Article

Isomeric 3-Pyridinylmethylcoumarins Differ in

Erk1/2-Inhibition and Modulation of BV2
Microglia-Mediated Neuroinflammation
Rami Schulzki 1,†, Matthias Apweiler 1,†, Caroline Röttger 2, Christoph W. Grathwol 3, Nora Struchtrup 2,
Sophia Abou El Mirate 2, Claus Normann 4, Stefan Bräse 2,3 and Bernd L. Fiebich 1,*

1 Neuroimmunology and Neurochemistry Research Group, Department of Psychiatry and Psychotherapy,

Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg,
D-79104 Freiburg, Germany; rischulzki@gmail.com (R.S.); matthias.apweiler@uniklinik-freiburg.de (M.A.)
2 Institute of Organic Chemistry (IOC), Karlsruhe Institute of Technology (KIT), Kaiserstraße 12,

D-76131 Karlsruhe, Germany; caroline.roettger@kit.edu (C.R.); noramarie@shaw.ca (N.S.);

sophia.mirate@kit.edu (S.A.E.M.); braese@kit.edu (S.B.)
3 Institute of Biological and Chemical Systems-Functional Molecular Systems (IBCS-FMS), Karlsruhe Institute

of Technology (KIT), Kaiserstraße 12, D-76131 Karlsruhe, Germany; christoph.grathwol@kit.edu

4 Mechanisms of Depression Research Group, Department of Psychiatry and Psychotherapy,

Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg,

D-79104 Freiburg, Germany; claus.normann@uniklinik-freiburg.de
* Correspondence: bernd.fiebich@uniklinik-freiburg.de
† These authors contributed equally to this work.

Abstract: Coumarins are known for their multiple biological effects and have been estab-
lished as anti-coagulative drugs for years. Furthermore, some coumarins can promote
anti-inflammatory effects via the GPR55 receptor, and dual target coumarins have been
synthesized. Anti-inflammatory drugs might be beneficial in the treatment of neuropsy-
Academic Editor: Grażyna Zgórka chiatric disorders, as the inflammatory hypothesis suggests. For the current study, we
Received: 30 April 2025
compared isomeric 3-pyridinylmethylcoumarins with altered N-atom position regarding
Revised: 28 May 2025 their effects on cytokine and chemokine synthesis and expression in LPS-stimulated BV2
Accepted: 29 May 2025 microglial cells. The 3-pyridin-4-yl-methylcoumarin showed the most potent anti-inflam-
Published: 3 June 2025 matory effects, followed by the 3-pyridin-2-ylmethylcoumarin analog. The observed ef-
Citation: Schulzki, R.; Apweiler, M.; fects might be mediated by an inhibition of ERK phosphorylation.
Röttger, C.; Grathwol, C.W.;
Struchtrup, N.; Abou El Mirate, S.; Keywords: coumarins; isomeric; Erk1/2; MAPK; microglia; neuroinflammation; cytokines;
Normann, C.; Bräse, S.; chemokines; GPR55
Fiebich, B.L. Isomeric
3-Pyridinylmethylcoumarins Differ
in Erk1/2-Inhibition and Modulation
of BV2 Microglia-Mediated
Neuroinflammation. Molecules 2025, 1. Introduction
30, 2452. https://doi.org/10.3390/ Coumarins have been known for their multiple biological effects for years. For ex-
molecules30112452
ample, the anti-coagulative effect of warfarin by the inhibition of the γ-carboxylation in
Copyright: © 2025 by the authors. the synthesis of the vitamin K-dependent clotting factors [1] established coumarins as val-
Licensee MDPI, Basel, Switzerland. uable pharmaceutics. Depending on the chemical residues added to the coumarin scaf-
This article is an open access article
fold, anti-microbial, anti-proliferative, anti-oxidative, and anti-inflammatory effects have
distributed under the terms and
conditions of the Creative Commons
been described in addition to its already mentioned anti-coagulative effects [2]. The cou-
Attribution (CC BY) license marin scaffold consists of a benzene ring fused to a α-pyrone, provoking biological activ-
(https://creativecommons.org/license ity through hydrogen bonding and hydrophobic interactions with target structures [2]. In
s/by/4.0/). an investigation on the selectivity of various coumarins towards the cannabinoid recep-
tors and G-protein-coupled receptor (GPR) 55, substituents on C-5 of the coumarin

Molecules 2025, 30, 2452 https://doi.org/10.3390/molecules30112452

Molecules 2025, 30, 2452 2 of 22

scaffold were found to be essential for bioactivity. Instead, substituents on C-7 had an
influence on the cannabinoid receptors (CB) affinity only and additional substituents on
C-8 lead to an antagonistic activity towards GPR55 [3]. In one of our previous works, we
showed that coumarins with residues on C-3, -5, and -8 have anti-inflammatory and anti-
oxidative effects [4–7] in primary microglial cells, most likely due to an inverse agonism
on GPR55. Another coumarin derivative, KIT 10, with substituents on C-3, a methoxy
group at C-5, and a 1-butylcyclohexyl residue at C-7, equally exerted anti-inflammatory
effects probably dependent on GPR55, highlighting the relevance of C-3 and C-5 substitu-
tion compared to C-8 [7]. Since specific substitution patterns on the coumarin scaffold al-
low us to adjust the selectivity and affinity towards different target receptors, further re-
search into this pharmacophore has been undertaken [8]. For example, a coumarin–dithi-
ocarbamate hybrid showed inhibitory effects on both acetylcholinesterase (AChE) and
monoaminoxidase B (MAO B) and reversed scopolamine-induced memory deficits in
mice, underlining the potential of this structural class for the treatment of neurodegener-
ative diseases, such as Alzheimer’s disease (AD) [9].
In recent years, GPR55 has gained importance in the treatment of neuropsychiatric
disorders. For example, O-1602, a synthetic GPR55 agonist reversed β amyloid-induced
cognitive impairment and release of pro-inflammatory cytokines as well as the production
of reactive oxygen species and cell death in mice, suggesting GPR55 as a promising target
to treat AD [10]. Similar effects were observed in streptozotocin-induced murine AD mod-
els [11]. Furthermore, the role of GPR55 in substance use disorders [12], depressive symp-
toms [13], suicide [14], and anxiety [15] has been proposed. Therefore, a pharmacological
intervention via GPR55 and the activation of associated anti-inflammatory pathways
might be a promising and new approach in the treatment of various neuropsychiatric dis-
orders.
Studies support the hypothesis that anti-inflammatory pharmaceutics might be ben-
eficial in the treatment of neuropsychiatric disorders. Elevated interleukin (IL)-6 plasma
concentrations were measured in elderly patients with major depression [16]. Another
study did not show significant differences in IL-6 levels between patients with depression
and healthy controls,; however, treatment with selective serotonin reuptake inhibitors
(SSRIs) significantly decreased peripheral IL-6 levels in both patients and controls [17].
Another study showed an association between poorer psychopharmaceutical response to
SSRIs or selective serotonin and noradrenalin reuptake inhibitors (SNRIs) treatment and
high peripheral IL-6 levels [18]. The relevance of inflammatory processes in AD was
shown in post-mortem brain analyses of patients showing IL-6 immunoreactivity in β am-
yloid plaques in AD but not in brain plaques of age-matched controls [19]. Interestingly,
plasma IL-6 levels were negatively correlated with hippocampal gray matter density and
scoring in the mini-mental state examination (MMSE) [19]. Targeting those inflammatory
processes has gained interest in the scientific community in recent years and lead to the
development of new compounds and conduction of clinical trials, underlining the rele-
vance of anti-inflammatory drug research in psychiatric disorders. For example, the inhi-
bition of intracellular cortisone reactivation via 11β-hydroxysteroid dehydrogenase (11β-
HSD1) is discussed as another promising way to ameliorate neuroinflammation in psy-
chiatric disorders, especially AD. In some studies, reduction in cognitive decline as well
as reduced inflammatory responses have been observed under treatment with 11β-HSD1
inhibitors in preclinical studies, however, the results in the subsequent clinical trials were
inconsistent over different studies [20]. Excessive cortisol levels lead to an internalization
of glucocorticoid-receptors responsible for the anti-inflammatory actions and—in contrast
to normal levels—do not inhibit pro-inflammatory cytokine release for that reason [21].
11β-HSD activity was associated with the severity of depressive symptoms measured us-
ing the Hamilton Depression Rating Scale (HDRS) in women but not men [22]. In AD,
Molecules 2025, 30, 2452 3 of 22

