Efficient Palladium-Triggered Release of Vorinostat from a Bioorthogonal Precursor

Article abstract of DOI:10.1021/acs.jmedchem.6b01426

Bioorthogonal uncaging strategies have recently emerged as an experimental therapeutic approach to control drug release. Herein we report a novel masking strategy that enables to modulate the metal chelating properties of hydroxamic acid groups by bioorth

Full text of DOI:10.1021/acs.jmedchem.6b01426

Brief Article
pubs.acs.org/jmc
Efficient Palladium-Triggered Release of Vorinostat from a
Bioorthogonal Precursor
Belen Rubio-Ruiz,† Jason T. Weiss,†,‡ and Asier Unciti-Broceta*,†
́
†Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South,
Edinburgh EH4 2XR, U.K.
S
* Supporting Information
ABSTRACT: Bioorthogonal uncaging strategies have recently emerged as an experimental therapeutic approach to control drug
release. Herein we report a novel masking strategy that enables to modulate the metal chelating properties of hydroxamic acid
groups by bioorthogonal chemistry using Pd-functionalized resins. This novel approach allowed to devise an inactive precursor of
the histone deacetylase inhibitor vorinostat that was efficiently uncaged by heterogeneous Pd catalysis in cell culture models of
glioma and lung cancer.
combinations and, potentially, overcome chemoresistance.
Encouraged by this, our lab is exploring new chemistries to
develop novel caged chemotherapeutic agents that are
specifically released by bioorthogonal means, particularly via
heterogeneous Pd catalysis.4−7 To design an efficient
bioorthogonal prodrug, it is fundamental to identify and mask
the drug’s functional group that is most essential for its
cytotoxic mode of action. Importantly, the designated masking
group has to be resistant to metabolic processing and cleavable
by the triggering mechanism of choice at physiological
conditions (water, 37 °C, pH ∼ 7, isotonicity). Although
several bioorthogonal uncaging reactions have been reported in
recent years,12 they all can be grouped in two classes: the
deprotection of carbamate-masked primary amino
groups1−5,13−15 and the N-depropargylation of endocyclic
nitrogen atoms with lactam−lactim tautomerism.4,6,7 As the
cytotoxicity of many therapeutic agents is modulated by other
chemical functionalities, there is a need for an expanded set of
bioorthogonal masking strategies that could be accommodated
to such functional groups and thus increase the diversity of
bioorthogonally activated prodrugs. This is, for example, the
case of hydroxamic acid-based drugs, which are currently used
in the clinic for the treatment of different disorders including
iron-overload disease,16 cancer,17 and infections,18 and whose
pharmacological activity is endowed by the metal-chelating
capacity of their hydroxamic acid group.
INTRODUCTION
■
The activation of prodrugs by benign chemical processes that
are not reliant on intrinsic biological mediators have the
potential to achieve a superior control over the area where
drugs are generated (e.g., tissues, organs, cells), thus minimizing
untoward drug release and consequent adverse effects. The
feasibility of such an approach has been recently demonstrated
in cancer cell culture with two different strategies: metal-free
click-to-release chemistry1−3 and transition metal-mediated
bioorthogonal deprotection chemistry.4−8 The latter strategy
provides a nonenzymatic method to trigger multiple drug-
uncaging cycles per catalyst molecule. It has also been shown
that in vivo surgical insertion of Pd-functionalized implants (=
extracellular heterogeneous catalysts) can be used to mediate
local uncaging of systemically administered drug surrogates in
zebra fish.4,5 As a molecularly targeted version of this strategy,
the use of Ru- and Pd-loaded nanodevices with cell targeting
capabilities has been proposed to track single cancer cells and
individually treat them following administration of transition
metal-activated drug precursors.8−11
One of the distinctive opportunities offered by the
application of bioorthogonal activation methods in cancer
therapy is that it could be exploited to uncage multiple
specifically designed chemotherapy precursors using the same
triggering stimulus, thus enabling the controlled release,
concurrent or successively, of synergistic therapeutics at the
same anatomical area (e.g., inside a tumor where a Pd-
functionalized device has been inserted). Such an approach may
offer a safer way to treat locally advanced cancers through drug
Received: September 28, 2016
Published: October 27, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.6b01426
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
a
Scheme 1. (a) Binding Interactions between 1 and HDAC8 and (b) Proposed Masking Strategy
a
Upon coordination, the pKa of the hydroxamic acid group drops and the OH proton transferred to the imidazole group of His142. The generated
anion further strengthens the coordinative bond between the drug and the Zn2+ atom of the enzyme.25
a
Scheme 2. (a) Synthesis of O-Alkyl SAHA Derivatives 2a−e and (b) FeCl3 Colorimetric Test
a
1 and 2a−e were independently dissolved in a water−ethanol mixture (concentration = 40 mM). Each solution was subsequently mixed with an
equal volume of FeCl3 (80 mM in water−ethanol) and imaged. C = DMSO + FeCl3.
