Pojamide: An HDAC3-Selective Ferrocene Analogue with Remarkably Enhanced Redox-Triggered Ferrocenium Activity in Cells

Article abstract of DOI:10.1021/acs.organomet.7b00437

A ferrocene containing o-aminoanilide, N¹-(2-aminophenyl)-N⁸-ferrocenyloctanediamide (2b, Pojamide) displayed nanomolar potency vs HDAC3. In comparison to RGFP966, a potent and selective HDAC3 inhibitor, Pojamide displayed superior a

Full text of DOI:10.1021/acs.organomet.7b00437

Article
pubs.acs.org/Organometallics
Pojamide: An HDAC3-Selective Ferrocene Analogue with Remarkably
Enhanced Redox-Triggered Ferrocenium Activity in Cells
,
†,#
‡
†
‡,#,∇
Rhiannon Jones, Justin M. Roberts, Thomas G. Scott,§
‡
§
Cory A. Ocasio,*
Supojjanee Sansook,
∥
∥
⊥
⊥
Nikolaos Tsoureas, Peter Coxhead, Matthew Guille, Graham J. Tizzard, Simon J. Coles,
Helfrid Hochegger, James E. Bradner, and John Spencer*
§
,‡
†
Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, U.K.
Department of Chemistry, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QJ, U.K.
‡
§
Department of Medical Oncology, Dana-Farber Cancer Institute, 360 Longwood Avenue, Boston, Massachusetts 02215, United
States
∥
School of Biological Sciences, University of Portsmouth, King Henry Building, Portsmouth PO1 2DT, U.K.
⊥
UK National Crystallography Service, School of Chemistry, University of Southampton, Highfield Campus, Southampton SO17 1BJ,
U.K.
*
S Supporting Information
1
ABSTRACT: A ferrocene containing o-aminoanilide, N -(2-
8
aminophenyl)-N -ferrocenyloctanediamide (2b, Pojamide)
displayed nanomolar potency vs HDAC3. In comparison to
RGFP966, a potent and selective HDAC3 inhibitor, Pojamide
displayed superior activity in HCT116 colorectal cancer cell
invasion assays; however, TCH106 and romidepsin, potent
HDAC1 inhibitors, outperformed Pojamide in cellular
proliferation and colony formation assays. Together, these
data suggest that HDAC1,3 inhibition is desirable to achieve
maximum anticancer benefits. Additionally, we explored
Pojamide-induced redox pharmacology. Indeed, treating
HCT116 cells with Pojamide, SNP (sodium nitroprusside),
and glutathione (GSH) led to greatly enhanced cytotoxicity
and DNA damage, attributed to activation to an Fe(III) species.
INTRODUCTION
of HDAC inhibition in colorectal carcinomas and possibly
other cancer cells.
■
Histone deacetylases (HDACs) are key targets in cancer and
1
neurodegeneration. Their overexpression leads to increased
RESULTS AND DISCUSSION
deacetylated lysine/arginine levels and a condensed chromatin
state, resulting in transcriptional silencing. Clinically useful
■
Transition-metal-based anticancer agents are interesting due to
their novel ligand exchange and redox chemistry, the ability of a
heavy metal atom to facilitate phasing in protein X-ray
crystallography, and the availability of geometries and oxidation
2
HDAC inhibitors (HDACis) include SAHA (Figure 1) and
3
romidepsin, which trigger growth arrest and apoptosis via
histone hyperacetylation by pan-inhibition principally of class I
and II HDACs and other targets (e.g., p53).
1
0−13
states unachievable with carbon-based therapeutics.
Our increased knowledge of HDAC biology has emphasized
Ferrocene-based JAHAs and other metal-based analogues,
many containing a hydroxamic acid zinc-binding group
the need for isoform-selective histone deacetylase inhibitors
4
(
HDACis). Selective inhibitors of HDAC3 (e.g., the
(ZBG), are effective HDACis with good activity vs class I
5
,6
14−25
benzamides TCH106 and RGFP966) (Figure 1)
are
HDACs.
Guided by docking studies of a standard “cap-
7
8
particularly attractive in CNS applications and cancer.
However, recent studies using HDAC3-overexpressing
HCT116 cells show that silencing individual HDACs
linker-ZBG” arrangement, we wished to extend the chemistry
of JAHAs to o-anilide analogues, anticipating that this may lead
to HDAC3 selectivity and alleviate toxicity issues documented
26
for hydroxamate ZBGs. Hence, compounds 1a and 2a were
(
HDAC1−3) through RNAi is insufficient to achieve similar
readily made by standard coupling reactions and were
levels of growth arrest and apoptosis induced by generic
9
HDACis such as Trichostatin A. It is therefore likely that the
inhibition of specific combinations of HDAC isoforms,
HDAC1−3 in particular, may help to achieve the full benefits
Received: June 12, 2017
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.organomet.7b00437
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of HDAC3 bound to corepressor and inositol tetraphosphate
29
(
Figure 3A,B). We found that Pojamide bound to the zinc
active site, forming hydrogen bonds between the N−H of the
amide and the carbonyl of Asp93, the benzamide amide N−H
and the carbonyl of Gly143, the aniline NH and the nitrogen
2
of His134, and the carbonyl of Asp170 as well as the
characteristic benzamide−zinc interaction.