11β-HSD1 knockout or inhibition prevented cognitive impairments and memory deficits,

but effects might only be relevant in age-related cognitive decline [23]. However, the ef-
fects in clinical studies are not consistent and some 11β-HSD1 inhibitors failed to achieve
the primary endpoints, though higher dosages might be necessary [23,24].
Targets of the anti-inflammatory treatment are the central nervous system’s residual
immune cells, the microglial cells. Microglia are responsible for the pro- and anti-inflam-
matory homeostasis of the CNS under physiological conditions [25]. However, in case of
pathogen exposition, damage or auto-immunological reactions, microglia modulate a pro-
inflammatory response as well as production of reactive oxygen species in order to attract
and differentiate further immune cells to restrict the consequences of the exposition. The
aim of this cellular response is to remove pathogens, dead cells, and to restore the brain’s
homeostasis [25]. As highlighted before, involvement of cytokines, such as IL-6, is dis-
cussed in the genesis and progression of neuropsychiatric disorders. However, also chem-
okines, such as C-C motif chemokine ligand (CCL) 2, also referred to as “monocyte chem-
oattractant protein 1” (MIP-1), were, for example, elevated in patients with mild or mod-
erate major depressive disorder (MDD) in comparison to healthy controls and normalized
after an internet-based cognitive-behavioral intervention [26]. In patients with Parkin-
son’s Disease (PD), peripheral concentrations of the chemokine C-X-C motif chemokine
ligand (CXCL) 10, also known as interferon gamma induced protein 10 (IP-10), were pos-
itively correlated with cognitive deficits, disease’s progression but not depressive symp-
toms [27]. Furthermore, higher CXCL10 levels were observed in patients with major de-
pressive disorders compared to healthy controls and were even significantly enhanced
after 8 weeks treatment with the SSRI fluoxetine in one study [28]. Another study sup-
ported the finding of higher peripheral CXCL10 concentrations in patients with MDD. In
contrast to the previously mentioned study, patients who responded to an antidepressive
treatment (fluoxetine or desipramine), showed a significant decrease in CXCL10 levels
instead [29]. Therefore, the role of cytokines and chemokines still needs further investiga-
tions to clarify their potential for diagnostic and therapeutic applications in neuropsychi-
atric disorders. While rare cases of auto-antibody-mediated organic psychiatric disorders
have already resulted in new therapeutic options [30], no inflammatory biomarkers have
been established so far [31].
Nevertheless, some patients with psychiatric disorders seem to benefit from anti-in-
flammatory treatment. A systematic review suggests an improvement in depressive
symptoms after treatment with different anti-inflammatory substances as monotherapy
or add-on therapy to antidepressants [32]. Another review reported benefits on depressive
symptoms after treatment with non-selective cyclooxygenase (COX) inhibitors as well as
COX-2 selective pharmaceutics [33]. Patients with an inflammatory comorbidity and de-
pression, in particular, might profit by including an anti-inflammatory drug in the thera-
peutic regime [33].
Since coumarins are known for their anti-inflammatory effects and might act on mul-
tiple molecular pathways and targets via different receptors, the current study focuses on
anti-inflammatory effects of three novel isomeric 3-pyridinylmethylcoumarins. We were
especially interested in the biological significance of small structural changes in the pyri-
dine subunit. The effects of X6905 (Figure 1A, represented as light blue color scheme in
result figures), X7732 (Figure 1B, represented as blue color scheme in result figures), and
X7625 (Figure 1C, represented as dark blue color scheme in result figures) on cytokine and
chemokine release were evaluated in lipopolysaccharide (LPS)-stimulated BV2 microglial
cells. Furthermore, pathways that were possibly involved were examined focusing on mi-
togen-activated protein kinases (MAPKs).
Molecules 2025, 30, 2452 4 of 22

Figure 1. Chemical structures of the investigated isomeric 3-pyridinylmethylcoumarins: (A) 3-pyridin-4-

ylmethylcoumarin (X6905), (B) 3-pyridin-2-ylmethylcoumarin (X7732), and (C) 3-pyridin-3-ylmethyl-
coumarin (X7625). The coumarin core is highlighted in yellow, along with the varying para-, ortho- and
meta-position of the nitrogen atom (blue) within the pyridinyl moiety.

2. Results
2.1. Effects of X6905, X7732, and X7625 on Cell Viability (ATP -Assay)
An ATP assay was performed to assess whether the compounds X6905, X7732, and
X7625 (1, 10, 20 µM) have an influence on cell viability in LPS-stimulated BV2 cells (Figure
2). Cell viability was not affected by the stimulation with LPS, while 20% ethanol signifi-
cantly induced cell death. DMSO was used as solvent for all compounds. Accordingly, a
0.2% DMSO group was included as a vehicle control. DMSO decreased cell viability to
approximately 70% of untreated BV2 cells. X6905 and X7732 similarly reduced cell viabil-
ity to around 70% at 20 µM, while X7625 had a rather promoting effect at lower concen-
trations. The only significant reduction in cell viability was observed for X6905 at 20 µM.
To exclude a DMSO-mediated effect, a second one-way ANOVA was performed using
0.2% DMSO as reference (indicated with #), which showed no significant reduction in cell
viability in BV2 cells for any compound.

Figure 2. Effects of X6905-light blue bars (A), X7732—blue bars (B), and X7625—dark blue bars (C)
on cell viability of LPS-stimulated BV2 cells after 24 h of stimulation. Cell viability was assessed
using an ATP assay. Values are shown as the mean of 3 (ethanol—dark bar, DMSO—striped bar,
Molecules 2025, 30, 2452 5 of 22

coumarins) or 6 (control—white bar and LPS group—black bar) individual measurements ± SDs. A
one-way ANOVA was performed for statistical analysis using the untreated cells (white bar) or the
0.2% DMSO-group (striped bar) as reference for Dunett’s post-hoc test. Asterisks compare to the
control-group with * p < 0.05 and **** p < 0.0001, while hashtags are used for 0.2% DMSO-reference
with # p < 0.05; ## p < 0.01 and #### p < 0.0001. Non-significant differences are labeled as “ns”.

