Potent histone deacetylase inhibitors derived from 4-(aminomethyl)-N- hydroxybenzamide with high selectivity for the HDAC6 isoform

Article abstract of DOI:10.1021/jm400385r

A screen for HDAC6 inhibitors identified acyl derivatives of 4-(aminomethyl)-N-hydroxybenzamide as potent leads with unexpected selectivity over the other subtypes. We designed and synthesized constrained heterocyclic analogues such as tetrahydroisoquinolines that show further enhanced HDAC6 selectivity and inhibitory activity in cellular assays. Selectivity may be attributed to the benzylic spacer more effectively accessing the wider channel of HDAC6 compared to other HDAC subtypes as well as hydrophobic capping groups interacting with the protein surface near the rim of the active site.

Full text of DOI:10.1021/jm400385r

Article
pubs.acs.org/jmc
Potent Histone Deacetylase Inhibitors Derived from
4‑(Aminomethyl)‑N‑hydroxybenzamide with High Selectivity for the
HDAC6 Isoform
Christopher Blackburn,* Cynthia Barrett, Janice Chin, Kris Garcia, Kenneth Gigstad, Alexandra Gould,
Juan Gutierrez, Sean Harrison, Kara Hoar, Chrissie Lynch, R. Scott Rowland, Chris Tsu, John Ringeling,
and He Xu
Discovery, Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: A screen for HDAC6 inhibitors identified acyl derivatives of 4-(aminomethyl)-N-
hydroxybenzamide as potent leads with unexpected selectivity over the other subtypes. We
designed and synthesized constrained heterocyclic analogues such as tetrahydroisoquinolines
that show further enhanced HDAC6 selectivity and inhibitory activity in cellular assays.
Selectivity may be attributed to the benzylic spacer more effectively accessing the wider channel
of HDAC6 compared to other HDAC subtypes as well as hydrophobic capping groups
interacting with the protein surface near the rim of the active site.
histones in multiple myeloma cells13−15 and to potentiate
INTRODUCTION
■
proteasome inhibitor-induced apoptosis.16
The histone deacetylases (HDAC) constitute a family of
amidohydrolases that remove acetyl groups from the ε-amino
group of lysine residues and can be subdivided into four groups
Numerous HDAC inhibitors have been reported17 that are
based on Zn2+ complexing agents including thiols, acylated
ortho-phenylene diamines, and, most commonly, hydroxamic
acids (Figure 1). Pan HDAC or class I selective HDAC
termed class I (types 1, 2, 3, 8), IIa (4, 5, 7, 9), IIb (6, 10), and
IV (11), of which the class I, II, and IV enzymes are Zn2+
dependent metalloamidases.1 Unlike other HDACs, HDAC6
inhibitors such as trichostatin are most common, and some,
such as suberoylanilide hydroxamic acid (SAHA) or vorinostat,
have been shown to have therapeutic benefits.18−22 To
investigate further potential synergistic effects of HDAC6 and
proteasome inhibitors, we required inhibitors with significant
selectivity for the HDAC6 isoform, at least comparable to
tubacin and with improved physical properties. Despite their
metabolic liabilities, hydroxamic acids exhibit unique binding
energetics23 whereby the uncharged form predominates in
aqueous solution and the conjugate base is formed in the active
site of the enzyme. As a result, these chemotypes pay a much
smaller desolvation penalty on binding than species that are
already ionized in the aqueous phase. The HDAC inhibitors
shown in Figure 1 comprise a hydroxamic acid Zn2+ chelator,
spacer (in the case of vorinostat, the aliphatic chain), and
capping group (acylaniline). We report here a series of C-
arylhydroxamic acids that potently inhibit the HDAC6 isoform
with unusually high selectivity. While this work was in progress,
Kozikowski and co-workers reported a highly potent HDAC6
inhibitor NOC-7 with moderate subtype selectivity24 and a
compound with high HDAC6 selectivity, denoted tubastatin
A,25 related to the compounds described herein (Figure 1).
resides predominantly in the cytosol and has two functional
catalytic domains as well as a carboxy-terminal zinc-finger
domain that binds ubiquitinated proteins.2 Substrates for
HDAC6 include the cytoskeletal proteins α-tubulin and
cortactin and the chaperone Hsp90.3,4 Thus, HDAC6 mediates
a wide range of cellular functions ranging from microtubule-
dependent transport, membrane remodeling and chemotactic
motility and cellular adhesion to sensing ubiquitin levels, as well
as regulating chaperone levels and activity, all of which are
important in tumorigenesis, tumor growth, and metastasis.5−8
Recently, HDAC6 has been linked to autophagy, an alternative
pathway for protein degradation that compensates for
deficiencies in the activity of the ubiquitin proteasome system
arising from treatment with a proteasome inhibitor.9−12
HDAC6 binds ubiquitin, or ubiquitin-like conjugated proteins,
which would otherwise induce proteotoxic stress and transports
them as ternary complexes with dynein to the microtubule
organizing center (MTOC). These perinuclear complexes are
then sequestered into the aggresome6 and degraded by fusion
with lysosomes as part of autophagy. Hence, targeting both the
proteasome-dependent pathways and the aggresome pathway
in tumor cells is expected to cause greater accumulation of
polyubiquitinated proteins with consequent cell stress and
apoptosis. Indeed, the HDAC6 selective inhibitor tubacin is
reported to induce selective hyper-acetylation of α-tubulin over
Received: March 15, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm400385r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Examples of hydroxamic acid inhibitors of histone deacetylases.
significant HDAC inhibitory activity, presumably because the
ring nitrogen adjacent to the hydroxamate interferes with
coordination to active site zinc. On the basis of crystal
structures27,28 and homology models29 of HDAC complexes,
hydroxamate anions have been shown to interact with active
site zinc in a bidentate fashion, with two Asp and one His
residue of the active site completing the primary coordination
sphere23 at the end of an 11 Å deep channel.30 The spacer
projects along the channel with the capping groups
participating in additional binding interactions with the rim
of the active site of the protein. We reasoned that the selectivity
of compounds 1−3 might derive, in part, from the differing
“width” of the (benzylic) spacer as compared to long chain
aliphatic series, and we sought to explore this hypothesis by
configurationally restraining such analogues in fused ring
systems.
RESULTS AND DISCUSSION
■
We began our search for HDAC6 selective inhibitors by
screening compounds in our screening collection containing
functionality known to interact with the metal ion of zinc
metalloproteases. The inhibitory activities of these compounds
on the rate of deacetylation of the substrate Ac-Arg-Gly-
Lys(Ac)-AMC (AMC: 7-aminocoumarin) by HDAC6 were
measured, and selectivity was initially assessed from inhibition
of the enzymes present in HeLa nuclear extract (NucEx),
predominantly the class I HDACs 1 and 2. Numerous
hydroxamic acids were included in the screen, most of which
exhibited the pan HDAC inhibitory activity shown by
vorinostat.26 In contrast, these screens identified a derivative
of 4-aminomethylbenzoic acid (compound 1a) as a selective
inhibitor of HDAC6 in the enzymatic assays (Table 1).
The first examples of fused ring analogues were prepared
according to Scheme 2 by acylation of tetrahydroisoquinoline-
6-carboxylic acid methyl ester followed by conversion to the
hydroxamic acid as described previously to give compounds
5a−e. Isoindoline homologues were obtained starting from the
bis-benzylic bromide 6,31 which was cyclized by reaction with p-
methoxybenzylamine and deprotected by hydrogenolysis to the
secondary amine 7b, which was acylated and then transformed
to hydroxamic acids 8.
Table 1. HDAC6 Inhibitory Activity and Selectivity of
Hydroxamates Derived from 4-Aminomethylaryl Acids
a
b
compd
HDAC6 IC50 (nM)
selectivity NucEx /HDAC6
1a
1b
1c
2
19
79
115
200
84
76
19
202
123
8
3
52
4
12000
Seven-membered homologues, viz. isomeric benzazepines 9
and 10, were synthesized starting from the previously
reported32 amines 13 and 14 (Scheme 3). Thus, Schmidt
rearrangement of 6-bromotetralone using a modification of the
published procedure32 afforded a mixture of the inseparable
isomeric lactams 11 and 12, which were reduced to the
corresponding amines 13 and 14 then Boc protected.
Derivatives 15 and 16 were then separated by chromatography,
and each isomer was then subjected individually to lithiation
and quenching with methyl chloroformate to give esters 15 and
16, which were characterized and converted to hydroxamic
acids by treatment with hydroxylamine hydrochloride in the
presence of base as described previously.
a
Enzymatic data obtained with recombinant HDAC6 or HeLa nuclear
extracts using acetylated-Arg-Gly-Lys(Ac)-AMC as substrate with
subsequent release of AMC by treatment with trypsin. IC50 values are
means of two or more determinations. Standard deviation is within
20% of the IC50. Nuclear extract expresses predominantly HDACs 1
and 2.
b
Hydroxamate 1a also inhibited the cleavage of the HDAC6
substrate acetyl tubulin in cells (IC50 = 0.46 μM) with little
inhibitory activity on the hydrolysis of acetyl histone (26%
inhibition @ 10 μM). Related compounds were prepared by
parallel syntheses as shown in Scheme 1. Thus, acylation of the
methyl ester of 4-aminomethylbenzoic acid followed by
treatment with hydroxylamine hydrochloride in the presence
of base gave compounds 1a−c. We also synthesized analogues
derived from aminomethyl-heteroaryl carboxylic acids such as
thiophene 2 and thiazole 3 that proved to be HDAC6 selective
(Table 1). In contrast, isomeric thiazole 4 showed no
Analogues in the thiophene series were accessed as illustrated
in Schemes 4 and 5. Thus, 3-fluoro-4-formylpyridine was
condensed with methyl 2-mercaptoacatate33 to give 17 which
was quaternized, hydrogenated, and demethylated to give the
tetrahydro derivative 18.34 The 5-aza series derived from
B
dx.doi.org/10.1021/jm400385r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of 4-(Acylaminomethyl)-N-hydroxybenzamides
a
Reagents and conditions: (i) RCOOH, HBTU, N-methylmorpholine, DMF; (ii) NH2OH·HCl, MeOH, KOH, 70 °C 4 h.
a
Scheme 2. Synthesis of Tetrahydroisoquinolines and Isoindolines
a
Reagents and conditions: (i) TFA, CH2Cl2; (ii) RCOOH, HBTU, N-methylmorpholine, dichloroethane or RSO2Cl, DMAP, DMF; (iii) NH2OH·
HCl, MeOH, KOH, 70 °C, 4 h; (iv) NBS, CCl4, 12 h, 90%; (v) para-methoxybenzylamine, THF; (vi) Pd−C, HCl, MeOH.
a
Scheme 3. Synthesis of Tetrahydro-1H-benzazepines
a
Reagents and conditions: (i) MeSO3H, NaN3, 0−25 °C, 2 h; (ii) BH3SMe2, DME, 80 °C; (iii) Boc2O, Et3N, THF; (iv) silica gel chromatography;
(v) (a) t-BuLi, THF, −78 °C, (b) ClCOOMe, THF, 25 °C; (vi) TFA, DCM; (vii) RCOOH, HATU, DIPEA, DMF; (viii) NH2OH·HCl, KOH,
MeOH.
heterocycle 21 was prepared starting with Vilsmier formylation
of Boc-piperidone35 to give 20. Condensation of 20 with
methyl 2-mercaptoacatate36 and deprotection afforded amine
21, which was acylated and transformed to the corresponding
hydroxamic acids as described previously.