The docked structure of 1b is docked slightly shifted from
Pojamide. It forms hydrogen bonds with Asp93 and Gly143.
However, the substitution of the six-carbon aliphatic chain for a
four-carbon chain results in a rotation of the benzamide group,
leading to a loss of key interactions, namely, hydrogen bonding
with His134 and Asp170, as well as forcing zinc coordination
from the benzamide aniline to the amide carbonyl (Figure
3
A,B). Comparison of the active sites of HDAC3,8 reveals that
ferrocenyl substitution for the archetypal aryl cap in HDAC
inhibitors in Pojamide clashes with Tyr100 and Lys33 in
HDAC8, assuming a binding mode similar to that of Pojamide
in HDAC3. Thus, it is assumed that ferrocenyl substitution in
Pojamide and 1b is responsible for HDAC3 selectivity (Figure
3
C).
In addition to structural evidence supporting selective
Figure 1. SAHA and known HDACis.
HDAC3 binding, we confirmed Pojamide’s cell permeation
and HDAC3-specificity using Xenopus laevis embryos (2-cell to
stage 14) as a model system for deacetylase activity. Xenopus
laevis embryos were incubated with 1b and Pojamide in order
to test their cellular permeability, and JAHA, a broad HDAC
characterized in the solid state by X-ray crystallography (Figure
2
7
2).
30
inhibitor, was used as a positive control. As expected,
Pojamide and 1b, which have some selectivity toward HDAC3
(
vide infra), did not affect α-tubulin acetylation, whereas JAHA
increased acetylation of α-tubulin (Figure S1A in the
Supporting Information). Acetylated H4K12 (H4K12ac) has,
however, been shown to be a target of HDAC3 and was
expected to increase if these HDAC3is were able to function in
31
the whole organism. Compound 1b gave no sign of affecting
H4K12 acetylation levels, unlike Pojamide, which demonstrated
a concentration-dependent accumulation of H4K12ac (Figure
S1B). In three separate experiments the level of H4K12ac, as
detected by Western blotting, increased in developing embryos
treated with Pojamide; however, embryo development was
severely affected and many died (as low as 10% survival rates).
For this reason, it was impossible to obtain a clear
concentration dependence for Pojamide; nonetheless, we
conclude that Pojamide is highly likely to be cell permeable
as an HDAC3 inhibitor but that compound 1b is not as good
an inhibitor.
Figure 2. Ferrocene-based HDACis and precursors.
To explore the binding modes of 1b and 2b (Pojamide),
which were formed by deprotection of 1a and 2a respectively,
in HDAC3, we performed docking studies using the structure
Figure 3. (A, B) Docking poses of 1b and Pojamide in HDAC3 (PDB code 4A69): top docking poses of 1b (teal, A) and Pojamide (slate, B) in
2
8
HDAC3. (C) Superposition of the active sites of HDAC3 (gray) and HDAC8 (PDB code 1T69, green) showing key residues overlaid with
Pojamide top docking pose in HDAC3. Color scheme: hydrogen bonds shown by green dashed lines, π−π interactions shown by orange dashed
lines.
B
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Next, we tested Pojamide on a panel of HDACs vs 1b,
SAHA, and HDAC3is (Table 1). Indeed both 1b and
formation at 10 μM in HCT116 colony formation assays in
comparison to 75 and 60% inhibition of colony formation by
Pojamide and RGFP966, respectively (Figure 5A−E and Figure
S2A−C in the Supporting Information). Interestingly, TCH106
is 1.6- and 2.8-fold more potent toward HDAC1 inhibition than
RGFP966 and Pojamide, respectively (Table 1) and proliferat-
ing RPE cells have been shown to overexpress HDAC 1, 2, and
32
Table 1. Biochemical Evaluation of HDAC Isoforms 1−8
IC50 (μM)
a
a
b
HDAC
SAHA
1b
2b
RGFP966 TCH106
37
1
2
3
4
5
6
7
8
0.006 ± 0.001
0.016 ± 0.001
0.008 ± 0.001
19 ± 1
9.7 ± 0.1
0.030 ± 0.001
>30
8.94
13.79
0.606
>30
1.10 ± 0.14
1.3 ± 0.4
0.09 ± 0.02
>30
0.61
0.58
0.015
>30
>30
>30
>30
0.39
1.2
5, suggesting that HDAC1 inhibition is in part responsible for
the antiproliferative effects of Pojamide and TCH106 in these
cell lines. In fact, cellular proliferation and colony formation
assays using romidepsin, a potent and exquisitely selective
HDAC1,2 inhibitor (reported IC values of 36 and 47 nM,
0.05
>30
>30
>30
>30
>30
>30
>30
5
0
38
>30
>30
respectively ), revealed a GC value of 0.52 ± 0.02 nM and
50
>30
>30
near-complete inhibition of colony formation at 0.5 nM, clearly
the most potent antiproliferative HDACi tested in this study
0.36 ± 0.10
>30
>30
>30
a
b
(
Figure 5F and Figure S3A,B in the Supporting Information).