2.2. Effects of X6905, X7732, and X7625 on LPS-Induced Cytokine Release

Next, a potential compound-dependent modulation of LPS-induced cytokine release
was investigated by measuring cytokine concentrations of IL-6, CCL2, CXCL2, CXCL10,
and TNF-α in cell supernatant after 24 h of simulation with LPS in the absence or presence
of the respective coumarin derivative at different concentrations (0.1, 1, 5, 10, 20 µM).

2.2.1. IL-6 Release

Figure 3 shows the effects of X6905 (A), X7732 (B), and X7625 (C) on LPS-induced
IL-6 release. IL-6 release of BV2 cells was strongly induced after 24 h of LPS stimulation,
being 9- (A), 4.5- (B), or 3.5- (C) times higher compared to the control group. X6905 showed
a significant and dose-dependent reduction in IL-6 to 35% of LPS at 20 µM. A steady re-
duction in IL-6 was visible, starting at 1 µM, where IL-6 was already reduced to 78% of
the LPS response. In higher concentrations, highly significant results were obtained. X7732
had a similar but slightly weaker effect compared to X6905. At 20 µM, X7732 reduced LPS-
induced IL-6 release by 56%. X7625 did not show a dose-dependent reduction in LPS-
induced IL-6 release. Only at 20 µM, X7625 reduced the release of IL-6 to 63% of the LPS
response. At lower concentrations (0.1 to 1 µM), a slight, though statistically not signifi-
cant, increase in IL-6 release, with 33% and 55% above the LPS group, was observed.

Figure 3. Effects of X6905—light blue bars (A), X7732—blue bars (B), and X7625—dark blue bars (C)
on LPS-induced release of IL-6 in BV2 cells. IL-6 concentrations were measured in the supernatant
using ELISA. The values were normalized to the LPS response (black bar) and are shown as the
mean of three to six (n = six for the control and LPS groups in A and n = three for all other groups)
individual measurements ± SDs. For statistical analysis, one-way ANOVA with Dunett’s post-hoc
test was performed using the LPS group as reference with ** p < 0.01 and **** p < 0.0001. Non-signif-
icant differences are labeled as “ns”.
Molecules 2025, 30, 2452 6 of 22

2.2.2. CCL2 Release

CCL2 release of BV2 cells (Figure 4) was studied after 24 h of treatment with the cou-
marins X6905 (A), X7732 (B), and X7625 (C) in the presence of LPS. LPS increased CCL2
release by 33% (A) or 58% (B and C) of LPS compared to untreated BV2 cells. X6905 and
X7732 showed comparable inhibitory effects: Both compounds showed a dose-dependent
and significant reduction in LPS-mediated CCL2 release. However, X6905 lowered CCL2
stronger than the corresponding concentrations of X7732. Both exhibited a dose-depend-
ent reduction in CCL2 release between 0.1 µM and 10 µM, followed by a marked drop at
20 µM, reaching 27% of the LPS response for X6905 and 35% for X7732. In contrast, X7625
had no significant effect on LPS-induced CCL2 release at concentrations between 0.1 and
10 µM, with comparably high levels of CCL2 observed. At 20 µM, however, X7625 signif-
icantly decreased LPS-stimulated CCL2 release to 70% of the LPS response.

Figure 4. Effects of X6905—light blue bars (A), X7732—blue bars (B), and X7625—dark blue bars (C)
on LPS-induced release of CCL2 in BV2 cells. CCL2 concentrations were measured in the superna-
tant using ELISA. The values were normalized to LPS (black bar) and are shown as the mean of
three to six (n = six for the control and LPS groups in A and n = three for all other groups) individual
measurements ± SDs. For statistical analysis, one-way ANOVA with Dunett’s post-hoc test was per-
formed using the LPS group as reference with ** p < 0.01; *** p < 0.001 and **** p < 0.0001. Non-
significant differences are labeled as “ns”.

2.2.3. CXCL2 and CXCL10 Release

We further studied the effects of X6905 (A), X7732 (B), and X7625 (C) on LPS-induced
synthesis of CXCL2 and CXCL10, another group of chemokines in BV2 cells (Figures 5
and 6). After 24 h of stimulation, LPS induced a 10-time (A) and 4-time (B,C) increase in
released CXCL2 compared to unstimulated cells. For CXCL10, LPS showed a 25- (A) and
11-times (B,C) induction in comparison to untreated cells. Both X6905 and X7732 exhibited
dose-dependent and significant inhibitory effects on the LPS-mediated secretion of
CXCL2 and CXCL10. The effect on CXCL10 release was slightly stronger than for CXCL2,
although initially an increased CXCL10 level was detected at the lowest concentration of
0.1 µM X7732.
Molecules 2025, 30, 2452 7 of 22

At the highest concentration of 20 µM, CXCL2 release decreased to 66% of the LPS-
stimulated cytokine levels for X6905 and to 76% for X7732, respectively. In the case of
CXCL10, X6905 decreased the LPS-stimulated response to 42% while X7732 reached 51%
of LPS at 20 µM. X7625 showed no dose-dependent effects in the range of 0.1 to 20 µM.
Interestingly, it was observed that X7625 rather enhanced LPS-stimulated CXCL10 release
in concentrations between 0.1 and 10 µM. Nevertheless, at 20 µM, X7625 decreased the
CXCL2 and CXCL10 responses to about 72% and 67% of the LPS-stimulated cells.

Figure 5. Effects of X6905 – light blue bars (A), X7732 – blue bars (B), and X7625 – dark blue bars (C)
on LPS-induced release of CXCL2 in BV2 cells. CXCL2 concentrations were measured in the super-
natant using ELISA. The values were normalized to LPS (black bar) and are shown as the mean of
three to six (n = six for the control and LPS groups in A and n = three for all other groups) individual
measurements ± SDs. For statistical analysis one-way ANOVA with Dunett’s post-hoc test was per-
formed using the LPS group as reference with * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.
Non-significant differences are labeled as “ns”.

Figure 6. Effects of X6905 – light blue bars (A), X7732 – blue bars (B), and X7625 – dark blue bars (C)
on LPS-induced release of CXCL10 in BV2 cells. CXCL10 concentrations were measured in the
Molecules 2025, 30, 2452 8 of 22

supernatant using ELISA. The values were normalized to LPS (black bar) and are shown as the mean
of three to six (n = six for the control and LPS groups in A and n = three for all other groups) indi-
vidual measurements ± SDs. For statistical analysis one-way ANOVA with Dunett’s post-hoc test
was performed using the LPS group as reference with * p < 0.05; ** p < 0.01 and **** p < 0.0001. Non-
significant differences are labeled as “ns”.

2.2.4. TNF-α Release

We next studied the effects of X6905 (A), X7732 (B), and X7625 (C) on LPS-induced
synthesis of TNF-α, a key inflammatory cytokine involved in neuroinflammation (Figure
7). LPS stimulation led to a four- (A) and two-fold (B,C) increase in TNF-α secretion. X6905
exhibited a modest, dose-dependent decrease in TNF-α levels compared to LPS stimula-
tion. Between 0.1 and 10 µM, a gradual decrease to 87% of the LPS-stimulated response
was observed. At 20 µM, the effect of X6905 reached statistical significance with 62% of
the LPS-stimulated level.
In contrast, neither X7732 nor X7625 affected LPS-induced TNF-α levels. However,
some significant effects were observed: X7732 significantly enhanced LPS-mediated TNF-
α release at 10 µM, and X7625 showed a similar effect at 0.1 µM.

