Thiazolidinedione "magic Bullets" Simultaneously Targeting PPARγand HDACs: Design, Synthesis, and Investigations of their in Vitro and in Vivo Antitumor Effects

Article abstract of DOI:10.1021/acs.jmedchem.1c00491

Monotargeting anticancer agents suffer from resistance and target nonspecificity concerns, which can be tackled with a multitargeting approach. The combined treatment with HDAC inhibitors and PPARγagonists has displayed potential antitumor effects. Based on these observations, this work involves design and synthesis of molecules that can simultaneously target PPARγand HDAC. Several out of 25 compounds inhibited HDAC4, and six compounds acted as dual-targeting agents. Compound 7i was the most potent, with activity toward PPARγEC50 = 0.245 μM and HDAC4 IC50 = 1.1 μM. Additionally, compounds 7c and 7i were cytotoxic to CCRF-CEM cells (CC50 = 2.8 and 9.6 μM, respectively), induced apoptosis, and caused DNA fragmentation. Furthermore, compound 7c modulated the expression of c-Myc, cleaved caspase-3, and caused in vivo tumor regression in CCRF-CEM tumor xenografts. Thus, this study provides a basis for the rational design of dual/multitargeting agents that could be developed further as anticancer therapeutics.

Full text of DOI:10.1021/acs.jmedchem.1c00491

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Article
Thiazolidinedione “Magic Bullets” Simultaneously Targeting PPARγ
and HDACs: Design, Synthesis, and Investigations of their In Vitro
and In Vivo Antitumor Effects
Kalpana Tilekar, Jessica D. Hess, Neha Upadhyay, Alessandra Lo Bianco, Markus Schweipert,
Antonio Laghezza, Fulvio Loiodice, Franz-Josef Meyer-Almes, Renato J. Aguilera,* Antonio Lavecchia,*
and Ramaa C S*
Cite This: J. Med. Chem. 2021, 64, 6949−6971
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ABSTRACT: Monotargeting anticancer agents suffer from
resistance and target nonspecificity concerns, which can be tackled
with a multitargeting approach. The combined treatment with
HDAC inhibitors and PPARγ agonists has displayed potential
antitumor effects. Based on these observations, this work involves
design and synthesis of molecules that can simultaneously target
PPARγ and HDAC. Several out of 25 compounds inhibited
HDAC4, and six compounds acted as dual-targeting agents.
Compound 7i was the most potent, with activity toward PPARγ
EC50 = 0.245 μM and HDAC4 IC50 = 1.1 μM. Additionally,
compounds 7c and 7i were cytotoxic to CCRF-CEM cells (CC50
=
2.8 and 9.6 μM, respectively), induced apoptosis, and caused DNA
fragmentation. Furthermore, compound 7c modulated the
expression of c-Myc, cleaved caspase-3, and caused in vivo tumor regression in CCRF-CEM tumor xenografts. Thus, this study
provides a basis for the rational design of dual/multitargeting agents that could be developed further as anticancer therapeutics.
regulation of gene transcription and cellular homeostasis
events.10−13 Also, aberrant expression of HDACs has been
linked to a variety of solid and hematological malignancies,
neurological disorders, and inflammation.14 There are various
FDA-approved HDAC inhibitors such as SAHA, belinostat,
panobinostat, and romidepsin, as well as various agents under
clinical studies.15 However, the use of these inhibitors as a
monotherapy against solid tumors has shown limited efficacy;
thus, the development of HDAC inhibitors with multitarget
specificity has been studied extensively.16−22
PPARs (peroxisome proliferator-activated receptors) are
ligand-activated transcription factors that play a key role in the
regulation of a large number of genes whose products are
directly or indirectly involved in glucose homeostasis and lipid
metabolism. The subtype PPARγ is the most widely studied
and acts as a key factor in various metabolic processes playing
an important role in the regulation of insulin tissue sensitivity
INTRODUCTION
■
Cancer is the most prevalent disease worldwide and is
regulated by genetic and epigenetic mechanisms. The major
challenge in anticancer drug discovery is to bring about a
therapeutic approach capable of attenuating malignant cells
without damaging normal cells. Thus, there exist two
fundamentals of a drug, viz., target specificity and potency.
The clinical effectiveness of recent monotargeted drugs are
transitory because of unexpected resistance and target
nonspecificity due to tumor heterogeneity.1,2 To overcome
these concerns, an alternative strategy of multitargeting can be
applied to hit multiple cancer hallmarks and achieve the
desired pharmacological efficiency with reduced detrimental
effects such as drug−drug interactions, unforeseen side-effects,
and poor patient compliance.3−6 Moreover, in a multitarget
approach, the concept of “magic bullets” existwhich are
single agents with target specificity and high potency.7
In the development and progression of several malignancies,
epigenetics contributes significantly. Tumor cell activation is
effectively regulated by epigenetic mechanisms which facilitate
escape from chemotherapy; thus, the recent focus of anticancer
drug discovery has been directed to epigenetic targets.8,9 There
are numerous enzymes involved in epigenetic regulation; for
example, histone deacetylases (HDACs) are indispensable in
Received: March 18, 2021
Published: May 18, 2021
https://doi.org/10.1021/acs.jmedchem.1c00491
© 2021 American Chemical Society
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Figure 1. Designing of (partial) PPARγ and HDAC dual targeting agents.
and in the management of glucose and lipid uptake and
storage.23 PPARγ is the receptor of well-known antidiabetic
insulin sensitizer drugs rosiglitazone and pioglitazone, which
belong to the thiazolidinedione (TZD) class.24 Apart from its
role in the cardiovascular system, PPARγ is vital to cancer cell
growth regulation. A large body of evidence shows that
agonistic PPARγ ligands exert antitumorigenic effects against a
variety of neoplastic cell types, both in vivo and in vitro.25−28
For instance, efatutazone is indicated in thyroid29−31 and
nonsmall cell lung cancers;32 additionally, a phase I clinical
study demonstrated acceptable tolerance in patients with
advanced malignancies,33 and a phase II clinical trial is
ongoing.34 Though there are undesirable side effects associated
with full agonist activity,35 there exist PPARγ partial agonists
endowed with reduced side effects.36−41 For example, in the
preclinical setting, PPARγ partial agonist balaglitazone
displayed a better safety profile than full agonists.34,42,43
Another partial agonist netoglitazone (MCC-555) has one-
tenth the affinity to PPARγ as rosiglitazone, has lesser side
effects,44 and is significantly more potent.45 Thus, the search
for novel PPARγ partial agonists with structural diversity and
safe biological profiles remains an attractive therapeutic
strategy to circumvent cancer.
Recent reports suggested that combination treatment with
HDAC inhibitors and PPARγ agonists increased cytotoxic
effects in a synergistic/additive manner against various cancer
cell lines, resulting in proliferation arrest and apoptosis. In
some cases, even low doses of a PPARγ ligand in combination
with a weak HDAC inhibitor resulted in more profound
growth arrest than treatment with either drug alone.46−48
On the basis of this literature, a series of 25 novel TZD-
based naphthylidene derivatives were rationally designed and
successfully synthesized in pursuit of a multitarget drug
approach and simultaneous targeting of HDAC/PPARγ.
Biological evaluation of compounds revealed their dual-
targeting ability by partial transactivation of PPARγ and
selective inhibition of HDAC4. To our knowledge, this is the
first ever attempt to develop agents which simultaneously
target HDACs and PPARγ, two highly desirable targets in
cancer drug discovery. An investigation of the antiproliferative
capacity of this series of compounds revealed uncompromised
cytotoxicity in a panel of hematological and solid cancer cells,
induction of apoptosis, and in vivo tumor regression in CCRF-
CEM (T-cell leukemia) xenografts.
RESULTS AND DISCUSSION
■
Rationale of Designing PPARγ and HDAC Dual-
Targeting Analogues. The main challenge in designing
dual HDAC inhibitors and PPARγ agonists was achieving a
unique framework which satisfied the binding requirements of
both targets. In our previous reports, a distinct pharmaco-
phoric drug-design was used to individually achieve both
classes of compounds.49−53 Thus, on the basis of our
experience with both targets, a multitargeting strategy was
conceptualized in consideration of HDAC and PPARγ
pharmacophore properties.
Traditional (full) PPARγ agonists consist of a hydrophobic
tail linked to a central aryl ring, which is further linked through
another small methylene bridge (linker 2) to the pharmaco-
phoric TZD head (Figure 1). Examples are antidiabetic drugs
pioglitazone and rosiglitazone. However, partial PPARγ
agonists display a broader range of structural diversity, some
being structurally similar to traditional agonists (e.g.,
netoglitazone), while others have an entirely different
structural framework. The major distinguishing feature
between full and partial PPARγ agonists is their binding
pattern at the active site, which has been reviewed well in the
literature. A key structural difference between partial agonist
netoglitazone and traditional glitazones is the central
naphthalene ring in place of a phenyl ring, suggesting a
major contribution of naphthalene in shifting the molecule
from full to partial agonist.
N-Substituted benzylidene TZDs (an aryl ring attached to
TZD through −CCH− instead of −CH−CH2−), including
our previously reported compounds and GQ-16, were found to
be partial PPARγ agonists.54 Both netoglitazone, with a
naphthalene central aryl ring, and GQ-16, with a benzylidene
TZD ring, have demonstrated antitumor potential.55 Given the
partial PPARγ transactivation and antitumor potential of these
compounds, we sought to synthesize compounds with a
naphthalene ring attached to TZD ring through a benzylidene
like linker (−CCH−)thus generating the naphthylidene
TZD derivatives which are evaluated herein (designed
molecule, Figure 1).
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Typical HDAC inhibitors share a broad pharmacophore
with a surface recognition cap (SRC, denoted CAP herein)
which binds with amino acids of the active site, a hydrophobic
linker which occupies the active site channel, and a zinc
binding group (ZBG) which chelates the catalytic zinc ion.56
Previously, we developed HDAC inhibitors by placing a TZD
ring centrally to obtain N-substituted TZD derivatives like P19
(Figure 1), or terminally to get naphthylidene TZDs like 3k
(Figure 1) with good to moderate HDAC inhibitory
activity.50,52,53 Both TZD series showed different activity
against HDAC4 and HDAC8, which are representative of class
IIa and class I human zinc-dependent HDACs. While N-
substituted TZD analogues were found to be inactive against
HDAC4 with moderate activity against HDAC8 (e.g., P19
IC50 = 9.3 μM), compound 3k with ortho-substituted
naphthalene linker showed increased inhibitory activity on
both HDAC4(IC50 = 15 μM) and HDAC8 (IC50 = 2.3 μM)52
(Figure 1). Despite significantly different chemical structures
of P19 and 3k, their docked binding poses revealed that the
carbonyl oxygen of carboxamide binds to the catalytic zinc ion
and not the TZD ring. Thus, in the quest of modifying the
structure of 3k series compounds to discover more potent
HDAC inhibitors, we shifted substitutions at the naphthalene
linker in the opposite ends to get a more extended shape than
3k. A primary docking study of these newly designed HDAC
inhibitors at the active sites of HDAC4 and HDAC8 revealed
that the TZD ring exhibited zinc binding interactions in
HDAC4, whereas the carbonyl oxygen of carboxamide was
bound to the zinc ion of HDAC8, which was a positive
indication that the designed compounds could be HDAC
inhibitors with differential potencies and selectivity. Similarly,
HDAC inhibitors such as panobinostat have substitutions and
a cyclic linker attached ZBG which are opposite to each other
(Figure 1). We retained the naphthalene ring as a cyclic linker
to help establish the structure−activity relationship (SAR) for
HDAC inhibitory activity and partial PPARγ agonistic activity.
Although compounds like 3k are structural isomers of the
series described herein, the CAP portion have been varied to a
great extentaromatic, heterocyclic, and heteroarylto
optimize for surface complementarity with HDAC active
sites. In addition, the position of the substituents is such that it
alters the shape of the molecules, thus orienting them
differently from “series 3” and potentially leading to distinct
HDAC selectivity and potency.
Figure 2. Fitting of designed dual PPARγ and HDAC targeting agents
in typical pharmacophoric models of both targets.
a
Scheme 1. Synthesis of Substituted Aromatic Compounds
a
Reagents and conditions: (a) chloroacetyl chloride, K2CO3, DCM,
stir, 0−5 °C, 1 h, rt, 3−6 h; (b) piperidinium benzoate, toluene, reflux
3−4 h; (c) DMF, K2CO3, stir 24 h.
available 6-hydroxy-2-naphthaldehyde (3) was reacted with
2,4-TZD (4) under Knoevenagel conditions to afford
intermediate 5, which was further condensed with phenyl
acetamides (2a−2l, 2o−2r, 2u−2v) under weakly basic
conditions. Phenyl acetamides, in turn, were prepared from
commercially available aromatic amines (1a−1l, 1o−1r, 1u−
1v) as previously reported. Target compounds with a
terminally substituted heterocyclic ring 7m, 7n, 7s, and 7t
were synthesized using procedures as described in Scheme 2.
The phenyl acetamides 2m, 2n, 2s, and 2t were condensed first
with 3 and then were reacted by Knoevenagel conditions but
using methoxy-ethanol as a solvent rather than traditional
knoevenagel solvents. Use of methoxy-ethanol drastically
improved the yield and purity of the final compounds. Target
compounds 7w−7y were prepared as per the procedure
detailed in Scheme 3 in a similar way to heterocyclic
derivatives, with the variation of the solvent and reaction
conditions.
This rational designing led to oppositely substituted
naphthylidene TZD structures in accordance with the
pharmacophoric model of both targets (Figure 2). To clarify,
the terminal aromatic/heterocyclic/heteroaromatic groups
correspond to the lipophilic group of PPARγ agonists and to
the CAP for HDAC8 inhibitors. Likewise, the carboxamide
alkoxy moiety reflects linker 1 of PPARγ and the connecting
unit between CAP and cyclic linker for HDAC4, while the
carbonyl of this carboxamide may function as a ZBG for
HDAC8. The naphthalene ring serves as the central aryl ring
for PPARγ and as cyclic linker for HDACs. The methylene
(−CCH−) group corresponds to linker 2 for PPARγ and
spacer for HDACs; and ultimately, the terminal TZD ring is a
pharmacophore for PPARγ and ZBG for HDAC4. Therefore,
we theorized that these compounds would have the capacity to
simultaneously target HDACs and PPARγ.