Profiling done in duplicate, n = 8; all others n = 4. This slow, tight-
binding inhibitor, with inverted IC50 and K values, causes the IC
HeLa and HT-29 cells were only mildly inhibited by Pojamide
and RGFP966, showing maximum growth inhibition of ∼40
and 30%, respectively, at 10 μM, and in all cases compound 1b
was ineffective at inhibiting cellular proliferation (Figure 5A−
D). This result was mirrored in Xenopus laevis embryo
development assays, whereby Pojamide caused a concentra-
tion-dependent increase in acetylated H4K12 levels and 1b did
not (Figure S1 in the Supporting Information).
i
50
6
value to drop over longer preincubation periods. The IC50 value was
defined as the amount of compound that caused 50% reduction in
HDAC activity in comparison with DMSO-treated control and was
calculated using GraphPad Prism version 6 software.
Pojamide displayed ca. 11- and 22-fold selectivity, respectively,
toward HDAC3 over HDAC1,2 and significantly greater
selectivity over HDAC4−8. Pojamide’s profile was on par
with other o-anilide (benzamide) inhibitors, being most similar
to that of TCH106, yet was outperformed by RGFP966 with
respect to HDAC3 selectivity. Only SAHA displayed activity vs
HDAC8 (Table 1).
To establish the anti-invasive properties of Pojamide against
known HDACis, we investigated the effects of RGFP966,
TCH106, and Pojamide on HCT116 cellular invasion. In this
assay, we decided to test compounds at 1× and 0.1× of their
GC value determined using the HCT116 cellular proliferation
50
With in vitro validation of the anti-HDAC3 activity of these
ferrocene analogues, we sought to explore their inhibitory
activity in HDAC3-overexpressing cervical and colorectal
cancer cell lines. HeLa, HT-29, and HCT116 cells are tumor-
forming cell lines that have been used previously as model
assay. At the lowest concentrations tested, only Pojamide
demonstrated robust inhibition of invasion, with about 70%
inhibitory activity; however, at the 1× concentrations both
RGFP966 and Pojamide, exhibiting ca. 40 and 11-fold
selectivity for HDAC3 inhibition, respectively, in comparison
to HDAC1 (Table 1), inhibited invasion by about 90% in
comparison to 70% for TCH106, which showed only 7-fold
selectivity for HDAC3 vs HDAC1 inhibition (Figure 5G−I).
On the basis of these data and taking into consideration the
activity of these compounds in the cellular proliferation assay,
we conclude that HDAC isoform synergistic effects can be
exploited using HDAC1- and HDAC3-selective HDACis,
proliferation being attenuated more so by HDAC1, and
possibly HDAC2, and inhibition and invasion blocked more
robustly through HDAC3 inhibition.
9
,33−35
systems to characterize HDACis.
We confirm in this
study that in comparison to hTERT immortalized retinal
pigment epithelium (RPE) cellscells with a longer lifespan,
36
but incapable of forming tumors the malignant cancer cell
lines HeLa, HT-29, and HCT116 all showed significantly
elevated HDAC3 expression levels (Figure 4).
Pojamide appears more efficacious in preventing HCT116
cellular invasion, particularly at lower concentrations (i.e., 1
μM). In addition, we wanted to explore the possibility that
engaging a different oxidation state of the iron atom in the
ferrocene moiety might offer a unique advantage in targeting
cancer cells. Seminal studies with Ehrlich ascites tumor (EAT)
Figure 4. Western blot analysis of HDAC3 and α-tubulin (Tub) in
Retinal Pigment Epithelium (RPE), HeLa, HT-29 and HCT116 cells.
cells and HPB (human leukemic T lymphocytes) showed that
III
On the basis of these data, this small panel of cell lines was
treated with increasing concentrations of 1b, Pojamide, and the
control HDACis RGFP966 and TCH106. Despite RGFP966
showing 6.5-fold greater potency than Pojamide in blocking
HDAC3 activity (Table 1), we found that RGFP966 and
Pojamide are equipotent at inhibiting HCT116 cellular
proliferation, displaying GC50 values of 8.9 and 8.6 μM,
respectively (Figure 5A and Table 2). RPE cells, on the other
hand, were as sensitive to Pojamide as HCT116 cells but were
not inhibited by RGFP966 (Figure 5B and Table 2). TCH106
displayed superior potency against all cell lines with GC50
values ranging from 1 to 2 μM and completely blocked colony
incubation with ferrocenium salts (e.g., Fe Cp
2
PF
6
) inhibited
tumor growth, whereas their ferrocene counterparts were
ineffective. Indeed, ferrocenium’s toxicity involves the gen-
eration of OH radicals and the rapid induction of DNA
damage, repeated later in MCF7 and MCF10A cells.
