Synthesis and structure-activity relationship of 3-hydroxypyridine-2- thione-based histone deacetylase inhibitors

Article abstract of DOI:10.1021/jm401225q

We previously identified 3-hydroxypyridine-2-thione (3HPT) as a novel zinc binding group for histone deacetylase (HDAC) inhibition. Early structure-activity relationship (SAR) studies led to various small molecules possessing selective inhibitory activity against HDAC6 or HDAC8 but devoid of HDAC1 inhibition. To delineate further the depth of the SAR of 3HPT-derived HDAC inhibitors (HDACi), we have extended the SAR studies to include the linker region and the surface recognition group to optimize the HDAC inhibition. The current efforts resulted in the identification of two lead compounds, 10d and 14e, with potent HDAC6 and HDAC8 activities that are inactive against HDAC1. These new HDACi possess anticancer activities against various cancer cell lines including Jurkat J.γ1 for which SAHA and the previously disclosed 3HPT-derived HDACi were inactive.

Full text of DOI:10.1021/jm401225q

Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationship of 3‑Hydroxypyridine-
2-thione-Based Histone Deacetylase Inhibitors
Quaovi H. Sodji,†,∥ Vishal Patil,†,∥ James R. Kornacki,§,∥ Milan Mrksich,*,§
and Adegboyega K. Oyelere*,†,‡
‡
†School of Chemistry and Biochemistry, Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of
Technology, Atlanta, Georgia 30332-0400, United States
§Departments of Chemistry and Biomedical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois
60208-3113, United States
S
* Supporting Information
ABSTRACT: We previously identified 3-hydroxypyridine-2-thione (3HPT) as a novel zinc
binding group for histone deacetylase (HDAC) inhibition. Early structure−activity relationship
(SAR) studies led to various small molecules possessing selective inhibitory activity against
HDAC6 or HDAC8 but devoid of HDAC1 inhibition. To delineate further the depth of the
SAR of 3HPT-derived HDAC inhibitors (HDACi), we have extended the SAR studies to
include the linker region and the surface recognition group to optimize the HDAC inhibition.
The current efforts resulted in the identification of two lead compounds, 10d and 14e, with
potent HDAC6 and HDAC8 activities that are inactive against HDAC1. These new HDACi
possess anticancer activities against various cancer cell lines including Jurkat J.γ1 for which
SAHA and the previously disclosed 3HPT-derived HDACi were inactive.
Previous work from our lab has established 3-hydroxypyr-
idine-2-thione (3HPT) as a nonhydroxamate ZBG for HDAC
inhibition.9 Initial structure−activity relationship (SAR) studies
led to aryl- and diaryl-3HPT analogues possessing selective
inhibitory activity against HDAC6 or HDAC8 but not against
HDAC1 (Figure 1, representative compounds 1, 2, and 3). In
the same study, we observed that the replacement of the
proximal phenyl ring of the lead biphenyl compound 2 with a
1,2,3-triazole ring resulted in corresponding triazolyl analogue 3
lacking HDAC6 inhibition activity but having improved
HDAC8 inhibition (Figure 1). This observation suggests a
possible divergence in the SARs of the triazole and biphenyl 3-
HPT compounds.9 To characterize such divergence further, we
have expanded the SAR studies on the 3-HPT compounds
bearing triazole-linked cap groups. The current efforts identified
two lead compounds, 10d and 14e, which are potent inhibitors
of HDAC6 and HDAC8 but are inactive against HDAC1.
These new HDACi possess anticancer activities against various
cancer cell lines including Jurkat J.γ1 against which SAHA and
the previously disclosed 3HPT-derived HDACi were inactive.
INTRODUCTION
■
Histone deacetylase (HDAC) inhibition is a promising
epigenetic strategy for cancer treatment, and several distinct
small-molecule histone deacetylase inhibitors (HDACi) have
been reported.1 Currently, two of these HDACi, suberoylani-
lide hydroxamic acid (SAHA) (Figure 1) and cyclic peptide
FK228 (Romidepsin), are approved for the treatment of
cutaneous T-cell lymphoma (CTCL).2 However, most HDACi,
including the clinically approved agents, nonselectively inhibit
the deacetylase activity of class I and II HDACs, and many
suffer from metabolic instability. These shortcomings have been
associated with reduced in vivo potency and toxic side effects.3
Currently, significant efforts are ongoing to address these and
other deficiencies of HDACi to bolster the potential of HDAC
inhibition in cancer treatment.
Most HDACi fit a three-motif pharmacophoric model
consisting of a zinc binding group (ZBG), a linker, and a
surface recognition cap group. Hydroxamic acid (hydroxamate)
is by far the most common ZBG moiety in HDACi owing to its
ability to reliably chelate active-site zinc ions.4 However,
pharmacodynamic and pharmacokinetic liabilities of the
hydroxamate moiety have prompted efforts to find better-
suited alternatives.5 Examples of nonhydroxamate ZBGs
investigated include thiols, α-ketoesters, benzamide, trifluor-
omethylketone, oxime, phosphonates, and mercaptoaceta-
mide.6,7,5,8 However, most of these alternative ZBGs have
thus far elicited reduced potency relative to hydroxamic acid.
RESULTS AND DISCUSSION
■
SAR on the Linker Moiety. The hydrophobic linker
moiety of most HDACi consists of flexible methylene spacer
groups that separate the ZBG from the cap group to tailor the
Received: August 7, 2013
Published: December 4, 2013
© 2013 American Chemical Society
9969
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981

Journal of Medicinal Chemistry
Article
Figure 1. (a) Representative examples of HDACi. (b) Representative aryl- and diaryl-3HPT-based HDACi with their HDAC inhibition activities
(IC50). For compound 3, the percent inhibition of at 10 μM is shown.9
a
Scheme 1. Synthesis of 3-HPT-Based HDACi 10 for SAR Studies
a
Conditions: (a) NaN3, DMF, 75 °C; (b) methanesulfonyl chloride, Et3N, THF; (c) 3-methoxypyridin-2-one or 3-benzyloxypyridin-2-one, K2CO3,
THF/DMF, reflux; (d) phenylacetylene, CuI, DIPEA, THF; (e) H2, Pd/C, THF for R = Bn and BBr3, DCM for R = Me; (f) for 7a only: Lawesson’s
reagent, toluene, reflux; (g) P4S10, 175 °C, neat; (h) BBr3, DCM.
9970
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981

Journal of Medicinal Chemistry
Article
Table 1. SAR on Linker of Triazolic Compounds
compound
n
HDAC1 IC50 (nM)
HDAC6 IC50 (nM)
HDAC8 IC50 (nM)
a
b
3a
1
2
3
4
5
6
7
NI
41%
1570 1067
6050 750
6400 450
3303 260
917 139
6751 910
1377 205
232 19
10a
10b
10c
10d
10e
10f
NI
NI
NI
NI
NI
NI
14%
3628 1363
1085 333
911 173
22%
955 150
144 23
SAHA
38
2
a
b
No significant inhibition (below 20% inhibition at 10 μM). Percent inhibition of the compounds at 10 μM are given if the IC50 was above 10 μM.
intramolecular span between the active-site Zn2+ ion and outer-
rim amino acid residues. Previous studies from our laboratory
have shown that SAHA-like HDACi containing a 1,2,3-triazole
ring within the linker region differentially inhibited HDACs as a
function of linker length.10 Lead compound 3 is an analogue
with one methylene spacer separating the triazole ring and the
3HPT ZBG. To probe the effect of the spacer length on HDAC
inhibition activity, we initially synthesized and investigated the
anti-HDAC activity of compounds 10a−f, analogues of 3 with
increasing methylene groups. The syntheses of target
compounds were accomplished as shown in Scheme 1. The
reaction of various bromoalkanols with sodium azide yielded
their corresponding azidoalkanols, 4a−f. Subsequent mesyla-
tion of 4a−f followed by N-alkylation with O-methyl-or O-
benzyl-protected 3-hydroxypyridin-2-one (3HP) gave the azido
intermediates 6a−f. The phenyl moiety, which serves as surface
recognition group, was introduced via Cu(I)-catalyzed Huisgen
cycloaddition reaction11,12 between phenylacetylene and azido
intermediates 6a−f to afford compounds 7a−f. The depro-
tection of the O-benzyl moiety of 7b−f was accomplished using
catalytic hydrogenation to afford compounds 8b−f, which,
upon treatment with P4S10 at 175 °C, gave corresponding
3HPT compounds 10b−f (Scheme 1).13 Although otherwise
facile, this chemistry did not work for the two methylene linker
compound because of an extensive degradation that resulted in
an intractable mixture when compound 8a was exposed to
P4S10 at 175 °C. To obtain the requisite 3HPT compound 10a,
O-methyl protected 3HP 7a was first converted to its thione
analogue, 9, using Lawesson’s reagent.14 Subsequent BBr3
deprotection of the methyl ether group yielded desired
compound 10a (Scheme 1).
We then assayed compounds 10a−f as well as their 3HP
congeners, 8a−f, against HDAC isoforms 1, 6 and 8 using
SAMDI mass spectrometry as previously described.15−17 3HP
compounds 8a−f are inactive against all HDAC isoforms tested
(data not shown), a result that is in agreement with our
previous observation on aryl- and diaryl-3HP analogues.9 As
previously reported for diaryl-3HPT-based HDACi, com-
pounds 10a−f are not active against HDAC1. However,
compounds 10a−f do inhibit HDAC6 and HDAC8 (Table
1), and they do so as a function of methylene linker length.
Such apparent selectivity may result from active-site structure
differences between the isoforms. The channel to HDAC1
active site is narrow as opposed to HDAC6 whose hydrophobic
channel entrance appears large enough to accommodate the
3HPT group.18 Relative to lead compound 3, a progressive
increase in the length of the methylene linker separating the
triazole ring and the 3HPT group results in a corresponding
increase in HDAC6 inhibition activity up to five methylene
units (n = 5) (Table 1, compare 10a−d). A single methylene
linker extension (from n = 5 to 6) is not tolerated, as resulting
analogue 10f is devoid of HDAC6 inhibition activity; however,
a further extension of the methylene linker length (n = 7) fully
restores anti-HDAC6 activity (Table 1, compare 10d−f).
Similar methylene-linker-length-dependence in HDAC8 inhib-
ition activity was observed. However, unlike what was obtained
with HDAC6, no complete loss and subsequent full restoration
of anti-HDAC8 occurred when n = 6 and 7, respectively. This
initial SAR study suggests that five methylene spacers (n = 5)
are optimum for HDAC inhibition, as corresponding
compound 10d is the most potent in this series, with a
markedly improved anti-HDAC activity relative to starting lead
compound 3. Additionally, 10d is equipotent against HDAC6
and HDAC8 with an IC50 of approximately 900 nM (Table 1).
Molecular Docking Analyses. To understand the
structural basis of dependence of methylene linker length on
the HDAC inhibition by compounds 10a−f, we docked
selected analogues (10a and 10d) against an HDAC6
homology model built from HDAC8 (PDB code: 3F0R) and
HDAC8 (PDB code: 3SFF) using AutoDock 4.2.15,19,20 We
observed that 10a and 10d adopt docked conformations on
HDAC6 such that their 3HPT ZBG are able to maintain Zn2+
chelation irrespective of the methylene linker length. Unlike
10a, however, five methylene compound 10d is further
stabilized by a π-stacking interaction between its phenyl ring
and that of Phe680 (Figure 2A). The docked poses found on
HDAC8 have the phenyl ring of 10d inserted into a solvent-
inaccessible hydrophobic pocket adjacent to the active site that
is otherwise inaccessible to 10a because of its shorter
methylene spacers. Instead, the phenyl ring of 10a is positioned
in a solvent-exposed outer-rim compartment of HDAC8
(Figure 2B). The favorable interactions of the phenyl ring
with the active-site amino acid side chains of HDAC6 and the
subpocket of HDAC8 support the preference for five
methylene compound 10d.
SAR on the Surface Recognition Group: Optimization
of the Five Methylene-Linked Compound. As demon-
strated by in vitro studies and corroborated by docking
analyses, the five methylene spacer groups induced optimal
inhibition of both HDAC6 and HDAC8. This linker, along with
9971
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981

Journal of Medicinal Chemistry
Article
one 11 gave corresponding triazolyl intermediates 12a−g.
Subsequent treatment of 12a−g with Lawessons’ reagent
followed by BBr3 deprotection of the O-methyl group resulted
in desired 3HPT compounds 14a−g. Similarly, BBr3 depro-
tection of the O-methyl group of 12a−g afforded correspond-
ing 3HP compounds 15a−g.
As expected, 3HP compounds 15a−g were inactive against
all HDAC isoforms tested (data not shown). Similar to 3HPT
compounds 10a−f, none of substituted 3HPT compounds
14a−g were active against HDAC1. Despite the diversity of the
surface recognition groups tested, none was more potent than
unsubstituted lead compound 10d against HDAC8 (Table 2).
Against HDAC6, however, we observed an intriguing effect on
inhibition activity. Among the methyl substituents (para 14a,
meta 14b, and ortho 14c), only ortho-substituted 14c offered a
potency enhancement against HDAC6 relative to 10d. The
inhibitory activity of para-substituted compound 14a is
comparable to that of 10d, whereas meta-substituted
compound 14b is somewhat less potent. Remarkably,
substitution of methyl at the meta position of 14b with an
electron-withdrawing cyano group yielded the most potent
HDAC6 inhibitor, compound 14e, among the series.
Compound 14e is 2.5- and 3-fold more effective than
unsubstituted lead compound 10d and meta-methyl compound
14b, respectively (Table 2). The placement of the cyano group
at the para position conferred no additional benefit to HDAC
inhibitory activity, as resulting compound 14d is equipotent as
lead compound 10d and para-methyl compound 14a. More-
over, switching to the electron-donating N,N-dimethylamino
group at the para position (compound 14f) did not enhance
HDAC6 inhibition despite structural similarity to TSA.
Addition of a different electron-withdrawing group, a
trifluoromethyl moiety, to the meta position in the presence
of the cyano substituent at the para position partially rescued
the HDAC6 inhibitory activity that was lost in the compound
that has only the cyano group at the para position (Table 2,
compare 14d and 14g). This observation further supports the
influence of electron-withdrawing groups at the meta position
on the potency of the substituted 3-HPT compounds.
Figure 2. Molecular docking studies on 10a and 10d. (A) HDAC6
homology model built from HDAC8 (PDB code: 3F0R). (i) Zn2+
chelation is observed for both 10a (green) and 10d (orange). (ii) π-
Stacking interaction of the phenyl ring of 10d with Phe680. (B)
HDAC8 (PDB code: 3SFF). (i) Zn2+ chelation of 10a (yellow) and
10d (green). (ii) The phenyl ring of 10d (green) is inserted into a
hydrophobic pocket adjacent to the active site, whereas that of 10a
(yellow) is in a solvent-exposed HDAC8 outer rim.
the triazole ring, traverses the hydrophobic channel of the
HDAC to present the 3-HPT to the active site for Zn2+
chelation. To optimize lead compound 10d further, we
performed a SAR study on the cap group by investigating the
effects of various phenyl-ring substituents and substitution
patterns on HDAC inhibition activity.
The synthesis of the desired phenyl-substituted compounds
followed a similar protocol as that described for the synthesis of
compound 10 (Scheme 2). Briefly, Cu(I)-catalyzed Huisgen
cycloaddition reaction11,12 of appropriately substituted phenyl-
acetylene with azido O-methyl-protected 3-hydroxypyridin-2-
Molecular Docking Analysis on Substituted 3HPT
Compounds. To gain insight into the binding interactions
a
Scheme 2. Optimization of the Surface Recognition Group: Synthesis of the Five Methylene-Linked 3HPT HDACi
a
(a) 3-Methoxypyridin-2-one, K2CO3, THF/MeOH, reflux; (b) substituted phenylacetylenes (a−g), CuI, DIPEA, DMSO/THF; (c) Lawesson’s
reagent, toluene, reflux; d) BBr3, DCM.
9972
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981

