Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth

Article abstract of DOI:10.1021/jm301098e

The incidence of malignant melanoma has dramatically increased in recent years thus requiring the need for improved therapeutic strategies. In our efforts to design selective histone deactylase inhibitors (HDACI), we discovered that the aryl urea 1 is a modestly potent yet nonselective inhibitor. Structure-activity relationship studies revealed that adding substituents to the nitrogen atom of the urea so as to generate compounds bearing a branched linker group results in increased potency and selectivity for HDAC6. Compound 5g shows low nanomolar inhibitory potency against HDAC6 and a selectivity of ～600-fold relative to the inhibition of HDAC1. These HDACIs were evaluated for their ability to inhibit the growth of B16 melanoma cells with the most potent and selective HDAC6I being found to decrease tumor cell growth. To the best of our knowledge, this work constitutes the first report of HDAC6-selective inhibitors that possess antiproliferative effects against melanoma cells.

Full text of DOI:10.1021/jm301098e

Article
pubs.acs.org/jmc
Selective Histone Deacetylase 6 Inhibitors Bearing Substituted Urea
Linkers Inhibit Melanoma Cell Growth
Joel A. Bergman,† Karrune Woan,‡ Patricio Perez-Villarroel,‡ Alejandro Villagra,‡ Eduardo M. Sotomayor,‡
and Alan P. Kozikowski*,†
†Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago,
Illinois 60612, United States
‡Department of Immunology and Malignant Hematology, H. Lee Moffitt Cancer Center, Tampa, Florida 33613, United States
ABSTRACT: The incidence of malignant melanoma has
dramatically increased in recent years thus requiring the need
for improved therapeutic strategies. In our efforts to design
selective histone deactylase inhibitors (HDACI), we discov-
ered that the aryl urea 1 is a modestly potent yet nonselective
inhibitor. Structure−activity relationship studies revealed that
adding substituents to the nitrogen atom of the urea so as to
generate compounds bearing a branched linker group results in
increased potency and selectivity for HDAC6. Compound 5g
shows low nanomolar inhibitory potency against HDAC6 and a selectivity of ∼600-fold relative to the inhibition of HDAC1.
These HDACIs were evaluated for their ability to inhibit the growth of B16 melanoma cells with the most potent and selective
HDAC6I being found to decrease tumor cell growth. To the best of our knowledge, this work constitutes the first report of
HDAC6-selective inhibitors that possess antiproliferative effects against melanoma cells.
uncover the biological significance of HDAC11 as a participant
in activating the immune response, and targeting one or both of
these enzymes may be of therapeutic value.6,7 Thus, HDAC6
has emerged as a possible target in the treatment of melanoma.
Such an approach may well be devoid of some of the
undesirable side effects of the pan-HDACI and thus of value to
explore in the context of finding safer cancer therapeutics.8
Our laboratory has previously published on the development
of a variety of HDAC6 inhibitors.9,10 A key feature of these
potent and selective agents derives from the presence of the
benzylic linker that is built into the canonical inhibitor
structure, which consists of the usual cap-linker-zinc binding
group scaffold (Figure 1). Herein we report on the use of
certain urea-based branched linkers that lead to potent and
selective HDAC6 inhibitors with cellular antimelanoma activity.
INTRODUCTION
■
Epigenetic regulation and subsequent gene expression or
silencing represents a tightly orchestrated interplay among
enzymes responsible for modifying the tails of histones, around
which nuclear DNA is wrapped. Among the various modifiers
of the histones, the cell is capable of balancing the activity of
both histone acetyltransferases (HAT) and histone deacetylases
(HDAC) to attach or remove acetyl groups, respectively, from
the lysine tails of these histone barrels. This particular
epigenetic marker masks the positive lysine residues from
interacting closely with the DNA phosphate-backbone,
resulting in a more “open” chromatin state, whereas the
deacetylases remove these acetyl groups, resulting in a more
“closed” or compacted DNA−histone state.
There are currently no selective HDAC6I approved for
