Light-controlled inhibition of BRAFV600E kinase

Article abstract of DOI:10.1016/j.ejmech.2019.06.042

Metastatic melanoma is amongst the most difficult types of cancer to treat, with current therapies mainly relying on the inhibition of the BRAF^V600E mutant kinase. However, systemic inhibition of BRAF by small molecule drugs in cancer patients results – paradoxically – in increased wild-type BRAF activity in healthy tissue, causing side-effects and even the formation of new tumors. Here we show the development of BRAF^V600E kinase inhibitors of which the activity can be switched on and off reversibly with light, offering the possibility to overcome problems of systemic drug activity by selectively activating the drug at the desired site of action. Based on a known inhibitor, eight photoswitchable effectors containing an azobenzene photoswitch were designed, synthesized and evaluated. The most promising inhibitor showed an approximately 10-fold increase in activity upon light-activation. This research offers inspiration for the development of therapies for metastatic melanoma in which tumor tissue is treated with an active BRAF^V600E inhibitor with high spatial and temporal resolution, thus limiting the damage to other tissues.

Full text of DOI:10.1016/j.ejmech.2019.06.042

Accepted Manuscript
Light-controlled inhibition of BRAFV600E kinase
Mark W.H. Hoorens, Maria E. Ourailidou, Theo Rodat, Petra E. van der Wouden,
Piermichele Kobauri, Malte Kriegs, Christian Peifer, Ben L. Feringa, Frank J. Dekker,
Wiktor Szymanski
PII:
S0223-5234(19)30570-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.042
EJMECH 11445
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 February 2019
Revised Date: 22 May 2019
Accepted Date: 15 June 2019
Please cite this article as: M.W.H. Hoorens, M.E. Ourailidou, T. Rodat, P.E. van der Wouden, P.
Kobauri, M. Kriegs, C. Peifer, B.L. Feringa, F.J. Dekker, W. Szymanski, Light-controlled inhibition of
BRAFV600E kinase, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.06.042.
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Light-controlled Inhibition of BRAFV600E Kinase
Mark W. H. Hoorens1,2, Maria E. Ourailidou3, Theo Rodat4, Petra E. van der Wouden3,
Piermichele Kobauri2, Malte Kriegs5, Christian Peifer4, Ben L. Feringa2, Frank J. Dekker3,
Wiktor Szymanski1,2*
Affiliations
1: University Medical Center Groningen, Department of Radiology, Medical Imaging Center,
University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands
2: Stratingh Institute for Chemistry, Faculty of Science and Engineering, University of
Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands
3: Department of Chemical and Pharmaceutical Biology, University of Groningen, Antonius
Deusinglaan 1, 9713 AV Groningen, The Netherlands
4: Institute of Pharmacy, Christian-Albrechts-University of Kiel, Gutenbergstr. 76, 24118 Kiel,
Germany
5: Laboratory of Radiobiology & Experimental Radiooncology and UCCH Kinomics Core
Facility, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg,
Germany

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Highlights
- Photochemical control over the inhibition of BRAFV600E kinase was achieved.
- Diaryl-sulfonamide motifs are promising targets for the azologization approach.
- Azologization of kinase inhibitors does not only alter their activity, but also their kinase
selectivity.
Keywords
- BRAFV600E
- Photopharmacology
- Photocontrolled inhibitor
- Metastatic melanoma
- Azobenzene

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Abstract
Metastatic melanoma is amongst the most difficult types of cancer to treat, with current
therapies mainly relying on the inhibition of the BRAFV600E mutant kinase. However, systemic
inhibition of BRAF by small molecule drugs in cancer patients results – paradoxically – in
increased wild-type BRAF activity in healthy tissue, causing side-effects and even the
formation of new tumors. Here we show the development of BRAFV600E kinase inhibitors of
which the activity can be switched on and off reversibly with light, offering the possibility to
overcome problems of systemic drug activity by selectively activating the drug at the desired
site of action. Based on a known inhibitor, eight photoswitchable effectors containing an
azobenzene photoswitch were designed, synthesized and evaluated. The most promising
inhibitor showed an approximately 10-fold increase in activity upon light-activation. This
research offers inspiration for the development of therapies for metastatic melanoma in
which tumor tissue is treated with an active BRAFV600E inhibitor with high spatial and temporal
resolution, thus limiting the damage to other tissues.
Graphical abstract:

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Introduction
Skin cancer is one the most frequently occurring types of cancer [1]. Of all skin cancers,
melanoma has been reported to be the most deadly and challenging to treat [2].
Approximately 40 to 50% of melanomas harbor a mutation in the BRAF kinase [3], which is a
cytosolic serine/threonine kinase belonging to the family of Rapidly Accelerated
Fibrosarcoma (RAF) kinases. RAF kinases are part of the RAS/RAF/MEK/ERK signal
transduction pathway which is involved in regulation of cell proliferation [4]. Increased activity
of this pathway is often involved in the formation of cancer (Fig. 1). Due to the high
frequency of the mutations and its role in the formation of cancer, the BRAF kinase became
of clinical interest [3].
The activity of BRAF in cells is tightly regulated to prevent too extreme proliferation. Current
hypothesis is that BRAF can be activated by dimerization and subsequent binding to RAS.
While in the homo-dimeric state, it can also inactivate its binding partner through
phosphorylation [5,6]. A single point mutation is believed to abolish the requirement of
dimerization and binding to RAS for activation, resulting in elevated BRAF kinase activity in
its monomeric form [7]. The most common BRAF mutation is V600E, which results in an
about 500 times increased activity of the BRAF kinase compared to the wild-type [8]. Since a
single mutation of the BRAF kinase drives the formation of a melanoma, BRAFV600E has
become a therapeutic target. Currently two FDA-approved BRAFV600E-selective ATP-
competitive inhibitors are of use in the clinical practice. Vemurafenib (whose name is derived
from V600E mutated BRAF inhibitor) has selectivity for BRAFV600E over wtBRAF and is
clinically used for the treatment of metastatic melanoma [9], alongside the other second FDA
approved BRAFV600E inhibitor, Dabrafenib (Fig. 2.) [10].

