Brain Penetrable Histone Deacetylase 6 Inhibitor SW-100 Ameliorates Memory and Learning Impairments in a Mouse Model of Fragile X Syndrome

Article abstract of DOI:10.1021/acschemneuro.8b00600

Disease-modifying therapies are needed for Fragile X Syndrome (FXS), as at present there are no effective treatments or cures. Herein, we report on a tetrahydroquinoline-based selective histone deacetylase 6 (HDAC6) inhibitor SW-100, its pharmacological a

Full text of DOI:10.1021/acschemneuro.8b00600

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Article
Brain Penetrable Histone Deacetylase 6 Inhibitor SW-100 Ameliorates
Memory and Learning Impairments in a Mouse Model of Fragile X Syndrome
ALAN KOZIKOWSKI, Sida Shen, Marta Pardo, Mauricio Temotheo Tavares, Dora
Szarics, Veronick Benoy, Chad A. Zimprich, Zsofia Kutil, GuiPing Zhang, Cyril Barinka,
Matthew B. Robers, Ludo Van Den Bosch, James H Eubanks, and Richard S Jope
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00600 • Publication Date (Web): 04 Dec 2018
Downloaded from http://pubs.acs.org on December 5, 2018
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Brain Penetrable Histone Deacetylase 6 Inhibitor SW-100 Ameliorates
Memory and Learning Impairments in a Mouse Model of Fragile X Syndrome
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Alan P. Kozikowski , Sida Shen2,‡,†, Marta Pardo3,‡,†, Maurício T. Tavares , Dora Szarics ,
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2,†
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Veronick Benoy , Chad A. Zimprich , Zsófia Kutil , Guiping Zhang , Cyril Bařinka , Matthew
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B. Robers , Ludo Van Den Bosch , James H. Eubanks , & Richard S. Jope
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StarWise Therapeutics LLC, Madison, WI 53719, United States
2
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of
Illinois at Chicago, Chicago, IL 60612, United States
3
Department of Psychiatry and Behavioral Sciences, Miller School of Medicine, University of
Miami, Miami, FL 33136, United States
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Division of Genetics and Development, Krembil Research Institute, University Health
Network, Toronto, Ontario M5G 2C4, Canada
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Laboratory of Neurobiology, Vesalius Research Center (VIB) and Leuven Research Institute
for Neuroscience and Disease (LIND), KU Leuven, B-3000 Leuven, Belgium.
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Promega Corporation, Madison, WI 53711, United States.
7
Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy of
Sciences, Prumyslova 595, 252 50 Vestec, Czech Republic
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ABSTRACT
Disease-modifying therapies are needed for Fragile X syndrome (FXS), as at present there are no
effective treatments or cures. Herein, we report on a tetrahydroquinoline-based selective histone
deacetylase 6 (HDAC6) inhibitor SW-100, its pharmacological and ADMET properties, and its ability
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to improve upon memory performance in a mouse model of FXS, Fmr1 mice. This small molecule
demonstrates good brain penetrance, low-nanomolar potency for the inhibition of HDAC6 (IC = 2.3
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nM), with at least a thousand-fold selectivity over all other class I, II, and IV HDAC isoforms.
Moreover, through its inhibition of the α-tubulin deacetylase domain of HDAC6 (CD2), in cells SW-
100 upregulates α-tubulin acetylation with no effect on histone acetylation and selectively restores the
-/-
impaired acetylated α-tubulin levels in the hippocampus of Fmr1 mice. Lastly, SW-100 ameliorates
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several memory and learning impairments in Fmr1 mice, thus modeling the intellectual
deficiencies associated with FXS, and hence providing a strong rationale for pursuing HDAC6-based
therapies for the treatment of this rare disease.
KEYWORDS phenylhydroxamate, permeability, Ames negative, acetylated α-tubulin, memory
and learning impairments
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INTRODUCTION
Fragile X Syndrome (FXS) is the most common form of inherited severe cognitive
impairment in males (1 in 3,000 to 4,000 individuals) and a significant cause of intellectual
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disability in females (1 in 7,000 to 8,000 individuals). This condition causes an array of
developmental problems including learning disability, cognitive impairment, and behavioral
characteristics. Other signs and symptoms may include autism spectrum disorders, seizures, and
characteristic physical features. In most cases, FXS is caused by transcriptional silencing of the
fragile X mental retardation 1 (FMR1) gene due to the methylation of a cytosine-guanine-guanine
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(CGG) trinucleotide repeat expansion (number of repeats in FXS > 200, normal < 50), resulting
in the absence of the gene product, fragile X mental retardation protein (FMRP) which regulates
mRNA trafficking and dendritic translation. FMRP deficiency affects the translational regulation
of multiple proteins, including several that are important for learning and memory.3-5 At present,
there are no effective treatments or cures for FXS. Metabotropic glutamate receptor 5 (mGlu5)
inhibitors were considered as a potential therapy for FXS based on the ‘mGluR theory of FXS’.6-8
Recent phase II clinical trials, however, with two different mGlu5 negative allosteric modulators
(basimglurant and mavoglurant) failed to induce significant improvement over placebo for primary
endpoints.9, 10 In addition to mGluR5, a diverse set of alternative therapeutic targets such as GSK-
β, ERK1/2, IGF1, and MMP9 have been investigated in animal studies or early clinical trials.11
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Among them, the pan-histone deacetylase (HDAC) inhibitor valproic acid (VPA) was found to
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ameliorate the symptoms of attention deficit hyperactivity disorder (ADHD) in FXS boys. After
VPA treatment in FXS lymphoblastoid cells, however, FMR1-mRNA levels remained low, FMRP
protein was undetectable, and the gene remained methylated, although a significant increase in
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histone acetylation was observed. Additionally, there is accumulating evidence that the down-
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regulation of class I and II HDAC levels leads to neuroprotection or improvement of learning and
memory in rodent models of various brain diseases.14-18 Some research studies have demonstrated,
for example, that the administration of siRNA targeting class I HDACs 1-3, which are
predominantly responsible for regulating histone acetylation, improves cognitive impairments in
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the FXS intellectual deficiency mouse model. However, to date no research has been published
on the activity of selective HDAC6 inhibitors in FXS animal models.
