Harnessing the Role of HDAC6 in Idiopathic Pulmonary Fibrosis: Design, Synthesis, Structural Analysis, and Biological Evaluation of Potent Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.1c00184

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by a progressive-fibrosing phenotype. IPF has been associated with aberrant HDAC activities confirmed by our immunohistochemistry studies on HDAC6 overexpression in IPF lung tissues. We herein developed a series of novelhHDAC6 inhibitors, having low inhibitory potency overhHDAC1 andhHDAC8, as potential pharmacological tools for IPF treatment. Their inhibitory potency was combined with lowin vitroandin vivotoxicity. Structural analysis of 6h and structure-activity relationship studies contributed to the optimization of the binding mode of the new molecules. The best-performing analogues were tested for their efficacy in inhibiting fibrotic sphere formation and cell viability, proving their capability in reverting the IPF phenotype. The efficacy of analogue 6h was also determined in a validated human lung model of TGF-β1-dependent fibrogenesis. The results highlighted in this manuscript may pave the way for the identification of first-in-class molecules for the treatment of IPF.

Full text of DOI:10.1021/acs.jmedchem.1c00184

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Article
Harnessing the Role of HDAC6 in Idiopathic Pulmonary Fibrosis:
Design, Synthesis, Structural Analysis, and Biological Evaluation of
Potent Inhibitors
Giuseppe Campiani,* Caterina Cavella, Jeremy D. Osko, Margherita Brindisi, Nicola Relitti,
Simone Brogi, A. Prasanth Saraswati, Stefano Federico, Giulia Chemi, Samuele Maramai,
Gabriele Carullo, Benedikt Jaeger, Alfonso Carleo, Rosaria Benedetti, Federica Sarno, Stefania Lamponi,
Paola Rottoli, Elena Bargagli, Carlo Bertucci, Daniele Tedesco, Daniel Herp, Johanna Senger,
Giovina Ruberti, Fulvio Saccoccia, Simona Saponara, Beatrice Gorelli, Massimo Valoti,
Breándan Kennedy, Husvinee Sundaramurthi, Stefania Butini,* Manfred Jung, Katy M. Roach,
Lucia Altucci, Peter Bradding, David W. Christianson, Sandra Gemma,∥∥ and Antje Prasse∥∥
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ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is an interstitial
lung disease characterized by a progressive-fibrosing phenotype.
IPF has been associated with aberrant HDAC activities confirmed
by our immunohistochemistry studies on HDAC6 overexpression
in IPF lung tissues. We herein developed a series of novel
hHDAC6 inhibitors, having low inhibitory potency over hHDAC1
and hHDAC8, as potential pharmacological tools for IPF
treatment. Their inhibitory potency was combined with low in
vitro and in vivo toxicity. Structural analysis of 6h and structure−
activity relationship studies contributed to the optimization of the
binding mode of the new molecules. The best-performing
analogues were tested for their efficacy in inhibiting fibrotic sphere formation and cell viability, proving their capability in
reverting the IPF phenotype. The efficacy of analogue 6h was also determined in a validated human lung model of TGF-β1-
dependent fibrogenesis. The results highlighted in this manuscript may pave the way for the identification of first-in-class molecules
for the treatment of IPF.
IPF accounts for 25% of all ILDs with about 35 000 new cases
diagnosed every year in Europe and 5 000 000 worldwide. IPF
has an annual incidence of 0.22−7.4 per 100 000 people with a
prevalence of 1.25−23.4 cases per 100 000 population.2 The
contribution of inflammation to the fibrotic process in IPF is
controversial, but conventional anti-inflammatory therapies
(e.g., glucocorticoids) are not efficacious.3,4 In the past decades,
many clinical trials have been designed to determine the safety
and efficacy of pharmacotherapies for patients with IPF.
However, so far, only two drugs (pirfenidone 1 and nintedanib
2, Figure 1) were found to have an impact on disease
progression. These two drugs have been approved for the
INTRODUCTION
■
Millions of people worldwide suffer from lung diseases that
affect the whole respiratory system including the airways, air sacs
(alveoli), lung interstitium, blood vessels, and pleura. Interstitial
lung diseases (ILDs) are a numerous and heterogeneous group
of diseases that affect the lung interstitium, a thin layer of cells,
and the tissue matrix between the alveoli, which contain blood
vessels and cells that help support the alveoli, allowing efficient
gas exchange. The pathological deposition of collagen and other
connective tissue proteins causes progressive scarring and
fibrosis. This disorganized damaged tissue impairs the vital
role of the lungs in respiration with devastating consequences in
terms of functional capacity, quality of life, and increased
mortality.1 The most relevant ILDs may be associated with a
progressive-fibrosing phenotype, and these ILDs are classified
with a term known as progressive-fibrosing rare diseases. Among
these progressive-fibrosing ILDs, the most common is idiopathic
pulmonary fibrosis (IPF), characterized by a very poor
prognosis.
Received: February 1, 2021
Published: July 12, 2021
https://doi.org/10.1021/acs.jmedchem.1c00184
© 2021 American Chemical Society
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HDAC9-10 possessing nucleocytoplasmic shuttling ability, also
observed in class IV HDAC (HDAC11). Class III HDAC
contains nonzinc-dependent enzymes, called sirtuins (SIRT 1−
7), requiring NAD+ for their activity.10 Overexpression of
HDACs is implicated in many pathological conditions including
cancer, neurodegeneration, and rare diseases such as IPF as
confirmed by us (see below).7,11−13 Currently, four pan-HDAC
inhibitors (HDACis) have been approved by the FDA for the
treatment of hematological malignancies, and many other
HDACis are under clinical investigation for cancer and other
diseases.14,15 However, none of them have been approved for
fibrotic diseases yet.
Figure 1. Representative structures of the FDA-approved drugs for the
Aberrant HDAC activities are observed in fibrotic diseases;
mounting evidence indicates the involvement of HDACs in the
initiation and progression of fibrosis occurring in organs such as
the lungs, heart, liver, and kidneys; preliminary studies
performed on animal models have shown that HDACi can
ameliorate various forms of fibrosis.16,17 Due to the limitations
of pirfenidone (1) and nintedanib (2) in IPF, and a lack of
promising clinical candidates, an unprecedented effort is
required to enrich the therapeutic arsenal available to tackle
IPF and other rare fibrotic disorders.18 In particular, the
regulation of transforming growth factor β1 (TGF-β1) by
HDAC6, a microtubule-associated deacetylase, is significant in
the pathogenesis and progression of fibrotic diseases through
epithelial−mesenchymal transitions (EMT).19 Fibrotic lesions
are associated with an aberrant expression of TGF-β1, which is a
potent EMT inducer.20 Recent reports suggest that HDAC6
inhibition by siRNA or tubacin (3, Figure 2) reduces the TGF-
β1-induced EMT markers and impairs SMAD3 activation in
response to TGF-β1. Since SMAD3 is a core element for TGF-
β1 signaling, its inactivation impairs HDAC6-dependent
deacetylation of α-tubulin highlighting the role of HDAC6 in
EMT through the TGF-β1-SMAD3 signaling pathway.17 Several
reports highlighted the efficacy of pan-HDACi (such as SAHA
(4) and panobinostat (5), Figure 2) against IPF and fibrotic lung
treatment of IPF.
treatment of IPF by regulatory agencies and are currently in
clinical use worldwide.5,6 Even though these drugs possess an
acceptable safety profile, they only delay disease progression and
fail to reverse lung damage.2,6,7
Epigenetic regulation modulates many cellular processes and
greatly influences key disease mechanisms. Histone deacetylase
(HDAC) enzymes play a crucial role either as biomarkers or
therapeutic targets owing to their involvement in specific
pathophysiological pathways. HDACs are a class of enzymes
responsible for the removal of acetyl groups from histones
leading to decreased gene transcription and are mostly involved
in biological processes related to chromatin.8 HDACs can
deacetylate both histone and nonhistone substrates, including
transcription factors such as p53, Rb, and others. They also
regulate key protein substrates such as α-tubulin, actin, and
cortactin and can thereby influence many cellular processes,
namely, apoptosis, cell signaling, and DNA repair, replication,
and recombination.9 HDACs represent a family of 11 zinc-
dependent enzymes (HDAC1-11) clustered into three groups
(classes I, II, and IV). Class I comprises nuclear enzymes
HDAC1-3 and HDAC8, while class II contains HDAC4-7 and
Figure 2. Representative structures of some HDACis as antifibrotic agents and title compounds ( )-6a−m (as described in Table 1).
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diseases,21 mainly based on the reduction in fibroblast-
myofibroblast differentiation and fibroblast proliferation in-
duced by TGF-β1.22,23 Recently, HDAC6 inhibitors showed to
protect mice from lung fibrosis by repressing TGF-β1-induced
collagen expression and diminished Akt phosphorylation.24
For many decades, HDACis have drawn widespread attention
as therapeutic agents for different diseases. However, due to
reports indicating several challenges encountered with the use of
pan-HDACi and their off-target effects, there is an urgent need
to develop isoform-selective inhibitors to be investigated in
fibrotic diseases.25,26 Based on these observations, further
studies pointed out how only HDAC6 enzyme could be
selectively targeted, mostly because of its cytoplasmic local-
ization.27
Table 1. Inhibitory Activity of Compounds 6a−m toward
a
hHDAC1 and hHDAC6 (as IC50, nM)
The general pharmacophoric model of HDACi comprises (i)
a cap-group, which interacts with the amino acid residues on the
surface of the enzyme, (ii) a zinc-binding group (ZBG), usually a
hydroxamic acid group, chelating the Zn2+ necessary for the
inhibitory activity, and (iii) a linker group between these
portions. Our research group has been actively involved in the
development of potent HDACi as therapeutic agents for the
treatment of cancers, rare disorders, and infectious dis-
eases.28−31 Based on reports outlining the HDACi (compound
3)-mediated impairment of the TGF-β1-EMT pathway, we
designed and synthesized novel HDAC6is (6a−m, Table 1)
selective over hHDAC1 and hHDAC8, as promising pharma-
ceutical tools for the treatment of IPF, with the aim of
investigating also the role of this enzyme isoform in fibrotic
processes. In this strategy, a key challenge was the rational
improvement of HDAC6 selectivity over other specific isoforms,
such as HDAC1 and HDAC8, class I nuclear HDACs. While
HDAC1 deacetylates histones and transcriptional regulators,
HDAC8 is fundamentally distinct from HDAC1-3 and is the
only isoform for which the gene lies in the X chromosome.
Moreover, because of similarities in the active site of HDAC6
and HDAC8, discriminating between these two isoforms is
challenging.
Following our previous experience on HDAC6 inhibition by
small molecules, we decided to investigate the effect of
introducing a bulkier cap-group to increase the number of
interactions with the lipophilic pocket of the HDAC6 enzyme
minimizing interactions with the catalytic sites of hHDAC1 and
hHDAC8 isoforms.31,32 This was achieved by decorating the
indoline scaffold with different aromatic and aliphatic moieties
at the C2 and C3 positions, respectively. Thereafter, we
determined the affinity of the newly developed series of
compounds on hHDAC6, hHDAC8, and hHDAC1. To
establish the binding mode on HDAC6 of the new series, we
cocrystallized compound 6h with zebrafish HDAC6
(zf HDAC6) (2.04 Å). The best-performing compounds were
then evaluated for their antifibrotic and pharmacokinetic
profiles. In addition, their potential toxicity, cytotoxicity,
cardiotoxicity, and mutagenicity were also evaluated in different
in vitro and in vivo models.
a
Each value is the mean of at least three determinations; results are
expressed with standard deviation (SD). When not specified, SD is
<10% of the IC50.
RESULTS AND DISCUSSION
■
Chemistry. The synthetic approach for the development of
the new molecules is based on three key steps: (i) the formation
of the suitable ketones to be subjected to interrupt Fischer
indolization and subsequent indolenine reduction, (ii) reductive
amination on the indoline nitrogen, and (iii) the conversion of
the methyl ester to hydroxamic acid.
The synthesis of tosylhydrazones 7, 21, and 26 was achieved
following the procedure applied in our previous work starting
from commercially available supplies.30 In Scheme 1, the
synthesis of the final compounds 6a−f is reported. The
tosylhydrazone 7 was converted to ketones 8a−f after treatment
with the opportune aldehyde and Cs2CO3. 8a−f were subjected
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a
Scheme 1. Synthesis of Compounds ( )-6a−f
a
Reagents and conditions: (a) Appropriate aryl aldehyde, Cs2CO3, 1,4-dioxane, 110 °C, 14 h, 40−100%; (b) phenylhydrazine, H2SO4, 1,4-dioxane,
70 °C, 2 h, then 25 °C, 14 h, 30−66%; (c) NaBH4, MeOH, 25 °C, 12 h or H2/Pd, MeOH, 25 °C, 12 h, 40−100%; (d) methyl 4-formylbenzoate,
NaBH3CN, EtOH, AcOH, 70 °C, 14 h, 25−43%; (e) NH2OH (50% in H2O), KOH, DCM/MeOH from 0 to 25 °C, 12 h, 33−73%.
a
Scheme 2. Synthesis of Aldehydes 16 and 20
a
Reagents and conditions: (a) MeOH, from 0 to 25 °C, 12 h, 100%; (b) p-cresol, Cs2CO3, MeCN, 90 °C, 14 h, 74%; (c) NBS, AIBN, CCl4, 90 °C,
1 h; (d) NBS, DMSO, 100 °C, 2 h, 30% over two steps; (e) SOCl2, MeOH, 0−25 °C, 12 h, 100%; (f) NBS, AIBN, CCl4, 80 °C, 4 h, 60%; (g)
NMO, MeCN, 25 °C, 12 h, 70%.
to a Fischer protocol with phenylhydrazine and sulfuric acid
affording spiroindolenines 9a−f. Catalytic hydrogenation of the
metastable indolenine followed by reductive amination with
methyl 4-formylbenzoate afforded spiroindolines 11a−f. These
intermediates were converted to their corresponding hydroxa-
mic acids ( )-6a−f after treatment with KOH and aqueous
NH2OH. All of the products were obtained as racemic mixtures.
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a
Scheme 3. Synthesis of Compounds ( )-6g−l
a
Reagents and conditions: (a) Appropriate aryl aldehyde, Cs2CO3, dioxane, 110 °C, 14 h, 89−100%; (b) for 23a−c: phenylhydrazine, AcOH, 80
°C, 14 h; for 23d: 4-methoxyphenylhydrazine, AcOH, 80 °C, 14 h, 30−48%; (c) NaBH4, MeOH, 25 °C, 12 h or H2, Pd/C, MeOH, 25 °C, 12 h,
50−79%; (d) for ( )-25a−c and 25f: methyl 4-formylbenzoate, EtOH, AcOH, NaBH3CN, 40 °C, 14 h; for ( )-25d: 20, EtOH, AcOH,
NaBH3CN, 40 °C, 14 h; for ( )-25e: 16, NaBH(OAc)3, DCM, 40 °C, 14 h, 20−84%; (e) NH2OH (50% in H2O), KOH, DCM/MeOH from 0 to
25 °C, 12 h, 18−79%.
The synthesis of the linker moieties 16 and 20 is described in
Scheme 2. 4-Bromobutyryl chloride was converted to its
corresponding methyl ester and then used to alkylate p-cresol
in the presence of Cs2CO3. The resulting compound 14 was
subjected to radical bromination, leading to the formation of
unstable intermediate 15 that was immediately oxidized by NBS
in the presence of DMSO. This reaction provided aldehyde 16 in
good yields.
For the synthesis of lateral chain 20, 5-methylthiophene-2-
carboxylic acid was converted into its corresponding methyl
ester 18. Compound 18 was subjected to a radical bromination
with NBS and AIBN affording the bromo-derivative 19. The
aldehyde 20 was obtained from intermediate 19 upon reaction
with 4-methylmorpholine N-oxide (NMO) in MeCN.
In Scheme 3, the synthesis of compounds 6g−l is described.
Following the synthetic approach previously shown in Scheme
1, tosylhydrazones 21 were reacted with suitable arylaldehydes
to obtain ketones 22b−c. The cyclization reaction between
phenylhydrazine and compounds 22a−c provided derivatives
23a−c. 22b was reacted with 4-methoxyphenylhydrazine to
furnish compound 23d. These intermediates were reduced with
NaBH4 or by catalytic hydrogenation affording 24a−d. Indoline
derivatives were subjected to a reductive amination protocol
with the appropriate aldehydes generating the compounds
( )-25a−f that were reacted with KOH and NH2OH to provide
hydroxamic acids ( )-6g−l as racemic mixtures.
In Scheme 4, the synthesis of compound 6m is reported.
Following the previously described procedure, tosylhydrazone
26 was reacted with 3-pyridinecarboxaldehyde and Cs2CO3 to
afford compound 27. This intermediate was subjected to a
Fischer reaction to get the corresponding indolenine. The loss of
the Boc protecting group due to the acidic conditions of the
Fischer reaction required subsequent treatment with Boc2O for
its reinstallation, providing indolenine 28. The reduction of this
intermediate by catalytic hydrogenation followed by a reductive
amination with methyl 4-formylbenzoate afforded ( )-30 as a
racemic mixture. This compound was converted to hydroxamic
acid ( )-6m after treatment with KOH and NH2OH.
To evaluate the stereoselective interaction with hHDAC6, we
decided to resolve the racemic mixture of ( )-6h. Analogue 6h
successfully cocrystallized with the enzyme (Figure 4). To this
end, we resolved the racemic mixture of esters ( )-25b by chiral
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a
Scheme 4. Synthesis of Compound ( )-6m
a
Reagents and conditions: (a) 3-Pyridinecarboxaldehyde, Cs2CO3, dioxane, 110 °C, 14 h, 100%; (b) phenylhydrazine, H2SO4, dioxane, 70 °C, 2 h,
14%; (c) Boc2O, THF, NaOH, 25 °C, 2 h, 100%; (d) H2, Pd/C, MeOH, 25 °C, 12 h, 100%; (e) methyl 4-formylbenzoate, NaBH3CN, EtOH,
AcOH, 70 °C, 14 h, 30%; (f) NH2OH (50% in H2O), KOH, DCM/MeOH from 0 to 25 °C, 12 h, 51%.
a
Scheme 5. Synthesis of (R)-(+)6h and (S)-(+)6h
a
Reagents and conditions: (a) NH2OH (50% in H2O), KOH, DCM/MeOH from 0 to 25 °C, 12 h, 34−38%.
high-performance liquid chromatography (HPLC) (see the
Supporting Information (SI)). The two enantiomers, (+)-25b
and (−)-25b, were isolated with an enantiomeric excess of >99%
(Figure S1 of the Supporting Information) and underwent the
final reaction giving the two enantiomers of 6h; in particular,
(+)-25b and (−)-25b yielded (+)-6h and (−)-6h, respectively
(Scheme 5). The absolute configuration of (−)-25b was
established by electronic circular dichroism (ECD) spectrosco-
py and time-dependent density functional theory (TD-DFT)
calculations. The comparison between the experimental and
theoretical spectra (Figure 3) allowed us to assign the (S)-
absolute configuration to (−)-25b (r = 0.7345 for the ECD
spectra) and, consequently, to (−)-6h.
asymmetric unit of a monoclinic unit cell was obtained. There
are no major conformational changes between the liganded and
unliganded (PDB 5EEM) enzyme structures, and the root-
mean-square deviation (RMSD) is 0.15 Å for 309 Cα atoms in
monomer A. By soaking experiments with racemic 6h, we
obtained cocrystallization of the (S)-enantiomer only. The
inhibitor hydroxamate group coordinates to the catalytic Zn2+
ion through only the ionized N−O− moiety (Figure 4). The
Zn2+-bound oxyanion also accepts a hydrogen bond from the
phenolic hydroxyl group of Y745. Weak electron density for a
Zn2+-bound water molecule is also observed. This water
molecule forms hydrogen bonds with H573, H574, and the
carbonyl group of the hydroxamate moiety.
Crystal Structure Determination of the zf HDAC6−6h
Complex. The 2.04 Å-resolution crystal structure of the
zf HDAC6−6h complex containing two monomers in the
The inhibitor capping group consists of a 3,3-dimethyl-2-
(pyridine-3-yl)indoline, and the pyridine nitrogen forms a
hydrogen bond with N645 at the mouth of the active site cleft.
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Figure 3. Comparison between the experimental spectra of (−)-25b (solid lines) and the theoretical spectra of (S)-25b, as determined by TD-DFT
calculations (dashed lines). (A) ECD spectra. (B) UV spectra.