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Unfortunately, BRAFV600E inhibitors Vemurafenib and Dabrafenib, used systemically, have
several disadvantages. BRAFV600E inhibitors can increase the activity of the wild-type BRAF
kinase in healthy cells, an effect known as “BRAF paradox” (See Fig. 1). This paradoxical
activation is hypothesized to be caused by sub-saturated state of BRAF as a homo-dimer, in
which the auto-inactivation mechanism is partially inhibited, resulting in a net increase of
BRAF activity [10–12]. In healthy cells, increased activity of the RAS/RAF/MEK/ERK pathway
promotes proliferation and development of new cancers, a fact that has been observed in
some melanoma patients treated with BRAFV600E inhibitors [10,13]. The side effects observed
with these kinase inhibitors illustrate that systemic exposure to chemotherapeutic agents can
tremendously increase the disease burden. This highlights the importance of developing new
concepts for spatial and temporal control over the exposure to or activity of
chemotherapeutic agents.
The disadvantages of systemically used chemotherapeutic agents inspire, amongst others,
the development of innovative solutions for local drug activation/inactivation by external
control. Such approaches have the potential to improve the exposure of diseased tissue and
to reduce the exposure of healthy tissue to active chemotherapeutic agents, whereas the
disease tissue can be treated with higher doses. The emerging field of photopharmacology
[14–17] offers a technology that enables local activation of chemotherapeutic agents by
using light to control drug activity (see Fig. 1). Irreversible control can be acquired by the
introduction of a photocleavable protecting group, which will result in a pro-drug that can be
light-activated. [18] This has been successfully achieved for many chemotherapeutic
agents,[19] including BRAFV600E inhibitor Vemurafenib[20]. Reversible photocontrol over drug
activity is being achieved through the introduction of a photoswitchable functional group,
such as the frequently used azobenzene, into the structure of bioactive compounds.
Azobenzene (See Fig. 2, highlighted in blue) is stable in the trans isomer and, upon
irradiation with UV light, can be switched to the cis isomer [21]. This process can be reversed

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thermally or via irradiation with visible light. The two photoisomers of azobenzene differ in
structure, polarity and solubility [22] and these differences can be employed to design
molecules with photoswitchable biological activity. Since isomerization from trans to cis can
only be achieved photochemically, while re-isomerization from cis to trans occurs both
photochemically and thermally, the fraction of cis isomer is more easily regulated. Therefore,
it is preferred to carefully design molecules in which the cis isomer is more active than the
trans isomer [15].
The photopharmacology approach has been successfully applied for molecules inspired by
anti-cancer drugs such as Vorinostat [23], Bortezomib [24,25] and Combretastatin A4 [26–
29]. Also for kinases, several photocontrolled inhibitors have been reported. For example, the
group of Grøtli acquired control over the activity of the RET kinase [30], the group of Branda
over the activity of Protein Kinase C [31] and the group of Peifer of the activity of vascular
endothelial growth factor receptor 2 (VEGFR2) [32]. This sets the stage to apply this concept
to develop molecules that enable reversible, spatial and temporal control over the activity of
BRAFV600E using light. Yet, the development of kinase inhibitors with photocontrolled activity
has been shown to be challenging [33]. This is supported by a review of Hüll et al., which
reports 123 photoswitchable bioactive compounds [17], with only three of them targeting a
kinase [30–32], even though kinase inhibitors are of high clinical interest. More recently, the
groups of Peifer and Herges reported photo-switchable kinase inhibitors for the p38α MAPK
and CK1β kinases. For these photo-switchable kinase inhibitors, small differences in activity
between photo-isomers was observed, probably due to conformational adaptation of the
kinase to either one of the photo-isomers of the ligand and/or irreversible reduction of diazo
as the key causes for small differences in activity between photo-isomers [34].

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Here we describe the design, synthesis and evaluation of BRAFV600E inhibitors with
photocontrolled activity, whose structure was inspired by an analog of Vemurafenib. In total,
eight BRAFV600E inhibitors were synthesized and their photochemical properties were
determined. Subsequently, both the activities of their respective trans and cis isomers were
determined in a cell-free BRAFV600E Western-blot-based activity assay, followed by cell
cytotoxicity studies and kinome off-target screening for the most promising inhibitor.
Fig. 1: Conceptual description of the comparison between the disease (left), the systemic
Vemurafenib treatment of Melanoma (middle) and the photopharmacological approach
(right). Left: The BRAFV600E mutation drives the cell proliferation, resulting in cancer. Middle:
For the treatment of Melanoma, Vemurafenib is active in both healthy and cancerous tissue,
where in healthy tissue it can paradoxically increase the activity of the downstream pathway
resulting in increased proliferation and potentially in the formation of new tumors. In
melanoma, Vemurafenib induces cell death. Right: The photopharmacological approach
aims to use an inactive inhibitor in healthy tissue that does not affect proliferation, while in
melanoma the BRAFV600E inhibitor can be locally switched on with light, inducing cell death.

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Results and discussion
Numerous inhibitors for the BRAFV600E kinase, including the FDA-approved drugs
Vemurafenib and Dabrafenib, harbor a common motif (Fig. 2, red), which consists of a
benzene ring with a sulfonamide and a fluorine substituents. Group X on the sulfonamide is
generally a propyl or benzyl group, while the Y group is usually hydrogen or fluorine and the
Z group consists of heterocycles, coupled either directly (such as in Dabrafenib) or by a
carbonyl linker (such as in Vemurafenib) on an amide [35–40]. As demonstrated by co-
crystal structures of BRAFV600E bound to its inhibitors [36–39], the common motif and the Z
group are flat or under a small angle in the ATP binding pocket, where the heterocycles of
the Z group form hydrogen bonds with Cys532. In contrast to the rest of the inhibitor, which is
planar, the sulfonamide is bent, resulting in an approximate 90 degrees angle between the
common motif and the X group. This X group binds in a small pocket, stabilized by
interactions between the sulfonamide and Gly596, Phe595 and Asp594.

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Fig. 2: Top: structure of Vemurafenib, Dabrafenib and a common motif found in many
BRAFV600E inhibitors. Bottom: Compound 1 has been reported as an improved Vemurafenib
analog. Inspired by compound 1, eight photoswitchable BRAFV600E inhibitors 2a-h were
designed. Upon irradiation with light of wavelength λ1, trans-2a-h can be switched to cis-2a-h
cis. This isomerization can be reversed by irradiation with light of wavelength λ2 or in a
thermal relaxation process.
A straightforward choice for the design of a photoswitchable BRAFV600E inhibitor would be the
replacement of the sulfonamide of Dabrafenib by a diazo group, introducing a
photoswitchable azobenzene, along the principles of azologization [41]. However, Dabrafenib
itself is already photochemically unstable. Upon UV light irradiation, a side-product is
irreversibly formed, which is biologically less active [42]. This process might compete with

