Zap-Pano: a Photocaged Prodrug of the KDAC Inhibitor Panobinostat

Article abstract of DOI:10.1002/cmdc.202100403

We report the synthesis and biological evaluation of a light-activated (caged) prodrug of the KDAC inhibitor panobinostat (Zap-Pano). We demonstrate that addition of the 4,5-dimethoxy-2-nitrobenzyl group to the hydroxamic acid oxygen results in an inactive prodrug. In two cancer cell lines we show that photolysis of this compound releases panobinostat and an unexpected carboxamide analogue of panobinostat. Photolysis of Zap-Pano causes an increase in H3K9Ac and H3K18Ac, consistent with KDAC inhibition, in an oesophageal cancer cell line (OE21). Irradiation of OE21 cells in the presence of Zap-Pano results in apoptotic cell death. This compound is a useful research tool, allowing spatial and temporal control over release of panobinostat.

Full text of DOI:10.1002/cmdc.202100403

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Zap-Pano: a Photocaged Prodrug of the KDAC
Inhibitor Panobinostat
Kathrin S. Troelsen,[a, b, c] Ewen D. D. Calder+,[a] Anna Skwarska+,[b] Deborah Sneddon,[a]
Ester M. Hammond,*[b] and Stuart J. Conway*[a]
We report the synthesis and biological evaluation of a light-
activated (caged) prodrug of the KDAC inhibitor panobinostat
(Zap-Pano). We demonstrate that addition of the 4,5-dimeth-
oxy-2-nitrobenzyl group to the hydroxamic acid oxygen results
in an inactive prodrug. In two cancer cell lines we show that
photolysis of this compound releases panobinostat and an
unexpected carboxamide analogue of panobinostat. Photolysis
of Zap-Pano causes an increase in H3K9Ac and H3K18Ac,
consistent with KDAC inhibition, in an oesophageal cancer cell
line (OE21). Irradiation of OE21 cells in the presence of Zap-
Pano results in apoptotic cell death. This compound is a useful
research tool, allowing spatial and temporal control over release
of panobinostat.
Introduction
and Zn2+-dependent histone/lysine deacetylases (H/KDACs)
that remove this mark.[5,6]
Protein post-translational modifications (PTMs) add a layer of
complexity to the proteome and provide mechanisms to fine-
tune protein function.[1] Lysine acetylation is a well-established
PTM[2] that is found in over 3600 locations on over 1750
proteins throughout the cellular environment.[3,4] Recently, there
has been a particular focus on lysine acetylation in histone
proteins, which play a key role in the organisation of DNA in
chromatin. Acetylation leads to the formation of an amide,
which removes the positive charge from the ɛ-nitrogen atom of
lysine, resulting in a weaker association with negatively charged
DNA, and the formation of euchromatin. This more relaxed
form of chromatin is associated with transcriptional activation.
Lysine acetylation state is regulated by histone/lysine acetyl
transferase (H/KATs) enzymes, which transfer an acetyl group
from AcCoA to lysine, and the mechanistically distinct sirtuin
Bromodomains are protein modules that bind to acetyl-
lysine (KAc) residues and in so doing mediate protein-protein
and protein-chromatin interactions enabling the assembly of
transcription complexes.[7–10]
Many examples exist of KDAC dysregulation being linked to
oncogenic states in tissues throughout the body.[11] These
observations have led to significant interest in molecules that
can inhibit these enzymes, culminating in the clinical approval
of five pan-KDAC inhibitors to date (belinostat, chidamide,
panobinostat (Pano, 2), romidepsin, and vorinostat).[11]
All KDAC inhibitors possess a group that binds to the Zn2+
ion found in the enzyme active site, which mimics KAc binding
in the active site, and inhibits enzyme function. For belinostat,
Pano and vorinostat this moiety is an hydroxamic acid. In
addition to Zn2+, this group can bind to other metals or inhibit
other off-target enzymes. Hydroxamic acids are also polar and
can hinder the diffusion of KDAC inhibitors into cells and
organs, for example, the brain. KDACs are also broad epigenetic
remodellers that affect the transcription of many genes, in both
healthy tissues and tumours.
For these reasons, KDAC inhibitors have a number of
documented side effects including anaemia, neutropenia,
thrombocytopenia, fatigue, diarrhoea, nausea and vomiting.[13,14]
In recent years, attempts have been made to increase the
selectivity of these inhibitors and therefore mitigate these
unwanted effects. Work to achieve this has included both
designing selective inhibitors of a given KDAC enzyme, or
developing prodrugs that selectively release inhibitors in a
target tissue.[15–18] Prodrugs of KDAC inhibitors have been
developed that are activated by hydrolysis,[19] thiols,[13] reactive
oxygen species,[20] hydrogen peroxide or peroxynitrite,[21]
esterases,[22,23] transition metals,[24] and light.[25–29] We have
recently reported prodrugs of the KDAC inhibitors vorinostat
(SAHA)[30] and Pano[12] that are selectively released in hypoxia.
We have previously used a caging approach to develop
photolabile precursors of capsaicin,[31,32] the mitochondrial
[a] Dr. K. S. Troelsen, Dr. E. D. D. Calder,+ Dr. D. Sneddon, Prof. S. J. Conway
Department of Chemistry,
Chemistry Research Laboratory
University of Oxford
Mansfield Road, Oxford, OX1 3TA (UK)
E-mail: stuart.conway@chem.ox.ac.uk
[b] Dr. K. S. Troelsen, Dr. A. Skwarska,+ Prof. E. M. Hammond
Department of Oncology
Oxford Institute for Radiation Oncology
University of Oxford,
Oxford, OX3 7DQ (UK)
E-mail: ester.hammond@oncology.ox.ac.uk
[c] Dr. K. S. Troelsen
Department of Drug Design and Pharmacology
University of Copenhagen
Jagtvej 162, 2100 Copenhagen (Denmark)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100403
This article belongs to the joint Special Collection with ChemBioChem,
“Chemical Epigenetics”.