Figure 7. Effects of X6905 – light blue bars (A), X7732 – blue bars (B), and X7625 – dark blue bars (C)
on LPS-induced release of TNF-α in BV2 cells after 24 h treatment. TNF-α concentrations were meas-
ured in the supernatant using ELISA. The values were normalized to LPS (black bar) and are shown
as the mean of three to six (n = six for the control and LPS groups in A and n = three for all other
groups) individual measurements ± SDs. For statistical analysis, one-way ANOVA with Dunett’s
post-hoc test was performed using the LPS group as reference with * p < 0.05 and **** p < 0.0001.
Non-significant differences are labeled as “ns”.

2.3. Effects of X6905 on LPS-Induced Cytokine Expression

To analyze whether the effects observed on the protein levels are also detectable on
the transcriptional level, we stimulated BV2 cells with different doses of X6905 (1, 5, 10,
and 20 µM) in presence of LPS and investigated the expression of IL-6 (A), CCL2 (B),
CXCL2 (C), CXCL10 (D), and TNF-α (E) using qPCR (Figure 8). For all cytokines, a con-
siderable induction of expression was observed after LPS stimulation.
Molecules 2025, 30, 2452 9 of 22

IL-6, CCL2, and TNF-α were the only cytokines/chemokines significantly affected by
X6905. Treatment with 1, 5, and 10 µM of X6905 led to a gradual decrease on LPS-induced
IL-6 expression with a significant reduction of 50% of LPS-stimulated cells at 10 µM. At a
concentration of 20 µM, LPS-induced IL-6 expression was significantly reduced to 58%.
The influence of X6905 on LPS-mediated CCL2 expression showed a comparable profile:
Between 1 and 10 µM, a gradual, dose-dependent decrease in CCL2 expression was ob-
served, reaching 54% of the LPS-stimulated CCL2 expression. At 20 µM, however, a re-
duction to only 65% compared to LPS was found. X6905 had only weak effects on LPS-
induced TNF-α expression, reducing mRNA levels to 82% of LPS-stimulated levels at con-
centrations of 1, 5, and 10 µM. A significant effect was observed at 20 µM, where the tran-
scription of TNF-α was decreased to 63% of LPS-stimulated levels. LPS-stimulated
CXCL10 expression was not affected by X6905.

Figure 8. Effects of X6905 (light blue bars) on LPS-induced gene expression of IL-6 (A), CCL2 (B),
CXCL2 (C), CXCL10 (D), and TNF-α (E). Gene expression was quantified using qPCR. The quantity
of copies was normalized to consecutively expressed GAPDH. The values were normalized to LPS
(black bar) and are shown as the mean of three individual values ± SDs. For statistical analysis, one-
way ANOVA with Dunett’s post-hoc test was performed using the LPS group (black bar) as refer-
ence with * p < 0.05 and ** p < 0.01. Non-significant differences are labeled as “ns”.
Molecules 2025, 30, 2452 10 of 22

2.4. Effects of X6905, X7732, and X7625 on LPS-Mediated Phosphorylation of Erk1/2

After investigating the anti-inflammatory potential of X6905, X7732, and X7625, fur-
ther research was carried out to reveal a possible inhibition of intracellular signaling path-
ways and kinases such as MAP-Kinases (MAPK) and others. X6905 affected LPS-induced
phosphorylation of Erk1/2 (Figure 9), which triggered further investigations of Erk1/2 in
response to a treatment with X7732 and X7625 (Figure 10).
The effect of X6905 on phospho-Erk1 (A) and phospho-Erk2 (B) are shown separately.
After LPS stimulation, a three- (A) and two-fold (B) increase in phosphorylated Erk1 (A)
and Erk2 (B) was detected. The levels of both phosphorylated proteins were significantly
decreased by X6905, particularly at higher concentrations. Interestingly, X6905 had differ-
ential effects on Erk1 and Erk2 inhibition: A much stronger effect was observed for Erk1,
where X6905 decreased phospho-Erk1 levels dose-dependently and significantly. At 1
µM, phospho-Erk1 was already reduced by 22% of the LPS-stimulated group and was
further decreased at 20 µM X6905 to almost the level of the unstimulated control to 40%
of the LPS-stimulated group. For phospho-Erk2, the effect of X6905 was less pronounced
but still significant at a concentration of 20 µM, showing a reduction to 60% of the LPS-
stimulated protein level.
To investigate if a similar effect on Erk1 phosphorylation is also caused by the other
compounds X7732 and X7625, further investigations were carried out (Figure 10). Phos-
pho-Erk1 (A,C) and phospho-Erk2 (B,D) were again analyzed individually. In all experi-
ments, phosphorylation of Erk1 and Erk2 was induced by LPS-stimulation.

Figure 9. Effects of X6905 (light blue bars) on phospho-Erk1 (A) and phospho-Erk2 (B), Vinculin
was used for reference for equal gel loading. The cells were stimulated with X6905 for 30 min before
LPS was added for another 30 min. The values were normalized to LPS (black bar) and are shown
as the mean of three–four individual Western blot measurements ± SDs. One-way ANOVA with
Dunett’s post-hoc test was performed using the LPS group as reference with * p < 0.05; ** p < 0.01;
*** p < 0.001. Non-significant differences are labeled as “ns”.

Interestingly, X7732 also exerted a mild inhibitory effect on phospho-Erk1. A de-

crease was observed at 1 µM and 5 µM, reaching 78% and 70% of LPS-stimulated levels,
respectively. At 10 and 20 µM, the levels of phospho-Erk1 increased but remained below
the LPS-stimulated levels. For phospho-Erk2, a similar effect was observed, namely a ra-
ther weak dose-dependent reduction in phospho-Erk2 to 73% of LPS-stimulated levels.
However, the LPS control group showed relatively high standard deviations for phospho-
Erk1 and phospho-Erk2. Contrarily, X7625 was observed to increase phospho-Erk1 levels
to approximately 120% of the LPS control at 10 and 20 µM, while not affecting phospho-
Erk2 levels.
Molecules 2025, 30, 2452 11 of 22

Figure 10. Effects of X7732 (blue bars) and X7625 (dark blue bars) on phosphorylation of Erk1 (A,C)
and Erk2 (B,D). Vinculin is used for reference for equal gel loading. The cells were stimulated with
the compounds for 30 min, before LPS was added for another 30 min. The values were normalized
to the LPS value (black bar) and are shown as the mean of three individual Western blot measure-
ments ± SDs. One-way ANOVA with Dunett’s post-hoc test was performed using the LPS group
(black bar) which showed no significant (ns) effects.