involved in oncogenesis.19 The characteristic presence of
RESULTS AND DISCUSSION
■
multiple expression defects in transformed cells20 and their
Rationale for Prodrug Design. Histone deacetylases
sensitivity to oxidative damage21 have been proposed as the
(HDACs) belong to a class of enzymes that catalyzes the
ε
removal of acetyl groups from N-acetylated lysine residues of
essential features for the preferential activity of HDAC
DNA-binding proteins called histones, resulting in a tight
inhibitors over cancer cells. Nevertheless, these inhibitors suffer
chromatin structure where gene expression is significantly
repressed.19−22 The antitumor activity of HDAC inhibitors is
mainly a consequence of transcriptional changes in genes
from dose-limiting side effects due to the central role of
HDACs in normal cells.22
B
DOI: 10.1021/acs.jmedchem.6b01426
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Journal of Medicinal Chemistry
Brief Article
Figure 1. Half-log dose response curves and calculated EC50 values of 1 and 2a−e after 5 d incubation with NSCLC A549 cells and glioma U87G
cells.
a
Scheme 3. Pd-Triggered Uncaging of Compound 2e
a
Conditions: (a) Treatment of 2e (4 mM) with Pd resins (10 mg/mL) and FeCl3 (8 mM) in a water/ethanol solution for 24 h resulted in no color
change under these conditions. LCMS proved formation of intermediate 3. (b) Treatment of 2e (0.1 mM) with Pd resins (1 mg/mL) in PBS at 37
°C for 24 h resulted in the formation of 1.
Suberoylanilide hydroxamic acid (SAHA), 1, aka vorinostat,
was the first HDAC inhibitor to be approved by the FDA to
treat cancer.21−25 It is currently indicated for the treatment of
cutaneous T cell lymphoma and under clinical investigation for
recurrent glioblastoma multiforme and advanced nonsmall-cell
lung carcinoma (NSCLC). X-ray analysis24 determined that the
main binding interactions between vorinostat and its target are
mediated through the hydroxamic acid group, which doubly
coordinates to a zinc atom at the catalytic site of the enzyme
(Scheme 1a).25
On the basis of this mode of binding, alkylation of the OH
group of the hydroxamic acid (Scheme 1b) is expected to result
in a significant reduction of its chelating properties and,
consequently, of the capacity of 1 to inhibit its target(s). In
addition, we envisaged that, as opposed to carboxylic esters and
amides, O-alkyl hydroxamates would be resistant to enzymatic
hydrolysis, as neither esterases nor amidopeptidases should
recognize this functional group. Consequently, this straightfor-
ward derivatization of bioactive hydroxamic acids could in
principle facilitate the generation of derivatives that are both
pharmacologically inactive and metabolically stable, two
essential features for the successful generation of bioorthogonal
prodrugs.
was prepared by direct treatment of 1 with the corresponding
alkyl bromides, including propargyl and 4-(propargyloxy)benzyl
bromides, and DBU (Scheme 2a).
Study of the Iron Chelating Capacity of Compounds 1
and 2a−e. A remarkable feature of hydroxamic acids is their
ability to form strongly colored coordination complexes with
ferric ions.16 Such a capacity should be either lost or greatly
diminished in compounds 2a−e. As the chelating properties of
1 are essential for its bioactivity, this reaction was used as a
colorimetric test to measure the efficacy of the masking
strategy. As expected, treatment of 1 with a solution of FeCl3
(water/ethanol solution) produced a dark-violet mixture
(Scheme 2b), proving its potent affinity for ferric cations
(siderochrome). On the contrary, 2a−e did not induce any
color change relative to the negative control (DMSO + FeCl3),
even at high concentrations (20 mM), thus indicating that the
chelating properties of these derivatives are abolished by the
alkyl promoiety attached to the hydroxamic OH group. Because
of the striking differences between the coordination properties
of 1 and 2a−e, we conveniently used a FeCl3 solution as a TLC
staining reagent during synthesis.