Journal of Medicinal Chemistry
Article
Table 2. HDAC Inhibition Profile of the Substituted 3HPT
Compounds
Figure 3. Molecular docking studies on substituted 3HPT compounds.
(i) HDAC6 homology model built from HDAC8 (PDB code: 3F0R).
The cyano group of compound 14e is positioned for potential H-
bonding interactions with the hydroxyl side chain and amide backbone
of Ser568. (ii) HDAC8 (PDB code: 3SFF). The cap groups of
monosubstituted compounds are inserted within HDAC8 subpocket,
whereas that of disubstituted compound 14g is excluded. (iii) HDAC8.
Space-filling model reveals a better fit of unsubstituted phenyl 10d
(green) within the HDAC8 subpocket relative to representative
monosubstituted compound 14b (orange).
3). Even though the size limitation may preclude the subpocket
from affecting enhanced inhibition through lead development,
it offers a distinctive feature not observed on HDAC6 that
accounts for the pronounced isoform selectivity of compound
14e. The lead candidates from the foregoing SAR studies are
compounds 10d and 14e. Compound 10d is equipotent against
HDAC6 and HDAC8, whereas 14e is 8-fold-selective for
HDAC6.
In Vitro Cell Growth Inhibition. We next tested the effect
of lead compounds 10d and 14e and comparable compounds
10f and 14c on the proliferation of selected cancer cell lines.
We investigated DU-145 (androgen-independent prostate
cancer), LNCaP (androgen-dependent prostate cancer), Jurkat
(T-cell leukemia cell line), and Jurkat J.γ1 (a mutant Jurkat cell
line resistant to HDAC inhibition).9,21
Table 3 shows the IC50 values of each compound against the
cancer cell lines studied. Lead meta-cyano compound 14e is
about 2-fold more potent against DU145 and LNCaP prostate
cancer cell lines relative to unsubstituted congener 10d. Such
improved sensitivity with 14e toward prostate cancer lines,
particularly LNCaP, may be rationalized by its HDAC6
selectivity.22 LNCaP is an androgen-dependent prostate cancer
line whose viability is linked to the interaction between HSP90
and its client proteins including the androgen receptor.23
Misregulation of HSP90−client protein interactions following
HDAC6 inhibition is detrimental to cell viability.22 Inhibition
of HSP90 has been shown to compromise the viability of
DU145, offering a similar rationale for the enhanced
cytotoxicity of 10d against this cell line.24,25 Compounds 10d
and 14e are equipotent against the wild-type Jurkat cell line
a
No significant inhibition (below 20% inhibition at 10 μM
concentration)).
that underlie the SAR for HDAC inhibition, we docked each of
the substituted 3HPT compounds against our HDAC6
homology model and HDAC8. For HDAC6, the phenyl ring
of all compounds mediates a potential π-stacking interaction
with the phenyl ring of Phe680, mimicking the interaction
observed for compound 10d (see Supporting Information
Figure S1). Most striking, however, is the observation that the
cyano group of compound 14e is ideally positioned for a
stabilizing H-bonding interaction with the hydroxyl side chain
and amide backbone of Ser568 and likely accounts for its
enhanced potency against HDAC6 (Figure 3). Moreover, the
concomitant reduction in electron density by the cyano group
could further enhance the stacking interaction with the phenyl
ring of Phe680.
Although phenyl ring substitution impaired HDAC8
inhibition, docking analyses offered useful insight into the
putative hydrophobic pocket in which the inhibitor’s phenyl
group is expected to reside. Monosubstituted compounds fit
within this subpocket, whereas disubstituted compound 14g is
excluded, indicating a restrictive size limitation (Figure 3 and
Supporting Information Figure S2). Space-filling models of all
of the docked compounds reveal that unsubstituted phenyl 10d
fit in the subpocket the best and that any substituent slightly
pushes the phenyl ring out of the hydrophobic pocket (Figure
9973
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981

Journal of Medicinal Chemistry
Article
a
Table 3. Cell Viability Assay
cellular IC50 (μM)
compound
10d
DU-145
LNCaP
Jurkat
Jurkat J.γ1
Vero
9.33 0.96
17.72 3.28
11.08 2.38
5.03 1.13
2.49 0.2
NT
5.43 0.47
10.95 1.92
4.95 0.43
3.49 0.34
2.31 0.74
3.27 0.60
9.04 1.31
5.18 1.18
3.44 0.57
1.49 0.10
1.86 0.21
>20
>20
b
10f
NT
NT
>20
14c
NT
14e
0.90 0.12
c
d
SAHA
Tubastatin A
NI
5.20 0.96
>20
10.88 1.49
3.38 0.26
NI
a
b
c
d
IC50 of selected compounds against various cancer cell lines. NT, not tested. NI, no inhibition at 20 μM. Ref 15.
despite the enhanced HDAC8 inhibition activity of the former
(Table 2), which was expected to favor cytotoxicity to the
Jurkat cell line.21,26 It is reasonable that the compensation of
the weaker HDAC8 inhibition activity of 14e by the 3-fold
increase in its anti-HDAC6 activity, relative to that of 10d, may
contribute to its cytotoxicity in the Jurkat cell. The cytotoxicity
of 10f, an analogue containing a seven methylene linker, and
ortho-methyl-substituted five methylene compound 14c do not
completely trend with the pattern of their HDAC inhibition
activities. Against both prostate cancer lines investigated, 10f is
2- and 3-fold less cytotoxic compared with lead compounds
10d and 14e, respectively, whereas it is 3-fold less cytotoxic
relative to both lead compounds against the wild-type Jurkat
cell line. Ortho-substituted compound 14c is 2-fold less
cytotoxic against DU145, whereas it is only marginally less
cytotoxic against LNCaP and the wild-type Jurkat cell line
compared with 14e.
Although 10d and 10f have similar IC50 against HDAC6, the
latter has a significantly higher cellular IC50 against LNCaP
cells. Against the Jurkat cell line, 14c and 14e also display
similar incongruity, which we first attributed to solubility
differences. To account partially for such pharmacokinetic
liabilities, we estimated the solubility of each tested
compound,27,28 but we found that the four compounds had
solubilities greater than the threshold proposed to affect cell
activity adversely (Supporting Information, Figure S3 and
Table S1). This suggests that the observed discrepancies may
be attributed to factors other than solubility.
We have shown previously that the Jurkat J.γ1 cell line, a
mutant Jurkat cell line lacking phospholipase C activity, is
resistant to SAHA.9 To check if the 3HPT compounds would
be similarly innocuous, we investigated the effect of lead
compounds 10d, 10f, and 14e on Jurkat J.γ1 cell growth. We
observed that compound 10f is significantly less cytotoxic to
Jurkat J.γ1 cells versus their wild-type counterpart. Surprisingly,
both 10d and 14e are potently cytotoxic to Jurkat J.γ1 cells with
IC50 values of 1 and 2 μM, respectively. Comparatively, the
IC50’s of 10d and 14e against the healthy mammalian cell line
Vero were estimated to be greater than 20 μM, the highest
tested concentration (Table 3). To evaluate indirectly the
contribution of HDAC6 inhibition to the anticancer activity of
the compounds, the antiproliferative activity of HDAC6-
selective inhibitor tubastatin A was evaluated against these
cell lines. Although tubastatin A was as potent as 10d and 14e
in Jurkat cells, it was 2- to 3-fold less potent against LNCaP and
was inactive against Jurkat J.γ1 (Table 3). Given that the pan-
inhibitor SAHA and HDAC6-selective tubastatin A are inactive
against Jurkat J.γ1, the cytotoxic activity of lead compounds
10d and 14e against against Jurkat J.γ1 could be through
inhibition of other yet to be identified cellular target(s).
Toward identifying other possible targets of the lead
compounds 10d and 14e, we screened for their effect on the
activity of a collection of matrix metalloproteases (MMPs) that
have been suggested to play roles in apoptosis.29 The rationale
for screening against MMPs is based on the fact that 3HPT has
been previously used as a ZBG in the design of MMP
inhibitors.30 At the tested concentration of 10 μM, 10d and 14e
were inactive against MMP1, 2, 3, 7, 9, and 14, whereas the
activity of MMP13 was halved at this very high concentration
(Figure S4). Despite the marginal effect on MMP13, these
proteins are not likely to be affected directly by the compounds
tested.
We also tested lead compounds against HDAC2, which has
been shown to be upregulated in certain malignancies and
whose inhibition could result in apoptosis.31,32 However, the
IC50 values of 10d and 14e were 5840 740 and 3680 800
nM, respectively, suggesting that HDAC2 is not likely a direct
target.
Intracellular Target Validation. The data presented
showed that the lead 3HPT compounds possess HDAC6
inhibition activities (Table 2); nevertheless, we sought to
confirm the involvement of HDAC6 inhibition in the
mechanism of action of compounds 10d and 14e. Using
western blotting, we examined the level of tubulin acetylation, a
common marker for intracellular HDAC6 activity, in LNCaP
cells following exposure to 10d and 14e.33 We used SAHA as a
positive control in this experiment. We observed that 10d and
14e increased tubulin acetylation but to a lesser extent than
SAHA (Figure 4). These data propose that lead compounds
10d and 14e may derive their antiproliferative effects from an
HDAC6-dependent pathway.
CONCLUSIONS
■
We have previously reported 3HPT as an advantageous ZBG
for HDAC inhibition. Early SAR studies led to aryl- and diaryl-
3HPT compounds that are devoid of HDAC1 inhibition
activity but possess inhibitory activity against HDAC6 or
HDAC8.9 Herein, we have delineated the depth of the SAR of
Figure 4. Western blot analysis of tubulin acetylation (HDAC6
inhibition) in the LNCaP cell line. Lane 1, 10d (20 μM); lane 2, 10d
(5.43 μM); lane 3, 14e (20 μM); lane 4, 14e (3.49 μM); lane 5, SAHA
(20 μM); lane 6, SAHA (2.31 μM); and lane 7, control.
9974
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981