HDAC4/8 Activity Assay. To determine the effects of
synthesized derivatives 7a−7y (Figure 2) on the activity of
HDACs and to detect if they exhibit selectivity against class I
or class II, all 25 compounds were initially screened at 35 μM
concentration on both isoforms, viz., HDAC4 from class II and
HDAC8 from class I. The test compounds showed exceptional
Chemistry. The procedures to synthesize the target
compounds with a terminally substituted aromatic ring (7a−
7l, 7o−7r, 7u−7v) were outlined in Scheme 1. Commercially
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a
Scheme 2. Synthesis of Substituted Heterocyclic Compounds
a
Reagents and conditions: (a) chloroacetyl chloride, DCM/chloroform, TEA, stir, 0−5 °C, 1 h, rt, 8−12 h; (b) 6-hydroxy naphthaldehyde (3),
DMF, K2CO3, stir 36 h; (c) 2,4-thiazolidinedione, methoxy ethanol, piperidine, reflux 3−4 h.
a
Scheme 3. Synthesis of Substituted Heteroaryl Compounds
a
Reagents and conditions: (a) chloroacetyl chloride, chloroform, K2CO3, stir, 0-5 °C, 1 h, rt, 24−48 h; (b) 6-hydroxy naphthaldehyde (3), DMF,
K2CO3, stir 48 h; (c) 2,4-thiazolidinedione, DMF, piperidine, reflux 3−4 h.
inhibitory effects on HDAC4, with markedly lower but
significant activity on HDAC8 (Figure 3; Table S1 in
Supporting Information). On the basis of the results gained
in primary screening, compounds inducing a residual enzyme
activity of <40% were evaluated at six concentrations to
establish a dose−response curve (Figure 2, Table 1). Few
compounds were found to be dual HDAC4 and HDAC8
inhibitors. Most of the compounds were more selective and
potent on HDAC4 and at least 2 times potent on HDAC4 over
HDAC8 at the tested concentrations. The best activities
against HDAC4 were comparable to “Cpd 6”57 (Table 1),
indicative of the therapeutic potential of this compound series.
Against HDAC8, 12 out of 25 compounds possessed
inhibition capacity. The remaining compounds showed no or
only moderate activity at tested concentrations. Compounds
7a, 7b, 7c, 7g, 7l, 7n, 7q, 7s, 7w, and 7x exhibited IC50 values
of <10 μM, 7o and 7y of < 20 μM. The most potent inhibitor
7r) were found to be inactive, thus suggesting that dihalo
substitution leads to loss of HDAC8 inhibition. Likewise,
substitution with Cl (7f), CF3 (7e) or disubstitution with these
two groups (7p) did not show HDAC8 inhibition. Addition-
ally, it was observed that compounds with disubstitution at
position 3- and 4- of the phenyl ring (7h, 7u, 7v) were not
active. When the Ar group is a heterocyclic ring such as
pyridinyl, halo-substituted pyridinyl (7s) and methyl sub-
stituted pyridinyl (7n) derivatives displayed greater HDAC8
inhibition than the unsubstituted compound (7m). Compar-
ison between benzothiazole containing compounds revealed
that an unsubstituted benzothiazole (7w) ring was preferred
over alkyl (7x) and alkoxy substitution (7y).
As far as HDAC4 inhibitory activity is concerned, all
derivatives exhibited HDAC4 inhibition with IC50 < 6.0 μM,
except 7m which had IC50 = 17 0.7 μM. The most potent
compounds exhibiting IC50 values in the submicromolar range
were 7l, 7s, and 7w (IC50 = 0.55 0.05 μM, 0.84 0.22 μM,
and 0.42 0.05 μM respectively). Among aromatic analogues
(Ar = substituted phenyl) compounds with no substitution
(7a), monohalo-substitution (7b, 7f, 7o), monoalkyl sub-
stitution (7c), and monoalkoxy substitution (7d, 7g) were
found to be more potent than dihalo substituted compounds
(7h, 7k, 7v except 7r). Even, disubstitution with a halogen and
methyl group (7l, 7u) was well tolerated with significant
activity. In the case of pyridinyl and benzothiazolyl derivatives,
was 7w, which had an IC50 = 2.7
0.2 μM. Among the
compounds with a substituted aromatic group (Ar), it was
observed that the unsubstituted (7a), monohalo-substituted
with F or Br (7b, 7o), and monoalkyl substituted (7c)
derivatives retained HDAC8 inhibitory activity. Compounds
7d and 7g are structural isomers with p-OCH3 and m-OCH3
respectively, but only 7g exhibited inhibitory potential,
indicating that the point of methoxy group attachment may
be important. Compounds with dihalo substitution (7h, 7i, 7k,
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Figure 3. Dose−response curves (DRCs) of indicated inhibitors (A−E) against HDAC4 and (F−H) against HDAC8. Residual enzyme activity of
HDAC4 or HDAC8 for compounds 7a−7m (I) and compounds 7n−7y (J) in the presence of 35 μM of the indicated compounds. Data represent
means and standard deviations, n = 3. Compounds with less than 40% residual enzyme activity were taken further to determine IC50 values.
the effect of substitution pattern on inhibitory activity was
found similar to that of HDAC8. Among pyridinyl derivatives,
halogen substitution (7s) and alkyl substitution (7n) led to
more potent activity than an unsubstituted pyridine ring (7m),
whereas in benzothiazole containing compounds, an unsub-
stituted benzothiazole ring (7w) was preferred over alkyl (7x)
and alkoxy substitution (7y). Since all compounds exhibited
HDAC4 inhibition, it can be assumed that an Ar group of all
three types, viz., aromatic, heterocyclic, and heteroaromatic, is
well tolerated with significant retention of activity.
Compounds 7d, 7e, 7f, 7h, 7i, 7j, 7k, 7m, 7p, 7r, 7t, 7u, and
7v were found more potent on HDAC4 over HDAC8, whereas
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a
Table 1. IC50 Values of the “Series 7” against HDAC4 and HDAC8
compound
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
7u
7v
7w
7x
Ar group
HDAC4 IC50 (μM)
HDAC8 IC50 (μM)
phenyl
2.1 0.3
1.6 0.3
1.7 0.3
4.7 0.1
2.5 0.4
3.8 0.1
1.6 0.2
5.5 0.3
1.1 1.7
5.2 0.5
3.9 0.1
0.55 0.05
17 0.7
1.1 1.2
2.9 0.1
4.0 0.2
1.7 0.4
1.9 0.1
0.84 0.22
1.7 0.8
1.1 0.4
4.9 0.3
0.42 0.05
1.2 0.4
3.6 0.3
0.22
4.5 0.2
6.6 0.2
9.0 0.2
>50
>50
>50
7.6 0.2
>50
>50
>50
>50
7.4 0.3
>50
7.5 0.2
16 0.5
>50
4.0 0.2
>50
7.2 0.2
>50
>50
>50
2.7 0.2
5.8 0.1
17 0.3
>50
4-fluorophenyl
4-methylphenyl
4-methoxyphenyl
3-trifluoromethylphenyl
3-chlorophenyl
3- methoxyphenyl
3,4-dichlorophenyl
2-bromo-4,6-difluorophenyl
2-phenoxyphenyl
2,4-difluorophenyl
4-bromo-2-methylphenyl
pyridin-2-yl
6-methylpyridin-2-yl
4-Br phenyl
3-chloro-5 (trifluoromethyl)phenyl
4- nitrophenyl
4-bromo-2-fluorophenyl
6-fluoropyridin-2-yl
5-methylisoxazol-3-yl
3-chloro-4-methylphenyl
3,4-dibromophenyl
benzo[d]thiazol-2-yl
4-methylbenzo[d]thiazol-2-yl
6-ethoxybenzo[d]thiazol-2-yl
-
7y
Cpd 657
PCI-3405159
-
10
0.024
a
Data represent mean sd (standard deviations), n = 3. Compounds with less than 40% residual enzyme activity were taken further to determine
IC50 values.
compounds 7a, 7b, 7c, 7g, 7l, 7n, 7o, 7q, 7s, 7w, 7x, and 7y
were found to be dual inhibitors of HDAC4 and HDAC8 with
at least 2 times more potency (as reflected from IC50 values)
toward HDAC4 than HDAC8.
P19 with an IC50 value of 9.3 μM,53 Figure 4). Docking results
suggested that P19 binds to the catalytic zinc ion through its
The difference between our previously reported “series 3”
and the current “series 7” is only the position of the TZD ring
on the central naphthalene ring. Thus, shifting the point of
TZD ring attachment to the opposite side on naphthalene ring
leads to an extreme change in HDAC potency and selectivity.
In the previously reported “series 3”, the arrangement of the
various groups in the molecule was such that they adopted an
L- shape which is an ideal fit at the active site of HDAC8 as per
the Hou et al. model; instead, in our current “series 7”, the
shifting of the TZD ring to the opposite site causes the
structure to deviate from an L-shape, which could be the
reason why the current series are less selective and potent
inhibitors of HDAC8.58 Nevertheless, the common element
between our previously reported “series 3” and current “series
7” is the naphthalene linker, thus supporting our hypothesis of
incorporating a naphthalene ring as cyclic linker to obtain
potent HDAC inhibitors.
Figure 4. Optimization of dual activity of TZD-analogues against
HDAC4 and HDAC8. P19 is a moderate HDAC8 selective
inhibitor,53 3k52 shows improved activity on both, HDAC4 and
HDAC8, and 7w shows high activity against both HDAC enzymes.
carbonyl oxygen and occupies the lower side pocket which
connects the canonic active site pocket and acetate release
channel.
TZD analogue 3k with ortho-substituted naphthalene linker
showed increased inhibitory activity on both HDAC4 and
HDAC852 (Figure 4). Despite their different chemical
structures, in spacer and substitution pattern at the TZD
group, P19 and 3k have similar features in their docked
Molecular Docking into HDAC4 and HDAC8. Different
TZD scaffolds show different activities against HDAC4 and
HDAC8, which are representatives of class IIa and class I of
human zinc-dependent HDACs. While N-substituted TZD
analogues are generally inactive against HDAC4, there are
some examples with moderate activity against HDAC8 (e.g.,
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Figure 5. (A) 3D docking pose of 7l within HDAC8, (B) 2D interactions between 7l and HDAC8. (C) Docked 3D binding pose of 7l within the
enlarged binding groove of HDAC4o. (D) 2D interactions between 7l and HDAC4o. Conventional hydrogen bonds are indicated by dotted green
lines, alkyl and π-alkyl interactions by dotted magenta or pink lines. A dotted gray line denotes metal binding, whereas a dotted orange line
highlights a π-sulfur interaction.
binding poses. Namely, the protrusion of the aromatic
carboxamide moiety into the transition area between active
site and acetate release channel and binding of the carbonyl
oxygen to the catalytic zinc ion. “Series 7” differentiate from
the branched 3k in that the substitutions at the naphthalene
linker are in the opposite position from the TZD group, thus
forming a more extended shape. This difference led to a
tremendous increase in activity against both HDAC
isoenzymes (Figure 4). Compound 7w also showed low
micromolar activity against HDAC8 and HDAC4, thus
resulting in a dual HDAC4/8 inhibitor with some preference
for HDAC4.
To rationalize the observed dual activity of “series 7”
analogues against HDAC8 and particularly HDAC4, a
comprehensive docking study was performed. At first, the
docking routine was validated by redocking of the respective
ligands into the used crystal structures of HDAC8- (PDB ID:
3SFF) and HDAC4-ligand complexes (PDB IDs: 2VQJ and
4CBY). The docked and crystallized poses of the ligand within
the binding pocket of HDAC8 (PDBID: 3SFF) showed
excellent overlap with an RMSD over 26 heavy atoms of 0.2 Å.
The thiophene linker and trifluoromethyl warhead of the
redocked ligand in HDAC4 (PDB ID: 2VQJ) show a good
RMSD (root-mean-square deviation) value of 0.4 Å with
respect to the crystal structure (Table S1 in Supporting
Information). Since the aromatic headgroup of the trifluor-
omethyl ketone ligand protrudes into free solution, this part of
the molecules is intrinsically flexible and thus not considered
for the calculation of RMSD. For HDAC8, the crystal structure
with PDB-ID 3SFF, which was previously utilized in docking of
3k, was selected.52 All compounds of “series 7” superposed
well and docked smoothly into the binding pocket of HDAC8,
showing a very similar binding mode to 3k (Tables S2, S3 in
Supporting Information). The aromatic ring of the carbox-
amide group of 7l fit perfectly into the lower binding pocket
adjacent to the catalytic center at the entrance of the acetate
release channel, and the carbonyl oxygen was bound to the
catalytic zinc ion (Figure 5A). Moreover, the naphthalene
linker forms a π-stacking interaction with Phe152 and Phe208
and a π-sulfur interaction with Met274 (Figure 5A,B).
The TZD group points out of the binding pocket and is
exposed to the outer solvent. The malleability of the HDAC8
binding pocket allows for accommodation of the branched
TZD-analogue 3k as well as the more linearly shaped 7l. This
exceptional flexibility of HDAC8 has been exploited previously
for the design of linkerless or branched isoenzyme selective
inhibitors.60,61 However, all of these active compounds contain
the potentially problematic hydroxamate group of most
canonical HDAC inhibitors. Overall, compounds of “series 7”
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show good docking scores that are similar to or better than 3k
(Table 2).
the naphthalene linker is supposed to undergo hydrophobic
interactions with Pro156. Two additional charge enhanced
hydrogen bonds, between theoxygen next to the naphthalene
moiety and Arg154 as well as the distal carbonyl oxygen and
Lys20, contribute significantly to the binding of 7l to HDAC4o
(Figure 5C,D). Possible hydrophobic interactions between
neighboring Arg37, Arg154, and the aromatic head group may
also increase affinity depending on the substitution pattern.
Altogether, the experimentally demonstrated dual activity of
TZD compounds from “series 7” against HDAC4 and HDAC8
agrees with favorable docking scores for both HDAC
isoenzymes. Furthermore, the docking results suggest two
distinct binding modes of 7l and analogues to HDAC8 and the
open conformation of HDAC4 characterized by different
complexation of the catalytic zinc ion.
PPARγ Transactivation Assay. Compounds 7a−7y were
analyzed via PPARγ-GAL4 transactivation assay on HepG2
cells in agonist mode at 25, 5, and 1 μM concentrations.
Rosiglitazone at a 2 μM concentration was used as a control,
while the blank contained 0.1% dimethyl sulfoxide (DMSO).
