full-width-at-half-
lase activity of sirtuin isotypes could be inhibited with nicoti- maximum of 0.3 eV [79-82].
namide and was shown to be NAD+-dependent.
Supporting Information
Bioassay: The inhibitory effect of compounds 2a–h, 4a/b, 8a
and 11 on Sirt1–3 was detected via a previously reported
fluorescence based assay [38]. The synthetic substrate
Z-Lys(acetyl)-AMC (ZMAL) is deacetylated by sirtuins, fol-
lowed by tryptic digestion and thereby release of 7-amino-
methylcoumarin, leading to a fluorescent readout. Inhibition
was determined by comparing percentage substrate conversion
to a DMSO control after subtraction of the blank fluorescence
signal. All compounds were tested at 100 µM, 50 µM and
10 µM, respectively. For compounds that showed more than
50% inhibition at 10 µM an IC50 value was determined. IC50
values were calculated with OriginPro 9.0 G using a non-linear
regression to fit the dose response curve. An enzyme-free blank
control and a 100% conversion control using AMC instead of
ZMAL were measured as well. Inhibition measurements were
performed in biological duplicates for all compounds.
The Supporting Information features experimental and
analytical data for the synthesis of intermediates and
compounds 4a, 4b, 2c–2h and 8a and 1H and 13C NMR
spectra for all synthesized compounds. Procedures of
photochemical experiments and their analysis are
described. Detailed summaries of electronic structure
calculations for two conformers (A and B) of each double
bond isomer ((E)-2b and (Z)-2b), photocyclization and
oxidation products 8a and 8b are given.
Supporting Information File 1
Experimental procedures, analytical data and quantum
chemical calulations.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-15-214-S1.pdf]
Photochemistry: All photoisomerization experiments were
conducted under ruby light of 630 nm. Illumination was Acknowledgements
executed using a Bio-Link 254 Crosslinker from Vilber- The Jung group thanks the Deutsche Forschungsgemeinschaft
Lourmat equipped with six Vilber-Lourmat T8-C lamps (8 W, (DFG, Ju295/14-1 and RTG1976) for funding.
2180

Beilstein J. Org. Chem. 2019, 15, 2170–2183.
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ORCID® iDs
Christoph W. Grathwol - https://orcid.org/0000-0003-0232-3279
Enrico Tapavicza - https://orcid.org/0000-0002-0640-0297
Robert K. Hofstetter - https://orcid.org/0000-0002-1077-9703
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Andreas Link - https://orcid.org/0000-0003-1262-6636
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