Journal of Medicinal Chemistry
Article
protocols.17 Deacetylation yields in each triplicate sample were
determined from the integrated peak intensities of the molecular
ions for the substrate and the deacetylated product ion by taking the
ratio of the former over the sum of both. Yields were plotted with
respect to inhibitor concentration and fitted to obtain IC50 values for
each isoform−inhibitor pair.
Isoform-optimized substrates were prepared by traditional FMOC
solid-phase peptide synthesis (reagents supplied by Anaspec) and
purified by semi-preparative HPLC on a reverse-phase C18 column
(Waters). The peptide GRKacFGC was prepared for HDAC1 and
HDAC8 experiments, whereas the peptide GRKacYGC was prepared
for HDAC6 and HDAC2 experiments. Isoform preference for the
indicated substrates was determined by earlier studies on peptide
arrays.36
HDAC1, HDAC6, and HDAC2 were purchased from BPS
Biosciences. The catalytic domain of HDAC8 was expressed as
previously reported.36 Briefly, an amplicon was prepared by PCR with
the following primers: forward (5′−3′) TATTCTCGAGGA-
CCACATGCTTCA and reverse ( 5′−3′) ATAAGCTAGCATG-
GAGGAGCCGGA. A pET21a construct bearing the genetic insert
between the NheI and XhoI restriction sites was transformed into
Escherichia coli BL21(DE3) (Lucigen) and expressed by standard
protocols. Following purification by affinity chromatography, the His-
tagged enzyme-containing fractions were purified by FPLC (AKTA)
on a superdex size-exclusion column (GE), spin-concentrated, and
stored at −80 °C in HDAC buffer with 10% glycerol.
3HPT-derived HDACi. The current efforts resulted in two lead
compounds, 10d and 14e, demonstrating potent HDAC6 and
HDAC8 activities without targeting HDAC1. Additionally,
these new HDACi’s enhanced tubulin acetylation in the
LNCaP cell line and possessed antiproliferative activities
against various cancer cell lines including Jurkat J.γ1 for
which SAHA, tubastatin A, and the previously disclosed 3HPT-
derived HDACi were inactive.
EXPERIMENTAL SECTION
■
Materials and Methods. Bromoalkanoic acid, benzyl bromide, 4-
bromobenzylbromide, 4-(bromomethyl)-1,1′-biphenyl, 3-methoxy-
2(1H)-pyridone, propargyl bromide, phenylacetylene, and representa-
tive boronic acids were purchased from either Sigma-Aldrich or Alfa
Aesar. Alkynes 16 and 18, which we could not obtain from commercial
sources, were synthesized using the Bestmann−Ohira reagent as
described previously (see the Supporting Information).10,34,35
Anhydrous solvents and other reagents were purchased and used
without further purification. Analtech silica gel plates (60 F254) were
used for analytical TLC, and Analtech preparative TLC plates (UV
254, 2000 μm) were used for purification. UV light was used to
examine the spots. Silica gel (200−400 mesh) was used in column
chromatography. NMR spectra were recorded on a Varian-Gemini 400
1
magnetic resonance spectrometer. H NMR spectra were recorded in
parts per million (ppm) relative to the peak of CDCl3, (7.24 ppm),
CD3OD (3.31 ppm), or DMSO-d6 (2.49 ppm). 13C spectra were
recorded relative to the central peak of the CDCl3 triplet (77.0 ppm),
CD3OD (49.0 ppm), or the DMSO-d6 septet (39.7 ppm) with
complete heterodecoupling. Multiplicities are described using the
abbreviations s, singlet; d, doublet, t, triplet; q, quartet; m, multiplet;
and app, apparent. High-resolution mass spectra were recorded at the
Georgia Institute of Technology mass spectrometry facility in Atlanta.
All final 3HPT-based compounds were established to be >95% pure
using HPLC. These HPLC analyses were done on a Beckman Coulter
instrument with a Phenomenex RP C-18 column (250 mm × 4.6 mm)
using a water (solvent A) and acetonitrile (solvent B) gradient starting
from 40 to 80% of B over 20 min and constant at 80% for 5 min. The
flow rate was 1 mL/min, and detection was at 379 nm. DU-145,
LNCaP, and Jurkat J.λ1 were obtained from ATCC (Manassas, VA,
USA). The Jurkat E6-1 cell line was kindly donated by Dr. John
McDonald and grown in recommended medium supplemented with
10% fetal bovine serum (Global Cell Solutions, Charlottesville, VA,
USA) and 1% pen/strep (Cellgro, Manassas, VA, USA) at 37 °C in an
incubator with 5% CO2. Mouse anti-acetylated α-tubulin antibody was
obtained from Invitrogen (Life Technologies, Grand Island, NY,
USA). Rabbit anti-actin and rabbit anti-tubulin α antibodies and
tubastatin A were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Secondary antibodies (goat anti-rabbit conjugated to IRDye680
and goat anti-mouse conjugated to IRDye800) were purchased from
LI-COR Biosciences (Lincoln, NE, USA). The CellTiter 96 aqueous
one solution cell proliferation assay (MTS) kit was purchased from
Promega (Madison, WI, USA).
Histone Deacetylase Inhibition. The HDAC activity in the
presence of various compounds was assessed by SAMDI mass
spectrometry. As a label-free technique, SAMDI is compatible with a
broad range of native peptide substrates without requiring potentially
disruptive fluorophores. To obtain IC50 values, we incubated isoform-
optimized substrates (50 μM, detailed below) with enzyme (250nM,
detailed below) and inhibitor (at concentrations ranging from 10 nM
to 1.0 mM) in HDAC buffer (25.0 mM Tris-HCl, pH 8.0, 140 mM
NaCl, 3.0 mM KCl, 1.0 mM MgCl2, and 0.1 mg/mL BSA) in 96-well
microtiter plates (60 min, 37 °C). Solution-phase deacetylation
reactions were quenched with trichostatin A (TSA) and transferred to
SAMDI plates to immobilize the substrate components. SAMDI plates
were composed of an array of self-assembled monolayers (SAMs)
presenting maleimide in standard 384-well format for high-throughput
handling capability. Following immobilization, plates were washed to
remove buffer constituents, enzyme, inhibitor, and any unbound
substrate and analyzed by MALDI mass spectrometry using automated
Molecular Docking analysis. The docking studies were
performed as previously reported with Autodock Vina through
PyRx.37,38 Following the 3D energy minimization of the ligand by
ChemBioDraw 3D, the docking was run in a 25 Å cubic space
encompassing the active site, the binding pocket, and its surrounding.
Cell Viability Assay. DU-145 and Vero cells were maintained in
EMEM and DMEM, respectively, supplemented with 10% FBS and
1% pen/strep, and all other cell lines were maintained in RPMI 1640
supplemented with 10% FBS and 1% pen/strep. DU-145, Vero, and
LNCaP cells were incubated on a 96-wells plate for 24 h prior to a 72
h drug treatment, whereas Jurkat and Jurkat J.γ1 cells were incubated
in media containing the various compounds for 72 h. Cell viability was
measured using the MTS assay according to the manufacturer’s
protocol. The DMSO concentration in the cell media during the cell
viability experiment was maintained at 0.1%.
Western Blot Analysis for Tubulin Acetylation. LNCaP cells
were plated for 24 h and treated with various concentrations of
compounds for 4 h. The cells were washed with PBS buffer and
resuspended in CelLyticM buffer containing a cocktail of protease
inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Following quantifi-
cation through a Bradford protein assay, equal amounts of protein
were loaded onto an SDS-page gel (Bio-Rad, Hercules, CA, USA) and
resolved by electrophoresis at a constant voltage of 100 V for 2 h. The
gel was transferred onto a nitrocellulose membrane and probed for
acetylated tubulin and tubulin as well as actin as a loading control.
MMP Assay. MMP inhibitor profiling kit was purchased from Enzo
Life Sciences (Farmingdale, NY, USA). The selection of MMPs tested
was guided by the reported antiproliferative activity of AG3340, an
MMP inhibitor selective for isoforms 1, 2, 3, 7, 9, 13, and 14.29 Each
MMP was incubated with the inhibitor (10 μM) for 30 min at 37 °C in
the assay buffer. The OmniMMP fluorogenic substrate peptide was
added, and the reaction was allowed to proceed at 37 °C for 30 min.
The fluorescence was measured using a fluorescence plate reader with
excitation at 328 nm and emission at 420 nm.39
Statistical Analysis. The values are reported as the mean SD
from at least two independent experiments performed in triplicate.
The Student’s t test was performed in Excel, and results with p value
less than 5% were considered statistically different.
Representative Procedure for the Conversion of Azidoalka-
nol to Azidoalkyl Methanesulfonate 5. Synthesis of 2-
Azidoethyl Methanesulfonate (5a). To a solution of compound
2-azidoethanol (1.00 g, 11.49 mmol) in THF (25 mL) and
triethylamine (Et3N) (2.418 mL, 17.24 mmol) was added mesyl
chloride (1.328 mL, 17.24 mmol) at 0 °C, and the mixture was allowed
9975
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981

Journal of Medicinal Chemistry
Article
to warm to room temperature. Stirring continued for 3 h, during which
TLC revealed a quantitative conversion into a higher Rf product.
CH2Cl2 (70 mL) and saturated sodium bicarbonate (50 mL) were
added, and the two layers were separated. The organic layer was
washed with sodium bicarbonate (2 × 50 mL) and saturated brine (45
mL) and dried over Na2SO4. Solvent was evaporated off to give crude
compound 5a (1.70 g) as a colorless oil, which was used for next step
without further purification. A similar procedure was used for the
synthesis of 5b−f.
Representative Procedure for the Synthesis of 1-(2-
Azidoalkyl)-3-methoxypyridin-2-ones. Synthesis of 6-1-(2-
Azidoethyl)-3-methoxypyridin-2-one (6a). A mixture of 3-
methoxypyridin-2-one (0.40 g, 3.20 mmol), 5a (0.79 g, 4.80 mmol),
and K2CO3 (1.32 g, 9.60 mmol) in THF was stirred under refluxing
conditions overnight. The reaction mixture was cooled to room
temperature and partitioned between CH2Cl2 (60 mL) and water (50
mL), and the two layers were separated. The organic layer was washed
with water (2 × 35 mL) and brine (1 × 30 mL), dried on Na2SO4, and
evaporated in vacuo. The crude product was purified by flash
chromatography, eluting with a step gradient of acetone in CH2Cl2
(max 15%) to give 0.34 g of 6a (55%) as a colorless oil. 1H NMR (400
MHz, CDCl3) δ 6.77 (dd, J = 6.9, 1.6 Hz, 1H), 6.48 (dd, J = 7.5, 1.6
Hz, 1H), 5.96 (m, 1H), 3.91 (m, 2H), 3.62 (s, 3H), 3.52 (m, 2H). 13C
NMR (100 MHz, CDCl3) δ 157.39, 149.41, 128.61, 112.16, 104.37,
55.36, 49.00, 48.91. HRMS (EI) calcd for C8H10N4O2 [M]+,
194.0804; found, 194.0816.
6f (0.55 g, 48%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.42
(dd, J = 7.8, 1.0 Hz, 2H), 7.31 (m, 3H), 6.85 (dd, J = 6.9, 1.7 Hz, 1H),
6.61 (dd, J = 7.4, 1.7 Hz, 1H), 5.99 (m, 1H), 5.09 (s, 2H), 3.93 (m,
2H), 3.23 (t, J = 6.9 Hz, 2H), 1.74 (m, 2H), 1.55 (m, 2H), 1.34 (m,
6H). 13C NMR (100 MHz, CDCl3) δ 158.32, 149.19, 136.63, 129.15,
128.74, 128.13, 127.52, 115.66, 104.69, 94.66, 70.96, 51.62, 50.03,
29.22, 28.98, 26.77, 26.72.
Representative Procedure for Synthesis of 7. Synthesis of 1-
Phenyltriazolylethyl-3-methoxypyridin-2-one (7a). Phenylacety-
lene (0.17 g, 1.67 mmol) and 6a (0.27 g, 1.36 mmol) were dissolved in
anhydrous THF (10 mL) and stirred under argon at room
temperature. Copper(I) iodide (0.01 g, 0.07 mmol) and Hunig’s
base (0.1 mL) were added to the reaction mixture, and stirring was
continued for 4 h. The reaction mixture was diluted with CH2Cl2 (40
mL) and washed with 1:4 NH4OH/saturated NH4Cl (3 × 30 mL) and
saturated NH4Cl (30 mL). The organic layer was dried over Na2SO4
and concentrated in vacuo. The crude product was triturated with
hexanes to give 366 mg (91%) of white solid 7a. 1H NMR (400 MHz,
CDCl3) δ 7.68 (s, 1H), 7.47 (d, J = 7.5 Hz, 2H), 7.11 (m, 3H), 6.43
(d, J = 7.4 Hz, 1H), 6.35 (dd, J = 6.8, 0.9 Hz, 1H), 5.78 (t, J = 7.2 Hz,
1H), 4.57 (t, J = 5.7 Hz, 2H), 4.25 (t, J = 5.6 Hz, 2H), 3.53 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 157.70, 149.08, 147.21, 129.40,
128.30, 128.08, 127.80, 125.00, 120.91, 113.02, 105.44, 55.15, 49.72,
47.44. HRMS (EI) calcd for C16H16N4O2 [M]+, 296.1273; found,
296.1268.
1-Phenyltriazolylpropyl-3-benzyloxypyridin-2-one (7b). Reaction
of phenylacetylene (0.13 g, 1.25 mmol) and 6b (0.30 g, 1.04 mmol)
within 4 h as described for the synthesis of 7a gave compound 7b
(0.31 g, 77%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.00 (s,
1H), 7.77 (d, J = 7.3 Hz, 2H), 7.27 (m, 8H), 6.88 (d, J = 5.7 Hz, 1H),
6.57 (d, J = 6.4 Hz, 1H), 5.95 (t, J = 7.1 Hz, 1H), 4.99 (s, 2H), 4.34 (t,
J = 6.4 Hz, 2H), 3.94 (t, J = 6.4 Hz, 2H), 2.32 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ 157.92, 148.47, 147.30, 135.73, 130.26, 128.81,
128.49, 128.22, 127.76, 127.71, 127.01, 125.33, 120.16, 114.95, 104.83,
70.33, 47.00, 46.48, 29.30. HRMS (EI) calcd for C23H22N4O2 [M]+,
386.1743; found, 386.1734.
1-(3-Azidopropyl)-3-benzyloxypyridin-2-one (6b). Reaction of 3-
benzyloxypyridin-2-one (0.40 g, 1.99 mmol) and 5b (0.53 g, 2.98
mmol) within 16 h as described for the synthesis of 6a gave compound
1
6b (0.30 g, 53%) as a colorless oil. H NMR (300 MHz, CDCl3) δ
7.28 (m, 5H), 6.84 (dd, J = 6.9, 1.6 Hz, 1H), 6.60 (dd, J = 7.4, 1.5 Hz,
1H), 5.98 (t, J = 7.1 Hz, 1H), 4.96 (s, 2H), 3.97 (t, J = 6.8 Hz, 2H),
3.28 (t, J = 6.5 Hz, 2H), 1.98 (p, J = 6.7 Hz, 2H). 13C NMR (75 MHz,
CDCl3) δ 157.78, 148.65, 135.93, 128.85, 128.24, 127.69, 127.03,
115.16, 104.53, 70.39, 48.14, 47.06, 27.66. HRMS (EI) calcd for
C15H16N4O2 [M]+, 284.1273; found, 284.1271.
1-(4-Azidobutyl)-3-benzyloxypyridin-2-one (6c). Reaction of 3-
benzyloxypyridin-2-one (0.40 g, 1.99 mmol) and 5c (0.58 g, 2.98
mmol) within 16 h as described for the synthesis of 6a gave compound
6c (0.36 g, 62%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.22
(m, 5H), 6.78 (dd, J = 6.9, 1.7 Hz, 1H), 6.54 (dd, J = 7.4, 1.7 Hz, 1H),
5.91 (m, 1H), 4.96 (s, 2H), 3.85 (t, J = 7.2 Hz, 2H), 3.17 (t, J = 6.8
Hz, 2H), 1.71 (m, 2H), 1.48 (m, 2H). 13C NMR (100 MHz, CDCl3) δ
157.52, 148.27, 135.78, 128.37, 127.98, 127.42, 126.82, 114.92, 104.32,
70.09, 50.43, 48.46, 25.83, 25.42. HRMS (EI) calcd for C16H18N4O2
[M]+, 298.1430; found, 298.1446.
1-Phenyltriazolylbutyl-3-benzyloxypyridin-2-one (7c). Reaction of
phenylacetylene (0.12 g, 1.18 mmol) and 6c (0.30 g, 1.01 mmol)
within 4 h as described for the synthesis of 7a gave compound 7c
(0.31 g, 77%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.83 (s,
1H), 7.76 (m, 2H), 7.26 (m, 8H), 6.76 (dd, J = 6.9, 1.5 Hz, 1H), 6.56
(dd, J = 7.4, 1.5 Hz, 1H), 5.91 (t, J = 7.1 Hz, 1H), 4.99 (s, 2H), 4.31
(m, 2H), 3.89 (t, J = 7.1 Hz, 2H), 1.85 (m, 2H), 1.67 (m, 2H). 13C
NMR (100 MHz, CDCl3) δ 157.71, 148.38, 147.20, 135.83, 130.29,
128.45, 128.41, 128.16, 127.68, 127.63, 126.96, 125.25, 119.87, 114.99,
104.60, 70.26, 49.11, 48.02, 26.78, 25.65. HRMS (EI) calcd for
C24H24N4O2 [M]+, 400.1899; found, 400.1888.
1-(5-Azidopentyl)-3-benzyloxypyridin-2-one (6d). Reaction of 3-
benzyloxypyridin-2-one (0.50 g, 2.49 mmol) and 5d (0.62 g, 2.98
mmol) within 16 h as described for the synthesis of 6a gave compound
1-Phenyltriazolylpentyl-3-benzyloxypyridin-2-one (7d). Reaction
of phenylacetylene (0.04 g, 0.39 mmol) and 6d (0.10 g, 0.32 mmol)
within 4 h as described for the synthesis of 7a gave compound 7d
1
6d (0.49 g, 63%) as a colorless oil. H NMR (400 MHz, CDCl3) δ
1
7.35 (d, J = 8.0 Hz, 2H), 7.23 (m, 3H), 6.81 (m, 1H), 6.56 (m, 1H),
5.94 (m, 1H), 5.01 (s, 2H), 3.85 (m, 2H), 3.18 (m, 2H), 1.71 (m, 2H),
1.54 (m, 2H), 1.32 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 157.68,
148.47, 135.98, 128.60, 128.12, 127.53, 126.93, 115.09, 104.32, 70.27,
50.77, 49.21, 28.19, 28.06, 23.37. HRMS (EI) calcd for C17H20N4O2
[M]+, 312.1586; found, 312.1589.
(0.09 g, 72%) as a white solid. H NMR (400 MHz, CDCl3) δ 7.78
(m, 3H), 7.30 (m, 8H), 6.79 (d, J = 6.8 Hz, 1H), 6.57 (d, J = 7.3 Hz,
1H), 5.94 (t, J = 7.1 Hz, 1H), 5.02 (s, 2H), 4.31 (t, J = 7.0 Hz, 2H),
3.87 (t, J = 7.2 Hz, 2H), 1.91 (m, 2H), 1.74 (m, 2H), 1.30 (m, 2H).
13C NMR (100 MHz, CDCl3) δ 157.80, 148.57, 147.39, 136.02,
130.44, 128.64, 128.56, 128.27, 127.81, 127.71, 127.05, 125.40, 119.57,
115.12, 104.49, 70.40, 49.71, 49.06, 29.42, 28.03, 23.05.
1-Phenyltriazolylhexyl-3-benzyloxypyridin-2-one (7e). Reaction of
phenylacetylene (0.08 g, 0.78 mmol) and 6e (0.21 g, 0.65 mmol)
within 4 h as described for the synthesis of 7a gave compound 7e
(0.16 g, 58%) as a white solid. NMR (400 MHz, CDCl3) δ 7.66 (m,
3H), 7.20 (m, 8H), 6.68 (dd, J = 6.9, 1.5 Hz, 1H), 6.46 (dd, J = 7.4, 1.5
Hz, 1H), 5.82 (t, J = 7.1 Hz, 1H), 4.92 (s, 2H), 4.18 (t, J = 6.8 Hz,
2H), 3.75 (t, J = 6.8 Hz, 2H), 1.76 (m, 2H), 1.56 (m, 2H), 1.19 (m,
4H). 13C NMR (100 MHz, CDCl3) δ 157.64, 148.46, 147.27, 135.95,
130.36, 128.57, 128.48, 128.19, 127.72, 127.60, 126.96, 125.32, 119.37,
115.02, 104.37 70.45, 50.03, 49.35, 29.90, 28.66, 25.87, 25.78. HRMS
(FAB) calcd for C26H29N4O2 [M + H]+, 429.2290; found, 429.2291.
1-Phenyltriazolylheptyl-3-benzyloxypyridin-2-one (7f). Reaction
of phenylacetylene (0.09 g, 0.88 mmol) and 6f (0.25 g, 0.74 mmol)
1-(6-Azidohexyl)-3-benzyloxypyridin-2-one (6e). Reaction of 3-
benzyloxypyridin-2-one (0.50 g, 2.49 mmol) and 5e (0.66 g, 2.98
mmol) within 16 h as described for the synthesis of 6a gave compound
1
6e (0.48 g, 60%) as a colorless oil. H NMR (400 MHz, CDCl3) δ
7.15 (m, 5H), 6.65 (dd, J = 7.4, 1.8 Hz, 1H), 6.45 (dd, J = 7.4, 1.8 Hz,
1H), 5.81 (t, J = 7.1 Hz, 1H), 4.92 (s, 2H), 3.76 (t, J = 6.8 Hz, 2H),
3.05 (t, J = 6.8 Hz, 2H), 1.58 (m, 2H), 1.40 (m, 2H), 1.90 (m, 4H).
13C NMR (100 MHz, CDCl3) δ 157.55, 148.25, 135.95, 128.60,
128.10, 126.95, 126.50, 115.05, 104.90, 70.50, 51.05, 49.20, 28.65,
28.50, 26.20, 25.80. HRMS (FAB) calcd for C18H23N4O2 [M + H]+,
327.1821; found, 327.1848.
1-(7-Azidoheptyl)-3-benzyloxypyridin-2-one (6f). Reaction of 3-
benzyloxypyridin-2-one (0.79 g, 3.38 mmol) and 5f (0.81 g, 4.05
mmol) within 16 h as described for the synthesis of 6a gave compound
9976
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981