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photoisomerization, which makes Dabrafenib unsuitable as a starting point for a
photoswitchable inhibitor. Our design of a photoswitchable BRAFV600E inhibitor was instead
based on compound 1 (Fig. 2), which was found upon optimization of Vemurafenib [43]. We
selected compound 1 as a starting point to design photoswitchable analogs. This compound
contains three aromatic moieties coupled by either an amide or a sulfonamide linker. Both
linkers provides a site for replacement by a diazo group to generate inhibitors with an
azobenzene photoswitch. Yet, when comparing into the binding mode of inhibitors in co-
crystals with the BRAFV600E mutant, it became apparent to us that the two aromatic rings
coupled by the amide are in one plane or deviate only slightly from this plane. Replacement
of this amide by a trans isomer of the azobenzene functionality would provide inhibitors that
retain the orientation of these rings. In crystal structures, the two aromatic rings coupled by
the sulfonamide adopt a bend conformation. Therefore we anticipate that replacement of the
sulfonamide by the cis isomer of the azobenzene functionality would retain the bio-active
conformation. Since the concept of photo-activation requires activity of the cis isomer and
inactivity of the trans isomer (vide supra), we chose to replace the sulfonamide functionality
of 1 by a diazo group. We note, however, that the sulfonamide is involved in binding to the
BRAFV600E kinase and we, therefore, anticipate on a decrease of activity.
For bio-active compounds with photocontrolled activity, every chemical modification to the
structure potentially changes both the biological and photochemical properties such as the
position of the absorption band, the rate of thermal relaxation of the cis isomer back to the
trans isomer and the highest ratio between trans and cis that can be achieved upon
irradiation at photo-stationary state (PSS). Optimizing photocontrolled inhibitors with respect
to all the parameters proved to be challenging, because a maximal difference in biological
activity between both photo-isomers had to be achieved, while retaining optimal trans-cis
ratios at PSS and half-life of the cis isomer. First, compound 2a was designed to determine
the effect of replacing the sulfonamide group with a diazo linker. While the percentages of
the cis isomer at PSS of unsubstituted azobenzenes are usually relatively low, installing a p-

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MeO group enable improvement to more than 95% of the cis isomer upon isomerization [44],
This idea inspired the design of compound 2b with a p-MeO on the azobenzene photoswitch.
The MeO group was moved to the ortho position in compound 2h, with the idea that this can
also improve the ratio of isomers at PSS. Compound 2c was designed to determine the
effect of both the fluorine atoms at the R1 and R2 position. Compounds 2d and 2f were
inspired by the SAR described by Wenglowsky et al.,[43] where fluorine at R3 increase the
activity of the inhibitor, while at R4 it showed opposite effects. To increase the steric bulk,
compound 2e and 2g were designed with a methyl substituent at either the ortho or para
position.
Scheme 1: Synthesis of BRAFV600E inhibitors with photocontrolled activity
Photoswitchable BRAFV600E inhibitors 2a-h were synthesized (Scheme 1) by condensing
anilines 3 and nitrosobenzenes 4a-h under standard Mills reaction conditions (see
Supporting Information). Subsequently, the methyl ester of 5a-h was hydrolyzed using LiOH
and the resulting acid was coupled to a previously reported [35] 3-methoxy-1H-pyrazolo[3,4-
b]pyridine-5-amine, giving the final compounds 2a-h. Thus we generated eight
photoswitchable BRAFV600E inhibitors.
Photochemical properties of compounds 2a-h
Next, the photochemical properties of these eight photoswitchable BRAFV600E inhibitors were
determined and the results are shown in Table 1. Compounds 2b and 2c containing a p-
MeO substituent have absorption maxima in DMSO at λ = 357 nm and 352 nm, respectively.
All other inhibitors have absorption maxima at λ’s ranging from 332 to 340 nm. When

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recording absorption spectra in the more biologically relevant BRAF assay buffer, the
absorption bands show small hypsochromic shifts, yet all retain their maxima between 328
and 345 nm.
Using NMR spectroscopy, the distribution of isomers at the photo-stationairy state (PSS)
upon irradiation with 365 nm light was determined, to quantify the efficiency of photochemical
trans-cis isomerization. Unsubstituted compound 2a can only reach 55% cis at PSS.
Methoxy substituents at the R3 and R4 positions of 2b, 2c and 2h resulted in near-
quantitative switching. Methyl and fluorine substituents (compound 2d-g) result in a PSS of
between 61 and 84%.
To determine the rate of thermal cis-trans relaxation, the half-lives of the cis isomers were
determined in both DMSO and the BRAF assay buffer at room temperature. In DMSO, for all
compounds relatively long half-lives were observed. The half-life of the cis isomer of
compounds 2a and 2c was determined in BRAF assay buffer to be shorter than in DMSO,
yet the half-lives are still in the hour range. Of all other compounds, the half-lifes of the
respective cis isomers were determined in a 1:1 mixture of BRAF assay buffer and
acetonitrile, for solubility reasons. In this mixture, long half-lives of over a day were observed
at room temperature. From the measured half-lives we concluded that the extend of thermal
relaxation from cis to trans during the 1 h reaction time used in the western blot BRAFV600E
activity assay (vide infra) is very limited.
Table 1: Photochemical properties
compound structure
λmax,trans
DMSO
t1/2 cis
At rt
% cis at λmax,trans
t1/2 cis2
PSS
BRAF assay
DMSO
buffer
2a
332 nm
> 10 h
55%
328 nm
5.4 h

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2b
2c
357 nm
> 10 h
92%
341 nm
> 24 h *
> 10 h
352 nm
> 10 h
94%
345 nm
2d
2e
336 nm
340 nm
> 20 h
> 20 h
71%
84%
331 nm
338 nm
̴ 5 h *
> 24 h *
2f
335 nm
335 nm
334 nm
> 20 h
> 70 h
> 24 h
62%
77%
88%
338 nm
334 nm
327 nm
> 24 h *
> 24 h *
> 24 h *
2g
2h
1 in DMSO
2 At room temperature in BRAF assay buffer * (50% ACN)
Biological evaluation
The activity of the BRAFV600E inhibitors was determined using an assay [45] with purified
recombinant BRAFV600E with inactive MEK-1 as a substrate, and the enzymatic activity was
determined by quantifying the phosphorylated-MEK-1 product using Western Blot. Prior to
o
the assay, the inhibitor was either heated for 30 min at 60 C to fully thermally adapt to the
trans isomer or irradiated with 365 nm light for 45 min to reach PSS (see Table 1)

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Fig. 3: BRAFV600E activity assay. A: IC50 values as determined using the BRAFV600E assay. B:
Top: dose-response curve of 2a in trans and cis. Bottom: Western Blot detection of p-MEK1.
C: Kinase activity after sequential switching, by irradiation from trans to cis, thermal
relaxation to trans and irradiation from trans to cis, at 1 µM of 2a (N=2, ± S.D.)
First, the IC50 values of the reference compounds Vemurafenib and compound 1 were
determined to be 9.6 ± 3.3 n
as substituents at R3 and R4, showed an IC50 in the dark of 1.68 ± 0.62 µ
to 156 ± 47 n upon light-induced switching to the cis isomer, resulting in an approximately
M
and 22 ± 10 nM, respectively (Fig. 3A). Compound 2a, with H
M, which decreased
M
10-fold increase in activity upon irradiation, as shown in Fig. 3B. Furthermore, the
reversibility of this activation was determined, as demonstrated in Fig. 3C: going through a
cycle of switching to cis with UV light and thermal re-isomerization, the original activity was
recovered, indicating that the irradiation leads to reversible isomerization of the azobenzene
moiety and does not result in an irreversible photodegradation. We have observed that, as
compared to 1, compound 2a lost some of the potency, likely due to the loss of the beneficial
interactions of the sulfonamide within the active site. Unfortunately, the photochemical
properties of compound 2a are suboptimal, with only 55% of the active cis isomer at PSS.
Compared to compound 2a, installing a para-alkyloxy substituent on azobenzenes usually
results in near quantitative isomerization from trans to cis, as was also observed for
compounds 2b and 2c, bearing a MeO substituent at the R4 position. Even though 2b and 2c
show improved photo-isomerization compared to 2a, no statistically significant difference in

