A toolbox of molecular photoswitches to modulate the CXCR3 chemokine receptor with light

Article abstract of DOI:10.3762/bjoc.15.244

We report a detailed structure–activity relationship for the scaffold of VUF16216, a compound we have previously communicated as a small-molecule efficacy photoswitch for the peptidergic chemokine GPCR CXCR3. A series of photoswitchable azobenzene ligands was prepared through various synthetic strategies and multistep syntheses. Photochemical and pharmacological properties were used to guide the design iterations. Investigations of positional and substituent effects reveal that halogen substituents on the ortho-position of the outer ring are preferred for conferring partial agonism on the cis form of the ligands. This effect could be expanded by an electron-donating group on the para-position of the central ring. A variety of efficacy differences between the trans and cis forms emerges from these compounds. Tool compounds VUF15888 (4d) and VUF16620 (6e) represent more subtle efficacy switches, while VUF16216 (6f) displays the largest efficacy switch, from antagonism to full agonism. The compound class disclosed here can aid in new photopharmacology studies of CXCR3 signaling.

Full text of DOI:10.3762/bjoc.15.244

A toolbox of molecular photoswitches to modulate the CXCR3
chemokine receptor with light
Xavier Gómez-Santacana1,2, Sabrina M. de Munnik1, Tamara A. M. Mocking1,
Niels J. Hauwert1, Shanliang Sun1, Prashanna Vijayachandran1, Iwan J. P. de Esch1,
Henry F. Vischer1, Maikel Wijtmans*1 and Rob Leurs*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 2509–2523.
1
Division of Medicinal Chemistry, Amsterdam Institute for Molecules
doi:10.3762/bjoc.15.244
Medicines and Systems (AIMMS), Vrije Universiteit Amsterdam, 1081
HZ, Amsterdam, The Netherlands and 2present address: Institute of
Functional Genomics, Université de Montpellier, Unité 5302 CNRS
and Unité U1191, INSERM, 34090 Montpellier, France
Received: 14 July 2019
Accepted: 27 September 2019
Published: 23 October 2019
Email:
This article is part of the thematic issue "Molecular switches".
Guest Editor: W. Szymanski
Maikel Wijtmans* - m.wijtmans@vu.nl; Rob Leurs* - r.leurs@vu.nl
*
Corresponding author
©
2019 Gómez-Santacana et al.; licensee Beilstein-Institut.
Keywords:
License and terms: see end of document.
azo compounds; chemokine receptor; efficacy photoswitching;
G protein-coupled receptors; photopharmacology
Abstract
We report a detailed structure–activity relationship for the scaffold of VUF16216, a compound we have previously communicated
as a small-molecule efficacy photoswitch for the peptidergic chemokine GPCR CXCR3. A series of photoswitchable azobenzene
ligands was prepared through various synthetic strategies and multistep syntheses. Photochemical and pharmacological properties
were used to guide the design iterations. Investigations of positional and substituent effects reveal that halogen substituents on the
ortho-position of the outer ring are preferred for conferring partial agonism on the cis form of the ligands. This effect could be
expanded by an electron-donating group on the para-position of the central ring. A variety of efficacy differences between the trans
and cis forms emerges from these compounds. Tool compounds VUF15888 (4d) and VUF16620 (6e) represent more subtle effi-
cacy switches, while VUF16216 (6f) displays the largest efficacy switch, from antagonism to full agonism. The compound class
disclosed here can aid in new photopharmacology studies of CXCR3 signaling.
Introduction
Photopharmacology is an emerging discipline at the interface of Photopharmacological approaches use light-sensitive photo-
medicinal chemistry and photochemistry. Classical medicinal chromic ligands that provide an advantageous and more precise
chemistry approaches make use of small-molecule ligands pharmacological alternative, especially with respect to spatial
binding a target protein, thereby modifying its activity. and temporal precision [1,2]. Photochromic ligands usually
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contain a molecular photoswitch (photoswitchable moiety) that based on azologization of a biaryl ligand class [24] and that
under certain wavelengths of illumination undergoes an isomer- activates the chemokine CXCR3 receptor (CXCR3), a GPCR
ization event, thereby changing the properties of the molecule endogenously activated by large peptides CXCL9, CXCL10
and the binding affinity for the target protein [3-5] or the and CXCL10 and involved in inflammatory responses. In fact,
intrinsic functional activity (efficacy) [6,7]. Despite the consid- the six compounds reported in that study represent the first pho-
erable number of photoswitches reported to date, such as tochromic small-molecule class that harbors a dynamic effi-
spiropyrans, diarylethenes, fulgides or azobenzenes, the most cacy photoswitch (from antagonism to agonism) on a
widely used moiety in the photopharmacology is the latter one. peptidergic GPCR (Figure 1B). Here, we report the rationale
One of the main reasons is that an azobenzene has a relatively and synthetic strategies behind this series of compounds, a
simple structure that can resemble various biaryl moieties of detailed analysis of the molecular determinants that control the
bioactive compounds: two aromatic rings linked with a bridge efficacy of the ligands (SAR, structure-activity relationship)
(
e.g., amide, ether, alkane or alkyne) [8]. In the case of azoben- and a toolbox of pharmacologically useful photoswitchable
zene the bridge is a diazene group (also called azo group) and small-molecule CXCR3 agonists.
depending on the wavelength of illumination, a linear trans-
isomer or a bent cis-isomer can be obtained [9]. If certain biaryl
moieties are replaced by an azobenzene (i.e., azologization ap- Azologization design
Results and Discussion
proach), there is a relatively good chance that one of the result- The chemokine receptor CXCR3 is endogenously activated by
ing photoisomers will have a spatial disposition similar to the the chemotactic peptides CXCL11, CXCL10 and CXCL9. Syn-
original biaryl unit and, therefore, a similar biological activity thetic small-molecule ligands can also bind to CXCR3 [25].