Journal of Medicinal Chemistry
Article
within 4 h as described for the synthesis of 7a gave compound 7f (0.23
g, 70%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.79 (m, 3H),
7.31 (m, 8H), 6.83 (dd, J = 6.9, 1.7 Hz, 1H), 6.60 (dd, J = 7.4, 1.7 Hz,
1H), 5.97 (t, J = 7.1 Hz, 1H), 5.07 (s, 2H), 4.35 (t, J = 7.1 Hz, 2H),
3.91 (m, 2H), 1.88 (m, 2H), 1.71 (m, 2H), 1.32 (d, J = 10.0 Hz, 6H).
13C NMR (100 MHz, CDCl3) δ 158.31, 149.13, 147.92, 136.57,
130.93, 129.15, 129.04, 128.74, 128.28, 128.15, 127.51, 125.90, 125.86,
119.75, 115.60, 104.79, 70.92, 50.52, 49.90, 30.42, 29.13, 28.72, 26.53,
26.46. HRMS (FAB) calcd for C27H31N4O2 [M + H]+, 443.2447;
found, 443.2494.
synthesis of 8b gave 8f (0.13 g, 74%). 1H NMR (400 MHz, DMSO) δ
8.91 (s, 1H), 8.57 (s, 1H), 7.82 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.6 Hz,
2H), 7.31 (t, J = 7.3 Hz, 1H), 7.09 (d, J = 6.6 Hz, 1H), 6.64 (d, J = 7.0
Hz, 1H), 6.04 (t, J = 6.9 Hz, 1H), 4.36 (t, J = 7.0 Hz, 2H), 3.86 (t, J =
7.2 Hz, 2H), 1.83 (m, 2H), 1.61 (m, 2H), 1.26 (m, 6H). 13C NMR
(100 MHz, CDCl3) δ 158.80, 147.89, 147.00, 130.88, 129.05, 128.30,
127.12, 125.87, 119.83, 114.13, 107.10, 50.48, 50.05, 30.37, 29.20,
28.67, 26.46, 26.42. HRMS (FAB) calcd for C20H25N4O2 [M + H]+,
353.1977; found, 353.1992.
1-Phenyltriazolylethyl-3-methoxypyridine-2-thione (9). To a
stirring solution of Lawesson’s reagent (0.08 g, 0.19 mmol) in toluene
(10 mL) was added starting material 7a (0.10g, 0.33 mmol), and the
reaction mixture was heated at refluxing temperature overnight. The
reaction mixture was cooled to room temperature, and the solvent was
evaporated under reduced pressure. The crude product was purified by
preparative TLC, eluting with CHCl3/acetone/EtOH (10:1:0.2) to
give 9 (0.07 g, 68%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ
7.69 (d, J = 7.4 Hz, 2H), 7.56 (s, 1H), 7.35 (t, J = 7.5 Hz, 2H), 7.28
(m, 1H), 6.97 (d, J = 6.6 Hz, 1H), 6.62 (d, J = 7.8 Hz, 1H), 6.37 (m,
1H), 5.13 (t, J = 5.6 Hz, 2H), 5.03 (t, J = 5.6 Hz, 2H), 3.86 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 171.92, 158.94, 147.52, 132.68,
129.92, 128.69, 128.14, 125.43, 120.96, 111.72, 110.22, 56.72, 56.63,
46.38. HRMS (EI) calcd for C16H16N4OS [M]+, 312.1045; found,
312.1039.
1-Phenyltriazolylethyl-3-hydroxypyridin-2-one (8a). To a solution
of 7a (0.10 g, 0.34 mmol) in dry CH2Cl2 (8 mL) was slowly added 1
M BBr3 (1.2 equiv) at −30 °C under an argon atmosphere. The
reaction mixture was stirred for 48 h at room temperature. The
mixture was again cooled to −30 °C, and MeOH (5 mL) was slowly
added to the mixture. After evaporation of the solvent, the residue was
adjusted to pH 7 with 1 M NaOH and then extracted with CHCl3 (30
mL × 3). The combined organic layers were dried over Na2SO4 to give
0.08 g (83%) of 8a as slightly brownish solid without any further
1
purification required. H NMR (400 MHz, DMSO) δ 9.11 (s, 1H),
8.51 (s, 1H), 7.78 (d, J = 7.3 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.31 (t,
J = 7.3 Hz, 1H), 6.75 (d, J = 5.9 Hz, 1H), 6.64 (d, J = 6.0 Hz, 1H),
5.95 (t, J = 7.0 Hz, 1H), 4.76 (t, J = 5.5 Hz, 2H), 4.42 (t, J = 5.6 Hz,
2H). 13C NMR (100 MHz, DMSO) δ 158.25, 147.17, 146.77, 131.11,
129.34, 128.41, 128.31, 125.54, 122.23, 115.38, 105.76, 49.42, 48.33.
HRMS (EI) calcd for C15H14N4O2 [M]+, 282.1117; found, 282.1120.
1-Phenyltriazolylpropyl-3-hydroxypyridin-2-one (8b). To a sol-
ution of 7b (0.29 g, 0.75 mmol) in CH2Cl2/EtOAc/MeOH (2:2:1, 10
mL) was added 10% Pd on carbon (15 mg). The reaction was stirred
under ballon hydrogen pressure for 5 h. The reaction mixture was
filtered, and the solvent was evaporated. Column chromatography
eluting with a step gradient of acetone in CH2Cl2 (max 20%) gave
1-Phenyltriazolylethyl-3-hydroxypyridine-2-thione (10a). To a
solution of 9 (0.06 g, 0.19 mmol) in dry CH2Cl2 (8 mL) at −30 °C
was slowly added 1 M BBr3 in CH2Cl2 (0.22 mmol) under an argon
atmosphere, and the reaction mixture was subsequently stirred for 48 h
at room temperature. The mixture was cooled to −30 °C, and MeOH
(5 mL) was slowly added. The solvent was evaporated off, and the
residue was adjusted to pH 7 with aqueous 1 M NaOH and then
extracted with CHCl3 (3 × 30 mL). The combined organic layers were
dried over Na2SO4, and the solvent was evaporated in vacuo to yield
1
pure 8b (0.19 g, 85%) as a slightly brownish solid. H NMR (400
1
MHz, DMSO) δ 9.03 (s, 1H), 8.62 (s, 1H), 7.82 (d, J = 7.6 Hz, 2H),
7.38 (m, 3H), 7.15 (d, J = 6.6 Hz, 1H), 6.68 (d, J = 6.4 Hz, 1H), 6.11
(t, J = 6.7 Hz, 1H), 4.43 (t, J = 6.4 Hz, 2H), 3.99 (m, 2H), 2.27 (m,
2H). 13C NMR (100 MHz, CDCl3) δ 147.49, 129.71, 128.52, 127.99,
125.29, 120.42, 107.98, 66.61, 47.10, 29.27. HRMS (EI) calcd for
C16H16N4O2 [M]+, 296.1273; found, 296.1274.
1-Phenyltriazolylbutyl-3-hydroxypyridin-2-one (8c). Reaction of
7c (0.25 g, 0.62 mmol) in anhydrous THF as described for the
synthesis of 8b gave 8c (0.12 g, 63%). 1H NMR (400 MHz, CDCl3) δ
7.79 (m, 3H), 7.41 (t, J = 7.6 Hz, 2H), 7.32 (t, J = 7.4 Hz, 1H), 6.76
(m, 3H), 6.13 (t, J = 7.2 Hz, 1H), 4.45 (t, J = 6.8 Hz, 2H), 4.02 (t, J =
7.1 Hz, 2H), 1.99 (m, 2H), 1.82 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 158.47, 147.63, 146.62, 130.41, 128.69, 127.99, 126.60,
125.53, 119.80, 114.15, 107.10, 49.38, 48.57, 27.02, 26.01. HRMS (EI)
calcd for C17H18N4O2 [M]+, 310.1430; found, 310.1425.
1-Phenyltriazolylpentyl-3-hydroxypyridin-2-one (8d). Reaction of
7d (0.09 g, 0.20 mmol) in anhydrous THF as described for the
synthesis of 8b gave 8d (0.05 g, 77%). 1H NMR (400 MHz, DMSO) δ
8.56 (s, 1H), 7.82 (d, J = 8.1 Hz, 2H), 7.43 (t, J = 7.7 Hz, 2H), 7.32 (t,
J = 7.4 Hz, 1H), 7.10 (dd, J = 6.8, 1.6 Hz, 1H), 6.65 (dd, J = 7.2, 1.5
Hz, 1H), 6.05 (t, J = 7.0 Hz, 1H), 4.38 (t, J = 7.0 Hz, 2H), 3.89 (t, J =
7.2 Hz, 2H), 1.89 (m, 2H), 1.67 (m, 2H), 1.25 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ 158.44, 147.67, 146.61, 130.51, 128.72, 128.01,
126.71, 125.57, 119.54, 113.81, 106.86, 49.86, 49.38, 29.58, 28.31,
23.19. HRMS (FAB) calcd for C18H21N4O2 [M + H]+, 325.1664;
found, 325.1683.
1-Phenyltriazolylhexyl-3-hydroxypyridin-2-one (8e). Reaction of
7e (0.15 g, 0.35 mmol) in anhydrous THF as described for the
synthesis of 8b gave 8e (0.081 g, 69%). 1H NMR (400 MHz, CDCl3)
δ 7.81 (m, 3H), 7.42 (m, 2H), 7.32 (m, 1H), 6.77 (m, 2H), 6.12 (t, J =
7.1 Hz, 1H), 4.38 (t, J = 7.1 Hz, 2H), 3.95 (m, 2H), 1.95 (m, 2H),
1.76 (d, J = 7.0 Hz, 2H), 1.39 (m, 4H). 13C NMR (100 MHz, CDCl3)
δ 158.20, 147.25, 146.40, 130.30, 128.60, 127.85, 126.65, 125.40,
119.65, 113.90, 106.80, 50.20, 49.60, 30.05, 28.95, 26.00, 25.90. HRMS
(FAB) calcd for C19H23N4O2 [M + H]+, 339.1821; found, 339.1814.
1-Phenyltriazolylheptyl-3-hydroxypyridin-2-one (8f). Reaction of
7f (0.22 g, 0.50 mmol) in anhydrous THF as described for the
compound 10a (0.06 g, 93%) as a dark violet solid. tR 8.1 min. H
NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 7.73 (d, J = 7.3 Hz, 2H),
7.51 (s, 1H), 7.40 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.3 Hz, 1H), 6.95
(dd, J = 9.2, 7.2 Hz, 2H), 6.46 (m, 1H), 5.08 (m, 4H). 13C NMR (100
MHz, CDCl3) δ 168.47, 155.10, 147.75, 132.00, 129.82, 128.77,
128.30, 125.53, 120.90, 113.66, 112.61, 57.45, 46.41. HRMS (EI) calcd
for C15H14N4OS [M]+, 298.0888; found, 298.0888.
1-Phenyltriazolylpropyl-3-hydroxypyridine-2-thione (10b). Com-
pound 8b (0.06 g, 0.20 mmol) was ground together with P4S10 (0.05g,
0.11 mmol) in a mortar and pestle to form a gray powder. The powder
was stirred under argon in a flask fitted with a condenser and heated at
175 °C for 2 h. The reaction flask was covered with aluminum foil
during the reaction. After 2 h of reaction, the flask was cooled to room
temperature, 10% MeOH in CH2Cl2 (25 mL) was added, and stirring
was continued for another 15 min. The reaction mixture then washed
with water (2 × 30 mL), the organic layer was dried with Na2SO4, and
the solvent was evaporated in vacuo. The crude product was purified
by preparative TLC, eluting with 8% MeOH in CH2Cl2 to give 10b
(0.04 g, 64%) as an olive green solid. tR 8.2 min. 1H NMR (400 MHz,
CDCl3) δ 8.47 (s, 1H), 7.82 (m, 3H), 7.50 (d, J = 6.5 Hz, 1H), 7.43 (t,
J = 7.5 Hz, 2H), 7.34 (t, J = 7.4 Hz, 1H), 6.97 (m, 1H), 6.66 (m, 1H),
4.62 (t, J = 6.9 Hz, 2H), 4.48 (m, 2H), 2.65 (m, 2H). 13C NMR (100
MHz, CDCl3) δ 168.59, 155.22, 148.03, 131.85, 130.17, 128.84,
128.29, 125.61, 119.84, 113.87, 112.28, 54.99, 46.95, 28.02. HRMS
(EI) calcd for C16H16N4OS [M]+, 312.1045; found, 312.1060.
1-Phenyltriazolylbutyl-3-hydroxypyridine-2-thione (10c). Reac-
tion of 8c (0.06 g, 0.18 mmol) and P4S10 (0.05 g, 0.11 mmol)
under neat conditions as described for the synthesis of 10b gave
1
compound 10c (0.03 g, 51%) as an olive green solid. tR 8.7 min. H
NMR (400 MHz, CDCl3) δ 8.50 (s, 1H), 7.81 (m, 3H), 7.36 (m, 4H),
6.94 (d, J = 7.6 Hz, 1H), 6.62 (t, J = 6.8 Hz, 1H), 4.54 (t, J = 7.6 Hz,
2H), 4.46 (t, J = 6.4 Hz, 2H), 2.03 (m, 4H). 13C NMR (100 MHz,
CDCl3) δ 168.51, 155.08, 147.73, 131.02, 130.36, 128.79, 128.13,
125.58, 119.91, 113.87, 112.10, 56.96, 49.41, 27.02, 25.08. HRMS (EI)
calcd for C17H18N4OS [M]+, 326.1201; found, 326.1200.
1-Phenyltriazolylpentyl-3-hydroxypyridine-2-thione (10d). Reac-
tion of 8d (0.03 g, 0.08 mmol) and P4S10 (0.02 g, 0.05 mmol) under
9977
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981