In order to take advantage of ferrocenium cytotoxicity, we
generated the ferrocenium species 3b (Fe -Poj) through
standard means by reacting Pojamide with nitrosonium (NO )
tetrafluoroborate (Scheme 1), which was confirmed by cyclic
•
39,40
III
+
41
voltammetry (Figure S4 in the Supporting Information).
Hence, we hypothesized that intracellular generation of NO in
the presence of Pojamide might also lead to Fe -Poj in cells.
+
III
C
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Figure 5. (A) HCT116, (B) RPE, (C) HeLa, and (D) HT-29 CellTiter-Blue proliferation assays. (E) HCT116 colony formation assays. The percent
colony formation (normalized to DMSO control) was quantitated manually and the average ±SD was plotted. (F) Inhibitory activity of romidepsin
on HCT116 cellular proliferation. HCT116 cellular invasion assays in the presence of (G) RGFP966, (H) TCH106, and (I) Pojamide.
Table 2. Cellular Characterization of HDACis
of SNP with GSH to form innocuous NO may further reduce
44
SNP cytotoxicity.
a
GC50 (μM)
After identifying the optimal conditions for SNP/GSH
treatment, we treated HCT116 cells with increasing concen-
trations of inhibitor in the presence and absence of SNP/GSH.
cell line
2b
TCH106
RFP966
na
romidepsin
nd
1b
RPE
9.0 ± 0.7
8.6 ± 1.2
na
∼1
na
na
na
na
b
III
HCT116
HeLa
2.2 ± 0.2
1.5 ± 0.2
∼1
8.9 ± 0.7
na
na
0.52 ± 0.02
nd
nd
The generation of Fe -Poj was monitored by blotting for the
45
DNA-damage marker phospho-γH2AX (pH2AX) (Figure
6A−D). Without SNP/GSH, increasing concentrations of
RGFP966 and Pojamide reduced pH2AX levels, but addition
of SNP/GSH to cells treated with Pojamide led to an initial
decrease in pH2AX, with recurrence of the DNA-damage
marker at concentrations greater than 2 μM (Figure 6A−D).
Over a longer time course (6 days) with 4 μM inhibitor
HT-29
na
a
The GC50 value was defined as the amount of compound that caused
50% reduction in cellular proliferation in comparison with DMSO-
treated control and was calculated using GraphPad Prism version 6
software. na = not applicable and nd = not determined. This GC50
value is in nM.
b
(
±SNP/GSH), only the Pojamide/SNP/GSH combination led
Scheme 1. Synthetic Ferrocenium Pojamides
to significant DNA damage nearly tantamount to the levels of
DNA damage caused by cisplatin (4 μM) (Figure 6E).
To further support that our SNP/GSH treatment in the
III
presence of Pojamide leads to production of Fe -Poj, we
assessed the levels of NAD+ in cells using the NAD/NADH-
Glo assay. Early studies on the one-electron transfer from
III
NADH to ferrocenium oxidants such as Fe Cp PF demon-
2
6
strated that ferrocenium salts can successfully oxidize NADH to
4
6
It has previously been reported that sodium nitroprusside
NAD+ at physiologic pH in a phosphate buffer in vitro. To
recapitulate this conversion in cells, we treated HCT116 cells
with DMSO/SNP/GSH and 2.5 μM Pojamide with and
without SNP/GSH. For each condition, total NAD+/NADH
levels remained the same, but Pojamide in the presence of
SNP/GSH led to an increase in NAD+ levels, similar to those
42
SNP) leads to intracellular NO+ release, yet studies in
(
neuronal PC12 cells have shown that SNP alone triggers
42
apoptosis at concentrations greater than 30 μM. To develop a
+
cell-based assay utilizing SNP as an NO donor, we conducted
cytotoxicity studies using the colony formation assay (Figure
S5A in the Supporting Information). At 25 μM SNP, just shy of
the concentration that triggers apoptosis in PC12 cells,
HCT116 colony formation is reduced by about 30%; however,
addition of 500 μM GSH completely eliminated SNP
cytotoxicity (Figure S5A,B). GSH is a free radical scavenger
and detoxifies H O in a glutathione peroxidase-1 dependent
III
obtained with synthetic Fe -Poj. In contrast, Pojamide
treatment alone decreased NAD+ levels (Figure 6F).