Bioorthogonality Study. Along with rendering the drug
precursor responsive to a specific triggering stimulus, an
optimal prodrug design should be capable of reducing a drug’s
bioactivity at significant levels, preferably 2 orders of
magnitude.4 To determine the bioorthogonality5 of the O-
alkyl hydroxamates in cancer cells, dose response studies were
performed for 1 and 2a−e in NSCLC A549 cells and glioma
U87G cells. As shown in Figure 1, O-alkyl SAHA derivatives
2a−e mediated patently lower cytotoxicity than the parent drug
Synthesis of Pd-Activated Prodrugs 2a−e. We and
others have shown that propargyl groups attached to
functionalities bearing moderately acidic hydrogen atoms
(pKa ∼ 9), such as aromatic δ-lactams4,6,7 and phenolic
alcohols,26,27 are readily cleaved by Pd species in biocompatible
environs. Because the reported pKa of aliphatic hydroxamic acid
groups is 9.4,25 a selection of O-alkylated hydroxamates of 1
C
DOI: 10.1021/acs.jmedchem.6b01426
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Journal of Medicinal Chemistry
Brief Article
1 in both cell lines, highlighting the low activity observed for
derivative 2e, with a projected EC50 (2e)/EC50 (1) value far
beyond 2 orders of magnitude. Interestingly, the difference in
activity between the O-propargyl derivative 2b and 1 was more
modest (EC50 (2b)/EC50 (1) = 20−30), which might be due to
limited intracellular stability. To the best of our knowledge, this
study is the first to show a qualitative correlation between the
metal chelating properties of the hydroxamic acid group and
the antiproliferative activity of the corresponding derivative and
further corroborates the role played by such a functional group
in the bioactivity of 1.
Prodrug Activation Studies: In Vitro and in Cell
Culture. The Pd-mediated release of 1 from nonchelating O-
alkyl SAHA derivatives 2a−e was tested next. FeCl3 was mixed
with each derivative and treated with a heterogeneous Pd
catalyst (Pd-resins containing 4.62% w/w in Pd0 nanoparticles
captured in a PEG-grafted polystyrene matrix of 150 μm in
diameter4). As shown in Supporting Information (SI), Figure
S1, no color change (= no release of 1) was observed after
incubating the benzyl derivative 2d with Pd resins for 24 h. In
contrast, the solution of the propargyl and 1-butyn-3-yl
derivatives 2b,c turned to violet just after 3 h and the color
intensity increased with time, suggesting formation of 1, while
the allyl derivative 2a showed much lower sensitivity to Pd
resins. Treatment of 2e and Pd resins did not result in color
variation, although discoloration of the mixture suggested that a
reaction did occur. Rather than due to the inability of Pd to
cleave the O-propargyl group, we presumed that the lack of
color change was due to the inhibited elimination of the self-
immolative linker of the depropargylated intermediate 3 under
the reaction conditions (Scheme 3, conditions a), a process that
is expected to be influenced by the pH.28 This was confirmed
by LCMS (SI, Figure S2).
neous Pd-mediated prodrug activations.4,6,7 Reaction of 2e with
Pd resins in mildly acidic media (pH = 6.0) resulted in the
deceleration, but not inhibition, of the pH-dependent 1,6-
elimination step (SI, Figures S8,S9). Interestingly, the acidic
environment showed no effect over the kinetics of the O-
depropargylation reaction. Because the 4-hydroxybenzyl motif
can be found in natural compounds such as the amino acid
tyrosine, a residue that is involved in the regulation of many
biochemical processes including ligand−receptor recognition
events or auto/trans-phosphorylation reactions, the scope of
the O-propargylation method herein described should be
extendable to additional applications of interest in medicinal
chemistry and chemical biology such as the bioorthogonal
regulation14 of peptides and proteins’ bioactivity (e.g., kinase
activity) by Pd chemistry in physiological environments.
Next, the Pd-triggered release of 1 from 2a−e was
investigated in cancer cell culture using A549 and U87G cells
as cancer cell models. Cells were treated with Pd resins (1 mg/
mL) and 2a−e (100 μM) separately (negative controls) or in
combination (activation assay) and unmodified 1 (100 μM)
used as the positive control.