Compounds 7i, 7l, and 7r were the most active and showed
efficacy of 20−30% followed by 7c, 7o, and 7q with an efficacy
around 10% compared with the control (Table 3) (Figure S4
Table 2. GBVI/WSA dG Docking Scores for Indicated
Molecules and HDAC Isoenzymes
ID
HDAC4o
HDAC8
ID
HDAC4o
HDAC8
7a
7b
7c
7d
7e
7f
7g
7h
7i
−8.7
−8.7
−9.1
−9.3
−8.9
−9.1
−10.3
−9.1
−9.4
−10.5
−9.0
−9.4
−9.2
−9.0
−8.8
−9.8
−10.3
−10.0
−9.9
−9.9
−9.8
−9.7
−10.1
−9.8
−10.2
−9.1
7n
7o
7p
7q
7r
−9.2
−9.7
−9.0
−10.3
−9.9
−8.2
−9.5
−8.5
−8.9
−9.6
−9.6
−10.5
−8.7
−9.6
−9.8
−10.2
−9.9
−10.2
−10.7
−10.5
−10.0
−10.2
−10.2
−10.3
−11.2
−9.5*
7s
7t
7u
7v
7w
7x
7y
3k
7j
7k
7l
7m
To understand the difference between 3k and 7l in
molecular recognition of HDAC4 and HDAC8 appears more
complex and challenging. It is of utmost importance to
recollect that different types of inhibitors have been shown to
stabilize either the open or the closed conformation of the
catalytic domain of HDAC4 because of its conformationally
flexible second zinc-binding domain.62,63 To explore the
impact of open (PDB ID: 2VQJ) and closed (PDB ID:
4CBY) conformations of HDAC4 on the binding affinity of the
most active analogues, 7l and 7w (IC50 0.55 0.05 and 0.42
0.05 μM, respectively), docking was performed using both
crystal structures. Binding to the enlarged pocket of open
conformation HDAC4 (HDAC4o) yielded more favorable
binding energies than closed HDAC4 (HDACc) (Table S2 in
Supporting Information). Consequently, all TZD analogues
have been docked into the large binding groove of HDAC4o,
yielding good scores and indicating favorable contacts between
protein and ligand (Table 2). A comparison with 3k reveals
that the compounds of “series 7” exhibit favorable interactions
with both HDAC4o and HDAC8, while 3k shows good affinity
toward HDAC8 but is poorly recognized by HDAC4o.52 Thus,
docking results are in good agreement with experimental data
and may provide insight into important contacts that
determine binding mode of action with HDAC4. Docking
suggested that “series 7” analogues bind to HDAC4o rather
than HDAC4c. The enlarged binding groove of HDAC4o is
complementary to the linear shape of the TZD analogues of
“series 7”, offering favorable contacts between the amino acids
at the surface of the binding pocket and the ligands (Figure
5C). The resulting binding poses were similar among all
analogues (Figure S1A,B in Supporting Information). In
contrast to the binding mode of the TZD analogues in
HDAC8, which is determined by an interaction between the
catalytic zinc ion and carbonyl oxygen of the ligand, “series 7”
compounds bind the corresponding zinc ion through the TZD
group in HDAC4o (Figure 5C,D). A closer look at the distinct
contacts between 7l and HDAC4o revealed multiple
interactions between the TZD group and binding pocket:
one carbonyl oxygen of the TZD ring interacts with the
catalytic zinc ion, the amide nitrogen serves as hydrogen donor
for His159, and the sulfur atom forms a π-sulfur interaction
with the aromatic ring of Phe168 (Figure 5C,D). Moreover,
a
Table 3. PPARγ Transactivation Assay
PPARγ
b
compound
Emax%
EC50 μM
“Ar” group
4-methylphenyl
2-bromo-4,6-difluorophenyl
4-bromo-2- methylphenyl
4-bromophenyl
7c
7i
7l
7o
7r
7q
13
2
1.220 0.260
0.245 0.006
0.359 0.031
0.729 0.027
0.791 0.025
22.4 1.8
32
13.0 0.2
27
8.5 1.5
3
6
4-bromo-2-fluorophenyl
4-nitrophenyl
1.610 0.280
a
b
EC50 values with mean SD. Efficacy values (Emax) were calculated
as a percentage of the maximum obtained fold induction with the
reference compounds. All transfection experiments were repeated at
least twice in triplicate.
in Supporting Information). Many compounds showed partial
activation of PPARγ, but because Emax was less than 10%, their
activity was considered negligible. However, we were successful
to determine EC50 (half maximal effective concentration)
values for the six compounds listed in Table 3. Thus, our
hypothesized design of naphthylidene TZD analogues as
partial PPARγ agonists was successful. The most potent partial
agonists were 7i and 7l with EC50 values of 0.245 0.006 and
0.359 0.031 μM, respectively. In spite of the small number of
PPARγ active compounds, some preliminary remarks can be
made about structure−activity relationships. It is very
interesting to note that five p-Br (bromo) substituted
compounds, 7i, 7l, 7o, 7r, and 7v, displayed PPARγ activity,
which indicates that this substitution has the appropriate
requisites to elicit the transactivation capacity of these
derivatives. For these compounds, other substituents can be
tolerated at the ortho position, particularly fluorine (7i, 7r) or
methyl (7l), whereas a methyl or a polar nitro group alone at
the para position of the phenyl ring led to less potent
derivatives (7c, 7q). In contrast, the presence of two bromine
atoms, as in the 3,4-disubstituted derivative 7v, led to poor
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Figure 6. In silico docking of compound 7i into the PPARγ binding pocket. (A) Binding mode of compound 7i (partial agonist, green sticks) into
the PPARγ binding site represented as a gray ribbon model. Only amino acids located within 4 Å of the bound ligand are displayed (white sticks)
and labeled. H12 is shown in slate. H-bonds discussed in the text are depicted as dashed yellow lines. The halogen bond is shown as dashed
magenta lines. The water molecules bridging the interaction of the ligand with the protein are displayed as red spheres. (B) 2D ligand-interaction
diagram of 7i. The plot was generated by LigPlot+. H-bonds are shown as dashed yellow lines, while the halogen bond is shown as dashed magenta
lines. The water molecules are displayed as red spheres. The spoked arcs represent residues making nonbonded contacts with the ligand. (C) Cα
superposition of the complexes of PPARγ with compound 7i and Rosiglitazone (full agonist, magenta sticks, PDB ID: 4EMA).
activity. This suggests that an excess steric bulk in this part of
the molecule is detrimental for the interaction with the
receptor. Given that all heterocyclic and pyridinyl derivatives
showed very low activity, it can be assumed that the phenyl
ring is more appropriate to achieve significant PPARγ
transactivation.
These findings illustrate the potential of 7i, 7q, and 7r as
dual targeting partial PPARγ agonist and selective HDAC4
inhibitor compounds, and of 7c, 7l, and 7o to target PPARγ,
HDAC4, and HDAC8 but with more efficacy (3- to 10-fold)
on HDAC4.
Molecular Docking into PPARγ. To understand the
structural basis for the observed partial agonism of 7i, 7l, 7o,
7r, 7c, 7q toward PPARγ, we undertook docking simulations
using the Glide module, which is part of the Maestro software
suite. Docking experiments were carried out into the X-ray
crystal structures of PPARγ ligand binding domain (LBD)
complexed to partial agonist CAY10638 (PDB ID: 6DGR).64
This structure was chosen as a template because of the good
resolution (2.15 Å) and the similarity of the cocrystallized
ligand with the candidate compounds, both containing a TZD
headgroup and an unsaturated linker, which restricts the
mobility of the TZD group. Analysis of the CAY10638/PPARγ
complex revealed that the ligand is H-bonded via two
crystallographically observed water molecules to the protein:
via HOH708 to L340 (β-sheet) and via HOH706 to S342 (β-
sheet). Accordingly, the two water molecules were included in
the docking experiments.
The LBD of PPARγ consists of a hydrophobic entrance (arm
III) that branches off into two subsites: the polar arm I, which
is extended toward H12, and the hydrophobic arm II, which is
located between helix H3 and β-sheet.
Compound 7i fit well within the PPARγ LBD, adopting a U-
shaped conformation like the known full agonist Rosiglitazone.
The interactions between 7i and the PPARγ LBD involved
many H-bonds, one halogen bond, and several hydrophobic
interactions (Figure 6A,B). The TZD headgroup is located in
arm I and establishes a bifurcated H-bond with Nε atom of
H323 (d = 2.8 Å) and Oγ atom of S289 (d = 3.2 Å) by one of
two carbonyl oxygens. Unlike full agonist Rosiglitazone, 7i
shows no direct H-bond with residue Y473 in the activation
function domain (AF-2) located on H12. Interestingly, the
superimposition of 7i on the cocrystal structure of
Rosiglitazone bound to PPARγ LBD (PDB ID: 4EMA)
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Table 4. Primary Single Dose DNS Screening of Selected Compounds for Cytotoxicity in a Panel of Seven Cell Lines at 10 μM
a
Concentration
% cell death at 10 μM
compound
CEM
Ramos
HL60
HeLa
MDA-MB-231
SH-SY5Y
HS-27
7c
7e
7f
7g
7i
7j
7l
7o
7q
7r
>40
25.87
>40
>40
>40
40.24
30.82
37.44
42.28
>40
>40
>40
>40
>40
>40
20.95
19.55
27.58
0
12.43
0
32.90
17.64
12.34
0
>40
39.09
29.68
>40
>40
>40
>40
>40
38.95
0
22.80
5.96
2.84
>40
>40
>40
31
0
>40
0
>40
0
10.79
0
>40
>40
>40
>40
0
13
>40
22.56
24.69
>40
0
24.90
0
15.32
>40
>40
29.78
34.53
35.18
3.91
>40
a
CEM: T cell leukemia, Ramos: Burkitt’s B cell lymphoma, HL60: promyelocytic leukemia (myeloblasts), HeLa: cervical adenocarcinoma
(epithelial), MDA-MB-231: triple negative breast (epithelial), SH-SY5Y: neuroblastoma (epithelial), HS-27: normal foreskin (fibroblasts).
revealed that the TZD headgroup of 7i undergoes a slight shift
toward H3, thus preventing the formation of a key H-bond
with residue Y473 (Figure 6C). Because stabilization of the
AF2 helix in the activated conformation is a key characteristic
of full agonism,65 the lack of direct interaction between this
domain of the receptor and 7i may explain the observed partial
agonist/modulator activities of this class of compounds. The
naphthalene moiety is placed in the center of the LBD and
makes hydrophobic interactions with C285, I326, Y327, L330,
F363, and M364. The phenyl amide tail lies in the subpocket
between H3, β-sheet, and Ω-loop (β-sheet subpocket),
establishing numerous nonpolar and polar interactions with
the participation of HOH708 and HOH706 in setting-up an
indirect H-bond network. Specifically, the oxygen atom bound
to the naphthalene moiety makes an H-bond with HOH708 (d
= 3.1 Å), which in turn interacts with the CO backbone of
L340 (d = 3.1 Å). The bromine atom in the ortho position of
the phenyl ring forms a halogen bond with the structural water
HOH706 (d = 3.1 Å), that in turn interacts with the NH
backbone of S342 (d = 3.1 Å). Hydrophobic contacts are also
observed between 7i and the subpocket residues V339, I341,
M348 (β-sheet); I281 (H3); and F264 (Ω-loop). These
interactions contribute to the stabilization of the H3/β-sheet/
Ω-loop region of the LBD, similar to other PPARγ partial
agonists such as BVT.13, MRL-24, and nTZDpa.66 It has in
fact been demonstrated that the activity of partial agonists,
which do not robustly stabilize H12, may stem from the
stabilization of the β-sheet subpocket.66−69 In addition, the
terminal phenyl ring appears to be optimally oriented for a
favorable edge-to-face π-stacking interaction with H266 on the
Ω-loop, which contributes to further increase the Ω-loop
stabilization. It is thus conceivable to suggest that ligands of
PPARγ can afford graded transcriptional responses by
employing a compensatory mechanism of β-sheet subpocket
interactions to balance the lack of H12 stabilization. However,
it is important to note that the Ω-loop is highly unstable and
the residues within this loop are quite flexible; thus, the
prediction of ligand/Ω-loop interactions might not be accurate
and must be interpreted accordingly.67,70
interacts with the CO backbone of S342 (d = 3.1 Å).
Moreover, the methyl group at the ortho position on the
terminal phenyl ring allows 7l to form additional hydrophobic
interaction with L353.
Further docking studies of 7o, 7r, 7c, 7q revealed that these
compounds were in the same position as 7i and 7l but were
unable to strongly stabilize the β-sheet, thus showing a
decrease in potency (Figure S5 in Supporting Information).
The low efficacy is due to the lack of the halogen bond with
HOH706, since bromine is placed in para position in 7o and
7r, and to steric clashes of methyl and nitro group in 7c and
7q, respectively, with H266 and F264 residues belonging to the
Ω-loop.
Cytotoxicity Assessment by DNS Assay. On the basis of
the prior experiments, it was discovered that derivatives 7c, 7i,
7l, 7o, 7r, and 7q were dual HDAC and PPARγ targeting
agents; hence, they were considered for further evaluation in
regard to their antitumor activity. Compounds 7e, 7f, and 7j
were also selected as they were selective for HDAC4, as was 7g
for its dual activity against HDAC4 and HDAC8. Thus, a total
of 10 compounds were screened via differential nuclear
staining (DNS) assay to assess their cytotoxic potential against
a panel of seven cell lines. The panel consisted of solid tumor
cells: HeLa (cervical adenocarcinoma, epithelial), MDA-MB-
231 (triple negative breast, epithelial), and SH-SY5Y (neuro-
blastoma, epithelial); hematological tumor cells: CCRF-CEM
(T-cell leukemia), Ramos (Burkitt’s B-cell lymphoma), and
HL60 promyelocytic leukemia, myeloblasts); and noncancer-
ous cells: HS-27 (normal foreskin, fibroblasts). A primary
single dose screening of all 10 compounds was performed at a
concentration of 10 μM, and those which elicited greater than
40% cell death (Table 4) were analyzed at several
concentrations to find their half maximal cytotoxic concen-
tration (CC50). CC50 values indicate the average concentration
of compound required to kill 50% of the sample population
and is reported as mean SD in Table 5.
Several experimental compounds were cytotoxic at <10.0
μM (Table 5) on nearly all cancer cell lines except SH-SY5Y;
however, compounds 7c and 7i were identified as the most
potent candidates from this series. The greatest potency of
compound 7c was against CEM (CC50 = 2.8 μM), Ramos
(CC50 = 7.0 μM), and HeLa (CC50 = 10.3 μM), whereas 7i
was active against CEM (CC50 = 9.6 μM), Ramos (CC50 = 9.6
μM), and MDA-MB-231 (CC50 = 9.8 μM). In comparing the
cytotoxicity of “series 7” compounds with existing HDAC or
Docking of 7l, which has a similar potency toward PPARγ as
7i, revealed a binding mode of this compound comparable to
that described above for 7i, with the only consistent difference
being the lack of the halogen bond, which is outweighed by the
formation of a H-bond between the carbonyl oxygen of the
phenylacetamide tail and HOH706 (3.5 Å), that in turn
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Compounds 7c and 7i Exhibit Selectivity toward
Tumorigenic Cells. To compare the cytotoxicity of
compounds 7c and 7i between tumorigenic and normal cells,
the CC50 value for each compound against HS-27 cells was
divided by the CC50 of each cancer cell line to generate
selective cytotoxicity index (SCI) values. SCI values greater
than 1.0 denote the ability of a given compound to kill cancer
cells more effectively than cells of noncancerous origin.