2.5. Effects of X6905 on Other Kinases and Signaling Proteins

In addition to Erk1/2, further kinases and signal-transducing proteins were investi-
gated for the potential inhibitory effects of X6905. PKC, p38 MAPK, and IkBα were not
modulated after X6905 treatment (Supplementary Materials). Phosphorylation of
SAPK/JNK was inhibited by X6905 to a small extent, but no statistical significance was
observed (Figure 11). JNK consists of two subunits 46 kDa (A) and 54 kDa (B), which were
both extensively phosphorylated after LPS-treatment. X6905 caused a dose-dependent re-
duction to 76% of the LPS-stimulated levels for the 46 kDa subunit. Interestingly, LPS-
induced phosphorylation of the 54 kDa subunit was inhibited by X6905: While X6905 did
not affect this subunit at 1 and 5 µM, a dose-dependent decrease to 61% was visible after
stimulation at 20 µM.
Molecules 2025, 30, 2452 12 of 22

Figure 11. Effects of X6905 (light blue bars) on the phosphorylated subunits of JNK: phospho-JNK
(46 kDa) (A) and phospho-JNK (54 kDa) (B). Vinculin was used for reference for equal gel loading.
The cells were stimulated with X6905 for 30 min, before LPS was added for another 30 min. The
values were normalized to LPS (black bars) and are shown as the mean of three–four individual
Western blot measurements ± SDs. One-way ANOVA with Dunett’s post-hoc test was performed
using the LPS group as reference that showed no significant effects (ns).

3. Discussion
In this study, we investigated the anti-inflammatory effects of three novel isomeric 3-
pyridinylmethylcoumarin derivatives in murine BV2 microglial cells. The aim was to eval-
uate the biological effects of the different orientations of the pyridine moiety. Only X6905,
slightly but significantly, reduced cell viability in the highest concentration of 20 µM.
X7732 showed a trend toward reduced cell viability at the highest concentration, although
this effect did not reach statistical significance. X6905 strongly inhibited LPS-induced IL-
6 release and expression as well as CCL2 release, probably mediated via the Erk1/2 path-
way. Furthermore, a significant reduction in CXCL2 and CXCL10 release was observed
for X6905. X7732 exhibited comparable effects to X6905 with a slightly reduced biological
potency and increased TNF-α release at a concentration of 10 µM, and with an enhance-
ment of CXCL10 release at 0.1 µM. X7625 enhanced TNF-α release at concentrations of 0.1
µM and significantly reduced CXCL10 release at 20 µM; however, the effects were less
concentration-dependent. Overall, these results support the anti-inflammatory potential
of coumarin derivatives and underline the enormous relevance of small chemical altera-
tions in modulating biological efficacy.
The applied ATP assay is based on a luciferase reaction, allowing the measurement
of cellular ATP levels by luminescence after cell lysis. It is known for its high sensitivity
and robustness against artifacts compared to other cell viability assays [34]. However, in-
tracellular ATP levels may depend on the cells’ metabolic state [35,36]; therefore, treat-
ment with compounds may influence intracellular ATP levels. Treatment with 0.2% of
DMSO, which was used as solvent of the tested coumarin derivatives, reduced cell viabil-
ity by approximately 30%. To address the cytotoxic effects of DMSO, 0.2% of DMSO was
included in all controls (untreated cells and LPS control) in the following experiments.
However, the cytotoxic effects of the tested compounds should be further investigated in
future studies, including more complex tissue models or in vivo experiments and the pos-
sible use of alternative solvents. Some novel aprotic polar solvents, such as zwitterionic
liquid [37], might be less cytotoxic than DMSO with comparable solubility of the couma-
rins and should be compared to DMSO in upcoming experiments.
Since X6905 showed the most potent inhibition of LPS-induced IL-6, TNF-α, CCL2,
CXCL2, and CXCL10 release, we further evaluated its effects on mRNA expression of
Molecules 2025, 30, 2452 13 of 22

those chemokines and cytokines. Interestingly, only the expressions of IL-6, CCL2, and
TNF-α were significantly reduced by pretreatment with X6905, while the expressions of
CXCL2 and CXCL10 remained unaffected. In contrast to the concentration-dependent ef-
fects on the cytokine and chemokine release, 20 µM of X6905 showed a weaker inhibition
of IL-6 and CCL2 release. A possible explanation for the observed differences between
effects on release and expression might be the different time points of assessment. The
effects of X6905 on mRNA expression were measured after 4 h of LPS stimulation, while
effects on cytokine and chemokine release were measured after 24 h. Therefore, differ-
ences might be explained due to time dynamic effects. However, as we have shown pre-
viously for COX-2, the expression and synthesis differed over time, and synthesis might
be modulated on post-transcriptional levels rather than by expression only [6].
When comparing the tested 3-pyridinylmethylcoumarins, X6905 exhibited the high-
est biological activity, followed by X7732. Interestingly, X7625 showed a tendency for con-
trary effects especially at lower concentrations, as observed for IL-6 and CXCL10. Due to
the high electronegativity of nitrogen, the position of the N-atom in the pyridine ring may
influence the affinity to receptors. This might be an explanation for the different biological
effects observed between X6905, X7732, and X7625 in the current study. It seems that the
pyridin-4-yl and pyridin-2-yl derivative enhance biological activity, as X6905 (pyridin-4-
yl) and X7732 (pyridin-2-yl) showed higher anti-inflammatory potency. In contrast to
GPR55 agonists, the antagonists at the GPR55 mostly contain a heteroaromatic or aromatic
moiety attached via a methylene linker to the 3-position of the coumarin scaffold, possibly
preventing receptor toggle switches. Also, the most electronegative region of commer-
cially available GPR55 antagonists is located at the end of the molecule’s central portion,
fitting vertically in GPR55’s binding pocket [38]. Regarding the coumarin scaffold, the
most electronegative portion is located at the molecule’s ester function. However, the pyr-
idine ring may be the part of the molecule protruding from the GPR55 binding pocket,
stabilizing the receptor’s “off” state [38]. Further modifications to other parts of the cou-
marin derivatives might be even more relevant. The pyridin-3-yl derivative (X7625) might
not be as effective in preventing toggle switches as the pyridin-4-yl or the pyridine-2-yl
derivative. Future studies with other coumarin derivatives might help us understand the
influence of chemical alterations in respect to GPR55-dependent effects as well as for cou-
marins acting on multiple molecular targets, referred to as “polypharmacy”. Furthermore,
experimental designs to measure the affinity to GPR55 and possible other molecular tar-
gets should be pursued in future research.
Regarding the structure of the isomeric 3-pyridinylmethylcoumarins X6905, X7732,
and X7625, the substitution in the C-3, C-5, and C-7 positions of the coumarin scaffolds
suggests an antagonistic activity of the three compounds on the GPR55 receptor as well
as an agonistic activity at CB2 due to the large lipophilic residue on C-7 [3]. Especially
long and bulky alkyl moieties have been shown to increase GPR55 affinity and introduc-
ing CB agonism, possibly changing the antagonistic mechanism from orthosteric to allo-
steric at GPR55 [3,39]. Therefore, the 1-butylcyclohexyl residue at C-7 of X6905, X7732,
and X7625 may lead to dual target molecules. For the 3-pyridinylmethylcoumarins used
in this study, CB-1 and CB-2 affinities have been shown, most likely with an agonistic
activity [39]. As we have shown previously, coumarin derivatives with different substitu-
tion patterns exhibited antioxidative effects, which were abolished after GPR55 knockout
[5]. We further demonstrated that these compounds activate GPR55 [6], which is likely
associated with their observed anti-inflammatory effects. In contrast to the coumarin de-
rivatives evaluated in the current study, those compounds were substituted with an iso-
propyl group on C-8 and had no chemical residues at C-7, so no CB receptor affinity was
expected [3]. However, further experiments are necessary to prove the GPR55-depend-
ency and the significance of the expected CB-affinity on the observed anti-inflammatory
Molecules 2025, 30, 2452 14 of 22