In order to correlate this increase in NAD+ with oxidative
stress, reactive oxygen species (ROS) levels were determined in
HCT116 cells treated with Pojamide, the non-transition-metal-
based HDAC3i RGFP966, and transition-metal-based com-
pounds JAHA and ruthenocene. Indeed, without SNP/GSH,
ferrocene ligands Pojamide and JAHA both caused an induction
in ROS, an effect documented with other ferrocene ligands
such as ferrocifens and aminoferrocene prodrugs, which
2
2
4
3
manner, and its cytoprotective effects were demonstrated in
our assay at concentrations of 50 and 500 μM, whereas GSH
treatments, in the presence of SNP, enhanced colony formation
by about 4 and 10%, respectively (Figure S5A,B). The reaction
D
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Figure 6. Western analysis of pH2AX and α-tubulin (Tub) in HCT116 cells treated for 3 days with (A) RGFP966, (B) Pojamde and (C) Pojamide
SNP/GSH. (D) pH2AX/Tub ratio as determined via densitometry and average ratio normalized to DMSO control plotted as the mean ±SD. (E)
Western analysis of pH2AX and α-tubulin (Tub) in HCT116 cells treated for 6 days as indicated above. NAD+ fold induction (F) and total NAD
/NADH (F) and reactive oxygen species (ROS) (G) levels normalized to the DMSO control. The average (n = 10) was plotted ±SD. The t test
+
+
statistical module of Prism 6.0 was used to determine p values (ns (not statistically significant), P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
Figure 7. (A) HCT116 colony formation assays. (B) Percent colony formation (normalized to DMSO control), quantitated manually with the
average ±SD plotted. The t test statistical module of Prism 6.0 was used to determine p values (ns (not statistically significant), P > 0.05; *, P ≤ 0.05;
*
*, P ≤ 0.01; ***, P ≤ 0.001).
produce quinone methides, and in the latter case, ferrocenium
catalysts for ROS production, in the absence of SNP.
CONCLUSIONS
■
47,48
In summary, the SNP/GSH combination treatment is an ideal
system for increasing the intracellular ferrocenium concen-
tration from a Pojamide precursor. In addition, when Fe -Poj
DMSO, RGFP966, and ruthenocene were assayed and showed
no ROS induction without SNP/GSH. In the presence of SNP/
GSH, ROS levels increased by 2.5-fold with and without
RGFP966 and, interestingly, all other conditions with SNP/
GSH, Pojamide, JAHA and ruthenocene led to greater ROS
levels, with Pojamide displaying the most significant increase,
III
is reduced back to the Fe(II) species, it would be available to
act upon HDACs. Due to having two separate modes of action,
one of which is SNP/GSH-dependent, Pojamide is advanta-
geous in comparison to other similarly potent HDACis: its
cytotoxicity is enhanced by its facile conversion to the cytotoxic
Fe(III) species in cells, while the reduced species inhibits
cellular invasion through potently targeting HDAC3 and
proliferation, to a lesser extent, due to its low micromolar
HDAC1 inhibitory activity (Figure 8). Finally, cotreatments of
3
.3-fold, despite cotreatment with excess GSH (Figure 6G).
With cellular validation of Fe -Poj generation, we tested our
III
system in the HCT116 colony formation assay (Figure 7A,B).
Pojamide alone caused a significant decrease in colony
formation, which was enhanced by addition of SNP/GSH: 27
and 53% inhibition, respectively. At 4 μM, RGFP966, with and
without SNP/GSH, displayed a 10−15% reduction in colony
formation; however, like Pojamide, both ferrocenium salts were
less effective without SNP/GSH. In combination with SNP/
III
III
GSH, Fe -Poj and Fe Cp PF caused a slight, about 10%,
2
6
reduction in colony formation (Figure 7A,B). Interestingly,
colonies resulting from treatment with ferrocenium salts and
SNP/GSH were larger, but overall fewer colonies formed, a
result similar to RGFP966 treatment. Although the cytotoxicity
of ferrocenium salts is well documented, their activity is
39
reduced ∼100-fold in comparison to Pojamide, perhaps due
to limited membrane permeability; this explains their meager
inhibitory activities at the 4 and 20 μM concentrations tested.
Figure 8. Model for the enhanced redox-triggered cytotoxicity of
Pojamide.
E
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intravenous SNP injection along with Pojamide administration
might offer a highly efficacious strategy for managing some
colon carcinomas, a strategy that might have broader,
mL). To this was added triethylamine (0.5 mL, 3.6 mmol), and the
mixture was cooled in an ice bath. Next, propanephosphonic acid
anhydride T3P (50% solution in DMF, 0.59 mL, 0.66 mmol) was
added and the reaction mixture was warmed to room temperature
overnight. Then the mixture was poured into a saturated solution of
generalizable applications when used with pharmacologically
II
distinct Fe Cp -containing drugs (e.g., aminoferrocenes,
2
47−50
K CO , stirred for 30 min, and extracted into CH Cl . The organic
2
3
2
2
ferrocifens, ferroquines).
Current studies are looking at
layer was dried (MgSO ), filtered, and evaporated in vacuo. The
4
ruthenium-based HDACis and will be reported in due course.