Figure 4. Stereoview of a Polder omit map of the zfHDAC6/(S)-6h complex (PDB 6V79; contoured at 2.5 σ). The Zn2+-bound water molecule is
contoured at 3.5 σ. Atoms are color-coded as follows: C = light blue (zfHDAC6 monomer A), light gray (zf HDAC6 monomer B), or wheat
(inhibitor), N = blue, O = red, Zn2+ = gray sphere, and Zn2+-bound water molecule = small red sphere. Metal coordination and hydrogen bond
interactions are indicated by solid and dashed black lines, respectively.
The pyridine NH group is presumably protonated in this
environment to donate the hydrogen bond to the side chain
carbonyl group of N645. The orientation of N645 is fixed by
other hydrogen bond interactions with the protein. This
hydrogen bond may rationalize the orientation of the capping
group into the L2 pocket, where relatively few HDAC6
inhibitors orient unless they possess bifurcated capping
groups.33 Notably, however, hHDAC6 has a methionine residue
at position 645 instead of an asparagine, so this interaction is
specific only to the zebrafish enzyme. No other hydrogen bond
interactions with the protein, either direct or water-mediated,
are observed for the inhibitor capping group.
Docking Studies and Structure−Activity Relationship
(SARs). Computational investigations were performed using
the crystal structure of zf HDAC6 in comparison with hHDAC6
applying the docking protocol described in the Experimental
Section, evaluating the performance of our docking protocol. All
molecules were docked on HDAC1 and HDAC6 by their (S)
and (R) enantiomers (see the Supporting Information). The
most relevant docking results are herein discussed for selected
molecules/enantiomers. Aiming at investigating the binding
modes of the developed compounds into the catalytic sites of
hHDAC1 and hHDAC6 enzymes and performing the SAR
analysis, we combined experimental data (Table 1) with in silico
investigation (human enzymes, Figures 5 and S3−S15).28,29
Generally, for the selected compounds, we observed a common
trend in their binding mode with the HDAC6 enzyme,
establishing a higher number of contacts compared to HDAC1.
In particular, taking into account (S)-(−)-6h, complexed with
zf HDAC6, when docked into hHDAC6 and zf HDAC6, it
similarly accommodates in both enzymes,28−30 see the SI for
details (Figure S2). This study confirms that zf HDAC6 could be
a valuable model for translating the results of potential inhibitors
to hHDAC6.31
When docked in zfHDAC6, both enantiomers of 6a, besides
the metal coordination, with their hydroxamate moiety target
G619 and Y782, the benzyl linker forms π−π stacking with
H651 and F680, while the benzyl substituent establishes π−π
stacking with F679. The cap-group establishes hydrophobic
contacts with F620 and L749. The higher number of contacts of
6a reflects its higher affinity for HDAC6 over HDAC1 (IC50
values: HDAC1 = 4290 nM; HDAC6 = 73.2 nM) (see the SI for
a detailed discussion of the docking results for HDAC1). The
introduction of Cl at the p- or m-position of the phenyl ring (6b
and 6c, respectively) does not substantially modify the binding
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Figure 5. Docked poses of (S)-(−)-6h (light blue sticks) into HDAC1 (PDB ID 4BKX; panel A) and HDAC6 (PDB ID 5EDU; panel B). The residues
in the active sites are represented as lines, and the proteins are represented as cartoons. Zn2+ is represented as a gray sphere. H-bonds are represented as
black dotted lines, while the metal coordination bond is represented by a red stick.
mode retrieved for 6a. The same was true after the introduction
of a p-F-phenyl (6e) or a pyridin-3-yl (6f) at C2 (see the SI).
Among the halogenated analogues, the o-F-phenyl substituent
at the chiral C2 of 6d (Figure S6) favored beneficial interactions
with F679 and H651 residues when in the R configuration. The
replacement of the spiro N-methylpiperidine moiety by two
geminal methyl groups at the 3-position of the indoline system
of 6a led to analogue 6g. 6g showed similar contacts to spiro-
fused analogues (6a−f, m) for both enantiomers ((S)-6g: π−π
stacking with H651 and F680; the cap-group with F620; (R)-6g
lacks the contact with F620). The introduction of a pyridin-3-yl
substituent at the 2-position of the indoline core of 6g led to
analogue 6h. The docking output for the 6h S-enantiomer on
HDAC1/6 isoforms is reported in Figure 5 (R-enantiomer in
Figure S10). In HDAC1 active site, 6h established critical π−π
stackings involving the benzyl linker (H141 and F150) and the
cap-group (F205) (Figure 5A). The R-enantiomer (Figure
S10A) establishes, in addition to the contacts of the
hydroxamate moiety, π−π stacking with H141 (benzyl linker)
and Y204 (cap-group). Figure 5B highlights the contacts of (S)-
6h with HDAC6: the benzyl linker interacts with H651 and
F680 (π−π stacking), while the pyridine at C2 interacts with
F680. Interactions of (R)-6h are shown in Figure S10B. Docking
data support the higher affinity of 6h for HDAC6 (IC50 HDAC1
= 5330 nM; IC50 HDAC6 = 41.9 nM).
The N-Me group of 6f was also replaced by a Boc group (6m),
establishing contacts into HDAC1 and HDAC6 similar to those
identified for other analogues (Figure S15), indicating that the
protonatable function is not critical for binding HDAC enzymes.
In addition to hHDAC1 and hHDAC6, the potency of three
representative inhibitors (6d,h,l, Table 2) was assessed on the
Table 2. Inhibitory Activity of Compounds 6d,h,l, as IC50
a
(μM), against the hHDAC8 Enzyme
compound
6d
6h
6l
TubA28
a
IC50 (μM)
3.49 1.54
2.17 0.70
4.64 1.28
0.695
a
The concentration range of all compounds (40 nM−50 μM) was
determined using the reference compound TubA and the results are
expressed as standard deviations.
hHDAC8 isoform, which represents a unique member of the
class I HDAC family. HDAC8 is endowed with the ability to
recognize both histone and nonhistone substrates. This isoform
is ubiquitously expressed, and it is localized either in the nucleus
or in the cytoplasm.34 Discrimination between hHDAC6 and
hHDAC8 binding sites was achieved and analogues 6d, 6h, and
6l demonstrated low potency against HDAC8 (IC50 in the range
of 2.17−4.64 μM, Table 2).
HDAC6 is a Privileged Target of This New Series:
Western Blot Analysis. The selectivity issues for HDACi is a
crucial point in HDAC research; however, the reliability of in
vitro enzymatic tests on all of the 11 HDAC isoforms is
debatable, as recently pointed out.35 Accordingly, for assessing
HDAC6 preferential interaction, the most potent and
interesting analogues (6a,6d,6e,6h, and 6l) were engaged in
experiments in living cells. We performed our cell-based assays
using three cancer cell lines including hematological (NB4,
A similar result was obtained when a methoxy substituent was
introduced on the cap-group of 6h, obtaining compound 6i
(Figure S11, IC50: HDAC1 = 5320 nM and HDAC6 = 67.2 nM).
The replacement of the benzyl linker of 6h with a
thienylmethyl linker (6j) or a propyloxybenzyl system (6k)
caused a large decrease in potency against the HDAC6 enzyme
(Figures S12−S13). The introduction of pyridin-4-yl at C2 (6l)
confirmed the same interactions observed for 6h.
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acute promyelocytic leukemia) and solid (U2OS, osteosarcoma
and U87, glioblastoma) cancer cells. In all of these cells, the
levels of tubulin acetylation (the client protein of HDAC6) and
acetylation of histone H3 (the primary substrate of HDAC1 and
other nuclear HDACs) were evaluated after incubation of our
compounds at 5 μM for 30 h (Figure 6). Under these conditions,
RNAseq analyses and immunohistochemistry data of IPF tissues
showed that ABC is shifted toward an EMT phenotype and
HDAC6 is crucial to TGF-β-induced EMT.37 Accordingly,
analysis of the BAL transcriptome from IPF patients showed a
marked increase in the expression of genes exclusively expressed
by ABC.37 Immunohistochemistry highlighted HDAC6 ex-
pression in three-dimensional (3D) organoids derived from
ABC from IPF patients, which build bronchospheres. HDAC6 is
highly expressed by the outer rim of ABC of the bronchosphere
structure. Immunohistochemistry for HDAC6 expression also
revealed high HDAC6 expression of ABC derived from IPF
patients. These data and cell RNA sequencing analyses of IPF
tissues showed that ABCs were shifted toward an EMT
phenotype that is induced by TGF-β, which in turn is regulated
by the activity of the HDAC6 enzyme.19 Further studies showed
a profibrotic effect of ABC derived from IPF patients,39 which
prompted us to analyze the expression of HDAC6 in normal and
lung tissues derived from IPF patients. Immunohistochemistry
of lung tissues derived from IPF patients confirmed HDAC6
overexpression in fibrotic lungs (Figure 7).
To investigate the potential of the new HDAC6 inhibitors,
functional and gene expression studies were performed on ABC
obtained by routine bronchoscopy using bronchial brushes in
accordance with a standardized protocol and lung tissues
derived from explants. In addition, we investigated the
functional role of TGF-β, EMT, and HDAC6 in our recently
established 3D organoid assay.39 Further details on 3D organoid
culture preparation are shown in Figure S16. Sphere formation
was counted by bright-field microscopy, and cell viability was
quantified with the aid of the MTT assay.
HDAC6 Overexpression as a Key Element in Cell
Migration, Proliferation, and Fibrotic Remodeling by
ABC. The best-performing compounds (6a, 6d, 6h, and 6l)
resulting from the enzymatic assay were chosen as potential hit
compounds for IPF treatment and tested in the 3D organoid
assay (Figure 8). The molecules were tested in the concentration
range of 0.1 to 50 nM for 14 days with ABC derived from IPF
patients (n = 3). The sphere counts (dots) and the MTT assay
demonstrated a strong reduction in sphere formation and cell
proliferation at 50 and 20 nM (Figures 9 to 10). The best
compounds of the series 6a (IC50 = 1.73 nM), 6d (IC50 = 3.31
nM), 6h (IC50 = 5.15 nM), and 6l (IC50 = 4.61 nM) were highly
effective in reverting the IPF phenotype and determined the
complete inhibition of sphere formation (Figure 9,A−D) and in
the cell proliferation assay (Figure 9, E−H). These data indicate
the pivotal role of HDAC6 inhibitors, such as 6a, 6d, 6h, and 6l,
that may represent promising hit compounds for the develop-
ment of novel small molecules as useful pharmacological tools
for IPF treatment.
6h Attenuates TGF-β1-Dependent Fibrogenesis in
Human Lung Tissue. Having identified HDAC6 expression
in healthy and IPF human lung tissue ex vivo, we tested the
efficacy of 6h in a validated human lung model of TGF-β1-
dependent fibrogenesis.40 mRNA RIN values were >8 in all
experimental conditions. As previously described, TGF-β1
significantly upregulated mRNA for the fibrosis-associated
molecules α-SMA, collagens I, and collagen III in ex vivo
human lung parenchyma over 7 days of culture (n = 6 donors,
Figure 11A−C). TGF-β1 also significantly upregulated mRNA
for the matrix protein fibronectin (Figure 11D). There was a
trend toward increased expression of HDAC6 with TGF-β1
stimulation, but this did not reach statistical significance (n = 3
donors, Figure 11E). Compound 6h at a concentration of 4.1
Figure 6. Western blot experiments to evaluate HDAC6 selectivity over
HDAC1. Acetylation levels of the α-tubulin (AcTub) and histone H3 in
NB4, U87, and U2OS cell lines treated with compounds 6a, 6d, 6e, 6h,
and 6l at 5 μM for 30 h. SAHA was used as a positive control at the same
time and concentration. GAPDH and H4 were used as loading controls.
in NB4 and U87 cells, the analogues 6a, 6e, 6h, and 6l efficiently
inhibited HDAC6, inducing marked acetylation of tubulin. At
the same dose, histone H3 acetylation was unchanged, hinting to
the preferential interaction of this set of compounds toward the
HDAC6 enzyme, with respect to HDACs1/8. Conversely,
compound 6d, although equally effective in inhibiting HDAC6,
also induced the acetylation of histone H3, proving to be slightly
less selective. When compounds 6h, 6l, and 6d were tested in the
U2OS cell line, we observed different results and only 6l
demonstrated to preferentially promote acetylation of α-tubulin.
In this case, it cannot be excluded that the specific characteristics
of the U2OS cell line (e.g., the alternative lengthening of
telomeres)36 may play a role in the response to HDACi.
HDAC6 Overexpression in IPF Lung Tissues. It was
recently demonstrated that the accumulation of airway basal
cells (ABCs) in lung tissues from IPF patients was associated
with a lower survival rate, suggesting that these cells play an
important profibrotic role in IPF progression.37 By the
bronchoalveolar lavage (BAL) transcriptome analysis of three
independent cohorts of IPF patients, we have recently shown
that genes, exclusively expressed by ABC, are enriched in the
BAL of patients with poor outcome.37,38 Lung tissue data
demonstrated the accumulation of ABC in IPF, and our own
data suggest a profibrotic role of these cells. Recent single-cell
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Figure 7. HDAC6 overexpression in IPF lung tissues. Immunohistochemistry of HDAC6 in normal (A) and IPF lung tissues (B−D). In IPF lungs,
HDAC6 is highly expressed in ABC covering fibroblasts foci or honeycomb cysts as well as by macrophages and lymphocytes. Notably, in normal lungs,
alveolar macrophages expressed HDAC6.
Figure 8. Newly generated HDAC6 inhibitors showed a concentration-dependent inhibitory effect on bronchosphere formation in 3D organoid
cultures derived from IPF ABC. (A−D) ABC of IPF patients (n = 3) in 3D organoid cultures were stimulated for 14 days with the newly developed
HDAC6 inhibitors 6a (A), 6d (B), 6h (C), and 6l (D) in the concentration range from 0.1 to 50 nM. Bright-field microscopy mosaic images of one
representative experiment were taken with an Observer.Z1 Zeiss microscope, and exemplary registrations are depicted.
μM significantly inhibited the mRNA expression for α-SMA,
collagen types I and III, fibronectin, and HDAC6 in TGF-β1-
stimulated lung tissue (Figure 11A−D,F). Taken together, these
data suggest that HDAC6 plays a critical role in profibrotic TGF-
β1-dependent signaling human lung parenchyma and that 6h
may be an effective tool for the treatment of IPF.
solubility at neutral pH was observed between the two
compounds, with compound 6h being less soluble. This fact
could be explained by the absence of the polar N-methyl-
piperidyl moiety on the structure of this compound. However,
both the solubility and chemical stability of these compounds
can be considered satisfactory.
Preliminary In Vitro Physicochemical Property Assess-
ment and Determination of Geno- and Cytotoxicity and
Metabolic Stability. We assessed the solubility and chemical
stability of compounds 6d and 6h. The solubility (at pH = 3 and
7.4, Table 2) and the chemical stability (at pH = 3, Table 3) of
these compounds were measured by HPLC methods, as
previously reported.41,42 From our analysis, it emerged that
both 6d and 6h exhibited favorable chemical stability and
solubility profile at acidic pH. Instead, a significant difference in
In vitro studies were conducted to assess the metabolic
stability of 6d and 6h in human liver microsomal (HLM)
preparations. The plot of nonmetabolized compound [natural
logarithm of % of compound recovery (100% at time 0 min)] as
a function of incubation time showed a monoexponential decay
relationship (first-order kinetics) for both substrates (Figure
12). The apparent decay constants (k), half-life time (t1/2), and
intrinsic clearance (CLint) are reported in Table 4. The
incubation of both compounds in HLM showed different
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Figure 9. Bronchosphere generation was completely blocked by newly developed HDAC6 inhibitors. Bronchosphere counts derived from ABC from
IPF patients (n = 3) were obtained (A−D, 6a, 50 nM: p = 0.044, 20 nM: p = 0.044; 6d, 50 nM: p = 0.0161, 20 nM: p = 0.0161; 6h, 50 nM: p = 0.0229, 20
nM: p = 0.0229; 6l, 50 nM: p = 0.0047, 20 nM: p = 0.0307), and cell proliferation was tested by the MTT assay (E−H, 6a, 50 nM: p = 0.023 20 nM: p =
0.044; 6d, 50 nM: p = 0.0113, 20 nM: p = 0.0228; 6h, 50 nM: p = 0.0163; 6l, 50 nM: p = 0.0053, 20 nM: p = 0.0441) in the presence of several HDAC6
inhibitors such as compounds 6a (A, E), 6d (B, F), 6h (C, G), and 6l (D, H) in the concentration range of 1−50 nM. Bronchosphere counts and optical
density values derived from the MTT assay correlated well. The data indicate mean standard error of the mean (SEM) from triplicate measurements,
n = 3. For statistical comparison, repeated measures analysis of variance (ANOVA) followed by Friedman multiple comparisons test was used.
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Figure 10. HDAC6 inhibitors show a concentration-dependent inhibitory effect on bronchosphere formation. IC50 values were determined from
bronchosphere counts and bronchosphere cell proliferation by GraphPad Prism 9 (mean SEM, n = 3). The mean value is determined from the IC50
value of the organoid counts and the MTT assay. Calculated IC50 values represent the concentration of the best HDAC6 inhibitor compounds 6a, 6d,
6h, and 6l at which they exert their half-maximal inhibitory effect on bronchosphere formation in the 3D organoid assay.
behavior as confirmed by the t1/2 values (51.68 and 53.47 min for
6d and 6h, respectively). The similar values of CLint indicate that
both 6d and 6h can be considered to possess intermediate
properties in terms of metabolic stability.
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Table 3. Solubility and Chemical Stability of Compounds 6d
and 6h
solubility (μM) after
24 h
chemical stability (%) after 24 h
pH = 3
compound
pH = 3
pH = 7.4
6d
6h
TubA
456
459
244
285
97
231
98
89
nc
a
a
nc: not calculated.
Figure 12. CYP-dependent metabolic depletion of 5 μM 6d (red line,
three time points, at 0, 30, and 60 min) and 6h (black line, six time
points, at 0, 5, 15, 30, 45, and 60 min) in HLM preparation. Results are
presented graphically as a percentage of compound recovery (100% at
time 0 min) as a function of incubation time. Data are presented as
mean SEM of three different experiments.
Table 4. Kinetic Parameters and Metabolic Stability of
Compounds 6d and 6h
compound
k (min−1
)
t1/2 (min)
CLint (μL/min/mg prot)
6d
6h
SAHA44
0.01297
0.01357
51.68
53.47
60
27.43
25.46
Potential mutagenicity associated with the use of hydroxamic
acid-based compounds poses a significant challenge in terms of
their druglike profile.45 To date, Givinostat is the only
compound under clinical evaluation that has exhibited no
mutagenic effect, while the FDA-approved drugs have shown
mutagenicity.46 Therefore, we confirmed for compound 6h a
lack of mutagenic effect in TA98 and TA100 strains of
Salmonella typhimurium. The Ames test was employed to detect
potential risks of mutagenicity at the early stages of drug
development. The assay can be performed with or without the
S9 fraction of the rat liver. This latter condition allows an in-
depth investigation for evaluating the risk of mutagenicity
derived from the metabolites of the compounds under study.
After applying both the experimental conditions, no mutagenic
effect was observed for compound 6h at all tested concen-
trations (1−75 μM) (Figure 13). Besides the physicochemical
parameters of the compounds, we experimentally determined
additional features that might contribute to designate the most
promising compound 6h as the potential hit of the series.
Accordingly, we evaluated its potential cytotoxicity profile after
incubation with mouse embryonic fibroblasts (NIH3T3 cell
line). The viability of these cells, after incubation with 6h, is
reported in Table 4 and expressed as IC50 (μM). We observed
Figure 11. HDAC6 inhibition attenuates TGF-β1-dependent profi-
brotic gene expression in ex vivo cultured human lung parenchyma. (A)
α-SMA actin mRNA expression is upregulated by TGF-β1 and
inhibited by 4.1 μM 6h (n = 6). **p < 0.01, *p < 0.05, paired t-test.
(B) Collagen type I mRNA expression is upregulated by TGF-β1 and
inhibited by 4.1 μM (n = 6) **p < 0.01, *p < 0.05, paired t-test. (C)
Collagen type III mRNA expression is upregulated by TGF-β1 and
inhibited by 4.1 μM (n = 6). *p < 0.05, paired t-test. (D) Fibronectin
mRNA expression is upregulated by TGF-β1 and inhibited by 4.1 μM
(n = 6). *p < 0.05, paired t-test. (E) HDAC6 mRNA is expressed in
human lung parenchyma (n = 3). (F) HDAC6 mRNA expression is
inhibited by 4.1 μM (n = 3), *p < 0.05, paired t-test. NS =
nonstimulated.
In silico analysis by means of MetaSite software,43 for
predicting the site of CYP-dependent metabolism, revealed
that the hydroxylation of the aromatic ring is the most probable
metabolite of both compounds. Moreover, these metabolites
seem to be formed, preferentially, by CYP3A4 and CYP2D6.