effects of X6905, X7732, and X7625. Additionally, it is essential to rule out other molecular
targets and potential receptor-independent effects.
Approaches to knock down the GPR55 in the BV2 microglial cells might help assign
the observed effects to molecular targets, such as receptors or direct intracellular mecha-
nisms. The anti-inflammatory effects might be mediated via the Erk-pathway, which is
activated by GPR55. Lysophosphatidylinositol (LPI), discussed as endogenous GPR55 ag-
onist, promoted phosphorylation of Erk1/2 in GPR55-HEK293 cells [40]. However, the
downstream signaling of GPR55 might differ depending on the specific ligand [6,40].
Therefore, the understanding of involved pathways is crucial for the drug design and us-
age of functional selectivity of receptors and its associated signaling.
LPS is known to activate toll-like receptor (TLR) 4, resulting in a phosphorylation of
the MAPK-pathway, leading to phosphorylation of Erk as downstream kinase and induc-
tion of pro-inflammatory gene expression via c-Fos [41]. Activation of the MAPK-Erk
pathway results in a pro-inflammatory response, such as IL-6 release [42]. As a possible
intracellular mechanism of the observed anti-inflammatory effects of X6905, a reduced
phosphorylation of Erk1 and Erk2 was shown. However, the activation of upstream ki-
nases of the MAPK pathway was not significantly modified by X6905 (Supplementary
Materials). X7732 showed a slight trend to reduce Erk1/2 phosphorylation while X7625
did not modulate Erk phosphorylation. This is in line with the smaller biological potency
of X7732 and noticeably different effects of X7625. The MAPK-Erk pathway is further ac-
tivated by TNF-α, resulting in the inhibition of IL-6 release [42]. Therefore, the direct ef-
fects of X6905 on the MAPK pathway might add up with the inhibitory effects of X6905
on TNF-α release, which was not shown for X7732 and X7625. Erk1 and Erk2 are discussed
as redundant isoforms, since the silencing of one gene leads to an overexpression of the
remaining form. However, some studies report isoform-specific phenotypes [43]. Erk2 is
expressed at higher levels in most tissues, and no isoform-specific signaling is described.
Rather, Erk1 and Erk2 compete with each other for phosphorylation by MEK1/2 and share
most amino acids of the enzymes’ region that interacts with the substrates [43]. Therefore,
the different effects observed on phosphorylation of Erk1 and Erk2 might be dependent
on the different expressional levels of both kinases.
In patients with depression, the addition of anti-inflammatory agents to an antide-
pressant drug enhanced therapeutic effects. Furthermore, the use of non-steroidal anti-
inflammatory drugs (NSAIDs) and cytokine-inhibitors showed antidepressant effects
when compared to placebo [32]. Ibuprofen, a well-known and established NSAID, is
known to reduce inflammatory parameters, such as IL-6 and CCL2 [44]. However, in clin-
ical practice, the long-term use of NSAIDs is especially associated with side-effects, such
as gastric ulcers and consecutive bleeding. Another possible involved pathway is the pros-
taglandin E2 (PGE2) pathway via COX-2, since specific COX-2 inhibitors show antidepres-
sant effects as adjunctive to antidepressive treatment [32,45]. However, these COX-2 in-
hibitors, such as celecoxib, are associated with increased cardiovascular risk and gastric
bleeding as well. Via the PGE-receptor 3 (EP3), the COX-2/PGE2 pathway is coupled to the
modulation of chemokine release, such as CCL-2 [46], and other coumarin derivatives
modulate COX-2 and PGE2 synthesis [6,7]. Therefore, the effects of the 3-pyridinylmethyl-
coumarins on the COX-2/PGE2 pathway should be investigated further. IL-6 antibodies,
such as tocilizumab, and TNF-α antibodies, such as infliximab, are already used in differ-
ent diseases linked to inflammatory processes. However, studies regarding anti-depres-
sant effects have not shown robust antidepressive effects so far [47,48], even though some
studies suggest a benefit regarding depressive symptoms [49]. Those antibodies, however,
are quite expensive and patients receiving those antibodies experience a higher risk of
infections. Coumarin derivatives, such as warfarin, are established in the clinical use for
decades as anti-coagulative drug. Beside bleeding as major complication during the
Molecules 2025, 30, 2452 15 of 22

warfarin treatment, the three tested 3-pyridinylmethylcoumarins might be possibly asso-

ciated with less side-effects substantiating further clinical studies regarding their anti-in-
flammatory effects. Furthermore, other already described possible effects of the coumarin-
scaffold, such as anti-microbial or anti-proliferative characteristics [2], should be evalu-
ated in cell cultures and more complex organoid models, such as organotypic hippocam-
pal slice cultures (OHSC), before evaluating the effects and possible side-effects in healthy
and disease in vivo models.
The neuroinflammation hypothesis assumes that inflammatory mechanisms might
be involved in the genesis and progression of neuropsychiatric disorders [33]. However,
clinical effects of pharmacological or psychotherapeutic interventions on cytokine or
chemokine levels are still on a correlative level [26,32,50,51], failing to show a causal asso-
ciation of central nervous inflammation and neuropsychiatric disorders. Based on the in-
flammatory hypothesis of depression and neuropsychiatric disorders and previous works
with coumarin derivatives, the coumarin scaffold is a promising structure for the devel-
opment of anti-inflammatory drugs. However, future clinical studies focusing on the in-
flammatory hypothesis and the potential of anti-inflammatory drugs in neuropsychiatric
disorders are necessary to draw further conclusions about the potential use of the couma-
rin scaffold in therapeutic regimes in those disorders. Those studies should include as-
sessment of additional effects and side-effects, since the coumarin scaffold is known for
its complex pharmacological activity and multiple molecular targets [52,53].

4. Materials and Methods

4.1. Novel Coumarin-Derivatives X6905, X7732, and X7625
X6905, X7732, and X7625 were synthesized as previously published [39] and pro-
vided by the Karlsruhe Institute of Technology (KIT) as a 10 mM stock solution in DMSO
(PAN-Biotech™ GmbH, Aidenbach, Germany). The stock solutions were stored and
shipped at −20 °C.

4.2. BV2 Microglial Cell Culture

BV2 microglial cells were kindly provided by Prof. Langmann (Department of Oph-
thalmology, University of Cologne, Cologne, Germany). BV2 cells were cultured in 1x
RMPI-Medium 1640 with 10% fetal calf serum (FCS) and 1% Penicillin/Streptomycin (all
from Thermo Fisher Scientific Inc., Waltham, MA, USA), and kept at 37 °C and 5% CO2. If
necessary, the medium was replaced or the cells were passaged.
For treatment, the cells’ medium was removed, rinsed with dPBS (Sigma-Aldrich
Corporation, St. Louis, MO, USA), and the cells were detached, adding 0.05% Trypsin-
EDTA (Thermo Fisher Scientific Inc., Waltham, MA, USA) for a few minutes. Afterwards,
the cells were re-seeded in 6-, 12-, 24- or 96-well plates at different concentrations (3 × 105,
2 × 105, 1 × 105 or 3 × 104 cells/well). After cell adherence around 24 h later, the medium
was replaced, and the cells were used for stimulation. Pro-inflammatory stimulation was
achieved by addition of 10 ng/mL LPS from Escherichia coli O111 B:4 (from Sigma-Al-
drich, Taufkirchen, Germany).