The HDAC6 enzyme and cellular inhibitory activities as well
as selectivity data for the configurationally constrained series
are listed in Table 2. Tetrahydroisoquinoline 5a exhibited
slightly reduced inhibitory activity for HDAC6 in the enzymatic
assay compared to the acyclic analogue 1a but improved
selectivity with respect to inhibition of class I enzymes present
in nuclear extract as well as improved cellular activity (Ac-Tub
assay). Further improvements to the selectivity were observed
with analogues 5b−e, in which the N-acetylpyrrole capping
group was replaced by alternative hydrophobes albeit with small
losses in HDAC6 inhibitory potency. The two examples of five-
membered homologues were found to be less active and
selective than their six-membered counterparts (compare 8a
with 5a and 8b with 5b). The enzymatic and cellular HDAC6
inhibitory activities and selectivity of the seven-membered ring
compounds 9a−b and 10a−b were lower than for series 5 and
8 and could not be compensated by further changes to the
C
dx.doi.org/10.1021/jm400385r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 4. Synthesis of Tetrahydrothieno[2,3-c]pyridines
a
Reagents and conditions: (i) HSCH2COOMe, K2CO3, DMF, 82%; (ii) MeI, MeCN, 40 °C; (iii) H2, PtO2, MeOH, 95%; (iv) (a)
MeCH(Cl)OCOCl, DIPEA, dioxane 0−70 °C, 3 h, (b) MeOH, reflux; (v) RCOOH, TBTU, DIPEA, DMF, DCM; (vi) NH2OH·HCl, KOH,
MeOH, 80 °C, 2 h.
a
Scheme 5. Synthesis of Tetrahydrothieno[3,2-c]pyridines
a
Reagents and conditions: (i) POCl3, DMF, DCM, 0 °C; (ii) HSCH2COOEt, Et3N, THF, reflux; (iii) TFA; (iv) RCOOH, HATU, Et3N, DMF; (v)
NH2OH·HCl, KOH, MeOH.
capping group (exemplified by 9e−f and 10e−f). Consistently,
the most potent HDAC6 inhibitors, in both the enzymatic and
cellular assays, were derived from tetrahydrothienopyridines
(series 19 and 22) with all the tabulated compounds showing
IC50 values <50 nM. For example, the pyrrole capped
compounds 19a and 22a are more potent than the analogous
compounds shown in Table 2, including 1a and 5a. The
improved potency was more dramatic in the case of the cellular
data where 19a and 22a show IC50 values of 5 and 18 nM,
respectively, while 5a and 1a exhibit IC50 values of 210 and 460
nM, respectively. However, compounds in series 19 and 22
were also more active inhibitors of class I HDACs as shown by
the relatively low 14- and 30-fold selectivity ratios, respectively
(compared to 500-fold for tetrahydroisoquinoline 5a).
To probe subtype selectivity in more detail, several
compounds were assessed across the full panel of individual
HDACs and their inhibitory activities are compared with
previously reported HDAC6 selective compounds in Table 3.
These data confirm the significant improvement in selectivity of
5a as compared to the acyclic analogue 1a observed in the
nuclear extract assay. Moreover, compound 5a shows a
selectivity profile considerably better than those reported in
the literature for both vorinostat26 and tubacin16,25 and, in
addition, shows negligible inhibition of matrix metalloproteases
such as MMPs 2, 9, and 4 (>100 μM). In fact, the profile is
comparable to that of tubastatin,25 with HDAC8 the only
isoform for which any significant inhibitory activity was
observed (ca. 50-fold selectivity). This general selectivity profile
was also observed for compounds 5b, 8a, 8b, and 10a (Table
3). Other properties of compounds in this series bode well for
their use as biological tools and for potential further
development, exemplified by compound 5a, which showed
high aqueous solubility (2 mM at pH 7.5)37 and high Caco-2
permeability (22 × 10−6 cm s−1 at pH 7.4 with efflux ratio =
1.5).
is shown in Figure 2, left panel, and compared with that of
HDAC7 (Figure 2, right panel) obtained from the published
crystal structure.27 While the HDACs are highly conserved,29
some differences in the loop regions have been noted38 which
affect the geometry of the active site with the channel of
HDAC6 that leads to the catalytic Zn2+ being wider and
shallower than that of HDAC 7 (as well as the other subtypes).
In consequence, HDAC6 can readily accommodate hydroxamic
acids with benzylic spacers such as compound 5b, whereas
significant steric clashes are observed when 5b is docked into
the crystal structure of HDAC7 (Figure 2). In contrast, the
flexibility of hydroxamic acids derived from long chain aliphatic
spacers facilitates access to the active site channels of all the
subtypes, accounting for the pan HDAC inhibitory activity of
vorinostat and related compounds. The more subtle SAR
trends summarized in Table 2 may be attributed to varying
effectiveness of interaction between the hydrophobic capping
group and amino acid residues in the rim of the catalytic
channel. For example the t-butyl capping group of 5b forms a
near optimal interaction with the rim of the HDAC6 channel as
shown in Figure 2, left panel).
CONCLUSION
■
A screen of our compound repository for HDAC6 inhibitory
activity and selectivity against other HDAC subtypes identified
derivatives of 4-(aminomethyl)-N-hydroxybenzamide as potent
inhibitors of HDAC6 with improved selectivity compared to
previously reported hydroxamates. Optimization of this chemo-
type in the case of fused ring analogues led to several subseries
with further improved selectivities (up to 1000-fold vs nuclear
extract and 40-fold vs HDAC8) compared to previously
reported HDAC6 selective agents. Selectivity may be attributed
to the benzylic spacer more effectively accessing the wider
channel of HDAC6 compared to other HDAC subtypes as well
as hydrophobic capping groups interacting with the protein
surface near the rim of the active site. These biological tools are
being investigated further to probe the function of HDAC6 in
proteotoxic stress and its interactions between the ubiquitin
proteasome system and aggresome pathways for protein
degradation.
Although the crystal structure of HDAC6 has not been
reported, homology models of HDAC6 and HDAC1 have been
employed to explain the selectivity of tubastatin A25 and
tubacin29 in terms of the interactions of capping groups with
the rim of the catalytic channels. We also developed a
homology model of HDAC6, the catalytic channel of which
D
dx.doi.org/10.1021/jm400385r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
with the appropriate carboxylic acid (0.15 mmol) followed by HBTU
(57 mg, 0.15 mmol) and N-methylmorpholine (33 μL, 0.3 mmol) in a
deep-well polypropylene plate. The wells were sealed and the plate
shaken at ambient temperature for 16 h then concentrated under
reduced pressure in a Genevac. The residues were partitioned between
CHCl3−THF (3:1) and aqueous 1N NaHCO3 and the organic layer
collected and combined with further extracts of the aqueous layer.
After evaporation, the residue in each well was dissolved in anhydrous
MeOH (2.5 mL) and treated with hydroxylamine hydrochloride (40
mg, 0.56 mmol) and powdered KOH (80 mg, 1.4 mmol) and heated
at 70 °C for 4 h. The reaction mixtures were concentrated under
reduced pressure and the residues treated with DMSO (1.4 mL),
filtered, and the solution of crude product subjected to preparative
HPLC on a C-18 column eluting with 10−60% MeCN−H2O to give
1a (7 mg, 19%). 1H NMR (300 MHz, CD3OD): δ 7.70 (m, 2H), 7.41
(m, 2H), 6.82 (m, 1H), 6.79 (dd, J = 4.0, 1.7 Hz, 1H), 6.05 (dd, J =
3.9, 2.6 Hz, 1H), 4.52 (s, 2H), 3.88 (s, 3H). 13C NMR (100 MHz,
CD3OD, ppm) δ 168.04, 164.48, 144.96, 132.18, 129.56, 128.40,
128.31, 126.53, 114.19, 108.33, 43.32, 36.88. HRMS (ESI+): calculated
for C14H15N3O3 [M + H] 274.1186; found 274.1182.
Table 2. HDAC6 Inhibitory Activity and Selectivity of Fused
Ring Hydroxamates
b
ring
system
capping
group
HDAC6 IC50 selectivity NucEx / Ac-Tub IC50
a
c
(nM)
HDAC6
(nM)
1
5
a
19
115
460
a
36
52
50
60
54
500
1500
730
210
430
b
c
540
d
e
610
1300
340
650
8
9
a
40
89
690
340
340
560
Compounds 1b−c and 2−3 were prepared similarly from the
respective methyl esters and compound 4 from the corresponding
ethyl ester.
b
a
b
e
f
130
120
120
59
480
340
340
340
1510
580
1b (9 mg, 26%). 1H NMR (400 MHz, CD3OD ppm): δ 8.21 (br, s,
1H), 7.71 (d, J = 9.0 Hz, 2H), 7.36 (d, J = 9.0 Hz, 2H), 4.42 (m, 2H),
1.25 (s, 9H). 13C NMR (100 MHz, CD3OD) δ 181.48, 168.04, 144.81,
132.26, 128.31, 128.27, 43.72, 39.72, 27.88. HRMS (ESI+): calculated
for C13H18N2O3 [M + H] 251.1390; found 251.1385.