that might change upon isomerization of the azobenzene [8]. Multiple small-molecule CXCR3 antagonist scaffolds have
The second reason for the success of azobenzene in the been disclosed but small-molecule CXCR3 agonists are scarce
photopharmacology field is the robust photoisomerization. It and are mostly limited to peptidomimetics [25], which makes
provides typically high yields of photoisomerization with rela- our published biaryl series a notable exception [24]. The
tively low intensity of light and minimal photobleaching even general scaffold of these biaryl ligands consists of a polycy-
over hundreds of cycles. A third reason is the relatively high cloaliphatic anchor and a biaryl moiety both linked to an ammo-
synthetic accessibility to azobenzenes. All these properties nium ion. Depending on the substitution pattern of this biaryl
make azobenzene compounds ideal molecular photoswitches to moiety, a broad spectrum of efficacies for CXCR3 can be ob-
control protein activity and physiological events with light.
tained, i.e., from antagonists to partial agonists and full agonists
Figure 1C) [24]. Meta and para-substitution yields antagonists
(
A number of protein targets have been explored with photo- (exemplified by 1a,b), while ortho-substitution with halogen
chromic small-molecule ligands, such as ion channels, micro- atoms provides agonists, exemplified by partial agonist 1c and
tubules, enzymes and GPCRs (G protein-coupled receptors) equal full agonists 1d and 1e (VUF11418, Figure 2A). A tenta-
[
[
1,10]. We focus our photopharmacology research on GPCRs tive explanation for this efficacy switch includes a variation of
3,7,11], which constitute a superfamily of membrane proteins the dihedral angle of the biaryl moiety, an increase of the elec-
that regulate many physiological processes [12]. Despite the tron density in the biaryl unit and/or a postulated halogen bond
high relevance of GPCRs both functionally and as a drug target of the halogen substituent to the binding site of the receptor
[
12], the first synthetic GPCR photochromic small-molecule [24].
ligands appeared only five years ago [13-15]. Since then,
photopharmacology has been explored on GPCRs targeted In order to obtain an efficacy photoswitching, we opted for
endogenously by small molecules [3,4,11,16-20], small replacing the biaryl moiety for an azobenzene in an azologiza-
peptides [5,13] and larger peptides [7,21,22]. Most of the tion approach (Figure 1D) with the expectation that the isomeri-
targeted GPCRs belong to the three rhodopsin-, secretin- and zation of the azobenzene would provide changes in 3D shape
glutamate-like subfamilies and involve GPCRs that endoge- that are similar to those observed in the biaryl series. To rein-
nously bind small-molecule ligands [10]. The ensuing photo- force this hypothesis, molecular alignments were performed
chromic GPCR ligands are usually orthosteric and the photo- with Molecular Operating Environment (MOE) software [26] in
switching generally affects the functional potency [4,11,23] which 1e was used as a model for full agonism (Figure 2). Its
and/or the binding affinity [3-5,11] of the ligand (Figure 1A). 3D structure was superposed with both the trans and cis-
However, as mentioned, GPCRs that endogenously bind large isomers of parent azobenzene compound 2a allowing flexi-
molecules (large peptides or proteins) can be also targeted by bility of the molecules except for the conformation of the trans
allosteric photochromic ligands. In an initial communication and cis-azobenzene moieties, which were fixed in the lowest
[
7], we recently reported a photochromic ligand class that is energy conformation to ensure a shape that has also been vali-
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Figure 1: Design of the CXCR3 efficacy photowitchable ligands. A,B) Schematic representation of a GPCR photochromic ligand that photoisomerizes
and thereby photoswitches (A) binding affinity and/or (B) functional efficacy. Red represents the inactive GPCR, while green represents the active
GPCR. C) General structure and exemplary functional dose-response curves of the parent biaryl family of CXCR3 ligands disclosed in Wijtmans et al.
[
24], in which ortho substitution on the outer aromatic ring gives partial or full agonists, while meta substitution provides antagonists. D) Azologization
of the biaryl moiety provides a family of photowitchable CXCR3 small-molecule ligands.
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Figure 2: Conformational alignment of a biaryl CXCR3 agonist with a designed azobenzene analogue. A) 2D structure of biaryl CXCR3 agonist 1e
and designed ligand trans-2a and cis-2a. B,C) Alignments of 1e (grey carbon atoms) and 2a, in which the trans and cis-isomers are shown in (B)
turquoise and (C) light magenta carbon atoms, respectively. The iodine atom is shown in red for clarity.
dated by crystallographic data [27]. The results show a reason- contains a bromine atom in the ortho-position, was also tested
able overall alignment between trans-2a and the agonist 1e since this atom type provides full agonist activity of parent 1d.
(
Figure 2B), since the planar azobenzene is partially overlap- The synthesis of the compounds 2a–e was performed according
ping with the biaryl moiety. However, the two aromatic rings of to the strategies depicted in Scheme 1. The intermediate 7 was
both compounds can evidently not be in exactly the same plane prepared as described previously by us [28] and was used in a
because the azobenzene moiety is planar while the tilting of the reductive amination with 4-nitrobenzaldehyde (8a) to give the
dihedral angle of the biaryl moiety of 1e was speculated to be corresponding tertiary amine 9a in high yield. The nitro group
associated with its agonist activity (vide supra) [24]. The align- of 9a was subsequently reduced by SnCl2 in high yield. The re-
ment of the cis-isomer of 2a with agonist 1e is very different. sulting aniline 10a was used to obtain the azo compounds
The outer aromatic ring of cis-2a goes out of plane and is now 12a–e in varying yields through a Mills reaction with the corre-
occupying the space that is also occupied by the iodine atom of sponding nitroso compounds 11a–e, which were commercially
1
e. These calculations indicate that CXCR3 agonism is more available or prepared as described in our previous communica-
likely to be associated with the cis-isomer than with the trans- tion [7]. A final methylation of the tertiary amine 12a–e with
isomer in our designed azobenzenes.
MeI in DCM and subsequent precipitation with MTBE (methyl
tert-butyl ether) gave 2a–e as orange powders with ≥99% trans-
isomer in moderate to high yield.