4.3. Cell Viability ATP Assay

An ATP assay (CellTiter-Glo® 2.0 Assay from Promega Corporation, Fitchburg, WI,
USA) was used to assess the cytotoxic effects of the coumarin derivatives. It works by
quantifying ATP through an ATP-dependent enzymatic reaction, which emits light. To
this end, the cells were plated with approximately 3 × 104 cells/well in a 96-well plate. After
adherence of cells, the medium was replaced before simulation. After incubation of the
compounds, DMSO, and ethanol for 30 min, LPS was added for 24 h. Afterwards, the plate
Molecules 2025, 30, 2452 16 of 22

was incubated for 30 min at room temperature and the ATP assay reagent was added
according to the manufacturer’s protocol. The plate was shaken for 2 min and incubated
for another 10 min at room temperature before luminescence was detected with a Modu-
lus™ Microplate Multimode Reader (from Promega Corporation, Fitchburg, WI, USA).
The control group was set to 100% viability of BV2 cells and used as a reference for the
following one-way ANOVA statistical analysis (*). Additionally, 0.2% DMSO was also
used as a reference for a second one-way ANOVA (#).

4.4. Cell Treatment and Determination of Cytokine Release with ELISA

The cells were plated with approximately 105 cells/well onto a 24-well plate. After
adherence, the medium was replaced and the cells were ready for stimulation with the
compounds dissolved in DMSO. To exclude the potential effects of DMSO, an equal
amount of DMSO was added to untreated cells and the LPS-stimulated group, which was
repeated for all following cell stimulations. Thirty minutes later, LPS was added to the
appropriate wells. After 24 h of incubation, supernatants were collected and centrifuged
at 11,000 rpm for 2 min to discard pellets. The samples were stored at −80 °C.
The concentrations of cytokines and chemokines in the supernatants (IL-6, CCL2,
CXCL2, CXCL10, and TNF-α) were assessed by performing ELISA according to the man-
ufacturer’s protocol (Bio-Techne GmbH, Wiesbaden, Germany).
Briefly, 96-well plates were coated with the respective capture antibody and incu-
bated overnight. The next day, the plates were washed and blocked. After 1 h of incuba-
tion and another washing step, the samples and standards were added for 2 h. The respec-
tive detection antibody was added after another washing step. After 2 h, Streptavidin-
HRP was added, and after application of 1 × TMB (Thermo Fisher Scientific Inc., Waltham,
MA, USA), the plate was read at 450 nm using a MRXe microplate absorbance reader
(Dynex® Technologies, Chantilly, VA, USA). The cytokine and chemokine release in the
LPS group was set to 100% and used as reference for one-way ANOVA statistical analysis.

4.5. RNA Isolation and Real-Time PCR

Quantitative PCR using SYBR Green as dye was performed to investigate effects on
cytokine and chemokine expression. The BV2 cells were seeded on a 6-well plate with
approximately 3 × 105 cells/well. The medium was replaced after cell adherence. One hour
later, the cells were stimulated with X6905 and DMSO for 30 min before LPS was applied
for an additional four hours. The sample collection and the RNA isolation were achieved
with the Universal RNA Purification Kit (Roboklon, Berlin, Germany), the protocol was
followed accordingly. After isolation, the RNA samples were solved in ddH2O, and the
concentration was measured using NanoDrop (ddH2O and NanoDrop both from Thermo
Fisher Scientific Inc., Waltham, MA, USA).
Subsequently, cDNA was synthesized from 500 ng purified RNA applying random
primers (Biomers.net GmbH, Ulm, Germany) to the RNA sample and placed into the
Thermocycler (Analytik Jena GmbH+Co. KG, Jena, Germany). After annealing, dNTPs
(Sigma-Aldrich Corporation, St. Louis, MO, USA), reverse transcriptase, 5X RT-Buffer,
and ribonuclease inhibitor (all from Promega Corporation, Fitchburg, WI, USA) were
added and placed into the Thermocycler again for cDNA-synthesis.
For qPCR, the samples were processed on 384-well plates and incubated with SYBR
green (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the corresponding primers for
each gene. The sequences of primers (all from Biomers.net GmbH, Ulm, Germany) were as
follows. IL-6: Forward (F): 5′-AGTTGCCTTCTTGGGACTGA-3′/Reverse (R): 5′-TTCTG-
CAAGTGCATCATCGT-3′, CCL2: F: 5′-TGATCCCAATGAGTAGGCTGG-3′/R: 5′-AC-
CTCTCTCTTGAGCTTGGTG-3′, CXCL2: F: 5′-CCCTCAACGGAAGAACCAAAG-3′/R: 5′-
GAGGCACATCAGGTACGATCCA-3′, CXCL10: F: 5′-CAGTGGATGGCTAGTCCTAATTG-
Molecules 2025, 30, 2452 17 of 22

3′/R: 5′-ACTCAGACCAGCCCTTAAAGAAT-3′, TNF-α: F: 5′-CCCACGTCGTAGCAAAC-

CACCA-3′/R: 5′-CCATTGGCCAGGAGGGCGTTG-3′ and GAPDH: F: 5′-TGGGAA-
GCTGGTCATCAAC-3′/R: 5′-GCATCACCCCATTTGATGTT-3′.
The plate was set up with two technical replicates for each sample; afterwards, the
mean of both values was used. To exclude self-replication of primers, an NTC containing
only ddH2O and forward and reverse primers was also added. Consecutively expressed
GAPDH was used as the reference for each sample. The qPCR was performed on the
CFX384 Real Time Detection System (Bio-Rad-Laboratories, Inc., Hercules, CA, USA), and
the LPS group was set to 100% of cytokine/GAPDH and used as reference for one-way
ANOVA statistical analysis.