Journal of Medicinal Chemistry
Article
neat conditions as described for the synthesis of 10b gave compound
10d (0.02 g, 73%) as an olive green solid. tR 9.6 min. H NMR (400
NMR (101 MHz, CDCl3) δ 157.53, 149.61, 147.34, 138.06, 130.18,
128.45, 128.34, 127.88, 125.94, 122.39, 119.49, 111.79, 104.55, 55.43,
49.58, 48.77, 29.31, 27.91, 22.87, 21.07.
1
MHz, DMSO) δ 8.55 (d, J = 2.9 Hz, 2H), 7.83 (t, J = 7.8 Hz, 3H),
7.43 (q, J = 7.4 Hz, 2H), 7.32 (t, J = 6.6 Hz, 1H), 7.08 (m, 1H), 6.92
(dd, J = 31.0, 6.8 Hz, 1H), 6.81 (m, 1H), 4.51 (m, 2H), 4.41 (t, J = 7.0
Hz, 2H), 1.89 (m, 4H), 1.32 (m, 2H). 13C NMR (100 MHz, CDCl3) δ
168.37, 155.10, 147.66, 131.04, 130.46, 128.75, 128.05, 125.57, 119.68,
113.69, 111.96, 57.73, 49.73, 29.47, 27.12, 23.04. HRMS (EI) calcd for
C18H20N4OS [M]+, 340.1358; found, 340.1364.
1-Phenyltriazolylhexyl-3-hydroxypyridine-2-thione (10e). Reac-
tion of 8e (0.18 g, 0.53 mmol) and P4S10 (0.12 g, 0.27 mmol)
under neat conditions as described for the synthesis of 10b gave
compound 10e (0.08 g, 43%) as an olive green semisolid. tR 10.8 min.
1H NMR (400 MHz, CDCl3) δ 8.57 (s, 1H), 7.83 (d, J = 7.5 Hz, 2H),
7.76 (s, 1H), 7.41 (t, J = 7.5 Hz, 2H), 7.32 (m, 2H), 6.94 (m, 1H),
6.72 (m, 1H), 4.49 (m, 2H), 4.40 (t, J = 6.9 Hz, 2H), 1.97 (m, 4H),
1.36 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 164.38, 162.90, 147.70,
130.54, 128.76, 128.03, 126.64, 125.61, 119.51, 118.75, 118.29, 59.74,
50.10, 29.91, 27.99, 25.82, 25.65. HRMS (ESI) calcd for C19H23N4OS
[M + H]+, 355.1587; found, 355.1632.
3-Methoxy-1-(5-(4-(o-tolyl)-1H-1,2,3-triazol-1-yl)pentyl)pyridin-2-
one (12c). 1-Ethynyl-2-methylbenzene (0.12 g, 1.06 mmol) and 11
(0.21 g, 0.89 mmol) were reacted as described for the synthesis of 12a
to give compound 12c (0.21 g, 68%). 1H NMR (400 MHz, CDCl3) δ
7.72−7.62 (m, 1H), 7.61 (s, 1H), 7.16 (d, J = 3.1 Hz, 3H), 6.75 (dd, J
= 6.9, 1.7 Hz, 1H), 6.48 (dd, J = 7.5, 1.6 Hz, 1H), 6.04−5.91 (m, 1H),
4.30 (t, J = 7.1 Hz, 2H), 3.85 (t, J = 7.2 Hz, 2H), 3.66 (s, 3H), 2.36 (s,
3H), 1.94−1.84 (m, 2H), 1.77−1.63 (m, 2H), 1.37−1.21 (m, 2H). 13C
NMR (101 MHz, CDCl3) δ 157.89, 149.98, 146.89, 135.44, 130.81,
130.01, 128.78, 128.00, 125.99, 121.93, 112.14, 104.87, 55.76, 49.86,
49.12, 31.91, 29.67, 28.25, 23.26, 21.37.
4-(1-(5-(3-Methoxy-2-oxopyridin-1(2H)-yl)pentyl)-1H-1,2,3-tria-
zol-4-yl)benzonitrile (12d). 4-Ethynylbenzonitrile (0.13 g, 1.05 mmol)
and 11 (0.21 g, 0.87 mmol) were reacted as described for the synthesis
1
of 12a to give compound 12d (0.30 g, 95%). H NMR (400 MHz,
CDCl3) δ 7.96 (s, 1H), 7.81 (d, J = 8.7 Hz, 2H), 7.53 (d, J = 8.7 Hz,
2H), 6.73 (dd, J = 6.9, 1.7 Hz, 1H), 6.46 (dd, J = 7.5, 1.6 Hz, 1H),
5.98−5.90 (m, 1H), 4.28 (t, J = 7.1 Hz, 2H), 3.80 (t, J = 7.2 Hz, 2H),
3.62 (s, 3H), 1.90−1.77 (m, 2H), 1.71−1.59 (m, 2H), 1.31−1.15 (m,
2H). 13C NMR (101 MHz, CDCl3) δ 157.42, 149.48, 145.25, 134.76,
132.15, 127.79, 125.57, 120.99, 118.37, 111.83, 110.57, 104.52, 55.36,
49.63, 48.56, 29.04, 27.79, 22.71.
1-Phenyltriazolylheptyl-3-hydroxypyridine-2-thione (10f). Reac-
tion of 8f (0.12 g, 0.35 mmol) and P4S10 (0.08 g, 0.17 mmol) under
neat conditions as described for the synthesis of 10b gave compound
1
10f (0.05 g, 43%) as an olive green semisolid. tR 12.1 min. H NMR
(400 MHz, DMSO) δ 8.55 (m, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.65 (d,
J = 6.2 Hz, 1H), 7.42 (m, 2H), 7.30 (m, 1H), 7.02 (m, 1H), 6.85 (m,
1H), 4.50 (m, 2H), 4.36 (t, J = 7.1 Hz, 2H), 1.85 (d, J = 7.0 Hz, 4H),
1.33 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 147.76, 130.66, 128.76,
130.39, 128.98, 128.80, 128.06, 125.97, 125.67, 119.46, 60.10, 50.25,
30.11, 29.67, 28.33, 26.13, 26.10. HRMS (FAB) calcd for C20H25N4OS
[M + H]+, 369.1749; found, 369.1762.
1-(5-Azidopentyl)-3-methoxypyridin-2-one (11). 3-Methoxypyri-
din-2-one (1.8g, 14.1 mmol) and 5-azidopentyl methanesulfonate 5d
(3.5g, 16.9 mmol) were dissolved in 2:1 (THF/MeOH) and stirred for
48 h at 80 °C. Upon completion of the reaction, EtOAc was added,
and the organic layer was washed with H2O and brine and dried to
yield the crude product (2.42 g, 73%). The crude product was washed
with petroleum ether, yielding 11 (1.96 g, 59%). 1H NMR (400 MHz,
CDCl3) δ 6.74 (dd, J = 6.9, 1.6 Hz, 1H), 6.46 (dd, J = 7.4, 1.6 Hz, 1H),
5.95 (t, J = 7.1 Hz, 1H), 3.81 (t, J = 7.3 Hz, 2H), 3.64 (s, 3H), 3.11 (t,
J = 6.8 Hz, 2H), 1.67−1.57 (m, 2H), 1.51−1.41 (m, 2H), 1.31−1.20
(m, 2H). 13C NMR (101 MHz, CDCl3) δ 157.46, 149.66, 127.87,
111.68, 104.34, 55.38, 50.72, 49.00, 28.14, 28.02, 23.28.
3-Methoxy-1-(5-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)pentyl)pyridin-2-
one (12a). 1-Ethynyl-4-methylbenzene (0.14 g, 1.22 mmol) and 11
(0.24 g, 1.02 mmol) were dissolved in a 1:1 anhydrous THF/DMSO
(10 mL) and stirred under argon at room temperature. Copper(I)
iodide (0.01 g, 0.07 mmol) and Hunig’s base (0.1 mL) were then
added to the reaction mixture, and stirring was continued for 4 h. The
reaction mixture was diluted with CH2Cl2 (40 mL) and washed with
1:4 NH4OH/saturated NH4Cl (3 × 30 mL) and saturated NH4Cl (30
mL). The organic layer was dried over Na2SO4 and concentrated in
vacuo. The crude product was purified by preparative TLC with 12:1
CH2Cl2/MeOH, resulting in 12a (0.39 g, quantitative yield). 1H NMR
(400 MHz, cdcl3) δ 7.69 (s, 1H), 7.58 (d, J = 8.2 Hz, 2H), 7.07 (d, J =
8.4 Hz, 2H), 6.70 (dd, J = 6.9, 1.6 Hz, 1H), 6.43 (dd, J = 7.4, 1.6 Hz,
1H), 5.92 (t, J = 7.1 Hz, 1H), 4.20 (t, J = 7.1 Hz, 2H), 3.77 (t, J = 7.2
Hz, 2H), 3.62 (s, 3H), 2.22 (s, 3H), 1.87−1.72 (m, 2H), 1.68−1.52
(m, 2H), 1.29−0.89 (m, 2H). 13C NMR (101 MHz, cdcl3) δ 157.77,
149.83, 147.52, 137.70, 129.38, 128.20, 127.80, 125.43, 119.54, 112.10,
104.80, 55.69, 49.81, 49.02, 29.57, 28.17, 23.14, 21.19.
3-(1-(5-(3-Methoxy-2-oxopyridin-1(2H)-yl)pentyl)-1H-1,2,3-tria-
zol-4-yl)benzonitrile (12e). 3-Ethynylbenzonitrile 16 (see the
Supporting Information) (0.13 g, 1.05 mmol) and 11 (0.21 g, 0.87
mmol) were reacted as described for the synthesis of 12a to give
1
compound 12e (0.31 g, 98%). H NMR (400 MHz, CDCl3) δ 8.01−
7.97 (m, 1H), 7.96 (s, 1H), 7.94 (dt, J = 7.7, 1.5 Hz, 1H), 7.43 (dt, J =
7.7, 1.5 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 6.74 (dd, J = 6.9, 1.7 Hz,
1H), 6.46 (dd, J = 7.5, 1.6 Hz, 1H), 6.02−5.83 (m, 1H), 4.29 (t, J = 7.1
Hz, 2H), 3.80 (t, J = 7.2 Hz, 2H), 3.62 (s, 3H), 1.94−1.78 (m, 2H),
1.74−1.57 (m, 2H), 1.31−1.15 (m, 2H). 13C NMR (101 MHz,
CDCl3) δ 157.74, 149.79, 145.29, 132.00, 131.09, 129.67, 129.60,
128.83, 128.14, 120.82, 118.50, 112.63, 112.14, 104.85, 55.68, 49.98,
48.93, 29.43, 28.14, 23.07.
1-(5-(4-(4-(Dimethylamino)phenyl)-1H-1,2,3-triazol-1-yl)pentyl)-
3-methoxypyridin-2-one (12f). 4-Ethynyl-N,N-dimethylaniline (0.15
g, 1.02 mmol) and 11 (0.20 g, 0.85 mmol) were reacted as described
for the synthesis of 12a to give compound 12f (0.27 g, 84%). 1H NMR
(400 MHz, CDCl3) δ 7.55 (d, J = 2.4 Hz, 2H), 7.53 (s, 1H), 6.68 (d, J
= 6.9 Hz, 1H), 6.59 (d, J = 9.0 Hz, 2H), 6.42 (d, J = 7.5 Hz, 1H),
5.93−5.87 (m, 1H), 4.15 (t, J = 7.1 Hz, 2H), 3.74 (t, J = 7.2 Hz, 2H),
3.60 (s, 3H), 2.80 (s, 6H), 1.81−1.71 (m, 2H), 1.64−1.53 (m, 2H),
1.22−1.07 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 157.40, 149.82,
149.43, 147.58, 127.87, 126.10, 118.38, 117.97, 111.94, 111.81, 104.44,
55.32, 49.35, 48.68, 39.94, 29.19, 27.80, 22.78.
4-(1-(5-(3-Methoxy-2-oxopyridin-1(2H)-yl)pentyl)-1H-1,2,3-tria-
zol-4-yl)-2-(trifluoromethyl)benzonitrile (12g). 4-Ethynyl-2-
(trifluoromethyl)benzonitrile (0.09 g, 0.45 mmol) 18 (see the
Supporting Information) and 11 (0.13 g, 0.54 mmol) were reacted
as described for the synthesis of 12a to give compound 12g (0.19 g,
1
98%). H NMR (400 MHz, CDCl3) δ 8.25 (s, 1H), 8.12 (d, J = 5.2
Hz, 2H), 7.83 (d, J = 8.1 Hz, 1H), 6.81 (dd, J = 6.9, 1.5 Hz, 1H), 6.55
(dd, J = 7.4, 1.5 Hz, 1H), 6.06 (t, J = 7.1 Hz, 1H), 4.41 (t, J = 7.1 Hz,
2H), 3.91 (t, J = 7.2 Hz, 2H), 3.73 (d, J = 5.8 Hz, 3H), 2.07−1.91 (m,
2H), 1.83−1.68 (m, 2H), 1.41−1.27 (m, 2H). 13C NMR (101 MHz,
CDCl3) δ 157.80, 149.88, 144.44, 135.56, 135.11, 128.51, 127.94,
123.40, 121.75, 120.79, 115.41, 111.99, 108.22, 104.87, 55.64, 50.05,
48.84, 29.25, 28.03, 23.55, 22.92.
3-Methoxy-1-(5-(4-(m-tolyl)-1H-1,2,3-triazol-1-yl)pentyl)pyridin-
2-one (12b). 1-Ethynyl-3-methylbenzene (0.12 g, 1.05 mmol) and 11
(0.21 g, 0.88 mmol) were reacted as described for the synthesis of 12a
1-(4-Tolyl)triazolylpentyl-3-methoxypyridine-2-thione (13a). A
suspension of 12a (0.24 g, 0.67 mmol) and Lawesson’s reagent
(0.16 g, 0.40 mmol) in toluene (12 mL) was heated at reflux for 12 h.
The reaction mixture was cooled to room temperature, and the solvent
was evaporated in vacuo. The crude solid was purified on preparative
TLC, eluting with CH2Cl2/ acetone/MeOH (5:1:0.2) to give 13a
1
to give compound 12b (0.3 g, 97%) as a yellow oil. H NMR (400
MHz, CDCl3) δ 7.74 (s, 1H), 7.59 (s, 1H), 7.52 (d, J = 7.7 Hz, 1H),
7.20 (t, J = 7.6 Hz, 1H), 7.04 (d, J = 7.6 Hz, 1H), 6.74 (dd, J = 6.9, 1.7
Hz, 1H), 6.48 (dd, J = 7.5, 1.6 Hz, 1H), 6.05−5.90 (m, 1H), 4.27 (t, J
= 7.1 Hz, 2H), 3.83 (t, J = 7.2 Hz, 2H), 3.67 (s, 3H), 2.29 (s, 3H),
1.92−1.79 (m, 2H), 1.74−1.62 (m, 2H), 1.34−1.17 (m, 2H). 13C
1
(0.23 g, 95%) as a yellow solid. H NMR (400 MHz, CDCl3) δ 7.77
(s, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.33 (m, 1H), 7.25 (dd, J = 11.4, 5.2
9978
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981