The other CYP isoforms, such as CYP belonging to the 2C
family, seem to drive the metabolism toward N-oxide formation.
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Figure 13. Ames test performed on S. Typhimurium TA98 and TA100 strains for compound 6h.
Table 5. Viability of Mouse Fibroblasts NIH3T3 after Incubation with Different Concentrations of 6h and Reference HDAC
a
Inhibitor SAHA (%)
[μM]
1
2
2.5
*49
5
7.5
*22
10
*17
15
94
20
*7
25
*87
50
*71
75
6h
SAHA
98
*31
6
5
9
5
9
*53 10
*83
6
*62
5
3
3
6
6
a
Cell viability was measured by the neutral red uptake (NRU) test, and data were normalized as % control. Data are expressed as mean s.d. of
three experiments repeated in six replicates. *Values are statistically different versus control, p ≤ 0.05.
Figure 14. (A) Toxicity screening of 6h in the zebrafish model and (B) measurement of visual capacity in 5-day postfertilization (dpf) old larvae at
different concentrations (1−100 μM).
that 6h showed toxicity only in the μM range with a TC50 of 75
μM, resulting in a highly safe profile for this compound (Table
5).
administration in humans, we decided to use this model to assess
the toxic potential of 6h. Three-day-old zebrafish larvae were
treated for 2 days with increasing doses of this compound
(Figure 14A). 6h was well tolerated up to 50 μM instead, and
only at 100 μM, some larvae failed to inflate their swim bladder
and presented mild cardiac edema, thus confirming the safety
profile of this compound. Additionally, visual behavior analysis
conducted on five-day-old larvae revealed that there were no
significant changes in larvae treated with compound 6h in
comparison to vehicle control (0.1% DMSO)-treated larvae,
thus confirming that the drug is well tolerated (Figure 14B).
Safety of 6h in a Zebrafish Model and in Langendorff-
Perfused Rat Hearts. There is a clear potential for zebrafish to
provide valuable new insights into chemical toxicity, useful in the
drug discovery trajectory, and human disease using recent
advances in forward and reverse genetic techniques coupled with
large-scale, high-throughput screening. Recently, there is
increasing use of zebrafish in toxicology.47 Since zebrafish
could provide a sound basis for the risk assessment of drug
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a
Table 6. Effects of 6h on HR, RR, PQ, QRS, QT, and QTc in Langendorff-Perfused Rat Hearts
6h (μM)
HR (BPM)
RR (ms)
PQ (ms)
QRS (ms)
QT (ms)
QTc (ms)
none
0.01
0.1
1
235.7 8.3
235.4 9.2
238.1 10.5
235.4 12.0
207.9 17.1**
257.2 8.1
255.9 9.3
253.7 10.0
257.7 11.2
294.1 20.9**
43.6 2.5
44.1 2.6
44.6 3.2
44.5 3.0
50.4 2.2**
15.2 0.2
15.6 0.5
15.4 0.4
15.8 0.4
18.0 0.5**
76.2 2.9
78.1 3.2
78.0 3.3
76.4 3.1
78.3 3.4
150.5 5.5
154.6 5.8
155.1 6.6
150.8 6.1
145.3 5.7
10
a
Each value represents mean SEM (n = 5). **P < 0.01, repeated measures ANOVA and Dunnett’s post-test. HR, frequency; RR, cycle length;
PQ, atrioventricular conduction time; QRS, intraventricular conduction time; QT, duration of ventricular depolarization and repolarization, i.e., the
action potential duration; QTc, corrected QT.
Cardiovascular adverse effects contribute disproportionately
to drug withdrawals from the market and represent one of the
major hurdles in the development of new drugs. To evaluate the
potential cardiovascular toxicity of compound 6h, its effect on
cardiac mechanical function and the electrocardiogram (ECG)
in Langendorff-isolated rat hearts was assessed, as previously
described.48,49 Under control conditions, left ventricle pressure
(LVP) and coronary perfusion pressure (CPP) values of 57.53
dependent fibrogenesis in human lung tissue with compound
6h. Overall, this work provides robust proof for HDAC6
inhibitors as potential therapeutic tools for the treatment of IPF.
In conclusion, we identified 6h as a promising and optimized
HDAC6i hit, with efficacy in reverting the IPF phenotype, which
may pave the way to the development of druglike leads.
EXPERIMENTAL SECTION
■
7.19 and 52.30
obtained. At the maximum concentration tested (10 μM), 6h
significantly increased LVP to 70.99 10.15 mmHg and
5.65 mmHg (n = 5), respectively, were
Chemistry. Unless otherwise specified, materials were purchased
from commercial suppliers and used without further purification.
Reaction progress was monitored by TLC using silica gel 60 F254
(0.040−0.063 mm) with detection by UV (254 nm). Silica gel 60
(0.040− 0.063 mm) or aluminum oxide 90 (0.063−0.200 mm) were
used for column chromatography. 1H NMR and 13C NMR spectra were
recorded on a Varian 300 MHz or Bruker 400 MHz spectrometer using
the residual signal of the deuterated solvent as the internal standard.
Splitting patterns are described as singlet (s), doublet (d), triplet (t),
quartet (q), quintet (p), and broad (br); the value of chemical shifts (δ)
is given in ppm, and coupling constants (J) are given in hertz (Hz).
Electrospray ionization mass spectrometry (ESI-MS) spectra were
performed by an Agilent 1100 series LC/MSD spectrometer. Optical
rotation values were measured at room temperature using a
PerkinElmer model 343 polarimeter operating at 589 nm, correspond-
ing to the sodium D line. Yields refer to purified products and are not
optimized. All moisture-sensitive reactions were performed under an
argon atmosphere using oven-dried glassware and anhydrous solvents.
ESI-MS spectra for exact mass determination were recorded on an LTQ
Orbitrap Thermo Fischer Scientific instrument. All final compounds
were purified by flash column chromatography. The purity of final
products (>95%) was determined by an analytical HPLC Merk
Purospher STAR RP-18e (5 μm) LiChroCART 250-4 column;
detection at 254 nm; flow rate = 1.0 mL/min; mobile phase A, 0.01%
trifluoroacetic acid (TFA) (v/v) in water; mobile B, acetonitrile;
gradient, 90/10−10/90 A/B in 20 min. The gradient was optimized
based on compound polarity.
decreased CPP to 44.11 3.53 mmHg. Moreover, at 10 μM, 6h
significantly increased RR, PQ, and QRS, though not QTc ECG
intervals (Table 6). Therefore, these findings highlight that at
the maximum concentration tested, which was however three
orders of magnitude higher than that effective in hDAC6
inhibition, 6h exhibited positive inotropic, negative chrono-
tropic, and coronary vasodilating activity and prolonged the
cardiac cycle length as well both the atrioventricular and
intraventricular conduction time.
CONCLUSIONS
■
Combining structural analysis, bioinformatics, and molecular
modeling efforts, we generated a series of new HDAC6
inhibitors, selective over hHDAC1 and hHDAC8. Synthetic
accessibility, high potency, and interesting preliminary pharma-
cokinetic profile, including low geno/cardio/cytotoxicity,
characterize the novel heterocyclic inhibitors of isoform 6,
exemplified by 6h. Using 6h, we confirmed the lack of a
stereoselective inhibition of HDAC6 since both enantiomers of
6h showed similar inhibition properties ((R)-6h IC50 hHDAC6
= 91 nM vs (S)-6h IC50 hHDAC6 = 71 nM), with the (R)-
enantiomer much more selective over hHDAC1 (Table 1). The
binding mode on HDAC6 was determined by X-ray
crystallography using 6h in complex with zf HDAC6. SARs of
the new inhibitors were analyzed by docking protocols using
human enzymes. Selected analogues were not mutagenic;
moreover, they were not cardiotoxic in Langendorff-isolated
rat hearts and not toxic in vivo. Additionally, selected analogues
6d and 6h showed favorable solubility, chemical stability at both
neutral and acidic pH, and metabolic stability to hCYP3A4. By
an immunohistochemistry analysis, we confirmed the over-
expression of HDAC6 in human IPF lung tissues. Our data
suggest that this fact may confer hyperproliferative and
profibrotic effects of ABC on IPF. Consecutively, we tested
our best-performing compounds, resulting from enzymatic assay
(6a, 6d, 6h, and 6l), in 3D organoid and MTT assays to evaluate
their efficacy in reverting the IPF phenotype and their
antifibrotic activity. The results emphasized the important role
that HDAC6 inhibition plays in the treatment of IPF. This
evidence was further confirmed by the attenuation of TGF-β1-
(1-Methylpiperidin-4-yl)(phenyl)methanone (8a). Tosylhydra-
zone 7 (538 mg, 1.91 mmol) and Cs2CO3 (932 mg, 2.86 mmol)
were placed in a tube. The tube was backfilled with nitrogen, before the
addition of 1,4-dioxane (7 mL) followed by the addition of
benzaldehyde (194 μL, 1.91 mmol). The tube was sealed with a
silicone/poly(tetrafluoroethylene) (PTFE) cap and heated to 110 °C
for 12 h. The reaction was cooled to 25 °C, quenched with a saturated
solution of NH4Cl (20 mL), and extracted with DCM (3 × 20 mL).
The combined organic layers were dried over Na2SO4, and the solvents
were removed in vacuo to give a residue that was purified by flash
column chromatography on silica gel (MeOH/DCM 1:15) to give 8a
(155 mg, 40% yield); 1H NMR (400 MHz, CDCl3) δ 7.90 (d, J = 7.2
Hz, 2H), 7.58−7.47 (m, 1H), 7.43 (t, J = 7.8 Hz, 2H), 3.28−3.11 (m,
1H), 2.92 (m, 2H), 2.29 (s, 3H), 2.17−2.00 (m, 2H), 1.97−1.73 (m,
4H); ESI-MS m/z: [M + H]+ 204.
(4-Chlorophenyl)(1-methylpiperidin-4-yl)methanone (8b). Start-
ing from 7 (1.0 g, 3.56 mmol) and 4-chlorobenzaldehyde (500 mg, 3.56
mmol), compound 8b was obtained following the procedure described
for the preparation of 8a. Purification by column chromatography on
1
silica gel (MeOH/DCM 1:15) afforded 8b (432 mg, 51% yield); H
NMR (300 MHz, CDCl3) δ 7.86 (d, J = 8.6 Hz, 2H), 7.43 (d, J = 8.8 Hz,
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2H), 3.19−3.10 (m, 1H), 2.96−2.94 (m, 2H), 2.32 (s, 3H), 2.17−2.03
(m, 2H), 1.91−1.80 (m, 4H); ESI-MS m/z: [M + H]+ 239.
(3-Chlorophenyl)(1-methylpiperidin-4-yl)methanone (8c). Start-
ing from 7 (1.0 g, 3.56 mmol) and 3-chlorobenzaldehyde (500 mg, 3.56
mmol), compound 8c was obtained following the procedure described
for the preparation of 8a. Purification by column chromatography on
silica gel (MeOH/DCM 1:15) afforded the title compound (633 mg,
75% yield); 1H NMR (300 MHz, CDCl3) δ 7.83−7.69 (m, 2H), 7.44 (s,
1H), 7.27−7.17 (m, 1H), 3.12 (m, 1H), 2.88 (m, 2H), 2.40 (t, J = 5.0
Hz, 2H), 2.25 (s, 3H), 1.88−1.70 (m, 4H); ESI-MS m/z: [M + H]+
238.
(2-Fluorophenyl)(1-methylpiperidin-4-yl)methanone (8d). Start-
ing from 7 (1.0 g, 3.56 mmol) and 2-fluorobenzaldehyde (442 mg, 3.56
mmol), compound 8d was obtained following the procedure described
for the preparation of 8a. Purification by column chromatography on
silica gel (MeOH/DCM 1:15) afforded the title compound (598 mg,
76% yield); 1H NMR (300 MHz, CDCl3) δ 7.78−7.65 (m, 1H), 7.70−
7.56 (m, 1H), 7.48−7.31 (m, 1H), 7.07−6.93 (m, 1H), 3.06−2.90 (m,
1H), 2.86−2.68 (m, 2H), 2.39−2.22 (m, 2H), 2.18 (s, 3H), 2.05−1.86
(m, 1H), 1.86−1.74 (m, 2H), 1.74−1.55 (m, 1H); ESI-MS m/z: [M +
H]+ 222.
2-(2-Fluorophenyl)-1′-methylspiro[indole-3,4′-piperidine] (9d).
Compound 9d was obtained from 8d (300 mg, 1.36 mmol) following
the procedure described for the preparation of 9a. Purification by
column chromatography on silica gel (MeOH/DCM 1:20) afforded
the title compound (120 mg, 30% yield); 1H NMR (300 MHz, CDCl3)
δ 7.83 (d, J = 7.5 Hz, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.46−7.38 (m, 2H),
7.32−7.23 (m, 3H), 7.23−7.07 (m, 1H), 2.81 (s, 1H), 2.69 (t, J = 11.6
Hz, 2H), 2.42 (s, 3H), 2.23 (t, J = 12.6 Hz, 2H), 1.57 (d, J = 13.2 Hz,
2H); ESI-MS m/z: [M + H]+ 295.
2-(4-Fluorophenyl)-1′-methylspiro[indole-3,4′-piperidine] (9e).
Compound 9e was obtained from 8e (627 mg, 2.8 mmol) following
the procedure described for the preparation of 9a. Purification by
column chromatography on silica gel (acetone/DCM 1:20) afforded
the title compound (412 mg, 50% yield); 1H NMR (300 MHz, CDCl3)
δ 8.26−8.11 (m, 1H), 7.85 (d, J = 7.5 Hz, 2H), 7.71 (d, J = 7.9 Hz, 2H),
7.41 (t, J = 7.7 Hz, 1H), 7.27−7.06 (m, 2H), 3.06−2.87 (m, 2H), 2.79−
2.60 (m, 4H), 2.50 (s, 3H), 1.51−1.39 (m, 2H); ESI-MS m/z: [M +
H]+ 295.
1′-Methyl-2-(pyridin-3-yl)spiro[indole-3,4′-piperidine] (9f). Start-
ing from 8f (200 mg, 0.98 mmol), the title compound 9f was obtained
following the procedure described for the preparation of 9a.
Purification by column chromatography on silica gel (MeOH/DCM
1:10) afforded the title compound (180 mg, 66% yield); 1H NMR (300
MHz, CDCl3) δ 9.37 (s, 1H), 8.71−8.62 (m, 1H), 8.40 (d, J = 8.1 Hz,
1H), 7.83 (d, J = 6.3 Hz, 1H), 7.76−7.63 (m, 1H), 7.47−7.32 (m, 2H),
7.28−7.14 (m, 1H), 3.03−2.91 (m, 2H), 2.80−2.56 (m, 4H), 2.50 (s,
3H), 1.46 (d, J = 12.6 Hz, 2H); δ ESI-MS m/z: [M + H]+ 279.
( )-1′-Methyl-2-phenylspiro[indoline-3,4′-piperidine] (( )-10a).
To a solution of 9a (110 mg, 0.40 mmol) in MeOH (5 mL), NaBH4
(76 mg, 2.00 mmol) was added. The reaction was kept stirring for 12 h
at 50 °C. Then, the reaction was quenched with a saturated solution of
NaHCO3 (10 mL) and MeOH was removed under reduced pressure.
The residue was dissolved with EtOAc and washed with H2O (3 × 10
mL). The organic phase was dried over Na2SO4, and solvents were
removed in vacuo to give a residue that was purified by flash column
chromatography on silica gel (MeOH/DCM 1:20) affording the title
compound (60 mg, 40% yield); 1H NMR (300 MHz, CDCl3) δ 7.32−
7.20 (m, 6H), 7.09 (td, J = 7.6, 1.1 Hz, 1H), 6.77 (td, J = 7.4, 0.8 Hz,
1H), 6.66 (d, J = 7.7 Hz, 1H), 4.59 (s, 1H), 4.14 (br, 1H), 2.85−2.73
(m, 1H), 2.66−2.55 (m, 1H), 2.52−2.41 (m, 1H), 2.30 (s, 3H), 2.13−
1.74 (m, 4H), 1.42 (m, 1H); ESI-MS m/z: [M + H]+ 279.
( )-2-(4-Chlorophenyl)-1′-methylspiro[indoline-3,4′-piperidine]
(( )-10b). Starting from 9b (110 mg, 0.35 mmol), compound ( )-10b
was obtained following the procedure described for the preparation of
( )-10a. Purification by column chromatography on silica gel
(MeOH/DCM 1:20) afforded the title compound (45 mg, 40%
yield); 1H NMR (300 MHz, CDCl3) δ 7.31−7.17 (m, 5H), 7.11 (t, J =
7.6 Hz, 1H), 6.78 (t, J = 7.5 Hz, 1H), 6.68 (d, J = 7.7 Hz, 1H), 4.07 (s,
1H), 2.82 (br, 1H), 2.65−2.52 (m, 2H), 2.35 (s, 3H), 2.19−1.90 (m,
4H), 1.41 (t, J = 9.8 Hz, 2H); ESI-MS m/z: [M + H]+ 313.
( )-2-(3-Chlorophenyl)-1′-methylspiro[indoline-3,4′-piperidine]
(( )-10c). Starting from 9c (102 mg, 0.33 mmol), compound ( )-10c
was obtained following the procedure described for the preparation of
( )-10a. The title compound was used in the next step without any
further purification. (Quantitative yield); 1H NMR (300 MHz, CDCl3)
δ 7.36−7.17 (m, 5H), 7.10 (t, J = 8.4 Hz, 1H), 6.77 (t, J = 7.4 Hz, 1H),
6.68 (d, J = 7.8 Hz, 1H), 4.56 (s, 1H), 4.09 (br, 1H), 2.80−2.71 (m,
1H), 2.67−2.52 (m, 1H), 2.51−2.36 (m, 1H), 2.31 (s, 3H), 2.19−1.85
(m, 4H), 1.83−1.65 (m, 1H); ESI-MS m/z: [M + H]+ 313.
(4-Fluorophenyl)(1-methylpiperidin-4-yl)methanone (8e). Start-
ing from 7 (1.0 g, 3.56 mmol) and 4-fluorobenzaldehyde (440 mg, 3.56
mmol), compound 8e was obtained following the procedure described
for the preparation of 8a. Purification by column chromatography on
silica gel (MeOH/DCM 1:20) afforded the title compound (597 mg,
75% yield); 1H NMR (300 MHz, CDCl3) δ 8.02−7.89 (m, 2H), 7.14 (t,
J = 8.6 Hz, 2H), 3.28−3.11 (m, 1H), 3.00−2.88 (m, 2H), 2.34 (s, 3H),
2.22−2.06 (m, 2H), 1.95−1.77 (m, 4H); ESI-MS m/z: [M + H]+ 222.
(1-Methylpiperidin-4-yl)(pyridin-3-yl)methanone (8f). Starting
from 7 (500 mg, 1.78 mmol) and 3-pyridinecarboxaldehyde (107 mg,
1.78 mmol), compound 8f was obtained following the procedure
described for the preparation of 8a. Title compound was obtained as
1
pure without any further purification (quantitative yield); H NMR
(300 MHz, CDCl3) δ 9.14 (s, 1H), 8.77 (d, J = 4.8 Hz, 1H), 8.20 (d, J =
8.0 Hz, 1H), 7.48−7.36 (m, 1H), 3.26−3.11 (m, 1H), 3.02−2.89 (m,
2H), 2.33 (s, 3H), 2.22−2.05 (m, 4H), 1.95−1.83 (m, 2H); ESI-MS m/
z: [M + H]+ 205.
1′-Methyl-2-phenylspiro[indole-3,4′-piperidine] (9a). Phenylhy-
drazine (143 μL, 1.45 mmol) and 8a (295 mg, 1.45 mmol) were
dissolved in 1,4-dioxane (5 mL) and cooled to 0 °C. Concentrated
sulfuric acid (700 μL) was added dropwise to the reaction at 0 °C. The
reaction was then heated at 70 °C for 2 h and stirred for an additional 12
h at 25 °C. The mixture was then treated with a saturated solution of
NaHCO3 (20 mL) and extracted with DCM (3 × 20 mL). The
combined organic layers were dried over Na2SO4, and solvents were
removed in vacuo to give a residue that was purified by flash column
chromatography on silica gel (MeOH/DCM 1:15) to give the title
compound (120 mg, 30% yield); 1H NMR (300 MHz, CDCl3) δ 8.27−
7.99 (m, 2H), 7.85 (d, J = 7.5 Hz, 2H), 7.73 (d, J = 7.7 Hz, 2H), 7.61−
7.32 (m, 4H), 7.22 (t, J = 8.1 Hz, 1H), 2.95 (br, 2H), 2.75 (br, 4H), 2.49
(s, 3H), 1.45 (br, 2H); ESI-MS m/z: [M + H]+ 277.