Synthesis of azobenzene analogues and
exploration of substitution pattern on the
outer aromatic ring
Next, we characterized the photochemical properties of 2a–e,
In addition to unsubstituted azobenzene analogue 2a, we including absorption maxima, wavelengths of illumination and
explored the substitution pattern of the outer aromatic ring with percentage of conversion from trans to cis in the photosta-
chlorine atoms in the ortho, meta and para-position (com- tionary state (PSS cis). First, UV–vis absorption spectra were
pounds 2b–d, respectively) to also assess the possibility of measured in the dark and after illumination with a wavelength
agonism provided by a halogen bond. Compound 2e, which of 360 nm. In all cases the spectrum measured in the dark
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Scheme 1: Synthetic strategies for compounds 2a–e, 3a–e, 4a–d, 4f–i and 5b,c (Y = H, Cl). Reagents and conditions: (a) i) Et3N (1.1–1.6 equiv),
DCM, rt, 10–30 min; ii) NaBH(OAc)3 (1.6 equiv), rt, 6–16 h, 91–97%; (b) SnCl2 (5.0 equiv), EtOH, 75 °C, 2 h, 87%–quant; (c) AcOH/DCM, rt, 1–5 d,
23–73%, (d) MeI (20 equiv), DCM, rt, 6–72 h, 41–97%.
shows a large band between 320 and 330 nm that corresponds dard Error of the Mean). These experiments were performed
to the π–π* transition of the trans-isomer (Figure S1A, Support- with samples under dark conditions to ensure ≥99% trans-
ing Information File 1). After irradiating with different wave- isomer and with samples previously illuminated with 360 nm
lengths, the proportion of trans and cis-isomer varies to reach light to obtain a high percentage of cis compound in the PSS360.
the PPS. For example, after irradiating with 360 nm the π–π* This assay setup is enabled by the bistable nature of the photo-
transition band of the trans-isomer can barely be observed, but switch (vide supra), maintaining integrity of both the trans and
a less intense wide band around 420 nm appears, which corre- cis-isomer for the duration of the assay. Compounds 2a–e under
sponds to the n–π* transition of the cis-isomer (Figure S1A, dark conditions (trans-isomers) bind CXCR3 with Ki values in
Supporting Information File 1). This indicates that, using the high nanomolar range. In contrast, compounds 2a–e after
3
60 nm light, a PPS of high percentage of cis-isomer (PPS360) 360 nm illumination bind CXCR3 with Ki values in the low
can be reached. Literature evidence [7,29,30] suggests that this micromolar range, although the observed photoinduced affinity
scaffold would have sufficiently long half-lives of the PSS shifts (PAS) are not large (<4.0-fold). To assess if the com-
state, i.e., the cis-isomer only slowly reverts to trans in the dark, pounds have agonist or antagonist activity on CXCR3-medi-
which was confirmed by early-stage analyses on key com- ated signaling, a single-dose functional [35S]-GTPγS accumula-
pounds (data not shown). In fact, due to this bistable nature, the tion assay was performed with the compounds 2a–e at 10 µM
percentage of each photoisomer can be quantified by analytical (Table 1). In this assay, we observed that most of the com-
chromatography (LC–MS). The integration wavelength pounds are not activating the CXCR3 receptor in either cis or
(
265 nm) was selected closely to the observed isosbestic point trans configuration, which indicates that the compounds bind to
for most compounds to get an indication of mole ratios from the the receptor as antagonists. However, cis-2b and, more notably,
UV area ratios. For 2a–e after illumination at 360 nm, PPS360 cis-2e show a small partial agonist activity (11% and 23%, re-
values of 79–90% of cis-isomer were obtained. We also tested spectively) that gives a hint of a slight photoswitching of their
4
34, 460 nm and/or 494 nm to revert the isomerization process efficacy. Both compounds have a halogen atom on the ortho-
to PSS trans (Figure S1A, Supporting Information File 1). After position of the outer ring, which seems to reaffirm the impor-
re-illuminating the samples at 434 nm or 460 nm, the π–π* tran- tance of the ortho-halogen atoms effect observed in the biaryl
sition band of the trans-isomer re-appears, indicating a high series [24]. However, the difference in efficacy between cis and
percentage of trans-isomer at that PSS. The use of 494 nm trans-isomers (defined as PDE - photoinduced difference of
affords less trans compound and is less efficient in achieving efficacy) needed to be improved.
PSS trans.
Optimization of positioning of azobenzene
Next, the CXCR3 binding properties of 2a–e were measured unit
(
Table 1) in a competition binding assay versus displacement Aiming to improve the position and directionality of the
of a radiolabeled small-molecule CXCR3 antagonist halogen atom, we next designed a subseries with the azo group
[3H]-VUF11211 [31]). Values reported are mean ± SEM (Stan- at the meta-position of the central ring (scaffold 3) instead of at
(
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Table 1: Structures and results of photochemical, binding and functional characterization of compounds 2a–e and 3a–h.
Photochemistry
CXCR3 binding affinity
Functional CXCR3 activity
λmax
λmax
PSS360
trans
cis
(area %
pKi
pKi
E (%)
E (%)
PDE
Cmpd
X
π–π* a n–π* a
cis)b
SEM transc SEM PSS360c SEM PASd transe SEM PSS360f SEM (%)g
2
2
2
2
2
a
b
c
d
e
H
321
323
320
330
324
422
416
421
423
421
88.3
84.9
79.2
89.7
87.2
0.8
0.5
0.9
0.5
0.5
6.3
6.3
6.5
6.6
6.4
0.0
0.0
0.0
0.0
0.0
5.7
5.8
6.3
6.0
5.9
0.1
0.1
0.0
0.0
0.1
4.0
3.2
1.6
4.0
3.2
−11.0
2.4
−10.3
−9.8
7.0
2.3
4.3
3.5
3.5
1.6
−8.3
11.1
−9.6
−2.7
22.7
0.7
2.0
2.7
3.6
2.7
8.7
0.7
7.1
2-Cl
3-Cl
4-Cl
2-Br
1.5 15.7
3
3
3
3
3
3
3
3
a
b
c
d
eh
f
g
h
H
320
323
317
325
323
323
318
337
423
419
421
424
421
422
422
422
82.0
85.0
83.4
92.0
88.9
80.9
82.5
91.1
1.5
0.5
0.4
0.3
0.4
0.2
1.5
1.1
6.0
6.3
6.3
6.4
6.3
6.2
6.1
6.0
0.1
0.0
0.0
0.0
0.0
0.0
0.1
0.0
5.4
5.6
5.8
5.7
5.7
5.8
6.1
5.7
0.1
0.0
0.0
0.0
0.1
0.1
0.0
0.0
4.0
5.0
3.2
5.0
4.0
2.5
1.0
2.0
−11.6
−4.7
−8.4
−8.1
−5.8
−4.0
−11.1
−15.3
2.2
1.7
2.2
2.6
1.7
1.2
0.6
1.1
−4.7
17.4
−0.4
1.7
25.1
15.8
−7.6
5.4
1.7
3.7 22.1
1.6
2.3
2.0 30.9
1.3 19.8
6.9
2-Cl
3-Cl
4-Cl
2-Br
2-I
8.0
9.8
3-I
4-I
2.3
1.5 20.7
3.5
aThe absorbance maxima were extracted from UV–vis spectra at 25 µM in PBS buffer with 1% DMSO. b% of cis-isomer at the photostationary state
PSS360) measured in 68% TRIS buffer and 32% DMSO (1 mM) after being pre-irradiated at 360 nm as obtained by LC–MS integration of the cis and
(
trans-isomer signals at 265 nm. The mean and SEM of at least two experiments are shown. cBinding affinity of trans-isomer or PSS360 as measured
using [3H]-VUF11211 displacement. The mean and SEM of at least three experiments are shown. dThe photoinduced affinity shift (PAS) is calculated
as the ratio of the Ki PSS360 and Ki trans. eNormalized CXCR3 functional activity of trans-isomer (10 µM) in the dark (efficacy of 1d set at 100% activi-
ty). The mean and SEM of at least three experiments are shown. fNormalized CXCR3 functional activity of a sample (10 µM) pre-irradiated at 360 nm
to reach the photostationary state (efficacy of 1d set at 100% activity). The mean and SEM of at least three experiments are shown. gThe photoin-
duced difference of efficacy (PDE) is obtained by subtracting E trans from E PSS360. hCompound was previously described by us [7].