2000
2600
1c (6 mg, 16%). 1H NMR (400 MHz, DMSO-d6, ppm): δ, 8.24 (s,
1H), 7.67 (d, J = 9 Hz, 2H), 7.27 (d, J = 9 Hz, 2H), 4.08 (s, 2H), 2.16
(m, 1H), 1.69−1.57 (m, 4H), 1.36−1.15 (m, 6H). 13C NMR (100
MHz, CD3OD, ppm) δ 175.27, 165.59, 143.14, 129.14, 127.05, 126.77,
44.89, 41.47, 29,21, 25.42, 25.24. HRMS (ESI+): calculated for
C15H20N2O3 [M + H] 277.1547; found 277.1541.
10
a
b
e
f
260
490
340
120
>380
>200
>200
60
1480
3680
2900
3090
19
22
a
b
c
28
35
20
14
71
50
5.0
158
1
2 (20 mg, 14%). H NMR (400 MHz, DMSO-d6, ppm): δ, 10.72
(br, 1H), 8.28 (m, 1H), 7.81 (s, 1H), 7.04 (s, 1H), 6.57 (m, 1H), 6.53
(m, 1H), 6.41 (dd, J = 3.8, 1.6 Hz, 1H), 5.62 (s, 1H), 4.13 (m, 2H),
3.43 (s, 3H). 13C NMR (100 MHz, CD3OD, ppm) δ 164.25, 149.82,
129.67, 129.41, 126.77, 126.35, 114.38, 108.37, 101.42, 96.04, 38.82,
36.88. HRMS (ESI+): calculated for C12H13N3O3S [M + H] 280.0750;
found 280.0747.
50
a
b
c
24
47
36
30
64
47
18
177
162
3 (7 mg, 9%). 1H NMR (400 MHz, CD3OD ppm): δ 8.11 (s, 1H),
6.84 (m, 2H), 6.08 (dd, J = 3.8, 1.6 Hz, 1H), 4.74 (s, 2H), 3.89 (s,
3H). 13C NMR (100 MHz, CD3OD, ppm) δ 176.58, 164.43, 161.10,
143.98, 132.93, 130.04, 125.98, 114.79, 108.50, 41.95, 36.91. HRMS
(ESI+): calculated for C11H12N4O3S [M + H] 281.0703; found
281.0699.
a
Enzymatic data obtained with recombinant HDAC6 or HeLa nuclear
extracts using acetylated-Arg-Gly-Lys(Ac)-AMC as substrate with
subsequent release of AMC by treatment with trypsin. IC50 values are
means of two or more determinations. Standard deviation is within
b
20% of the IC50. Nuclear extract (NucEx) expresses predominantly
c
HDACs 1 and 2. Cellular data: Ac-tubulin accumulation was
4 (8 mg, 20%). 1H NMR (400 MHz, CD3OD, ppm) δ 7.58 (s, 1H),
6.90 − 6.69 (m, 2H), 6.06 (dd, J = 8.9, 4.4 Hz, 1H), 4.61 (s, 2H), 3.88
(s, 3H). 13C NMR (100 MHz, CD3OD, ppm) δ 164.50, 162.77,
159.49, 157.44, 129.63, 126.46, 121.14, 114.34, 108.34, 40.12, 36.86.
HRMS (ESI+): calculated for C11H12N4O3S [M + H] 281.0703; found
281.0698.
determined by immunofluorescence with an anti-Ac-tubulin antibody
in cytosol. Ac-Lys accumulation in nuclear histones was measured by
immunofluorescence in HeLa cells with all compounds showing <20%
inh @ 20 μM.
EXPERIMENTAL SECTION
General Synthesis of N-Hydroxy-2-(substituted-phenylsulfonyl)-
1,2,3,4-tetrahydroisoquinoline-6-carboxamide Analogues (5a−c,
5e). A suspension of methyl 1,2,3,4-tetrahydroisoquinoline-6-carbox-
ylate hydrochloride (47 mg, 0.2 mmol) and the appropriate carboxylic
acid (0.23 mmol) in anhydrous 1,2-dichloroethane (1.5 mL) was
treated with HBTU (109 mg, 0.28 mmol) and N-methylmorpholine
(34 μL, 0.3 mmol). The reaction mixtures in a polypropylene deep-
well plate were sealed and shaken at ambient temperature for 16 h
then concentrated under reduced pressure in a Genevac. The residues
were partitioned between CHCl3−THF (3:1) and aqueous 1N
NaHCO3 and the organic layer collected and combined with further
extracts of the aqueous layer. After evaporation, the residue in each
well was dissolved in anhydrous MeOH (2.5 mL) and the presence of
the desired methyl ester confirmed by LC-MS analysis before
treatment with hydroxylamine hydrochloride (30 mg, 0.44 mmol)
and powdered KOH (50 mg, 0.87 mmol) and heating at 70 °C for 4 h.
The reaction mixtures were concentrated under reduced pressure and
■
General. NMR spectra were recorded on a Bruker 300 MHz
Avance1 or 400 MHz Avance2 using residual solvent peaks as the
reference. Compound purity was determined by integration of the
diode array UV trace of LC-MS chromatograms acquired on an
Agilent 1100 LC interfaced to a micromass Waters Micromass Zspray
mass detector (ZMD). Analyses were conducted using a water−
MeCN gradient containing 0.1% formic acid (FA); all compounds
were determined to be >95% pure. High-resolution mass spectra were
recorded using a QSTAR XL quadrupole time-of-flight mass
spectrometer (Applied Biosystems) coupled to an 1100 series HPLC
system (Agilent Technologies). For each analysis, approximately five
scans were summed and the centroid m/z value of the protonated
monoisotopic molecular ion [M + H]+ was recorded.
Synthetic Procedures. N-(4-(Hydroxycarbamoyl)benzyl)-car-
boxamides (1a−c, 2, 3, 4). A solution of methyl 4-amino-
methylbenzoate (40 mg, 0.14 mmol) in DMF (3 mL) was treated
E
dx.doi.org/10.1021/jm400385r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 3. Comparison of HDAC Isoform Inhibitory Activity of Benzylic Spacer Derived Inhibitors with Reference Compounds
a
IC50 (μM) isoform
b
compd
1
2
3
4
5
6
7
8
10
11
Ac-Tub IC50 (μM)
c
vorinostat
0.119
1.40
0.164
6.27
0.106
1.27
NR
17.3
>30
15.0
>50
>50
>50
>50
>50
NR
3.35
>30
3.30
>50
>50
>50
>50
>50
0.090
0.004
0.015
0.019
0.036
0.052
0.040
0.089
0.260
NR
9.70
>30
4.60
>50
>50
>50
>50
>50
1.50
1.27
0.854
0.69
2.10
2.10
1.30
1.30
0.890
0.072
3.71
NR
3.79
>30
ND
>50
>50
>50
>50
>50
NR
2.9
d,e
tubacin
e
tubastatin
16.4
5.80
45.0
>50
>30
16.0
>30
4.50
46.0
>50
>30
3.70
>50
ca. 2.5
0.460
0.21
0.43
0.34
0.56
1.48
1a
5a
>50
>50
>50
>50
>50
5b
8a
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
8b
10a
a
Enzymatic data obtained with recombinant HDAC6 or HeLa nuclear extracts using acetylated-Arg-Gly-Lys(Ac)-AMC as substrate with subsequent
b
release of AMC by treatment with trypsin. Cellular data: Ac-tubulin accumulation was determined by immunofluorescence with an anti-Ac-tubulin
antibody in cytosol. Ac-Lys accumulation in nuclear histones was measured by immunofluorescence in HeLa cells. Data from refs 24 and 26. Data
from ref 16. Data from ref 25; NR, not reported. ND, not determined.
c
d
e
Figure 2. Compound 5b docked into the active sites of HDAC6 from a homology model (left) and HDAC7 (right) from a crystal structure (PDB:
2PQO, 2.9 Å).27 For the homology model, HDAC7 (PDB: 2PQO) was used as the primary template and additional crystal structures HDAC2
(PDB: 3MAX 2.0 Å), HDAC8 (PDB: 1T69 2.9 Å), and HDAH (PDB: 1ZZ1 1.57 Å) were used to improve loop modeling. Electrostatic color
coding is red positive, blue negative, and gray neutral. See Supporting Information for further details.
the residues treated with DMSO (1.3 mL), filtered, and the solution of
crude product subjected to preparative HPLC on a C-18 column
eluting with 10−60% MeCN−H2O to give:
127.91, 127.63, 126.20, 126.15, 44.38, 43.22, 43.07, 41.26, 32.49,
32.37, 30.62, 29.30, 29.20, 27.57, 27.70 (doubling of some resonances
due to rotamers). HRMS (ESI+): calculated for C18H24N2O3 [M + H]
317.1860; found 317.1852.
1
5a (20 mg, 32%). H NMR (400 MHz, CD3OD, ppm) δ 7.57−
7.54 (m, 2H), 7.24 (br, 1H), 6.86 (br, 1H), 6.50 (dd, J = 3.8, 1.6 Hz,
1H), 6.12 (dd, J = 3.7, 2.7 Hz, 1H), 4.91 (s, 2H), 3.97 (t, J = 6.1 Hz,
2H), 3.74 (t, 3H), 3.00 (t, J = 6.1 Hz, 2H). 13C NMR (100 MHz,
CD3OD, ppm) δ 167.94, 165.58, 138.40, 136.40, 132.05, 128.68,
128.20, 127.75, 126.19, 126.08, 114.31, 108.27, 35.76, 29.96. HRMS
(ESI+): calculated for C16H17N3O3 [M + H] 300.1348; found
300.1340.