Histone deacetylases (HDACs) are a family of proteins responsible for catalyzing the hydrolysis
of acetylated histone lysine residues to produce deacylated products. There are 18 known
mammalian HDACs, which are divided into 4 classes, based on their sequence similarity to yeast
homologs: class I (HDAC1, 2, 3, and 8), class IIa (HDAC4, 5, 7, and 9), class IIb (HDAC6 and
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0), class III (SIRT1-7), and class IV (HDAC11). Among these isoforms, HDAC6 has attracted
particular attention due to its relative uniqueness within its family: (a) unlike its related class
members, HDAC6 contains two tandem protein deacetylase motifs (CD1 and CD2) and has an N-
terminal nuclear export sequence and a C-terminal cytoplasmic retention sequence;21, 22 (b) unlike
other HDAC members, which are present in the nucleus, HDAC6 is found primarily within the
cytosol and is responsible for regulating the acetylation state of specific cytosolic proteins,
although it is also able to shuttle into the nucleus and thus can also potentially regulate
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transcription. Its regulation of α-tubulin acetylation has sparked considerable interest because
microtubule-dependent transport rates are facilitated by higher levels of acetylated α-tubulin.24-27
This effect presumably stems from the increased association of the motor proteins kinesin-1 and
dynein with acetylated α-tubulin, which in turn regulates both anterograde and retrograde transport
activities.28 In addition to facilitating anterograde transport of new cargo to synaptic zones,
acetylated α-tubulin also increases the ability of damaged organelles or misfolded proteins to leave
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synaptic zones. Selective inhibition of HDAC6 thus appears to offer a potential therapeutic
approach for treating a number of neurological disorders such as depression,30 stroke,31
Parkinson’s disease,32 Alzheimer’s disease,33-35 Charcot-Marie-Tooth disease,36-38 Rett
Syndrome,33, 39 and amyotrophic lateral sclerosis (ALS).40 Intriguingly, significantly fewer
mitochondria and greatly reduced mitochondrial mobility have been observed in hippocampal
neurites of Fragile-X-associated tremor/ataxia syndrome (FXTAS, 50 < number of CGG repeats
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<
200).41, 42 Since HDAC6 inhibitors have been shown to increase mitochondrial movement
including vesicular trafficking of brain-derived neurotrophic factor (BDNF),43-45 we postulated
that HDAC6 inhibition could provide a useful therapeutic intervention for FXS. Therefore, an
early aim of the present study was to investigate whether selective inhibition of HDAC6 could
increase the acetylation of α-tubulin in brain, and if so whether that modification would improve
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the FXS-like phenotypes in Fmr1 mice.
A number of broad-spectrum HDAC inhibitors, such as vorinostat (SAHA), romidepsin,
panobinostat, and belinostat, are being used as anti-cancer agents due to their ability to enhance
gene expression by increasing histone acetylation, and in turn promoting cell-cycle arrest,
differentiation, and apoptosis. However, significant concerns have emerged that pan-HDAC
inhibitors may be too broadly acting for clinical use beyond oncology, especially with respect to
their potential neurotoxicity.46, 47 In the last decade, selective HDAC6 inhibitors (HDAC6is) have
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been reported, and two (poorly selective) HDAC6is (rocilinostat and citarinostat) are in clinical
trials for several types of cancer.49-51 However, only a handful of HDAC6is have been reported
that are brain-penetrant and display appropriate in vitro and in vivo activities in models of
neurological diseases.30, 52-55
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One selective HDAC6i discovered by our group, tubastatin A (TubA), has been investigated for
its potential to be used in central nervous system (CNS) disorders.30-35, 39, 44 TubA exhibits four of
the six desirable physicochemical properties for CNS drugs within the predicted optimal ranges:
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tPSA (40 < tPSA ≤ 90 Å ), pK (≤ 8.0, basic moiety), clogP (≤ 3), and MW (≤ 360), whereas two
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parameters including its HBD (optimally = 0) and clogD (optimally ≤ 2) are in less favorable
ranges. As such, TubA receives a relatively high CNS multi-parameter optimization (MPO)
desirability score of 5.33 (>5).56-58 Moreover, the positive calculated logBB value of TubA (0.18,
determined by ACD/Percepta software) suggests that TubA should cross the blood-brain-barrier
(BBB) effectively. However, its measured ratio of brain to plasma levels has been reported to be
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only modest (AUCbrain/AUCplasma = 0.18). Thus, prior to exploring the effects of an HDAC6i in
the FXS mouse model, we needed to identify a compound with improved brain penetrance.
To better understand the reasons for the poor brain penetrance of TubA, and to further guide the
identification of future novel brain penetrant analogs, we present here a set of HDAC6i analogs
selected from our compound library. Using a series of design modifications and in vitro
experiments we were able to identify the novel brain-penetrant HDAC6i, designated SW-100. This
compound is easy to synthesize, and it possesses low nanomolar potency against HDAC6,
excellent isozyme selectivity over other HDACs, selective engagement of HDAC6-CD2 as
measured in cells, as well as α-tubulin/histone acetylation selectivity in neurons. Moreover, we
were able to show that selective HDAC6i increases the levels of acetylated α-tubulin in the
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hippocampus of Fmr1 mice, and that this correlates with improved learning and memory
performances in this animal model of FXS.