the para-position as in 2a–e. The analogue without halogen benzene cannot be accessed through oxidation of the corre-
substitution (3a) as well as Cl/Br analogues comparable to the sponding aniline owing to oxidation sensitivity of the iodine
first series (3b–e) were prepared. Moreover, since the impor- atom. Therefore, an alternative route had to be used to synthe-
tance of the presence of a halogen in the outer ring was sug- size 3f–h (Scheme 2). The route began with the oxidation of
gested in 2, compounds 3f–h, which include an iodine atom on methyl 3-aminobenzoate (17a) using Oxone® to obtain a crude
the ortho, meta and para-positions, respectively, were synthe- nitroso product 18a, which was used in a Mills reaction with an
sized.
iodoaniline (19a–c) at 100 °C to obtain azobenzenes 20g,h in
high yields and ortho-analogue 20f in a decreased yield presum-
The synthesis of compounds 3a–e was performed following the ably due to steric hindrance. The methyl ester was selectively
strategies shown in Scheme 1 as disclosed for compounds 2a–e. reduced with DIBAL-H to benzyl alcohols 23f–h, which were
Briefly, a reductive amination of 7 and 8b gave nitro com- oxidized with Dess–Martin periodinane to the corresponding
pound 9b, which after reduction to 10b, coupling with nitroso benzaldehyde 26f–h. Reductive amination of 26f–h with 7 gave
compounds 11a–e to 13a–e and methylation gave iodide salts the tertiary amines 13f–h. Methylation with iodomethane and
3
a–e with purities of trans-isomers ≥99% and overall yields subsequent precipitation gave 3f–h as orange powders with
similar to the ones obtained for 2a–e. However, 2-iodonitroso- ≥99% trans-isomer in high yields.
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Scheme 2: Synthetic strategies for compounds 3f–h, 4e, 6b, and 6d (Y = H, F, Cl, Br). Reagents and conditions: (a) Oxone® (2.0 equiv), DCM/H2O
1
:4, rt, 2 h, 91–98%; (b) AcOH, 100 °C, 16–20 h, 61–96%; (c) i) DIBAL-H (3.0–4.0 equiv), THF, 0–5 °C to rt, 2–4 h; ii) NH4Cl (aq), Rochelle salt (10%
aq), EtOAc, 1–2 h, 76–99%; or for 23h i) DIBAL-H (1.2 equiv) , DCM, −78 °C, 1 h; ii) MeOH, −78 °C to rt, 0.5 h, iii) Rochelle salt (10% aq), 3 h, 45%;
d) Dess–Martin periodinane (1.0 equiv), DCM, rt, 1–2 h, 68–97%; (e) i) Et3N (1.1–1.6 equiv), DCM, rt, 10–30 min; ii) NaBH(OAc)3 (1.6 equiv), rt,
–16 h, 69–96%; (f) MeI (20 equiv), DCM, rt, 6–72 h, 79–95%.
(
6
The photochemical properties of 3a–h are very similar to those Substituent effects on the outer ring
of 2a–e. For the trans-isomers, the maximum of the π–π* band One of the postulated contributors to the CXCR3 agonism
is located around 317–325 nm, with the exception of 3h which effect of parent biaryls such as 1d and 1e is the increased
has an iodine atom on the para-position and arguably confers a π-electron density of the aromatic rings [24]. A way to translate
larger electron delocalization of π-electrons that is translated to this effect to the azobenzene system is by including a π-donat-
a bathochromic shifting of the band to lower energy wave- ing substituent on the aromatic system. For synthetic access and
length (337 nm). PSS values of 81–92% are obtained when illu- thus rapid exploration, we chose a Cl atom as mildly π-EDG
minating with 360 nm light. The binding properties of 3a–h (electron-donating group) even though it is σ-EWG (electron-
also result in outcomes similar to those of 2a–e. That is, Ki withdrawing group) on the para-position of the inner ring with
values are in the high nanomolar range for the trans-isomers respect to the azo bond (i.e., the ortho-position with respect to
with no or low PAS values (1.0–5.0-fold) after isomerization. In the benzylic position) to afford subseries 4. This π-electron de-
single-dose functional assays, all trans-isomers do not substan- localization would increase the electron density of the azoben-
tially activate CXCR3, whereas three of the cis-isomers weakly zene unit, and was also expected to have an effect on the
to substantially activate CXCR3 (3b: 17%, 3e: 25% and 3f: trans–cis azobenzene isomerization and PSS value. In terms of
1
6%). Interestingly, these three compounds are the only ones to the outer ring, and given that ortho halogen atoms in subseries 2
include a halogen atom on the ortho-position of the outer and 3 play an important role in conferring the cis-isomer with
azobenzene ring, as is the case with para-compounds 2b (11%) partial agonism, in subseries 4 we explored halogen atoms and
and 2e (23%). However, compared to the latter, meta-com- groups differing in, e.g., steric and electronic properties (Me,
pounds 3b,e,f display an agonist effect that is slightly higher. CF3, OMe, OCF3). The electron-donating groups in this series
Increasing the size of the ortho-halogen does not guarantee a (Me and OMe) also increase the electron density of the azoben-
maximal agonist effect, as the absolute activity of 3f is lower zene system.