4.6. Electrophoresis and Immunoblotting

Western blot was used to study the effects of the compounds on intracellular signal-
ing. The BV2 cells were plated at approximately 2 × 105 cells/well onto a 12-well cell culture
plate. After adherence, the medium was replaced, and the cells were stimulated after 1 h.
The compounds and DMSO were added for 30 min before stimulation with LPS for an-
other 30 min. The medium was removed, and all wells were rinsed with dPBS twice. To
prevent protein-lysis, all steps were performed on ice. A total of 100 µL of lysis buffer
(LB)++ (containing 42 mM Tris-HCl, 1.3% sodium dodecyl sulfate, 6.5% glycerin, 100 µM
sodium orthovanadate, and 2% phosphatase/0.2% protease inhibitors) was added to each
well, and cell lysis was achieved through scratching the cells with an upside-down pipette
tip for 1 min. After the sample collection, the lysate was centrifuged and heated at 90 °C
for 5 min.
To determine the protein concentration of each sample, a BCA Protein Assay (Thermo
Fisher Scientific Inc., Waltham, MA, USA) was performed after the sample collection. The
protocol was followed accordingly, and the samples were read using a microplate reader
at 570 nm. DTT (Sigma-Aldrich Corporation, St. Louis, MO, USA) was added to each sam-
ple before dilution in a loading buffer to a concentration of 0.5 µg/µL. For SDS-PAGE, 7.5
µg of each sample was run on a 10% acrylamide gel. Afterwards, the proteins were blotted
onto a 3 mm transfer membrane (Sigma-Aldrich Corporation, St. Louis, MO, USA),
blocked for 1 h with Roti-Block (Carl Roth GmbH+Co. KG, Karlsruhe, Germany) and
washed with TBS-T for 10 min before adding the primary antibody and incubating at 4 °C
overnight.
The following primary antibodies were used: mouse anti-vinculin (Cat. No.: V9264-
25UL, diluted 1:20 000, Sigma-Aldrich Corporation, St. Louis, MO, USA); rabbit anti-phos-
pho-p44/42 MAPK (Cat. No.: 45899, diluted 1:1000, Cell Signaling Technology, Cam-
bridge, UK); Phospho-SAPK/JNK (Cat. No.: 9251S, diluted 1:1000, Cell Signaling Technol-
ogy, Cambridge, UK); Phospho-PKC (pan) (Cat. No.: 9371T, diluted 1:1000, Cell Signaling
Technology, Cambridge, UK); Phospho-p38 MAPK (Cat. No.: 4511S, diluted 1: 1000, Cell
Signaling Technology, Cambridge, UK), and IkBα antibody (Cat. No.: 9242S, diluted 1:
1000, Cell Signaling Technology, Cambridge, UK). After washing three times with TBS-T
for 10 min, the respective secondary antibody (anti-mouse secondary antibody: Cat. No.:
401253-2ML, diluted 1:10,000, Merck KGaA, Darmstadt, Germany, or anti-rabbit second-
ary antibody: Cat. No.: 5450-0010, diluted 1:10,000, LGC Ltd., Teddington, UK) was added
and incubated for at least 1 h. After another three washing steps with TBS-T, quantifica-
tion of the protein-bound antibodies through the application of WesternBright chemilu-
minescence substrate (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) was per-
formed using the ChemiDoc™ MP Imaging System (Bio-Rad-Laboratories, Inc., Hercules,
CA, USA; Image Lab Touch software version 2.2.0.08). ImageJ 1.54g (National Institute of
Health, Bethesda, MD, USA) was used for the densiometric analysis of the chemilumines-
cence bands. Vinculin, an underlying constitutive expression, was used for the
Molecules 2025, 30, 2452 18 of 22

normalization of the quantity of each sample, and the values are shown as protein/vincu-
lin. The LPS group was set to 100% of protein/vinculin and used as reference for one-way
ANOVA statistical analysis.

4.7. Statistical Analysis

The raw values were first converted into percentages of the corresponding reference
group (untreated cells for ATP-assay or LPS for ELISA, qPCR, and Western blot). All data
are presented as the mean of each value of the group ± SDs of three or more biological
replicates. The statistical analysis was performed using one-way ANOVA with GraphPad
Prism 10.2.3 (Graphpad Software, Inc., La Jolla, CA, USA). The level of significance is
shown as * p < 0.05, ** p < 0.01, *** p 0.001, **** p < 0.0001 and indicated above the respective
bar.

5. Conclusions
Anti-inflammatory effects have been described for C3-, C5-, and C7-substituted cou-
marins and are most likely dependent on an inverse agonistic activity at GPR55. In previ-
ous studies, we already showed the relevance of different chemical residues on the anti-
inflammatory potency. However, in this study, we proved that the position of nitrogen in
the pyridine ring is crucial for the biological effects of the isomeric coumarin derivatives.
These findings might offer new insights for the development of a new class of anti-inflam-
matory pharmaceutics that might open new therapeutic options in the treatment of psy-
chiatric disorders.

Supplementary Materials: The following are available online at https://www.mdpi.com/arti-

cle/10.3390/molecules30112452/s1, Figure S1. Effects of X6905 (light blue bars) on phospho p38
MAPK, Vinculin was used as reference for equal gel loading. Cells were stimulated with X6905 for
30 minutes, before LPS was added for another 30 minutes. Values were normalized to LPS and are
shown as the mean of 3 individual Western Blot measurements with ± SDs. One-way-ANOVA with
Dunett’s post hoc test was performed using the LPS-group (black bar) as reference; Figure S2. Effects
of X6905 (light blue bars) on phospho PKC beta, Vinculin was used as reference for equal gel loading.
Cells were stimulated with X6905 for 30 minutes, before LPS was added for another 30 minutes.
Values were normalized to LPS and are shown as the mean of 3 individual Western Blot measure-
ments with ± SDs. One-way-ANOVA with Dunett’s post hoc test was performed using the LPS-
group (black bar) as reference; Figure S3. Effects of X6905 (light blue bars) on IkBa, Vinculin was
used as reference for equal gel loading. Cells were stimulated with X6905 for 30 minutes, before LPS
was added for another 30 minutes. Values were normalized to LPS and are shown as the mean of 3
individual Western Blot measurements with ± SDs. One-way-ANOVA with Dunett’s post hoc test
was performed using the LPS-group (black bar) as reference.

Author Contributions: B.L.F., M.A., and R.S. participated in the design of the research. R.S. carried
out the experiments and analyzed the respective data. C.R., N.S. and, S.A.E.M. performed the syn-
thesis of X6905, X7732, and X7625. B.L.F., M.A., and R.S. wrote or contributed to writing the manu-
script. In addition, R.S., M.A., C.R., C.W.G., C.N., S.B., and B.L.F. reviewed all the data and discussed
the manuscript. The experimental procedures and the data of the coumarin derivative synthesis
were uploaded to the Chemotion Repository by C.R. All authors have read and agreed to the pub-
lished version of the manuscript.

Funding: The article processing charge was funded by the Baden-Württemberg Ministry of Science,
Research and Art and the University of Freiburg Library in the “Open Access Publishing” funding
program.

Institutional Review Board Statement: Not applicable.

Molecules 2025, 30, 2452 19 of 22

Informed Consent Statement: Not applicable.

Data Availability Statement: All data on the synthesis and analysis of the investigated compounds,
X6905, X7732, and X7625, are available via the Chemotion repository (https://www.chemotion-re-
pository.net/, accessed on 18 January 2025) and can be accessed via the following collection DOI:
https://dx.doi.org/10.14272/collection/CSR_2024-10-07 [54].

Acknowledgments: This project was supported by the core facility “Molecule Archive” of the Ger-
man Research Foundation (Deutsche Forschungsgemeinschaft, DFG project number: 284178167)
and the Repository Chemotion (DFG, grant number 266379491). The skillful technical assistance of
Ute Wering in general and in qPCR is greatly acknowledged. Furthermore, the assistance of
Dominik Klett in questions to ELISA and cell culture is appreciated. The authors thank the Baden-
Württemberg Ministry of Science, Research and Art and the University of Freiburg Library for their
support via the Open Access Publishing funding program. Moreover, C.R. acknowledges the sup-
port from the Karlsruhe School of Optics and Photonics (KSOP), the BioInterface International Grad-
uate School (BIF-IGS), and the Jürgen Manchot Foundation. We also gratefully acknowledge
Thomas Hurrle for his valuable contribution to the development of the synthetic route and his foun-
dational work on this topic. Moreover, we appreciate the support of the Deutscher Akademischer
Austauschdienst (DAAD).

Conflicts of Interest: The authors declare no conflicts of interest.

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