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH. This is
an open access article under the terms of the Creative Commons Attribution
License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
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uncoupler AG10,[33] and the neurotransmitters glutamate and Results and Discussion
GABA.[34] This technique provides temporal and spatial control
over the release of the active component. Building on our work
developing a hypoxia-activate prodrug of Pano,[12] here we
report the design, synthesis, and initial validation of a light-
activated prodrug of Pano, Zap-Pano (1, Figure 1). This com-
pound releases Pano with temporal control in the oesophageal
cancer cell line, OE21; it will be a useful research tool for those
studying lysine acetylation and has the potential to allow the
release of Pano while minimising off-target effects.[35]
Chemistry
We, and others, have previously demonstrated that substitution
on the hydroxamic acid oxygen atom is an effective strategy for
developing prodrugs of KDAC inhibitors. This modification can
prevent the interaction between the compound and the active
site Zn2+ ion, leading to an inactive compound.[12,30] Therefore,
we adopted this approach to develop light-activated derivatives
of Pano and selected the 4,5-dimethoxy-2-nitrobenzyl group as
33,36,37]
the photo-labile component (Scheme 1).[31–
In addition,
we also employed two negative control compounds a 4-
nitrobenzyl analogue (NB, 13) and an unsubstituted benzyl
analogue (Bn, 14), which should not photorelease Pano.
The synthesis of Pano was carried out as has been
previously published.[12,38] The photolabile DMNB derivative of
Pano (Zap-Pano, 1) and both control compounds were
synthesised from the substituted hydroxylamines using a route
previously developed within the group (Scheme 1).[12,30] Briefly,
the nitrobenzyl halides (3 and 6) were reacted with N-
hydroxyphthalimide, before deprotection with hydrazine, to
give the substituted hydroxylamines (5 and 8). Both of the
synthesised hydroxylamines (5 and 8), and the commercially
available O-benzylhydroxylamine, were then coupled to the di-
Boc protected core fragment of Pano (9) using PyBOP. The Boc
groups were removed using trifluoroacetic acid, to yield Zap-
Pano (1), and the two control compounds (13 and 14). We also
synthesised the carboxamide analogue of Pano, 15, as this
compound was detected as a by-product in the photolysis
reaction (vide infra).
Zap-Pano does not inhibit KDAC enzymes
Analysis of DMNB-Pano in a commercial KDAC inhibition assay
showed that this compound does not inhibit KDAC1-9 or
KDAC11 to any significant degree (Table 1). This is consistent
with our previous work on prodrugs of SAHA and Pano,[12,30] and
encouraged us to progress Zap-Pano for further analysis.
Figure 1. The light-activated derivative of Pano (2), Zap-Pano (1), is photo-
lysed to release the KDAC inhibitor Pano (2).
Table 1. Zap-Pano shows little KDAC inhibition in vitro.
IC50 values (nM) for Pano and Zap-Pano against KDAC1-9 and KDAC11. The colour scale represents a heat map, with ‘hot’ colours showing effective enzyme
inhibition. [a] Data obtained by Reaction Biology Corporation, NI=no inhibition observed at concentrations up to 10 μM. [b] Data taken from Arts et al.[39]
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Scheme 1. Synthesis of Zap-Pano (1) and the benzylated Pano derivatives 13 and 14, and the amide 15. Reagents and conditions: (A) (a) N-
[12]
°
°
Hydroxyphthalimide, DIPEA, DMF, 0 C to rt, 2 h, 76–84%, n=2, (b) N2H4·H2O, CH2Cl2, rt, 2 h, 72–95%, n=4. (B) (c) N-Hydroxyphthalimide, DIPEA, DMF, 70 C,
2 h, 88%, n=1; (d) N2H4·H2O, CH2Cl2, rt, 3 h, 75–99%, n=2. (C) (e) RONH2, PyBOP, NEt3, THF, rt, 18 h, 10, R=DMNB, 62–74%, n=2, RONH2, PyBOP, NEt3, THF, rt,
18 h, 11, R=NB, 71–92%, n=2, RONH2, PyBOP, NEt3, THF, rt, 18 h, 12, R=Bn, 73%, n=1; (f) TFA, TIPSÀ H, CH2Cl2, rt, 1 h, 1, R=DMNB, 51–65%, n=2, TFA, TIPSÀ H,
CH2Cl2, rt, 1 h, 13, R=NB, 68–69%, n=2, TFA, TIPSÀ H, CH2Cl2, rt, 1 h, 14, R=Bn, 79%, n=1. (D) (g) (i) PyBOP, NEt3, NH4Cl, THF, rt, 18 h, 84%, n=1; (ii) TFA,
TIPSÀ H, CH2Cl2, rt, 1 h, 65%, n=1.
Photolysis of Zap-Pano releases Pano in PBS buffer
two control compounds, NB-Pano (13) and Bn-Pano (14), were
treated with UV-light and analysed using HPLC.