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kinase inhibitory activity between the dark and irradiated state was observed for either
compound. The difference between 2b and 2c is the presence of two fluorine substituents at
the R1 and R2 position of compound 2b. However, having the two fluorines replaced by
hydrogen in compound 2c did not affect the biological activity.
Additional fluorine substituents were placed at the R3 position in compound 2d and at the R4
position of compound 2f. Both for compound 2d and 2f no statistically significant difference in
activity between the dark and irradiated state was observed and the fluorine substituents
resulted in less active inhibitors. In contrast to placing a small fluorine substituent at the R4
position – surprisingly – a methyl substituent on this position of compound 2e resulted in
improved activity. Even though no difference in activity between the dark and irradiated state
of 2e was observed, the inhibitor has an IC50 value comparable to reference compound 1 and
Vemurafenib. Furthermore, the influence of ortho substituents was further determined with
compound 2g with a methyl at the R3 position and compound 2h with a methoxy substituent
at the R3 position, again showing no statistically significant difference between the dark and
irradiated state a further loss of potency.
In the enzymatic assay, the activity of compound 2a increased approximately 10-fold upon
irradiation. To determine if the observed difference in activity from the enzyme assay could
be translated to a difference in cytotoxicity, cell viability experiments were performed in A375
cells, a melanoma cell line - harboring the BRAFV600E mutation – which is commonly used to
study the cytotoxicity of BRAFV600E inhibitors [12,38,40,46]. However, upon incubation for 24h
(see Fig. 4a), no cytotoxicity was observed for both 2a in the dark and irradiated state, where
both reference compounds resulted in lower cell viability. The inhibition of BRAFV600E by
compound 2a in the enzymatic inhibition assay could not be translated to cytotoxicity in A375
cells, possibly due to off-target activity for other kinases or escape routes to the
RAS/RAF/MEK/ERK proliferation pathway.
In the development of kinase inhibitors, it has proven to be very challenging to develop
inhibitors with high selectivity towards other kinases [47]. This implies that azologization of a

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selective kinase inhibitor can potentially result in a loss of selectivity among kinase
isoenzymes. To acquire a deeper understanding of other effects of the photoswitchable
BRAFV600E kinase inhibitor 2a, the kinome inhibition profile was investigated using Pamgene
STK Chips, which is a well-established method for target evaluation [48,49]. Cell lysates from
SK-Mel-28 cell were treated with DMSO as a control, compound 1 on which the
photoswitchable inhibitors were based and compound 2a (both dark and irradiated) (See Fig.
4B). Compared to the DMSO control, compound 1 resulted in a decrease in kinase activity in
the cell lysates. Interestingly, treatment with compound 2a (dark) resulted in a general
increase in phosphorylation activity (See. Fig. 4C). In line with the expectation, treatment of
2a (irradiated) showed a decrease in phosphorylation activity compared to the 2a (dark),
which corresponds to increased kinase inhibition upon irradiation. However, compared to the
DMSO control, the general phosphorylation level upon 2a (irradiated) treatment is still
elevated. This could be explained by the presence of 2a trans in the irradiated state, where
irradiation with λ = 365 nm light results in only 55% of the more active cis isomer. Yet, the
unexpected increased phosphorylation level in lysates SK-MEL28 observed for compound 2a
in both the dark and irradiated state fit with the lack of cytotoxicity of compound 2a in A375
cells.

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Fig. 4: A: Cell viability MTS assay of A375 cells after 24 h incubation with 10µM
Vemurafenib, compound 1, compound 2a thermally adapted and irradiated, all compared to
DMSO. B-C: Kinome profiling using cell lysates from SK-Mel-28 cells. The lysates were
treated with a DMSO control, reference compound 1, compound 2a dark and compound 2a
irradiated with 365 nm light (in vitro inhibition). B: Heatmap showing log2-transformed signal
intensities of the phosphorylated peptides included on the STK arrays. The signals were
sorted from high (red) to low (blue) intensity. C: Box plots summarizing the overall peptide
phosphorylation levels depicted in C.

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Fig. 5: Binding site of BRAFV600E cocrystallized with compound 1 (PDB ID: 3SKC, protein in
grey and ligand in cyan), superimposed on the docking poses of (A) compound 2a trans
(green) and (B) compound 2a cis (orange). Hydrogen bonds are depicted as yellow dashed
lines.
As an important prerequisite for any future in vivo applications, we have further tested the
plasma stability of compound 2a. First, photo-isomerization of compound 2a in bovine
plasma was studied (see Fig. S7.1). Irradiation with 365 nm results in a decrease of the
absorption band of 2a trans, however, surprisingly, irradiation with white light does not result
in photo-isomerization back to the trans isomer. The same photochemical behavior is
o
observed after incubation of 1h at 37 C prior to photo-isomerization (see Fig S7.2), which
suggests at least partial preservation of the azobenzene fragment. When compound 2a was

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o
pre-irradiated with 365 nm light in DMSO, added to plasma and incubated at 37 C for 1 h,
reversible photo-isomerization was observed (See Fig. S7.3). Finally, compound 2a was
incubated for 20 h in bovine plasma at 37 oC, after which by LCMS masses of both isomers
(See Fig S.7.4 to S7.7) of the fully intact molecules were detected. An additional peak of an
unknown product was observed, of which the mass doesn’t match with products of
azobenzene reduction. Altogether this further supports that 20h of plasma incubation does
not fully degrade photoswitchable BRAFV600E inhibitor 2a
Finally, to rationalize the 10-fold difference in BRAF inhibitory potency between the two
isomers of inhibitor 2a, both photo-isomers were docked into the crystal structure of
BRAFV600E. As shown in Fig. 5A, compound cis-2a shows a nearly perfect overlap with
reference compound 1. However, due to the replacing the sulfonamide for a diazo bridge, the
interactions with the Asp594, Phe595 and Gly596 backbone are no longer present, which
could explain the drop in potency of compound 2a compared to compound 1. Still, cis-2a and
1 are spectacularly close in conformation, demonstrating that cis-azobenzenes are suitable
for mimicking diarylsulfonamide moieties in drugs. Also trans-2a was docked into the same
crystal structure, as shown in Fig. 5B. Compound trans-2a docked into a conformation that is
completely different from the original compound 1, thus indicating binding of this isomer to
the kinase active site is perturbed. The docking data of 2a trans suggest that bulky groups at
the R1 position might disfavor binding of the trans isomer and decrease the activity, which
could potentially improve the difference in activity between trans and cis of 2a.