As shown in Figure 2, 2a,d showed low or null bioactivity
regarless of the presence and absence of Pd resins. Interestingly,
We then investigated the deprotection of derivatives 2b,c and
2e in the presence of Pd resins under physiological conditions,
i.e., 37 °C in PBS (pH = 7.2−7.4). Reactions were monitored
for 24 h by HPLC (UV detector). In agreement with the
colorimetric tests, 2b,c rapidly reacted with Pd resins under
these conditions. However, the reactions did not only result in
the generation of 1. The disappearance of 2b,c and the
formation of two uncharacterized products were observed just
after 3 h, with 1 being a minor product of the reaction (see SI,
Figures S3,S4). Remarkably, reaction of 2e with Pd resins under
such conditions (Scheme 3, conditions b) resulted in the
quantitative generation of 1 (SI, Figure S5). As shown in SI,
Figure S6, most of 2e was cleaved after 3 h and two new species
appeared in the chromatogram: O-(4-hydroxybenzyl) SAHA, 3,
and 1 (identified by LCMS, SI, Figure S5). After 6 h
incubation, the major product was 1, showing that the self-
immolative linker of 3 is labile at physiological pH. This is
noteworthy because it represents the first successful use of a
self-immolative linker capable of undergoing a 1,6-benzyl
elimination as a masking strategy for hydroxamic acids. In
fact, previous work from Cohen and co-workers reported that
O-hydroxybenzyl benzohydroxamate (an aromatic hydroxamic
acid) is stable in water (HEPES buffer, pH = 7.5),29 which may
have discouraged further investigations in this direction.
The disappearance in absorbance of 2e at 254 nm at different
time points was used to make a kinetics plot (SI, Figure S7)
and to calculate the rate constant of the O-depropargylation
Figure 2. Antiproliferative activity of 2a−e in A549 cells after
incubation with or without Pd resins for 5 d. Controls: untreated cells
(dark green), cells treated with Pd resins (light green) or 1 (red).
2b and 2c reduced the viability of cancer cells in combination
with the Pd source, indicating that the unknown products
formed in the process might also possess bioactivity. As
expected (see Figure 2), 2e elicited a potent cytotoxic effect
only in the presence of the heterogeneous catalyst (=
implantable device). The observed antiproliferative effect was
equivalent to that mediated by 1, suggesting that the active drug
is rapidly released in standard cell culture conditions. These
results were further corroborated by time lapse imaging using
an IncuCyte ZOOM device. As shown in Figure 3a,b, cells
incubated with either 2e or Pd resins showed a growth curve
equal to that of untreated cells (DMSO), whereas the 2e/Pd
resins combination (in blue) displayed a cytotoxic effect
identical to that of cells incubated with the parental drug (in
red).
Dose response study of 2e alone or in combination with Pd
resins demonstrated concentration dependent cytotoxic effect
in the presence of the catalyst, with complete innocuity in its
absence (Figure 3c and SI, Figure S10).
Target Engagement Study. In situ formation of 1 was
verified by studying intracellular target displacement of the
fluorescent HDAC inhibitor coumarin−SAHA, 4 (Figure 4a).
As reported by Srivastava et al.,31 the blue-emitting fluorescent
properties of this closely related analogue of 1 are quenched in
cells after binding to HDACs. Competitive displacement from
reaction in physiological conditions: 3.8 × 10−4 M−1 s−1 (t1/2
=
30 min), which is slightly slower than classical homogeneous
CuAAC reactions30 but much faster than previous heteroge-
D
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Figure 3. (a) Real-time growth curves of A549 cells under treatment for 5 d. Drug (1)/prodrug (2e) concentrations: 100 μM. Cell growth was
monitored using an IncuCyte system in an incubator (5% CO2, 37 °C). Error bars: SD from n = 3. (b) Images of A549 cells after 5 d treatment
with (from left to right) 1 mg/mL of Pd resins, 100 μM of 2e, 100 μM of 1, or combination of both. Pd resins are identified with arrows. Scale bar:
300 μm. (c) Half-log dose response study (1−100 μM) of 2e cytotoxic activity in the presence and absence of Pd resins in A549 cells. Controls:
untreated cells (dark green), cells treated with Pd-resins (light green), or 1 (red). Cell viability was measured at day 5 using PrestoBlue reagent. Error
bars: SD from n = 3.