The uncaging of Zap-Pano and release of Pano was first
evaluated in PBS buffer. Upon irradiation with light of wave-
length 405 nm for 15 min, we observed approximately 20%
Pano being released. However, we were surprised to also
observe approximately 20% of an amide derivative of Pano by-
product (Pano-NH2, 15, Figure 2D), which was formed during
irradiation (Figure 2A, B). The structure of 15 was confirmed
using LCMS analysis (see Supporting Information) and synthesis
of an authentic sample for comparison (Scheme 1D). To
determine whether the by-product was formed as a result of
photolysis, or resulted from instability of Zap-Pano in PBS, the
Interestingly, both compounds were stable to UV irradiation,
and did not release any by-products that could be detected
with HPLC (Figure 1C). Based on these observations, a plausible
mechanism for the formation of 15 is shown in Scheme 2. Given
that this product is only formed when 1 is irradiated with light,
we suggest that the first step is excitation to the singlet state,
followed by intersystem crossing to give the triplet state, as is
the case for the expected photolysis pathway.[37] However, the
relatively weak NÀ O bond (201 kJmolÀ 1) of the hydroxamic acid
presents an alternative pathway for the radical reaction to
proceed. We suggest that the benzylic radical can react to form
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Figure 2. Photolysis of Zap-Pano in PBS buffer. (A) Zap-Pano 10 μM in PBS was treated with UV-light (405 nm) for up to 15 min, aliquots were taken at desired
time points and analysed by HPLC (10 μL injections) to determine the concentration of Zap-Pano, Pano and Pano-NH2. (B) Overlaid HPLC traces of Zap-Pano
(blue), Pano (red), Pano-NH2 (black) Zap-Pano treated with UV light for 5 min (green). Retention times are 6.25 min for Zap-Pano, 4.08 min for Pano and
4.33 min for Pano-NH2. (C) Zap-Pano, Bn-Pano (14) and NB-Pano (13) were irradiated with UV-light for 5 minutes then the products quantified using HPLC
analysis. (D) Chemical structures of Zap-Pano (1), Pano (2) and the unexpected by-product of photolysis Pano-NH2 (15).
the nitrobenzaldehyde 16, and the amide product 15, as shown
in Scheme 2.
Zap-Pano has a UV-dependent effect on H3K9Ac and
H3K18Ac levels
Numerous studies have confirmed that nanomolar doses of
Pano lead to increases in acetylation of histone H3 including at
lysines 9 and 18.[40–43] To determine the effects of Zap-Pano on
histone acetylation, OE21 cells were treated with Zap-Pano for
3 hours then exposed to UV (5 minutes) before being returned
to normal culture conditions (24 hours). Cells were then
harvested, and western blotting carried out for H3K18Ac and
H3K9Ac (Figure 4A). Importantly, in the absence of exposure to
UV light there was no change in H3K18Ac or H3K9Ac,
confirming that the prodrug does not act as a KDAC inhibitor in
cells. However, after UV exposure a clear dose dependency was
observed for both acetylation marks. Having determined that a
dose of 1 μM Zap-Pano was sufficient to increase H3K18Ac and
H3K9Ac, we further investigated the time after UV exposure
required to observe these changes. OE21 cells were again
treated with Zap-Pano for 3 hours followed by UV exposure.
The cells were then harvested after 24 hours (as previously) or
after 5 hours. A clear increase in H3K18Ac and H3K9Ac was
observed at both 5 and 24 hours after UV exposure (Figure 4B).
Zap-Pano releases Pano in response to UV-treatment of
cancer cells
Next, we investigated the uptake and light-activation of Zap-
Pano. We chose to use two cancer cell lines OE21 (oesophageal)
and HCT116 (colorectal) as both originate from cancer types
that are accessible with a UV light i.e., the oesophagus and
colon/rectum. To demonstrate that Zap-Pano is cell permeable,
the cells were treated with 10 μM Zap-Pano followed by HPLC
analysis to determine the cellular concentration of Zap-Pano,
Pano and Pano-NH2. Pleasingly, Zap-Pano was found to be cell
permeable and stable over a 24-hour period (Figure 3A). Most
importantly, when OE21 or HCT116 cells were treated with Zap-
Pano (10 μM for 3 hours) followed by UV treatment, the levels
of Zap-Pano decreased and there was a corresponding increase
in Pano (Figure 3B, C, D). The released Pano was still detectable
24 hours after UV treatment; 1.65�0.8 nM in OE21 cells (data
not shown).
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indicate that Zap-Pano has the potential to decrease cancer cell
viability in a light-dependent manner.
Conclusion
In conclusion, we have designed and synthesised a DMNB-
derivative of Pano, Zap-Pano 1. We have shown that this
compound does not inhibit KDAC enzymes in vitro or in cells.
Photolysis of Zap-Pano causes release of Pano and an
unexpected amide derivative 15. Photolysis of Zap-Pano in the
OE21 cell line causes an increase in acetylation at H3 K9 and
K3 K18, consistent with light-dependent KDAC inhibition by the
released Pano. Photolysis of Zap-Pano in OE21 cells also causes
cell death, and an increase in PARP cleavage, which indicates
that this results from KDAC inhibition and subsequent apopto-
sis. These data show that Zap-Pano compound is a powerful
research tool that places KDAC inhibition, and therefore lysine
acetylation state, under the control of light, providing temporal
and spatial control over their inhibition. As the cellular testing
of this compound was conducted using cell lines derived from
cancers that are accessible to light, our data suggest that Zap-
Pano has the potential to be used in photodynamic therapy,
where the release of Pano is controlled by light, minimising the
effects of Pano on non-cancerous tissue.
Experimental Section
Biology Experimental section
Cell lines. OE21 (oesophageal squamous carcinoma, PHE) and
HCT116 (colorectal carcinoma, gift from Professor Bert Vogelstein,
Johns Hopkins Medical School, USA) cancer cells were cultured in
RPMI and DMEM medium, respectively, supplemented with 10%
FBS, penicillin (100 μg/mL) and streptomycin (100 μg/mL). Cells
Scheme 2. Proposed mechanism for the formation of the carboxamide
derivative of Pano, Pano-NH2 (15). ISC=intersystem crossing.
°
were grown at 37 C in a humidified atmosphere of 5% of CO2. Cells
were routinely tested for mycoplasma and found to be negative.
LED photolysis experiments were performed with 405 nm LED,
800 mA, 3.3 V, WL-SUMW SMT Ultraviolet Ceramic Waterclear,
Würth Elektronik.
Zap-Pano leads to loss of cell viability in a UV-dependent
manner
High-performance liquid chromatography (HPLC) analysis was
carried out using HPLC (Waters 2695, Watford, UK) with a photo-
diode array detector (Waters 2996). Separation was achieved using
a HiChrom RPB column (C18, 5 μm, 3.2×100 mm) maintained at
Next, we determined the toxicity of Zap-Pano (1, Figure 5D)
both with and without UV-exposure and compared to Pano-NH2
(15, Figure 5D). OE21 cells were exposed to a range of doses of
Zap-Pano (0–10 μM) or Pano-NH2 and a colony survival assay
carried out (Figure 5A, B).