These results revealed substantial selectivity of experimental
compounds 7c and 7i toward CEM and other cancer cell lines
within our panel. Compound 7c was the most selective, with a
14.4 SCI in the CEM cell line, whereas compound 7i had an
SCI value of 4.2. Additional selectivity values for these
compounds (and others) are presented in Table 5. Based on
the potency and selectivity of 7c and 7i, CEM cells were the
chosen model for several experiments that are described
herein.
Apoptosis Assay. To determine whether cell death elicited
by experimental compounds 7c and 7i was occurring via
apoptosis or necrosis, an Annexin V-fluorescein isothiocya-
nate/propidium iodide (AnnexinV-FITC/PI) assay was
performed via flow cytometry. In this experiment, CEM cells
were treated in triplicate with the CC50 or 2xCC50 of 7c, 7i, or
SAHA (for comparison) and incubated for 72 h. 4% DMSO
(dimethyl sulfoxide), 1 mM H2O2(hydrogen peroxide), and
untreated cells were utilized as vehicle, positive, and negative
controls respectively (Figure S6 in Supporting Information).
Phosphatidylserine (PS) is a phospholipid which flips from the
inner to the outer leaflet of the plasma membrane and serves as
an “eat me” signal in apoptotic cells. Annexin V is a cellular
protein with high affinity for PS that is commonly conjugated
to the fluorochrome FITC and used as a probe for PS
translocation. Propidium iodide, however, is permeable to all
cells with compromised membranes. Apoptotic cell popula-
tions in this assay are those which stain positive for AnnexinV-
FITC, whereas necrotic cells are those which stain positively
for PI but negative for AnnexinV-FITC. This analysis identified
significant PS externalization in 7c (21.46 0.33%; p < 0.001)
and 7i (26.53 0.96%; p < 0.001) CC50-treated samples when
compared with the vehicle control, implying the stimulation of
apoptosis by these compounds (Figure 8). Dose (CC50 vs
2×CC50) displayed negligible effect on the proportion of
apoptotic cells. Approximately 40 000 events (cells) were
analyzed per sample, and statistical significance was deter-
mined by Student’s t test (Tables S3−S6 in Supporting
Information).
Table 5. CC50 Values of Selected Compounds (7c, 7e, 7f, 7g,
7i, 7j, 7l, 7o, 7q, 7r) and Standard Drugs (SAHA,
Pioglitazone)
a
compounds
7c
cell line
CEM
Ramos
HeLa
SH-SY5Y
HS-27
Ramos
CEM
Ramos
CEM
Ramos
HeLa
MDA-MB-231
CEM
Ramos
CC50 (μM)
SCI
2.8 0.31
7.0 1.17
10.3 0.56
49.5 1.09
40.2 4.75
6.2 0.28
10.6 0.22
9.1 0.08
10.7 0.41
9.3 0.01
10.4 0.58
9.8 0.22
9.6 0.15
9.6 0.14
10.4 0.16
9.8 0.1
21.8 0.41
39.9 1.95
10.0 0.39
10.2 0.52
10.3 0.12
52.3 3.09
9.9 0.25
91.1 4.26
10.1 0.04
55.8 1.56
40.9 3.16
16.0 0.69
57.0 8.45
0% @ 50 μM
0% @ 10 μM
2.5 0.13
2.8 1.47
14.4
5.8
3.9
0.8
-
7e
7f
>10
>10
>10
>10
>10
>10
>10
4.2
4.2
3.8
4.1
1.8
-
>10
>10
>10
>10
>10
>10
>10
>10
1.4
3.6
-
7g
7i
HeLa
MDA-MB-231
SH-SY5Y
HS-27
Ramos
HeLa
MDA-MB-231
SH-SY5Y
HeLa
SH-SY5Y
HeLa
MDA-MB-231
CEM
SH-SY5Y
HS-27
7j
7l
7o
7q
7r
Pioglitazone
SAHA
CEM
HeLa
CEM
HeLa
-
-
-
-
a
CC50 reported as average concentration std. deviation (μM); SCI
= CC50HS‑27/CC50cancer; Nd*-Not determined
PPARγ-targeted therapeutics, class I and II HDAC inhibitor
suberoylanilide hydroxamic acid (SAHA; CEM CC50 = 2.5 μM
and HeLa CC50 = 2.8 μM) displayed similar potency to 7c, and
highly selective PPARγ agonist pioglitazone had no cytotoxic
effect even at 50 μM. Dose−response curves (DRCs) for
compounds 7c and 7i against the CEM cell line are shown in
Figure 7.
Figure 7. Dose−response curves of compounds 7c and 7i toward CCRF-CEM; n = 3.
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Figure 8. AnnexinV-FITC/PI Assay in CCRF-CEM after 72 h exposure to 7c, 7i, and controls (SAHA, H2O2); n = 3, ***p < 0.001.
Figure 9. Cell cycle distributions in CCRF-CEM after 72 h exposure to 7c, 7i, and control SAHA (A). Graphical representation of distribution of
the cells treated with 7c, untreated control, DMSO, H2O2, and SAHA (B). Graphical representation of distribution of the cells treated with 7i
untreated control, DMSO, H2O2, and SAHA. Histograms of vehicle (C), compound 7i (D), compound 7c (E); n = 3, *p < 0.05, **p < 0.01, ***p
< 0.001.
Cell Cycle Analysis. To evaluate the effect of 7c and 7i on
the cell cycle, flow cytometry was used to quantify DNA
content within each cell sample. CEM cells were exposed, in
triplicate, to the CC50 or 2×CC50 of each compound for 72 h.
HDAC inhibitor SAHA was likewise included for comparison.
4% v/v DMSO, 1 mM H2O2, and untreated cells were utilized
as vehicle, positive, and negative controls repectively. After the
incubation period, cells were collected and resuspended in a
nuclear isolation medium containing DNA-intercalating dye
DAPI (NIM-DAPI) immediately prior to analysis. DAPI
fluorescent signal was used to quantify the replication status of
cells within each sample, as its intensity is directly proportional
to the amount of DNA. Approximately 40 000 events (cells)
were analyzed per sample, and statistical significance was
determined by Student’s t test (Tables S7−S9 in Supporting
Information).
DNA fragmentation, represented by the sub-G0/G1
population, is a key feature of apoptosis. Despite significant
apoptosis in our AnnexinV-FITC/PI assay at the same time
point (Figure 8), just 6% of sub-G0/G1 accumulation was
evident after treatment with 7c and 7i at either concentration.
Compound 7c did not have a significant effect on the cell
cycle, whereas G0/G1 arrest (p = 0.0103) was evident in 7i
CC50-treated samples. Thus, identifying a distinct effect of each
compound on the cell cycle which also differs from SAHA
(Figure 9).
Western Blotting. The c-Myc oncogene is known as a
master controller of tumor cell growth regulation and
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metabolism, which are closely inter-related.71 The metabolic
changes driven by c-Myc overexpression, which occurs often in
tumor cells, are essential to support the synthesis of nucleic
acids, and various proteins/lipids necessary for rapid cell
proliferation.72 Thus, downregulation of the c-Myc gene could
help to control proliferation of tumor cells. Treatment with
both PPARγ agonists and HDAC inhibitors have shown to
decrease expression of c-Myc. The PPARγ agonist class
glitazones, which include insulin-sensitizing drugs like
pioglitazone, have demonstrated an ability to downregulate
this gene in various cancer cells.73 When treated with HDAC
inhibitor SAHA, c-Myc is acetylated at L-323, and its
expression decreases, leading to TRAIL activation and
apoptosis.74 Thus, downregulation of c-Myc is an important
aspect of antiproliferative effects exerted by these classes of
compounds. Often, c-Myc is activated in different types of
leukemia and plays a vital role in the induction and progression
of leukemogenesis.75−78 To ascertain the effects of PPARγ and
HDAC dual targeting compound 7c in CEM (T-cell leukemia)
cells, immunoblotting was performed. Western blot analysis of
compound 7c in CEM cells revealed downregulation of the c-
Myc protein much greater than positive control pioglitazone
(Figure 10). To assess if the observed suppression of c-Myc
(like SAHA) and PPARγ agonists (like pioglitazone) are linked
to enhanced expression of cleaved caspases and apoptotic cell
death.82−84 In agreement with these observations, Western
blotting revealed an increased expression of cleaved caspase-3
with compound 7c treatment compared with positive control
pioglitazone (Figure 10).
The compounds of “series 7” are the very first evidence of
molecules simultaneously targeting HDAC and PPARγ. The
overall aim of these experiments was an initial evaluation of the
antitumor potential of this unique class of molecules. Several
analogues, particularly compound 7c, demonstrated effective
antitumor activity at micromolar concentrations and sub-
stantial selective cytotoxicity. Though further analyses are
needed to fully elucidate their mechanism of action,
preliminary data on “series 7” strongly supports apoptotic
cell death. This is evident by the translocation of PS
(AnnexinV-FITC/PI assay), DNA fragmentation (cell cycle
analysis), and cleavage of caspase-3 (Western blotting). In
addition, c-myc is an oncogene essential for cell cycle
progression in human tumor cells, and its suppression has
been associated with decreased cell proliferation and arrest,
predominantly in G0/G1 phase of the cell cycle.85,86 The
downregulation of c-myc that was detected in 7c-treated CEM
cells opens the possibility that other analogues in the “series 7”
may undergo c-myc dependent exit from the cell cycle and
subsequent (apoptotic) death. Such may be the case with
compound 7i, for example, considering the observation of G0/
G1 phase arrest.
In Vivo Evaluation. In vivo preclinical tumor growth
inhibition has been regarded as an indispensable experiment to
understand the expected activity of antitumor agents in
humans and thus serves as important milestone in anticancer
drug development. The most promising dual targeting
derivative 7c, was further evaluated in vivo using a standard
subcutaneous xenograft model produced using the CEM cell
line. To investigate the capacity of compound 7c to inhibit
murine tumor growth, SCID mice were implanted with CEM-
derived tumor cells and the tumor allowed to grow to an
approximate volume of 100 mm3. The animals were then
assigned to three groups (n = 6 mice per treatment group) for
treatment with control, Doxorubicin (20 mg/kg), or 7c (50
mg/kg). Treatments were given as described in the methods.
As shown in Figure 11, administration of compound 7c
significantly retarded tumor growth. This was reflected by
reduction in tumor volume by ∼48% compared with the
control group (Figure 11A) and a %TGI (tumor growth
inhibition) of 24% on day 18. Body weights of the mice were
not significantly affected by 7c or doxorubicin administration
when compared to the control group, and there were no
behavioral changes observed in any of the 7c treated mice.
These results suggest that 7c significantly inhibits tumor
growth with no signs of severe toxicity.
Figure 10. Western blotting of CEM cells incubated with compound
7c (10 μM) and positive control, pioglitazone (10 μM), and
untreated cells (UT) for 5 h; n = 2, *p < 0.05, **p < 0.0001. The
graph shows the normalized data with respect to TBP band.
Many HDAC inhibitors and PPARγ agonists with antitumor
capacity have been evaluated for in vivo efficacy, wherein both
classes of drugs have showed effective tumor regression at
relatively high doses from 50 mg/kg to 400 mg/kg, %TGI
ranging from 9% (for low doses) to 40% (at higher doses),
with animals showing no sign of severe toxicity. For example,
the recently studied HDAC inhibitor belinostat has shown in
vivo efficacy in pancreatic cancer at doses of 100 mg/kg in
nude mice. Also, its combination with gemcitabine had
synergistic effects.87 Similarly, vorinostat was used at a
concentration of 150 mg/kg in urothelial carcinoma.88 Several
was a cascading effect associated with upregulation of tumor
suppressor gene p53,79 this protein was also evaluated;
however, no upregulated expression was observed with
exposure to compound 7c. This p53-independence suggests
that c-Myc suppression could be a feature of the dual HDAC
inhibition and PPARγ agonism exerted by compound 7c.
Caspases are also known as key mediators of apoptosis,80
and among them, caspase-3 is a frequently activated death
protease, which catalyzes the cleavage of many important
cellular proteins leading to cell death.81 Both HDAC inhibitors
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Figure 11. (A) Growth curve of tumor volume vs time in CEM (T cell leukemia) xenografts treated with doxorubicin 20 mg/kg i.p.,7c 50 mg/kg
i.p., untreated saline i.p. Error bars represent standard error of the mean (SEM) n = 6. p < 0.001. (B) Excised tumor image of the animals treated
with doxorubicin 20 mg/kg i.p., 7c 50 mg/kg i.p., untreated saline i.p.
other newer HDAC inhibitors have also been evaluated in vivo
in various cancer models.89−94 In the case of in vivo evaluation
of PPARγ agonists in various cancer models, a similar trend is
observed.95−98 Hence, dual-targeting compound 7c with %TGI
of 24% appears more potent than single-targeted agents.
Considering its in vivo efficacy at higher doses, 7c analogues
and other HDAC inhibitors or PPARγ agonists might be used
as an adjuvant to existing chemotherapy to help reduce
resistance/toxicity issues and possibly enhance antitumor
effects in a synergistic manner. Hence, the in vivo
combinatorial effects of compound 7c should be further
evaluated.
were supported by in vivo tumor regression by compound 7c in
CCRF-CEM tumor xenografts. Taken together, this study
provides a theoretical basis for rational design of dual/
multitargeting agents that could be developed further as
anticancer therapeutics.
EXPERIMENTAL SECTION
■
Chemistry. Commercial grade reagents of make S D Fine, Sigma-
Aldrich or Research Lab were acquired from dealers in India. Thin-
layer chromatography (TLC) was done on precoated Merck Silica
Gel 60 F254. Melting points were determined by thermo-analytical
technique using DSC (Differential scanning calorimetry) device II
Nanotechnology (SEIKO) exstar DSC 6220. Infrared (FTIR) spectra
were found using Schimadzu FT/IR-8400S with use of direct
sampling procedure. 1H and 13C NMR spectra were noted on a
Bruker instrument at 400 MHz, and chemical shift values (δ) are
described in ppm. J values (Coupling constants) are stated in hertz
(Hz). Abbreviations in NMR interpretation are, s- singlet, d-doublet,
dd - doublet of doublet, t-triplet, m- multiplet, bs- broad singlet and q
- quartet. Mass spectra were recorded with LC-MS Agilent
Technologies 1260 Infinity instrument. The conditions of HPLC
chromatography: column - Hemochrome C18 (4.6*250 mm),
detector- PDA detector, detection wavelength - up to 400 nm, flow
rate - 1 mL/min, sample concentration - 10 ppm, oven temperature -
30 °C; technique - gradient elution with a run time of 15 min, mobile
phase - methanol:formic acid (0.1%) in 70:30 ratio, mass range-100 to
650 m/z. All final compounds achieved a minimum of 95% purity and
molecular ions (of fraction of HPLC elute sent in mass spectrometer)
detected in MS of was corresponding to the mass of the individual
compounds.