2-(4-Chlorophenyl)-1′-methylspiro[indole-3,4′-piperidine] (9b).
Compound 9b was obtained from 8 (268 mg, 1.13 mmol) following
the procedure described for the preparation of 9a. Purification by
column chromatography on silica gel (MeOH/DCM 1:30) afforded
the title compound (112 mg, 32% yield); 1H NMR (300 MHz, CDCl3)
δ 8.16 (d, J = 8.6 Hz, 2H), 7.86 (d, J = 7.1 Hz, 1H), 7.73 (d, J = 7.8 Hz,
1H), 7.44 (d, J = 8.5 Hz, 2H), 7.41 (m, 1H), 7.29−7.16 (m, 1H), 3.02−
2.97 (m, 2H), 2.81−2.62 (m, 4H), 2.51 (s, 3H), 1.53−1.21 (m, 2H);
ESI-MS m/z: [M + H]+ 311.
( )-2-(2-Fluorophenyl)-1′-methylspiro[indoline-3,4′-piperidine]
(( )-10d). A mixture of 9d (113 mg, 0.38 mmol) and a catalytic amount
of 10% palladium on carbon in MeOH (2 mL) was stirred under a
hydrogen atmosphere at 25 °C. The disappearance of the starting
material was monitored by TLC, and the Pd/C was filtered and washed
with MeOH (5 mL). The solvent was removed in vacuo, and the title
compound was used in the next step without any further purification
2-(3-Chlorophenyl)-1′-methylspiro[indole-3,4′-piperidine] (9c).
Compound 9c was obtained from 8c (476 mg, 2.0 mmol) following
the procedure described for the preparation of 9a. Purification by
column chromatography on silica gel (MeOH/DCM 1:20) afforded
the title compound (187 mg, 30% yield); 1H NMR (300 MHz, CDCl3)
δ 8.16 (s, 1H), 7.97 (d, J = 7.1 Hz, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.74 (d,
J = 7.7 Hz, 1H), 7.50−7.36 (m, 3H), 7.30−7.20 (m, 1H), 3.00−2.92
(m, 2H), 2.81−2.59 (m, 4H), 2.51 (s, 3H), 1.50−1.42 (m, 2H); ESI-
MS m/z: [M + H]+ 311.
1
(quantitative yield); H NMR (300 MHz, CDCl3) δ 7.35−7.20 (m,
4H), 7.15−6.96 (m, 2H), 6.79 (t, J = 7.5 Hz, 1H), 6.68 (d, J = 7.8 Hz,
1H), 5.10 (s, 1H), 4.00 (br, 1H), 3.06−2.87 (m, 1H), 2.80−2.55 (m,
9975
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Article
2H), 2.44 (s, 3H), 2.28−1.93 (m, 4H), 1.62−1.43 (m, 2H); ESI-MS m/
z: [M + H]+ 297.
16.1 Hz, 1H), 3.87 (s, 3H), 3.86 (d, J = 16.3 Hz, 1H), 2.87−2.73 (m,
1H), 2.59−2.45 (m, 2H), 2.31 (s, 3H), 2.11−1.87 (m, 4H), 1.56−1.43
(m, 1H); ESI-MS m/z: [M + H]+ 445.
( )-2-(4-Fluorophenyl)-1′-methylspiro[indoline-3,4′-piperidine]
(( )-10e). Starting from 9e (300 mg, 1.02 mmol), compound ( )-10e
was obtained following the procedure described for the preparation of
( )-10a. Purification by column chromatography on silica gel
(acetone/DCM 1:20) afforded the title compound (176, 58% yield);
1H NMR (300 MHz, CDCl3) δ 7.31−7.20 (m, 3H), 7.10 (t, J = 7.6 Hz,
1H), 6.96 (t, J = 8.7 Hz, 2H), 6.77 (t, J = 7.5 Hz, 1H), 6.67 (d, J = 7.9
Hz, 1H), 4.58 (s, 1H), 4.09 (br, 1H), 2.89−2.73 (m, 1H), 2.70−2.55
(m, 1H), 2.55−2.41 (m, 1H), 2.33 (s, 3H), 2.15−2.04 (m, 1H), 2.04−
1.89 (m, 2H), 1.84−1.79 (m, 1H), 1.45−1.29 (m, 1H); ESI-MS m/z:
[M + H]+ 297.
( )-1′-Methyl-2-(pyridin-3-yl)spiro[indoline-3,4′-piperidine]
(( )-10f). Starting from 9f (180 mg, 0.64 mmol), compound ( )-10f
was obtained following the procedure described for the preparation of
( )-10a. The title compound was used in the next step without any
further purification (quantitative yield); 1H NMR (300 MHz, CDCl3) δ
8.56 (d, J = 2.3 Hz, 1H), 8.54−8.48 (m, 1H), 7.59 (d, J = 7.9 Hz, 1H),
7.28 (d, J = 7.9 Hz, 1H), 7.25−7.14 (m, 1H), 7.11 (t, J = 7.6 Hz, 1H),
6.79 (t, J = 7.4 Hz, 1H), 6.69 (d, J = 7.7 Hz, 1H), 4.62 (d, J = 1.4 Hz,
1H), 4.10 (br, 1H), 2.87−2.72 (m, 1H), 2.69−2.54 (m, 1H), 2.54−2.40
(m, 1H), 2.31 (s, 3H), 2.16−1.86 (m, 4H), 1.88−1.71 (m, 1H), 1.42−
1.24 (m, 1H); ESI-MS m/z: [M + H]+ 280.
( )-Methyl 4-((2-(4-Fluorophenyl)-1′-methylspiro[indoline-3,4′-
piperidin]-1-yl)methyl)benzoate (( )-11e). Starting from ( )-10e
(178 mg, 0.60 mmol), ( )-11e was obtained following the procedure
described for the preparation of ( )-11a. Purification by column
chromatography on silica gel (MeOH/DCM 1:20) afforded the title
compound (122 mg, 46% yield); 1H NMR (300 MHz, CD3OD) δ 7.92
(d, J = 6.7 Hz, 2H), 7.30 (d, J = 6.7 Hz, 2H), 7.26−7.11 (m, 3H), 7.07−
6.98 (m, 3H), 6.70 (t, J = 7.4 Hz, 1H), 6.43−6.34 (m, 1H), 4.46 (s, 1H),
4.37 (d, J = 16.1 Hz, 1H), 3.91 (d, J = 11.0 Hz, 1H), 3.86 (s, 3H), 2.92−
2.77 (m, 1H), 2.68−2.45 (m, 2H), 2.30 (s, 3H), 2.13−1.95 (m, 2H),
1.95−1.75 (m, 2H), 1.46−1.33 (m, 1H); ESI-MS m/z: [M + H]+ 445.
( )-Methyl 4-((1′-Methyl-2-(pyridin-3-yl)spiro[indoline-3,4′-pi-
peridin]-1-yl)methyl)benzoate (( )-11f). Starting from ( )-10f (189
mg, 0.68 mmol), compound ( )-11f was obtained following the
procedure described for the preparation of ( )-11a. Purification by
column chromatography on silica gel (EtOAc/n-hexane 1:2) afforded
the title compound (73 mg, 25% yield); 1H NMR (300 MHz, CDCl3) δ
8.53 (d, J = 6.4 Hz, 1H), 8.36 (m, 1H), 7.96 (d, J = 8.3 Hz, 2H), 7.48−
7.37 (m, 1H), 7.32−7.21 (m, 3H), 7.19 (d, J = 6.5 Hz, 1H), 7.09 (t, J =
7.7 Hz, 1H), 6.76 (t, J = 6.9 Hz, 1H), 6.39 (d, J = 7.8 Hz, 2H), 4.45−
4.29 (m, 1H), 3.90 (s, 3H), 2.88−2.68 (m, 2H), 2.57−2.40 (m, 2H),
2.09−1.80 (m, 4H), 1.46−1.30 (m, 2H); ESI-MS m/z: [M + H]+ 428.
( )-N-Hydroxy-4-((1′-methyl-2-phenylspiro[indoline-3,4′-piperi-
din]-1-yl)methyl)benzamide (6a). To a solution of ( )-11a (66 mg,
0.155 mmol) in DCM/MeOH 1:2 cooled at 0 °C, NH2OH (50% H2O
solution, 1.02 mL, 15.50 mmol) and a 4 M solution of KOH (1.94 mL,
7.75 mmol) were added, and the reaction mixture was left stirring at 25
°C for 4 h. Then, the pH was adjusted to 7 with 6 N HCl and the solvent
was removed under reduced pressure. The residue was purified by
column chromatography on silica gel (MeOH/DCM 1:20) affording
the final product (45 mg, 68% yield); 1H NMR (300 MHz, CD3OD) δ
7.68 (d, J = 8.2 Hz, 2H), 7.40−7.16 (m, 8H), 7.08 (t, J = 7.7 Hz, 1H),
6.73 (t, J = 7.3 Hz, 1H), 6.45 (d, J = 7.9 Hz, 1H), 4.53−4.36 (m, 2H),
3.90 (d, J = 15.9 Hz, 1H), 3.18 (m, 1H), 3.05 (m, 1H), 2.86 (m, 1H),
2.57 (s, 3H), 2.43 (m, 1H), 2.15 (m, 1H), 1.97 (m, 2H), 1.53 (m, 1H).
13C NMR (75 MHz, DMSO) δ 164.7, 151.4, 142.5, 138.1, 136.9, 132.2,
( )-Methyl 4-((1′-Methyl-2-phenylspiro[indoline-3,4′-piperidin]-
1-yl)methyl)benzoate (( )-11a). To a solution of ( )-10a (121 mg,
0.438 mmol) and methyl 4-formylbenzoate (72 mg, 0.438 mmol) in
EtOH (5 mL), AcOH (700 μL) and NaBH3CN (55 mg, 0.876 mmol)
were added. The reaction was then heated to 70 °C for 2 h and stirred
for additional 12 h at 25 °C. The mixture was then treated with a
saturated solution of NaHCO3 (20 mL) and extracted with EtOAc (3 ×
20 mL). The combined organic phases were dried over Na2SO4, and
solvents were removed in vacuo to give a residue that was purified by
flash column chromatography on silica gel (MeOH/DCM 1:15) (65
1
mg, 35% yield); H NMR (300 MHz, CDCl3) δ 7.95 (d, J = 8.2 Hz,
2H), 7.32−6.97 (m, 9H), 6.73 (t, J = 7.4 Hz, 1H), 6.35 (d, J = 7.8 Hz,
1H), 4.43−4.28 (m, 2H), 3.96−3.79 (m, 4H), 2.98−2.81 (m, 1H),
2.75−2.46 (m, 2H), 2.36 (s, 3H), 2.18−1.84 (m, 4H), 1.60−1.40 (m,
1H); ESI-MS m/z: [M + H]+ 427.
128.8, 128.6, 128.4, 127.7, 124.0, 118.0, 106.8, 77.2, 52.4, 49.9, 46.2,
( )-Methyl 4-((2-(4-Chlorophenyl)-1′-methylspiro[indoline-3,4′-
piperidin]-1-yl)methyl)benzoate (( )-11b). Starting from ( )-10b
(40 mg, 0.13 mmol), compound ( )-11b was obtained following the
procedure described for the preparation of 11a. Purification by column
chromatography on silica gel (acetone/DCM 1:30) afforded the title
compound (26 mg, 43% yield); 1H NMR (300 MHz, CDCl3) δ 7.96 (d,
J = 8.0 Hz, 2H), 7.34−7.20 (m, 5H), 7.10 (t, J = 7.6 Hz, 3H), 6.75 (t, J =
7.4 Hz, 1H), 6.38 (d, J = 7.8 Hz, 1H), 4.43−4.31 (m, 2H), 3.91 (s, 3H),
3.85 (d, J = 16.1 Hz, 1H), 2.89 (q, J = 5.8, 5.4 Hz, 2H), 2.68−2.54 (m,
2H), 2.38 (s, 3H), 2.18−1.91 (m, 2H), 1.58−1.39 (m, 2H); ESI-MS m/
z: [M + H]+ 461.
( )-Methyl 4-((2-(3-Chlorophenyl)-1′-methylspiro[indoline-3,4′-
piperidin]-1-yl)methyl)benzoate (( )-11c). Starting from ( )-10c
(100 mg, 0.32 mmol), compound ( )-11c was obtained following the
procedure described for the preparation of ( )-11a. Purification by
column chromatography on silica gel (MeOH/DCM 1:20) afforded
the title compound (47 mg, 32% yield); 1H NMR (300 MHz, CD3OD)
δ 7.94 (d, J = 7.5 Hz, 2H), 7.33 (d, J = 8.2 Hz, 2H), 7.32−7.17 (m, 4H),
7.14−7.00 (m, 2H), 6.73 (t, J = 7.5 Hz, 1H), 6.50−6.35 (m, 1H), 4.47
(s, 1H), 4.40 (d, J = 16.3 Hz, 1H), 3.93 (d, J = 16.8 Hz, 1H), 3.88 (s,
3H), 2.96−2.81 (m, 1H), 2.73−2.52 (m, 2H), 2.34 (s, 3H), 2.18−1.98
(m, 2H), 1.97−1.82 (m, 2H), 1.39 (d, J = 30.0 Hz, 1H); ESI-MS m/z:
[M + H]+ 461.
46.1, 31.4; ESI-MS m/z: [M + H]+ 428; HPLC RT: 13.0 min.
( )-4-((2-(4-Chlorophenyl)-1′-methylspiro[indoline-3,4′-piperi-
din]-1-yl)methyl)-N-hydroxybenzamide (6b). Compound 6b was
obtained from ( )-11b (26 mg, 0.056 mmol) following the procedure
described for the preparation of 6a. Purification by column
chromatography on silica gel (NH4OH/MeOH/DCM 0.1:1:10)
afforded the title compound as a transparent oil (9 mg, 33% yield);
1H NMR (300 MHz, CD3OD) δ 11.13 (br, 1H), 8.98 (br, 1H), 7.67 (d,
J = 8.3 Hz, 2H), 7.44−7.30 (m, 2H), 7.28 (d, J = 8.3 Hz, 2H), 7.19 (d, J
= 7.2 Hz, 2H), 6.99 (t, J = 7.7 Hz, 1H), 6.63 (t, J = 6.9 Hz, 1H), 6.34 (d,
J = 6.9 Hz, 2H), 4.51 (s, 1H), 4.31 (d, J = 16.3 Hz, 1H), 3.83 (d, J = 16.3
Hz, 1H), 2.68−2.53 (m, 2H), 2.14 (s, 3H), 1.76−1.63 (m, 4H), 1.22−
1.08 (m, 2H); 13C NMR (75 MHz, DMSO) δ 164.7, 151.1, 142.3,
137.2, 136.9, 133.0, 132.2, 128.9, 128.4, 127.8, 127.7, 124.1, 118.1,
106.8, 76.2, 52.4, 52.1, 49.9, 46.6, 46.2, 37.6, 31.5; ESI-MS m/z: [M +
H]+ 462; calcd for C27H29N3O2Cl 462.1943; found 462.1929; HPLC
RT: 12.2 min.
( )-4-((2-(3-Chlorophenyl)-1′-methylspiro[indoline-3,4′-piperi-
din]-1-yl)methyl)-N-hydroxybenzamide (6c). Compound 6c was
obtained from ( )-11c (37 mg, 0.08 mmol) following the procedure
described for the preparation of 6a. Purification by column
chromatography on silica gel (NH4OH/MeOH/DCM 0.1:1:10)
afforded the title compound (21 mg, 57% yield); 1H NMR (300
MHz, CD3OD) δ 11.15 (br, 1H), 8.99 (br, 1H), 7.67 (d, J = 8.2 Hz,
2H), 7.34 (s, 1H), 7.29 (d, J = 6.5 Hz, 2H), 7.20 (d, J = 7.4 Hz, 2H),
7.00 (t, J = 7.6 Hz, 2H), 6.64 (t, J = 8.1 Hz, 1H), 6.43−6.22 (m, 2H),
4.54 (s, 1H), 4.32 (d, J = 14.0 Hz, 1H), 3.86 (d, J = 16.3 Hz, 1H), 2.74−
2.58 (m, 1H), 2.49−2.32 (m, 1H), 2.17 (s, 3H), 2.00−1.82 (m, 2H),
1.79−1.57 (m, 2H), 1.29−1.09 (m, 2H); 13C NMR (75 MHz,
CD3OD) δ 164.9, 149.5, 139.6, 138.3, 137.7, 132.9, 130.6, 128.9, 128.5,
( )-Methyl 4-((2-(2-Fluorophenyl)-1′-methylspiro[indoline-3,4′-
piperidin]-1-yl)methyl)benzoate (( )-11d). Starting from ( )-10d
(156 mg, 0.53 mmol), compound ( )-11d was obtained following the
procedure described for the preparation of ( )-11a. Purification by
column chromatography on silica gel (MeOH/DCM 1:20) afforded
the title compound (59 mg, 25% yield); 1H NMR (300 MHz, CDCl3) δ
7.97 (d, J = 8.3 Hz, 2H), 7.31−7.19 (m, 4H), 7.17−6.90 (m, 3H),
6.78−6.68 (m, 2H), 6.35 (d, J = 7.8 Hz, 1H), 4.95 (s, 1H), 4.34 (d, J =
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127.8, 127.7, 126.5, 124.2, 121.3, 110.3, 84.4, 52.1, 51.3, 50.6, 46.0,
34.7; ESI-MS m/z: [M + H]+ 462; HPLC RT: 10.4 min.
Methyl 4-(4-Formylphenoxy)butanoate (16). Methyl 4-(4-
(Bromomethyl)phenoxy)butanoate (15). To a solution of 14 (420
mg, 2.01 mmol) in CCl4 were added NBS (77 mg, 0.9 mmol) and AIBN
(33 mg, 0.20 mmol), and the reaction mixture was kept stirring at 80 °C
for 4 h. The solvent was evaporated to obtain compound 15 that was
immediately used in the next reaction without further purification.
To a solution of 15 (100 mg, 0.35 mmol) in DMSO (1.5 mL) was
added NBS (87 mg, 0.49mmol), and the reaction mixture was kept
stirring at 60 °C for 1 h and then at 100 °C for an additional hour. After
cooling, H2O was added (15 mL) and organic products were extracted
with EtOAc (3 × 15 mL). The organic phase was dried over Na2SO4
and reduced in vacuo. Purification by column chromatography on silica
gel (EtOAc/n-hexane 1:4) afforded the title compound (23 mg, 30%
yield over two steps); 1H NMR (300 MHz, CDCl3) δ 9.87 (s, 1H), 7.87
(d, J = 7.6 Hz, 2H), 7.15 (d, J = 7.5 Hz, 2H), 4.02 (t, J = 7.5 Hz, 2H),
3.64 (s, 3H), 2.48 (t, J = 8.1 Hz, 2H), 2.16−2.06 (m, 2H).
Methyl 5-Methylthiophene-2-carboxylate (18). To a solution of 5-
methyl-2-thiophenecarboxylic acid (1.0 g, 7.0 mmol) in MeOH (15
mL), SOCl2 (2 mL) was added dropwise at 0 °C. The reaction mixture
was allowed to reach 25 °C and then was left stirring for 12 h. The
solvent was removed, and the residue was dissolved in EtOAc and
washed with a saturated solution of NaHCO3. The title compound was
used in the next step without any further purification (quantitative
yield). 1H NMR (300 MHz CDCl3) δ 7.60 (d, J = 3.7 Hz, 1H), 6.76 (d, J
= 3.7 Hz, 1H), 3.85 (s, 3H), 2.51 (s, 3H). ESI-MS m/z: [M + H]+ 157.
Methyl 5-(Bromomethyl)thiophene-2-carboxylate (19). To a
solution of 18 (570 mg, 3.65 mmol) in CCl4 (8 mL), NBS (584 mg,
mmol) and AIBN (60 mg, 0.37 mmol) were added and the reaction
mixture was stirred at 80 °C for 4 h. CCl4 was removed under reduced
pressure, and the residue was diluted with DCM and purified by column
chromatography on silica gel (EtOAc/n-hexane 1:30) (512 mg, 60%
yield); 1H NMR (300 MHz, CDCl3) δ 7.63 (d, J = 3.8 Hz, 1H), 7.08 (d,
J = 3.8 Hz, 1H), 4.66 (s, 3H), 3.87 (s, 3H); ESI-MS m/z: [M + H]+ 235.