As expected, Pano-NH2 and Zap-Pano in the absence of UV
light did not significantly affect clonogenic potential of OE21
cancer cells. However, when Zap-Pano was combined with UV
light treatment, a significant decrease in OE21 colony forming
and survival was observed. As Pano has been shown to
promote apoptosis,[41] we investigated whether Zap-Pano could
also cause apoptotic cell death in the presence of UV light. To
investigate this hypothesis western blotting was carried out for
PARP cleavage, a well-characterised marker of apoptosis (Fig-
ure 5C). PARP cleavage was observed only in the cells treated
with Zap-Pano that were exposed to UV. Together these results
°
35 C and flow rate of 0.5 mL/min. The system was employed with
0.01% v/v TFA in water as eluent A and acetonitrile as eluent B.
Samples were injected in methanol, water or acetonitrile. The
method was used as shown in Table 2.
Liquid chromatography mass spectrometry (LCMS) was carried
out using an Agilent 1260 Infinity II system equipped with a vial
Table 2. HPLC conditions.
[A]
[B]
Time [Min]
Slope
80%
30%
80%
80%
20%
70%
20%
20%
0
6
6.1
10
0
1
0
0
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Figure 3. Photo-uncaging of Zap-Pano in cancer cell lines. (A) OE21 cells were treated with Zap-Pano (10 μM) and incubated for 3 or 27 h Cell lysis was
performed in MeCN:MeOH (1:1) with a fixed volume of 100 μL. Cell lysate was analysed using HPLC to determine the concentration of Zap-Pano, Pano and
Pano-NH2. Quantification was made using a calibration curve for Zap-Pano, Pano and Pano-NH2 (n=3). (B) A schematic representation of the photo-uncaging
experiments is shown. (C) OE21 cells and (D) HCT116 cells were treated with Zap-Pano (10 μM). After 3 h, the media containing Zap-Pano was removed and
replaced with fresh media. The cells were then exposed to UV-light for the time periods indicated (0–10 minutes). Cell lysis was performed in MeCN:MeOH
(1:1) with a fixed volume of 100 μL. Cell lysates were analysed using HPLC to determine the concentration of Zap-Pano, Pano and Pano-NH2.
Figure 4. OE21 cells treated with Zap-Pano and exposed to UV light have increased H3 K18 and H3 K9 acetylation. (A) OE21 cells were treated with the doses
of Zap-Pano indicated (0–10 μM) for 3 h and were then either kept in the dark (Con) for 24 h or treated with UV light for 5 min followed by incubation for
24 h. The levels of H3 K9 and H3 K18 acetylation are shown, H3 was included as a loading control. (B) OE21 cells were treated with 1 μM Zap-Pano for 3 h
followed by UV light (5 min) or no light (labelled C), the cells were allowed to grow for 5 h or 24 h before lysing and immunoblotting for H3K9Ac and
H3K18Ac.
sampler, diode array detector, fraction collector and single quadra-
pole LC/MSD. A sample (10 μL) of Pano-NH2 (15) spiked with Pano
acid was injected onto an Agilent Zorbax RRHT StableBond SBÀ C18
and Pano acid (2.263 min) was confirmed using mass spectrometry.
The method was used as shown in Table 3.
Photorelease experiments Zap-Pano (10 μM in PBS, 4 mL) was
placed in a 6 cm plastic dish under the LED (405 nm, 800 mA).
Aliquots (50 μL) were taken at the indicated time points and diluted
°
column (2.1×50 mm, 1.8 μM) at 40 C following the specified
gradient profile below. The identity of Pano-NH2 (15, 1.071 min)
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Figure 5. Zap-Pano leads to loss of cell viability in a UV-dependent manner. (A) Colony survival assay of OE21 cells treated with Zap-Pano and Pano-NH2.[44]
Cells were treated at the concentrations of Zap-Pano shown (grey), Zap-Pano followed by 5 min exposure to UV light (purple), and Pano-NH2 (grey). Mean �
SEM of three independent experiments performed in triplicate are shown. Statistical significance was determined using two-way ANOVA, ****p<0.0001. (B)
Representative images from part A are shown. (C) OE21 cells were exposed to the doses indicated of Zap-Pano for 3 h followed by UV or mock treatment
(Control). Cells were harvested 24 h after UV and western blotting carried out. β-actin is shown as a loading control. (D) The chemical structures of Zap-Pano
(1) and Pano-NH2 (15).
Table 3. LCMS conditions.
Immunoblotting. Following Zap-Pano/UV treatment cells were
washed in PBS, lysed in UTB buffer (9 M urea, 75 mM Tris-HCl
pH 7.5, 0.15 M β-mercaptoethanol). Cell lysates were briefly soni-
cated, centrifuged for 15 mins at 13500 rpm, and the supernatant
was collected for western blotting. 50 μg of total proteins were
separated by SDS-gel electrophoresis and electroblotted onto
nitrocellulose membranes, which were subsequently probed with
the following primary antibodies: anti-PARP (9542 Cell Signaling),
anti-H3K18Ac (9675 Cell Signaling), anti-H3K9Ac (949S Cell Signal-
ing), anti-H3 total (3638S Cell Signaling), and anti-β-actin (sc-69876
Santa-Cruz biotechnology). Secondary antibodies were IRDye®
680RD Goat anti-Mouse IgG (H+L) and IRDye® 800CW Donkey anti-
Rabbit IgG (H+L) from LI-COR Biosciences. Odyssey IR imaging
technology (LI-COR Biosciences) was used for imaging.