N-Hydroxy-2-((4-(trifluoromethyl)phenyl)sulfonyl)-1,2,3,4-tetra-
hydroisoquinoline-6-carboxamide (5d). A solution of methyl 1,2,3,4-
tetrahydroisoquinoline-6-carboxylate hydrochloride (50 mg, 0.22
mmol) and N,N-dimethylaminopyridine (110 mg, 0.9 mmol) in
DMF (3.2 mL) was treated with the appropriate sulfonyl chloride
(0.22 mmol) and stirred at ambient temperature for 16 h. The solvent
was removed under reduced pressure and the residue partitioned
between DCE and saturated aqueous NaHCO3 and the organic layer
collected and combined with further extracts of the aqueous layer. The
organic layer was dried (MgSO4) and the presence of the desired
methyl ester confirmed by LC-MS analysis [ES+ (FA) 400] and treated
with hydroxylamine hydrochloride (80 mg, 1.00 mmol) and powdered
KOH (200 mg, 4.0 mmol) and heated at 70 °C for 4 h. The solvent
was removed under reduced pressure and the residue was treated with
DMSO (1.3 mL), filtered, and the solution of crude product subjected
to preparative HPLC on a C-18 column eluting with 10−60%
MeCN−H2O to give 5d (24 mg, 27%). 1H NMR (400 MHz, CD3OD,
ppm) δ 8.08 (m, 2H), 7.88 (m, 2H), 7.50 (m, 2H), 7.20 (m, 1H), 4.40
(s, 2H), 3.44 (m, 1H), 2.95 (m, 2H). 13C NMR (100 MHz, CD3OD,
ppm) δ 167.74, 141.98, 136.82, 135.65, 135.33, 134.99, 132.18, 129.63,
128.75, 127.86, 127.58, 126.07, 123.53, 48.61, 40.44, 29.60. HRMS
calculated for C17H15F3N2O4S [M + H] 401.0777; found 401.0771.
Methyl 2,3-Dihydro-1H-isoindole-5-carboxylate Hydrochloride
(7b). A mixture of methyl 3,4-dimethylbenzoate (1.33g, 8.1 mmol),
N-bromosuccinimide (3.17g, 17.8 mmol), and benzoyl peroxide
(0.14g, 0.58 mmol) in CCl4 (5 mL) was heated under reflux for 1.5
h. After cooling to ambient temperature, the suspension was filtered
1
5b (8 mg, 12%). H NMR (400 MHz, CD3OD, ppm) δ 7.57 (m,
2H), 7.27 (d, J = 5.5 Hz, 1H), 4.82 (s, 2H), 3.91 (t, J = 6.1 Hz, 2H),
2.94 (t, J = 6.1 Hz, 2H), 1.32 (s, 9H). 13C NMR (100 MHz, CD3OD,
ppm) δ 178.96, 168.04, 138.63, 136.27, 132.03, 128.62, 127.73, 126.10,
44.69, 39.96, 29.84, 28.58. HRMS (ESI+): calculated for C17H19N3O3
[M + H] 314.1499; found 314.1498.
1
5c (14 mg, 20%). H NMR (400 MHz, CD3OD, ppm) δ 7.58 (m,
2H), 7.32−7.27 (m, 2H), 4.82 and 4.73 (s, 2H combined, rotamers),
3.82 (m, 2H), 2.99 and 2.89 (s, 2H combined, rotamers), 2.76 (m,
1H), 1.84−1.72 (m, 4H), 1.51−1.23 (m, 6H). 13C NMR (100 MHz,
CD3OD, ppm) δ 177.68, 177.43, 168.00, 138.60, 138.40,
136.73,136.25, 132.20, 131.92, 128.57, 128.41, 127.92, 127.66,
126.15, 45.47, 44.19, 42.08, 41.90, 42.13, 30.70, 30.57, 30.44, 29.33,
27.04, 26.75, 26.70 (doubling of some resonances due to rotamers).
HRMS (ESI+): calculated for C15H20N2O3 [M + H] 277.1547; found
277.1548.
5e (10 mg, 10%). 1H NMR (400 MHz, CD3OD) δ 7.59 (t, J = 8.0
Hz, 2 H), 7.32 (m, 1H), 4.67 (s, 2H), 3.87 (m, 2H), 3.01 (m, 1H),
2.91 (m, 2H), 1.67 (m, 12H). 13C NMR (100 MHz, CD3OD) 178.74,
167.94, 138.67, 138.38, 136.75, 136.28, 132.24, 131.94, 128.58, 128.39,
F
dx.doi.org/10.1021/jm400385r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
and evaporated to give the crude bis-bromomethyl derivative which
was redissolved in THF (30 mL) and treated with Et3N (2.26 mL, 16.3
mmol), followed by 4-methoxybenzylamine (1.05 mL, 8.13 mmol) in
THF (20 mL), and stirred at ambient temperature for 16 h. The
reaction mixture was filtered, the solvent evaporated, and the crude
product purified by flash chromatography on silica gel (hexane−
EtOAc gradient) to give 7a as a pale-yellow oil (1.19g, 49%). LC-MS
4.45 (br, 1H), 4.40 (br, 1H), 3.90 (s, 3H), 3.68 (br, 2H), 3.00 (br,
2H), 1.78 (m, 2H), 1.37 (s, 9H).
3-tert-Butyl 7-Methyl-4,5-dihydro-1H-benzo[d]azepine-3,7(2H)-
1
dicarboxylate (15). 15 was prepared similarly. H NMR (400 MHz,
CDCl3, ppm) δ 7.82−7.79 (m, 2H), 7.24 (d, J = 7.6 Hz, 1H), 3.90 (s,
3H), 3.55 (br, 4H), 2.95 (br, 4H), 1.48 (s, 9H).
N-Hydroxy-2-pivaloyl-2,3,4,5-tetrahydro-1H-benzo[c]azepine-7-
carboxamide (10b). A solution of 16 (0.44g, 0.145 mmol) in DCM (5
mL) was cooled in an ice bath and treated with 4 M HCl in dioxane
(1.5 mL). The reaction mixture was allowed to warm to ambient
temperature, stirred for 1 h, and the solvent removed to give the
corresponding hydrochloride salt. LC-MS (FA) ES+ 206. A solution of
trimethylacetic acid (15.5 mg, 0.152 mmol), HATU (58 mg, 0.52
mmol), and Et3N (60 μL, 0.43 mmol) in DCM (2 mL) and DMF (0.2
mL) was treated with a solution of the amine hydrochloride (35 mg,
0.145 mmol) in DMF (0.5 mL). The reaction mixture was stirred at
ambient temperature for 6 h and partitioned between DCE and
saturated aqueous NaHCO3 solution. The aqueous phase was further
extracted with DCE and the combined organic layers dried (MgSO4)
and evaporated. The crude ester was treated with MeOH (2.0 mL),
KOH (56 mg, 1.0 mmol), and hydroxylamine hydrochloride (30 mg,
0.43 mmol) and heated at 80 °C for 1 h. The reaction mixture was
then neutralized by addition of acetic acid, evaporated, and the crude
product dissolved in DMSO and subjected to reverse phase
chromatography on a C-18 column eluting with 20−45% MeCN in
water to give 10b (19 mg, 45%) as a white solid. 1H NMR (400 MHz,
CD3OD, ppm) δ 7.55 (m, 1H), 7.49 (dd, J = 7.8, 1.8 Hz, 1H), 7.39 (d,
J = 7.8, 1h), 4.57 (br, 2H), 4.01 (br, 2H), 3.08 (br, 2H), 1.86 (br, 2H),
1.21 (s, 9H). 13C NMR (100 MHz, CD3OD, ppm) δ 178.62, 168.11,
143.90, 143.21, 132.56, 131.25, 128.90, 125.75, 53.60, 53.45, 39.95,
35.49, 30.64, 28.73. HRMS calculated for C16H22N2O3 [M + H]
291.1709; found 291.1706.
1
ES+ 298. H NMR (400 MHz, CDCl3, ppm) δ 7.89 (dd, J = 7.8, 1.5
Hz, 1H), 7.84 (s, 1H), 7.74 (d, J = 8.7 Hz, 2H), 7.31 (d, J = 8.7 Hz,
2H), 7.22 (d, J = 7.2 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 3.93 (s, 3H),
3.89 (s, 4H), 3.85 (s, 2H), 3.82 (s, 3H).
A solution of 7a (0.82 g, 2.76 mmol) in MeOH (25 mL) was
treated with 20% Pd on carbon (0.19 g) and conc HCl (0.41 mL) and
stirred under an atmosphere of hydrogen gas for 48 h. The reaction
mixture was filtered and the solvent evaporated to give 7b (0.53 g,
91%). LC-MS ESI+ 179. 1H NMR (400 MHz, DMSO-d6, ppm) δ 9.71
(br, s, 2H), 8.01 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 7.7 Hz,
2H), 4.56 (d, J = 6.0 Hz, 4H), 3.87 (s, 3H).
N-Hydroxy-2-(1-methyl-1H-pyrrole-2-carbonyl)isoindoline-5-car-
boxamide (8a). A suspension of 7b (40 mg, 0.19 mmol) and N-
methylpyrrole-2-carboxylic acid (26 mg, 0.21 mmol) in DCM (2.5
mL) was treated with HATU (78 mg, 0.21 mmol) and N-
methylmorpholine (0.1 mL, 0.91 mmol) and stirred at ambient
temperature for 16 h. The resulting solution was washed with
saturated aqueous NaHCO3 and the organic layer dried (MgSO4) and
evaporated. The residue was redissolved in MeOH (3 mL) and treated
with hydroxylamine hydrochloride (39 mg, 0.56 mmol) and powdered
KOH (100 mg, 1.8 mmol) and heated at 70 °C for 2 h. The solvent
was removed under reduced pressure, and the residue was suspended
in DMSO, filtered, and subjected to preparative HPLC on a C-18
column eluting with 10−60% MeCN−H2O to give 8a (27 mg, 50%).
1H NMR (400 MHz, CD3OD, ppm) 7.72 (m, 2H), 7.45 (br, 1H), 6.88
The following compounds were prepared and purified similarly:
(t, J = 2.1 Hz, 1H), 6.81 (dd, J = 3.8, 1.6 Hz, 1H), 6.17 (dd, J = 3.8, 2.6
Hz, 1H), 5.13, (br, 2H), 4.99 (br, 2H), 3.85 (s, 3H). 13C NMR (100
MHz, CD3OD, ppm) δ 167.88, 164.57, 133.32, 128.91, 127.78, 126.19,
124.09, 122.66, 115.73, 108.28, 55.83, 53.56, 36.73. HRMS (ESI+):
calculated for C15H15N3O3 [M + H] 286.1192; found 286.1183.
1
10a (42%). H NMR (400 MHz, CD3OD, ppm) δ 8.08 (s, 1H),
7.57 (s, 1H), 7.48 (m, 2H), 6.75 (m, 1H), 6.28 (m, 1H), 6.06 (m, 1H),
4.78 (s, 2H), 4.00 (br, 2H), 3.11 (br, 2H), 1.88 (br, 2H). 13C NMR
(100 MHz, CD3OD, ppm) δ 167.95, 143.87, 143.07, 132.88, 130.87,
129.28, 126.92, 125.90, 112.63, 108.13, 35.63, 35.21, 30.65. HRMS
calculated for C17H19N3O3 [M + H] 314.1499; found 314.1497.