residue was purified by trituration with DCM to give the orange solid
(
233.1 mg, 71%). Crystallization by solvent evaporation of DCM
1
EXPERIMENTAL SECTION
provided yellow crystals. H NMR (DMSO-d ): δ 9.40 (1H, s, NH),
9
7
CHAr), 4.55 (2H, s, 2CH (Cp)), 4.06 (5H, s, unsusbt Cp), 3.91 (2H,
s, 2CH (Cp)), 2.33 (2H, t, J = 7.4 Hz, CH ), 2.14 (2H, t, J = 7.4 Hz,
CH ), 1.62−1.54 (4H, m, 2CH ), 1.44 (9H, s, 3CH ), 1.38−1.26 (4H,
m, 2CH
■
6
.15 (1H, s, NH), 8.26 (1H, s, NH), 7.54−7.48 (1H, m, CHAr), 7.42−
.35 (1H, m, CHAr), 7.13−7.09 (1H, m, CHAr), 7.08−7.03 (1H, m,
Solvents and reagents were purchased from commercial suppliers and
were used without purification. Ferrrocenylamine was purchased from
TCI UK and used as such. All reactions were performed in a fume
hood. NMR spectra were recorded on Varian 500 and 400 MHz
spectrometers, and chemical shifts are reported in ppm, usually
referenced to TMS as an internal standard. LCMS were performed
with a Shimadzu LCMS-2020 instrument equipped with a Gemini 5
μm C18 110 Å column, and percentage purities were run over 30 min
in water/acetonitrile with 0.1% formic acid (5 min at 5%, 5%−95%
over 20 min, 5 min at 95%) with the UV detector at 254 nm. High-
resolution mass spectrometry (HRMS) was performed by the EPSRC
National Mass Spectrometry Facility, University of Swansea. Elemental
analyses were conducted by Stephen Boyer (London Metropolitan
University). FT-IR spectra were recorded on a PerkinElmer Spectrum
Version 10.03.06 instrument.
2
2
2
3
13
). C NMR (DMSO-d , 126 MHz): δ 171.6, 171.2, 142.3,
2
6
126.1, 125.7, 124.1, 116.6, 116.3, 96.1, 69.1, 64.1, 61.0, 39.7, 39.5, 36.4,
36.2, 29.0, 28.9, 25.7, 25.6.
1
8
N -(2-Aminophenyl)-N -ferrocenyloctanediamide (2b, Poja-
mide). tert-Butyl-2-(8-oxo-8-(phenylamino)octanamido)ferrocenyl
carbamate (136.8 mg, 0.25 mmol, 1 equiv) was suspended in
dichloromethane (10 mL) and MeOH (1 mL). To this mixture was
added 4 N HCl/dioxane (2 mL), and the mixture was stirred at room
temperature overnight. The volatiles were removed in vacuo, and then
saturated Na CO (aq) was added to the residue and the mixture was
2 3
tert-Butyl-2-(6-oxo-6-phenylamino)hexanamido)ferrocenyl
sonicated. The precipitate was collected by suction and washed on the
frit with water, dried, and triturated with CH Cl to give the title
1
4
Carbamate (1a). 6-Oxo-6-(ferrocenylamino)hexanoic acid (493.6
mg, 1.5 mmol, 1 equiv) and N-Boc-o-phenylenediamine (343.4 mg,
1
2
2
1
compound as a brown solid (82 mg, 73%). H NMR (DMSO-d ): δ
6
.65 mmol, 1.1 equiv) were dissolved in dichloromethane (18 mL). To
this was added triethylamine (1.17 mL, 9 mmol), and the mixture was
cooled in an ice bath. Next, propanephosphonic acid anhydride (T3P)
9.18 (1H, s, NH), 9.05 (1H, s, NH), 7.14 (1H, dd, J = 8.0, 1.5 Hz,
CHAr), 6.91−6.84 (1H, m, CHAr), 6.70 (1H, dd, J = 8.0, 1.5 Hz,
CHAr), 6.54−6.48 (1H, m, CHAr), 4.78 (2H, s, NH ), 4.57 (2H, t, J =
2
(
50% solution in DMF, 1.38 mL, 1.1 mmol) was added and the
1.9 Hz, 2CH (Cp)), 4.08 (5H, s, unsubst Cp), 3.92 (2H, t, J = 1.9 Hz,
reaction mixture was warmed to room temperature overnight. Then
the mixture was poured into a saturated solution of K CO , stirred for
2CH (Cp)), 2.31 (2H, t, J = 7.4 Hz, CH ), 2.15 (2H, t, J = 7.4 Hz,
2
1
3
2
3
CH ), 1.68−1.53 (4H, m, 2CH ), 1.35−1.29 (4H, m, 2CH ).
NMR (DMSO-d , 126 MHz): δ 171.6, 171.2, 142.3, 126.1, 125.7,
C
2
2
2
3
0 min, and extracted into CH Cl (DCM). The organic layer was
2 2
6
dried (MgSO ), filtered, and evaporated in vacuo. The residue was
4
124.1, 116.6, 116.3, 96.1, 69.1, 64.1, 61.0, 39.7, 39.5, 36.4, 36.2, 29.0,
purified by trituration with DCM to give an orange solid (576.3 mg,
2
8.9, 25.7, 25.6. HRMS-ESI (m/z): found 448.1675, calcd for
7
4%). Crystallization by solvent evaporation of DCM provided yellow
+
[
C H FeN O ] 448.1682. Anal. Calcd for C H FeN O : C,
24 30 3 2 24 29 3 2
1
crystals. H NMR (DMSO-d , 500 MHz): δ 9.44 (1H, s, NH), 9.22
6
64.44; H, 6.53; N, 9.39. Found: C, 64.23; H, 6.60; N, 9.29.