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RESULTS AND DISCUSSION
SW-100 is a highly selective and potent HDAC6 inhibitor
The structure of a typical HDACi consists of a cap that interacts with the surface of the catalytic
cavity, a linker that occupies the hydrophobic channel, and a zinc binding group (ZBG) that
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2+
coordinates with the zinc ion (Zn ) at the deep bottom of the pocket. In general, the
phenylhydroxamate motif has been demonstrated to impart HDAC6 selectivity to a host of ligands,
as first revealed by the invention of TubA. X-ray crystal structures of several related compounds
in complex with HDAC6 typically display similar monodentate phenylhydroxamate-Zn2+
coordination geometry, while a canonical bidentate Zn2+ coordination was observed for the
hydroxamate inhibitors that bear an alkyl chain in the linker region, as exemplified by the pan-
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HDAC inhibitor TSA and the partially selective, HDAC6i ricolinostat. The two residues Phe583
and Phe643 located within the hydrophobic channel engage in a double π-stacking interaction with
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the phenyl group present in the linker. A previous publication has reported that even the capless
parent compound, phenylhydroxamate (1) (Figure 1a), shows an IC of 115 nM against HDAC6
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and more than 10-fold selectivity over class I HDACs. In this case, the crystal structure of the
enzyme-inhibitor complex containing phenylhydroxamate also displays bidentate zinc
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coordination, which is believed to underly its high potency. Thus, the capping group attached to
the phenylhydroxamate moiety is able to affect its degree of coordination with the catalytic Zn2+
ion leading to either bidentate or monodentate chelation. It has been calculated that the
monodentate coordination mode is slightly less stable by ~0.5 kcal/mol compared to bidentate
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coordination. Appendage of an N,N-diethylaminomethylene group or a pyrrolidinomethylene
group on the phenylhydroxamic acid core leads to compounds 2 and 3, respectively, both of which
show reduced HDAC6 activity. On the other hand, the heteroaromatic pyrrolomethylene (4) and
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indolylmethylene (5) derivatives, which can be viewed as structurally simplified analogs of TubA,
provide analogs of nanomolar activity against HDAC6 and high selectivity over HDAC1 (Figure
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1a). Docking simulations of compounds 2–5 and TubA to the active site of HDAC6-CD2 (Figure
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b-f) have been carried out, and these studies reveal that 2, 4, and TubA engage in monodentate
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coordination with Zn , with binding being further enhanced by hydrogen bonding interactions
with Gly582 and Tyr745 (2, Figure 1c); Gly582 and His614 (4, Figure 1d); or Gly582, Cys584,
and Tyr745 (TubA, Figure 1e), respectively. Within the narrow hydrophobic channel of HDAC6-
CD2, Phe583 and Phe643 engage in double π-stacking interactions with the bulky phenyl group
present in the linker; this binding mode is consistent with the interactions observed from the X-ray
structure of the phenylhydroxamate 1 in complex with HDAC6.60 The N,N-diethylamino group
of 2 did not show any hydrophobic interactions with the rim region, while the pyrrole cap of 4 was
found to engage in a hydrophobic interaction with the Pro464 side chain (Figure 1c), which is
similar to that observed for the tricyclic moiety of TubA (Figure 1e). Overall, hydrophobic
interactions between the aromatic caps and the residues present on the rim region of HDAC6
provide additional binding energies, regardless of the size of these cap groups. This information
may be helpful in the design of other novel ligands that may in turn show an improved ability to
penetrate the blood-brain-barrier.
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Figure 1. Hydrophobic caps are valuable for enhancing HDAC6 potency. (a) HDAC1/6 enzyme
potencies and structures of compounds 1-5; (b) Empirical conformation of 1 (green) in HDAC6-
CD2 (PDB: 6CSR); (c-e) Docked pose of 2 (yellow), 4 (magenta), and TubA (orange) in HDAC6-
CD2; (f) Superimposed structures of 2, 4, and TubA in the catalytic site of HDAC6-CD2.
Interatomic distances of both polar interactions and coordination are depicted in green dashed lines
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distance in Å). Zn2+ ions are depicted as dark grey spheres. The mesh molecular surface is
depicted in orange (wires) and pale green (surface).
Next we selected several HDAC6is containing different types of monocyclic or bicyclic
aromatic caps from our work for further investigation (Figure 2a).62, 63 The majority of these
compounds exhibit desirable calculated CNS physicochemical properties and MPO scores that
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would suggest ready brain permeability (Table S1). TubA and another well-known HDAC6i
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NexA were included as positive controls. A number of these potent HDAC6is including TubA
and NexA exhibit high selectivity against the class I HDACs 1, 2, and 3 but poorer selectivity
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against HDAC8 (Figure 2b and 2c). The benzimidazole analogs 8 and 9 were also found to show
strong activity against the class IIa isoforms. On the other hand, based upon these data and other
SAR data available to us, we designed the tetrahydroquinoline (THQ)-based analog SW-100. The
synthetic route to SW-100 starts from 4-chloroaniline (13) which was first converted to the N-
phenylpropanamide 14 by treatment with 3-chloropropanoyl chloride. Next, intramolecular
Friedel-Crafts cyclization using aluminum trichloride followed by reduction of the intermediate
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lactam with LiAlH afforded the tetrahydroquinoline 15. The building block 15 was then alkylated
4
with commercially available methyl 4-(bromomethyl)benzoate to provide the corresponding ester,
which upon treatment with an aqueous NH OH/NaOH solution afforded the desired hydroxamate
2
analog SW-100 (Scheme 1). The chemical route has been used to prepare multigram amounts of
this HDAC6i.
Cl
Cl
a
O
N
H
Cl
NH2
13
14
O
Cl
OH
b
c
N
H
N
N
H
Cl
15
SW-100
Scheme 1. Reagents and conditions. a) 3-chloropropanoyl chloride, acetone, reflux; b) (i) AlCl3,
140 °C (neat), (ii) LiAlH , THF, 0 °C to reflux; c) (i) methyl 4-(bromomethyl)benzoate, K CO ,
4
2
3
DMF, 100 °C, (ii) NaOH, 50 wt% aq. NH OH, THF/MeOH (1:1), 0 °C to rt.
2
As apparent from the data presented in Figure 2, SW-100 exhibits a 1000-fold to 10000-fold
selectivity for HDAC6 relative to all other HDAC isozymes, including HDAC8 (SI: 1280-fold;
data from Reaction Biology Corp.).
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Figure 2. SW-100 is a highly potent and selective HDAC6 inhibitor. (a) Structures of compounds
6
-12, and SW-100; (b) Heatmap summary of HDAC activities; (c) Full HDAC potency table of
SW-100, TubA, and NexA. IC values are the mean of two experiments ± SD obtained from
5
0
curve-fitting of a 10-point enzymatic assay starting from 30 µM with 3-fold serial dilution against
all HDAC isoform; TSA was tested in singlet 10-dose IC mode with 3-fold serial dilution starting
5
0
at a concentration of 10 µM against HDAC class I, IIb, and IV isoforms; TMP269, a selective
HDAC class IIa inhibitor, was tested in singlet 10-dose IC mode with 3-fold serial dilution
5
0
starting at a concentration of 10 µM against HDAC class IIa isoforms.
The docking results of SW-100 with HDAC6-CD2 shown in Figure 3 indicate that the
2
+
hydroxamate group engages in the expected monodentate interaction with the Zn ion, which is
further reinforced by hydrogen bonding between its NH and the Gly582 backbone as well as the
carbonyl and Tyr745/His614, respectively. Both residues Phe583 and Phe643 located in the
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hydrophobic channel engage in double π-stacking interactions with the phenyl ring present in the
linker of SW-100. Also, an additional interaction was observed between the chlorine atom on the
THQ cap and Asn645 present on the rim of the HDAC6-CD2 pocket (Figure 3a). Superimposed
and surface views of SW-100 and TubA in Figure 3b-d show that TubA engages in hydrophobic
interactions with Leu712 while SW-100 is turned toward residues Phe642 and Asn645. From
these static images, it is clear that the cap groups add to inhibitor potency through interactions with
different residues comprising the large rim area. Moreover, in comparison with the parent THQ
analog 16 [blue ligand in Figure 3b which lacks the chlorine atom; structure shown in Figure S2,
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IC (HDAC6) = 15 nM], SW-100 rotates within the binding pocket to allow for the interaction
50
with Asn645 at the rim surface which may in turn be responsible for the improved potency of SW-
1
00.