Methyl 5-Formylthiophene-2-carboxylate (20). To a solution of 4-
methylmorpholine N-oxide (449 mg, 3.83 mmol) in MeCN (5 mL),
cooled at 0 °C and in the presence of 3 Å molecular sieves, a solution of
19 (300 mg, 1.28 mmol) in MeCN was added. The reaction mixture
was stirred at 25 °C for 12 h, and then, it was purified by column
chromatography on silica gel (EtOAc/n-hexane 1:20) obtaining the
title compound (152 mg, 70% yield); 1H NMR: (300 MHz, CDCl3) δ
9.96 (s, 1H), 7.82 (d, J = 1.1 Hz, 1H), 7.72 (d, J = 2.8 Hz, 1H), 3.93 (s,
3H); ESI-MS m/z: [M + H]+ 171.
( )-4-((2-(2-Fluorophenyl)-1′-methylspiro[indoline-3,4′-piperi-
din]-1-yl)methyl)-N-hydroxybenzamide (6d). Compound 6d was
obtained from ( )-11d (30 mg, 0.07 mmol) following the procedure
described for the preparation of 6a. Purification by column
chromatography on silica gel (NH4OH/MeOH/DCM 0.1:1:10)
1
afforded the title compound (12 mg, 40%); H NMR (300 MHz,
CD3OD) δ 11.15 (br, 1H), 8.99 (br, 1H), 7.66 (d, J = 8.3 Hz, 2H),
7.41−7.27 (m, 2H), 7.31−7.15 (m, 2H), 7.17−7.04 (m, 2H), 7.02 (t, J
= 8.3 Hz, 2H), 6.65 (t, J = 6.9 Hz, 1H), 6.43 (d, J = 7.8 Hz, 1H), 4.84 (s,
1H), 4.41 (d, J = 16.0 Hz, 1H), 3.78 (d, J = 16.0 Hz, 1H), 2.74−2.57 (m,
1H), 2.44−2.24 (m, 2H), 2.15 (s, 3H), 1.91−1.67 (m, 4H), 1.29−1.09
(m, 1H); 13C NMR (75 MHz, DMSO) δ 164.7, 162.5, 159.2, 151.2,
141.9, 136.5, 132.2, 130.5, 129.4, 128.6, 127.9, 127.7, 125.1, 124.8,
124.1, 118.2, 116.7, 116.4, 106.8, 67.5, 52.4, 51.9, 49.7, 46.5, 46.4, 37.7,
31.7; ESI-MS m/z: [M + H]+ 446; HRMS-ESI m/z: [M + H]+ calcd for
C27H29FN3O2 446.2238; found 446.2225; HPLC RT: 10.6 min.
( )-4-((2-(4-Fluorophenyl)-1′-methylspiro[indoline-3,4′-piperi-
din]-1-yl)methyl)-N-hydroxybenzamide (6e). Compound 6e was
obtained from ( )-11e (60 mg, 0.13 mmol) following the procedure
described for the preparation of 6a. Purification by column
chromatography on silica gel (NH4OH/MeOH/DCM 0.1:1:10)
afforded the title compound (32 mg, 56% yield); 1H NMR (300
MHz, CD3OD) δ 11.12 (br, 1H), 8.99 (br, 1H), 7.67 (d, J = 8.0 Hz,
2H), 7.28 (d, J = 8.0 Hz, 2H), 7.21−7.04 (m, 4H), 6.98 (t, J = 7.6 Hz,
2H), 6.63 (t, J = 7.3 Hz, 1H), 6.33 (d, J = 7.8 Hz, 1H), 4.50 (s, 1H), 4.30
(d, J = 16.2 Hz, 1H), 3.83 (d, J = 16.3 Hz, 1H), 2.60 (s, 1H), 2.35−2.23
(m, 1H), 2.12 (s, 3H), 1.95−1.79 (m, 2H), 1.79−1.53 (m, 2H), 1.33−
1.07 (m, 2H); 13C NMR (75 MHz, CD3OD) δ 166.7, 164.4, 161.1,
150.9, 142.7, 136.0, 133.4, 132.0, 131.2, 129.1, 127.9, 127.6, 127.1,
123.3, 117.8, 114.7, 106.4, 76.2, 51.9, 51.8, 49.6, 45.6, 44.9, 36.6, 30.8;
ESI-MS m/z: [M + H]+ 446; HRMS-ESI m/z: [M + H]+ calcd for
C27H29FN3O2 446.2238; found 446.2227; HPLC RT: 11.4 min.
( )-N-Hydroxy-4-((1′-methyl-2-(pyridin-3-yl)spiro[indoline-3,4′-
piperidin]-1-yl)methyl)benzamide (6f). Compound 6f was obtained
from ( )-11f (45 mg, 0.11 mmol) following the procedure described
for the preparation of 6a. Purification by column chromatography on
silica gel (NH4OH/MeOH/DCM 0.1:1:10) afforded the title
1
compound (34 mg, 73% yield); H NMR (300 MHz, CD3OD) δ
11.16 (br, 1H), 8.99 (br, 1H), 8.53 (s, 1H), 7.68 (d, J = 7.9 Hz, 2H),
7.35 (s, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.07 (t, J = 7.6 Hz, 1H), 6.71 (t, J
= 7.4 Hz, 1H), 6.44 (d, J = 7.8 Hz, 1H), 4.59 (s, 1H), 4.36 (d, J = 16.1
Hz, 1H), 3.85 (d, J = 16.1 Hz, 2H), 3.05−2.87 (m, 1H), 2.64 (s, 3H),
2.14−1.86 (m, 4H), 1.30−1.16 (m, 2H); 13C NMR (75 MHz, CD3OD)
δ 166.6, 150.7, 149.3, 142.1, 136.3, 133.8, 133.4, 131.3, 128.8, 127.7,
127.3, 124.2, 123.3, 118.7, 107.4, 73.9, 51.4, 49.9, 44.9, 42.9, 34.2, 34.1,
29.1; ESI-MS m/z: [M + H]+ 429; HRMS-ESI m/z: [M + H]+ calcd for
C26H29N4O2 429.2285; found 429.2271; HPLC RT: 7.1 min.
Methyl 4-Bromobutanoate (13). 4-Bromobutyryl chloride (1.6 mL,
13.82 mmol) was treated with MeOH (7 mL) at 0 °C. The reaction
mixture was stirred at 0 °C for 2 h and then at 25 °C for 12 h. The
solvent was removed under reduced pressure. The title compound was
obtained as pure compound without further purification (quantitative
yield); 1H NMR (300 MHz, CDCl3) δ 3.65 (s, 3H), 3.43 (t, J = 5.7 Hz,
2H), 2.48 (t, J = 6.4 Hz, 2H), 2.22−2.04 (m, 2H).
2-Methyl-1-(pyridin-3-yl)propan-1-one (22b). Starting from 21
(513 mg, 2.27 mmol) and 3-pyridinecarboxaldehyde (214 μL, 2.27
mmol), compound 22b was obtained following the procedure
described for the preparation of 8a. Purification by column
chromatography on silica gel (EtOAc/EtPet 1:2) afforded the title
compound (301 mg, 89% yield); 1H NMR (300 MHz, CDCl3) δ 9.11
(s, 1H), 8.72 (d, J = 4.8 Hz, 1H), 8.23−8.13 (m, 1H), 7.44−7.33 (m,
1H), 3.60−3.36 (m, 1H), 1.20 (s, 3H), 1.18 (s, 3H); ESI-MS m/z: [M +
H]+ 150.
2-Methyl-1-(pyridin-4-yl)propan-1-one (22c). Starting from 21
(1.0 g, 4.41 mmol) and 4-pyridinecarboxaldehyde (474 μL, 4.41
mmol), compound 22c was obtained following the procedure described
for the preparation of 8a. The title compound was used in the next step
1
without any further purification (quantitative yield); H NMR (300
Methyl 4-(p-Tolyloxy)butanoate (14). To a solution of p-cresol
(500 mg, 2.76 mmol) in dry MeCN (2.5 mL), Cs2CO3 (900 mg, 2.76
mmol) and the mixture were stirred at 95 °C for 30 min. Then, a
solution of 13 (149 mg, 1.38 mmol) in dry MeCN (2.5 mL) was added
and the reaction mixture was left at 95 °C for 12 h. The reaction was
cooled down to 25 °C; then, the solvent was removed under reduced
pressure. The residue was dissolved in EtOAc, washed with a saturated
solution of NaHCO3, dried over Na2SO4, and evaporated in vacuo.
Purification by column chromatography on silica gel (EtOAc/n-hexane
1:8) afforded the title compound (425 mg, 74% yield); 1H NMR (300
MHz, CDCl3) δ 7.07 (d, J = 8.1 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 3.97
(t, J = 6.1 Hz, 2H), 3.68 (s, 3H), 2.52 (t, J = 7.3 Hz, 2H), 2.28 (s, 3H),
2.17−1.99 (m, 2H).
MHz, CDCl3) δ 8.79 (d, J = 4.5 Hz, 2H), 7.70 (d, J = 4.5 Hz, 2H), 3.53−
3.43 (m, 1H), 1.23 (s, 3H), 1.21 (s, 3H); ESI-MS m/z: [M + H]+ 150.
3,3-Dimethyl-2-phenyl-3H-indole (23a). To a solution of the
commercially available 2-methyl-1-phenylpropan-1-one (22a, 506 μL,
3.37 mmol) in AcOH (5 mL), phenylhydrazine (331 μL, 3.37 mmol)
was added. The reaction was heated at 80 °C for 12 h; then, it was
concentrated to dryness, extracted with EtOAc, and washed with
Na2CO3. The organic layer was dried over Na2SO4, evaporated, and
purification by column chromatography on silica gel (EtOAc/n-hexane
1:30) afforded the title compound (357 mg, 48% yield); 1H NMR (300
MHz, CDCl3) δ 8.22−8.09 (m, 2H), 7.77−7.65 (m, 1H), 7.55−7.45
(m, 3H), 7.40−7.21 (m, 3H), 1.60 (s, 6H); ESI-MS m/z: [M + Na]+
222.
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Article
3,3-Dimethyl-2-(pyridin-3-yl)-3H-indole (23b). Starting from 22b
(300 mg, 2.01 mmol), compound 23b was obtained following the
procedure described for the preparation of 23a. Purification by column
chromatography on silica gel (EtOAc/n-hexane 1:2) afforded the title
compound (134 mg, 30% yield); 1H NMR (300 MHz, CDCl3) δ 9.31
(s, 1H), 8.70 (d, J = 4.7 Hz, 1H), 8.48 (d, J = 8.1 Hz, 1H), 7.70 (d, J =
7.6 Hz, 1H), 7.45−7.25 (m, 4H), 1.59 (s, J = 0.6 Hz, 6H); ESI-MS m/z:
[M + H]+ 223.
3,3-Dimethyl-2-(pyridin-4-yl)-3H-indole (23c). Compound 23c
was obtained from 22c (745 mg, 5.0 mmol) following the procedure
described for the preparation of 23a. Purification by column
chromatography on silica gel (EtOAc/ n-hexane 1:1) afforded the
title compound (333 mg, 30% yield); 1H NMR (300 MHz, CDCl3) δ
8.76 (d, J = 4.7 Hz, 2H), 7.96 (d, J = 6.2 Hz, 2H), 7.79−7.69 (m, 1H),
7.48−7.29 (m, 3H), 1.59 (s, 6H); ESI-MS m/z: [M + H]+ 223.
5-Methoxy-3,3-dimethyl-2-(pyridin-3-yl)-3H-indole (23d). Start-
ing from 22b (500 mg, 3.36 mmol) and 4-methoxyphenylhydrazine
(587 mg, 3.36 mmol), compound 23d was obtained following the
procedure described for the preparation of 23b. Purification by column
chromatography on silica gel (EtOAc/ n-hexane 1:1) afforded the title
compound (322 mg, 38% yield); 1H NMR (300 MHz, CDCl3) δ 9.27
(s, 1H), 8.67 (d, J = 3.2 Hz, 1H), 8.51−8.36 (m, 1H), 7.61 (d, J = 9.2
Hz, 1H), 7.46−7.34 (m, 1H), 6.95−6.80 (m, 2H), 3.87 (s, 3H), 1.58 (s,
6H); ESI-MS m/z: [M + H]+ 253.
4.05 (d, J = 16.4 Hz, 1H), 3.92 (s, 3H), 1.41 (s, 3H), 0.84 (s, 3H); ESI-
MS m/z: [M + H]+ 372, [M + Na]+ 394.
( )-Methyl 4-((3,3-Dimethyl-2-(pyridin-3-yl)indolin-1-yl)methyl)-
benzoate (( )-25b). Starting from ( )-24b (130 mg, 0.585 mmol),
compound ( )-25b was obtained following the procedure described
for the preparation of ( )-11a. Purification by column chromatog-
1
raphy on silica gel (EtOAc/ n-hexane 1:3) (43 mg, 20% yield); H
NMR (300 MHz, CDCl3) δ 8.57 (br, 1H), 7.97 (d, J = 8.2 Hz, 2H), 7.70
(br, 1H), 7.33 (d, J = 8.0 Hz, 2H), 7.06 (dd, J = 12.1, 4.4 Hz, 2H), 6.82
(t, J = 7.4 Hz, 1H), 6.43 (d, J = 7.9 Hz, 1H), 4.37 (d, J = 16.2 Hz, 1H),
4.28 (s, 1H), 4.04 (d, J = 16.4 Hz, 1H), 3.91 (s, 3H), 1.40 (s, 3H), 0.83
(s, 3H); ESI-MS m/z: [M + H]+ 372, [M + Na]+ 394;
(+)-Methyl 4-((3,3-Dimethyl-2-(pyridin-3-yl)indolin-1-yl)methyl)-
benzoate ((+)-25b). 1H NMR data are identical to those of the racemic
mixture ( )-25b; [α]2D0 = +51.0 (c 0.58, CHCl3);
(−)-Methyl 4-((3,3-Dimethyl-2-(pyridin-3-yl)indolin-1-yl)methyl)-
benzoate ((−)-25b). 1H NMR data are identical to those of the racemic
mixture ( )-25b; [α]2D0 = −52.0 (c 0.63, CHCl3).
( )-Methyl 4-((5-Methoxy-3,3-dimethyl-2-(pyridin-3-yl)indolin-1-
yl)methyl)benzoate (( )-25c). Starting from ( )-24c (102 mg, 0.40
mmol), compound ( )- 25c was obtained following the procedure
described for the preparation of ( )-21a. Purification by column
chromatography on silica gel (EtOAc/ n-hexane 1:2) afforded the title
compound (39 mg, 24% yield); 1H NMR (300 MHz, CDCl3) δ 8.57 (s,
1H), 7.97 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 7.9 Hz, 2H), 7.33−7.18 (m,
2H), 6.70 (d, J = 2.5 Hz, 1H), 6.61−6.53 (m, 1H), 6.30 (d, J = 8.5 Hz,
2H), 4.28 (d, J = 16.4 Hz, 1H), 4.22 (s, 1H), 3.98 (d, J = 16.2 Hz, 1H),
3.91 (s, 3H), 3.76 (s, 3H), 1.39 (s, 3H), 0.83 (s, 3H); ESI-MS m/z: [M
+ H]+ 403 [M + Na]+ 426.
( )-3,3-Dimethyl-2-phenylindoline (( )-24a). To a solution of 23a
(500 mg, 2.26 mmol) in MeOH (25 mL), NaBH4 (513 mg, 13.56
mmol) was added. The reaction was stirred for 12 h at 50 °C. Then, the
reaction was quenched with a saturated solution of NaHCO3 (10 mL),
MeOH was removed under reduced pressure, and the residue was
dissolved with EtOAc and washed with H2O (3 × 20 mL). The organic
phase was dried over Na2SO4, and solvents were removed in vacuo to
give a residue that was purified by flash column chromatography on
( )-Methyl 5-((3,3-Dimethyl-2-(pyridin-3-yl)indolin-1-yl)methyl)-
thiophene-2-carboxylate (( )-25d). To a solution of ( )-24b (140
mg, 0.63 mmol) in dry DCM (3 mL) was added a solution of 20 (106
mg, 0.63 mmol) in dry DCM (3 mL). After 1 h at 25 °C, NaBH(OAc)3
(668 mg, 2.52 mmol) was added and the reaction mixture was stirred at
50 °C for 12 h. The reaction was treated with a saturated solution of
NaHCO3, and organic products were extracted with DCM. The
combined organic layers were washed with brine, dried over Na2SO4,
and concentrated in vacuo. Purification by column chromatography on
silica gel (PetEt/Et2O 2:1) afforded the title compound (126 mg, 53%
yield); 1H NMR (300 MHz, CDCl3) δ 8.64−8.49 (m, 2H), 7.81 (br,
1H), 7.62 (d, J = 3.8 Hz, 1H), 7.39−7.28 (m, 1H), 7.15−6.98 (m, 2H),
6.87−6.75 (m, 2H), 6.61 (d, J = 7.8 Hz, 1H), 4.50 (d, J = 16.2 Hz, 1H),
4.22 (s, 1H), 4.16 (d, J = 16.3 Hz, 1H), 1.35 (s, 3H), 0.79 (s, 3H); ESI-
MS m/z: [M + H]+ 379.
1
silica gel (MeOH/DCM 1:30) (304 mg, 60% yield); H NMR (300
MHz, CDCl3) δ 7.45−7.38 (m, 1H), 7.34−7.26 (m, 3H), 7.26−7.13
(m, 2H), 6.80−6.71 (m, 1H), 6.63−6.56 (m, 1H), 4.69 (s, 1H), 3.71
(br, 1H), 1.36 (s, 3H), 0.98 (s, 3H).
( )-3,3-Dimethyl-2-(pyridin-3-yl)indoline (( )-24b). Starting from
23b (500 mg, 2.26 mmol), ( )-24a was obtained following the
procedure described for ( )-24a. Purification by column chromatog-
raphy on silica gel (MeOH/DCM 1:30) (305 mg, 60% yield); 1H NMR
(300 MHz, CDCl3) δ 8.67 (s, 1H), 8.56 (d, J = 6.4 Hz, 1H), 7.92−7.80
(m, 1H), 7.84−7.73 (m, 1H), 7.36−7.21 (m, 1H), 7.14−6.99 (m, 1H),
6.81 (t, J = 8.0 Hz, 1H), 6.74 (d, J = 7.4 Hz, 1H), 4.61 (s, 1H), 4.08 (br,
1H), 1.44 (s, 3H), 0.74 (s, 3H).
( )-3,3-Dimethyl-2-(pyridin-4-yl)indoline (( )-24c). Starting from
23c (300 mg, 1.02 mmol), compound ( )-24c was obtained following
the procedure described for the preparation of ( )-24a. Purification by
column chromatography on silica gel (MeOH/DCM 1:20) afforded
the title compound (115 mg, 50% yield); 1H NMR (300 MHz, CDCl3)
δ 8.57 (d, J = 4.9 Hz, 2H), 7.39 (d, J = 5.1 Hz, 2H), 7.17−6.96 (m, 2H),
6.85−6.77 (m, 1H), 6.73 (d, J = 7.7 Hz, 1H), 4.57 (s, 1H), 4.10 (br,
1H), 1.46 (s, 3H), 0.72 (s, 3H); ESI-MS m/z: [M + H]+ 225.
( )-5-Methoxy-3,3-dimethyl-2-(pyridin-3-yl)indoline (( )-24d).
Starting from 23d (327 mg, 1.29 mmol), compound ( )-24d was
obtained following the procedure described for the preparation of
( )-24a. Purification by column chromatography on silica gel
(MeOH/DCM 1:30) afforded the title compound (259 mg, 79%
yield); 1H NMR (300 MHz, CDCl3) δ 8.67 (s, 1H), 8.56 (d, J = 5.5 Hz,
2H), 7.85 (d, J = 7.9 Hz, 1H), 7.44−7.31 (m, 1H), 6.90−6.80 (m, 1H),
6.73−6.52 (m, 2H), 4.59 (s, 1H), 3.87 (s, 3H), 1.42 (s, 3H), 0.74 (s,
3H); ESI-MS m/z: [M + H]+ 255.
( )-Methyl 4-(4-((3,3-Dimethyl-2-(pyridin-3-yl)indolin-1-yl)-
methyl)phenoxy)butanoate (( )-25e). Starting from ( )-24b (61
mg, 0.27 mmol) and 16 (60 mg, 0.27 mmol), compound ( )-25f was
obtained following the procedure described for the preparation of
( )-25e. Purification by column chromatography on silica gel (PetEt/
1
Et2O 1:1) and afforded the title compound (23 mg, 20% yield); H
NMR (300 MHz, CDCl3) δ 8.56 (d, J = 4.8 Hz, 2H), 7.71 (br, 1H),
7.32−7.18 (m, 2H), 7.16−6.99 (m, 4H), 6.85−6.71 (m, 2H), 6.55 (d, J
= 7.8 Hz, 1H), 4.35 (d, J = 15.5 Hz, 1H), 4.22 (s, 1H), 3.97 (t, J = 6.1
Hz, 2H), 3.90 (d, J = 15.5 Hz, 1H), 3.69 (s, 3H), 2.52 (t, J = 7.3 Hz,
2H), 2.15−2.01 (m, 2H), 1.37 (s, 3H), 0.79 (s, 3H); ESI-MS m/z: [M +
H]+ 431.