Figure 4. (a) Fluorescent assay designed to prove the in situ synthesis of 1 from 2e via Pd chemistry. Upon release of 1, quenched compound 4 is
competitively displaced from the catalytic site of the enzyme, resulting in a fluorescent signal (λex/em = 405/450 nm). (b) Mean fluorescence of A549
cells analyzed by flow cytometry after treatment with 4 (6 h) followed by addition of either DMSO, 1, 2e, or 2e/Pd resin combinations (12 h). Error
bars: SD from n = 3; p < 0.001, *** (ANOVA).
the target’s binding site restores its fluorescent properties and
thus can be used to study specific target engagement. A549 cells
were incubated with 4 (10 μM) for 6 h, followed by addition
(100 μM) of either 1, 2e, or 2e/Pd resins combination and
incubation for 12 h. Cell fluorescence intensity was studied by
flow cytometry (λex/em = 405/450 nm). As shown in Figure 4b,
cotreatment of 4 with nonchelating precursor 2e did not induce
any change in fluorescence emission, thus demonstrating that
this derivative does not interact with HDACs and is
biochemically stable. In contrast, cells pretreated with 4 and
incubated with 2e in the presence of Pd resulted in a highly
significant increase of fluorescence signal, which was equivalent
to the increment observed by direct cotreatment with
unmodified compound 1.
CONCLUSIONS
■
HDAC inhibitors are a distinct class of pharmaceuticals that
target the abnormal epigenetic states found in different
disorders, including cancer. 1 was the first HDAC inhibitor
to reach the clinic for the treatment of cutaneous T cell
lymphoma, and it has shown great promise in other
malignancies.21−25 However, both its systemic side effects
(e.g., hematological problems, cardiotoxicity, etc.) and its poor
pharmacokinetics properties (t1/2 in human serum is 1.5 h)32
have limited its clinical use. Consequently, a number of
research groups have attempted to circumvent the liabilities of
1 by developing prodrugs designed to be activated through a
range of biochemical processes.32−34
Herein we have reported a novel strategy that enabled, for
the first time, to devise a completely inactive precursor of 1 that
is rapidly uncaged by a bioorthogonal activation method in cell
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O-1-Butyn-3-yl Suberoylanilide Hydroxamate (2c). White solid
(32 mg, 45% yield). H NMR (500 MHz, DMSO) δ 10.97 (s, 1H),
models of cancer. The deprotection proceeds via a tandem
mechanism triggered by the Pd-catalyzed depropargylation of a
phenolic ether group and followed by 1,6-elimination of a 4-
hydroxybenzyl group directly attached to the OH of the drug’s
hydroxamic acid group, a previously unreported process that
takes place at physiological pH. While important challenges
remain to be solved before translating this experimental
approach to the clinic (e.g., quantity of Pd required to achieve
bioactive drug concentrations, long-term stability of the catalyst
in vivo, use of extra vs intracellular Pd devices, etc.), extending
the scope of functional groups amenable to activation by Pd
chemistry will provide future therapeutic alternatives upon
which to capitalize once such issues are overcome. Additionally,
because of the versatility of the linker,35 the scope of this
strategy extends beyond Pd and affords manifold opportunities
to modulate the chelating/bioactive properties of hydroxamic
acids through bioorthogonal and biochemical stimuli.
1
9.82 (s, 1H), 7.58 (d, J = 7.6 Hz, 2H), 7.27 (m, 2H), 7.01 (t, J = 7.4
Hz, 1H), 4.61 (q, J = 6.5 Hz, 1H), 3.51 (d, J = 1.6 Hz, 1H), 2.28 (t, J =
7.5 Hz, 2H), 1.98 (t, J = 7.3 Hz, 2H), 1.57 (m, 2H), 1.49 (m, 2H),
1.34 (d, J = 6.7 Hz, 3H), 1.27 (m, 4H). 13C NMR (126 MHz, DMSO)
δ 171.2, 169.6, 139.3, 128.6 (2 × CH), 122.9 (CH), 119.0 (2 × CH),
83.0 (C), 76.6 (CH), 69.4 (CH), 36.3 (CH2), 32.1 (CH2), 28.4
(CH2), 28.3 (CH2), 25.0 (CH2), 24.8 (CH2), 19.9 (CH3). HRMS
(ESI) m/z [M + Na]+ calcd for C18H24O3N2Na, 339.1679; found,
339.1682.