Time
(min)
Flow
rate
[mL/
%A
%B
H2O+0.1% v/v formic
acid
MeCN+0.1% v/v formic
acid
min]
0
6
6.1
10
0.5
0.5
0.5
0.5
80
30
80
80
20
70
20
20
in MeCN (50 μL). Quantification was made using a calibration curve
for Zap-Pano, Pano, and Pano-NH2 measuring absorbance at
280 nm, and are not scaled from the HPLC injection volume. OE21
or HCT116 cells (0.5×106, 6 cm dish, 4 mL media) were seeded
overnight and treated with Zap-Pano (10 μM) for 3 h allowing
sufficient time for the drug to accumulate within cells. Next, the
drug containing medium was removed and replaced with drug-free
medium. Cells were then placed under the LED for the indicated
time. Immediately after UV treatment cells were washed twice with
PBS, scraped into 1 mL of PBS, and centrifuged at 3000 g for
5 minutes. Cell pellets were lysed in ice-cold MeOH:MeCN (1:1,
100 μL), briefly sonicated and cell debris were removed by
centrifugation at 13300 g (15 min). Resulting supernatants were
analysed by HPLC (10 μL injections) to determine the concentration
of Zap-Pano, Pano and Pano-NH2. Quantification was performed
using a calibration curve for Zap-Pano, Pano, and Pano-NH2 at
280 nm and were not scaled from the HPLC injection volume.
Colony survival assay. OE21 cells were seeded at low density in
3.5 cm Petri dishes (500-1000 cells/dish), allowed to attach and
treated with Zap-Pano for 3 h. The drug containing media was
removed and replaced with drug-free medium, cells were irradiated
for 5 minutes and then the media was again replaced with fresh
media. Cells were incubated in normal conditions for 6–10 days to
form colonies (>50 cells). Colonies were fixed in 75% methanol
and stained with 0.5% crystal violet. Colonies were counted
manually to quantify the surviving fraction.
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Tensor 27 spectrometer. Absorption maxima are reported in wave-
Chemistry Experimental Section
numbers (cmÀ 1). Only the main, relevant peaks have been assigned.
UV/vis spectra were obtained as 10 μM solutions in water on a
Shimadzu UV-1800 spectrophotometer and raw data were proc-
essed in GraphPad Prism 7.
General chemistry procedures
Chemicals were purchased from Acros Organics, Alfa Aesar, Apollo
Scientific, Fisher Scientific, Fluka, Fluorochem, Merck or Sigma
Aldrich and were used without further purification. Where appro-
priate and if not otherwise stated, all non-aqueous reactions were
carried out under an inert atmosphere of argon, using flame-dried
glassware. Anhydrous solvents were obtained under the following
conditions: THF, acetonitrile, dichloromethane, diethyl ether and
DMF were dried by passing them through a column of active basic
alumina according to Grubbs’ procedure and stored over activated
3 Å molecular sieves under argon.[45] Anhydrous methanol and
ethanol was purchased from Sigma Aldrich UK in SureSeal™ bottles
and used without further purification. Analytical thin layer
chromatography (TLC) was performed on normal phase Merck
silica gel 60 F254 aluminium-supported thin layer chromatography
sheets. Spots were visualised by either absorption under UV light
(254 nm), exposure to iodine vapor or thermal development after
dipping into a solution of ammonium molybdate in sulfuric acid, an
aqueous solution of potassium permanganate or an ethanolic
solution of ninhydrin. Reaction progress was monitored at appro-
priate times using TLC analysis. Normal phase silica gel flash
column chromatography was performed manually using Geduran
Silica gel 60 (40–63 μm) under a positive pressure of compressed
nitrogen or on a Biotage SP1 automated column chromatography
Purity of all novel compounds was determined using analytical
high-performance liquid chromatography (HPLC; Table 4) on a
PerkinElmer Flexar system with a Binary LC Pump and UV/vis LC
Detector. All biologically tested compounds were of >95% purity
as determined by HPLC. For determination of compound purity on
reversed phase (RP) 2–10 μL of sample was injected a Dionex
Acclaim® 120 column (C18, 5 μm, 12 Å, 4.6×150 mm). The gradient
profile, unless otherwise stated is shown in Table 3. Method A
refers to the solvents shown without modifiers. Method B refers to
the solvents shown with the addition of 0.1% v/v TFA.
Experimental methods
N-Phthalimido-O-(4, 5-dimethoxy-2-nitrobenzyl)-hydroxylamine
(7)
The reaction, work-up and purification were carried out in the dark
as far as possible. N,N-Diisopropylethylamine (345 μL, 1.98 mmol,
1.2 eq) was added to a stirred solution of N-hydroxyphthalimide
(269 mg, 1.65 mmol, 1.0 eq) in N,N-dimethylformamide (8 mL). 4,5-
Dimethoxy-2-nitrobenzyl bromide (6, 500 mg, 1.81 mmol, 1.1 eq)
1
system using KP-Sil® SNAP Flash Silica Cartridges. H NMR spectra
°
was added and the solution was heated to 70 C for 2 h. The
were recorded on a Bruker AVIIIHD 400 (400 MHz) a Bruker AVII 500
with dual 13C(1H) cryoprobe (500 MHz) or a Bruker AVIIIHD 500
(500 MHz) spectrometer with the stated solvents as a reference for
the internal deuterium lock. Chemical shifts are reported as δH in
parts per million (ppm) relative to tetramethyl silane (TMS). The
spectra are calibrated using the solvent peak with the data
provided by Fulmer et al.[46] Identical proton coupling constants are
averaged in each spectrum and reported to the nearest 0.1 Hz. The
coupling constants were determined by analysis using Bruker
TopSpin software (versions 3.2 and 4.0) or Mestrenova software
reaction solution was cooled to rt, diluted with ethyl acetate
(200 mL), and washed with aqueous 0.5 M lithium chloride (4×
200 mL) then dried (MgSO4), filtered, and concentrated in vacuo.