Figure 3 (a) Binding interactions of SW-100 (orange) within the HDAC6-CD2; (b) Superimposed
structures of SW-100 (orange), 16 (blue), and TubA (cyan) with HDAC6-CD2; (c) Surface pose
of SW-100 (orange) within HDAC6-CD2; (d) Surface pose of TubA (cyan) within HDAC6-CD2.
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The Zn ion is depicted as a grey sphere. Molecular surfaces are depicted in in grey, yellow
(
hydrophobic interaction area) and green (polar interaction area).
It is well appreciated that the ability of a small molecule to inhibit an enzyme in its pure/isolated
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state may not properly translate to its predicted outcomes in the cellular context. It thus became of
interest to further assess the potency and selectivity of SW-100 in live cells using the NanoBRET
target engagement assay, which is based on the competitive displacement of a luminescent reporter
6
5
complex utilizing a cell-permeable SAHA tracer. These results are summarized in Figures 4a-d,
and demonstrate that SW-100 maintains high selectivity over HDAC1 in cells. The measured
HDAC6 potency of SW-100 was slightly higher when the α-tubulin preferring deacetylase domain
of HDAC6, the CD2 domain (IC = 97 nM) was used in place of full-length HDAC6 (IC = 279
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nM). Results are also provided for SAHA for comparison purposes (Figure 4a-d).
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Figure 4. SW-100 selectively inhibits HDAC6-CD2 and enhances α-tubulin acetylation in cells.
(a-d) HDAC1/6 NanoBRET target engagement assay for compound SW-100 in HEK293. IC50
values are the mean of four experiments ± SEM obtained from curve-fitting of a 12-point
engagement assay starting from a concentration of 20 µM with 3-fold serial dilution. SAHA was
used as a positive control.
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SW-100 displays a significantly improved ability to cross the blood-brain-barrier
A compound’s brain uptake is largely determined by its ability to diffuse over cellular
membranes and its affinity for drug efflux transporters like P-glycoprotein (P-gp), which is
6
6
extensively expressed at the BBB. Compounds with permeability coefficients [Papp (A–B)] higher
−
6
than 3 × 10 cm/s generally show high brain uptake, while compounds with P
less than 1
app (A–B)
−
6
67
× 10 cm/s in the MDCK-MDR1 cell assay are less likely to penetrate the BBB. The data in
Table 1 reveal that all compounds studied can be classified as moderately permeable (2 × 10−6
−
6
cm/s < Papp (A–B) < 20 × 10 cm/s) except for the benzimidazole analog 12 that shows both a modest
Papp (A–B) and Papp (B–A). All three benzimidazole analogs (8, 9, and 12) display unfavorable efflux
ratios (>2.5) for CNS access as results from their higher Papp (B–A).68 Notably, the extremely high
Papp (B–A) of TubA results in an efflux ratio of 9.83, while compounds 6, 7, 10, 11, and SW-100
exhibit more favorable efflux ratios (<1.0). The predicted physicochemical properties and
desirable CNS-MPO scores suggest that all these compounds should exhibit similar permeability
properties (Table S1). However, diverse P-gp efflux ratios were obtained which may be related to
the amphoteric properties of these inhibitors. The calculated pK values of the acidic hydroxamate
a
groups are in the range of 9.0-9.2, while the pK values of basic functional group in the cap groups
a
range from 0.84 to 7.14 (Table 1 and Figure S1). A zwitterionic form is a preferred protonation
form for many amphoteric drugs at physiological pH, while a neutral status may become
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predominant when the pK values of the basic moiety are lower than 3. Moreover, the
a
zwitterionic character of a compound usually leads to poor membrane permeability. However, this
is amenable to improvement by replacing a more basic moiety with a less basic one.70, 71 We thus
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plotted the efflux ratios against the cap group’s pK , and the observed correlation is consistent
a
with the foregoing postulate (Figure S1). In light of the favorable cell line permeability data for
SW-100, we next carried out brain/plasma PK studies in vivo. As apparent from the data presented
in Table 2, SW-100 affords a reasonably good brain/plasma ratio of 2.44 at 1 h and 4.54 at 4 h
upon IP administration in wild-type (WT) mice, data that are consistent with its low efflux ratio.
In contrast, TubA displays a poor brain/plasma ratio of 0.15 at 8 min and 0.86 at 60 min after IV
administration in WT mice which is consistent with its permeability properties (Table S2).
SW-100 was also evaluated in both liver microsomal and hepatocyte stability assays, as well as
for hERG and CYP inhibition. The data in Table 2 indicate that its metabolic stability is low, while
inhibition of the hERG and CYPs are generally in an acceptable range. In addition, to investigate
the possible mutagenicity of SW-100,72 the compound was incubated with four strains of
Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and one strain of Escherichia coli
(
WP2 uvrA) in the presence and absence of mammalian microsomal enzymes (S9 mix). The results
in Table 2 show that SW-100 is negative under the conditions of this Ames assay.
pKa
MDCK-MDR1 cell line
Compd.
-
6
-6
acidic
9.05
9.20
9.20
9.20
basic
2.91
2.07
5.12
5.95
Papp (A-B) (10 , cm/s) P
(10 , cm/s) Efflux Ratio
app (B-A)
SW-100
4.58
7.35
2.33
0.51
0.70
0.63
3.23
6
7
8
5.24
11.04
13.45
17.61
4.44
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10.46
0.66
2.00
13.17
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TubA
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Table 1. Permeability determination for 6-12, SW-100, and TubA.a
a
pK values were obtained by Marvin Sketch version 18.5. MDCK-MDR1 cell line assays were
a
carried out by Pharmaron, Inc. (Irvine, CA).