1
Compound 8b was prepared similarly (13 mg, 27%). H NMR (400
MHz, CD3OD) δ 7.60 (s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.32 (d, J =
7.7 Hz, 1H), 5.05 (br, 2H), 4.74 (br, 2H), 1.25 (s, 9H). 13C NMR
(100 MHz, CD3OD, ppm) δ 179.16, 167.94, 133.27, 127.71, 123.95,
122.63, 55.78, 54.35, 40.28, 27.81. HRMS (ESI+): calculated for
C14H18N2O3 [M + H] 263.1390; found 263.1385.
Synthesis of Carboxymethylbenzazepines 15 and 16. The
Schmidt rearrangement of 6-bromo-2-tetralone to give the mixture
of lactams 11 and 12 was conducted using a modification of the
method of Schultz and co-workers32 as follows:
1
10e (21%). H NMR (400 MHz, DMSO-d6, ppm) δ 11.13 (d, J =
13.7 Hz, 1H), 8.98 (s, 1H), 7.69−7.15 (m, 3H), 4.65 (s, 1H), 4.49 (s,
1H), 3.76 (m, 2H), 2.98 (m, 2H), 2.88−2.59 (m, 1H), 1.84−1.27 (m,
14H). 13C NMR (100 MHz, DMSO-d6) 175.4, 164.0, 141.8, 131.6,
129.5, 128.3, 127.8, 124.4, 51.2, 49.0, 34.3, 31.1, 29.2, 27.7, 27.0, 26.0.
HRMS (ESI+): calculated for C19H26N2O3 [M + H] 331.2016; found
331.2012.
1
10f (27%). H NMR (400 MHz, CD3OD, ppm) δ 7.53 (m, 12H),
4.64 (s, 1H), 4.06 (m, 1H), 3.80 (s, 2H), 3.13 (m, 3H), 1.98 (s, 1H),
1.81 (m, 2H). 13C NMR (100 MHz, CD3OD, ppm) δ 172.73, 167.72,
143.96, 143.89, 142.65, 141.35, 136.01, 132.94, 131.25, 130.05, 129.23,
128.98, 128.61, 128.24, 128.07, 125.98, 62.69, 54.70, 51.62, 35.83,
35.49, 30.50, 28.28, 20.05 (multiple signals due to rotamers). HRMS
(ESI+): calculated for C24H22N2O3 [M + H] 387.1703; found
387.1701.
A solution of 6-bromo-2-tetralone (10 g, 0.044 mol) in MeSO3H
(50 mL) was cooled in an ice bath and treated with sodium azide
(3.61g, 0.055 mol) with stirring. The reaction mixture was allowed to
warm to ambient temperature and stirred for a further 2 h then poured
into a cold solution of 1 M aqueous KOH (800 mL) and extracted
thoroughly with ethyl acetate. The extracts were washed with brine
and evaporated to give a mixture of 11 and 12, which was used without
further purification. Reduction to amines 13 and 14, Boc protection,
and separation was also conducted as described.32
2-tert-Butyl 7-Methyl 4,5-Dihydro-1H-benzo[c]azepine-2,7(3)-di-
carboxylate (16). A solution of the N-Boc-derivative of bromo
compound 14 (0.37g, 1.13 mmol) in anhydrous THF (4 mL) was
added dropwise to a solution of tert-butyllithium (0.92 mL of 1.6 M
solution in pentane) in anhydrous THF (15 mL) at −78 °C. After 10
min, methylchloroformate (1.75 mL, 22.7 mmol) was added and the
reaction mixture allowed to warm to ambient temperature and stirred
for 30 min. The solvent was evaporated under reduced pressure and
the residue partitioned between ethyl acetate (50 mL) and water (10
mL). The organic layer was combined with further extracts of the
aqueous phase, washed with brine, dried (MgSO4), and evaporated to
give an oil that was purified by chromatography on silica gel eluting
with 0−10% EtOAc in hexane to give 16 (0.14g, 41%). 1H NMR (400
MHz, CDCl3, ppm): δ 7.83−7.80 (m, 2H), 7.24 (d, J = 7.6 Hz, 1H),
9a (20%), White Solid. 1H NMR (400 MHz, CD3OD, ppm) δ 8.06
(s, 1H), 7.52 (m, 2H), 7.25 (d, J = 7.8 Hz, 1H), 6.78 (m, 1H), 6.36
(m, 1H), 6.07 (m, 1H), 3.84 (m, 4H), 3.56 (s, 3H), 3.06 (m, 4H). 13C
NMR (100 MHz, CD3OD, ppm) δ 168.01, 166.18, 145.61, 142.06,
131.94, 130.96, 129.22, 127.66, 126.60, 126.52, 113.52, 108.27, 37.57,
35.41. HRMS calculated for C17H19N3O3 [M + H] 314.1499; found
314.1498.
9b (20%). 1H NMR (400 MHz, CD3OD, ppm) δ 8.07 (s, 1H), 7.51
(m, 2H), 7.23 (d, J = 7.2 Hz, 1H), 3.75 (m, 4H), 3.02 (m, 4H), 1.25
(s, 9H). 13C NMR (100 MHz, CD3OD, ppm) δ 179.23, 168.07,
145.67, 142.04, 131.82, 130.94, 129.24, 126.43, 62.87, 40.17, 37.84,
37.73, 29.04. HRMS calculated for C16H22N2O3 [M + H] 291.1709;
found 291.1712.
1
9e (17%). H NMR (400 MHz, CD3OD, ppm) δ 7.53 (m, 2H),
7.25 (t, J = 8.3 Hz, 1H), 3.93 (m, 4H), 3.11- 2.91 (m, 4H), 2.84 (m,
1H), 1.87−1.35 (m, 12H). 13C NMR (100 MHz, CD3OD) 179.1,
G
dx.doi.org/10.1021/jm400385r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
168.0, 145.5, 141.9, 131.1, 129.4, 126.5, 45.5, 42.8, 38.3, 37.3, 32.7,
29.3, 27.8. HRMS (ESI+): calculated for C19H26N2O3 [M + H]
331.2016; found 331.2015.
temperature, and the solvent evaporated under reduced pressure. The
crude product was dissolved in DMSO and subjected to preparative
reverse phase HPLC on a C-18 column eluting with 15−45% MeCN
1
1
9f (11%). H NMR (400 MHz, CD3OD, ppm) δ 7.84−7.16 (m,
in H2O to give 19b (3.51 mg, 12%) as a white solid. H NMR (400
12H), 3.91 (m, 2H), 3.63 (m, 2H), 3.16 (m, 2H), 3.00 (m, 2H). 13C
NMR (100 MHz, CD3OD) δ 173.40, 167.99, 143.99, 141.39, 136.19,
131.06, 130.05, 129.34, 128.99, 128.42, 128.28, 128.09, 126.59, 51.14,
45.62, 37.96, 37.12. HRMS (ESI+): calculated for C24H22N2O3 [M +
H] 387.1703; found 387.1705.
MHz, DMSO) δ 7.35 (s, 1H), 4.74 (s, 2H), 3.80 (t, J = 5.7 Hz, 2H),
2.66 (t, J = 5.3 Hz, 2H), 1.22 (s, 9H). 13C NMR (100 MHz, DMSO-
d6, ppm) δ 175.50, 159.55, 136.50, 134.58, 134.52, 127.22, 44.00,
42.58, 38.29, 27.95, 25.22. HRMS (ESI+): calculated for C13H18N2O3S
[M + H] 283.1111; found 283.1107.
Methyl Thieno[2,3-c]pyridine-2-carboxylate (17). A solution of 3-
fluoro-4-pyridinecarboxaldehyde (0.80 mL, 8.0 mmol) in DMF (15
mL) was cooled to 0 °C and treated with potassium carbonate (1.22 g,
8.83 mmol) and methyl 2-mercaptoacetate (0.75 mL, 8.43 mmol).
After 30 min at 0 °C, the reaction mixture was allowed to warm to
room temperature and stirred for 48 h. Water was added, and the
precipitate that formed on cooling to 0 °C was collected and washed
with cold water, affording 17 (1.55 g, 82%) as a white solid. LCMS:
(FA) ES+ 194. 1H NMR (DMSO-d6, 300 MHz, ppm) δ 9.37 (s, 1 H),
8.56 (d, J = 5.4 Hz, 1 H), 8.26 (d, J = 0.6 Hz, 1 H), 7.97 (dd, J = 5.4
Hz, 1.2 Hz, 1 H), 3.92 (s, 3 H).
Methyl 6-Methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-2-car-
boxylate (18). A solution of methyl thieno[2,3-c]pyridine-2-
carboxylate 17 (370 mg, 1.9 mmol) and methyl iodide (0.34 mL,
5.5 mmol) in acetonitrile (7.0 mL) was stirred at 40 °C for 6 h.
Volatiles were then removed under reduced pressure and the residue
treated with methanol (10 mL) and platinum dioxide (100 mg, 0.4
mmol) and stirred under an atmosphere of hydrogen gas (balloon
pressure) for 4 h. The catalyst and solvent were then removed and the
residue partitioned between EtOAc and saturated aqueous NaHCO3
and the aqueous layer extracted with EtOAc three additional times.
The combined organic phases were washed with saturated aqueous
NaHCO3, water, and brine, dried over anhydrous MgSO4, and
concentrated to give methyl 6-methyl-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-2-carboxylate (0.400 g, 95%) as an off-white solid. 1H NMR
(CDCl3, 300 MHz, ppm) δ 7.50 (s, 1 H), 3.85 (s, 3 H), 3.64 (m, 2 H),
2.73 (m, 4 H), 2.48 (s, 3 H). LCMS: (FA) ES+ 212.