Journal of Medicinal Chemistry
Article
Hz, 2H), 6.66 (d, J = 7.8 Hz, 1H), 6.59 (dd, J = 7.8, 6.5 Hz, 1H), 4.60
(m, 2H), 4.43 (t, J = 6.9 Hz, 2H), 3.91 (s, 3H), 2.38 (s, 3H), 2.01 (m,
4H), 1.43 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 171.03, 158.50,
147.11, 137.37, 131.77, 129.02, 127.33, 125.03, 119.32, 111.60, 109.78,
56.27, 49.28, 30.48, 29.05, 26.51, 22.57, 20.82. HRMS (EI) calcd for
C20H24N4OS [M]+, 368.1671; found, 368.1667.
1-(3-Tolyl)triazolylpentyl-3-methoxypyridine-2-thione (13b). Re-
action of 12b (0.14 g, 0.39 mmol) and Lawesson’s reagent (0.09 g,
0.22 mmol) in toluene (10 mL) within 12 h as described for the
synthesis of 13a gave compound 13b (0.10 g, 73%) as a yellow solid.
1H NMR (400 MHz, CDCl3) δ 7.78 (s, 1H), 7.62 (s, 1H), 7.54 (d, J =
7.7 Hz, 1H), 7.26 (m, 2H), 7.07 (d, J = 7.6 Hz, 1H), 6.59 (m, 1H),
6.52 (dd, J = 7.8, 6.5 Hz, 1H), 4.50 (m, 2H), 4.34 (t, J = 7.0 Hz, 2H),
3.81 (s, 3H), 2.32 (s, 3H), 1.90 (m, 4H), 1.33 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ 171.47, 158.81, 147.45, 138.19, 131.83, 130.21,
128.58, 128.46, 126.03, 122.48, 119.68, 111.72, 109.80, 56.55, 56.47,
49.50, 29.24, 26.70, 22.77, 21.18. HRMS (EI) calcd for C20H24N4OS
[M]+, 368.1671; found, 368.1682.
8.12 (d, J = 6.7 Hz, 2H), 7.86 (d, J = 8.1 Hz, 1H), 7.36 (d, J = 6.5 Hz,
1H), 6.68 (d, J = 7.8 Hz, 1H), 6.61 (t, J = 7.2 Hz, 1H), 4.59 (m, 2H),
4.47 (m, 2H), 3.88 (s, 3H), 2.02 (m, 4H), 1.43 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ 171.84, 159.14, 144.54, 135.55, 135.22, 133.51,
133.18, 131.81, 128.59, 121.86, 120.86, 115.46, 112.00, 109.97, 108.47,
77.31, 76.99, 76.67, 56.69, 49.87, 29.19, 26.77, 22.77. HRMS (EI)
calcd for C21H20N5OSF3 [M]+, 447.1341; found, 447.1341.
1-(4-Tolyl)triazolylpentyl-3-hydroxypyridine-2-thione (14a). Re-
action of 13a (0.08 g, 0.20 mmol) and 1 M BBr3 (0.24 mL) in CH2Cl2
(8 mL) within 48 h as described for the synthesis of 10a gave
1
compound 14a (0.04 g, 62%) as an olive green solid. tR 11.0 min. H
NMR (400 MHz, CD3OD) δ 8.18 (s, 1H), 7.66 (d, J = 8.0 Hz, 2H),
7.55 (d, J = 5.7 Hz, 1H), 7.20 (d, J = 7.7 Hz, 2H), 6.97 (d, J = 6.7 Hz,
1H), 4.55 (m, 2H), 4.43 (t, J = 6.7 Hz, 2H), 2.34 (s, 3H), 2.01 (m,
4H), 1.39 (t, J = 22.5 Hz, 2H). 13C NMR (100 MHz, CD3OD) δ
149.25, 139.68, 130.93, 129.13, 127.01, 122.27, 51.41, 30.95, 24.51,
21.83. HRMS (EI) calcd for C19H22N4OS [M]+, 354.1514; found,
354.1512.
1-(2-Tolyl)triazolylpentyl-3-methoxypyridine-2-thione (13c). Re-
action of 12c (0.12 g, 0.35 mmol) and Lawesson’s reagent (0.09 g,
0.21 mmol) in toluene (10 mL) within 12 h as described for the
synthesis of 13a gave compound 13c (0.09 g, 72%) as a yellow solid.
1H NMR (400 MHz, CDCl3) δ 7.72 (m, 1H), 7.27 (m, 2H), 6.60 (m,
1H), 4.57 (m, 1H), 4.42 (t, J = 7.0 Hz, 1H), 3.86 (s, 1H), 2.44 (s, 1H),
1.99 (m, 2H), 1.41 (dt, J = 15.2, 7.6 Hz, 1H). 13C NMR (100 MHz,
CDCl3) δ 171.81, 159.04, 146.84, 135.33, 131.86, 130.74, 129.81,
128.68, 127.94, 125.93, 121.86, 111.78, 109.79, 104.88, 56.75, 56.61,
49.60, 29.45, 26.85, 22.97, 21.36. HRMS (EI) calcd for C20H24N4OS
[M]+, 368.1671; found, 368.1662.
1-(3-Tolyl)triazolylpentyl-3-hydroxypyridine-2-thione (14b). Re-
action of 13b (0.10 g, 0.27 mmol) and 1 M BBr3 (0.33 mL) in CH2Cl2
(8 mL) within 48 h as described for the synthesis of 10a gave
1
compound 14b (0.05 g, 56%) as an olive green solid. tR 11.1 min. H
NMR (400 MHz, CD3OD) δ 8.20 (s, 1H), 7.61 (s, 1H), 7.55 (m, 2H),
7.26 (t, J = 7.6 Hz, 1H), 7.12 (d, J = 7.5 Hz, 1H), 6.92 (m, 2H), 4.55
(m, 2H), 4.44 (t, J = 6.7 Hz, 2H), 2.36 (s, 3H), 2.01 (m, 4H), 1.41 (m,
2H). 13C NMR (100 MHz, CD3OD) δ 149.29, 140.05, 131.87, 131.78,
130.38, 130.20, 127.90, 127.63, 124.13, 122.51, 51.40, 31.07, 30.93,
28.97, 24.49, 22.02. HRMS (EI) calcd for C19H22N4OS [M]+,
354.1514; found, 354.1523.
1-(4-Benzonitrile)triazolylpentyl-3-methoxypyridine-2-thione
(13d). Reaction of 12d (0.21 g, 0.55 mmol) and Lawesson’s reagent
(0.13 g, 0.33 mmol) in toluene (12 mL) within 12 h as described for
the synthesis of 13a gave compound 13d (0.15 g, 67%) as a yellow
1-(2-Tolyl)triazolylpentyl-3-hydroxypyridine-2-thione (14c). Reac-
tion of 13c (0.07 g, 0.20 mmol) and 1 M BBr3 (0.30 mL) in CH2Cl2
(8 mL) within 48 h as described for the synthesis of 10a gave
1
compound 14c (0.05 g, 67%) as an olive green solid. tR 10.6 min. H
1
solid. H NMR (400 MHz, CDCl3) δ 7.64 (m, 3H), 7.28 (m, 1H),
NMR (400 MHz, CD3OD) δ 7.94 (s, 1H), 7.61 (m, 1H), 7.47 (s, 1H),
7.21 (m, 1H), 6.97 (m, 1H), 6.76 (m, 1H), 4.52 (m, 1H), 4.45 (t, J =
6.8 Hz, 1H), 2.38 (s, 1H), 2.01 (m, 2H), 1.43 (m, 1H). 13C NMR
(100 MHz, CD3OD) δ 146.74, 135.53, 130.65, 129.46, 128.69, 128.21,
125.88, 122.79, 53.41, 49.83, 29.42, 27.32, 22.98, 20.55. HRMS (EI)
calcd for C19H22N4OS [M]+, 354.1514; found, 354.1509.
1-(4-Benzonitrile)triazolylpentyl-3-hydroxypyridine-2-thione
(14d). Reaction of 13d (0.10 g, 0.271 mmol) and 1 M BBr3 (0.33 mL)
in CH2Cl2 (8 mL) within 48 h as described for the synthesis of 10a
gave compound 14d (0.053 g, 56%) as an olive green solid. tR 9.4 min.
1H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H), 8.27 (s, 1H), 7.82 (d,
J = 7.1 Hz, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 7.5 Hz, 1H),
6.93 (m, 1H), 6.77 (m, 2H), 4.52 (d, J = 6.9 Hz, 2H), 4.36 (t, J = 7.0
Hz, 2H), 3.21 (s, 6H), 1.88 (m, 4H), 1.34 (m, 2H). 13C NMR (100
MHz, CD3OD) δ 145.3, 132.0, 131.1, 129.7, 129.6, 128.6, 121.7, 112.7,
105.0, 49.8, 29.1, 27.0, 22.7. HRMS (EI) calcd for C19H19N5OS [M]+,
365.1310; found, 365.1310.
1-(3-Benzonitrile)triazolylpentyl-3-hydroxypyridine-2-thione
(14e). Reaction of 13e (0.10 g, 0.25 mmol) and 1 M BBr3 (0.30 mL)
in CH2Cl2 (8 mL) within 48 h as described for the synthesis of 10a
gave compound 14e (0.06 g, 60%) as an olive green solid. tR 9.3 min.
1H NMR (400 MHz, DMSO) δ 8.76 (s, 1H), 8.01 (d, J = 8.1 Hz, 2H),
7.87 (m, 2H), 7.65 (d, J = 6.4 Hz, 1H), 7.00 (m, 1H), 6.87 (d, J = 8.2
Hz, 1H), 6.70 (m, 1H), 4.44 (m, 4H), 1.89 (m, 4H), 1.31 (m, 2H).
13C NMR (100 MHz, CDCl3) δ 168.23, 155.01, 145.75, 134.86,
132.56, 131.00, 125.91, 121.05, 118.68, 113.76, 112.04, 111.17, 57.64,
49.81, 29.31, 27.06, 22.91. HRMS (EI) calcd for C19H19N5OS [M]+,
365.1310; found, 365.1313.
1-(4-(N,N-Dimethylaniline)triazolylpentyl-3-hydroxypyridine-2-
thione (14f). Reaction of 13f (0.11 g, 0.25 mmol) and 1 M BBr3 (0.33
mL) in CH2Cl2 (8 mL) within 48 h as described for the synthesis of
10a gave compound 14f (0.07 g, 66%) as an olive green solid. tR 4.6
min. 1H NMR (400 MHz, CD3OD) δ 8.38 (s, 1H), 8.15 (s, 1H), 8.10
(d, J = 7.9 Hz, 1H), 7.63 (m, 3H), 6.95 (d, J = 7.5 Hz, 1H), 6.67 (m,
1H), 4.57 (m, 2H), 4.48 (t, J = 7.0 Hz, 2H), 2.03 (m, 4H), 1.43 (m,
2H). 13C NMR (100 MHz, CD3OD) δ 145.34, 132.04, 131.14, 129.72,
6.73 (d, J = 8.3 Hz, 2H), 6.61 (d, J = 7.6 Hz, 1H), 6.52 (t, J = 7.1 Hz,
1H), 4.50 (m, 2H), 4.31 (t, J = 6.7 Hz, 2H), 3.81 (s, 3H), 2.92 (s, 6H),
1.89 (m, 4H), 1.32 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 171.44,
158.81, 149.78, 147.77, 131.91, 126.37, 119.05, 118.34, 112.52, 111.73,
109.84, 56.60, 56.49, 49.44, 40.45, 29.29, 26.74, 22.81. HRMS (EI)
calcd for C20H21N5OS [M]+, 379.1467; found, 379.1469.
1-(3-Benzonitrile)triazolylpentyl-3-methoxypyridine-2-thione
(13e). Reaction of 12e (0.19 g, 0.52 mmol) and Lawesson’s reagent
(0.13 g, 0.31 mmol) in toluene (10 mL) within 12 h as described for
the synthesis of 13a gave compound 13e (0.13 g, 67%) as a yellow
solid. 1H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 7.84 (d, J = 8.4 Hz,
2H), 7.56 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 6.5 Hz, 1H), 6.60 (d, J = 7.0
Hz, 1H), 6.52 (dd, J = 7.7, 6.6 Hz, 1H), 4.48 (m, 2H), 4.34 (t, J = 6.9
Hz, 2H), 3.76 (s, 3H), 1.88 (m, 4H), 1.31 (m, 2H). 13C NMR (100
MHz, CDCl3) δ 171.23, 158.70, 145.36, 134.76, 132.30, 131.78,
125.68, 121.20, 118.49, 111.83, 110.73, 109.92, 56.43, 49.50, 29.01,
26.56, 22.56. HRMS (EI) calcd for C20H21N5OS [M]+, 379.1467;
found, 379.1466.
1-(4-(N,N-Dimethylaniline))triazolylpentyl-3-methoxypyridine-2-
thione (13f). Reaction of 12f (0.15 g, 0.40 mmol) and Lawesson’s
reagent (0.10 g, 0.24 mmol) in toluene (10 mL) within 12 h as
described for the synthesis of 13a gave compound 13f (0.15 g, 98%) as
a yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.12 (t, J = 1.4 Hz, 1H),
8.06 (m, 1H), 7.90 (s, 1H), 7.59 (dt, J = 7.7, 1.4 Hz, 1H), 7.52 (t, J =
7.8 Hz, 1H), 7.33 (dd, J = 6.5, 1.4 Hz, 1H), 6.67 (dd, J = 7.9, 1.3 Hz,
1H), 6.60 (dd, J = 7.8, 6.5 Hz, 1H), 4.59 (m, 2H), 4.46 (t, J = 6.9 Hz,
2H), 3.89 (s, 3H), 2.02 (m, 4H), 1.43 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 171.30, 158.73, 145.13, 131.82, 131.71, 130.97, 129.52,
129.44, 128.64, 120.61, 118.28, 112.49, 111.81, 109.91, 56.44, 49.62,
29.11, 26.64, 22.68. HRMS (EI) calcd for C21H27N5OS [M]+,
397.1936; found, 397.1928.
1-(4-(3-Trifluomethyl)-benzonitrile)triazolylpentyl-3-methoxypyr-
idine-2-thione (13g). Reaction of 12g (0.09 g, 0.21 mmol) and
Lawesson’s reagent (0.05 g, 0.13 mmol) in toluene (10 mL) within 12
h as described for the synthesis of 13a gave compound 13g (0.08 g,
1
81%) as a yellow solid. H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H),
9979
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981