Table 2. Brain/plasma pilot PK studies and initial ADMET evaluation of SW-100.a
Brain/plasma PK studies
route
dose
mg/kg)
time brain concentration
plasma concentration
(ng/mL)
brain/plasma
ratio
(
(h)
(ng/mL)
141.8 ± 52.3
11.5 ± 0.30
IP
IP
20
20
1
58.2 ± 24.9
2.52 ± 0.13
2.44
4.54
4
ADMET parameters
Human
Mouse
23
23
Liver microsomal stability (t min)
1
/2
Human
30
Hepatocyte stability (t , min)
1
/2
Mouse
10
hERG test (IC , µM)
HEK293 cells
12.23
20.53
18.84
83.21
2.51
-0.63
5
0
1
A2
C9
2C19
2
CYP inhibition (%@10 µM)
2
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D6
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TA98, TA100, TA1535,
TA1537, WP2 uvrA
Ames test
negative
aSW-100 was administered to C57BL/6 male mice by IP administration at the dose of 20 mg/kg.
Blood and brain samples were collected at 1 h and 4 h time-points. Brain tissues were homogenized
at a 1:4 ratio of tissue weight (g) to volume of PBS (mL). The data found (ng/mL) was multiplied
by 5 to obtain the concentration (ng/mL) in brain tissues. PK studies and ADMET profiles were
conducted by Pharmaron, Inc., Irvine, CA.
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SW-100 selectively enhances the levels of acetylated α-tubulin in vitro.
Compounds 6, 7, 10, 11, and SW-100, having reasonable efflux ratios, were further evaluated
for their ability to induce α-tubulin acetylation (Figure 5a-c) in cells. HEK293 cells were treated
with the HDAC6i at concentrations of 0.01, 0.1, 1, and 10 µM. As is apparent from the data
displayed in Figure 5a the monocylic analogs 6 and 7 were able to elevate the levels of acetylated
α-tubulin only at concentrations of 10 µM (one-way ANOVA F(10,32) = 8.425, p < 0.0001), while
the indole analogs 10 and 11 began to show reasonable activity at 1 µM (one-way ANOVA
F(10,22) = 5.973, p = 0.0002, Figure 5b). In contrast, SW-100 treatment led to a more obvious
increase in the acetylated α-tubulin levels in a dose-dependent manner that was similar to the
results shown by the comparator compound NexA (one-way ANOVA F(10,45) = 4.537, p =
0.0002, Figure 5c). Further, in order to ascertain the functional selectivity of SW-100, we assessed
its ability to induce histone acetylation in comparison to tubulin acetylation in the N2a cells. As is
apparent from the bar graphs in Figures 5d-f, SW-100 increased the ratio of acetylated α-tubulin
to α-tubulin (>8-fold compared to vehicle, p = 0.07) while inducing only modest changes in the
levels of acetylated histone 3 to histone 4 (<1.5-fold compared to vehicle). These effects are similar
to those found for the reference HDAC6i TubA (one-way ANOVA, F(3,12) = 3.272, p = 0.05) at
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acetylated histone H3 significantly compared to vehicle (p < 0.01, Figure 5a and 5c).
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HEK293 cells (a-c) and selectively increases the levels of acetylated α-tubulin in N2a cells (d-f).
(a-c) The α-tubulin acetylation assay was carried out in HEK293 cells treated with the indicated
drug and dose for a period of 48 h. Densitometric analysis of the acetyl-α-tubulin to total tubulin
in these HEK293 cells was performed and the resulting ratios normalized to the 10 µM dose of
TubA. Graphs are depicted as mean ± SEM of the resulting ratiometric values (test compounds: n
=
3 or 4; TubA: n = 4, vehicle: n = 4). Statistical significance was analyzed by one-way ANOVA
in comparison with the vehicle group. *p < 0.05, **p < 0.01, ***p < 0.001. (d-f) α-Tubulin/histone
acetylation assays in N2a cells. N2a cells were treated with 1 µM or 10 nM of SW-100. TubA and
TSA were used as positive controls. The ratios of acetylated α-tubulin to total α-tubulin and
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acetylated histone 3 to histone 4 were quantified by western blot. Tubulin acetylation and histone
acetylation were repeated and normalized to the vehicle group. Graphs represent mean ± SEM. n
= 4. Statistical significance was analyzed by one-way ANOVA in comparison with the vehicle
group. **p < 0.01. n/d, not determined.
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-/-
Efficacy studies of HDAC6i SW100 in Fmr1 mice modeling the learning and memory
deficits of FXS.
Given its excellent potency, target selectivity, as well as its improved brain penetrance, SW-100
was deemed a suitable candidate for studies in animal models of CNS disorders such as the FXS
mouse model. We advanced SW-100 into preliminary behavioral experiments to assess if this class
of HDAC6i could show the potential for ameliorating learning and memory impairments in Fmr1-/-
mice that display several phenotypes characteristic of FXS.73, 74 Fmr1 mice were previously
shown to have learning disability and cognitive impairments in a variety of tasks including novel
object recognition, temporal ordering, and coordinate and categorical spatial processing.75
Furthermore, these mice also exhibit elevated locomotor activity, but, this did not interfere with
-/-
-/-
76
the ability of Fmr1 mice to explore the objects. 20 mg/kg of SW-100 was administered twice a
day in order to compensate for its short metabolic half-life.
In the coordinate spatial processing task, the distance between two identical objects is altered
between the habituation and testing periods. The categorical spatial processing task involves
interchanging the positions of two new identical objects following the habituation phase without
altering the distance between them. Exploration ratios were calculated as time exploring during
the 5 min test session divided by time exploring during the 5 min test session plus the last 5 min
of the habituation session.
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No significant differences between groups was found in total time exploring objects, when first
-/-
exposed to the objects, in all behavioral tests (data not shown). Fmr1 mice displayed reduced
exploration ratios in both the coordinate [F(3,31) = 2.92, p < 0.05; Figure 6a] and categorical
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[
F(3,31) = 4.22, p < 0.05; Figure 6b] spatial processing tasks compared to WT mice. While
administration of SW-100 did not significantly alter the behavior of WT mice in these tests, it
-/-
tended to reverse the impairment in Fmr1 mice in coordinate spatial processing and in categorical
-/-
spatial processing, so after SW-100 treatment the Fmr1 mice no longer exhibited deficits
compared with vehicle-treated WT mice.
In the novel object recognition test, WT littermate mice spent significantly more time exploring
the novel object than the familiar object, [t(14) = 7.50, p < 0.01; Figure 6c]. In contrast, Fmr1-/-
-/-
mice spent less time exploring the novel object, indicating that Fmr1 mice do not remember the
familiar object [t(14) = 8.40, p < 0.01]. The exploration ratio (calculated by dividing the difference
between the time spent with the novel object versus the familiar object divided by total time
-/-
exploring) was significantly lower in Fmr1 mice than WT mice [F(3,31) = 22.49, p < 0.01; Figure
6
d]. Administration of SW-100 did not alter the novel object recognition of WT mice [t(16) = 6.43,
-/-
p < 0.01], but improved the abilities of Fmr1 mice, which after treatment spent significantly more
time exploring the novel object than the familiar object [t(18) = 7.24, p < 0.01; Figure 6c].