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Discussion
In the emerging field of photopharmacology, the development of kinase inhibitors has been
very challenging and only a few examples have been reported [30–32]. Herein, we show a
proof of concept for the development of a BRAFV600E inhibitor with photocontrolled activity,
which is a reversibly photocontrolled inhibitor for a therapeutically relevant kinase. We have
identified compound 2a, which has a difference in activity between the thermal and irradiated
state of 10-fold, even though a PSS of only a 55% cis was achieved upon irradiation. Using
docking studies, we rationalized the binding of both cis and trans, which indicates how photo-
isomerization can change activity. Using kinome screening, it was found that azologization of
compound 1 to provide compound 2a resulted in increased kinase activity for both dark and
irradiated samples, which could explain the observed lack of cytotoxicity for the BRAFV600E
inhibitor photo-isomers in A375 cells, despite their 10-fold difference in activity in the ATP-
competitive enzyme inhibition studies. This demonstrates the importance of off-target
screening in the development of photoswitchable kinase inhibitors.
Selective BRAFV600E inhibitors with light-controlled activity will provide opportunities for local
and precise treatment of BRAFV600E-driven tumors, without harming the activity of the
RAS/RAF/MEK/ERK pathway in healthy cells and tissues. Selective UV light irradiation of
BRAFV600E-driven tumors activates the inhibitor and can besides that possibly be of additional
therapeutic value by inducing phototoxicity. Indeed, UV light irradiation is used for treating
other types of skin cancer such as Cutaneous T-Cell lymphoma in which irradiation results in
apoptosis [50]. Furthermore, in a research setting, a BRAFV600E inhibitor with light-controlled
activity will be a powerful research tool [51] to study the kinase and its interplay with other
proteins that control the activity of the RAS/RAF/MEK/ERK pathway. Photocontrolled
inhibitors allow for short-term BRAF activation and inactivation in a fully reversible manner.
Besides that, spatial control allows for local modulation of BRAF activity with high resolution.
Such insights are particularly relevant for the BRAF kinase because of the complex
mechanisms involved in its regulation, which will contribute to develop better therapies for
melanoma in the future.

ACCEPTED MANUSCRIPT
Acknowledgements
The support of the Netherlands Organization for Scientific Research (NWO-CW VIDI grants
723.014.001 to W.S. and 723.012.005 to F.D.) is kindly acknowledged. We would like to
thank Konstantin Hoffer (UCCH Kinomics Hamburg) for technical support and acknowledge
SFB 677 “Function by Switching” (Collaborative Research Center financial Support by DFG)
for financial support.

ACCEPTED MANUSCRIPT
Synthetic procedures.
3: Methyl 2,6-difluoro-3-(phenyldiazenyl)benzoate.
A solution of methyl 3-amino-2,6-difluorobenzoate (6.60 mmol, 1.23 g, Combi-Blocks) and
nitrosobenzene (6.00 mmol, 642 mg) in acetic acid (30 mL) was heated at 40 °C overnight.
Afterwards, the reaction mixture was diluted with diethyl ether (200 mL) and washed with 1N
aq. HCl (2 x 150 mL), sat. aq. NaHCO3 (150 mL) and brine (150 mL). The organic phase was
dried (MgSO4) and filtered through a plug of silica. The volatiles were removed in vacuo and
to the residue was added hot pentane (200 mL). The resulting suspension was filtered and
the volatiles were removed to provide product as red powder (1.27 g, 77%). Rf = 0.50
(pentane/Et2O, 1:1, v/v); Mp. 78-79 °C; 1H NMR (400 MHz, CDCl3):
(m, 1H), 7.50-7.55 (m, 3H), 7.90-7.96 (m, 3H); 13C NMR (100 MHz, CDCl3):
δ 4.02 (s, 3H), 7.01-7.06
δ
53.0, 112.2 (dd,
JC–F = 4.2 Hz, JC–F = 23.2 Hz), 120.8 (dd, JC–F = 2.0 Hz, JC–F = 10.7 Hz), 123.2, 129.2, 131.8,
137.5 (dd, JC–F = 4.1 Hz, JC–F = 6.3 Hz), 152.2, 160.0 (dd, JC–F = 6.5 Hz, JC–F = 268 Hz),
161.5, 161.8 (dd, JC–F = 5.7 Hz, JC–F = 262 Hz); 19F NMR (376 MHz, CDCl3): -120.5 (m), -
105.9 (m); HRMS (ESI+) calc. for [M+H]+ (C14H11F2N2O2): 277.0783, found: 277.0783.
4: 2,6-difluoro-3-(phenyldiazenyl)benzoic acid.
To a solution of compound 3 (1.80 mmol, 500 mg) in THF (15 mL) was added sat. aq. LiOH
(10 mL). The resulting biphasic mixture was vigorously stirred for 5h, after which the volatiles
were evaporated. 1N aq. HCl (30 mL) was added to the residue, the resulting precipitate was
filtered off and redissolved in AcOEt (60 mL). The solution was dried (MgSO4) and the
volume was reduced. The product was precipitated by addition of pentane, filtered off and
1
washed with pentane to give orange solid (186 mg, 40%). Mp. 175-176 °C; H NMR (400
3
MHz, DMSO-d6):
(100 MHz, DMSO-d6):
JC–F = 21.1 Hz), 120.8 (d, JC–F = 10.9 Hz), 123.3, 130.0, 132.7, 137.3 (dd, JC–F = 3.9 Hz, JC–F
δ
7.36 (t, J = 8.8 Hz, 1H), 7.56-7.65 (m, 3H), 7.85-7.94 (m, 3H); 13C NMR
δ
113.4 (dd, JC–F = 3.8 Hz, JC–F = 23.4 Hz), 114.0 (dd, JC–F = 20.3 Hz,
=