N-Hydroxy-6-(1-methyl-1H-pyrrole-2-carbonyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-2-carboxamide (19a). 19a was
prepared similarly. Yield: 20%. 1H NMR (DMSO-d6, 400 MHz,
ppm) δ 7.37 (s, 1 H), 6.94 (m, 1 H), 6.44 (m, 1H), 6.06 (m, 1H), 4.83
(s, 2 H), 3.86 (t, J = 5.9 Hz, 2 H), 3.68 (s, 3H), 2.51 (t, J = 5.9 Hz, 2
H). 13C NMR (100 MHz, DMSO-d6, ppm) δ 162.43, 159.69, 136.07,
134.70, 127.26, 126.80, 124.67, 112.23, 106.70, 99.50, 45.62, 35.04,
25.35. HRMS (ESI+): calculated for C14H15N3O3S [M + H] 306.0912;
found 306.0901.
6-(Cyclohexylcarbonyl)-N-hydroxy-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-2-carboxamide (19c). 19c was prepared similarly and
purified by preparative HPLC on a C-18 column eluting with 20−50%
1
MeCN−H2O. Yield: 36%. H NMR (CD3OD, 300 MHz, ppm): δ
7.30 (s, 1 H), 4.75 (s, 2 H), 3.83 (t, J = 6.0 Hz, 2 H), 2.65 (m, 3 H),
1.74 (m, 6 H), 1.41 (m, 4 H). 13C NMR (100 MHz, CD3OD, ppm) δ
177.44, 163.15, 138.47, 138.07, 136.89, 135.87, 129.35, 129.21, 46.00,
44.14, 42.15, 41.86, 41.06, 30.73, 30.48, 27.48, 27.02, 26.72, 26.63,
26.07 (multiple signals due to rotamers). HRMS (ESI+): calculated for
C15H20N2O3S [M + H] 309.1267; found 309.1265.
Ethyl 4,5,6,7-Tetrahydrothieno[3,2-c]pyridine-2-carboxylate (21).
N-Boc protected piperidone was chloroformylated using a Vilsmer−
Hack reaction to afford compound 20, which was treated with
ethylmercaptoacetate in the presence of excess triethylamine to give
21.22,23 Removal of the Boc-protecting group under acidic conditions
yields was conducted as follows:
A solution of 5-tert-butyl 2-ethyl-6,7-dihydrothieno[3,2-c]pyridine-
2,5(4H)-dicarboxylate (1.00 g, 3.21 mmol) in DCM (15 mL) was
treated with trifluoroacetic acid (1.48 mL, 19.3 mmol) dropwise at 0
°C. The solution was allowed to warm to ambient temperature then
stirred for 1 h. The reaction mixture was partitioned between saturated
aqueous NaHCO3 solution (10 mL) and DCM (10 mL) and the
aqueous phase extracted further with DCM. The combined organic
layers were washed with brine, dried over anhydrous MgSO4, and
concentrated to give ethyl 4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-
carboxylate 21 (0.547 g, 81%) as an oil. LCMS: (FA) ES+ 212.
N-Hydroxy-5-pivaloyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-
carboxamide (22b). A solution of HATU (104.0 mg, 0.274 mmol)
and trimethylacetic acid (28.0 mg, 0.274 mmol) in methylene chloride
(2.0 mL) was treated with triethylamine (104 μL, 0.203 mmol) and
stirred at room temperature for 30 min. A solution of ethyl 4,5,6,7-
tetrahydrothieno[3,2-c]pyridine-2-carboxylate (21) (20.0 mg, 0.101
mmol) in DMF (0.5 mL) was next added and the solution stirred at
ambient temperature for 16 h. The reaction mixture was diluted with
DCM and washed with saturated aqueous NaHCO3. The aqueous
layer was extracted with further with DCM, and the combined organic
phase was washed with water, brine, dried over anhydrous MgSO4, and
concentrated to afford an oil which was treated with methanol (2.0
mL), hydroxylamine hydrochloride (52.1 mg, 0.75 mmol), and
powdered potassium hydroxide (84.2 mg, 1.5 mmol). The reaction
mixture was heated at 80 °C for 3 h and the solvent evaporated under
reduced pressure. The residue was dissolved in DMSO (1.4 mL) and
purified by preparative reverse phase HPLC on a C-18 column eluting
Methyl 6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-2-carboxy-
late (9.84 g, 46.6 mmol) in 1,4-dioxane (400 mL) was treated with
N,N-diisopropylethylamine (81.1 mL, 0.47 mol). The solution was
cooled to 0 °C and treated dropwise with α-chloroethyl chloroformate
(50.2 mL, 0.47 mol). The reaction mixture was allowed to warm to
ambient temperature and stirred for 3 h. The solvent was removed
under reduced pressure, and the residue obtained was dissolved in
methanol (300 mL) and heated to reflux for 3 h. Removal of the
solvent under reduced pressure afforded a solid which was partitioned
between dichloromethane and saturated aqueous NaHCO3. The
aqueous layer was further extracted with dichloromethane three times.
The combined organic phases were washed with brine, dried over
anhydrous MgSO4, and concentrated. Purification by flash chromatog-
raphy on silica gel (50% EtOAc−hexanes to 1% triethylamine−99%
EtOAc to 1% MeOH−1% triethylamine−98% EtOAc) afforded 18
1
(5.08 g, 55%). LCMS: (FA) ES+ 198. H NMR (CDCl3, 300 MHz,
ppm) δ 7.50 (s, 1 H), 4.04 (s, 2 H), 3.86 (s, 3 H), 3.12 (t, J = 6.0 Hz, 2
H), 2.66 (t, J = 6.0 Hz, 2 H).
6-(2,2-Dimethylpropanoyl)-N-hydroxy-4,5,6,7-tetrahydrothieno-
[2,3c]pyridine-2 carboxamide (19b). To a solution of HBTU (38.4
mg, 0.101 mmol) and trimethylacetic acid (10.4 mg, 0.101 mmol) in
methylene chloride (2.0 mL) was added triethylamine (28 μL, 0.203
mmol). The reaction mixture was stirred at room temperature for 30
min. A solution of methyl 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-2-
carboxylate (20.0 mg, 0.101 mmol) in methylene chloride added, and
the solution was then stirred at room temperature for 16 h then heated
at 36 °C for 1 h. Upon cooling to room temperature, the reaction
mixture was diluted with DCM and washed with saturated aqueous
NaHCO3. The aqueous layer was extracted with DCM, and the
combined organic phases were washed with water, brine, dried over
anhydrous MgSO4, and concentrated to afford an oil which was treated
with methanol (1.5 mL), hydroxylamine hydrochloride (28 mg, 0.41
mmol), and powdered potassium hydroxide (46 mg, 0.81 mmol). The
reaction mixture was heated at 80 °C for 2 h, cooled to room
1
with 15−40% MeCN in H2O to give a white solid (16 mg, 24%). H
NMR (DMSO-d6, 400 MHz, ppm): δ 11.13 (s, 1 H), 9.08 (s, 1H),
7.37 (s, 1 H), 4.56 (s, 2 H), 3.83 (t, J = 5.8 Hz, 2 H), 2.83 (t, J = 5.4
Hz, 2 H), 1.22 (s, 9 H). 13C NMR (100 MHz, DMSO-d6, 100 MHz,
ppm) δ 175.69, 159.57, 137.69, 134.37, 133.39, 125.60, 44.50, 42.75,
38.29, 27.97, 25.05. HRMS (ESI+): calculated for C13H18N2O3S [M +
H] 283.1111; found 283.1108.
N-Hydroxy-5-(1-methyl-1H-pyrrole-2-carbonyl)-4,5,6,7-
tetrahydrothieno[3,2-c]pyridine-2-carboxamide (22a). 22a was
H
dx.doi.org/10.1021/jm400385r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
prepared similarly. H NMR (400 MHz, CD3OD) δ 7.30 (br, 1H),
at 5 μM. IC50 values were extracted by curve-fitting the dose/response
slopes.
6.87 (m, 1H), 6.50 (m, 1H), 6.13 (m, 1H), 4.79 (s, 2H), 4.04 (t, 2H),
3.75 (s, 3H), 3.02 (t, 2H). 13C NMR (100 MHz, CD3OD) 169.68,
165.82, 140.30, 135.00, 134.18, 128.35, 127.23, 126.03, 114.27, 108.32,
47.90, 35.74, 26.44. HRMS (ESI+): calculated for C14H15N3O3S [M +
H] 306.0912; found 306.0903.
5-(Cyclohexanecarbonyl)-N-hydroxy-4,5,6,7-tetrahydrothieno-
[3,2-c]pyridine-2-carboxamide (22c). 22c was prepared similarly (15
mg, 19%). H NMR (400 MHz, CD3OD, ppm) δ 7.32 (s, 1H), 4.67
(s, 2H), 3.87 (s, 2H), 2.96 (s, 1H), 2.82 (m, 1H), 2.74 (m, 1H), 1.74
(m, 4H), 1.57−1.07 (m, 6H). 13C NMR (100 MHz, DMSO-d6),
174.0, 159.5, 138.0, 134.5, 133.0, 125.6, 45.8, 44.7, 41.9, 29.1, 25.7,
25.6, 25.1. HRMS (ESI+): calculated for C15H20N2O3S [M + H]
309.1267; found 309.1266.
Cellular Immunofluorescence Assays. HDAC6 activity was
determined by measuring K40 hyperacetylation of α-tubulin with an
acetylation selective monoclonal antibody. Selectivity against class I
HDAC activity was determined similarly using an antibody that
recognizes nuclear acetylation in the immunofluorescence assay as
follows:
Cell Culture. HeLa human tumor cells were obtained from ATCC
and were maintained in a humidified chamber at 37 °C with 5% CO2.
HeLa cells were grown in Minimum Essential Media (MEM)
(Invitrogen), supplemented with 10% FBS, 1% glutamine, 1 mM
sodium pyruvate, and 1 mM nonessential amino acids.
1
Acetylated Tubulin and Acetylated Histone Assays. HeLa cells
(8000/well) were grown on 96-well cell culture dishes. The cells were
treated with compound diluted in DMSO in 3-fold serial dilutions with
concentrations ranging from 20 to 0.003 μM for the acetylated tubulin
and acetylated histone for 6 and 24 h, respectively. Compounds at
each dilution were added as triplicates across three plates. Cells treated
with DMSO (three wells per plate, N = 9; 0.2% final concentration)
served as the untreated control, while cells treated with 20 μM tubacin
served as the maximum inhibition control (three wells per plate, N =
9). Following treatment, the cells were fixed with 4% paraformalde-
hyde in PBS for 10 min, permeabilized with 0.5% Triton X-100 in PBS
for 10 min, and washed in PBS. Fixed cells were treated with blocking
reagent (Roche no. 11096176001) for 1 h at room temperature. Cells
were stained with antiacetylated tubulin (1:5000; Sigma no. T6793) or
antiacetyl lysine (abcam no. ab21623) in blocking reagent for 60 min
at room temperature. The cells were washed with PBS and PBS with
0.05% Tween 20 and stained with Alexa 488-conjugated goat
antimouse IgG (1:500, Molecular Probes no. A11029) or Alexa 488-
conjugated antirabbit IgG (1:500, Molecular Probes no. A11034, and
Hoechst (1:50,000; Molecular Probes no. H-3570) in blocking reagent
for 1 h at room temperature. The cells were washed with PBS.