-/-
Furthermore, the exploration ratio was significantly increased in Fmr1 mice treated with SW-
00 [F(3,31) = 22.49, p < 0.01; Figure 6d], demonstrating that HDAC6 inhibition effectively
1
-/-
reversed the novel object recognition deficit in Fmr1 mice.
WT mice displayed temporal order recognition as more time was spent exploring the initial
-/-
object [t(14) = 7.10, p < 0.01; Figure 6e], whereas Fmr1 mice displayed a deficit in temporal
order recognition [t(14) = 2.20, p = 0.05; Figure 6e]. Thus, the object exploration ratio (calculated
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by dividing the difference between the time spent with the initial object (object 1) versus the more
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recent object (object 3) by total time exploring), differed significantly between Fmr1 and WT
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mice, revealing a temporal order recognition deficit in Fmr1 mice [F(3,31) = 9.24, p < 0.01;
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Figure 6f]. Fmr1 mice treated with SW-100 spent significantly more time exploring the first
object compared to the most recent object presented, similarly to untreated or treated WT mice
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[
WT: t(16) = 5.00, p < 0.01; Fmr1 : t(18) = 7.53, p < 0.01; Figure 6e]. Furthermore, the
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exploration ratio was significantly increased in Fmr1 mice treated with SW-100 [F(3,31) = 9.24,
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p < 0.05; Figure 6f]. These results demonstrate that temporal ordering is impaired in Fmr1 mice
-/-
and that this deficit is corrected by inhibition of HDAC6. Overall, we observed that Fmr1 mice
displayed significant deficits in these four cognitive tasks, and that administration of the HDAC6i
SW-100 ameliorated all of these cognitive deficits.
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Figure 6. SW-100 effects on performance of Fmr1 mice in four cognitive tasks. (a) Performance
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of WT mice (n = 17) and Fmr1 mice (n = 18) in the coordinate spatial processing task (means ±
SEM; *p < 0.05 compared to vehicle-treated WT mice); (b) Performance of WT mice (n = 17) and
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Fmr1 mice (n = 18) in the categorical spatial processing task (Means ± SEM; *p < 0.05 compared
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to vehicle-treated WT mice). (c, d) Performance of WT mice (n = 18), and Fmr1 mice (n = 16)
in novel object recognition. (c) Percent time spent exploring the novel (N) and familiar (F) object.
(
(
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Means ± SEM; *p < 0.01 compared to time spent with familiar object); (d) Discrimination index
means ± SEM; *p < 0.01 compared to vehicle-treated WT mice). (e, f) Performance of WT mice
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n = 17) and Fmr1 mice (n = 18) on the temporal order task. (e) Percent time spent exploring the
first (1) and last (3) object presented. (means ± SEM; *p < 0.05 compared to time spent with object
); (f) Discrimination index (means ± SEM; *p < 0.05 compared to vehicle-treated WT). (g)
Scheme of treatment and testing. Arrows indicate SW-100 treatment.
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α-Tubulin acetylation effects of SW-100 in the Fmr1 mice.
-/-
We examined the levels of acetylated α-tubulin levels in the hippocampus of the Fmr1 mice
and WT mice. We found that the acetylated α-tubulin levels were 30% lower in the hippocampus
-/-
of Fmr1 mice than that of WT mice [t(18) = 2.72, p < 0.05; Figure 7a]. The impaired acetylated
-/-
α-tubulin levels were significantly increased in Fmr1 mice [t(16) = 4.38, p < 0.01; Figure 7c] by
treatment with SW-100, while no significant influence on hippocampal α-tubulin acetylation was
found in the WT mice [t(15) = 0.08 p = 0.93; Figure 7b]. The lack of any significant changes in
the acetylated α-tubulin levels in the WT mice results from the fact that estimated acetylated α-
tubulin levels in WT brains are approximately 75% of the total tubulin (i.e., only 25% of brain α-
tubulin is non-acetylated). Consequently, any putative increase (of theoretical maximum of 25%
to reach 100% acetylated α-tubulin) would be only marginal and difficult to quantify accurately.
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To the contrary, lower basal levels of acetylated α-tubulin in Fmr1 brains increase the dynamic
range of the assay and allow for more accurate quantification upon inhibitor treatment.
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Figure 7. SW-100 electively increases the levels acetylated α-tubulin in the hippocampus of Fmr1-
/
-
mice. 20 mg/kg SW-100 was administered twice a day on days 1 and 2 and an additional injection
was given on day 3 (1 hour prior to cognition assessments) and another injection was given on day
(1 hour prior to cognition assessments). (a) Basal levels of acetylated α-tubulin in the
4
hippocampus of WT and Fmr1-/- mice. Data were normalized relative to the WT mice.
Immunoblots from representative mice in each group, at 2 h after treatment, showing acetylated
-/-
α-tubulin in the hippocampus of (b) WT mice, and (c) Fmr1 mice. Data were normalized to the
vehicle group (0 mg/kg). Graphs represent mean ± SEM. *p < 0.05 (a), **p < 0.01 (c) compared
to control condition. (n = 6-10 per group).
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CONCLUSION
The main goal of this study was to identify a potent HDAC6 inhibitor with excellent isoform
selectivity and improved BBB permeability, and then to test its ability to improve a cardinal
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phenotype in Fmr1 mice that model intellectual deficits present in FXS patients.
In general, it would appear reasonable that a compound claimed to be selective for HDAC6
would exhibit selectivity for HDAC6 over HDAC1 in the range of 100 to a 1000-fold. However,
many of the published HDAC6is exhibit relatively low selectivity versus other HDACs, and in
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particular against HDAC8 and the class IIa isozymes. In contrast, the novel compound SW-100,
displayed at least 1000-fold selectivity against all class I, II, and IV HDAC isoforms. These results
position SW-100 as one of the most selective HDAC6is reported to date. Moreover, cellular target
engagement assays revealed that SW-100 is able to displace a SAHA-based tracer from HDAC6-
CD2 at low nanomolar concentrations, the key catalytic site that has been linked to the tubulin
deacetylase function of HDAC6. The cellular selectivity of compound SW-100 was further
investigated through α-tubulin and histone acetylation assays, and the results obtained are
consistent with its HDAC isoform selectivity determined from the isolated enzyme assays.