Journal of Medicinal Chemistry
Article
129.58, 128.63, 121.75, 112.74, 104.99, 49.79, 29.08, 27.04, 22.73.
HRMS (EI) calcd for C20H25N5OS [M]+, 383.1780; found, 383.1776.
1-(4-(3-Trifluomethyl)-benzonitrile)triazolylpentyl-3-hydroxypyri-
dine-2-thione (14g). Reaction of 13g (0.11 g, 0.25 mmol) and 1 M
BBr3 (0.33 mL) in CH2Cl2 (8 mL) within 48 h as described for the
synthesis of 10a gave compound 14g (0.072 g, 66%) as an olive green
solid. tR 12.4 min. 1H NMR (400 MHz, CD3OD) δ 8.59 (s, 1H), 8.35
(s, 1H), 8.21 (d, J = 7.8 Hz, 1H), 8.00 (t, J = 9.5 Hz, 1H), 7.58 (d, J =
6.2 Hz, 1H), 6.96 (d, J = 7.5 Hz, 1H), 6.72 (m, 1H), 4.57 (m, 4H),
2.04 (m, 4H), 1.42 (m, 2H). 13C NMR (100 MHz, CD3OD) δ 146.09,
137.48, 137.24, 134.79, 130.46, 125.55, 124.87, 123.60, 122.77, 116.84,
109.75, 55.10, 51.63, 30.84, 28.90, 24.47. HRMS (EI) calcd for
C19H19N5OS [M]+, 365.1310; found, 365.1310. HRMS (EI) calcd for
C20H18N5OS [M]+, 433.1184; found, 433.1189.
1-(4-Tolyl)triazolylpentyl-3-hydroxypyridin-2-one (15a). Reaction
of 12a (0.10 g, 0.28 mmol) and 1 M BBr3 (0.34 mL) in CH2Cl2 (8
mL) within 48 h as described for the synthesis of 10a gave compound
15a (0.05 g, 52%) as a light brown solid. 1H NMR (400 MHz, CDCl3)
δ 7.71 (m, 3H), 7.24 (m, 2H), 6.76 (t, J = 6.4 Hz, 2H), 6.12 (t, J = 7.0
Hz, 1H), 4.40 (t, J = 6.9 Hz, 2H), 3.96 (t, J = 7.1 Hz, 2H), 2.38 (s,
3H), 2.00 (m, 2H), 1.82 (m, 2H), 1.39 (m, 2H). 13C NMR (100 MHz,
DMSO) δ 171.80, 146.31, 137.02, 129.40, 128.08, 125.02, 120.79,
114.36, 105.20, 49.27, 48.22, 29.17, 28.01, 22.85, 20.82. HRMS (EI)
calcd for C19H22N4O2 [M]+, 338.1743; found, 338.1750.
1-(4-(N,N-Dimethylaniline))triazolylpentyl-3-hydroxypyridin-2-
one (15f). Reaction of 12f (0.08 g, 0.22 mmol) and 1 M BBr3 (0.26
mL) in CH2Cl2 (6 mL) within 48 h as described for the synthesis of
10a gave compound 15f (0.05 g, 64%) as a light brown solid. 1H NMR
(400 MHz, CD3OD) δ 8.39 (s, 1H), 8.16 (s, 1H), 8.12 (d, J = 7.5 Hz,
1H), 7.68 (d, J = 7.5 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.04 (m, 1H),
6.76 (m, 1H), 6.21 (m, 1H), 4.45 (m, 2H), 3.99 (m, 2H), 2.01 (m,
2H), 1.78 (m, 2H), 1.33 (m, 2H). 13C NMR (100 MHz, CDCl3) δ
145.61, 131.97, 131.31, 129.74, 129.65, 129.10, 120.30, 118.49, 112.99,
104.90, 50.07, 49.40, 29.52, 28.37, 23.17. HRMS (EI) calcd for
C20H25N5O2 [M]+, 367.2008; found, 367.2007.
1-(4-(3-Trifluomethyl)-benzonitrile)triazolylpentyl-3-hydroxypyri-
din-2-one (15g). Reaction of 12g (0.04 g, 0.10 mmol) and 1 M BBr3
(0.17 mL) in CH2Cl2 (6 mL) within 48 h as described for the
synthesis of 10a gave compound 15g (0.04 g, 85%) as a light brown
solid. 1H NMR (400 MHz, CDCl3) δ 8.24 (s, 1H), 8.12 (d, J = 7.9 Hz,
1H), 8.00 (s, 1H), 7.86 (d, J = 7.9 Hz, 1H), 6.75 (t, J = 5.8 Hz, 2H),
6.12 (t, J = 7.0 Hz, 1H), 4.44 (t, J = 6.9 Hz, 2H), 3.96 (t, J = 7.1 Hz,
2H), 2.02 (m, 2H), 1.81 (m, 2H), 1.37 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 158.58, 146.54, 144.71, 135.49, 135.26, 128.57, 126.70,
123.59, 123.54, 121.46, 115.47, 113.82, 108.63, 107.02, 50.19, 49.31,
29.45, 28.30, 23.12. HRMS (EI) calcd for C20H18N5O2F3 [M]+,
417.1413; found, 417.1425.
1-(3-Tolyl)triazolylpentyl-3-hydroxypyridin-2-one (15b). Reaction
of 12b (0.07 g, 0.21 mmol) and 1 M BBr3 (0.24 mL) in CH2Cl2 (8
mL) within 48 h as described for the synthesis of 10a gave compound
15b (0.05 g, 74%) as a light brown solid. 1H NMR (400 MHz, CDCl3)
δ 7.74 (s, 1H), 7.67 (s, 1H), 7.59 (d, J = 7.7 Hz, 1H), 7.30 (t, J = 7.6
Hz, 1H), 7.14 (d, J = 7.4 Hz, 1H), 6.76 (t, J = 6.2 Hz, 2H), 6.12 (t, J =
7.1 Hz, 1H), 4.39 (t, J = 6.9 Hz, 2H), 3.96 (t, J = 7.2 Hz, 2H), 2.39 (s,
3H), 1.99 (m, 2H), 1.80 (m, 2H), 1.38 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 158.58, 147.87, 146.52, 138.46, 130.38, 128.86, 128.67,
126.75, 126.31, 122.72, 119.48, 113.61, 106.85, 49.91, 49.45, 29.65,
28.38, 23.27, 21.38. HRMS (EI) calcd for C19H22N4O2 [M]+,
338.1743; found, 338.1741.
1-(2-Tolyl)triazolylpentyl-3-hydroxypyridin-2-one (15c). Reaction
of 12c (0.07 g, 0.18 mmol) and 1 M BBr3 (0.27 mL) in CH2Cl2 (8
mL) within 48 h as described for the synthesis of 10a gave compound
15c (0.05 g, 87%) as a light brown solid. 1H NMR (400 MHz, dmso)
δ 8.92 (s, 1H), 8.36 (s, 1H), 7.71 (m, 1H), 7.25 (m, 1H), 7.09 (d, J =
6.8 Hz, 1H), 6.64 (d, J = 6.9 Hz, 1H), 6.04 (t, J = 7.0 Hz, 1H), 4.39 (t,
J = 6.9 Hz, 1H), 3.88 (t, J = 7.0 Hz, 1H), 2.41 (s, 1H), 1.90 (m, 1H),
1.67 (m, 1H), 1.25 (m, 1H). 13C NMR (100 MHz, CD3OD) δ 146.28,
135.00, 130.15, 128.96, 128.21, 127.72, 125.38, 122.09, 106.72, 49.43,
48.98, 29.05, 27.78, 22.69, 20.09. HRMS (EI) calcd for C19H22N4O2
[M]+, 338.1743; found, 338.1741.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectral information, solubility data, and
molecular modeling. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(M.M.) E-mail: milan.mrksich@northwestern.edu. Phone:
847-467-0472. Fax: 847-467-3057.
*(A.K.O.) E-mail: aoyelere@gatech.edu. Phone: 404-894-4047.
Fax: 404-894-2291.
■
Author Contributions
∥These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Professor Olaf Wiest for providing us with
the HDAC 1 homology model. This work was financially
supported by NIH grant R01CA131217 (A.K.O.) and
R01GM084188 (M.M.). Q.H.S. is a recipient of a Southern
Regional Education Board Dissertation Fellowship and a
GAANN predoctoral fellowship from the Georgia Tech Center
for Drug Design, Development, and Delivery. We are grateful
to the ACS for a Division of Medicinal Chemistry travel award
to V.P.
1-(4-Benzonitrile)triazolylpentyl-3-hydroxypyridin-2-one (15d).
Reaction of 12d (0.08 g, 0.20 mmol) and 1 M BBr3 (0.24 mL) in
CH2Cl2 (8 mL) within 48 h as described for the synthesis of 10a gave
1
compound 15d (0.04 g, 60%) as a light brown solid. H NMR (400
MHz, CDCl3) δ 7.69 (d, J = 8.8 Hz, 2H), 7.61 (s, 1H), 6.77 (m, 4H),
6.11 (t, J = 7.1 Hz, 1H), 4.37 (t, J = 6.9 Hz, 2H), 3.96 (t, J = 7.2 Hz,
2H), 2.99 (s, 6H), 1.99 (m, 2H), 1.81 (m, 2H), 1.39 (m, 2H). 13C
NMR (100 MHz, CDCl3) δ 158.57, 150.35, 148.26, 146.61, 126.84,
126.61, 118.87, 118.11, 113.72, 112.49, 106.86, 49.83, 49.52, 40.46,
29.70, 28.41, 23.34. HRMS (EI) calcd for C19H19N5O2 [M]+,
349.1539; found, 349.1539.
ABBREVIATION USED
■
1-(3-Benzonitrile)triazolylpentyl-3-hydroxypyridin-2-one (15e).
Reaction of 12e (0.09 g, 0.25 mmol) and 1 M BBr3 (0.30 mL) in
CH2Cl2 (8 mL) within 48 h as described for the synthesis of 10a gave
HDAC, histone deacetylase; HDACi, histone deacetylase
inhibitors; ZBG, zinc binding group; TSA, trichostatin A;
3HPT, 3-hydroxypyridine-2-thione; MMP, matrix metallopro-
teinase; CTCL, cutaneous T-cell lymphoma
1
compound 15e (0.06 g, 72%) as a light brown solid. H NMR (400
MHz, DMSO) δ 8.93 (s, 1H), 8.76 (s, 1H), 8.01 (d, J = 7.7 Hz, 2H),
7.90 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 6.4 Hz, 1H), 6.64 (d, J = 7.1 Hz,
1H), 6.04 (t, J = 7.0 Hz, 1H), 4.41 (t, J = 6.5 Hz, 2H), 3.88 (t, J = 6.9
Hz, 2H), 1.89 (m, 2H), 1.67 (m, 2H), 1.24 (m, 2H). 13C NMR (100
MHz, CDCl3) δ 146.62, 145.80, 134.92, 132.57, 126.69, 125.93,
120.88, 118.70, 114.06, 111.19, 107.06, 50.00, 49.29, 29.43, 28.28,
23.08. HRMS (EI) calcd for C19H19N5O2 [M]+, 349.1539; found,
349.1544.
REFERENCES
■
(1) Minucci, S.; Pelicci, P. G. Histone deacetylase inhibitors and the
promise of epigenetic (and more) treatments for cancer. Nat. Rev.
Cancer 2006, 6, 38−51.
(2) Jain, S.; Zain, J.; O’Connor, O. Novel therapeutic agents for
cutaneous T-cell lymphoma. J. Hematol. Oncol. 2012, 5, 24.
9980
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981