than that of its Br analogue 3e. Nevertheless, evidence emerged
that the ortho-position of the outer aromatic ring in scaffold 3 is The synthesis of 4a–d and 4f–i was performed following the
important to achieve agonist activity of the cis-isomer, but it route shown in Scheme 1. Briefly, 7 was used in a reductive
should be complemented with other strategies to further amination with 2-chloro-3-nitrobenzaldehyde (8c) to give nitro
increase intrinsic activity.
compound 9c which was reduced to aniline 10c and used to
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obtain the azo compounds 14a–d,f–i in variable yields through amination of 27 with 7 to 14e and subsequent methylation gave
a Mills reaction with the corresponding nitroso compounds 11a- 4e as an orange powder with ≥99% trans-isomer.
b,e–j. Methylation of 14a–d,f–i with MeI yielded compounds
4
a–d,f–g,i as orange powders with ≥99% trans-isomer in mod- Photochemical characterization of 4a–i (Table 2) gives similar
erate to high yields. Salt 4h did not precipitate after treatment results as observed for subseries 2 and 3 (Figure S2 (Support-
with MTBE and was isolated as an oil, which retained substan- ing Information File 1) shows an exemplary time-resolved
tial amounts of MTBE solvate even after extensive drying. For NMR and LC–MS analysis). That is, trans π–π* bands have a
4
e, we used the strategy as explained for iodo compounds 3f–h maximum between 326 and 332 nm and PSS values generally
(
vide supra). Briefly, the route (Scheme 2) consists of the oxi- amount to over 90% of cis-isomer after illuminating with
dation of methyl 5-amino-2-chlorobenzoate (17c) with Oxone® 360 nm light, as predicted from the electron localization provi-
to 18c, which was used in a Mills reaction with 2-iodoaniline ded by the chlorine atom in the central aromatic ring. Com-
(
19a) to yield azobenzene 21. The methyl ester was selectively pounds 4e, 4i and most notably 4g deviate from this trend, since
reduced with DIBAL-H and the resulting alcohol 24 oxidized the percentage of cis-isomer in the PSS is significantly lower
with Dess–Martin periodinane to benzaldehyde 27. Reductive (58–80%). Trans-4a–i show a slight decrease in binding affinity
Table 2: Structure and results of photochemical, binding and functional characterization of compounds 4a–i and 5b,c.
Photochemistry
CXCR3 binding affinity
Functional CXCR3 activity
E (%) E (%) PDE
λmax
λmax
cis
PSS360
(area %
trans
π–π* a n–π* a
pKi
pKi
Cmpd
X
cis)b
SEM transc SEM PSS360c SEM PASd transe SEM PSS360f SEM (%)g
4
4
4
4
4
4
4
4
4
a
b
c
dh
e
f
g
h
i
H
2-F
2-Cl
2-Br
2-I
2-Me
2-CF3
2-OMe
2-OCF3
326
330
330
328
330
332
326
328
326
424
420
419
422
424
428
424
427
422
91.8
93.0
92.8
92.6
79.9
94.9
58.0
92.6
78.8
1.2
0.2
0.1
0.2
1.2
0.1
1.1
0.2
1.2
5.8
5.6
5.8
5.9
5.7
5.9
5.9
5.4
5.9
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
5.2
5.0
5.3
5.6
5.7
5.3
5.7
5.0
5.6
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
4.0
4.0
3.2
2.0
1.0
4.0
1.6
2.5
2.0
−2.9
−4.9
2.3
13.6
16.0
6.9
10.5
4.9
−3.0
1.0
0.5
1.6
2.4
1.4
3.1
1.6
1.2
2.0
12.1
14.6
36.9
49.6
37.1
32.2
8.3
0.7 15.0
0.8 19.5
2.0 34.6
2.6 36.0
0.5 21.1
1.1 25.3
1.5 −2.2
1.1 13.8
18.7
-2.4
3.1
0.6
4
5
5
dh
b
c
2-Br
3-Br
4-Br
328
324
335
422
420
427
92.6
87.4
93.8
0.2
1.2
0.5
5.9
5.7
6.2
0.0
0.1
0.0
5.6
5.6
5.6
0.0
0.0
0.0
2.0
1.3
4.0
13.6
−1.7
−7.2
2.4
1.9
1.8
49.6
10.5
1.8
2.6 36.0
0.5 12.2
2.5
9.0
aThe absorbance maxima were extracted from UV–vis spectra at 25 µM in PBS buffer with 1% DMSO. b% of cis-isomer at the photostationary state
PSS360) measured in 68% TRIS buffer and 32% DMSO (1 mM) after being pre-irradiated at 360 nm as obtained by LC–MS integration of the cis and
trans-isomer signals at 265 nm. The mean and SEM of at least two experiments are shown. cBinding affinity of trans-isomer or PSS360 in PBS
25 µM) as measured using [3H]-VUF11211 displacement. The mean and SEM of at least three experiments are shown. dThe photoinduced affinity
(
(
shift (PAS) is calculated as the ratio of the Ki PSS360 and Ki trans. eNormalized CXCR3 functional activity of trans-isomer 10 µM in the dark (efficacy
of 1d set at 100% activity). The mean and SEM of at least three experiments are shown. fNormalized CXCR3 functional activity of a sample (10 µM)
pre-irradiated at 360 nm to reach the photostationary state (efficacy of 1d set at 100% activity). The mean and SEM of at least three experiments are
shown. gThe photoinduced difference of efficacy (PDE) is obtained by subtracting E trans from E PSS360. hCompound was previously described by us
[7].