Brain penetrance is one major challenges for the development of HDAC6is as potential
therapeutics for CNS disorders due in part to the polarity of hydroxamate ZBG. Only a limited
number of brain penetrable selective HDAC6is have been published in studies with animals.30, 33,
5
3, 55
While the tool compound TubA displays excellent HDAC6 selectivity, it is not particularly
useful for study in animal models of CNS disorders, for as we have found it exhibits a high efflux
ratio (9.83) in the MDCK-MDR1 cell line assay and thus a poor brain/plasma ratio. To overcome
this weakness, we identified SW-100 that has a higher lipophilicity but lower pK value and thus
a
an acceptable brain/plasma ratio.
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Defects in α-tubulin acetylation are associated with age-related neurodegenerative diseases such
as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease, amongst others.78
Increasing the levels of acetylated α-tubulin by Hdac6 deletion were beneficial in a mouse model
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of ALS treatment, while other articles have reported that HDAC6 inhibition in mouse models
of Alzheimer’s disease can improve performance in various memory tasks.33, 54 Although the
exact role of α-tubulin acetylation in these diseases is still being studied, the hypothesis has
emerged that the defect in α-tubulin acetylation is associated with dysfunctional microtubule-
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mediated axonal and/or dendritic transport. As Fernandez-Barreda et al. stated, proteins that
modulate the levels of microtubules are potential pharmacological targets for therapeutic strategies
aimed at correcting the defects in α-tubulin acetylation found in a variety of neurodegenerative
diseases, because alterations in α-tubulin acetylation can lead to disorganization of neuronal
networks.81
-/-
The mouse model of FXS, Fmr1 mice, display cognitive impairments that can be detected in
measurements of novel object recognition, temporal ordering of objects, and coordinate and
categorical spatial processing.75, 82-85 Intellectual disability may be the most difficult characteristic
-/-
of FXS to treat, but it is critical for patients. Treatment of Fmr1 mice with the HDAC6i SW-100
improved their performance in all four of these tasks but did not alter the performance of WT mice
in any of these tasks. These findings demonstrate that cognitive impairments are not irreversible
in this mouse model of FXS and raise the possibility that HDAC6 inhibition may be a feasible
therapeutic intervention for FXS.
Overall, we report the development of a new HDAC6i, SW-100, that is highly selective, potent, and
-/-
penetrates the CNS. In vivo treatment with SW-100 to Fmr1 mice reversed deficits in the levels of
acetylated α-tubulin in the hippocampus and ameliorated several cognitive impairments. We believe
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that this research offers a truly innovative approach to the treatment of FXS, and that further
improvements in compound design will lead to drug candidates that can be tested in humans.
EXPERIMENTAL SECTION
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Chemistry.
General information.
1
13
H and C NMR spectra were obtained on 400/100 MHz or 300 MHz Bruker spectrometers,
except where noted otherwise, using the solvent residual peak as the internal reference (chemical
shifts: CD OD, 3.31/49.15; DMSO-d , 2.50/39.52; D O, 4.80). The following abbreviations for
3
6
2
multiplicities were used: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = double
doublet, dt = double triplet, td = triple doublet, ddd = double double doublet, and br s= broad
singlet. TLC plates (Merck silica gel 60 F , 250 µm thickness) were used to monitor reaction
2
54
progress, and spots were visualized under UV (254 nm). Low resolution mass spectrometry
LRMS) was carried out on a Shimadzu LCMS2020-PDA+ELSD+MS, and high-resolution mass
(
spectrometry (HRMS) on a Shimadzu IT-TOF instrument under the following conditions: column,
ACE 3AQ (50 × 2.1 mm i.d.); mobile phase, 8 – 100% CH CN/H O containing 0.1% formic acid
3
2
at a flow rate of 0.5 mL/min for 4 min. Flash chromatography was performed on a Combi-Flash
Rf system (Teledyne ISCO) with silica gel cartridges. Preparative HPLC was performed using a
Shimadzu preparative LC under the following conditions: column, ACE 5AQ (150 × 21.2 mm
i.d.); mobile phase: 8 – 100% MeOH (CH CN)/H O containing 0.05% TFA at a flow rate of 17
3
2
mL/min for 30 min; UV detection at 254 and 280 nm. Analytical HPLC was carried out on an
Agilent 1260 series instrument under the following conditions: column, ACE 3 (150 × 4.6 mm
i.d.); mobile phase, 8 – 100% MeOH (CH CN)/H O containing 0.05% TFA at a flow rate of 1.0
3
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mL/min for 25 min; UV detection at 254 nm. The purities of all tested compounds were > 98%, as
determined by analytical HPLC.
t
4-[(Diethylamino)methyl]-N-hydroxybenzamide (2). (i) General procedure A: To KO Bu (536.5
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mg, 4.79 mmol) in anhydrous DMF (15 mL) under argon was added diethylamine (350 mg, 4.79
mmol) dissolved in anhydrous DMF (10 mL). The reaction was stirred for 15 min at 0 °C, then
methyl 4-(bromomethyl)benzoate (1.097 g, 4.79 mmol) was added as a solution in anhydrous DMF
(8 mL). The reaction was stirred for 2 h at 70 °C, and then quenched and neutralized with 2N HCl.
The residue was extracted with EtOAc (3 × 30 mL), and the combined organic layers were washed
with H O (2 × 30 mL) and brine (15 mL), dried over sodium sulfate, filtered, and concentrated
2
under vacuum. The crude product was purified via Combi-Flash column chromatography
(
EtOAc/hexane = 0 − 100%) to afford methyl 4-[(diethylamino)methyl]benzoate as a yellow oil
790 mg, 75%). ii) General Procedure B: Solid NaOH (374 mg, 9.36 mmol) was dissolved in a 50%
(
aqueous solution of NH OH (4 mL) at
0
°C.
A
solution of methyl 4-
2
[(diethylamino)methyl]benzoate (518 mg, 2.34 mmol) in 1:1 THF/MeOH (6 mL) was added
dropwise with vigorous stirring. Upon complete addition, the ice bath was removed, and the
reaction was allowed to stir for 15 min. The reaction was then neutralized with 2N HCl solution,
and the mixture was concentrated under vacuum. The crude product was purified by preparative
HPLC to yield the title compound 2 as a white solid after neutralization and lyophilization (448
1
mg, 83%). H NMR (400 MHz, CD OD) δ 7.72 and 7.42 (AA′XX′ multiplet, J + JAX′ = 8.2 Hz,
3
AX
1
3
4H), 3.65 (s, 2H), 2.55 (q, J = 7.2 Hz, 4H), 1.08 (t, J = 5.0 Hz, 6H). C NMR (100 MHz, CD OD)
3
δ 166.3, 139.5, 131.9, 129.7 (2C), 127.0 (2C), 56.1, 46.4 (2C), 9.3 (2C). ESI HRMS calc. for
+
C H N O : [M + H] , m/z 233.1441; found: 233.1441.