Immunofluorescent cells were visualized using an Opera high content
imaging system (Perkin-Elmer). Images from nine sites per well were
captured at 20× magnification. Inhibition of HDAC6 was determined
by measuring fluorescent intensity of acetylated tubulin using Pipeline
Pilot software. Concentration response curves were generated by
calculating the increase of acetylated-tubulin fluorescent intensity in
compound treated samples relative to the DMSO and tubacin treated
controls, and 50% inhibitory concentrations (IC50) values were
determined from those curves.
Biological Assays. HDAC Enzymatic Assays. Enzymatic data were
obtained using recombinant HDAC6 or HeLa nuclear extract effecting
deacetylation of Ac-Arg-Gly-Lys(Ac)-AMC substrate with subsequent
release of AMC with trypsin in a modification of the procedure of
Wegener39 as follows:
Recombinant HDAC6 (N-GST-tagged, BPS Bioscience, San Diego,
CA) was prepared at a concentration of 1 nM in 25 mM Tris-Cl, 137
mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 0.1 mg/mL BSA at pH 8.0
and was incubated in black 384-well plates with 8 μM Ac-Arg-Gly-
Lys(Ac)-AMC substrate (Bachem Biosciences, King of Prussia, PA)
and a 10-point 1:3 serial dilution of test compound in DMSO (in
duplicate, 2% final DMSO concentration) for 1 h at 30 °C. The
reaction was stopped by the addition trichostatin A (Sigma) and
bovine trypsin (tosyl phenylalanyl chloromethyl ketone-treated,
Sigma) to final concentrations of 1 μM and 10 μg/mL, respectively,
in 50 mM Tris-Cl, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2, pH
8.0. After incubation for an additional 30 min at 30 °C, the release of
AMC from the deacetylated lysine by trypsin was quantitated on a
Pherastar fluorescence plate reader (BMG Labtech, Cary, NC) set at
λex 340 nm and λem 460 nm. Concentration−response curves were
generated from the fluorescence decrease in test compound-treated
samples relative to DMSO-treated controls, and IC50 values were
calculated using Pipeline Pilot (Accelrys, San Diego, CA) and
Condoseo (Genedata, Lexington, MA) software.
Similarly, 30 μg/mL HeLa nuclear extract (Enzo Life Sciences,
BML KI-140, containing primarily class I enzymes HDAC 1 and 2 as
per manufacturer) was incubated with 20 μM of the acetylated
substrate as described above, using a modified reaction buffer
consisting of 50 mM Tris-Cl, 137 mM NaCl, 2.7 mM KCl, and 1
mM MgCl2, pH 8.0, and the assay conducted as described previously.
Counter-screen enzyme inhibition assays for the individual isoforms
were performed by the Reaction Biology Corporation, Malvern, PA
(www.reactionbiology.com). The HDACs 1, 2, 4, 5, 7, 8, 9, 10, and 11
assays used baculovirus expressed recombinant human protein;
HDAC1 full length GST tag, HDAC2 and three full length C-
terminal 6 X His tagged; HDAC4 aa 627-1085, N-terminal GST tag;
HDAC5 aa 657-1123, C-terminal 6 X His tag; HDAC7, aa 518-end, N-
terminal GST tag; HDAC8 full length, C-terminal 6 X His tag;
HDAC9 aa 604-1066, C-terminal 6 X His tag; HDAC10, aa1-481, N-
terminal GST tag; HDAC 11, full length, N-terminal GST tag. The
substrate used for HDACs 1, 2, 3, 10, and 11 assays is a fluorogenic,
acetylated peptide substrate based on residues 379−382 of p53, i.e.,
Arg-His-Lys-Lys(Ac). The substrate used for HDAC8 is a fluorogenic,
diacetylated peptide substrate based on residues 379−382 of p53, i.e.,
Arg-His-Lys(Ac)-Lys(Ac). The substrate used for HDACs 4, 5, 7, and
9 assays was fluorogenic, acetyl-Lys(trifluoroacetyl)-AMC. Com-
pounds were dissolved in DMSO and tested in 10-dose IC50 mode
with 3-fold serial dilution starting at 30 μM in 50 mM Tris-HCl, pH
8.0, 127 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2 containing 1 mg/
mL BSA. Plates were sealed and incubated at 30 °C for 2 h. Developer
was then added to stop the reaction, and kinetic measurements were
recorded for 1.5 h at 15 min intervals on a fluorescence plate reader set
at λex 360 nm and λem 460 nm. End point data (after plateau of signal)
were used for subsequent data analysis. The control trichostatin A
(TSA) was tested in a 10-dose IC50 with 3-fold serial dilution starting
ASSOCIATED CONTENT
* Supporting Information
■
S
IC50 curves for enzymatic experiments involving compound 5a.
Experimental procedures for the Caco-2 and turbidometric
solubility assays. Description of homology and molecular
modeling. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 617 761 6811. E-mail: blackburn@mpi.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Tal Perlov, Rhiannan Thomas, Liting Ma,
Matthew Jones, David Lok, Nina Molchanova, Mingkun Fu,
Ashok Patil, and Cindy Xia for technical assistance.
ABBREVIATIONS USED
■
HDAC, histone deacetylase; MTOC, microtubule organizing
center; NucEx, HeLa nuclear extract; Ac-Tub, acyl tubulin; Boc,
I
dx.doi.org/10.1021/jm400385r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(19) Mahboobi, S.; Sellmer, A.; Hocher, H.; Garhammer, C.;
Pongratz, H.; Maier, T.; Ciossek, T.; Beckers, T. 2-Aroylindoles and 2-
Aroylbenzofurans with N-hydroxyacrylamide Substructures as a Novel
Series of Rationally Designed Histone Deacetylase inhibitors. J. Med.
Chem. 2007, 50, 4405−4418.
tert-butoxycarbonyl; DIEA, N,N-diisopropylethylamine;
DMAP, 4-(dimethylamino)pyridine; HBTU, O-benzotriazole,
N′,N′,N′,N′-tetramethyluronium hexafluorophosphate; DCE,
1,2-dichloroethane
(20) Kozikowski, A. P.; Chen, Y.; Gaysin, A. M.; Savoy, D. N.;
Billadeau; Kim, K. H. Chemistry, Biology, and QSAR Studies of
Substituted Biaryl Hydroxamates and Mercaptoacetamides as HDAC
Inhibitors-Nanomolar-Potency Inhibitors of Pancreatic Cancer Cell
Growth. ChemMedChem 2008, 3, 487−501.
(21) Coiffier, B.; Pro, B.; Prince, H. M.; Foss, F.; Sokol, L.;
Greenwood, M.; Caballero, D.; Borchmann, P.; Morschhauser, F.;
Wilhelm, M.; Pinter-Brown, L.; Padmanabhan, S.; Shustov, A.;
Nichols, J.; Carroll, S.; Baiser, J.; Balser, B.; Horwitz, S. Results from
a Pivotal, Open-Label, Phase II Study of Romidepsin in Relapsed or
Refractory Peripheral T-Cell Lymphoma after Prior Systemic Therapy.
J. Clin. Oncol. 2012, 30, 631−636.
(22) Kelly, W. K.; O’Connor, O. A.; Krug, L. M.; Chiao, J. H.; Heany,
M.; Culey, T.; MacGregore-Cortelli, B.; Tong, W.; Secrist, J. P.;
Schwartz, L.; Richardson, S.; Chu, E.; Olgac, S.; Marks, P. A.; Scher,
H.; Richon, V. M.; Phase, I. Study of an Oral Histone Deacetylase
Inhibitor, Suberoylanilide Hydroxamic Acid, in Patients with Advanced
Cancer. J. Clin. Oncol. 2005, 23, 3923−3931.
(23) Wang, D.; Helquist, P.; Wiest, O. Zinc Binding in HDAC
Inhibitors: A DFT Study. J. Org. Chem. 2007, 72, 5446−5449.
(24) Kozikowski, A. P.; Tapadar, S.; Luchini, D. N.; Kim, K. H.;
Billadeau, D. D. Use of Nitrile Oxide Cycloaddition (NOC) Reaction
for Molecular Probe Generation: A New Class of Enzyme Selective
Histone Deacetylase Inhibitors (HDACIs) Showing Picomolar
Activity at HDAC6. J. Med. Chem. 2008, 51, 4370−4373.
(25) Butler, K. V.; Kalin, J.; Brochier, C.; Vistoli, G.; Langley, B.;
Kozikowski, A. P. Rational Design and Simple Chemistry Yield a
Superior Neuroprotective HDAC6 Inhibitor, Tubastatin A. J. Am.
Chem. Soc. 2010, 132, 10842−10846.
(26) Yannini, A.; Volpari, C.; Filocamo, G.; Casavola, E. C.; Brunetti,
M.; Renzoni, D.; Chakravrti, P.; Paolini, C.; De Francesco, R.;
Gallinari, P.; Steinkuhler, C.; Di Marco, S. Crystal Structure of a
eukaryotic zinc-dependent histone deacetylase, human HDAC8,
complexed with a hydroxamic acid inhibitor. Proc. Natl. Acad. Sci. U.
S. A. 2004, 101, 15064−15069.
(27) Schuetz, A.; Min, J.; Allali-Hassani, A.; Schapira, M.; Shuen, M.;
Loppnau, P.; Mazitschek, R.; Kwiatkowski, N. P.; Lewis, T. A.;
Maglathin, R. L.; McLean, T. H.; Bochkarev, A.; Plotnikov, A. N.;
Vedadi, M.; Arrowsmith, C. H. Human HDAC7 harbors a class IIa
histone deacetylase-specific zinc binding motif and cryptic deacetylase
activity. J. Biol. Chem. 2008, 283, 11355−11363.