Crystallisation from hot ethanol yielded the title compound (7)
(520 mg, 88%) as a colourless solid. Rf 0.40 (50% ethyl acetate:
ĭ
petroleum ether); mp 188–190 C (from EtOH); νmax (thin film)/cmÀ 1
°
1
2950, 1740, 1525, 1498, 1281, 1066; H NMR (400 MHz, CDCl3) δH
7.88–7.81 (2H, m), 7.80–7.73 (2H, m), 7.70 (1H, s), 7.61 (1H, s), 5.68
(2H, s), 4.08 (3H, s), 3.96 (3H, s); 13C NMR (101 MHz, CDCl3) δC 163.5,
153.9, 148.5, 139.8, 134.8, 128.9, 126.4, 123.8, 110.5, 108.0, 76.3,
56.8, 56.5; HRMS m/z (ESI+) Found: 381.06925, C17H14N2O7Na
requires [M+Na]+ 381.06932; LRMS m/z (ESI+) 381 (100%, [M+
Na]+), 196 (30%); HPLC Method A, Retention time - 9.5 min, 92%.
1
(version 11). H spectra were assigned using 2D NMR experiments
including 1H-1H COSY, 13C-1H HSQC and 13C-1H HMBC. 13C NMR
spectra were recorded on a Bruker AVIIIHD 400 (101 MHz) or a
Bruker AVII 500 with dual 13C(1H) cryoprobe (126 MHz) spectrometer
in the stated solvents with broadband proton decoupling and an
internal deuterium lock. Chemical shifts are reported as δC in parts
per million (ppm) relative to tetramethyl silane (TMS). The spectra
are calibrated using the solvent peak with the data provided by
Fulmer et al.[46] The shift values of resonances are quoted to 1
decimal place unless peaks have similar chemical shifts, in which
case 2 decimal places are used. 13C spectra were assigned using 2D
NMR experiments including HSQC and 13C-1H HMBC. Electrospray
ionisation (ESI) mass spectra were acquired using an Agilent 6120
Quadrupole spectrometer or Waters LCT Premier spectrometer,
operating in positive or negative mode, as indicated, from solutions
of MeOH or MeCN. Chemical ionisation (CI) mass spectra were
acquired using a Waters GCT spectrometer. MS data was processed
using Mestrenova software (version 11). m/z values are reported in
Daltons and followed by their percentage abundance in parenthe-
ses. Accurate mass spectra were obtained using Bruker μTOF
O-(4, 5-Dimethoxy-2-nitrobenzyl)-hydroxylamine (8)
The reaction, work-up and purification were carried out in the dark
as much as possible. Hydrazine monohydrate (65% v/v in water)
(167 μL, 2.23 mmol, 4.0 eq) was added to a suspension of N-
phthalimido-O-(4’,5’-dimethoxy-2’-nitrobenzyl)-hydroxylamine (7)
(200 mg, 0.559 mmol, 1.0 eq) in dichloromethane (11 mL) and
stirred at rt for 3 h. The suspension was diluted with dichloro-
methane (20 mL) and filtered, then concentrated in vacuo to yield
the title compound (8) (126 mg, 99%) as a yellow solid which was
used without further purification. Rf 0.10 (50% ethyl acetate:
1
°
petroleum ether); mp 82–84 C (from dichloromethane/hexane); H
NMR (400 MHz, CDCl3) δH 7.68 (1H, s), 7.12 (1H, s), 5.58 (2H, br s),
5.10 (2H, s), 4.00 (3H, s), 3.95 (3H, s); 13C NMR (101 MHz, CDCl3) δC
spectrometer. m/z values are reported in Daltons. When
a
compound was not observed by LRMS, only HRMS is quoted.
Melting points were determined using either a Griffin capillary
tube melting point apparatus or a Kofler hot stage and are
uncorrected. The solvent(s) from which the sample was crystallised
is given in parentheses. Infrared (IR) spectra were obtained either
from neat samples, either as liquids or solids, or as a thin film using
a diamond ATR module. The spectra were recorded on a Bruker
Table 4. HPLC conditions.
[A]
[B]
Time [Min]
Slope
95%
5%
5%
5%
95%
95%
5
10
5
0
1
0
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ChemMedChem
153.7, 147.9, 140.2, 129.8, 110.0, 108.1, 74.7, 56.6, 56.5; Could not be
observed by MS; HPLC Method B, Retention time - 4.8 min, 88%.
545.23946; LRMS (ESI+) 567 ([M+Na]+, 100%), 545 ([M+H]+,75%),
528 (53%), 384 (47%), 371 (35%), 174 (27%), 129 (47); HPLC Method
B, Retention time – 7.2 min, 96–98% at 3 wavelengths, mean purity
97%. All batches used for biological testing were >95% purity. *Signal
at 49.0 ppm overlaps with solvent peaks, see SI for expansion.