1
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N-Hydroxy-4-(pyrrolidin-1-ylmethyl)benzamide (3). (i) The ester intermediate was synthesized
according to General Procedure A from pyrrolidine (0.12 mL, 1.52 mmol) and methyl 4-
(bromomethyl)benzoate (336 mg, 1.52 mmol) and isolated as a viscous clear oil (166 mg, 50%).
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(ii) The title compound 3 was synthesized according to General Procedure B from methyl 4-
(pyrrolidin-1-ylmethyl)benzoate (120 mg, 0.55 mmol) and afforded as an off-white solid after
1
preparative-HPLC purification and lyophilization (38 mg, 31%). H NMR (400 MHz, DMSO-d )
6
δ 11.33 (s, 1H), 10.69 (br s, 1H), 7.81 and 7.58 (AA′XX′ multiplet, JAX + JAX′ = 8.0 Hz, 4H), 4.39
1
3
(
s, 2H), 3.22 (br s, 4H), 1.94 (br s, 4H). C NMR (100 MHz, CD OD) δ 165.6, 134.2, 133.6, 130.3
3
+
(
2C), 127.6 (2C), 57.2, 53.6 (2C), 22.4 (2C). ESI HRMS calc. for C H N O : [M + H] , m/z
12 17
2
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221.1285; found: 221.1286.
4
-(1-Pyrrolylmethyl)-N-hydroxybenzamide (4).62 (i) The ester intermediate was synthesized
from pyrrole (150 mg, 2.24 mmol) and methyl 4-(bromomethyl)benzoate (495 mg, 1.52 mmol)
according to General Procedure A and isolated as a viscous clear oil (432 mg, 90%). (ii) The title
compound 4 was synthesized from methyl 4-(1-pyrrolylmethyl)benzoate (400 mg, 1.86 mmol)
according to General Procedure B and afforded as a white solid after preparative-HPLC
1
purification and lyophilization (225 mg, 56%). H NMR (400 MHz, CD OD) δ 7.69 and 7.19
3
(AA′XX′ multiplet, JAX + JAX′ = 8.3 Hz, 4H), 6.74 − 6.72 (m, 2H), 6.12 − 6.11 (m, 2H), 5.16 (s,
1
3
2
1
H). C NMR (100 MHz, CD OD) δ 168.0, 144.6, 132.8, 128.5 (2C), 128.2 (2C), 122.3 (2C),
3
+
09.6 (2C), 53.6. ESI HRMS calc. for C H N O : [M + H] , m/z 217.0972; found: 217.0974.
12 13
2
2
4-(1-Indolylmethyl)-N-hydroxybenzamide (5).62 (i) The ester intermediate was synthesized
t
according to General Procedure A (substituting NaH for KO Bu) from methyl 4-
(bromomethyl)benzoate (978 mg, 4.27 mmol) and indole (500 mg, 4.27 mmol) and isolated as a
white solid (860 mg, 76%). (ii) The title compound 5 was synthesized according to General
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Procedure B from methyl 4-(1-indolylmethyl)benzoate (840 mg, 3.17 mmol) and afforded as a
1
white solid after preparative-HPLC purification and lyophilization (443 mg, 53%). H NMR (400
MHz, DMSO-d ) δ 11.14 (s, 1H), 9.0 (br s, 1H), 7.67, 7.23 (AA′XX′ multiplet, J + JAX′ = 8.1
6
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hz, 4H), 7.56 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 3.1 Hz, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.09 (t, J =
1
3
7
.4 Hz, 1H), 7.02 (t, J = 7.3 Hz, 1H), 6.50 (d, J = 3.0 Hz, 1H), 5.48 (s, 2H). C NMR (100 MHz,
DMSO-d ) δ 163.9, 141.4, 135.7, 129.2 (2C), 128.3, 127.1 (2C), 126.8 (2C), 121.2, 120.5, 119.2,
6
+
1
10.1, 101.1, 48.7. ESI HRMS calc. for C H N O : [M+ H] , m/z 267.1128; found: 267.1137.
1
6
15
2
2
6
2
4
-(1-Pyrazolylmethyl)-N-hydroxybenzamide (6). (i) The ester intermediate was synthesized
according to General Procedure A from pyrazole (8.85 g, 0.13 mol) and methyl 4-
(bromomethyl)benzoate (30 g, 0.13 mol), and then isolated as a viscous yellow oil (24.5 g, 86%).
(ii) The title compound 6 was synthesized according to General Procedure B from methyl 4-(1-
pyrazolylmethyl)benzoate (20 g, 0.09 mmol) and afforded as a white solid after column
1
chromatography and recrystallization (11.17 g, 56%). H NMR (300 MHz, DMSO-d ) δ 11.19 (br
6
s, 1H), 9.09 (br s, 1H), 7.84 (d, J = 1.8 Hz, 1H), 7.70 and 7.24 (AA′XX′ multiplet, JAX + JAX’ = 8.1
Hz, 4H), 7.48 (d, J = 1.2 Hz), 6.29 (t, J = 2.1 Hz, 1H), 5.38 (s, 2H). ESI HRMS calc. for
+
C H N O : [M + H] , m/z 218.0924. Found: 218.0917. Purity: 99.86% (254 nm), 99.45%
11 12
3
2
(ELSD).
4
-[4-(Dimethylamino)benzyl]-N-hydroxybenzamide (7).62 (i) To
a
solution of 4-
(dimethylamino)phenylboronic acid (12.4 g, 75.0 mmol) and methyl 4-(bromomethyl)benzoate
(20 g, 90 mmol) in acetonitrile/water (500/50 mL) were added Pd(PPh ) (866 mg, 0.75 mmol)
3
4
and K CO (20.7 g, 150 mmol) under an argon atmosphere. The mixture was heated to reflux and
2
3
stirred for 6 h. Then, the mixture was diluted with H O (200 mL), and the organic products were
2
extracted with EtOAc (3 × 200 mL). The combined organic layers were washed with brine (200
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