ACCEPTED MANUSCRIPT
7.2 Hz), 152.3, 157.1 (dd, JC–F = 7.6 Hz, JC–F = 264 Hz), 161.2 (dd, JC–F = 6.7 Hz, JC–F = 258
Hz), 162.0; 19F NMR (376 MHz, DMSO-d6): -123.0 (m), -107.1 (m); HRMS (ESI+) calc. for
[M+H]+ (C13H9F2N2O2): 263.0627, found: 263.0625.
Trans-2a: 2,6-difluoro-N-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-3-phenyldiazenyl)-
benzamide.
A solution of compound 4 (0.38 mmol, 100 mg), 3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-
amine (0.37 mmol, 60 mg, prepared by a published procedure [52]) and EDC (0.42 mmol, 80
mg) in DMF (5 mL) was stirred at rt overnight. Afterwards, the reaction mixture was diluted
with ethyl acetate (80 mL) and washed with brine (2 x 60 mL) ,1N aq. HCl (2 x 60 mL), sat.
aq. NaHCO3 (60 mL) and again brine (60 mL). The organic phase was dried (MgSO4). The
volatiles were partially removed in vacuo and to the residue was added pentane (200 mL).
The resulting precipitate was filtered to provide product as orange powder (65 mg, 43%). Rf =
0.62 (pentane/AcOEt, 1:1, v/v); Mp. >250 °C; 1H NMR (400 MHz, DMSO-d6):
7.44 (t, 3J = 8.8 Hz, 1H, ArH), 7.55-7.66 (m, 3H), 7.85-7.97 (m, 3H), 8.52 (d, 4J = 2.0 Hz, 1H),
8.61 (d, 4J = 2.0 Hz, 1H), 11.22 (s, 1H), 12.61 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
δ 4.01 (s, 3H),
δ
56.1,
102.9, 113.4 (dd, JC–F = 3.5 Hz, JC–F = 23.3 Hz), 116.7 (dd, JC–F = 21.0 Hz, JC–F = 23.6 Hz),
119.0, 120.4 (d, JC–F = 10.7 Hz), 123.3, 128.4, 130.1, 132.8, 137.3 (dd, JC–F = 3.8 Hz, JC–F
=
7.2 Hz), 143.9, 150.1, 152.4, 155.1, 156.6 (dd, JC–F = 8.1 Hz, JC–F = 260.4 Hz), 158.1, 160.8
(dd, JC–F = 7.1 Hz, JC–F = 254.8 Hz); 19F NMR (376 MHz, DMSO-d6): -124.0 (m), -108.6 (m);
HRMS (ESI+) calc. for [M+H]+ (C20H15F2N6O2): 409.1219, found: 409.1211.
Cis-2a:
A solution of compound trans-2a in DMSO-d6 (0.5 mL, ~2 mg/mL) was irradiated at λ = 365
nm (distance ~1 cm) to provide a sample of 55% cis-2a. 1H NMR (400 MHz, DMSO-d6):
δ
3.99 (s, 3H), 6.94 (d, 3J = 8.0 Hz, 2H), 7.08-7.14 (m, 1H), 7.18-7.32 (m, 2H), 7.39 (app t, 3J =
8.0 Hz, 2H), 8.42 (d, 4J = 2.4 Hz, 1H), 8.54 (d, 4J = 2.0 Hz, 1H), 11.05 (s, 1H), 12.58 (s, 1H);
19F NMR (376 MHz, DMSO-d6): -122.4 (m), -114.1 (m).

ACCEPTED MANUSCRIPT
5: Methyl 2,6-difluoro-3-((4-hydroxyphenyl)diazenyl)benzoate.
Methyl 3-amino-2,6-difluorobenzoate (2.00 mmol, 374 mg) was dissolved in 1N aq. HCl (5.0
mL) and the solution was cooled in an ice-water bath. A solution of NaNO2 (2.50 mmol, 173
mg) in water (1.0 mL) was added, and the reaction mixture was stirred for 10 min. A solution
of phenol (1.80 mmol, 169 mg) and KOH (3.60 mmol, 202 mg) in MeOH (2.5 mL) was added
dropwise and the cooling was removed. After 10 min stirring, the reaction mixture was diluted
with AcOEt (80 mL) and washed with 1H HCl (2 x 60 mL) and brine (80 mL). The organic
phase was dried (MgSO4) and the solvent was evaporated. The product was purified by flash
chromatography (Silicagel, 40-63 µm, pentane:Et2O, 9:1 to 1:1, v/v) to give orange powder
(123 mg, 23%). Rf = 0.60 (pentane/Et2O, 1:1, v/v). Compound 5 was used in the following
step without further characterization.
6: Methyl 2,6-difluoro-3-((4-methoxyphenyl)diazenyl)benzoate.
To a solution of compound 5 (0.34 mmol, 100 mg) and methyl iodide (1.50 mmol, 94 µL) in
acetone (5 mL) was added potassium carbonate (0.50 mmol, 69 mg). The resulting
suspension was stirred at rt overnight, after which it was diluted with Et2O (50 mL) wand
washed with 1H HCl (2 x 40 mL), sat. aq. NaHCO3 (2 x 40 mL) and brine (40 mL). The
organic phase was dried (MgSO4) and the solvent was evaporated. The product was purified
by flash chromatography (Silicagel, 40-63 µm, pentane:Et2O, 9:1, v/v) to give orange powder
(100 mg, 96%). Rf = 0.64 (pentane/Et2O, 7:3, v/v); Mp. 60-62 °C; 1H NMR (400 MHz, CDCl3):
3
δ
3.88 (s, 3H), 4.02 (s, 3H), 6.95-7.05 (m, 3H), 7.84-7.92 (m, 1H), 7.91 (d, J = 9.2 Hz, 2H);
13C NMR (100 MHz, CDCl3):
δ 53.0, 55.6, 112.1 (dd, JC–F = 4.1 Hz, JC–F = 23.1 Hz), 114.3,
120.8 (dd, JC–F = 2.3 Hz, JC–F = 10.5 Hz), 125.3, 129.4, 137.7 (dd, JC–F = 4.1 Hz, JC–F = 7.3
Hz), 147.0, 157.8 (dd, JC–F = 6.4 Hz, JC–F = 267 Hz), 161.3 (dd, JC–F = 5.7 Hz, JC–F = 261 Hz),
19
161.6, 162.7; F NMR (376 MHz, CDCl3): -121.2 (m), -107.2 (m); HRMS (ESI+) calc. for
[M+H]+ (C15H13F2N2O3): 307.0888, found: 307.0885.