O-Benzyl Suberoylanilide Hydroxamate (2d). White solid (21.5
mg, 27% yield). 1H NMR (500 MHz, DMSO) δ 10.91 (s, 1H), 9.82 (s,
1H), 7.58 (d, J = 7.6 Hz, 2H), 7.38 (d, J = 4.4 Hz, 4H), 7.34 (m, 1H),
7.27 (m, 2H), 7.01 (t, J = 7.4 Hz, 1H), 4.77 (s, 2H), 2.28 (t, J = 7.4
Hz, 2H), 1.94 (t, J = 7.3 Hz, 2H), 1.57 (m, 2H), 1.49 (m, 2H), 1.26
(m, 4H). 13C NMR (126 MHz, DMSO) δ 171.2, 169.3, 139.3, 136.1,
128.7, 128.6 (2 × CH), 128.2 (2 × CH), 128.2 (2 × CH), 122.9
(CH), 119.0 (2 × CH), 76.7 (CH2), 36.3 (CH2), 32.2 (CH2), 28.4
(CH2), 28.3 (CH2), 25.0 (CH2), 24.8 (CH2). HRMS (ESI) m/z [M +
Na]+ calculated for C21H26O3N2Na, 377.1835; found, 377.1836.
O-(4-Propagyloxybenzyl) Suberoylanilide Hydroxamate (2e).
EXPERIMENTAL SECTION
■
General. Chemicals were purchased from Fisher Scientific, Sigma-
Aldrich, or VWR. 1 and 3 were purchased from Cayman Chemical
Company. NMR spectra were recorded at rt on a 500 MHz Bruker
Avance III spectrometer. Chemical shifts are reported in ppm relative
to the solvent peak. High resolution mass spectrometry was measured
in a Bruker MicrOTOF II. Analytical TLC was performed using Merck
TLC Silica Gel 60 F254 plates and visualized by UV light. Purifications
were carried out by flash column chromatography using commercially
available silica gel (220−440 mesh, Sigma-Aldrich). All compounds
used in the biological experiments were >95% pure, as measured by
HPLC using an UV−vis 254 nm detector. Method: eluent A, water
and trifluoroacetic acid (0.4%); eluent B, acetonitrile, A/B = 95:5 to
20:80 in 6 min, isocratic 1 min, 20:80 to 95:5 in 0.1 min, and isocratic
2 min. Stock solutions (100 mM) were prepared in DMSO.
Synthesis of 4-(Propargyloxy)benzyl Bromide. 4-Hydroxyl-
benzyl alcohol was treated with propargyl bromide and K2CO3 to give
rise 4-(propargyloxy)benzyl alcohol, which was then converted in the
corresponding halogenated intermediate using CBr4/PPh3 (67%, 2
steps).36
1
White solid (30 mg, 32% yield). H NMR (500 MHz, DMSO) δ
10.86 (s, 1H), 9.82 (s, 1H), 7.58 (d, J = 7.7 Hz, 2H), 7.32 (d, J = 8.6
Hz, 2H), 7.27 (m, 2H), 7.01 (t, J = 7.4 Hz, 1H), 6.98 (d, J = 8.6 Hz,
2H), 4.79 (d, J = 2.4 Hz, 2H), 4.70 (s, 2H), 3.55 (t, J = 2.4 Hz, 1H),
2.28 (t, J = 7.4 Hz, 2H), 1.94 (t, J = 7.3 Hz, 2H), 1.57 (m, 2H), 1.49
(m, 2H), 1.27 (m, 4H). 13C NMR (126 MHz, DMSO) δ 171.2, 169.3,
157.2, 139.3, 130.4 (2 × CH), 128.8, 128.6 (2 × CH), 122.9 (CH),
119.0 (2 × CH), 114.6 (2 × CH), 79.2, 78.2 (CH), 76.3 (CH2), 55.4
(CH2), 36.3 (CH2), 32.2 (CH2), 28.4 (CH2), 28.3 (CH2), 25.0 (CH2),
24.8 (CH2). HRMS (ESI) m/z [M + Na]+ calculated for
C24H28O4N2Na, 431.1941; found, 431.1949.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01426.