( )-Methyl 4-((3,3-Dimethyl-2-(pyridin-4-yl)indolin-1-yl)methyl)-
benzoate (( )-25f). Starting from ( )-24d (150 mg, 0.67 mmol),
compound ( )- 25d was obtained following the procedure described
for the preparation of ( )-11a. Purification by column chromatography
on silica gel (EtOAc/n-hexane 1:2) afforded the title compound (62
1
mg, 25% yield); H NMR (300 MHz, CDCl3) δ 8.57 (d, J = 5.1 Hz,
2H), 7.98 (d, J = 8.4 Hz, 2H), 7.38−7.21 (m, 4H), 7.11−6.96 (m, 2H),
6.88−6.72 (m, 1H), 6.43 (d, J = 8.2 Hz, 1H), 4.39 (d, J = 16.4 Hz, 1H),
4.25 (s, 1H), 4.05 (d, J = 16.4 Hz, 1H), 3.91 (s, 3H), 1.42 (s, 3H), 0.81
(s, 3H); ESI-MS m/z: [M + Na]+ 395.
( )-4-((3,3-Dimethyl-2-phenylindolin-1-yl)methyl)-N-hydroxy-
benzamide (6g). Starting from ( )-25a (100 mg, 0.27 mmol),
compound 6g was obtained following the procedure described for the
preparation of 6a. Purification by column chromatography on silica gel
(MeOH/DCM NH4OH 1:20:0.1) afforded the title compound (18
( )-Methyl 4-((3,3-Dimethyl-2-phenylindolin-1-yl)methyl)-
benzoate (( )-25a). Starting from ( )-24a (506 mg, 3.37 mmol),
compound ( )-25a was obtained following the procedure described
for the preparation of ( )-11a. Purification by column chromatog-
raphy on silica gel (EtOAc/n-hexane 1:20) afforded the title compound
(1050 mg, 84% yield); 1H NMR (300 MHz, CDCl3) δ 7.99 (d, J = 8.3
Hz, 2H), 7.44−7.15 (m, 6H), 7.14−6.88 (m, 2H), 6.79 (t, J = 7.9 Hz,
1H), 6.39 (d, J = 7.8 Hz, 1H), 4.43 (d, J = 16.4 Hz, 1H), 4.29 (s, 1H),
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mg, 18% yield); 1H NMR (300 MHz, DMSO) δ 7.67 (d, J = 8.2 Hz,
2H), 7.43−7.21 (m, 7H), 7.12−6.84 (m, 2H), 6.71 (t, J = 7.4 Hz, 1H),
6.43 (d, J = 7.8 Hz, 1H), 4.45 (d, J = 16.2 Hz, 1H), 4.24 (s, 1H), 4.00 (d,
J = 16.3 Hz, 1H), 1.35 (s, 3H), 0.76 (s, 3H); 13C NMR (75 MHz,
DMSO) δ 164.2, 150.8, 141.7, 138.7, 137.9, 132.4, 129.0, 128.4, 127.4,
127.9, 127.6, 122.7, 119.0, 108.7, 79.7, 50.7, 44.6, 27.2, 25.8; ESI-MS
m/z: [M + H]+ 373, [M + Na]+ 395; HRMS-ESI m/z: [M + Na]+ calcd
for C24H24N2O2Na 395.1730; found 395.1727; HPLC RT: 19.0 min.
( )-4-((3,3-Dimethyl-2-(pyridin-3-yl)indolin-1-yl)methyl)-N-hy-
droxybenzamide (6h). Starting from ( )-25b (40 mg, 0.114 mmol),
compound ( )-6h was obtained following the procedure described for
the preparation of 6a. Purification by column chromatography on silica
gel (MeOH/DCM/ NH4OH 1:8:0.1) afforded the title compound (14
mg, 33% yield); 1H NMR (300 MHz, CD3OD) δ 11.01 (br, 1H), 9.06
(br, 1H), 8.52 (dd, J = 4.8, 1.5 Hz, 2H), 7.75 (d, J = 7.8 Hz, 1H), 7.67
(d, J = 8.2 Hz, 2H), 7.44−7.37 (m, 1H), 7.29 (d, J = 8.2 Hz, 2H), 7.07
(d, J = 7.2 Hz, 1H), 6.99 (dd, J = 8.2, 7.1 Hz, 1H), 6.70 (t, J = 7.3 Hz,
1H), 6.44 (d, J = 7.8 Hz, 1H), 4.46−4.29 (m, 2H), 3.97 (d, J = 16.4 Hz,
1H), 1.32 (s, 3H), 0.71 (s, 3H); 13C NMR (75 MHz, CD3OD): δ 167.3,
166.7, 150.5, 148.9, 148.4, 142.5, 138.0, 134.5, 131.2, 127.6, 127.5,
127.2, 123.9, 122.0, 119.3, 108.6, 77.8, 53.6, 44.6, 25.9, 24.5; ESI-MS
m/z: [M + H]+ 374; HRMS-ESI m/z: [M + H]+ calcd for C23H24N3O2
374.1869; found 374.1862; HPLC RT: 10.4 min.
for the preparation of 6a. Purification by column chromatography on
silica gel (NH4OH/MeOH/DCM 0.1:1:12) afforded the title
compound (48 mg, 63% yield); H NMR (300 MHz, CD3OD) δ
1
8.61−8.45 (m, 2H), 7.92 (br, 1H), 7.50−7.45 (m, 1H), 7.43−7.35 (m,
1H), 7.08−7.01 (m, 2H), 6.86−6.74 (m, 2H), 6.70 (d, J = 7.8 Hz, 1H),
4.60 (d, J = 16.3 Hz, 1H), 4.25 (s, 1H), 4.17 (d, J = 16.3 Hz, 1H), 1.33
(s, 3H), 0.74 (s, 3H); 13C NMR (75 MHz, CD3OD) δ 161.7, 149.4,
149.0, 148.5, 145.9, 138.2, 137.2, 134.9, 134.0, 128.1, 127.5, 126.4,
124.3, 122.0, 119.2, 109.1, 76.9, 45.7, 44.7, 25.4, 24.6. ESI-MS m/z: [M
+ H]+ 380; HRMS-ESI m/z: [M + H]+ calcd for C21H22N3O2S
380.1427; found 380.1422; HPLC RT: 11.7 min.
(6l). Compound 6l was obtained from ( )-25f (20 mg, 0.047 mmol)
following the procedure described for the preparation of 6a.
Purification by column chromatography on silica gel (NH4OH/
MeOH/DCM 0.1:1:12) afforded the title compound (16 mg, 79%
yield); 1H NMR (300 MHz, CD3OD) δ 8.47 (d, J = 4.9 Hz, 2H), 7.81
(s, 1H), 7.48−7.33 (m, 1H), 7.15−6.94 (m, 4H), 6.80 (d, J = 8.6 Hz,
2H), 6.73 (t, J = 7.9 Hz, 1H), 6.60 (d, J = 8.8 Hz, 1H), 4.36 (d, J = 15.4
Hz, 1H), 4.23 (s, 1H), 4.06−3.80 (m, 3H), 2.27 (t, J = 7.3 Hz, 2H),
2.10−1.99 (m, 2H), 1.34 (s, 3H), 0.74 (s, 3H); 13C NMR (75 MHz,
CD3OD) δ 171.1, 158.3, 150.6, 148.9, 148.1, 137.9, 137.1, 134.7, 129.9,
128.9, 127.4, 123.8, 121.8, 118.8, 114.3, 108.6, 77.0, 66.7, 50.4, 44.4,
29.1, 25.9, 25.2, 24.5; ESI-MS m/z: [M + H]+ 432; HRMS-ESI m/z: [M
+ Na]+ calcd for C26H29N3O3Na 454.2101; found 454.2099; HPLC
RT: 10.0 min.
tert-Butyl 4-Nicotinoylpiperidine-1-carboxylate (27). Compound
27 was obtained from 26 (1.9 g, 5.09 mmol) following the procedure
described for the preparation of 8a. Purification by column
chromatography on silica gel (DCM/MeOH 30:1) afforded the title
compound (quantitative yield); 1H NMR (300 MHz, CDCl3) δ 9.23−
9.03 (m, 1H), 8.79 (dd, J = 4.8, 1.7 Hz, 1H), 8.22 (dt, J = 8.0, 2.0 Hz,
1H), 7.52−7.33 (m, 1H), 4.36−3.89 (m, 2H), 3.52−3.20 (m, 1H), 2.91
(t, J = 12.7 Hz, 2H), 1.97−1.79 (m, 2H), 1.79−1.64 (m, 2H), 1.46 (s,
9H); ESI-MS m/z: [M + H]+ 291.
tert-Butyl 2-(Pyridin-3-yl)spiro[indole-3,4′-piperidine]-1′-carbox-
ylate (28). Compound 28 was obtained from 27 (116 mg, 0.40 mmol)
following the procedure described for the preparation of 9a. The crude
of this reaction was immediately dissolved in THF (5 mL) and 0.5 M
NaOH (5 mL) and then treated with Boc2O (134 mg, 0.62 mmol). The
mixture was stirred at 25 °C for 2 h and then H2O was added, and the
crude was extracted with EtOAc (3 × 10 mL). The combined organic
layers were dried over Na2SO4, evaporated, and purified by column
chromatography on alumina (EtOAc/ n-hexane 1:4) affording the title
compound (20 mg, 14% yield, over two steps); 1H NMR (300 MHz,
CDCl3) δ 9.26 (dd, J = 2.3, 1.0 Hz, 1H), 8.70 (dd, J = 4.8, 1.5 Hz, 1H),
8.46−8.23 (m, 1H), 7.78 (t, J = 8.0 Hz, 2H), 7.55−7.35 (m, 2H), 7.35−
7.13 (m, 1H), 4.25 (br, 2H), 3.47 (t, J = 13.4 Hz, 2H), 2.61−2.29 (m,
2H), 1.63−1.31 (m, 11H); ESI-MS m/z: [M + H]+ 364.
(+)-4-((3,3-Dimethyl-2-(pyridin-3-yl)indolin-1-yl)methyl)-N-hy-
droxybenzamide ((+)-6h). Starting from (+)-25b (23 mg, 0.062
mmol), compound (+)-6h was obtained following the procedure
described for the preparation of 6a. Purification by column
chromatography on silica gel (MeOH/DCM/NH4OH 1:8:0.1)
afforded the compound (9 mg, 38% yield); 1H NMR data are identical
to those of the racemic mixture (( )-6h); [α]2D0 = +35.0 (c 0.16,
CHCl3);
(−)-4-((3,3-Dimethyl-2-(pyridin-3-yl)indolin-1-yl)methyl)-N-hy-
droxybenzamide ((−)-6h). Starting from (−)-25b (20 mg, 0.054
mmol), compound (−)-6h was obtained following the procedure
described for the preparation of 6a. Purification by column
chromatography on silica gel (MeOH/DCM/NH4OH 1:8:0.1)
1
afforded the title compound (8 mg, 34% yield); H NMR data are
identical to those of the racemic mixture ((−)-6h); [α]2D0 = −36.0 (c
0.39, CHCl3);
( )-N-Hydroxy-4-((5-methoxy-3,3-dimethyl-2-(pyridin-3-yl)-
indolin-1-yl)methyl)benzamide (6i). Compound 6i was obtained from
( )-25c (32 mg, 0.08 mmol) following the procedure described for the
preparation of 6a. Purification by column chromatography on silica gel
(DCM/ MeOH/NH4OH 0.1:1:8) afforded the title compound (16
mg, 49% yield); 1H NMR (300 MHz, CD3OD) δ 11.12 (br, 1H), 8.98
(br, 1H), 8.61−8.44 (m, 2H), 7.67 (d, J = 8.1 Hz, 2H), 7.43−7.33 (m,
1H), 7.32 (d, J = 8.1 Hz, 2H), 6.75 (d, J = 2.5 Hz, 1H), 6.62−6.51 (m,
2H), 6.30 (d, J = 8.4 Hz, 1H), 4.32−4.21 (m, 2H), 3.90 (d, J = 16.3 Hz,
1H), 3.64 (s, 3H), 1.30 (s, 3H), 0.71 (s, 3H); 13C NMR (75 MHz,
CD3OD) δ 166.7, 154.4, 148.9, 148.3, 144.6, 142.7, 139.6, 137.2, 134.5,
131.1, 127.6, 127.1, 123.8, 112.0, 109.4, 109.2, 78.6, 55.1, 52.3, 44.7,
25.2, 24.3; ESI-MS m/z: [M + H]+ 404; HPLC RT: 9.5 min.
( )5-((3,3-Dimethyl-2-(pyridin-3-yl)indolin-1-yl)methyl)-N-hy-
droxythiophene-2-carboxamide (6j). Compound 6j was obtained
from ( )-25d (65 mg, 0.17 mmol) following the procedure described
for the preparation of 6a. Purification by column chromatography on
silica gel (NH4OH/MeOH/DCM 0.1:1:10) afforded the title
compound (52 mg, 77% yield); 1H NMR (300 MHz, DMSO) δ
11.14 (br, 1H), 8.99 (br, 1H), 8.54 (d, J = 5.2 Hz, 2H), 7.67 (d, J = 8.2
Hz, 2H), 7.37−7.22 (m, 4H), 7.06 (d, J = 7.3 Hz, 1H), 6.99 (t, J = 7.7
Hz, 1H), 6.70 (t, J = 7.4 Hz, 1H), 6.45 (d, J = 7.8 Hz, 1H), 4.40 (d, J =
16.4 Hz, 1H), 4.31 (s, 1H), 3.98 (d, J = 16.4 Hz, 1H), 1.34 (s, 3H), 0.69
(s, 3H); 13C NMR (75 MHz, DMSO) δ 172.7, 164.7, 150.6, 150.3,
147.2, 141.7, 138.3, 132.2, 128.0, 127.7, 123.8, 122.7, 119.3, 108.9, 78.5,
55.5, 44.8, 27.3, 25.6; ESI-MS m/z: [M + H]+ 374 [M + Na]+ 396;
HRMS-ESI m/z: [M + H]+ calcd for C23H24N3O2 374.1863; found
374.1853; HPLC RT: 10.1 min.
tert-Butyl 2-(Pyridin-3-yl)spiro[indoline-3,4′-piperidine]-1′-car-
boxylate (( )-29). Compound ( )-29 was obtained from 28 (24
mg, 0.29 mmol) following the procedure described for the preparation
of 10d. The crude material was used in the next step without further
purification, affording the title compound (24 mg, quantitative yield);
1H NMR (300 MHz, CDCl3) δ 8.69−8.42 (m, 2H), 7.64 (d, J = 8.1 Hz,
1H), 7.35−7.18 (m, 2H), 7.12 (td, J = 7.6, 1.2 Hz, 1H), 6.90−6.54 (m,
2H), 4.61 (s, 1H), 4.19 (br, 1H), 3.88−3.53 (m, 2H), 3.48−3.27 (m,
1H), 3.27−2.97 (m, 1H), 1.98−1.75 (m, 2H), 1.75−1.55 (m, 1H), 1.42
(s, 10H); ESI-MS m/z: [M + H]+ 366.
tert-Butyl 1-(4-(Methoxycarbonyl)benzyl)-2-(pyridin-3-yl)spiro-
[indoline-3,4′-piperidine]-1′-carboxylate (( )-30). Compound
( )-30 was obtained from ( )-29 (24 mg, 0.07 mmol) following the
procedure described for the preparation of 11a. Purification by column
chromatography on silica gel (PetEt/EtOAc 4:1) afforded the title
1
compound (10 mg, 30% yield); H NMR (300 MHz, CDCl3) δ 8.56
(dd, J = 4.9, 1.7 Hz, 1H), 8.34 (br, 1H), 7.97 (d, J = 8.3 Hz, 2H), 7.52
(br, 1H), 7.37−7.16 (m, 4H), 7.11 (td, J = 7.7, 1.2 Hz, 1H), 6.78 (td, J =
7.5, 1.0 Hz, 1H), 6.42 (d, J = 7.8 Hz, 1H), 4.50−4.21 (m, 2H), 4.02−
3.83 (m, 4H), 3.83−3.66 (m, 1H), 3.66−3.50 (m, 1H), 3.50−3.31 (m,
1H), 3.09 (s, 1H), 1.97−1.80 (m, 2H), 1.80−1.60 (m, 2H), 1.42 (s,
9H).; ESI-MS m/z: [M + H]+ 514.
( )-4-(4-((3,3-Dimethyl-2-(pyridin-3-yl)indolin-1-yl)methyl)-
phenoxy)-N-hydroxybutanamide (6k). Compound 6k was obtained
from ( )-25e (74 mg, 0.20 mmol) following the procedure described
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tert-Butyl 1-(4-(Hydroxycarbamoyl)benzyl)-2-(pyridin-3-yl)spiro-
[indoline-3,4′-piperidine]-1′-carboxylate (6m). Compound ( )-6m
was obtained from ( )-30 (10 mg, 0.02 mmol) following the procedure
described for the preparation of 6a. Purification by column
chromatography on silica gel (NH4OH/DCM/MeOH 0.1:30:1)
afforded the title compound (5 mg, 51% yield); 1H NMR (300 MHz,
CD3OD) δ 8.48 (s, 1H), 8.38 (s, 1H), 7.68 (d, J = 8.1 Hz, 3H), 7.50−
7.17 (m, 4H), 7.09 (t, J = 7.7 Hz, 1H), 6.75 (t, J = 7.4 Hz, 1H), 6.48 (d, J
= 7.9 Hz, 1H), 4.57 (s, 1H), 4.41 (d, J = 16.1 Hz, 1H), 3.95 (d, J = 15.9
Hz, 1H), 3.87−3.68 (m, 1H), 3.68−3.40 (m, 2H), 3.13−2.86 (m, 1H),
2.06−1.63 (m, 4H), 1.41 (s, 9H); 13C NMR (75 MHz, CD3OD) δ
166.5, 155.1, 150.5, 148.7, 142.2, 135.2, 134.0, 131.0, 129.5, 129.3,
128.0, 127.4, 127.0, 123.4, 123.3, 118.2, 106.9, 79.7, 49.8, 36.1, 31.0,
29.4, 29.1, 27.2; ESI-MS m/z: [M + H]+ 515; HRMS-ESI m/z: [M +
Na]+ calcd for C30H34N4O4Na 537.2472; found 537.2463; HPLC RT:
12.3 min.
Stereochemical Characterization of Compound 25b. Enan-
tioselective HPLC. The racemic mixture of compound ( )-25b (1 mg/
mL) was resolved by enantioselective HPLC using a Daicel Chiralcel
OD column (cellulose tris(3,5-dimethylphenylcarbamate), 250 mm ×
10 mm I.D.), n-hexane/2-propanol (60:40 v/v) mixture as the mobile
phase, 4.5 mL/min flow rate, and 50 μL injection volume. The two
enantiomers were collected and analyzed by polarimetry to determine
their specific rotation ([α]2D0). The enantiomeric excess (e.e.) of both
fractions was then determined by HPLC analysis using the same
enantioselective method used for the resolution of the racemic mixture
(Figure S1).
Experimental Spectroscopy. The experimental chiroptical proper-
ties of 25b were determined by spectroscopic analysis on a sample of
(−)-25b (300 μM in 2-propanol). Ultraviolet (UV) absorption and
ECD spectra were measured in the 400−215 nm spectral range at 25 °C
on a Jasco J-810 spectropolarimeter (Tokyo, Japan) using a QS-grade
quartz cell (Hellma Analytics, Germany) with a 1 mm optical
pathlength, a 2 nm spectral bandwidth, a 2 s data integration time, a
50 nm/min scanning speed, a 0.2 nm data pitch, and an accumulation
cycle of three runs. Spectra were then blank-corrected and converted to
molar units.
Computational Spectroscopy. The theoretical chiroptical proper-
ties of 25b were determined by quantum mechanical (QM) calculations
based on TD-DFT. A preliminary conformational search on (S)-25b
was performed by molecular mechanics (MM) calculations using the
MMFF94s force field50 and Spartan’0251 software. DFT geometry
optimization was carried out on 35 conformers having relative MM
energy (ΔEMM) below 5 kcal mol−1 using Gaussian 09 software;52 the
B97D functional,53 the def2-TZVP basis set,54,55 and the IEFPCM
solvation model56 for 2-propanol (2-PrOH) were employed.