(
(
=
3
1H, s, NH), 8.31 (1H, s, NH), 7.53 (1H, d, J = 7.8 Hz, CHAr), 7.41
1H, d, J = 7.8 Hz, CHAr), 7.12−7.04 (1H, m, CHAr), 7.06 (1H, d, J
7.8 Hz, CHAr), 4.57 (2H, s, 2CH (Cp)), 4.08 (5H, s, unsusbst. Cp),
tert-Butyl-2-(8-oxo-8-(phenylamino)octanamido)-
ferroceniumcarbamate Tetrafluoroborate (3a). tert-Butyl-2-(8-
oxo-8-(phenylamino)octanamido)ferrocenyl carbamate (109.5 mg, 0.2
mmol, 1 equiv) was suspended in dry DCM (5 mL). To this was
.93 (2H, s, 2CH (Cp)), 2.37 (2H, d, J = 8.3 Hz, CH ), 2.19 (2H, t, J
2
13
=
6.1 Hz, CH ), 1.65−1.56 (4H, m, 2CH ), 1.45 (9H, s, 3CH ). C
NMR (DMSO-d , 126 MHz): δ 171.0, 153.5, 130.1, 125.4, 125.3,
2
2
3
added NOBF (37.4 mg, 0.32 mmol, 1.6 equiv). The mixture was
4
6
stirred for 2 h at RT and the solution changed from yellow to dark
1
24.3, 124.1, 96.1, 79.8, 69.2, 64.1, 61.1, 36.2, 28.7, 28.5, 25.3.
brown. Filtration afforded the title compound as a dark brown solid
1
6
N -(2-Aminophenyl)-N -ferrocenyladipamide (1b). The pre-
19
11
(
89 mg, 70%). F NMR (DMSO-d ): δ −29.53. B NMR (DMSO-
6
vious compound, tert-butyl-2-(6-oxo-6-(phenylamino)hexanamido)-
ferrocenyl carbamate (520 mg, 1.00 mmol, 1 equiv), was suspended
in dichloromethane (40 mL) and MeOH (4 mL). To this mixture was
added 4 N HCl/dioxane (8 mL), and the mixture was stirred at room
temperature overnight. The volatiles were removed in vacuo, and then
d ): δ −2.56. Anal. Calcd for C H BF FeN O : C, 54.92; H, 5.88; N,
.62. Found: C, 55.08; H, 5.84; N, 6.57. FTIR (cm ): 992 (BF ).
N -(2-Aminophenyl)-N -ferroceniumoctanediamide Tetra-
fluoroborate (3b, Fe -Poj). N -(2-Aminophenyl)-N -ferrocenyloc-
6
29 37
4
3
4
−1
−
6
4
1
8
III
1
8
tanediamide (100 mg, 0.2 mmol, 1 equiv) was suspended in dry DCM
saturated Na CO (aq) was added to the residue and the mixture was
2
3
(5 mL). To this was added NOBF (37.4 mg, 0.32 mmol, 1.6 equiv).
4
sonicated. The precipitate was collected by suction and washed on the
frit with water, dried, and triturated with CH Cl to give the title
The mixture was stirred for 2 h at RT and the solution changed from
2
2
yellow to dark brown. Filtration afforded the title compound as a dark
1
compound as a brown solid (318 mg, 76%). H NMR (DMSO-d ): δ
19
11
6
brown solid (78 mg, 73%). F NMR (DMSO-d ): δ −29.53.
B
6
9
6
.21 (1H, s, NH), 9.09 (1H, s, NH), 7.15 (1H, d, J = 7.8 Hz, CHAr),
.78−6.73 (1H, m, CHAr), 6.70 (1H, dd, J = 7.8, 1.4 Hz, CHAr), 6.53
NMR (DMSO-d ): δ −1.34. Anal. Calcd for C H BF FeN O : C,
7.12; H, 5.80; N, 9.49. Found: C, 66.97; H, 5.84; N, 9.31. FTIR
6
24 29
4
3
2
6
(
(
(
1H, dd, J = 7.8, 1.4 Hz, CHAr), 4.80 (2H, s, NH ), 4.56 (2H, s, 2CH
−1
2
(cm ): 1038 (BF ).
4
Cp)), 4.08 (5H, s, unsubst Cp), 3.92 (2H, s, 2CH (Cp)), 2.36−2.32
2H, m, CH ), 2.25−2.10 (2H, m, CH ), 1.68−1.54 (4H, m, 2CH ).
2
2
2
1
3
C NMR (DMSO-d , 126 MHz): δ 171.4, 171.1, 142.3, 126.1, 125.7,
ASSOCIATED CONTENT
6
■
1
(
24.1, 116.6, 116.3, 96.1, 69.2, 64.1, 61.1, 36.3, 36.1, 25.5. HRMS-ESI
* Supporting Information
S
+
m/z): found 420.1366, calcd for [C H FeN O ] 420.1369. Anal.
22
26
3
2
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.organo-
met.7b00437.