Journal of Medicinal Chemistry
Article
(3) Gryder, B. E.; Sodji, Q. H.; Oyelere, A. K. Targeted cancer
therapy: Giving histone deacetylase inhibitors all they need to succeed.
Future Med. Chem. 2012, 4, 505−524.
inhibitor PCI-34051 induces apoptosis in T-cell lymphomas. Leukemia
2008, 22, 1026−1034.
(22) Aldana-Masangkay, G. I.; Sakamoto, K. M., The Role of
HDAC6 in Cancer. J. Biomed. Biotechnol. 2011, 2011.
(23) Yamaki, H.; Nakajima, M.; Shimotohno, K. W.; Tanaka, N.
Molecular basis for the actions of Hsp90 inhibitors and cancer therapy.
J. Antibiot. 2011, 64, 635−644.
(24) Williams, C. R.; Tabios, R.; Linehan, W. M.; Neckers, L.
Intratumor Injection of the Hsp90 Inhibitor 17AAG Decreases Tumor
Growth and Induces Apoptosis in a Prostate Cancer Xenograft Model.
J. Urol. 2007, 178, 1528−1532.
(4) Kontogiorgis, C. A.; Papaioannou, P.; Hadjipavlou-Litina, D. J.
Matrix metalloproteinase inhibitors: A review on pharmacophore
mapping and (Q)SARs results. Curr. Med. Chem. 2005, 12, 339−355.
(5) Botta, C. B.; Cabri, W.; Cini, E.; De Cesare, L.; Fattorusso, C.;
Giannini, G.; Persico, M.; Petrella, A.; Rondinelli, F.; Rodriquez, M.;
Russo, A.; Taddei, M. Oxime amides as a novel zinc binding group in
histone deacetylase inhibitors: Synthesis, biological activity, and
computational evaluation. J. Med. Chem. 2011, 54, 2165−2182.
(6) Suzuki, T.; Ando, T.; Tsuchiya, K.; Fukazawa, N.; Saito, A.;
Mariko, Y.; Yamashita, T.; Nakanishi, O. Synthesis and histone
deacetylase inhibitory activity of new benzamide derivatives. J. Med.
Chem. 1999, 42, 3001−3003.
(7) Suzuki, T.; Nagano, Y.; Matsuura, A.; Kohara, A.; Ninomiya, S.-i.;
Kohda, K.; Miyata, N. Novel histone deacetylase inhibitors: Design,
synthesis, enzyme inhibition, and binding mode study of SAHA-based
non-hydroxamates. Bioorg. Med. Chem. Lett. 2003, 13, 4321−4326.
(8) Chen, B.; Petukhov, P. A.; Jung, M.; Velena, A.; Eliseeva, E.;
Dritschilo, A.; Kozikowski, A. P. Chemistry and biology of
mercaptoacetamides as novel histone deacetylase inhibitors. Bioorg.
Med. Chem. Lett. 2005, 15, 1389−1392.
(9) Patil, V.; Sodji, Q. H.; Kornacki, J. R.; Mrksich, M.; Oyelere, A. K.
3-Hydroxypyridin-2-thione as novel zinc binding group for selective
histone deacetylase inhibition. J. Med. Chem. 2013, 56, 3492−3506.
(10) Chen, P. C.; Patil, V.; Guerrant, W.; Green, P.; Oyelere, A. K.
Synthesis and structure-activity relationship of histone deacetylase
(HDAC) inhibitors with triazole-linked cap group. Bioorg. Med. Chem.
2008, 16, 4839−4853.
(11) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A
stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regiose-
lective “ligation” of azides and terminal alkynes. Angew. Chem., Int. Ed.
2002, 41, 2596−2599.
(12) Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on
solid phase: [1,2,3]-Triazoles by regiospecific copper(I)-catalyzed 1,3-
dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem.
2002, 67, 3057−3064.
(13) Ozturk, T.; Ertas, E.; Mert, O.; Berzelius, A. Reagent,
phosphorus decasulfide (P4S10), in organic syntheses. Chem. Rev.
2010, 110, 3419−3478.
(14) Brayton, D.; Jacobsen, F. E.; Cohen, S. M.; Farmer, P. J. A novel
heterocyclic atom exchange reaction with Lawesson’s reagent: A one-
pot synthesis of dithiomaltol. Chem. Commun. 2006, 0, 206−208.
(15) Mwakwari, S. C.; Guerrant, W.; Patil, V.; Khan, S. I.; Tekwani,
B. L.; Gurard-Levin, Z. A.; Mrksich, M.; Oyelere, A. K. Non-peptide
macrocyclic histone deacetylase inhibitors derived from tricyclic
ketolide skeleton. J. Med. Chem. 2010, 53, 6100−6111.
(16) Gurard-Levin, Z. A.; Mrksich, M. The activity of HDAC8
depends on local and distal sequences of its peptide substrates.
Biochemistry 2008, 47, 6242−6250.
(25) Namdar, M.; Perez, G.; Ngo, L.; Marks, P. A. Selective
inhibition of histone deacetylase 6 (HDAC6) induces DNA damage
and sensitizes transformed cells to anticancer agents. Proc. Natl. Acad.
Sci. U.S.A. 2010, 107, 20003−20008.
(26) Hackanson, B.; Rimmele, L.; Benkißer, M.; Abdelkarim, M.;
Fliegauf, M.; Jung, M.; Lubbert, M. HDAC6 as a target for
̈
antileukemic drugs in acute myeloid leukemia. Leuk. Res. 2012, 36,
1055−1062.
(27) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Experimental and computational approaches to estimate solubility and
permeability in drug discovery and development settings. Adv. Drug
Delivery Rev. 1997, 23, 3−25.
(28) Kerns, E. H.; Di, L.; Carter, G. T. In vitro solubility assays in
drug discovery. Curr. Drug Metab. 2008, 9, 879−885.
(29) Shalinsky, D. R.; Brekken, J.; Zou, H.; McDermott, C. D.;
Forsyth, P.; Edwards, D.; Margosiak, S.; Bender, S.; Truitt, G.; Wood,
A.; Varki, N. M.; Appelt, K. Broad Antitumor and Antiangiogenic
Activities of AG3340, a Potent and Selective MMP Inhibitor
Undergoing Advanced Oncology Clinical Trials. Ann. N.Y. Acad. Sci.
1999, 878, 236−270.
(30) Jacobsen, J. A.; Fullagar, J. L.; Miller, M. T.; Cohen, S. M.
Identifying chelators for metalloprotein inhibitors using a fragment-
based approach. J. Med. Chem. 2010, 54, 591−602.
(31) Ropero, S.; Esteller, M. The role of histone deacetylases
(HDACs) in human cancer. Mol. Oncol. 2007, 1, 19−25.
(32) Huang, B. H.; Laban, M.; Leung, C. H. W.; Lee, L.; Lee, C. K.;
Salto-Tellez, M.; Raju, G. C.; Hooi, S. C. Inhibition of histone
deacetylase 2 increases apoptosis and p21Cip1/WAF1 expression,
independent of histone deacetylase 1. Cell Death Differ. 2005, 12,
395−404.
(33) Haggarty, S. J.; Koeller, K. M.; Wong, J. C.; Grozinger, C. 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.
(34) Chinchilla, R.; Najera, C. Recent advances in Sonogashira
reactions. Chem. Soc. Rev. 2011, 40, 5084−5121.
(35) Chen, P. C.; Wharton, R. E.; Patel, P. A.; Oyelere, A. K. Direct
diazo-transfer reaction on β-lactam: Synthesis and preliminary
biological activities of 6-triazolylpenicillanic acids. Bioorg. Med. Chem.
2007, 15, 7288−7300.
(36) Gurard-Levin, Z. A.; Kilian, K. A.; Kim, J.; Bahr, K.; Mrksich, M.
Peptide arrays identify isoform-selective substrates for profiling
endogenous lysine deacetylase activity. ACS Chem. Biol. 2010, 5,
863−873.
(37) Trott, O.; Olson, A. J. AutoDock Vina: Improving the speed and
accuracy of docking with a new scoring function, efficient
optimization, and multithreading. J. Comput. Chem. 2010, 31, 455−
461.
(38) Gryder, B. E.; Rood, M. K.; Johnson, K. A.; Patil, V.; Raftery, E.
D.; Yao, L.-P. D.; Rice, M.; Azizi, B.; Doyle, D. F.; Oyelere, A. K.
Histone deacetylase inhibitors equipped with estrogen receptor
modulation activity. J. Med. Chem. 2013, 56, 5782−5796.
(39) Day, J. A.; Cohen, S. M. Investigating the selectivity of
metalloenzyme inhibitors. J. Med. Chem. 2013, 56, 7997−8007.
(17) Gurard-Levin, Z. A.; Scholle, M. D.; Eisenberg, A. H.; Mrksich,
M. High-throughput screening of small molecule libraries using
SAMDI mass spectrometry. ACS Comb. Sci. 2011, 13, 347−350.
(18) Di Micco, S.; Chini, M. G.; Terracciano, S.; Bruno, I.; Riccio, R.;
Bifulco, G. Structural basis for the design and synthesis of selective
HDAC inhibitors. Bioorg. Med. Chem. 2013, 21, 3795−3807.
(19) 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.
(20) Whitehead, L.; Dobler, M. R.; Radetich, B.; Zhu, Y.; Atadja, P.
W.; Claiborne, T.; Grob, J. E.; McRiner, A.; Pancost, M. R.; Patnaik,
A.; Shao, W.; Shultz, M.; Tichkule, R.; Tommasi, R. A.; Vash, B.;
Wang, P.; Stams, T. Human HDAC isoform selectivity achieved via
exploitation of the acetate release channel with structurally unique
small molecule inhibitors. Bioorg. Med. Chem. 2011, 19, 4626−4634.
(21) Balasubramanian, S.; Ramos, J.; Luo, W.; Sirisawad, M.; Verner,
E.; Buggy, J. J. A novel histone deacetylase 8 (HDAC8)-specific
9981
dx.doi.org/10.1021/jm401225q | J. Med. Chem. 2013, 56, 9969−9981