ACCEPTED MANUSCRIPT
7: 2,6-difluoro-3-((4-methoxyphenyl)diazenyl)benzoic acid.
To a solution of compound 6 (0.36 mmol, 110 mg) in THF (3 mL) was added sat. aq. LiOH (2
mL). The resulting biphasic mixture was vigorously stirred for 5h, after which the volatiles
were evaporated. 1N aq. HCl (30 mL) was added to the residue, the resulting precipitate was
filtered off and redissolved in AcOEt (60 mL). The solution was dried (MgSO4) and the
volume was reduced. The product was precipitated by addition of pentane, filtered off and
washed with pentane to give compound 7 (51 mg, 48%). Compound 7 was used in the
following step without further characterization.
Trans-2b:
2,6-difluoro-N-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-3-((4-
methoxyphenyl)-diazenyl)benzamide.
A solution of compound 7 (0.116 mmol, 34 mg), 3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-
amine (0.15 mmol, 25 mg, prepared by a published procedure [52]) and EDC (0.16 mmol, 31
mg) in DMF (2 mL) was stirred at rt overnight. Afterwards, the reaction mixture was diluted
with ethyl acetate (50 mL) and washed with brine (2 x 40 mL), 1N aq. HCl (2 x 40 mL), sat.
aq. NaHCO3 (40 mL) and again brine (40 mL). The organic phase was dried (MgSO4). The
volatiles were partially removed in vacuo and to the residue was added pentane (50 mL).
The resulting precipitate was filtered to provide product as orange powder (14 mg, 28%). Rf =
1
0.72 (pentane/AcOEt, 1:1, v/v); Mp. = 248-250 °C; H NMR (400 MHz, DMSO-d6):
δ 3.97 (s,
3H), 4.01 (s, 3H), 7.15 (d, 3J = 8.8 Hz, 2H), 7.41 (t, 3J = 8.8 Hz, 1H), 7.85-7.95 (m, 3H), 8.51
4
4
(d, J = 2.0 Hz, 1H), 8.61 (d, J = 2.0 Hz, 1H), 11.19 (s, 1H), 12.60 (s, 1H); 13C NMR (100
MHz, DMSO-d6): 56.1, 56.2, 102.9, 113.3 (dd, JC–F = 3.7 Hz, JC–F = 23.0 Hz), 115.3, 116.6
(dd, JC–F = 21.1 Hz, JC–F = 23.6 Hz), 118.9, 120.2 (d, JC–F = 10.3 Hz), 125.5, 128.4, 137.4 (dd,
δ
JC–F = 3.8 Hz, JC–F = 7.3 Hz), 143.8, 146.8, 150.1, 155.1, 156.1 (dd, JC–F = 8.0 Hz, JC–F
=
258.3 Hz), 158.2, 160.2 (dd, JC–F = 7.1 Hz, JC–F = 254.0 Hz), 163.2; 19F NMR (376 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6): -124.6 (m), -109.9 (m); HRMS (ESI+) calc. for [M+H]+ (C21H17F2N6O3): 439.1325,
found: 439.1318.
Cis-2b:
A solution of compound trans-2b in DMSO-d6 (0.5 mL, ~2 mg/mL) was irradiated at λ = 365
nm (distance ~1 cm) to provide a sample of 92% cis-2b. 1H NMR (400 MHz, DMSO-d6):
3.75 (s, 3H), 3.99 (s, 3H), 6.94 (d, 3J = 8.8 Hz, 2H), 7.02 (d, 3J = 8.8 Hz, 2H), 7.12-7.18 (m,
δ
3
4
4
1H) 7.27 (t, J = 8.8 Hz, 1H), 8.42 (d, J = 2.0 Hz, 1H), 8.54 (d, J = 2.0 Hz, 1H), 11.04 (s,
1H), 12.58 (s, 1H); 19F NMR (376 MHz, DMSO-d6): -123.1 (m), -114.8 (m).
8: Ethyl (E)-3-((4-hydroxyphenyl)diazenyl)benzoate.
Ethyl 3-aminobenzoate (3.0 mL, 2.7 g, 17 mmol) was dissolved in aq. 1N HCl (50 mL) and
NaNO2 (1.60 g, 23 mmol) was added. The reaction mixture was stirred at 0oC for 10 min.
MeOH (25 mL) was added to the reaction mixture and a solution of PhOH (1.93 g, 14.6
mmol) and KOH (2.14 g, 38.1 mmol) in MeOH (20 mL) was added dropwise. The reaction
mixture was stirred at room temperature for 1h. After completion, aq. 1N HCl (50 mL) and
EtOAc (50 mL) were added to the reaction mixture and the water layer was extracted with
EtOAc (3 x 50 mL). The combined organic layers were concentrated in vacuo, Et2O was
added (100 mL) and the product was filtered off and washed with pentane. The product was
o
1
obtained as an orange solid (1.58 g, 5.8 mmol, 34% yield). Mp: 141-146 C. H NMR (400
MHz, CDCl3) 1.44 (t, J = 7.1 Hz, 3H), 4.38 – 4.48 (m, 2H), 5.65 (s, 1H), 6.98 (d, J = 6.9 Hz,
δ
2H), 7.58 (t, J = 7.8 Hz, 1H), 7.91 (d, J = 6.9 Hz, 2H), 8.05 (d, J = 7.9 Hz, 1H), 8.12 (d, J =
7.7 Hz, 1H), 8.52 (s, 1H). Compound 8 was used in the following step without further
characterization.
9: Ethyl (E)-3-((4-methoxyphenyl)diazenyl)benzoate.
Compound 8 (0.70 g, 2.6 mmol) was dissolved in acetone (20 mL) and MeI (3.0 mL, 1.3 g,
9.3 mmol) and K2CO3 (3.7 g, 26.8 mmol) were added. The reaction mixture was stirred at 40

ACCEPTED MANUSCRIPT
oC overnight. After completion, Et2O (50 mL) and water (50 mL) were added and the organic
layer was separated, dried with MgSO4 and concentrated in vacuo. The product was purified
by flash chromatography (Silicagel 40 – 63 nm, pentane; 0-10% EtOAc in pentane). The
o
1
product was obtained as an orange solid (0.62 g, 2.2 mmol, 85% yield). Mp: 40 - 42 C. H
NMR (400 MHz, CDCl3) 1.42 (d, J = 14.3 Hz, 3H), 3.85 (s, 3H), 4.42 (q, J = 7.1 Hz, 2H),
δ
6.99 (d, J = 9.0 Hz, 2H), 7.54 (t, J = 7.8 Hz, 1H), 7.94 (d, J = 9.0 Hz, 2H), 8.11 (d, J = 7.9 Hz,
1H), 8.53 (s, 1H). 13C NMR (101 MHz, CDCl3)
δ
14.4, 55.5, 61.2, 76.8, 77.1, 77.4, 114.2,
123.9, 125.0, 126.3, 129.0, 131.0, 131.6, 146.8, 152.7, 162.4, 166.1. HRMS (ESI+) calc. for.
[M+H+] (C16H17N2O3): 285.1234, found: 285.1232.
10: 3-((4-methoxyphenyl)diazenyl)benzoic acid.
Compound 9 (3.52 mmol, 1.00 g) was dissolved in a mixture of methanol (40 mL) and
tetrahyrofuran (40 mL). Aqueous sodium hydroxide (2N, 20 mL) was added and the mixture
was heated under reflux for 3 hours. The volatiles were evaporated, the residue was
dissolved in aqueous HCl (1N, 20 mL) and extracted with ethyl acetate (2 x 20 mL). Organic
phases were collected, dried and the product was purified by precipitation from
AcOEt/pentane. Yield: 66%; orange powder. Mp. 171-173 °C; 1H NMR (400 MHz, DMSO-d6)
3
δ
3.85 (s, 3H), 7.12 (d, 3J = 9.0 Hz, 2H), 7.68 (t, 3J = 7.8 Hz, 1H), 7.91 (d, J = 8.9 Hz, 2H),
8.06 (dd, 3J = 7.9, 4J = 1.6 Hz, 2H), 8.32 (d, 4J = 1.9 Hz, 1H), 13.25 (br s, 1H); 13C NMR (100
MHz, DMSO-d6)
δ 56.1, 115.1, 122.4, 125.3, 127.5, 130.2, 131.6, 132.5, 146.5, 152.4, 162.8,
167.2. HRMS (ESI+) calc. for [M+H]+ (C14H13N2O3): 257.0921, found: 257.0923.