CONCLUSIONS
■
The clinical effectiveness of recent single target anticancer
drugs has been limited because of unexpected resistance and
target nonspecificity. Among the attempts to tackle this
concern, a strategy of multitargeting approach could be
applied which would be able to hit multiple cancer hallmarks
to achieve the desired pharmacological effectiveness with
reduced detrimental effects. The combination treatment of
HDAC inhibitors and PPARγ agonists increased the cytotoxic
effects in a synergistic/additive manner against different cancer
cell lines resulting in the arrest of proliferation and increased
apoptosis. On the basis of this literature evidence, in an
attempt to bring about a multitarget drug approach and
simultaneously target HDAC/PPARγ, a series of 25 novel
TZD-based naphthylidene derivatives were rationally designed
and successfully synthesized. Complete biological evaluation of
all compounds revealed their dual targeting ability by partial
activation of PPARγ and selective inhibition of HDAC4. In our
knowledge, this is the first ever attempt to develop agents that
simultaneously target HDAC and PPARγ, two potential targets
in cancer drug discovery. Out of 25 compounds synthesized
several were found to inhibit HDAC4 effectively and Six
compounds showed dual targeting activity with submicromolar
potencies; the most potent dual targeting agent 7i had PPARγ
EC50 = 0.245 μM and HDAC4 IC50 = 1.1 μM. Out of these six
compounds, 7c and 7i were also found to exhibit
antiproliferative effects against CEM cells with CC50 values
of 2.8 and 9.6 μM, respectively. Both of them also induced
apoptosis and caused a significant DNA fragmentation in cell
cycle analysis. Further, compound 7c was found to modulate
the expression of c-Myc and cleave caspase-3 in Western
blotting experiments. These in vitro antiproliferative effects
Synthesis and Structural Characterization Details. The
phenyl acetamide intermediates, 2a−2y, were prepared as per the
reported procedures and the details of the spectral data and structural
characterization could be found in our previous reports.99−101
Synthesis of 5-((6-Hydroxynaphthalen-2-yl)methylene)-
thiazolidine-2,4-dione (5). Compound 5 was synthesized as
previously described.52 In brief, toluene (10 mL), commercially
available 6-hydroxy-2-naphthaldehyde (3) (6.88 g, 0.04 mmol), and
2,4-thiazolidinedione (4) (4.68 g, 0.04 mmol) was mixed together. To
this mixture, was added a catalytic amount of piperidinium benzoate
and refluxed in Dean stark apparatus. The reaction was monitored for
completion by TLC, after 4−5 h reaction was stopped and mixture
was cooled to RT (room temperature). Solid precipitated was
collected by filtering under vacuum and washed with water to
obtained intermediate 5 (Scheme 1) in crystalline form, which was
further recrystallized from methanol. Yellow shiny crystals. Yield 8.8 g
(87%). M.P. charred at 300 °C. FTIR (cm−1) 3390, 3119, 1672, 1662,
1
1587. H NMR (400 MHz, DMSO-d6) δ ppm 7.16−7.19 (m, 2H),
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7.55 (d, J = 8.6 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.88−7.90 (m,
2H), 8.04 (d, J = 4.0 Hz, 1H), 10.21 (s, 1H), 12.59 (bs, 1H).
Synthesis of Compounds Containing “Ar” as Substituted
Aromatic Ring (7a−7l, 7o−7r, 7u−7v). To the stirred solution of
2a−2l, 7o−7r, and 7u−7v (0.004 mol) in dimethylformamide (DMF,
10 mL) along with potassium carbonate (0.007 mol) was added, 5-
((6-hydroxynaphthalen-2-yl)methylene)thiazolidine-2,4-dione, (5)
(0.008 mol) and stirred for 24−48 h at RT. The reaction was
immobilized with addition of 20 mL of water to get precipitates,
which were separated by vacuum filtration, washed several times with
water to remove solvent, and purified by TLC column chromatog-
raphy using ethyl acetate:hexane (30:70) as mobile phase and F254
silica gel as stationary phase by the continuous elution method.
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-phenylacetamide (7a). Yellow solid; Yield 49.5%; M.P.
(DSC) 330.3 °C; IR (cm−1) 3377, 1737, 1687, 1330; 1H NMR (400
MHz, DMSO-d6, δ ppm) 4.48 (s, 2H), 7.10 (t, J = 7.4 Hz, 1H), 7.34
(t, J = 8.0 Hz, 2H), 7.40−7.41 (m, 2H), 7.65 (d, J = 4.0 Hz, 3H),
7.91−7.92 (m, 1H), 7.92−7.95 (m, 1H), 8.02 (d, J = 8.0 Hz, 1H),
8.13 (s, 1H), 10.16 (s, 1H), 12.61 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) 67.173 (−CH2), 107.325−138.282 (Aromatic), 157.408
(CC, benzylidene), 166.135, 167.978 (CO); LC-MS (% Area,
RT, Theoretical mass, m/z, I%) 98.81%, 3.60 min, 355, 354 [(M−
H)+, 100%].
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(4-fluorophenyl)acetamide (7b). Yellow solid; Yield 55.7%;
M.P. (DSC) 340.9 °C; IR (cm−1) 3032, 1737, 1680, 1329; 1H NMR
(400 MHz, DMSO-d6, δ ppm) 4.86 (s, 2H), 7.18 (t, J = 8.0 Hz, 2H),
7.39−7.41 (m, 2H), 7.63−7.69 (m, 3H), 7.90−7.95 (m, 1H), 8.01−
8.03 (m, 1H), 8.13 (s, 1H), 10.23 (s, 1H), 12.64 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) 67.132 (−CH2), 107.343−134.687 (Ar-
omatic), 157.347 (CC, benzylidene), 166.118, 167.364, 167.968
(CO); LC-MS (% Area, RT, Theoretical mass, m/z, I%) 96.19%,
12.36 min, 422.07, 421.0 [(M−H)+, 100%].
(m, 1H), 7.35−7.41 (m, 3H), 7.55−7.58 (m, 1H), 7.63−7.66 (m,
1H), 7.85−7.86 (m, 2H), 7.90−7.94 (m, 1H), 8.00−8.03 (m, 1H),
8.13 (s, 1H), 10.35 (s, 1H), 12.62 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) 66.981 (−CH2), 107.151−134.680 (Aromatic), 157.875
(CC, benzylidene), 166.565, 167.553, 167.863 (CO); LC-MS
(% Area, RT, Theoretical mass, m/z, I%) 98.98%, 8.54 min, 438.04,
437.0 [(M−H)+, 100%].
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(3-methoxyphenyl)acetamide (7g). Yellow solid; Yield
48.0%; M.P. (DSC) 277.6 °C; IR (cm−1) 3016, 1743, 1681, 1334;
1H NMR (400 MHz, DMSO-d6, δ ppm) 3.74 (s, 3H), 4.86 (s, 2H),
6.69−6.69 (m, 1H), 7.23−7.23 (m, 2H), 7.36−7.40 (m, 3H), 7.63−
7.65 (m, 1H), 7.90−8.02 (m, 3H), 8.00−8.03 (m, 1H), 8.12 (s, 1H),
10.16 (s, 1H), 12.62 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
66.981 54.988 (−OCH3), 67.153 (−CH2), 105.423−139.477
(Aromatic), 157.384 (CC, benzylidene), 159.482, 166.182,
167.321, 167.947 (CO); LC-MS (% Area, RT, Theoretical mass,
m/z, I%) 95.83%, 6.16 min, 434.09, 433.1 [(M−H)+, 100%].
N-(3,4-Dichlorophenyl)-2-((6-((2,4-dioxothiazolidin-5-ylidene)-
methyl)naphthalen-2-yl)oxy)acetamide (7h). Yellow solid; Yield
36.7%; M.P. (DSC) 321.5 °C; IR (cm−1) 3076, 1735, 1685, 1325; 1H
NMR (400 MHz, DMSO-d6, δ ppm) 4.90 (s, 2H), 7.39−7.41 (m,
1H), 7.61−7.66 (m, 3H), 7.90−7.95 (m, 3H), 8.01−8.04 (m, 2H),
8.14 (s, 1H), 10.46 (s, 1H), 12.64 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) 66.505 (−CH2), 104.987−134.507 (Aromatic), 157.790
(CC, benzylidene), 166.630, 167.630 (CO); LC-MS (% Area,
RT, Theoretical mass, m/z, I%) 100.0%, 14.14 min, 471.01, 471.1
[(M−H)+, 100%].
N-(2-Bromo-4,6-difluorophenyl)-2-((6-((2,4-dioxothiazolidin-5-
ylidene)methyl)naphthalen-2-yl)oxy)acetamide (7i). Yellow solid;
Yield 25.6%; M.P. (DSC) 265.8 °C; IR (cm−1) 3230, 1735, 1685,
1
1348; H NMR (400 MHz, DMSO-d6, δ ppm) 4.94 (s, 2H), 7.43−
7.49 (m, 3H), 7.62−7.67 (m, 2H), 7.92−8.04 (m, 3H), 8.14 (s, 1H),
10.08 (s, 1H), 12.60 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
66.790 (−CH2), 104.491−134.630 (Aromatic), 157.138 (CC,
benzylidene), 166.987, 167.987 (CO); LC-MS (% Area, RT,
Theoretical mass, m/z, I%) 95.72%, 5.94 min, 517.97, 518.9 [(M
+H)+, 100%].
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(p-tolyl)acetamide (7c). Yellow solid; Yield 52.6%; M.P.
(DSC) 298.7 °C; IR (cm−1) 3074, 1737, 1693, 1349; 1H NMR (400
MHz, DMSO-d6, δ ppm) 2.26 (s, 3H), 4.85 (s, 2H), 7.14−7.15 (m,
2H), 7.40−7.41 (m, 2H), 7.53−7.55 (m, 3H), 7.91−7.94 (m, 3H),
8.13 (s, 1H), 10.09 (s, 1H), 12.64 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) 20.420 (−CH3), 67.168 (−CH2), 107.308−135.727
(Aromatic), 157.398 (CC, benzylidene), 165.912, 167.372,
167.991 (CO); LC-MS (% Area, RT, Theoretical mass, m/z, I%)
98.73%, 6.70 min, 418.1, 417.1 [(M−H)+, 100%].
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(2-phenoxyphenyl)acetamide (7j). Yellow solid; Yield 49.0%;
M.P. (DSC). 260.0 °C; IR (cm−1) 3074, 1737, 1680, 1330; 1H NMR
(400 MHz, DMSO-d6, δ ppm) 4.87 (s, 2H), 6.95−7.00 (m, 3H),
7.12−7.20 (m, 3H), 7.24 (d, J = 8.0 Hz, 1H), 7.34−7.39 (m, 3H),
7.63 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.90 (s, 1H), 7.96
(d, J = 8.0 Hz, 1H), 8.1 (m, 2H), 10.16 (s, 1H), 12.61 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) 66.790 (−CH2), 104.491−134.630
(Aromatic), 157.138 (CC, benzylidene), 166.987, 167.987 (C
O); LC-MS (% Area, RT, Theoretical mass, m/z, I%) 95.72%, 5.94
min, 517.97, 518.9 [(M+H)+, 100%].
N-(2,4-Difluorophenyl)-2-((6-((2,4-dioxothiazolidin-5-ylidene)-
methyl)naphthalen-2-yl)oxy)acetamide (7k). Yellow solid; Yield
50.3%; M.P. (DSC). 306.1 °C; IR (cm−1) 3290, 1755, 1685, 1329; 1H
NMR (400 MHz, DMSO-d6, δ ppm) 4.92 (s, 2H), 7.10 (m, 1H),
7.34−7.41 (m, 3H), 7.65−7.67 (m, 1H), 7.73−7.75 (m, 1H), 7.89−
7.94 (m, 2H), 8.02 (d, J = 8 Hz, 1H), 8.13 (s, 1H), 10.02 (s, 1H),
12.64 (s, 1H); 13C NMR (100 MHz, DMSO-d6) 66.987 (−CH2),
107.621−134.680 (Aromatic), 157.875 (CC, benzylidene),
166.565, 167.553, 167.863 (CO); LC-MS (% Area, RT,
Theoretical mass, m/z, I%) 99.00%, 6.44 min, 440.06, 439.1 [(M−
H)+, 100%].
N-(4-Bromo-2-methylphenyl)-2-((6-((2,4-dioxothiazolidin-5-
ylidene)methyl)naphthalen-2-yl)oxy)acetamide (7l). Yellow solid;
Yield 55.9%; M.P. (DSC) 301.7 °C; IR (cm−1) 3288, 1732, 1687,
1348; 1H NMR (400 MHz, DMSO-d6, δ ppm) 2.18 (s. 3H), 4.90 (s,
2H), 7.38−7.46 (m, 5H), 7.64−7.67 (m, 1H), 7.90−7.94 (m, 2H),
8.01 (d, J = 12 Hz, 1H), 8.13 (s, 1H), 9.65 (s, 1H), 12.64 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) 19.096 (−CH3), 79.658 (−CH2),
114.429−134.589 (Aromatic), 157.952 (CC, benzylidene),
162.611 (CO); LC-MS (% Area, RT, Theoretical mass, m/z, I%)
98.07%, 10.63 min, 496.01, 497.0 [(M+H)+, 100%].
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(4-methoxyphenyl)acetamide (7d). Yellow solid; Yield
53.6%; M.P. (DSC) 290.6 °C; IR (cm−1) 3041, 1739, 1683, 1327;
1H NMR (400 MHz, DMSO-d6, δ ppm) 3.72 (s, 3H), 4.83 (s, 2H),
6.89−6.94 (m, 2H), 7.33−7.41 (m, 2H), 7.55−7.57 (m, 2H), 7.63−
7.65 (m, 1H), 7.90−7.94 (m, 2H), 8.00−8.03 (m, 1H), 8.13 (s, 1H),
10.02 (s, 1H), 12.68 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
54.947 (−OCH3), 67.182 (−CH2), 105.153−139.947 (Aromatic),
157.549 (CC, benzylidene), 166.856, 167.420, 167.526 (CO);
LC-MS (% Area, RT, Theoretical mass, m/z, I%) 99.40%, 5.71 min,
434.09, 433.1 [(M−H)+, 100%].
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(3-(trifluoromethyl)phenyl) acetamide (7e). Yellow solid;
Yield 59.2%; M.P. (DSC) 292.8 °C; IR (cm−1) 3055, 1749, 1687,
1
1332; H NMR (400 MHz, DMSO-d6, δ ppm) 4.91 (s, 2H), 7.39−
7.46 (m, 3H), 7.57−7.61 (m, 1H), 7.64−7.66 (m, 1H), 7.90−7.95
(m, 3H), 8.02 (d, J = 8.0 Hz, 1H), 8.14−8.15 (m, 2H), 10.16 (s, 1H),
12.61 (s, 1H); 13C NMR (100 MHz, DMSO-d6) 66.670 (−CH2),
107.065−135.851 (Aromatic), 157.501 (CC, benzylidene),
166.506, 166.562, 167.868 (CO); LC-MS (% Area, RT,
Theoretical mass, m/z, I%) 95.54%, 7.37 min, 472.1, 471.1 [(M−
H)+, 100%].