General Method for the Synthesis of 2a−e. Vorinostat, 1 (60
mg, 0.23 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.27 mmol)
were dissolved in dry acetonitrile (1 mL) under N2 atmosphere and
cooled to 4 °C. Either allyl bromide, propargyl bromide, 3-bromo-1-
butyne, benzyl bromide, or 4-(propargyloxy)benzyl bromide (0.23
mmol) were dissolved in dry acetonitrile (0.5 mL). The solution was
added dropwise to the mixture and the resulting mixture stirred at
room temperature for 24 h. Solvent was then removed under reduced
pressure and the crude purified via flash chromatography eluting with
ethyl acetate/hexane (2:1).
NMR spectra of derivatives 2a−e, in vitro conversion
analyses, biological methods (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: 0044 1316518702. Fax: 0044 1317773520. E-mail:
Asier.Unciti-Broceta@igmm.ed.ac.uk.
■
O-Allyl Suberoylanilide Hydroxamate (2a). White solid (28 mg,
41% yield). H NMR (500 MHz, DMSO) δ 10.85 (s, 1H), 9.82 (s,
Present Address
1
‡For J.T.W.: Centre for Innovative Cancer Therapeutics,
Ottawa Hospital Research Institute, Ottawa, Ontario K1H
8L6, Canada.
1H), 7.58 (d, J = 7.6 Hz, 2H), 7.27 (m, 2H), 7.01 (t, J = 7.4 Hz, 1H),
5.90 (ddt, J = 16.6, 10.5, 6.0 Hz, 1H), 5.26 (m, 2H), 4.25 (d, J = 6.0
Hz, 2H), 2.28 (t, J = 7.4 Hz, 2H), 1.93 (t, J = 7.3 Hz, 2H), 1.57 (m,
2H), 1.48 (m, 2H), 1.27 (m, 4H). 13C NMR (126 MHz, DMSO) δ
171.2, 169.2, 139.3, 133.2 (CH), 128.6 (2 × CH), 122.9 (CH), 119.0
(2 × CH), 118.9 (CH2), 75.8 (CH2), 36.3 (CH2), 32.2 (CH2), 28.4
(CH2), 28.3 (CH2), 25.0 (CH2), 24.8 (CH2). HRMS (ESI) m/z [M +
H]+ calcd for C17H25O3N2, 305.1859; found, 305.1856.
O-Propargyl Suberoylanilide Hydroxamate (2b). White solid (27
mg, 40% yield). 1H NMR (500 MHz, DMSO) δ 11.09 (s, 1H), 9.82 (s,
1H), 7.58 (d, J = 7.6 Hz, 2H), 7.27 (m, 2H), 7.01 (t, J = 7.4 Hz, 1H),
4.43 (d, J = 2.4 Hz, 2H), 3.55 (t, J = 2.4 Hz, 1H), 2.28 (t, J = 7.5 Hz,
2H), 1.96 (t, J = 7.3 Hz, 2H), 1.57 (m, 2H), 1.49 (m, 2H), 1.28 (m,
4H). 13C NMR (126 MHz, DMSO) δ 171.2, 169.5, 139.3, 128.6 (2 ×
CH), 122.9 (CH), 119.0 (2 × CH), 78.9, 78.4 (CH), 62.3 (CH2), 36.3
(CH2), 32.1 (CH2), 28.4 (CH2), 28.3 (CH2), 25.0 (CH2), 24.7 (CH2).
HRMS (ESI) m/z [M + H]+ calcd for C17H23O3N2, 303.1703; found,
303.1693.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare the following competing financial
interest(s): The authors declare that a patent application (GB
1608716.5) is filed on compound 2e.
ACKNOWLEDGMENTS
■
́
We are grateful to the Alfonso Martin Escudero Foundation
(B.R.-R.), the EC (H2020-MSCA-IF-2014-658833, Chemo-
BOOM), and the EPSRC (EP/N021134/1) for financial
support.
F
DOI: 10.1021/acs.jmedchem.6b01426
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
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ABBREVIATIONS USED
■
CuAAC, copper(I)-catalyzed alkyne−azide cycloaddition;
DBU, 1,8-diazabicyclo(5.4.0)undec-7-ene; DMSO, dimethyl
sulfoxide; EC50, half-maximal effective concentration; HDAC,
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carcinoma; PBS, phosphate buffered saline; PEG, polyethylene
glycol; ppm, parts per million; rt, room temperature; SAHA,
suberoylanilide hydroxamic acid; SD, standard deviation; TLC,
thin layer chromatography
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DOI: 10.1021/acs.jmedchem.6b01426
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