ACCEPTED MANUSCRIPT
Trans-2c:
Trans-N-(3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-yl)-3-((4-methoxyphenyl)-
diazenyl)benzamide.
A solution of compound 10 (0.60 mmol, 154 mg), 3-methoxy-1H-pyrazolo[3,4-b]pyridin-5-
amine (0.55 mmol, 90 mg, prepared by a published procedure [52] ) and EDC (0.60 mmol,
115 mg) in DMF (6 mL) was stirred at rt overnight. Afterwards, the reaction mixture was
diluted with ethyl acetate (80 mL) and washed with brine (2 x 60 mL), 1N aq. HCl (60 mL),
sat. aq. NaHCO3 (60 mL) and again brine (60 mL). The organic phase was dried (MgSO4).
The volatiles were partially removed in vacuo and to the residue was added pentane (80
mL). The resulting precipitate was filtered to provide product as orange powder (110 mg,
50%). Rf = 0.38 (pentane/AcOEt, 1:1, v/v); Mp. = 233-235 °C; 1H NMR (400 MHz, DMSO-d6):
3
δ
3.87 (s, 3H), 4.01 (s, 3H), 7.15 (d, J = 8.9 Hz, 2H), 7.74 (t, 3J = 7.8 Hz, 1H), 7.95 (d, 3J =
8.9 Hz, 2H), 8.04 (d, 3J = 7.9 Hz, 1H), 8.11 (d, 3J = 8.1 Hz, 1H), 8.44 (s, 1H), 8.49 (d, 4J = 2.3
Hz, 1H), 8.74 (d, 4J = 2.3 Hz, 1H), 10.65 (s, 1H), 12.52 (s, 1H). 13C NMR (100 MHz, DMSO-
d6):
δ 56.0, 56.1, 102.9, 115.2, 120.1, 121.9, 125.2, 125.5, 128.9, 130.1, 130.2, 136.2, 145.3,
146.6, 150.1, 152.4, 155.0, 162.8, 165.5. HRMS (ESI+) calc. for [M+H]+ (C21H19N6O3):
403.1513, found: 403.1502.
Cis-2c:
A solution of compound trans-2c in DMSO-d6 (0.5 mL, ~1 mg/mL) was irradiated at λ = 365
nm (distance ~1 cm) to provide a sample of 94% cis-2c. 1H NMR (400 MHz, DMSO-d6):
3.71 (s, 3H), 3.99 (s, 3H), 6.78 – 7.11 (m, 4H), 7.46 (t, 3J = 7.8 Hz, 1H), 7.61 (t, 4J = 1.9 Hz,
δ
3
4
4
1H), 7.79 (d, J = 7.4 Hz, 1H), 8.44 (d, J = 2.3 Hz, 1H), 8.68 (d, J = 2.4 Hz, 1H), 10.48 (s,
1H), 12.51 (s, 1H).

ACCEPTED MANUSCRIPT
11: Methyl 2,6-difluoro-3-(2-fluoro-phenyldiazenyl)benzoate.
A solution of methyl 3-amino-2,6-difluorobenzoate (5.00 mmol, 935 mg, Combi-Blocks) and
2-fluoro-nitrosobenzene (4.24 mmol, 530 mg, prepared according to a published procedure
[53] ) in acetic acid (15 mL) was heated at 40 °C overnight. Afterwards, the reaction mixture
was diluted with diethyl ether (100 mL) and washed with 1N aq. HCl (2 x 80 mL), sat. aq.
NaHCO3 (80 mL) and brine (80 mL). The organic phase was dried (MgSO4) and filtered
through a plug of silica. The volatiles were removed in vacuo and to the residue was added
pentane (100 mL). The resulting suspension was filtered and the volatiles were removed to
provide product as red powder (700 mg, 56%). Rf = 0.66 (pentane/Et2O, 4:1, v/v); Mp. 92-93
°C; 1H NMR (400 MHz, CDCl3):
δ 4.02 (s, 3H), 7.04 (td, J = 9.0, 1.7 Hz, 1H), 7.18 – 7.34 (m,
2H), 7.50 (dddd, J = 8.7, 6.9, 5.0, 1.8 Hz, 1H), 7.79 (td, J = 7.8, 1.8 Hz, 1H), 7.96 (ddd, J =
9.2, 8.1, 5.9 Hz, 1H). 13C NMR (100 MHz, CDCl3):
δ
53.04 , 112.3 (dd, J = 23.3, 4.1 Hz),
117.1 (d, J = 19.7 Hz), 117.7 , 121.0 (dd, J = 10.8, 1.9 Hz), 124.4 (d, J = 3.8 Hz), 130.0 (d, J
= 7.4 Hz), 133.5 (d, J = 8.4 Hz), 137.7 (dd, J = 7.1, 4.3 Hz), 140.5 (d, J = 6.7 Hz), 158.5 (dd,
J = 269.0, 6.7 Hz), 159.2 , 161.8 , 162.2 (dd, J = 262.7, 5.9 Hz); 19F NMR (376 MHz, CDCl3):
-104.8 (m), -120.1 (m), -123.8 (m); HRMS (ESI+) calc. for [M+H]+ (C14H10F3N2O2): 295.0689,
found: 295.0688.
12: 2,6-difluoro-3-(2-fluoro-phenyldiazenyl)benzoic acid.
To a solution of compound 11 (1.77 mmol, 520 mg) in THF (15 mL) was added sat. aq. LiOH
(10 mL). The resulting biphasic mixture was vigorously stirred for 5h, after which the volatiles
were evaporated. 1N aq. HCl (30 mL) was added to the residue, the resulting precipitate was
filtered off and redissolved in AcOEt (60 mL). The solution was dried (MgSO4) and the
volume was reduced. The product was precipitated by addition of pentane, filtered off and
1
washed with pentane to give product (240 mg, 48%). Mp. 206-208 °C; H NMR (400 MHz,
DMSO-d6):
δ 7.29 – 7.39 (m, 1H), 7.49 (dd, J = 11.0, 8.3 Hz, 1H), 7.66 (dtd, J = 16.3, 7.9, 3.6
Hz, 2H), 7.85 (td, J = 8.8, 5.9 Hz, 1H); 13C NMR (100 MHz, DMSO-d6):
δ
113.6 (dd, J = 23.5,
3.8 Hz), 117.8, 118.0, 120.9 (d, J = 10.9 Hz), 125.6 (d, J = 3.7 Hz), 134.9 (d, J = 8.5 Hz),