Conformational clustering was performed with an RMSD threshold
value of 0.01 Å for heavy atoms; details on the resulting 27 optimized
conformers, including relative electronic energies (ΔEQM), are reported
in the Supporting Information (Table S1). TD-DFT calculations were
performed on all optimized conformers using Gaussian 09 software;52
the PBE0 functional57,58 was used in combination with the def2-TZVP
basis set and the IEFPCM(2-PrOH) solvation model. Oscillator
strengths (fj), rotational strengths in dipole length formalism (Rj), and
excitation energies (expressed as wavelengths (Table S2)). The
theoretical UV and ECD spectra of conformers were determined by
approximation of fj and Rj values to Gaussian bands (Δσ = 0.4 eV)59
and sum over all states; the theoretical spectra of (S)-25b were derived
by conformational averaging, according to the Boltzmann populations
of conformers at 298.15 K and 1 atm based on ΔEQM values (χQM) and
then compared to the experimental spectra of (−)-25b by means of the
Pearson correlation coefficient (r).
mM 6h] was combined with a 100 nL drop of precipitant solution [0.2
M ammonium phosphate dibasic and 20% PEG 3350] and equilibrated
against 80 μL of precipitant solution in the well reservoir surrounding
the sitting drop. Crystals formed within 2−3 days at 4 °C were soaked in
mother liquor augmented with 20% ethylene glycol for cryoprotection
before flash-cooling.
X-ray diffraction data were collected on the Northeastern
Collaborative Access Team (NE-CAT) beamline 24-ID-C at the
Advanced Photon Source (APS). The CCP4 program suite was
employed for data reduction:61 iMosflm62 was used to index the data,
and Aimless63 was used to scale the data. To phase the initial electron
density map of the enzyme−inhibitor complex by molecular
replacement, Phaser64 was utilized; the structure of unliganded
zf HDAC6 (PDB 5EEM) was used as a search probe. The atomic
model of the enzyme−inhibitor complex was built and manipulated
using the interactive graphics program Coot,65 and the structure was
refined using Phenix.66 The inhibitor was added to well-defined
electron density in the final stages of refinement. Occasional spurious
electron density peaks were left unmodelled. MolProbity67 was used to
validate the final structure prior to deposition of the atomic coordinates
in the Protein Data Bank (www.rcsb.org). All data reduction and
refinement statistics are recorded in Table S3.
Molecular Docking Simulations. Molecular docking simulations
were performed using a multicore workstation (72 Intel Xeon E5-2695
v4@2.10 GHz processors and two NVIDIA GeForce 1070 GTX GPU)
̈
with Ubuntu 18.04 OS, running Maestro release 2016 (Schrodinger,
LLC, New York, NY, 2016). Figures illustrating docking outputs were
prepared using PyMOL (The PyMOL Molecular Graphics System,
̈
v1.8.4.0, Schrodinger LLC, New York, 2015).
Protein and Ligand Preparation. Crystal structures of human
HDAC1 (PDB ID 4BKX)68 and HDAC6 (PDB ID 5EDU)33 were
taken from PDB, while zf HDAC6 was provided by our co-workers. The
proteins were submitted to Protein Preparation Wizard (PPW)
protocol implemented in Maestro suite release 2016 to obtain suitable
protein structures for molecular docking calculations as previously
reported for the same proteins.28,29 Ligands, water molecules, and
compounds used in the crystallization process were removed,
maintaining the Zn2+.28,29 By following the PPW protocol, we
performed a series of computational steps to (1) generate a metal-
binding state for the enzymes; (2) add hydrogens; (3) optimize the
orientation of hydroxyl groups, Asn, and Gln, and the protonation state
of His; and (4) perform a constrained minimization refinement with the
impref utility. The refined proteins were used in molecular docking
calculation as reported in the next paragraph.
Ligands were built in Maestro and minimized by MacroModel
̈
(MacroModel, Schrodinger Release 2016) software using OPLS-2005
as the force field. Moreover, the resulting compounds were treated by
̈
LigPrep application (LigPrep, Schrodinger Release 2016) to generate
the most probable ionization state at the cellular pH (7.4 0.2) as
reported by us.69,70 Moreover, according to the evidence reported in the
literature,71−74 we used a neutral hydroxamic acid moiety of the
compounds since the hydroxamic acid proton should not be transferred
in HDAC isoforms containing histidine residues in the binding site,
close to the reactive metal center as in the case of HDAC1 and
HDAC6.28,31
̈
Computational Procedure. Glide software (Glide, Schrodinger
Release 2016) has been employed to perform the docking studies
presented in this paper, using the ligands and proteins prepared as
above-mentioned, applying Glide extra precision (XP) scoring
function. Energy grids were prepared using the default value of the
protein atom scaling factor (1.0 Å) within a cubic box centered on the
zinc ion which roughly represents the center of the active sites.28,29
After grid generation with the introduction of metal constrains, the
ligands were docked into the enzymes. The number of poses entered to
post-docking minimization was set to 50. Glide SP score was evaluated.
In order to assess the validity of the docking protocol, SAHA and
Trichostatin A were used as reference compounds for a redocking
procedure. The docking results revealed a similar accommodation for
the above-mentioned reference compounds with respect to the
previously published results (data not shown).33,75
Crystal Structure Determination of the zf HDAC6 Complex.
The expression, purification, and crystallization of histone deacetylase 6
catalytic domain 2 (CD2) from Danio rerio (zebrafish; here, referred to
simply as “zfHDAC6”) were achieved as recently described.33,60 Using
a Mosquito crystallization robot (TTP Labtech), a 100 nL drop of
protein solution [10 mg/mL zfHDAC6, 50 mM 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) (pH 7.5), 100 mM KCl, 5%
glycerol (v/v), 1 mM tris(2-carboxyethyl)phosphine (TCEP), and 2
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In Vitro Testing of HDAC1, HDAC6, and HDAC8. OptiPlate-96
black microplates (PerkinElmer) were employed with an assay volume
of 60 μL. Human recombinant HDAC1 (BPS Bioscience, Catalog #:
50051) or human recombinant HDAC6 (BPS Bioscience, Catalog #:
50006) was diluted in incubation buffer (50 mM Tris−HCl, pH 8.0,
137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2 and 1 mg/mL bovine serum
albumin (BSA)). A total of 52 μL of this dilution was incubated with 3
μL of increasing concentrations of inhibitors in DMSO and 5 μL of
fluorogenic substrate ZMAL (Z-(Ac)Lys-AMC)76,77 (126 μM) at 37
°C. After 90 min incubation time, 60 μL of the stock solution (33 μM
Trichostatin A and 6 mg/mL trypsin in trypsin buffer [Tris−HCl 50
mM, pH 8.0, NaCl 100 mM]) was added. After the following
incubation at 37 °C for 30 min, the fluorescence was measured on a
BMG LABTECH POLARstar OPTIMA plate reader (BMG
Labtechnologies, Germany) at an excitation wavelength of 390 nm
and an emission wavelength of 460 nm.77,78
Recombinant human HDAC8 was purchased as part of the Fluor de
Lys HDAC8 fluorometric drug discovery kit (Enzo Life Sciences, No.
BMLAK518). Inhibition assays were performed as previously described
(Marek et al., 2018) with minor modifications.30,79 The DMSO
concentration was kept constant to 0.5%; the enzyme incubated with
DMSO only was used as the control. To keep safe from the possibility of
slow-binding inhibition, the enzyme was preincubated with the selected
compounds 15 min before substrate addition to the mixture. The Fluor
de Lys substrate was added at a final concentration of 50 μM, whereas
the enzyme was added at a concentration of 0.45U/reaction; the
reaction was allowed to proceed for 1 h at 30 °C. TSA (2 μM) within 50
μL of 1× Developer II was added to quench the reaction, and the
mixture was further incubated for 1 h at 30 °C. Fluorescence was
measured by a plate reader (Varioskan Lux, Thermo Fisher Scientifica)
with excitation wavelength at λ = 370 nm and emission wavelength at λ
= 450 nm. IC50 was estimated by the nonlinear regression curve fit
performed by means of GNU/Octave, according to a generalized form
of the dose−response curve equation, as reported by Copeland.80
Cellular Studies. NB4 cells were grown in RPMI-1640 (Sigma-
Aldrich, Milan, Italy) culture media, supplemented with 1% L-glutamine
(EuroClone, Milan, Italy), 10% heat-inactivated fetal bovine serum
(FBS) (GIBCO, Monza, Italy), and antibiotics (100 U/mL penicillin,
100 μg/mL streptomycin, and 250 ng/mL amphotericin-B). The U87
cell line was maintained in Eagle’s minimum essential medium (Sigma-
Aldrich, Milan, Italy) in the presence of 10% FBS. U2OS cells were
cultured in high-glucose Dulbecco’s modified Eagle’s medium
(DMEM; Gibco; Thermo Fisher Scientific) enriched with 10% FBS
and 1% glutamine. All cell lines were maintained in an incubator at 37
°C and 5% CO2.
Western Blot Analysis Methods. Cancer cells were treated with
6a, 6d, 6e, 6h, and 6l at 5 μM for 30 h. SAHA was used as a positive
control at the same time and concentration. For protein extraction, lysis
buffer (50 mmol/L Tris−HCl, pH 7.4, 150 mmol/L NaCl, 1% NP40,
10 mmol/L NaF, 1 mmol/L PMSF, and protease inhibitor cocktail) was
used. Samples were then centrifuged at 13 000 rpm for 30 min at 4 °C,
and the protein concentration quantified by the Bradford assay (Bio-
Rad). For histone extraction, cells were collected and resuspended in
triton extraction buffer [TEB; PBS containing 0.5% Triton X 100 (v/v),
2 mmol/L PMSF, 0.02% (w/v) NaN3], for 10 min at 4 °C. Samples
were centrifuged at 2000 rpm for 10 min at 4 °C, and pellets were
washed with TEB (half-volume). Samples were then resuspended in 0.2
N HCl, and acid histone extraction was carried out overnight at 4 °C.
Protein concentration was determined by the Bradford assay (Bio-
Rad). Each protein extract (35 μg) was loaded on 10% polyacrylamide
gels, and 2 μg of histone extract was instead used on 15%
polyacrylamide gel. Samples were then transferred on the nitrocellulose
membrane (Trans-blot turbo, Bio-Rad catalog: 1704150) and revealed
with anti-acetylated tubulin (clone 6-11B-1, Sigma) and anti-acetyl-
histone H3 antibody (cod: 06599, Millipore). GAPDH (cod: 14C10,
Cell Signaling) and H4 (ab31830, Abcam) antibodies were used as
loading controls.
multidisciplinary board according to the American Thoracic Society/
European Respiratory Society criteria and was later determined to be
consistent with recent guidelines.81−83 All IPF patients signed informed
consent prior to inclusion in the study. The studies were approved by
the local ethics committees.
Bronchoscopy. Bronchial epithelial cells were harvested by
bronchial brushes of subsegmental bronchi of the right lower lobe
during flexible bronchoscopy within the routine diagnostic work-up at
initial diagnosis. None of the patients received antifibrotic treatment
prior to bronchoscopy. None of the included subjects was currently
smoking.
Isolation of ABC. ABCs were isolated from bronchial brushes of
subsegmental bronchi of the right lower lobe using a similar protocol as
recently described.39,84,85 Bronchial brushes were placed in 2 ml of
prewarmed (37 °C) Clonetics bronchial epithelial cell growth medium
(BEGM) (Lonza, CC-3170). Then, airway epithelial cells were pelleted
by centrifugation (250g, 5 min) and disaggregated by resuspension in
trypsin/EDTA solution (0.05%/0.02%) (Merck, L2143) for 5 min at
37 °C. Afterward, the cell pellet was resuspended in 5 ml of BEGM and
seeded in T25 flasks (Merck, CLS3056) in BEGM, supplemented with
growth factors according to the manufacturer’s instructions. Cultures
were maintained in a humidified atmosphere of 5% CO2 at 37 °C. The
medium was changed every 7 days, and cells were harvested at day 21
when the cells were 90% confluent. Therefore, cells were trypsinized,
harvested, and counted. The purity of the ABC was determined by
immunocytology (cytokeratin-5 staining) of cytospins and always
exceeded 98%.
3D Organoid Assay. The bronchosphere formation assay was
performed as recently described.39 Briefly, IPF-ABCs (104 cells) were
added to 50 μL of ice-cold matrigel Corning matrigel matrix (Corning,
356231) in transwell inserts (Corning Lifesciences, Costar 3470) and
cultured for 30 min in the incubator (5% CO2, 37 °C) until the matrigel
became stiff. Then, 600 μL of a 1:1 ratio of BEGM and DMEM was
added below the inserts and additional 100 μL on top of the inserts.
Plates (Corning Lifesciences, Costar 3470) were cultured at 5% CO2
and 37 °C w/wo treatment with compounds 6a, 6d, 6h, and 6l. The
molecules were tested in the concentration range of 1−50 nM for 14
days. Medium exchange was done every 7 days.
Detection of Organoid Counts and Measurement of Cell
Proliferation. Mosaic photomicrographs were taken from HDAC6
inhibitor-treated 3D organoid assays by microscopy using an Axio
Observer Inverted microscope/Zeiss and ZEN microscope navigation
software. The numbers of organoids per well were counted by bright-
field microscopy on day 14. Only organoids with a size of 5 μm or larger
were counted. Cell proliferation was quantified using the colorimetric
MTT assay (Sigma-Aldrich, CT01) on day 14 according to the
manufacturer’s instructions. IC50 values were determined from
bronchosphere counts and the cell proliferation MTT assay by IC50
Calculator software (AAT Bioquest, Inc., Sunnyvale/USA). The mean
value was determined from the IC50 values of the organoid counts and
the MTT assay.
Immunohistochemistry. Formalin-fixed IPF lung and normal
tissues were used for immunohistochemistry as described.86 Three
micrometer thick sections of paraffin blocks were deparaffinized in
xylene and rehydrated with a descending alcohol row (as described
above). Heat-induced antigen retrieval was performed at 110 °C for 2
min in citrate buffer (pH 6.0) using a pressure cooker. First, staining
slides were washed for 5 min with Tris−NaCl buffer and then blocked
for 20 min with normal goat serum blocking solution 1:20 (Vector,
S1000). For the staining procedure, rabbit monoclonal anti-HDAC6
antibody [EPR1698(2)] (Abcam, ab133493) was incubated for 1 h at
room temperature (1:100). Isotype control was performed with
Universal Negative Control for IS-Series Rabbit Primary Antibodies
(Dako/Agilent Technologies, IS60061-2) (1:1000 dilution) overnight
at 4 °C.
Slides were washed with Tris−NaCl buffer. The incubation time of
the Goat IgG antirabbit IgG (H + L)-biotin (Dianova, 111-065-003)
(1:800 dilution) was 30 min at 25 °C. Slides were washed with Tris−
NaCl buffer. Activation was done using alkaline phosphatase Strept AP
(1:800 dilution) (Vector, SA-5100) for 30 min, and slides were washed
3D Organoid Model Assay and MTT Assay. Study Population.
ABCs from 12 patients with IPF were obtained during bronchoscopy at
routine diagnostic work-up. IPF diagnosis was established by a
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with Tris−NaCl buffer. Visualization was performed by DAKO REAL
Chromogen Red (Dako Real Kit) (Dako/Agilent Technologies,
K500311-2) (incubation time 20 min). Slides were washed with aqua
dest.
Slides were counterstained with Mayer′s hemalum solution (Merck,
109249) 1:10 for 90 s. Slides were washed shortly with aqua dest and for
90 s with Shandon Bluing Reagent (ThermoScientific, 6769001).
Before mounting, slides were washed once with aqua dest followed with
three short washing steps in 90% ethanol (CG Chemikalien), six short
washing steps in 100% ethanol (CG Chemikalien), and six short
washing steps in xylol (CG Chemikalien). Finally, coverslips were
placed over slides with the Eukitt Quick-hardening mounting medium
(Merck, 03989). All samples were digitalized using Mirax Scan 150 BF/
FL (Zeiss, Germany).
Human Lung Tissue. Human lung tissue samples were obtained
from healthy areas of lungs from patients undergoing lung resection for
carcinoma at Glenfield Hospital, Leicester, U.K. All patients gave
written informed consent, and the study was approved by the National
Research Ethics Service (reference 10/H0402/12 and 17/EM/0231).
Samples obtained were anonymized and coded before use.
Human Lung Tissue Explant Culture Model. Pieces of the
human lung tissue (2 mm3) were generated as described previously40
The tissue was cultured in DMEM + vehicle control (0.1% DMSO)
TGF-β1 (10 ng/mL) as described40 or DMEM + TGF-β1 (10 ng/mL)
+ 6h (0.41 and 4.1 μM) or vehicle control (0.1% DMSO). The tissue
was collected on day 7 for RNA extraction.
3.0, and the dilution of the stock solution in 50 mM aqueous PBS
afforded the sample solution at pH 7.4. These suspensions/solutions
were sealed and left for 24 h at 25 °C under orbital shaking to achieve
“pseudothermodynamic equilibrium”. After that time, the solutions
were filtered using PTFE filters and successively diluted 1:2 with the
buffer solution used for the preparation of the samples. Then, they were
analyzed by HPLC/UV/ DAD using UV detection at 254 nm for
quantitation. Solubility was calculated by comparing areas of the sample
and the standard
Asmp × FD × Cst
S =
Ast
S = solubility of the compound (μM); Asmp = UV area of the sample
solution; FD = dilution factor (2); Cst = standard concentration (250
μM); and Ast = UV area of the standard solution.
For each sample, the analysis was performed in triplicate, and the
solubility result reported was obtained from the average of the three
values. The same sample solutions were prepared to evaluate the
chemical stability of the compounds after 24 h at 25 °C and analyzed by
HPLC/UV/DAD using UV detection at 254 nm for quantitation.
Stability was calculated by comparing the area of the peak at T0 with the
area of the peak of the same solution after 24 h. A stability percentage
value was calculated by this method at pH 3.0 and pH 7.4 for each
compound by applying the following formula
AC24
RNA extraction was performed as described previously.40 The tissue
was dissociated using a Precellys 24 tissue homogenizer (Bertin
Technologies, Montigny-le-Bretonneux, France), and total RNA was
purified using the automated QIAcube with RNeasy Fibrosis Mini kit
(Qiagen, CA) according to the manufacturer’s instructions. RNA
integrity was assessed with a Bioanalyzer 2100 system (Agilent, CA),
and RIN values >8 were accepted as suitable for PCR profiling. RNA
concentrations were then measured using a Nanodrop 2000 (Labtech
International, East Sussex, U.K.).
% remaining =
× 100
ACT
0
Ac24 = area of the sample after 24 h at 25 °C; AcT0 = area of the sample
at T0. For each sample, the analysis was performed in triplicate and the
stability result reported was obtained from the average of the three
values.
Analysis of In Vitro Metabolic Stability of 6d and 6h in HLM.
The tested compound (6d or 6h), dissolved in MeCN, was incubated at
37 °C at 5 μM concentration in 100 mM phosphate buffer (pH 7.4)
with 0.5 mg/mL rat and human liver microsomal proteins as previously
reported.88 CYP-dependent reactions were started by addition of
NADPH-GS (2 mM NADPH), 66 mM glucose-6-phosphate, 0.4 U/
mL glucose-6-phosphate dehydrogenase to 66 mM MgCl2. Reactions
were terminated at regular time intervals (overall range 0−60 min) by
adding 1 mL of MeCN. All incubations were performed in triplicate.
HPLC analysis was performed on a Shimadzu Prominence apparatus
equipped with a Chromolith HPLC column RP-18 and coupled with a
UV−vis detector, set at λ = 254 nm. The analysis was carried out as
described above. The intrinsic clearance (Clint) was calculated by the
equation
Quantitative real-time PCR (qRT-PCR) was used to measure
mRNA expression levels of α-smooth muscle actin (αSMA), collagen
types I and III, and fibronectin using a Quantstudio 5 Real-Time PCR
machine (Applied Biosystems). Primer sequences for collagen types I
and III and αSMA have previously been described by Roach et al.87
Qiagen QuantiTect primer assays (QT00038024) were used for
fibronectin, and Taqman assays for HDAC6 (Hs00997427_m1) were
acquired from Thermo Fisher. Gene expression was quantified using
Brilliant SYBR Green QRT-PCR 1-Step master mix (Stratagene, the
Netherlands). All expression data were normalized to β2-microglobulin
using QuantiTect primer assay primers (Qiagen, Germany),
HS_B2M_1_SG, and corrected using the reference dye ROX. PCR
products were run on 1.5% agarose gel to confirm the product size, and
each product was sequenced to confirm the specificity of the primers.
The relative expression was calculated using the 2−ΔΔCt method.