tert-Butyl-(E)-3-(2-((tert-butoxycarbonyl)(4-(3-(((4,5-dimeth-
oxy-2-nitrobenzyl)oxy)amino)-3-oxoprop-1-en-1-yl)benzyl)
amino)ethyl)-2-methyl-1H-indole-1-carboxylate (10)
(E)-3-(4-((tert-Butyloxycarbonyl-(2-(1-(tert-butyloxycarbonyl)-2--
methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)
prop-2-enamide (S1)
Working in the dark as far as possible, PyBOP (209 mg, 0.402 mmol,
1.1 eq) was added to a solution of (E)-3-(4-((tert-butyloxycarbonyl-(2-
(1-(tert-butyloxycarbonyl)-2-methyl-1H-indol-3-yl)ethyl)amino)methyl)
phenyl)prop-2-enoic acid (9) (196 mg, 0.365 mmol, 1.0 eq) and triethyl-
amine (152 μL, 1.10 mmol, 3.0 eq) in dry tetrahydrofuran (3.7 mL). The
reaction mixture was stirred for 15 min at rt before O-(4,5-dimethoxy-
2-nitrobenzyl)-hydroxylamine (8) (100 mg, 0.439 mmol, 1.2 eq) was
added. Stirring was continued at rt for 18 h, then the reaction mixture
was diluted with ethyl acetate (20 mL) and quenched with aqueous
1 M solution of hydrochloric acid (10 mL). The reaction mixture was
extracted with ethyl acetate (20 mL), the combined organic compo-
nents were washed with a saturated solution of sodium hydrogen
carbonate (40 mL), water (40 mL), and brine (40 mL) then dried
(MgSO4), filtered, and concentrated in vacuo. Purification using column
chromatography (elution with 0–1% ethanol:chloroform) yielded the
PyBOP (32 mg, 0.062 mmol, 1.1 eq) was added to a solution of (E)-3-(4-
([tert-butyloxycarbonyl-(2-(1-(tert-butyloxycarbonyl)-2-methyl-1H-indol-
3-yl)ethyl)amino)methyl)phenyl)prop-2-enoic
acid
(9)
(30 mg,
0.056 mmol, 1.0 eq) and triethylamine (0.12 mL, 0.84 mmol, 15 eq) in
dry tetrahydrofuran (0.6 mL). The reaction mixture was stirred for
15 min at rt before ammonium chloride (30 mg, 0.56 mmol, 10 eq)
was added. Stirring was continued at rt for 18 h, then the reaction
mixture was diluted with ethyl acetate (20 mL) and quenched with
aqueous 1 M solution of hydrochloric acid (10 mL). The reaction
mixture was extracted with ethyl acetate (20 mL), the organic
components were washed with a saturated solution of sodium
hydrogen carbonate (40 mL), water (40 mL), and brine (40 mL), then
dried (MgSO4), filtered, and concentrated in vacuo. Purification using
column chromatography (elution with 80% ethyl acetate:petroleum
title compound (10) (201 mg, 74%) as a yellow solid. Rf 0.29 (50%
ĭ
°
ethyl acetate:petroleum ether); mp 88–92 C (from CHCl3); νmax (thin
film)/cmÀ 1 3192, 2977, 2932, 1728, 1686, 1521, 1460, 1366, 1323, 1276,
1220, 1159, 1137, 1117, 1068; H NMR(500 MHz, D6-DMSO) δH 11.00
ether) yielded the title compound (S1) (25 mg, 84%) as a colourless
ĭ
1
°
solid. Rf 0.55 (100% ethyl acetate); mp 97–100 C (from CHCl3); νmax
(1H, br s), 8.00 (1H, br d, J 8.1), 7.67 (1H, s), 7.50 (2H, d, J 8.0), 7.46 (1H,
d, J 15.5), 7.42 (1H, br d, J 7.5), 7.42 (1H, s), 7.24 (2H, d, J 8.0), 7.21 (1H,
ddd, J 8.1, 7.1, 1.3), 7.16 (1H, ddd, J 7.5, 7.1, 1.2), 6.46 (1H, d, J 15.5),
5.25 (2H, s), 4.41 (2H, s), 3.95 (3H, s), 3.33 (2H, t, J 7.2), 3.02 (2H, s), 2.83
(2H, t, J 7.2), 2.46 (3H, s), 1.64 (9H, s), 1.34 (9H, s); 13C NMR (126 MHz,
(D6-DMSO) δC 163.6*, 154.4, 153.0, 149.5, 147.9, 140.0, 139.2, 134.9,
133.2, 132.9, 129.1, 127.4, 127.2, 126.2, 122.7, 121.7, 117.8, 117.1, 114.8,
114.4, 112.1, 108.3, 83.1, 78.5, 73.2, 56.0, 55.9, 49.6, 46.1, 27.6, 27.5,
27.4, 21.4, 12.7; HRMS m/z (ESIÀ ) Found: 743.33043, C40H47N4O10
requires [MÀ H]À 743.32977; LRMS (ESIÀ ) 743 ([MÀ H]À , 100%); HPLC
Method A, Retention time – 13.4 min, >99%. *Peak at 163.6 ppm
confirmed by comparison to spectra of related compounds.
(thin film)/cmÀ 1 3340, 3189, 2976, 2930, 1729, 1670, 1607, 1460, 1393,
1323, 1253, 1159, 1137, 1117; H NMR (500 MHz, D6-DMSO) δH 8.01
1
(ddd, J=8.3, 1.3, 0.6 Hz, 1H), 7.49 (d, J 8.1, 2H), 7.41 (d, J 7.1, 1H), 7.41
(d, J 15.9, 1H), 7.24 (d, J 8.1, 2H), 7.21 (ddd, J 8.3, 7.3, 1.5, 1H), 7.17
(ddd, J 7.3, 7.1, 1.3, 1H), 6.58 (d, J 15.9, 1H), 4.41 (s, 2H), 3.33 (dd, J 8.1,
6.4, 2H), 2.83 (dd, J 8.1, 6.4, 2H), 2.46 (s, 3H), 1.64 (s, 9H), 1.34 (s, 9H);
13C NMR at (126 MHz, D6-DMSO) δC 166.3, 154.5, 149.6, 139.6, 138.3,
135.0, 133.6, 132.9, 129.1, 127.5, 127.1, 122.7, 122.0, 121.8, 117.2, 114.9,
114.4, 83.2, 78.5, 49.6, 46.0, 27.5, 27.4, 22.1, 12.7; HRMS m/z (ESI+)
Found: 556.2780, C31H39N3O5Na requires [M+Na]+ 556.2782; LRMS
(ESI+) 556 ([M+Na]+, 83%), 534 ([M+H]+, 24%), 478 (27%), 378
(15%), 361 (42%), 317 (100%), 300 (56%); HPLC Method A, Retention
time – 11.5 min, >99%.