N-(3-Chlorophenyl)-2-((6-((2,4-dioxothiazolidin-5-ylidene)-
methyl)naphthalen-2-yl)oxy)acetamide (7f). Yellow solid; Yield
42.4%; M.P. (DSC) 322.5 °C; IR (cm−1) 3093, 1737, 1679, 1330,
677; 1H NMR (400 MHz, DMSO-d6, δ ppm) 4.89 (s, 2H), 7.14−7.17
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N-(4-Bromophenyl)-2-((6-((2,4-dioxothiazolidin-5-ylidene)-
methyl)naphthalen-2-yl)oxy)acetamide (7o). Yellow solid; Yield
51.2%; M.P. (DSC) 316.3 °C; IR (cm−1) 3028, 1730, 1691, 1352; 1H
NMR (400 MHz, DMSO-d6, δ ppm) 4.87 (s, 2H), 7.38−7.41 (m,
2H), 7.51−7.53 (m, 2H), 7.64−7.66 (m, 3H), 7.91−7.94 (m, 2H),
8.02 (d, J = 12 Hz, 1H), 8.13 (s, 1H), 10.31 (s, 1H), 12.61 (s, 1H);
13C NMR (100 MHz, DMSO-d6) 67.159 (−CH2), 107.355−137.682
(Aromatic), 157.338 (CC, benzylidene), 166.364, 167.306,
167.928 (CO); LC-MS (% Area, RT, Theoretical mass, m/z, I%)
98.84%, 10.55 min, 481.99, 482.9 [(M+H)+, 100%].
2-((6-Formylnaphthalen-2-yl)oxy)-N-(pyridin-2-yl)acetamide
(6m). Brown solid; Yield 63.3%; M.P. 188.9 °C; IR (cm−1) 2918,
1
1722, 1678, 1301; H NMR (400 MHz, DMSO-d6, δ ppm) 4.92 (s,
2H), 7.02 (d, J = 16.0 Hz, 2H), 7.41−7.44 (m, 2H), 7.67−7.70 (m,
1H), 7.83−7.86 (m, 3H), 7.91−7.94 (m, 1H), 8.10−8.12 (m, 1H),
8.53 (s, 1H), 10.06 (s, 1H), 10.62 (s, 1H).
2-((6-Formylnaphthalen-2-yl)oxy)-N-(6-methylpyridin-2-yl)-
acetamide (6n). Brown solid; Yield 75.6%; M.P. 148.9 °C; IR (cm−1)
1
2922, 1722, 1678, 1303; H NMR (400 MHz, DMSO-d6, δ ppm)
2.43 (s, 3H), 4.98 (s, 2H), 7.01 (d, J = 8.0 Hz, 2H), 7.40−7.43 (m,
2H), 7.66−7.70 (m, 1H), 7.84−7.88 (m, 2H), 7.92−7.94 (m, 1H),
8.11−8.13 (m, 1H), 8.51 (s, 1H), 10.08 (s, 1H), 10.59 (s, 1H).
N-(6-Fluoropyridin-2-yl)-2-((6-formylnaphthalen-2-yl)oxy)-
acetamide (6s). Brown solid; Yield 56.4%; M.P. 202.9 °C; IR (cm−1)
N-(3-Chloro-5-(trifluoromethyl)phenyl)-2-((6-((2,4-dioxothiazoli-
din-5-ylidene)methyl)naphthalen-2-yl)oxy)acetamide (7p). Yellow
solid; Yield 44.4%; M.P. (DSC) 313.9 °C; IR (cm−1) 2968, 1726,
1
1691, 1327; H NMR (400 MHz, DMSO-d6, δ ppm) 4.97 (s, 2H),
1
3066, 1721, 1678, 1334; H NMR (400 MHz, DMSO-d6, δ ppm)
7.36−7.38 (m, 2H), 7.64 (t, J = 4.0 Hz, 3H), 7.91−7.94 (m, 2H),
8.01 (d, J = 8.0 Hz, 2H), 8.30 (s, 1H), 11.11 (s, 1H), 12.68 (s, 1H);
13C NMR (100 MHz, DMSO-d6) 67.851 (−CH2), 107.132−134.554
(Aromatic), 157.521 (CC, benzylidene), 166.029, 167.687,
167.347 (CO); LC-MS (% Area, RT, Theoretical mass, m/z, I%)
99.44%, 9.38 min, 506.03, 505.0 [(M−H)+, 100%].
4.99 (s, 2H), 7.01−7.03 (m, 2H), 7.41−7.43 (m, 2H), 7.69−7.72 (m,
1H), 7.84−7.86 (m, 2H), 7.92−7.94 (m, 1H), 8.11−8.13 (m, 1H),
8.51 (s, 1H), 10.06 (s, 1H), 10.58 (s, 1H).
2-((6-Formylnaphthalen-2-yl)oxy)-N-(5-methylisoxazol-3-yl)-
acetamide (6t). White solid; Yield 56.1%; M.P. 213.5 °C; IR (cm−1)
3300, 1722, 1678, 1334; 1H NMR (400 MHz, DMSO-d6, δ
ppm)2.26(s, 3H), 4.85 (m, 1H), 7.13 (d, J = 8.0 Hz,1H), 7.43−
7.44(m, 1H), 7.53−7.57 (m, 1H), 7.85−7.87 (m, 1H), 7.93−7.95(m,
1H), 8.13(d, J = 8.0 Hz, 1H), 8.51 (s, 1H), 10.04 (s, 1H), 10.08 (s,
1H).
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(4-nitrophenyl)acetamide (7q). Yellow solid; Yield 39.0%;
M.P. (DSC) 332.0 °C; IR (cm−1) 3010, 1734, 1689, 1329; 1H NMR
(400 MHz, DMSO-d6, δ ppm) 4.96 (s, 2H), 7.39−7.42 (m, 2H), 7.65
(d, J = 8.0 Hz, 2H), 7.92−7.95 (m, 3H), 8.03 (d, J = 8.0 Hz, 1H),
8.14 (s, 1H), 8.25−8.27 (m, 2H), 10.80 (s, 1H), 12.61 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) 67.073 (−CH2), 119.389−131.760
(Aromatic), 143.684 (C-NO2 str.), 157.338 (CC, benzylidene),
162.367 (CO); LC-MS (% Area, RT, Theoretical mass, m/z, I%)
98.10%, 8.60 min, 449.07, 448.0 [(M−H)+, 100%].
N-(Benzo[d]thiazol-2-yl)-2-((6-formylnaphthalen-2-yl)oxy)-
acetamide (6w). Brownish solid; Yield 41.1%; M.P. 285.2 °C; IR
1
(cm−1) 3383, 1726, 1680, 1334; H NMR (400 MHz, DMSO-d6, δ
ppm) 4.82 (s, 2H), 7.02−7.05 (m, 1H), 7.20−7.23 (m, 1H), 7.36−
7.38 (m, 2H), 7.47−7.48 (d, J = 4.0 Hz,1H), 7.67−7.69 (d, J = 8.0
Hz, 1H), 7.80 (m, 1H), 7.82 (m, 1H), 7.88−7.89 (m, 1H), 8.08−8.10
(d, J = 8.0 Hz, 1H), 8.49 (s, 1H), 10.06 (s, 1H).
N-(4-Bromo-2-fluorophenyl)-2-((6-((2,4-dioxothiazolidin-5-
ylidene)methyl)naphthalen-2-yl)oxy)acetamide (7r). Yellow solid;
Yield 32.6%; M.P. (DSC) 314.6 °C; IR (cm−1) 3317, 1732, 1687,
2-((6-Formylnaphthalen-2-yl)oxy)-N-(4-methylbenzo[d]thiazol-
2-yl)acetamide (6x). Brownish solid; Yield 28.4%; M.P. 243.1 °C; IR
1
1319; H NMR (400 MHz, DMSO-d6, δ ppm) 4.95 (s, 2H), 7.37−
1
(cm−1)3161, 1693, 1658, 1332; H NMR (400 MHz, DMSO-d6, δ
7.43 (m, 3H), 7.63−7.66 (m, 2H), 7.79−7.83 (m, 1H), 7.91−7.94
(m, 2H), 8.02 (d, J = 8 Hz, 1H), 8.14 (s, 1H), 10.16 (s, 1H), 12.61 (s,
1H); 13C NMR (100 MHz, DMSO-d6) 66.670 (−CH2), 107.236−
135.584 (Aromatic), 157.433 (CC, benzylidene), 166.622,
166.710, 167.921 (CO); LC-MS (% Area, RT, Theoretical mass,
m/z, I%) 99.23%, 11.00 min, 499.98, 500.9[(M+H)+, 100%].
N-(3-Chloro-4-methylphenyl)-2-((6-((2,4-dioxothiazolidin-5-
ylidene)methyl)naphthalen-2-yl)oxy)acetamide (7u). Yellow solid;
Yield 44.4%; M.P. (DSC) 304.0 °C; IR (cm−1) 3223, 1737, 1678,
1323; 1H NMR (400 MHz, DMSO-d6, δ ppm) 2.28 (s, 3H), 4.86 (s,
2H), 7.30−7.32 (m, 1H), 7.39−7.41 (m, 2H), 7.47−7.49 (m, 1H),
7.65 (d, J = 8.0 Hz, 1H), 7.84 (s, 1H), 7.91−7.95 (m, 2H), 8.02 (d, J
= 8.0 Hz, 1H), 8.29 (s, 1H), 10.24 (s, 1H), 12.63 (s, 1H); 13C NMR
(100 MHz, DMSO-d6) 19.658 (−CH3), 79.096 (−CH2), 114.294−
134.611 (Aromatic), 157.333 (CC, benzylidene), 162.429 (C
O); LC-MS (% Area, RT, Theoretical mass, m/z, I%) 100.0%, 11.92
min, 452.06, 451.1 [(M−H)+, 100%].
N-(3,4-Dibromophenyl)-2-((6-((2,4-dioxothiazolidin-5-ylidene)-
methyl)naphthalen-2-yl)oxy)acetamide (7v). Yellow solid; Yield
51.9%; M.P. (DSC) 318.4 °C; IR (cm−1) 3061, 1735, 1680, 1325; 1H
NMR (400 MHz, DMSO-d6, δ ppm) 4.90 (s, 2H), 7.40−7.42 (m,
2H), 7.61−7.67 (m, 3H), 7.91−7.95 (m, 3H), 8.02−8.05 (m, 1H),
8.30 (s, 1H), 10.45 (s, 1H), 12.62 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) 79.120 (−CH2), 117.474−134.671 (Aromatic), 157.429
(CC, benzylidene), 162.290, 167.063 (CO); LC-MS (% Area,
RT, Theoretical mass, m/z, I%) 100.0%, 13.32 min, 559.9, 558.9
[(M−H)+, 100%].
ppm) 2.60 (s, 3H), 5.09 (s, 2H), 7.31 (t, J = 8.0 Hz,1H), 7.34 (m,
1H), 7.45 (t, J = 8.0 Hz,1H), 7.61 (d, J = 4.0 Hz, 1H),), 7.78 (d, J =
8.0 Hz, 1H), 7.88 (s, 1H), 7.91−9.32 (m, 1H), 7.97−8.02 (m, 2H),
8.11 (s, 1H), 10.57 (s, 1H).
N-(6-Ethoxybenzo[d]thiazol-2-yl)-2-((6-formylnaphthalen-2-yl)-
oxy)acetamide (6y). Brownish solid; Yield 33.2%; M.P. 263.8 °C; IR
1
(cm−1) 3342, 1680, 1384; H NMR (400 MHz, DMSO-d6, δ ppm)
1.30−1.33 (m, 3H), 3.98−4.07 (m, 2H), 4.79 (s, 2H), 6.80−6.82 (m,
1H), 7.26 (m, 1H), 7.35−7.37 (m, 3H), 7.80−7.82 (m, 1H), 7.87−
7.89 (m, 1H), 8.07−8.09 (m, 1H), 8.48 (s, 1H), 10.06 (s, 1H).
Synthesis of Compounds Containing “Ar” as Substituted
Heterocyclic Ring: 7m, 7n, 7s, and 7t. Final compound 7m was
synthesized by adopting Scheme 2, wherein 2,4-thiazolidinedione (3)
(0.005 mol) was dissolved in 2-methoxyethanol (10 mL). To this
solution was added piperidine (0.4 mL) dropwise followed by
addition of intermediate, 6m (0.002 mol). The reaction mixture was
refluxed for 3−5 h and monitored by TLC, after which the reaction
was stopped, cooled, and the precipitated solid was collected by
filtration and washed with water (3 × 50 mL) followed by methanol
(3 × 10 mL). The crude product thus obtained was purified by TLC
column chromatography by using hexane:ethyl acetate (50:50 to
25:75 ratio) to give the final products 7m. The other products 7n, 7s,
and 7t were obtained by following the same procedure.
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(pyridin-2-yl)acetamide (7m). Light brown solid; Yield
46.2%; M.P. (DSC) 324.8 °C; IR (cm−1) 3078, 1718, 1681, 1301;
1H NMR (400 MHz, DMSO-d6, δ ppm) 4.97 (s, 2H), 7.14−7.17 (m,
Synthesis of Intermediate 6m, 6n, 6s, 6t, 6w−6y. To the
stirred solution of 6-hydroxynaphthaldehyde (3) (0.008 mol) along
with potassium carbonate (0.007 mol) in DMF was added the
appropriate phenyl acetamides (2m, 2n, 2s, 2t, 2w−2y) (0.004 mol),
and the reaction mixture was stirred on magnetic stirrer for 36 h for
6m, 6n, 6s, 6t and for 48 h for 6w−6y. After completion of the
reaction water was added to terminate the reaction and generate solid.
The solid obtained was washed with water (3 × 50 mL) and dried. It
was purified by recrystallization from the appropriate solvents.
1H), 7.36−7.38 (m, 2H), 7.64 (d, J = 8.0 Hz, 1H), 7.81 (t, J = 8.0 Hz,
1H), 7.90−7.93 (m, 2H), 8.05−8.07 (m, 2H), 8.13 (s, 1H), 8.35−
8.36 (m, 1H), 10.57 (s, 1H), 12.64 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) 71.033 (−CH2), 107.295−138.393 (Aromatic), 148.148
(CC, benzylidene), 167.001 (CO); LC-MS (% Area, RT,
Theoretical mass, m/z, I%) 97.19%, 5.78 min, 405.08, 404.0 [(M−
H)+, 100%].