Calcd for C H FeN O : C, 63.02; H, 6.01; N, 10.02. Found: C,
22
25
3
2
6
2.85; H, 6.05; N, 9.84.
tert-Butyl-2-(8-oxo-8-(phenylamino)octanamido)ferrocenyl
Carbamate (2a). Methyl-8-oxo-8-(ferrocenylamino)octanoic acid
215 mg, 0.6 mmol, 1 equiv) and N-Boc-o-phenylenediamine (137.4
mg, 0.66 mmol, 1.1 equiv) were dissolved in dichloromethane (7.7
Experimental details, characterization data, and crystallo-
graphic data (PDF)
(
F
DOI: 10.1021/acs.organomet.7b00437
Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
Accession Codes
3 Containing Chiral Oxazoline Capping Groups and a N-(2-
Aminophenyl)-benzamide Binding Unit. J. Med. Chem. 2015, 58
CCDC 1528185−1528186 contain the supplementary crystal-
lographic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif, or by email-
ing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
(
17), 6803−18.
(
9) Wilson, A. J.; Byun, D.-S.; Popova, N.; Murray, L. B.; L’Italien, K.;
Sowa, Y.; Arango, D.; Velcich, A.; Augenlicht, L. H.; Mariadason, J. M.
Histone Deacetylase 3 (HDAC3) and Other Class I HDACs Regulate
Colon Cell Maturation and p21 Expression and Are Deregulated in
Human Colon Cancer. J. Biol. Chem. 2006, 281 (19), 13548−13558.
(10) Meggers, E. Exploring biologically relevant chemical space with
AUTHOR INFORMATION
Corresponding Authors
C.A.O.: e-mail, co211@sussex.ac.uk; tel, + 44 (0)1273
78976.
■
metal complexes. Curr. Opin. Chem. Biol. 2007, 11 (3), 287−92.
(11) Meggers, E. Targeting proteins with metal complexes. Chem.
Commun. (Cambridge, U. K.) 2009, No. 9, 1001−10.
*
(12) Kilpin, K. J.; Dyson, P. J. Enzyme inhibition by metal complexes:
6
concepts, strategies and applications. Chemical Science 2013, 4 (4),
410.
13) Gasser, G.; Ott, I.; Metzler-Nolte, N. Organometallic Anticancer
*J.S.: e-mail, j.spencer@sussex.ac.uk; tel, + 44 (0)1273 877374.
1
(
ORCID
Cory A. Ocasio: 0000-0002-4957-4131
Justin M. Roberts: 0000-0001-6112-7476
John Spencer: 0000-0001-5231-8836
Compounds. J. Med. Chem. 2011, 54, 3−25.
(14) Spencer, J.; Amin, J.; Wang, M.; Packham, G.; Alwi, S. S.;
Tizzard, G. J.; Coles, S. J.; Paranal, R. M.; Bradner, J. E.; Heightman, T.
D. Synthesis and Biological Evaluation of JAHAs: Ferrocene-Based
Histone Deacetylase Inhibitors. ACS Med. Chem. Lett. 2011, 2 (5),
Present Address
∇
Supojjanee Sansook, Faculty of Science and Technology,
3
(
58−362.
Princess of Naradhiwas University, Thailand 96000.
15) Spencer, J.; Amin, J.; Boddiboyena, R.; Packham, G.; Cavell, B.
Author Contributions
C.A.O. and S.S. contributed equally to this paper.
E.; Syed Alwi, S. S.; Paranal, R. M.; Heightman, T. D.; Wang, M.;
Marsden, B.; Coxhead, P.; Guille, M.; Tizzard, G. J.; Coles, S. J.;
Bradner, J. E. Click JAHAs: conformationally restricted ferrocene-
based histone deacetylase inhibitors. MedChemComm 2012, 3 (1), 61.
#
Notes
The authors declare no competing financial interest.
(16) Leonidova, A.; Mari, C.; Aebersold, C.; Gasser, G. Selective
Photorelease of an Organometallic-Containing Enzyme Inhibitor.
Organometallics 2016, 35 (6), 851−854.
ACKNOWLEDGMENTS
We thank the Royal Thai Government for support (S.S.), the
European Community’s Seventh Framework Programme
FP7/2007-2013) under grant agreement no: PIIF-GA-2011-
01062 (C.A.O.), the CRUK grant C28206/A14499 (H.H.)
■
(17) Cross, J. M.; Blower, T. R.; Gallagher, N.; Gill, J. H.; Rockley, K.
L.; Walton, J. W. Anticancer RuII and RhIII Piano-Stool Complexes
that are Histone Deacetylase Inhibitors. ChemPlusChem 2016, 81,
(
3
1
(
276−1280.
18) Cazares-Marinero, J. d. J.; Buriez, O.; Labbe, E.; Top, S.;
́
́
and the EPSRC UK National Mass Spectrometry Facility at
Swansea University.
Amatore, C.; Jaouen, G. Synthesis, Characterization, and Antiprolifer-
ative Activities of Novel Ferrocenophanic Suberamides against Human
Triple-Negative MDA-MB-231 and Hormone-Dependent MCF-7
Breast Cancer Cells. Organometallics 2013, 32 (20), 5926−5934.
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H
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