2
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Beilstein J. Org. Chem. 2019, 15, 2509–2523.
compared to the subseries 2 and 3, amounting to low micro- benzene (11k,l) to form azobenzenes 15b,c, which were
molar values (Table 2). Moreover, PSS affinity values are also methylated to obtain 5b,c. The binding affinities (Table 2) ob-
modest, with only four compounds that have a PAS > 2.5 tained for the trans and cis-isomers are all in the micromolar
(
4a–c,f). However, functional data from [35S]-GTPγS assays range. More importantly, when comparing functional results of
provide encouraging results. While all trans-isomers of 4a–i do 4d, 5b and 5d, the preference for a Br atom on the ortho-posi-
not or only weakly activate the receptor, some of the cis- tion can also be reaffirmed for subseries 4, because cis-5c is an
isomers clearly behave as partial agonists. The highest efficacy antagonist, while cis-5b shows only a weak activity (Table 2).
is exerted by cis-4c–f with E values between 30–50% at 10 µM.
Interestingly, one of these compounds (4f) includes a methyl Substituent effects on the central ring
group as ortho-substituent on the outer ring and its agonist As shown, substitution of the outer ring with an ortho-bromine
effect at PSS amounts to 32%, possibly questioning one of our in conjunction with a Cl substituent on the central ring appeared
hypotheses that a halogen bond is involved in inducing CXCR3 to pave the way for efficacy photoswitching but there was still
agonism.
room for improvement. Our strategy in the final optimization
round was to replace the mildly π-EDG Cl atom with other
Similar to subseries 3, the substituent on the ortho-position of groups. Thus, to aim for a full-agonist cis compound, the sub-
the outer aromatic ring appears to be a driver for the agonist ac- series 6 was synthesized. In this subseries, different groups at
tivity of the corresponding cis-isomer. Starting from weak the para-position of the central ring (Y) in combination with the
partial agonist 4a (X = H), the agonist response of the cis in- ortho-Br atom on the outer aromatic ring are used to to explore
creases with increasing halogen atom (4b–d) to a maximal optimal electron densities in the azobenzene system. Besides
response with 4d (X = Br) having an agonist effect of 50%. the H and Cl already explored (3e and 4d, respectively), other
However, when X = I (4e) the agonist activity is reduced, as groups including an EWG halogen atom (F, 6b), EDG halogen
also observed in the 3 subseries (compare 3d to 3e). In general, atoms (Br, 6d and I, 6e) and stronger EDG moieties such as
the agonist effects and PDE values exerted by subseries 4 are OMe (6f), OiPr (6g), SMe (6h) and NMe2 (6i) were introduced.
larger than those of 3 (Figure 3). This could be explained by the
effect of the Cl atom present in the 4 subseries, that may give a Compounds 6b and 6d were synthesized by the route shown in
rise in electron density to the azobenzene necessary to increase Scheme 2, using in this case halogenated methyl aminoben-
the efficacy of the cis-isomer.
zoates 17b,c and 2-bromoaniline 19d. The synthetic route for
compound 6e proved more challenging. The route of Scheme 1
In both subseries 3 and 4, a bromine atom on the ortho-position could not be readily used because the corresponding iodinated
of the outer ring gives optimal results in providing CXCR3 effi- nitro precursor 8 is not commercially available, nor could the
cacy photoswitching (Figure 3). To confirm this for subseries 4, second route (Scheme 2) since the iodine would likely be sensi-
the analogues of 4d with the bromine on the meta and para po- tive to the first oxidation step. Therefore, we designed a new
sition (5b and 5c, respectively) were also synthesized. The syn- route (Scheme 3). The starting point was the advanced interme-
thetic route (Scheme 1) utilized 10c and 3- and 4-bromonitroso- diate 28b (Scheme 3), which was subjected to an aromatic
Figure 3: Comparison of compounds belonging to the subseries 3 or 4 with a halogen substitution on the ortho-position of the outer ring (X). (A) Activ-
ities of cis-isomers at 10 µM on CXCR3-mediated G protein activation. (B) PDE values at 10 µM.
2
517

Beilstein J. Org. Chem. 2019, 15, 2509–2523.
Scheme 3: Synthetic strategy for compound 6e. Reagents and conditions: (a) i) K2CO3 (2.0 equiv), DMF, µW, 65 °C, 3 h; ii) AcOH, reflux, 1 h, 67%;
(
3
b) i) pTsOH·H2O (3.0 equiv), MeCN, 10–15 °C; ii) NaNO2 (2.0 equiv), KI (2.5 equiv), H2O, 10–15 °C to rt, 2 h, 13%; (c) i) Et3N (1.2 equiv), DCM, rt,
0 min; ii) NaBH(OAc)3 (1.6 equiv), rt, 16 h, 54%; (f) MeI (20 equiv), DCM, rt, 20 h, 57%.
nucleophilic substitution with potassium phthalimide prepared The synthetic strategies for the synthesis of compound 6i were
in situ from 29 and K2CO3. Presumably due to the alkaline me- reported in our previous communication [7].
dium, the phthalimide ring was partially opened as detected by
HPLC–MS. Upon attempted re-closing under reflux in AcOH, In the subseries depicted in Table 3, several notable differences
completely deprotection took place and, after purification, in both the UV–vis spectra (Figure S1, Supporting Information
aniline 30 was obtained with high purity. Compound 30 was File 1) and the photoisomerization are observed. When the sub-
used to introduce the iodine atom through a Sandmeyer reac- stituent Y is a halogen atom, we observe a slight bathochromic
tion to give 28e albeit in low yield. Reductive amination with 7 shift of the π–π* band to higher wavelengths with increasing
afforded amine 16e followed by methylation to give trans-6e as size of the heteroatom, from 323 nm for Y = H (3e) to 339 nm
an orange solid with 97% purity.
for Y = I (6e) (Table 3, Figure 4A). When the group Y is OMe
6f), this shift is larger due to the higher EDG properties of
(
The synthesis of compounds 6f–h was performed following a MeO (λmax = 352 nm) and this is slightly increased with
nucleophilic aromatic substitution route on 28b (Scheme 4) Y = OiPr (6g, λmax = 355 nm). The shift is highest for
since precursors 8 or 17 with the required substituent Y were Y = NMe2 (6i, λmax = 387 nm). When the oxygen atom of 6f is
not available. We performed nucleophilic aromatic substitu- replaced by a sulfur atom, the bathochromic shift of the π–π*
tions with the corresponding sodium salts of MeOH, 2-PrOH band is also increased (6h, λmax = 373 nm, Table 3, Figure 4A).