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(6-methylpyridin-2-yl)acetamide (7n). Light brown solid;
6964
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Article
1
Yield 42.2%; M.P. (DSC) 274.3 °C; IR (cm−1) 3030, 1726, 1689,
1348; 1H NMR (400 MHz, DMSO-d6, δ ppm) 2.43 (s, 3H), 4.95 (s,
2H), 7.00 (d, J = 4.0 Hz, 1H), 7.35−7.37 (m, 2H), 7.62−7.65 (m,
1H), 7.69 (d, J = 8.0 Hz, 1H), 7.90−7.92 (m, 3H), 7.99−8.01 (m, 1),
8.13 (s, 1H), 10.56 (s, 1H), 12.61 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) 22.497 (−CH3), 71.033 (−CH2), 107.393−138.952
(Aromatic), 148.001 (CC, benzylidene), 167.148 (CO); LC-
MS (% Area, RT, Theoretical mass, m/z, I%) 97.41%, 7.88 min,
419.09, 418.1 [(M−H)+, 100%].
1332; H NMR (400 MHz, DMSO-d6, δ ppm) 2.83−2.88 (m, 3H),
4.05−4.06 (m, 2H), 5.06 (s, 2H), 7.03−7.04 (m, 1H), 7.39−7.46 (m,
2H), 7.55 (s, 1H), 7.62−7.69 (m, 1H), 7.86−7.94 (m, 2H), 7.99−
8.01 (m, 1H), 8.11−8.14 (m, 1H), 8.51 (s, 1H), 10.08 (s, 1H), 12.54
(s, 1H); 13C NMR (100 MHz, DMSO-d6) 14.624 (−CH3), 64.552
(O−CH2−CH3), 67.347 (−CH2), 107.398−132.003 (Aromatic),
157.302 (CC, benzylidene), 192.785 (CO); LC-MS (% Area,
RT, Theoretical mass, m/z, I%) 100.0%, 13.36 min, 505.08, 504.1
[(M−H)+, 100%].
HDAC4/8 Activity Assay. HDAC8 was produced by recombinant
technique as described recently.52,102 Recombinant cHDAC4 was
expressed using a pET14b vector (Novagen, EMD Millipore)
containing the codon-optimized catalytic domain of human
HDAC4. The assay was performed as described recently.52
Consecutive dilutions of test compounds in a buffer (75 mM KCl,
25 mM Tris-HCl, pH 8.0, 0.001% Pluronic F-127) were made. Test
compound solutions were added and allowed to incubate for 1 h with
HDAC8 and HDAC4 isoforms, in a 96-well black microtiter half a rea
plate (Greiner) at 30 °C. Then the reaction was started by adding 20
μM of Boc-Lys(trifluoroacetyl)-AMC (Bachem) as a substrate for
HDAC4 and HDAC8. After incubation for 60 min at 30 °C, the
reaction was terminated by adding 1.7 μM SATFMK for both
HDAC4 and HDAC8. The deacetylated substrate was transformed
into a fluorescent product by adding 0.4 mg/mL trypsin (Applichem).
The release of AMC was traced in a microplate reader (PheraStar
Plus, BMG Labtech) at 450 nm (λEx = 350 nm) and were correlated
to the activity of enzyme. Dose−response curves (DRCs) were
plotted, and IC50 values were calculated.
Docking Method for HDAC4 and HDAC8. Modeling,
preparation, and visualization of structural data as well as molecular
docking was performed using MOE 2019 software (Chemical
Computing Group ULC, Canada). Three crystal structures, PDB
IDs 3SFF, 4CBY and 2VQJ, were obtained from RCSB protein data
bank and subjected to the Quickprep procedure of MOE 2019
including 3D protonation for subsequent docking. The partial charges
of all protein and ligand atoms were calculated using the implemented
Amber14 force field. The docking site was defined by the ligand
within the binding pocket of the respective crystal structure. This
approach was not permissible for the open conformation of HDAC4
(PDB ID: 2VQJ) since the ligand covered only part of the
significantly enlarged binding groove. In this special case, the binding
site was analyzed using the Computed Atlas of Surface Topography of
proteins (CASTp) (http://sts.bioe.uic.edu/castp/index.
html?201l).103 The largest identified pocket with an estimated volume
of 1019,7 Å3 was identical with the widely opened binding groove of
HDAC4o. The flanking amino acids of this pocket (G36, R37, G331,
G330, H198, H158, H159, F227, P156, P155, F168, S123, R154)
were used to define the binding site of HDAC4o for the subsequent
docking procedure. Molecular docking was performed choosing the
triangle matcher for placement of the ligand in the binding site and
ranked with the London dG scoring function. The best 50 poses were
passed to the refinement and energy minimization in the pocket using
the induced fit method, and the 10 best poses were rescored using the
GBVI/WSA dG scoring function.
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(6-fluoropyridin-2-yl)acetamide (7s). Yellow solid; Yield
43.9%; M.P. (DSC) 307.6 °C; IR (cm−1) 3115, 1712, 1680, 1352;
1H NMR (400 MHz, DMSO-d6, δ ppm) 4.96 (s, 2H), 7.36−7.38 (m,
2H), 7.63−7.65 (m, 1H), 7.71−7.81 (m, 1H), 7.90−7.93 (m, 2H),
8.00 (d, J = 8.0 Hz, 1H), 8.10−8.12 (m, 2H), 8.37(s, 1H), 10.74 (s,
1H), 12.62 (s, 1H); 13C NMR (100 MHz, DMSO-d6) 66.981
(−CH2), 107.372−134.692 (Aromatic), 147.009 (CN str.),
157.448 (CC, benzylidene), 166.364, 167.306, 167.928 (CO);
LC-MS (% Area, RT, Theoretical mass, m/z, I%) 97.77%, 6.02 min,
423.07, 422.0 [(M−H)+, 100%].
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(5-methylisoxazol-3-yl)acetamide (7t). Yellow solid; Yield
36.7%; M.P. (DSC) 292.4 °C; IR (cm−1) 3010, 1743, 1676, 1350; 1H
NMR (400 MHz, DMSO-d6, δ ppm) 2.38 (s, 3H), 4.93 (s, 2H), 6.64
(s, 1H), 7.36−7.38 (m, 2H), 7.63−7.66 (m, 1H), 7.91−7.94 (m, 2H),
8.01 (d, J = 8.0 Hz, 1H), 8.3 (s, 1H), 11.18 (s, 1H), 12.61 (s, 1H);
13C NMR (100 MHz, DMSO-d6) 17.898 (−CH3), 66.196 (−CH2),
107.415−134.649 (Aromatic), 157.332 (CC, benzylidene),
167.333, 168.172 (CO); LC-MS (% Area, RT, Theoretical mass,
m/z, I%) 98.32%, 5.28 min, 409.07, 408.0 [(M−H)+, 100%].
Synthesis of Compounds Containing “Ar” as Substituted
Heteroaryl Ring: 7w, 7x, and 7y. Final compound 7w was
synthesized by the route depicted in Scheme 3, wherein 2,4-
thiazolidinedione (3) (0.005 mol) was dissolved in DMF (10 mL).
To this solution, was added piperidine (0.8 mL) dropwise followed by
addition of intermediate, 6w (0.002 mol). The reaction mixture was
refluxed for 3−5 h and monitored by TLC, after which the reaction
was stopped, cooled, and 3.0 mL of 30% acetic acid was added to
generate the solid which was collected by filtration and washed with
water (3 × 50 mL) followed by methanol (3 × 10 mL). The crude
product thus obtained was purified by TLC column chromatography
by using hexane:ethyl acetate (50:50 to 25:75 ratio) to give the final
products 7w.
The other products 7x and 7y were obtained with a similar
procedure.
N-(Benzo[d]thiazol-2-yl)-2-((6-((2,4-dioxothiazolidin-5-ylidene)-
methyl)naphthalen-2-yl)oxy)acetamide (7w). Yellow solid; Yield
25.5%; M.P. (DSC) 342.0 °C; IR (cm−1) 3171, 1735, 1685, 1325; 1H
NMR (400 MHz, DMSO-d6, δ ppm) 5.10 (s, 2H), 7.34 (t, J = 6.0 Hz,
1H), 7.40−7.42 (m, 2H), 7.47 (t, J = 8.0 Hz, 1H), 7.65 (d, J = 8.0 Hz,
1H), 7.79 (d, J = 8.0 Hz, 1H), 7.91−7.95 (m, 2H), 7.99−8.04 (m,
2H), 8.14 (s, 1H), 10.24 (s, 1H), 12.61 (s, 1H); 13C NMR (100
MHz, DMSO-d6) 66.245 (−CH2), 107.235−134.605 (Aromatic),
148.279 (CN str.), 157.429 (CC, benzylidene), 162.331,
167.485, 168.136 (CO); LC-MS (% Area, RT, Theoretical mass,
m/z, I%) 99.27%, 9.19 min, 461.05, 460.0 [(M−H)+, 100%].
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(4-methylbenzo[d]thiazol-2-yl)acetamide (7x). Yellow solid;
Yield 20.5%; M.P. (DSC) 331.2 °C; IR (cm−1) 3113, 1737, 1689,
1325; 1H NMR (400 MHz, DMSO-d6, δ ppm) 2.60 (s, 3H), 4.87 (s,
2H), 7.21−7.24 (m, 2H), 7.27−7.29 (m, 2H), 7.65 (d, J = 8.0 Hz,
1H), 7.80 (d, J = 8.0 Hz, 1H), 7.91−7.95 (m, 2H), 8.03 (d, J = 8.0
Hz, 1H), 8.14 (s, 1H), 10.24 (s, 1H), 12.78 (s, 1H); 13C NMR (100
MHz, DMSO-d6) 17.898 (−CH3), 66.196 (−CH2), 107.415−
134.649 (Aromatic), 157.332 (CC, benzylidene), 167.333,
168.172 (CO); LC-MS (% Area, RT, Theoretical mass, m/z, I%)
100.0%, 8.58 min, 475.07, 474.1 [(M−H)+, 100%].
PPARγ Transactivation Assay. Plasmids. The expression vector
expressing the chimeric receptor containing the yeast Gal4 DNA
binding domain fused to the human PPARγ ligand binding domain
(LBD), and the reporter plasmid for this Gal4 chimeric receptor
(pGal5TKpGL3) containing five repeats of the Gal4 response
elements upstream of a minimal thymidine kinase promoter that is
adjacent to the luciferase gene were described previously.104
Cell Culture and Transfections. Human hepatoblastoma cell line
HepG2 (Interlab Cell Line Collection, Genoa, Italy) was cultured in
minimum essential medium (MEM) containing 10% heat-inactivated
fetal bovine serum, penicillin G (100 U mL−1), and streptomycin
sulfate (100 μg mL−1) at 37 °C in a humidified atmosphere of 5%
CO2. For transactivation assays, 105 cells per well were seeded in a 24-
well plate, and transfections were performed after 24 h with
CAPHOS, a calcium-phosphate method, according to the manufac-
turer’s guidelines. Cells were transfected with expression plasmids
2-((6-((2,4-Dioxothiazolidin-5-ylidene)methyl)naphthalen-2-yl)-
oxy)-N-(6-ethoxybenzo[d]thiazol-2-yl)acetamide (7y). Yellow solid;
Yield 20.7%; M.P. (DSC). 347.5 °C; IR (cm−1) 3288, 1732, 1681,
6965
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Article
encoding the fusion protein Gal4−PPARγ LBD (30 ng),
pGal5TKpGL3 (100 ng), pCMVβgal (250 ng). Four hours after
transfection, cells were treated for 20 h with the indicated ligands in
triplicate. Luciferase activity in cell extracts was then determined by a
luminometer (VICTOR3 V Multilabel Plate Reader, PerkinElmer). β-
Galactosidase activity was determined using ortho-nitrophenyl-β-D-
galactopyranoside as described previously.105,106 All transfection
experiments were repeated at least twice. Reference compounds,
media, and other cell culture reagents were purchased from Sigma-
Aldrich (Milan, Italy).
Cytotoxicity Assessment by DNS Assay. Cell Lines and
Culture Conditions. In this study, seven human cell lines were
utilized: three leukemia/lymphoma, CCRF-CEM, Ramos, HL-60; one
cervical, HeLa; one triple negative breast, MDA-MB-231; one
neuroblastoma, SH-SY5Y; and one noncancerous foreskin fibroblast
for comparison, HS-27. Cell lines CCRF-CEM, Ramos, and HL-60
were cultured in RPMI-1640 medium supplemented with 10% heat-
inactivated fetal bovine serum (HI FBS), 100 U/mL penicillin, and
100 μg/mL streptomycin−except for HL-60, which required 20% HI
FBS. Similarly, cell lines MDA-MB-231, HeLa, and HS-27 were
cultured in DMEM (dulbecco’s modified eagle medium) medium,
and SH-SY5Y in DMEM/F12, supplemented with antibiotics and
10% HI FBS as described above. Cells were continuously maintained
in a 37 °C with 5% CO2 humidified atmosphere.
Assessment of cell viability and seeding into multiwell plates was
performed when cultures reached 60−75% confluence in the
exponential growth phase. Adherent cell lines (HeLa, MDA-MB-
231, SH-SY5Y, HS-27) were detached from their vessel by
trypsinization. Cell viability was examined by trypan blue exclusion,
and only cultures with ≥90% viable cells were seeded for
experimentation.
Docking Method for PPARγ. Computational Chemistry.
Molecular modeling and graphics manipulations were performed
using Maestro 11.0 (Schrodinger, LLC, New York, NY, 2020)107 and
̈
UCSF-Chimera 1.14 (http://www.cgl.ucsf.edu/chimera) software
packages108 running on a E4 Computer Engineering E1080
workstation provided of a Intel Core i7-930 Quad-Core processor.
̈
Figures were generated using Pymol 2.4 (Schrodinger, LLC, New
York, NY, 2020). 2D ligand-interaction diagram was made using
LigPlot+ v.2.2 (https://www.ebi.ac.uk/thornton-srv/software/
LigPlus/).109,110
Protein and Ligand Preparation. The starting coordinates of
PPARγ in complex with CAY10638 (PDB ID: 6DGR),64 retrieved
from Brookhaven Protein Database, were employed for the docking
calculations. The protein was processed with the Protein Preparation
Wizard implemented in Maestro. The appropriate bond orders as well
as charges and atom types were assigned. Hydrogen atoms were
added to the protein consistent with the neutral physiologic pH. The
guanidine and ammonium groups of arginine and lysine side chains
were considered cationic, whereas the carboxylate groups of the
aspartic and glutamic residues were considered anionic. Two well-
defined water molecules, HOH708 and HOH706, bridging between
CAY10638 and the β-sheet were retained and included in the docking
experiments, while the remaining crystallographic water molecules
were deleted. The H-bond network was optimized by exhaustive
sampling of rotamers, tautomers, and protonation states of titratable
amino acids at neutral pH. Imidazole rings H323 and H449 into
PPARγ were set in their Nε 2-H (N tau-H) tautomeric state. Finally,
the protein hydrogens only were minimized using the Impref module
of Impact with the OPLS_2005 force field.