Solubility and Chemical Stability Studies. HPLC Analysis of
Compounds 6d and 6h. For the HPLC analysis, a Chromolith HPLC
column RP-18 was employed. The runs were performed by gradient
elution starting from a mixture 0% MeCN (0.1% TFA as the phase
modifier) in H2O (0.1% TFA as the phase modifier) to 20% MeCN
(0.1% TFA) in H2O (0.1% TFA) in 4 min and then up to 50% MeCN
(0.1% TFA) in H2O in 3 min. The flow speed was set at 0.8 mL/min,
and the temperature was maintained at 25 °C. The volume of injection
of the sample was 10 mL, and the wavelength selected for the detection
was 254 nM. The retention times obtained following this protocol for
compounds 6d and 6h were 6.7 and 6.4 min, respectively.
k(min−1) × [V]
CLint
=
[P]
where k is the rate constant for the depletion of the substrate, V is the
volume of incubation in μL, and P is the amount of microsomal proteins
as reported elsewhere.43
Cytotoxicity and Mutagenicity Assays. Materials. Dulbecco’s
modified Eagle’s medium, trypsin solution, and all of the solvents used
for cell culture were purchased from Lonza (Switzerland). Mouse
immortalized fibroblasts NIH3T3 were purchased from American Type
Culture Collection. The mutagenicity assay was supplied by Biologik
s.r.l. (Trieste, Italy).
Cell Cultures and Cytotoxicity Assays. NIH3T3 cells were utilized
for cytotoxicity experiments. Cells were maintained in DMEM at 37 °C
in a humidified atmosphere containing 5% CO2. The culture media
were supplemented with 10% fetal calf serum (FCS), 1% L-glutamine--
penicillin−streptomycin solution, and 1% MEM nonessential amino
acid solution. Once at the confluence, cells were washed with 0.1 M
PBS, taken up with the trypsin−EDTA solution, and then centrifuged at
1000 rpm for 5 min. The pellet was resuspended in the medium solution
(dilution 1:15). The stock solution for each compound was prepared in
pure DMSO and diluted with the complete culture medium. The
solution/suspension obtained was then added to the cell monolayer.
Solubility Assay and Chemical Stability at 25 °C. A stock solution
for each tested compound was prepared by dissolving the sample in
DMSO to a final concentration of 10 mM. From the stock solution,
three samples were prepared: one was used as the standard solution and
the other two as the test solutions at pH 3.0 and pH 7.4. The
concentration of these solutions was 250 μM with a DMSO content of
2.5% (v/v). The standard solution was prepared by dilution of the stock
solution in PBS solution (MeCN/water, 60:40), the dilution of the
stock solution in 50 mM acetic acid afforded the sample solution at pH
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Cell viability after 24 h of incubation with the different concentrations
of each test compound was evaluated by neutral red uptake by the
procedure previously reported.89 Data processing included the
Student’s t-test with p < 0.05 taken as the significance level. First, the
following solutions were prepared to determine the percentage of viable
cells:
Isolated Rat Heart Preparation and Perfusion. All animal care
and experimental protocols conformed to the European Union
Guidelines for the Care and Use of Laboratory Animals (European
Union Directive 2010/63/EU) and were approved by the Italian
Department of Health (666/2015-PR). Male Sprague−Dawley rats
(350 g; Charles River Italia, Calco, Italy; n = 5) were used for this
experiment. Rats were anesthetized (ip) with a mixture of Zoletil 100
(7.5 mg kg−1 tiletamine and 7.5 mg kg−1 zolazepam; Virbac Srl, Milano,
Italy) and Xilor (4 mg kg−1 xylazine; Bio 98, San Lazzaro, Italy)
containing heparin (5000 U/kg), decapitated, and bled. The hearts,
spontaneously beating, were rapidly explanted and mounted on a
Langendorff apparatus for retrograde perfusion via the aorta at a
constant flow rate of 10 mL/min with the Krebs−Henseleit solution of
the following composition (mM): NaCl 118, KCl 4.7, CaCl2 2.5,
MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11.5, Na pyruvate 2, and
EDTA 0.5, bubbled with a 95% O2−5% CO2 gas mixture (pH 7.4), and
kept at 37 °C, as described elsewhere.91 The hearts were allowed to
equilibrate for at least 20 min before drug exposure. Heart contractility
was measured as left ventricle pressure (LVP) by means of a latex
balloon, inserted into the left ventricle via the mitral valve and
connected to a pressure transducer (BLPR, WPI, Berlin, Germany).
The balloon was inflated with deionized water from a microsyringe until
a left ventricular end diastolic pressure of 10 mmHg was obtained.
Alterations in the coronary perfusion pressure (CPP), arising from
changes in coronary vascular resistance, were recorded by the pressure
transducer (BLPR, WPI, Berlin, Germany) placed in the inflow line.92 A
surface electrocardiogram (ECG) was recorded at a sampling rate of 1
kHz by means of two steel electrodes, one placed on the apex and the
other on the left atrium of the heart. The ECG analysis included the
following measurements: RR (cycle length), HR (frequency), PQ
(atrioventricular conduction time), QRS (intraventricular conduction
time), and QT (overall action potential duration). LVP, CPP, and ECG
were recorded with a digital PowerLab data acquisition system
(PowerLab 8/30; ADInstruments, Castle Hill, Australia) and analyzed
by Chart Pro for Windows software (PowerLab; ADInstruments,
Castle Hill, Australia). LVP was calculated by subtracting the left
ventricular diastolic pressure from the left ventricular systolic
pressure.48 As the QT interval is affected by heart rate changes (e.g.,
it shortens with rapid heart rate), Bazett’s formula (QTc = QT/(RR)1/
2) was routinely used to avoid confounding effects. Compound 6h was
dissolved in DMSO. Solvents failed to alter the response of the
preparations (data not shown).
1. Neutral red (NR) stock solution: 0.33 g of NR dye powder in
100 mL of sterile H2O
2. NR medium: 1.0 mL of NR stock solution + 99.0 mL routine
culture medium prewarmed to 37 °C
3. NR desorb solution: 1% glacial acetic acid solution + 50%
ethanol + 49% H2O
At the end of incubation, the routine culture medium was removed
from each well, and cells were carefully rinsed with 1 mL of prewarmed
D-PBS. Multiwells were then gently blotted with paper towels. The NR
medium (1.0 mL) was added to each well and further incubated at 37
°C, 95% humidity, and 5.0% CO2 for 3 h. The cells were checked during
NR incubation for NR crystal formation. After incubation, the NR
medium was removed, and cells were carefully rinsed with 1 mL of
prewarmed D-PBS. Then, the PBS was decanted and blotted from the
wells and 1 mL of NR desorb solution was added to each sample.
Multiwells were then put on a shaker for 20−45 min to extract NR from
the cells and form a homogeneous solution. During this step, the
samples were covered to protect them from light. After 5 min, the
multiwells were removed from the plate shaker and the absorbance was
read at 540 nm by a UV/visible spectrophotometer (Lambda 25,
PerkinElmer).
Mutagenicity Assay: Ames Test. The TA100 and TA98 strains of
Salmonella Typhimurium and the S9 fraction were utilized for the
mutagenicity assay. Approximately 107 bacteria were exposed to six
concentrations of each test compound, as well as a positive and a
negative control, for 90 min in a medium containing sufficient histidine
to support approximately two cell divisions. After 90 min, the exposure
cultures were diluted with the pH indicator medium lacking histidine
and aliquoted into 48 wells of a 384-well plate. Within two days, cells
that have undergone reversion to His grew into colonies. Metabolism
by the bacterial colonies reduced the pH of the medium, changing the
color of that well. This color change can be detected visually or by a
microplate reader. The number of wells containing revertant colonies
were counted for each dose and compared to a zero-dose control. Each
dose was tested in six replicates. The test was performed both with and
without the S9 fraction.
Statistical Analysis. Data are reported as mean SEM; n (indicated
in parentheses) represents the number of rat hearts. Analysis of data was
accomplished using GraphPad Prism version 5.04 (GraphPad
Software). Statistical analyses and significance as measured by repeated
measures ANOVA (followed by Dunnett’s post-test or Friedman test)
were obtained using GraphPad InStat version 3.06 (GraphPad
Software). In all comparisons, P < 0.05 was considered significant.
Toxicity Screening of 6h in Zebrafish Larvae. All experiments
with in-house wild-type zebrafish larvae (Danio rerio) were performed
according to ethical exemptions granted by the UCD Animal Research
Ethics Committee, University College Dublin (AREC-Kennedy). No
animals were used as per European Union Directive 2010/63/EU;
larval forms of zebrafish that are not independently feeding and free-
living are not classified as animals. Zebrafish larval experiments were
performed with approval from the UCD Animal Research Ethics
ASSOCIATED CONTENT
■
Committee, Ireland. Wild-type (Tubingen) larvae were reared in
̈
embryo medium (0.137 M NaCl, 5.5 mM Na2HPO4, 5.4 mM KCl, 1.3
mM CaCl2, 0.44 mM KH2PO4, 1.0 mM MgSO4, and 4.2 mM NaHCO3,
conductivity 1200 μS, pH 7) containing methylene blue at 28.5 °C
under a 14 h light/10 h dark cycle (parameters measured in the
zebrafish facility for the year 2018: the pH, temperature, and
conductivity were reported).90 Adult zebrafish were maintained in a
recirculating water system at 28 °C under a 14 h light/10 h dark cycle
and fed daily with brine shrimp and dry pellet food. Wild-type zebrafish
larvae were obtained by incrosses of wild-type adults. The embryos
were raised and treated until 3 days old with either increasing
concentrations (1, 10, 25, 50, and 100 μM) of compound 6h or 0.1%
DMSO (vehicle control) for 2 days. Four larvae were placed per well in
400 μL of respective drug concentrations prepared in embryo media.
Eight larvae were used in total per treatment group, and the experiment
was performed in a 48-well plate. The visual behavioral assay
optokinetic reflex assaywas performed 2 days post-treatment as
described previously90 to determine the effect of compound 6h on
visual function.
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00184.
Figure S1, HPLC separation of the racemic mixture
( )-25b; Table S1, Relative MM energies (ΔEMM),
electronic energies (EQM and ΔEQM), free energies (G and
ΔG), and corresponding Boltzmann populations (χ) for
the conformers of (S)-25b; Table S2, Oscillator strengths
(fj), rotational strengths in dipole length formalism (Rj),
and excitation wavelengths (λj) for the conformers of (S)-
25b; Table S3, X-ray crystallographic data collection and
refinement statistics for the zf HDAC6−6h complex;
Figures S2−S15, Docking studies of compounds 6a−m;
Figure S16, Isolation of human airway basal cells; Figure
S17, Bronchosphere generation blocked by compounds
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6a, 6d, 6h, and 6l; 1H, 13C NMR spectra and HPLC purity
of final compounds ( )-6a−m (PDF)
Molecular formula strings (CSV)
Recommended compound characterization checklist
(XLSX)
Recommended compound characterization checklist
(XLS)
Samuele Maramai − Department of Biotechnology, Chemistry
and Pharmacy, DoE Department of Excellence 2018-2022,
University of Siena, 53100 Siena, Italy; orcid.org/0000-
0001-7499-6961
Gabriele Carullo − Department of Biotechnology, Chemistry
and Pharmacy, DoE Department of Excellence 2018-2022,
University of Siena, 53100 Siena, Italy; orcid.org/0000-
0002-1619-3295
Benedikt Jaeger − Klinik fu
Hochschule Hannover, Hannover 30625, Germany
Alfonso Carleo − Klinik fur Pneumologie, Medizinische
̈
r Pneumologie, Medizinische
Accession Codes
The atomic coordinates and crystallographic structure factors of
the HDAC6 complex with inhibitor 6h has been deposited in the
Protein Data Bank (www.rcsb.org) with accession code 6V79.
Authors will release the atomic coordinates and experimental
data upon article publication.
̈
Hochschule Hannover, Hannover 30625, Germany
Rosaria Benedetti − Department of Precision Medicine,
University of Campania Luigi Vanvitelli, 80138 Naples, Italy;
orcid.org/0000-0001-5517-5519
Federica Sarno − Department of Precision Medicine, University
of Campania Luigi Vanvitelli, 80138 Naples, Italy
Stefania Lamponi − Department of Biotechnology, Chemistry
and Pharmacy, DoE Department of Excellence 2018-2022,
University of Siena, 53100 Siena, Italy
Paola Rottoli − Specialization School of Respiratory Diseases,
Department of Medical Sciences, Surgery and Neurosciences,
Centro didattico Le Scotte, University of Siena, 53100 Siena,
Italy
Elena Bargagli − Department of Medical Sciences, Surgery and
Neurosciences, Respiratory Diseases Unit, AOUS, Centro
didattico Le Scotte, University of Siena, 53100 Siena, Italy
Carlo Bertucci − Department of Pharmacy and Biotechnology,
University of Bologna, Bologna 40126, Italy
AUTHOR INFORMATION
Corresponding Authors
■
Giuseppe Campiani − Department of Biotechnology, Chemistry
and Pharmacy, DoE Department of Excellence 2018-2022,
University of Siena, 53100 Siena, Italy; orcid.org/0000-
0001-5295-9529; Phone: (+39) 0577 232239;
Email: campiani@unisi.it
Stefania Butini − Department of Biotechnology, Chemistry and
Pharmacy, DoE Department of Excellence 2018-2022,
University of Siena, 53100 Siena, Italy; orcid.org/0000-
0002-8471-0880; Phone: (+39) 0577 234161;
Email: butini3@unisi.it
Authors
Daniele Tedesco − Department of Pharmacy and
Biotechnology, University of Bologna, Bologna 40126, Italy;
Present Address: Institute for Organic Synthesis and
Photoreactivity (ISOF), National Research Council
(CNR), via Piero Gobetti 101, 40129 Bologna, Italy;
orcid.org/0000-0003-2585-7791
Daniel Herp − Institute of Pharmaceutical Sciences, Albert-
Ludwigs-Universität Freiburg, 79104 Freiburg, Germany
Johanna Senger − Institute of Pharmaceutical Sciences, Albert-
Ludwigs-Universität Freiburg, 79104 Freiburg, Germany
Giovina Ruberti − Institute of Biochemistry and Cell Biology
(IBBC), National Research Council (CNR), 00015
Monterotondo (Rome), Italy; orcid.org/0000-0003-2367-
9709
Fulvio Saccoccia − Institute of Biochemistry and Cell Biology
(IBBC), National Research Council (CNR), 00015
Monterotondo (Rome), Italy
Simona Saponara − Department of Life Sciences, University of
Siena, I-53100 Siena, Italy
Beatrice Gorelli − Department of Life Sciences, University of
Siena, I-53100 Siena, Italy
Massimo Valoti − Department of Life Sciences, University of
Siena, I-53100 Siena, Italy
Breándan Kennedy − UCD School of Biomolecular and
Biomedical Science, UCD Conway Institute, University College
Dublin, D04 V1W8 Dublin, Ireland; orcid.org/0000-
0001-7991-4689
Husvinee Sundaramurthi − UCD School of Biomolecular and
Biomedical Science, UCD Conway Institute, University College
Dublin, D04 V1W8 Dublin, Ireland
Manfred Jung − Institute of Pharmaceutical Sciences, Albert-
Ludwigs-Universität Freiburg, 79104 Freiburg, Germany;
orcid.org/0000-0002-6361-7716
Caterina Cavella − Department of Biotechnology, Chemistry
and Pharmacy, DoE Department of Excellence 2018-2022,
University of Siena, 53100 Siena, Italy
Jeremy D. Osko − Roy and Diana Vagelos Laboratories,
Department of Chemistry, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6323, United States
Margherita Brindisi − Department of Biotechnology, Chemistry
and Pharmacy, DoE Department of Excellence 2018-2022,
University of Siena, 53100 Siena, Italy; Present
Address: Department of Excellence of Pharmacy,
University of Naples Federico II, Via D. Montesano, 49,
Naples 80131, Italy; orcid.org/0000-0001-9119-3773
Nicola Relitti − Department of Biotechnology, Chemistry and
Pharmacy, DoE Department of Excellence 2018-2022,
University of Siena, 53100 Siena, Italy; Present
Address: IRBM Science Park, Via Pontina km 30,600,
00071 Pomezia, Rome, Italy; orcid.org/0000-0001-
9783-8966
Simone Brogi − Department of Pharmacy, University of Pisa,
56126 Pisa, Italy; orcid.org/0000-0001-9375-6242
A. Prasanth Saraswati − Department of Biotechnology,
Chemistry and Pharmacy, DoE Department of Excellence
2018-2022, University of Siena, 53100 Siena, Italy
Stefano Federico − Department of Biotechnology, Chemistry
and Pharmacy, DoE Department of Excellence 2018-2022,
University of Siena, 53100 Siena, Italy; orcid.org/0000-
0002-7478-6128
Giulia Chemi − Department of Biotechnology, Chemistry and
Pharmacy, DoE Department of Excellence 2018-2022,
University of Siena, 53100 Siena, Italy; Present
Address: Wellcome Centre for Anti-Infectives Research,
Drug Discovery Unit, Division of Biological Chemistry and
Drug Discovery, University of Dundee, DD1 5EH Dundee,
United Kingdom; orcid.org/0000-0002-3868-6752
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Article
Katy M. Roach − Department of Respiratory Sciences,
University of Leicester, UK, Institute of Lung Health and
NIHR Leicester BRC-Respiratory, LE5 4PW Leicester, U.K.
Lucia Altucci − Department of Precision Medicine, University of
Campania Luigi Vanvitelli, 80138 Naples, Italy
Peter Bradding − Department of Respiratory Sciences,
University of Leicester, UK, Institute of Lung Health and
NIHR Leicester BRC-Respiratory, LE5 4PW Leicester, U.K.
David W. Christianson − Roy and Diana Vagelos Laboratories,
Department of Chemistry, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6323, United States;
orcid.org/0000-0002-0194-5212
Sandra Gemma − Department of Biotechnology, Chemistry and
Pharmacy, DoE Department of Excellence 2018-2022,
University of Siena, 53100 Siena, Italy; orcid.org/0000-
0002-8313-2417
Antje Prasse − Klinik fur Pneumologie, Medizinische
̈
Hochschule Hannover, Hannover 30625, Germany
idiopathic pulmonary fibrosis; k, apparent decay constant; LVP,
left ventricle pressure; NBS, N-bromosuccinimide; NMO, 4-
methylmorpholine N-oxide; NRU, neutral red uptake; PQ,
atrioventricular conduction time; QRS, intraventricular con-
duction time; RR, cycle length; t1/2, half-life time; TD-DFT,
time-dependent density functional theory; TGF-β1, trans-
forming growth factor β1; THF, tetrahydrofuran; ZBG, zinc-
binding group; zfHDAC6, zebrafish HDAC6
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00184
Author Contributions
∥∥S.G. and A.P. joined as Co-senior Authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the Regione Toscana-IT grant HIDE-IPF
(Bando Salute 2018) and NIH for grant GM49758 (D.W.C.) in
support of this research. This research was also funded by AIRC-
17217; Campania Regional Government Technology Platform
Lotta alle Patologie Oncologiche iCURE-B21C17000030007;
“Epigenetic Hallmarks of Multiple Sclerosis” (acronym Epi-MS)
(id: 415, Merit Ranking Area ERC LS) in VALERE 2019
Program; V:ALERE 2020“CIRCE” D.R. n. 138 del 17/02/
2020 Program; Campania Regional Government FASE2:
IDEAL; MIUR, Proof of Concept-EPICUREPOC01_00043-
B64I19000290008; and P.O.R. CAMPANIA Campania ESF
Regional Operational Programme 2014/2020 Axis III-
B27D18001070006. This work is based on the research
conducted at the Northeastern Collaborative Access Team
beamlines, which are funded by the National Institute of General
Medical Sciences of the National Institutes of Health (P30
GM124165). This research used resources of the Advanced
Photon Source, a U.S. Department of Energy (DOE) Office of
Science User Facility operated for the DOE Office of Science by
Argonne National Laboratory under Contract No. DE-AC02-
06CH11357. Prof. Riccardo Zanasi (Department of Chemistry
and Biology, University of Salerno, Italy) kindly provided access
to the computing cluster used to perform MM and QM
calculations for the stereochemical characterization of com-
pound 25b. The authors thank K. Schmidtkunz for technical
assistance in IC50 determination.
ABBREVIATIONS
■
ABC, airway basal cell; AIBN, azobisisobutirronitrile; BAL,
bronchoalveolar lavage; CLint, intrinsic clearance; CPP,
coronary perfusion pressure; DCM, dichloromethane; DMSO,
dimethyl sulfoxide; ECD, electronic circular dichroism; ECG,
electrocardiogram; EMT, epithelial−mesenchymal transition;
HDAC, histone deacetylase; HDACi, HDAC inhibitor; HLM,
human liver microsomal; ILDs, interstitial lung diseases; IPF,
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