and MeSH under µW irradiation. Both 28f (Y = OMe) and 28h This high capacity of sulfur substituents to induce a
(
Y = SMe) were formed in high yields, but the conversion of bathochromic effect has already been reported in the azoben-
the reaction with NaOiPr was very low and partially gave zene field [33]. The trans–cis photoisomerization for 3e, 4d, 6b
reduction of the benzaldehyde. An alternative route utilized a and 6d–h in general gives a high percentage of cis-isomer
method from Engle et al. proceeding through a tert-butylimine (89–93%) with two exceptions. Compound 6h shows a PSS of
intermediate (31) formed under Dean–Stark conditions [32]. only 65% cis-isomer due to a poor separation of the π–π* and
This imine was reacted with NaOiPr to form the ether 32, which n–π* bands as a result of the red-shifting by the SMe group
was subsequently hydrolyzed to obtain the desired aldehyde (Figure S1A,B, Supporting Information File 1), whereas 6i
2
8g in high yield. Reductive amination of the aldehydes 28f–h decomposes upon irradiation with 360 nm light (as previously
with 7 furnished amines 16f–h which was followed by methyla- reported by us [7]) leading to its exclusion from further charac-
tion to afford 6f–h as orange solids with ≥98% trans-isomer. terization. The PSS360 forms of exemplary compounds 4d, 6e
2
518

Beilstein J. Org. Chem. 2019, 15, 2509–2523.
Scheme 4: Synthetic strategies for compounds 6f–h (Y = OMe, OiPr, SMe). Reagents and conditions: (a) NaOMe or NaSMe (1.0–1.2 equiv), MeOH
or DMF, 65 °C, 30–60 min, 88–90%. (b) PhMe, 110 °C, Dean–Stark, 20 h, 99%; (c) NaH (1.0 equiv), DMSO, 100 °C, 1 h; (d) THF/H2O/AcOH
5
0:15:1, rt, 16 h, 79% (two steps); (e) i) Et3N (1.3–1.4 equiv), DCM, rt, 10–30 min; ii) NaBH(OAc)3 (1.6 equiv), rt, 6–16 h, 84–95%; (f) MeI (20 equiv),
DCM, rt, 20 h, 86–90%.
and 6f have thermal half-lives of 55 [7], 28 and 29 [7] days, re- The binding affinity of trans-3e, 4d, 6b and 6d–h (Table 3) is
spectively, at 10 μM in HEPES (4-(2-hydroxyethyl)-1-pipera- in the low micromolar range with a low PAS value upon illumi-
zineethanesulfonic acid) buffer with 1% DMSO at 25 °C and nation. Initial pilot studies on [35S]-GTPγS binding after
we consider this to be in line with the expectations (vide supra). CXCR3 stimulation with single concentrations of subseries 6
Table 3: Structure and results of photochemical and binding characterization of compounds 3e, 4d, 6b and 6d–i.
Photochemistry
CXCR3 binding affinity
λmax trans
π–π*a
λmax cis
n–π*a
PSS360
(% area cis)b
pKi
transc
pKi
PSS360c
Compound
Y
SEM
SEM
SEM
PASd
3
6
ee
be
H
F
323
325
421
417
88.9
92.7
0.4
0.1
6.3
6.0
0.0
0.0
5.7
5.4
0.1
0.0
4.0
4.0
2
519

Beilstein J. Org. Chem. 2019, 15, 2509–2523.
Table 3: Structure and results of photochemical and binding characterization of compounds 3e, 4d, 6b and 6d–i. (continued)
4
6
6
6
6
6
6
de
de
e
fe
g
Cl
Br
I
OMe
OiPr
SMe
NMe2
328
333
339
352
355
373
387
422
420
424
424
424
92.6
92.6
90.5
92.1
89.0
64.5
dec.g
0.2
0.2
1.0
0.1
0.4
1.7
5.9
5.8
5.5
5.4
5.1
5.3
0.0
0.0
0.1
0.0
0.2
0.0
5.6
5.4
5.5
5.0
5.0
5.5
0.0
0.0
0.0
0.0
0.0
0.1
2.0
2.5
1.0
2.5
1.3
0.6
h
ie
≈425f
dec.g
aThe absorbance maxima were extracted from UV–vis spectra at 25 µM in PBS buffer with 1% DMSO. b% of cis-isomer at the photostationary state
PSS360) in 68% TRIS buffer and 32% DMSO (1 mM) measured after being pre-irradiated at 360 nm as obtained by LC–MS integration of the cis and
(
trans-isomer signals at 265 nm. The mean and SEM of at least two experiments are shown. cBinding affinity of trans-isomer or PSS360 as measured
using [3H]-VUF11211 displacement. The mean and SEM of at least three experiments are shown. dThe photoinduced affinity shift (PAS) is calculated
as the ratio of the Ki PSS360 and Ki trans. eCompound was previously described by us [7]. fCould not be determined accurately due to partial overlap-
ping of the π–π* and n–π* bands. gCompound decomposes under illumination.
Figure 4: Properties of subseries 3e, 4d, 6b and 6d-h. (A) UV–vis absorption spectra of (top) trans-isomers of 6b, 4d, 6d and 6e (having substituent
Y = F, Cl, Br and I, respectively) and (bottom) 6f, 6g and 6h (having Y = OMe, OiPr and SMe, respectively). (B) Functional dose-response curves
using [35S]-GTPγS assay exemplified for 4d, 6f and 6e, respectively. (C) Summary of the efficacies of compounds 3e, 4d, 6b and 6d–h at PSS360.
(
D) Correlation between the bathochromic shifting of the π–π* band and the intrinsic activity of PSS360 for the compounds in Table 4.
(
data not shown) showed substantial levels of CXCR3 agonism lated the intrinsic activity (α) and potency (EC50). As reported
in this group of compounds. For subseries 6 (and associated 3e in our previous communication for some of these compounds
and 4d) we therefore generated dose-response curves for the [7], the PSS360 forms give agonism with high nanomolar poten-
trans and PSS360 forms (Figure S4, Supporting Information cies while most trans compounds are antagonists or partial
File 1) using the same [35S]-GTPγS functional assay and calcu- agonists with very low efficacies (Table 4). However, when the
2
520

Beilstein J. Org. Chem. 2019, 15, 2509–2523.