Preparation of Compounds for In Vitro Assays. To prepare stock
solutions, aliquots of dried experimental or reference compound were
dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mM
and stored at −20 °C prior to use. For experiments, fresh aliquots of
compounds stocks were thawed and diluted with DMSO to a 100× or
250× working concentration to minimize dose volume and possible
vehicle-induced cytotoxicity.
Cytotoxicity Screening via the Differential Nuclear Staining
(DNS) Assay. To evaluate the cytotoxicity of “series 7” compounds
(7c, 7e, 7f, 7g, 7i, 7j, 7l, 7o, 7q, 7r) the live cell imaging-based
differential nuclear staining (DNS) assay was utilized.113 For this
experiment, cells were seeded in 96-well plates at a density of 5000
cells/well, incubated overnight, and exposed to experimental or
control treatments in quadruplicate for 72 h. In each plate, 1%
DMSO, 1 mM H2O2, and untreated cells were utilized as controls.
One hour prior to imaging, a mixture of fluorescent nuclear dyes
Hoechst 33342 and propidium iodide (PI) were added to wells at a
concentration of 1 μg/mL each. Cytotoxicity was determined as the
percentage of Hoechst 33342+/PI+ cells. For each cell-containing well,
a 2 × 2 montage of images were captured and segmented using a GE
InCell Analyzer 2000 high-content analysis system and associated
software. Additionally, for comparative purposes, the cytotoxicity of
reference compounds SAHA and pioglitazone were assessed in an
identical manner.
Calculation of CC50 and SCI Values. Individual data points were
normalized by subtracting the average percentage of cytotoxicity in
vehicle-treated cells from experimental values. Cell death was then
reported as a cytotoxicity percentage after 10 μM treatment or CC50
value for each individual compound. The concentration of
experimental compound which kills 50% of the cell population
(cytotoxic concentration 50%; CC50), was calculated by linear
interpolation of normalized data points using the two concentrations
nearest 50% cytotoxicity and reported as an average of 3−4 replicates.
Selective cytotoxicity index (SCI) values were calculated by dividing
the CC50 of noncancerous HS-27 cells by the CC50 of individual
cancer cell lines.
Apoptosis Assay. To evaluate the type of cell death (apoptosis or
necrosis) elicited by experimental compounds 7c, and 7i, the Annexin
V-FITC/PI assay was performed. In this experiment, CCRF-CEM
cells were seeded in a 24-well plate at a density of 50 000 cells/well in
1 mL of complete RPMI-1640 culture medium. Cells were then
treated in triplicate with the CC50 or 2×CC50 of compounds 7c, 7i, or
HDAC inhibitor SAHA for comparison and incubated for 72 h. 4%
DMSO, 1 mM H2O2, and untreated cells were utilized as vehicle,
positive, and negative controls respectively. Following treatment, cells
were collected and centrifuged at 500g for 5 min at 4 °C. Meanwhile,
reagents were collected and prepared on ice. The supernatant of each
sample was discarded, cell pellets resuspended in 103.5 μL of a
AnnexinV-FITC/PI/binding buffer mixture and incubated (covered)
The core structure of compounds 7i, 7l, 7o, 7r, 7c, 7q were
sketched using the Molecular Builder module in Maestro. The ligands
̈
were then preprocessed with LigPrep 3.3 (Schrodinger, LLC, New
York, NY, 2020) and optimized by means of Macromodel 11.5
̈
(Schrodinger, LLC, New York, NY, 2020), employing the MMFFs
force field with 1000 steps of steepest descent; the resulting molecules
were then submitted to 500 steps of truncated Newton conjugate
gradient method. Partial atomic charges were assigned using the
OPLS-AA force field.
Docking Calculations. Docking studies were performed using the
111,112
̈
Schrodinger Glide algorithm
implemented in Maestro. The
docking site was defined as a 10 × 13 × 18 Å inner box centered on
the average of coordinates of the native ligand present in the PPARγ
crystal structure (PDB ID: 6DGR).64 A scaling factor of 0.8 was set
for van der Waals radii of receptor atoms. Ligand sampling was
allowed to be flexible. Default docking parameters were used, and no
constraints were included. Redocking experiments were run in order
to validate the accuracy of Glide at reproducing the position of
CAY10638 in the active site. On comparing the conformation of the
cocrystallized ligand with the docked poses, it was observed that the
SP (standard precision) mode reproduced the bioactive conformation
of the cognate ligand with RMSD less than 2 Å. Thus, further
molecular docking studies were performed at the SP level. Ligand
sampling was allowed to be flexible. At most 10 docking ligand poses
were retained per run and ranked using the GlideScore function.111,112
The pose results were analyzed for accuracy through the RMSD of the
common core to the original cocrystal ligand core. Binding poses were
selected on the basis of the scoring, the similarity to the cocrystallized
ligand binding mode, and the consistency of protein−ligand
interactions with the experimental data.
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on ice for 30 min. Afterward, an additional 300 μL of ice-cold binding
buffer was added and samples were analyzed by beckman coulter
cytomics FC 500 flow cytometer and associated kaluza software.
Approximately 40 000 events (cells) were analyzed per sample. Total
apoptosis was calculated by summing early (AnnexinV+/PI−) and late
(AnnexinV+/PI+) apoptotic events. Statistical significance was
assessed by comparison of vehicle control samples to other treatments
via independent samples t-test (p < 0.05).
mg/kg i.p.) and standard clinical agent doxorubicin (20 mg/kg i. p.).
Treatments were administered on days 1−5, 8−12, 15−18. Tumor
volume was calculated by formula: volume = (width)2 × length/2. At
the end of the experiment, the animals were sacrificed by cervical
dislocation. The animals were dissected, and tumors were excised and
immediately imaged.
ASSOCIATED CONTENT
■
Cell Cycle Analysis. To evaluate the effect of experimental
compounds 7c and 7i on cell cycle progression, CCRF-CEM cells
were seeded in 24-well plates at a density of 50 000 cells/well in 1 mL
of complete culture medium. Cells were then treated in triplicate with
the CC50 or 2×CC50 of compounds 7c, 7i, or HDAC inhibitor SAHA
for comparison and incubated for 72 h. 4% DMSO, 1 mM H2O2, and
untreated cells were utilized as vehicle, positive, and negative controls.
Following treatment, cells were collected and centrifuged at 262 g for
5 min at 20 °C. For each sample, the supernatant was discarded, and
cells resuspended in 100 μL of PBS and 200 μL of nuclear isolation
medium (NIM)-DAPI solution to permeabilize cell membranes and
stain DNA. Then they were immediately analyzed by beckman coulter
cytomics FC 500 flow cytometer and associated kaluza software.
Approximately 40 000 events (cells) were analyzed per sample.
Statistical significance was assessed by comparison of vehicle control
samples to other treatments via independent samples t-test (p < 0.05).
Western Blotting. CCRF-CEM Cells were grown in multiwell
plate which were washed twice with cold PBS, lysed in Laemmli buffer
(62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS) supplemented
with Complete protease inhibitors (Roche), sonicated before protein
quantification (DC BioRad Protein Assay cat no. 500-0114). Samples
with equal protein quantity were supplemented with 5% β-
mercaptoethanol, heated (95 °C; 12 min), size fractionated on a
9% SDS-PAGE gel, and transferred to nitrocellulose membranes. All
Blue Precision Plus Protein Standard (Bio-Rad cat. no. 161-0373) was
used as protein size marker. Membranes were blocked for 45 min in
PBS-T-milk (0.05% Tween, 5% dried fat-free milk); incubated with
primary antibody (3 h; RT; di luted in PBS-T milk), washed (PBS-T)
and incubated with secondary antibodies (1 h; RT; diluted in PBS-T
milk). After removal of unbound secondary antibodies, signals were
revealed using Super signal west femto maximum sensitivity substrate
(Pierce). Antibodies were purchased from cell signaling (p53 no.
9282, cMYC no. 9402, Cleaved caspase no. 9661, Ac Tub no. 5335).
All the bands were quantified and normalized with respect to TBP
band by using ImageJ software, version 1.2.
In Vivo Evaluation. In vivo tumor growth inhibition has served as
demonstrative model of achieving expected activity of antitumor
agents in human. Hence, the most promising dual targeting
compound 7c was further evaluated in vivo using standard
subcutaneous xenograft model produced using CEM cell line. All
the experiments were approved by the concerned IAEC (institutional
animal ethics committee, Approval No. CPCSEA/IAEC/KT/05/20/
5). 8−10 weeks old SCID mice were housed individually under
pathogen-free conditions in ventilated cage system with a 12 h light/
dark cycle. The area was controlled for noise and humidity. Animals
were fed autoclaved commercial pellets and water ad libitum and were
handled in a laminar air flow during the experimentation. Primary
dose escalation was performed with 5 animals which were
administered 500 mg/kg and 1000 mg/g single dose of 7c i.p.
Animal receiving 1000 mg/kg displayed symptoms of distress and
mortality in 2 animals. Animals getting 500 mg/kg did not exhibit any
mortality, and clinical distress signs were recovered within 6 h. Hence
1/10th of 500 mg/kg dose was selected for efficacy study. Briefly
CEM, ATCC (T cell leukemia) cells (1 × 105) suspended in serum
free medium were injected on the back of the mice and allowed to
form palpable tumors. Tumors were minced and regrafted in
experimental animals. Administration of test sample were done
when the tumors attained a volume of nearly 100 mm3, which were
measured using digital vernier calipers (Mitutoyo JAPAN) and
computed by utilizing the formula: volume = [(length × width2)/2].
Animals were randomly divided in to three groups based on body
weight and tumor volume and allocated to treatments: vehicle, 7c (50
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00491.
Additional docking figures of HDAC and PPARγ
docking studies; PPARγ activation assay images;
apoptosis histograms, value tables, and statistical data;
cell cycle value tables and statistical data; HDAC4 and
HDAC8 residual activity values tables; compounds
1
spectral details such as H NMR and 13C NMR, IR
spectra, DSC graph, LCMS PDA chromatograms, and
mass spectra of all target compounds (PDF)
Molecular formula strings and some data (CSV)
3D structure graphics (ZIP)
3D structure graphics (ZIP)
AUTHOR INFORMATION
■
Corresponding Authors
Ramaa C S − Department of Pharmaceutical Chemistry,
Bharati Vidyapeeth’s College of Pharmacy, Navi Mumbai
400614, India; orcid.org/0000-0002-5814-3616;
Phone: +91 9833915955; Email: sinharamaa@yahoo.in;
Fax: +9122-27572131
Antonio Lavecchia − Department of Pharmacy, “Drug
Discovery” Laboratory, University of Napoli “Federico II”,
80131 Napoli, Italy; orcid.org/0000-0002-2181-8026;
Phone: +39-081-678613/623; Email: antonio.lavecchia@
unina.it; Fax: +39-081-678012
Renato J. Aguilera − Cellular Characterization and
Biorepository Core Facility, Border Biomedical Research
Center, Department of Biological Sciences, The University of
Texas at El Paso, El Paso, Texas 79968, United States;
Phone: +1-915-747-6852; Email: raguilera@utep.edu
Authors
Kalpana Tilekar − Department of Pharmaceutical Chemistry,
Bharati Vidyapeeth’s College of Pharmacy, Navi Mumbai
400614, India
Jessica D. Hess − Cellular Characterization and Biorepository
Core Facility, Border Biomedical Research Center,
Department of Biological Sciences, The University of Texas at
El Paso, El Paso, Texas 79968, United States
Neha Upadhyay − Department of Pharmaceutical Chemistry,
Bharati Vidyapeeth’s College of Pharmacy, Navi Mumbai
400614, India
Alessandra Lo Bianco − Department of Pharmacy, “Drug
Discovery” Laboratory, University of Napoli “Federico II”,
80131 Napoli, Italy
Markus Schweipert − Department of Chemical Engineering
and Biotechnology, University of Applied Science, 64295
Darmstadt, Germany
Antonio Laghezza − Department of Pharmacy-Drug Sciences,
University of Bari “Aldo Moro”, 70126 Bari, Italy;
orcid.org/0000-0001-6221-6155
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Fulvio Loiodice − Department of Pharmacy-Drug Sciences,
University of Bari “Aldo Moro”, 70126 Bari, Italy;
orcid.org/0000-0003-3384-574X
Franz-Josef Meyer-Almes − Department of Chemical
Engineering and Biotechnology, University of Applied Science,
64295 Darmstadt, Germany; orcid.org/0000-0002-
1001-3249
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00491
Author Contributions
The manuscript was written through contributions of all
authors who gave approval to the final version of the
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by Indo-Poland joint research
program grant from Department of Science and Technology
(DST) with reference number DST/INT/Pol/P-27/2016 (to
CSR). Part of the Funding for this work was provided by the
Research Centers in Minority Institutions (RCMI) program
grants 5G12MD007592 and 5U54MD007592 to the Border
Biomedical Research Center (BBRC) at UTEP from the
National Institute on Minority Health and Health Disparities, a
component of NIH. The authors thank the personnel of the
Genomic Analysis and the Cell Characterization and
Biorepository Core Facilities at the University of Texas at El
Paso (UTEP). We also acknowledge funding through the
LOEWE priority program TRABITA, State of Hesse,
Germany.
ABBREVIATIONS USED
■
TZD, 2,4-thiazolidine dione; HDAC, histone deacetylase;
PPARγ, peroxisome proliferator-activated receptor gamma;
ZBG, zinc binding group; SRC, surface recognition cap; SAR,
structure activity relationship; DMF, dimethyl formamide;
DCM, dichloromethane; TEA, triethanolamine; LBD, ligand
binding domain; PDB, protein data bank; DNS, differential
nuclear staining; SAHA, suberoylanilide hydroxamic acid;
CC50, half maximal cytotoxic concentration; IC50, half maximal
inhibitory concentration; EC50, half maximal effective concen-
tration; DMEM, dulbecco’s modified eagle medium; HI FBS,
heat-inactivated fetal bovine serum; SCI, selective cytotoxicity
index; AnnexinV-FITC/PI, annexin V-fluorescein isothiocya-
nate/propidium iodide; PS, phosphatidylserine; DMSO,
dimethyl sulfoxide; NIM-DAPI, nuclear isolation medium
containing DNA intercalating dye DAPI; DNA, deoxyribonu-
cleic acid; TGI, total growth inhibition; TLC, thin layer
chromatography; FTIR, Fourier transform infrared spectros-
copy; NMR, nuclear magnetic resonance; PDA, photodiode
array detector; DSC, differential scanning calorimetry; HPLC,
high performance liquid chromatography; MP, melting point;
DRC, dose−response curve; CASTp, computed atlas of surface
topography of proteins.
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