(28) Somoza, J. R.; Skene, R. J.; Katz, B. A.; Mol, C.; Ho, J. D.;
Jennings, A. J.; Luong, C.; Arvai, A.; Buggy, J. J.; Chi, E.; Tang, J.; Sang,
B.-C.; Verner, E.; Wynands, R.; Leahy, E. M.; Dougan, D. R.; Snell, G.;
Naver, M.; Knuth, M. W.; Swanson, R. V.; McRee, D. E.; Tari, L. W.
Structural Snapshots of Human HDAC8 Provide Insights into the
Class I Histone Deacetylases. Structure 2004, 12, 1325−1334.
(29) Estiu, G.; Greenberg, E.; Harrison, C. B.; Kwiatkowski, N. P.;
Mazitschek, R.; Bradner, J. E.; Wiest, O. Structural Origin of Selectivity
in Class II-Selective Histone Deacetlyase Inhibitors. J. Med. Chem.
2008, 51, 2898−2906.
(30) Wang, D. Computational Studies on the Histone Deacetylases
and the Design of Selective Histone Deacetylase Inhibitors. Curr. Top.
Med. Chem. 2009, 9, 241−256.
(31) Frederickson, M.; Lyons, J. F.; Thompson, N. T.; Vinkovic, M.;
Williams, B.; Woodhead, A.; Woolford, A. J. Preparation of
Hydroxybenzamide Derivatives as Inhibitors of HSP90. PCT Int.
Appl. WO 2008044034, 2008,.
REFERENCES
■
(1) Gregoretti, I. V.; Lee, Y.-M.; Goodson, H. V. Molecular Evolution
of the Histone Deacetylase Family: Functional Implications of
Phylogenetic Analysis. J. Mol. Biol. 2004, 338, 17−31.
(2) Kalveram, B.; Schmidtke, G.; Groettrup, M. The ubiquitin-like
modifier FAT10 interacts with HDAC6 and localizes to aggresomes
under proteasome inhibition. J. Cell Sci. 2008, 121, 4079−4088.
(3) Boyault, C.; Sadoul, K.; Pabion, M.; Khochibin, S. HDAC6, at the
crossroads between cytoskeleton and cell signaling by acetylation and
ubiquitination. Oncogene 2007, 26, 5468−5476.
(4) Matthias, M. P.; Yoshida, M.; Khochbin, S. HDAC6 a new
cellular stress surveillance factor. Cell Cycle 2008, 7, 7−10.
(5) Simms-Waldrip, T.; Rodriguez-Gonzalez, A.; Lin, T.; Ikeda, A. K.;
Fu, C.; Skamoto, K. M. The aggresome pathway as a target for therapy
in hematologic malignancies. Mol. Genet. Metab. 2008, 94, 283−286.
(6) Rodriguez-Gonzalez, A.; Lin, T.; Ikeda, A. K.; Simms-Waldrip, T.;
Fu, C.; Sakamoto, K. M. Role of the Aggresome Pathway in Cancer:
Targeting Histone Deacetylase 6-Dependent Protein Degradation.
Cancer Res. 2008, 68, 2557−2560.
(7) Kapoor, S. Inhibition of HDAC6-dependent carcinogenesis:
emerging, new therapeutic options besides belinostat. Int. J. Cancer
2009, 124, 509.
(8) Lee, Y. S.; Lim, K. H.; Guo, X.; Kawaguchi, Y.; Gao, Y.;
Barrientos, T.; Ordentlich, P.; Wang, X. F.; Counter, C. M.; Yao, T. P.
The Cytoplasmic Deacetylase HDAC6 Is Required for Efficient
Oncogenic Tumorigenesis. Cancer Res. 2008, 68, 7561−7569.
(9) Kawaguchi, Y.; Kovacs, J. J.; McLaurin, A.; Vance, J. M.; Ito, A.;
Yao, T. P. The Deacetylase HDAC6 Regulates Aggresome Formation
and Cell Viability in Response to Misfolded Protein Stress. Cell 2003,
115, 727−738.
(10) Iwata, A.; Riley, B. E.; Johnston, J. A.; Kopito, R. R. HDAC6 and
Microtubules Are Required for Autophagic Degradation of Aggregated
Huntingtin. J. Biol. Chem. 2005, 280, 40282−40292.
(11) Ding, W. X.; Ni, H. M.; Gao, W.; Yoshimori, T.; Stolz, D. B.;
Ron, D.; Yin, X. M. Linking of Autophagy to Ubiquitin-Proteasome
System Is Important for the Regulation of Endoplasmic Reticulum
Stress and Cell Viability. Am. J. Pathol. 2007, 171, 513−524.
(12) Pandey, U. B.; Nie, Z.; Batlevi, Y.; McCray, B. A.; Ritson, G. P.;
Nedelsky, N. B.; Schwartz, S. L.; DiProspero, N. A.; Knight, M. A.;
Schuldiner, O.; Padmanabhan, R.; Hild, M.; Berry, D. L.; Garza, D.;
Hubbert, C. C.; Yao, T. P.; Baehrecke, E. H.; Taylor, J. P. HDAC6
rescues neurodegeneration and provides an essential link between
autophagy and the UPS. Nature 2007, 447, 859−863.
(13) Haggarty, S. J.; Koeller, K. M; Wong, J. C.; Grozinger, G. M.;
Schreiber, S. L. Domain-selective small-molecule inhibitor of histone
deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc. Natl.
Acad. Sci. U. S. A. 2003, 100, 4389−4394.
(14) Marks, P. A.; Richon, V. M.; Miller, T.; Kelly, W. K. Histone
Deacetylase Inhibitors. Adv. Cancer Res. 2004, 91, 137−168.
(15) Wong, J. C.; Hong, R.; Schreiber, S. L. Structural Biasing
Elements for In-Cell Histone Deacetylase Paralog Selectivity. J. Am.
Chem. Soc. 2003, 125, 5586−5587.
(16) Hideshima, T.; Bradner, J. E.; Wong, J.; Chauhan, P.;
Richardson, P.; Schreiber, S. L.; Anderson, K. C. Small-molecule
inhibition of proteasome and aggresome function induces synergistic
antitumor activity in multiple myeloma. Proc. Natl. Acad. Sci. U. S. A.
2005, 102, 8567−8572.
(17) Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. Histone
Deacetylase Inhibitors: From Bench to Clinic. J. Med. Chem. 2008, 51,
1505−1529.
(18) Minucci, S.; Pellicci, P. G. Histone Deacetylase Inhibitors and
the Promise of Epigenetic (And More) Treatments for Cancer. Nature
Rev. Cancer 2006, 6, 38−51.
(32) Cho, Y. S.; Jiang, L.; Shultz, M. Anti-tumor heterocycle
compounds. PCT Int. Appl. WO 2008076954, 2008,.
(33) Pessoa-Mahana, H.; Kosche, C.; Nadia, R. H.; Recabarren-
Gajardo, G.; Claudio, S. B.; Araya-Maturana, R.; Pessoa-Mahana, C. D.
Solvent-free microwave synthesis of 3-(4-benzo[b]thiophene-2-car-
bonyl)-1-piperazinyl-1-benzo[b]thiophen-2-yl-1-propanones. New het-
J
dx.doi.org/10.1021/jm400385r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
ero bis-ligands with potential 5-HT1A serotonergic activity. Hetero-
cycles 2008, 75, 1913−1929.
(34) Wang, Q.; Lucien, E.; Hashimoto, A.; Godwin, C.; Pais, G.;
Nelson, D. M.; Song, Y.; Thanassi, J. A.; Marlor, C. W.; Thoma, C. L.;
Cheng, J.; Podos, S. D.; Ou, S.; Deshpande, M.; Pucci, M. J.; Buechter,
D. D.; Bradbury, B. J.; Wiles, J. A. Isothiazoloquinolones with
Enhanced Antistaphylococcal Activities against Multidrug-Resistant
Strains: Effects of Structural Modifications at the 6-, 7-, and 8-
Positions. J. Med. Chem. 2007, 50, 199−210.
(35) Grunewald, G. L.; Seim, M. R.; Seema, R. B.; Wilson, M. E.;
Criscione, K. R. Synthesis of 4,5,6,7-tetrahydrothieno[3,2-c]pyridines
and comparison with their isosteric 1,2,3,4-tetrahydroisoquinolines as
inhibitors of phenylethanolamine N-methyltransferase. Bioorg. Med.
Chem. 2008, 16, 542−559.
(36) Venkatesan, A. M.; Agarwal, A.; Abe, T.; Ushirogochi, H.;
Yamamura, I.; Ado, M.; Tsuyoshi, T.; Dos Santos, O.; Gu, Y.; Sum, F.-
W.; Francisco, G.; Lin, Y.-I.; Peterson, P. J.; Yang, Y.; Kumagai, T.;
Wiess, W. J.; Shlaes, D. M.; Knox, J. R.; Mansour, T. S. Structure−
Activity Relationship of 6-Methylidene Penems Bearing 6,5 Bicyclic
Heterocycles as Broad-Spectrum β-Lactamase Inhibitors: Evidence for
the 1,4-Thiazepine Intermediates with C7 R Stereochemistry by
Computational Methods. J. Med. Chem. 2006, 49, 4623−4637.
(37) Thermodynamic solubility determination (see Supporting
Information). With the exception of compound 5d (25 μM), all
compounds also exhibited turbidometric solubilities > 200 μM (the
top concentration in the assay).
(38) Bottomley, M. J.; Surdo, P. L.; Di Giovine, P.; Cirillo, A.;
Scarpelli, R.; Ferrigno, F.; Jones, P.; Neddermann, P.; De Francesco,
R.; Steinkuhler, C.; Gallinari, P.; Carfi, A. Structural and Functional
Analysis of the Human HDAC4 Catalytic Domain Reveals a
Regulatory Structural Zinc-Binding Domain. J. Biol. Chem. 2008,
283, 26694−26704.
(39) Wegener, D.; Wirsching, F.; Riester, D.; Schwienhorst, A. A
Fluorogenic Histone Deacetylase Assay Well Suited for High-
Throughput Activity Screening. Chem. Biol. 2003, 10, 61−68.
K
dx.doi.org/10.1021/jm400385r | J. Med. Chem. XXXX, XXX, XXX−XXX