(E)-N-(4-(3-((4,5-Dimethoxy-2-nitrobenzyl)
oxy)-3-oxoprop-1-en-1-yl)benzyl)-2-(2-methyl-1H-indol-3-yl)
ethan-1-aminium trifluoroacetate (1, Zap-Pano·TFA)
(E)-3-(4-(((2-(2-Methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)
prop-2-enamide (15)
Trifluoroacetic acid (0.20 mL, 20% v/v) was added dropwise to a
rapidly stirred solution of (E)-3-(4-((tert-butyloxycarbonyl-(2-(1-(tert-
butyloxycarbonyl)-2-methyl-1H-indol-3-yl)ethyl)amino)methyl)phenyl)
prop-2-enamide (S1) (15 mg, 0.020 mmol, 1.0 eq) and triisopropylsi-
lane (0.83 μL, 4.0 μmol, 0.2 eq) in dichloromethane (1 mL). The
reaction mixture was stirred for 60 min, until TLC analysis indicated
complete consumption of starting material, then diluted with toluene
(1 mL) and dried by azeotroping with toluene (3×1 mL) in vacuo.
Purification using column chromatography (elution with 1:3:40 H2O:
isopropyl alcohol: ethyl acetate) yielded the title compound (15)
Trifluoroacetic acid (2.7 mL, 20% v/v) was added dropwise to a rapidly
stirred solution of tert-butyl-(E)-3-(2-((tert-butoxycarbonyl)(4-(3-(((4,5-
dimethoxy-2-nitrobenzyl)oxy)amino)-3-oxoprop-1-en-1-yl)benzyl)
amino)ethyl)-2-methyl-1H-indole-1-carboxylate (10) (0.10 g, 0.13 mmol,
1.0 eq) and triisopropylsilane (5.9 μL, 0.027 mmol, 0.2 eq) in dichloro-
methane (13 mL). The reaction mixture was stirred for 75 mins, until
TLC analysis showed complete consumption of starting material. The
reaction mixture was then diluted with toluene (1 mL) and dried by
azeotrope with toluene (3×1 mL) in vacuo. Purification using column
chromatography (elution with 1:3:40 water: isopropanol: ethyl
acetate) yielded the title compound (1) (46 mg, 51%) as a yellow solid.
(7.1 mg, 65%) as a pale-yellow solid. Rf 0.26 (1:2:10 H2O: isopropyl
ĭ
°
°
alcohol: ethyl acetate); mp 82–88 C (from CHCl3); νmax (thin film)/cmÀ 1
Rf 0.26 (1:2:10 water: isopropanol: ethyl acetate); mp 106–110 C
ĭ
(from ethanol); νmax (thin film)/cmÀ 1 2967, 2849, 1669, 1626, 1521,
3394, 3196, 2922, 2852, 1665, 1628, 1593, 1461, 1425,1397, 1202, 1181,
1135; H NMR (400 MHz, CD3OD) δH 7.53 (1H, d, J 15.7), 7.50 (2H, d, J
1
1461, 1331, 1277, 1201, 1136, 1068; 1H NMR (500 MHz, CD3OD) δH 7.68
(1H, s), 7.59 (2H, d, J 8.2), 7.59 (1H, d, J 15.8), 7.46 (2H, d, J 8.2), 7.39
(1H, br d, J 7.8), 7.39 (1H, s), 7.24 (1H, br d, J 8.0), 7.02 (ddd, J 8.0, 7.1,
1.0), 6.95 (1H, ddd, J 7.8, 7.1, 1.0), 6.48 (1H, d, J 15.8), 5.31 (2H, s), 4.18
(2H, s), 3.96 (3H, s), 3.88 (3H, s), 3.19–3.16 (2H, m), 3.09–3.06 (2H, m),
2.37 (3H, s); 13C NMR (126 MHz, CD3OD) δC 165.8, 155.0, 149.9, 141.6,
141.4, 137.2, 137.0, 135.1, 133.9, 131.3, 129.5, 129.3, 128.0, 121.8, 119.9,
119.3, 118.0, 112.5, 111.6, 109.2, 106.0, 75.5, 57.0, 56.8, 52.0, 49.0,* 22.5,
11.3; HRMS m/z (ESI+) Found: 545.23854, C30H33N4O6 requires [M+H]+
8.1), 7.38 (1H, ddd, J 7.9, 1.1, 1.0), 7.28 (2H, d, J 8.1), 7.23 (1H, ddd, J
8.1, 1.1, 1.0), 6.99 (1H, ddd, J 8.1, 7.1, 1.1), 6.92 (1H, ddd, J 7.9, 7.1, 1.1),
6.62 (1H, d, J 15.7), 3.80 (2H, s), 2.98–2.90 (2H, m), 2.89–2.80 (2H, m),
2.34 (3H, s); 13C NMR (126 MHz, CD3OD) δC 170.9, 142.2, 140.5, 137.1,
135.6, 133.4, 130.3, 129.7, 129.2, 121.5, 121.5, 119.6, 118.3, 111.4, 108.0,
53.3, 49.9, 24.2, 11.4; HRMS m/z (ESI+) Found: 334.19135, C21H24N3O
requires [M+H]+ 334.19139; LRMS (ESI+) 334 ([M+H]+, 100%), 158
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FULL PAPERS
Dr. K. S. Troelsen, Dr. E. D. D. Calder,
Dr. A. Skwarska, Dr. D. Sneddon,
Prof. E. M. Hammond*, Prof. S. J.
Conway*
1 – 11
Lights on, cancer out: We report the
synthesis and biological evaluation of
a light-activated (caged) prodrug of
the KDAC inhibitor panobinostat
(Zap-Pano). In two cancer cell lines we
show that photolysis of this
of panobinostat. Photolysis of Zap-
Pano causes an increase in H3K9Ac
and H3K18Ac, consistent with KDAC
inhibition, in an oesophageal cancer
cell line (OE21). Irradiation of OE21
cells in the presence of Zap-Pano
results in apoptotic cell death.
Zap-Pano: a Photocaged Prodrug
of the KDAC Inhibitor Panobino-
stat
compound releases panobinostat and
an unexpected carboxamide analogue