Table 4: Structure and results of functional characterization of compounds 3e, 4d, 6b and 6d–h.
pEC50
transa
pEC50
PSS360b
Compound
Y
SEM
SEM
α transc
SEM
α PSS360d
SEM
PDEe
1
3
6
4
6
6
6
6
6
d
–
H
F
Cl
Br
I
OMe
OiPr
SMe
6.9
0.0
6.9
6.5
6.2
6.3
6.2
6.3
6.4
6.0
6.1
0.0
0.1
0.1
0.1
0.1
0.2
0.1
0.0
0.1
1.04
0.05
0.12
0.11
0.14
0.49
0.16
n.m.h
0.85
0.08
0.03
0.00
0.01
0.02
0.04
0.01
n.m.h
0.08
1.03
0.30
0.41
0.54
0.58
1.00
0.99
1.19
1.29
0.07
0.01
0.03
0.01
0.05
0.04
0.04
0.07
0.03
−0.01
0.25
0.29
0.43
0.44
0.51
0.83
n.m.h
0.44
ef
bf
df
df
e
ff
g
h
n.m.g
n.m.g
n.m.g
n.m.g
5.5
n.m.g
n.m.h
5.7
n.m.g
n.m.g
n.m.g
n.m.g
0.3
n.m.g
n.m.h
0.3
aPotency of trans-isomer in the dark. n.m. = not measurable. The mean and SEM of at least three experiments are shown. bPotency of a sample pre-
irradiated at 360 nm to reach the photostationary state. The mean and SEM of at least three experiments are shown. cIntrinsic activity of trans-isomer
in the dark (CXCL11 efficacy set at α = 1). The mean and SEM of at least three experiments are shown. dIntrinsic activity of a sample pre-irradiated at
3
60 nm to reach the photostationary state (CXCL11 efficacy set at α = 1). The mean and SEM of at least three experiments are shown. eThe photoin-
duced difference of efficacy (PDE) is obtained by subtracting α trans from α PSS360. fCompound was previously described by us [7]. gToo low
window. hThe curve for trans-6g shows anomalous behavior at 10−5 M and higher.
size and/or EDG properties of Y increase, remarkably partial erally assumed to have an intrinsically higher electron density
agonism with substantial efficacies appears even for the trans- due to the disruption of the conjugation between the two rings
isomers, such as for 6e, 6g and 6h. The compounds illuminated of the azobenzene through the N=N bond. Moreover, the elec-
to PSS360 follow a similar qualitative trend: when the Y substit- tronic properties of the inner substituent Y have been proven to
uent is H or F (3e and 6b), the cis-isomer behaves as a partial be of strategic use in increasing the intrinsic activity (α) of the
agonist with medium efficacy (α = 0.30–0.41), but when the Y cis-isomers (Figure 4C). This capacity of the Y substituent to
substituent increases in size and/or EDG properties, the effi- alter the electron density is conceivably also related to its
cacy increases to full efficacy with compounds 6e–h (Y = I, capacity to induce a bathochromic shift of the π–π* band of the
OMe, OiPr and SMe). The trans-form of 6g shows anomalous trans-isomer. Potential evidence for this can be extracted from
behavior at 10−5 M and higher (Figure S4, Supporting Informa- the significant correlation between the bathochromic shift and
tion File 1), preventing the extraction of accurate functional intrinsic activity of PSS360 (Figure 4D) for the subseries 3e, 4d,
values. The PDE value, which reflects efficacies of both the 6b and 6d–h, which only differ in the nature of Y group
trans and PSS form, appears to have an optimum for 6f (Figure 4C, Table 4).
(
Figure 4B). Figure 4B also shows more subtle behavior for
compounds 4d and 6e, which harbor a PDE value of 0.43 Conclusion
and 0.51, respectively, but in different parts of the efficacy We report a toolbox of 31 photochromic small-molecule
window.
CXCR3 receptor ligands based on the modeling-assisted azolo-
gization of a biaryl series reported previously by us [24]. All
The results reveal that the electron density of the aryl rings, compounds show affinity for CXCR3 from the high nanomolar
especially of the inner one, plays a key role in inducing to the low micromolar range. Our efforts, however, were
agonism in CXCR3. In general, the cis-isomers of the series focused on exploring the landscape in functional efficacy. To
2
–6 are better CXCR3 agonists. Indeed, the cis-isomers are gen- this end, the scaffold was subjected to positional and substitu-
2
521

Beilstein J. Org. Chem. 2019, 15, 2509–2523.
tion changes in structure, necessitating extensive synthetic References
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nist behavior. The presence of a substituent on the para-posi-
tion of the central ring (substituent Y) capable of delocalizing
π-electrons increases the efficacy of the cis-isomer. The cis-
isomers of compounds with Y = I, OMe, OiPr or SMe are all
full agonists of CXCR3, however, the corresponding trans-
isomers also activate the receptor to varying degrees. In all, our
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Supporting Information
4
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ORCID® iDs
Xavier Gómez-Santacana - https://orcid.org/0000-0001-8830-0494
Tamara A. M. Mocking - https://orcid.org/0000-0001-6490-4429
Niels J. Hauwert - https://orcid.org/0000-0002-1217-1670
Prashanna Vijayachandran - https://orcid.org/0000-0002-1047-538X
Henry F. Vischer - https://orcid.org/0000-0002-0184-6337
Maikel Wijtmans - https://orcid.org/0000-0001-8955-8016
Rob Leurs - https://orcid.org/0000-0003-1354-2848
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doi:10.1016/j.chembiol.2016.06.013
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Ferrazzo, S.; Bosch, D.; Asede, D.; Malhaire, F.; Acher, F.; Giraldo, J.;
Valjent, E.; Ehrlich, I.; Ferraguti, F.; Pin, J.-P.; Llebaria, A.; Goudet, C.
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Preprint
A non-peer-reviewed version of this article has been previously published
as a preprint doi:10.3762/bxiv.2019.69.v1
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