clinical use in oncology. The development of such small
molecules may be advantageous as a therapeutic approach, for
they may possibly result in fewer side effects, which is an
apparent problem associated with the pan-HDACIs.1 Recent
preclinical efforts are being directed toward the use of HDAC6I
for certain cancers, specifically in combination with known
drugs.2 Literature reports suggest that HDACIs may be useful
as possible therapeutics for melanoma; however, many such
studies to date have focused on using pan-HDACIs, such as
suberoylanilide hydroxamic acid (SAHA).3 While SAHA
exhibits activity against all Zn2+-dependent HDAC isozymes,
it has been approved solely for the treatment of cutaneous T-
cell lymphoma.4
CHEMISTRY
■
Upon the basis of our knowledge of the structural parameters
essential for selective HDAC6 inhibitory activity, we chose to
investigate the design of some new hydroxamate-based
HDACIs that might retain the desired isozyme selectivity
while at the same time optimizing druglike properties.
Interestingly, a recent publication reported on the discovery
of HDACIs without a zinc-binding group (ZBG).11 These
agents possess modest activity against the class 1 enzymes;
however, our experience with HDAC6I design indicates that
inclusion of the ostensibly problematic hydroxamic acid group
It has previously been reported that HDAC6 forms an
Received: July 26, 2012
association with HDAC11.5 Recent efforts have begun to
© XXXX American Chemical Society
A
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ZBG. Finally, the hydroxamic acid group was installed using
hydroxylamine under basic conditions to provide the
hydroxamic acids 5a−h.
To evaluate the activity of compounds that possess a
branched linker positioned at the nitrogen atom distal to the
ZBG, we carried out the chemistry displayed in Scheme 2. A
copper-mediated Buchwald coupling reaction12 was used to
assemble anilines 6a,b from iodobenzene, as these intermedi-
ates were not commercially available. Triphosgene chemistry
was implemented to convert methyl 4-(aminomethyl)benzoate
into the corresponding isocyanate in situ, which underwent
reaction with secondary amines 6a−c to afford the penultimate
esters 7a−c. Final conversion to the hydroxamic acids was
accomplished in the usual fashion to complete the synthesis of
8a−c.
Figure 1. Structure of 1, a urea-containing HDACI. Appendage of
substitutions to the nitrogen atoms of the urea generates a branched
linker motif that may interact with a side cavity at the enzyme surface.
is not a liability. Our previous efforts toward developing
selective HDAC6Is were assisted by the development of a
homology model.9 This model showed the entrance to the
binding site was wider and shallower for HDAC6 than that of
HDAC1. Further examination of this model shows a lipophilic
cavity suitable for exploration in compound design. We
proposed that the introduction of certain branched-elements
to the linker would improve both the potency and selectivity by
accessing this side cavity, thereby perhaps resulting in better
interactions with the surface of HDAC6. These efforts
originated by generating analogues based on compound 1, a
modestly potent inhibitor of HDAC6 that is bereft of any
selectivity relative to HDAC1. With our knowledge of the
HDAC6 binding site, in combination with our previous SAR
data, we hypothesized that access to the HDAC6 lipophilic side
cavity could most efficiently be accomplished by appending
functionality to the nitrogen atoms of the urea within the linker
region of the canonical HDACI scaffold.
The synthesis of these branched ureas was readily
accomplished as outlined in Scheme 1. A variety of amines
were subjected to a reductive amination reaction employing
methyl 4-formylbenzoate 2 to form the desired secondary
amines 3a−h. Subsequent reaction of 3a−h with the
appropriate isocyanates afforded the branched urea esters
4a−h. This chemistry generated a series of ureas displaying
branched substitutions on the nitrogen atom proximal to the
RESULTS AND DISCUSSION
■
HDAC Isoform Activity. Our preliminary screening efforts
identified 1 to possess submicromolar HDAC inhibitory
activity; however, it was not selective against representative
members of the Zn2+-dependent classes 1 and 2. In an effort to
improve upon these compounds, we thus chose to investigate
the role of substitution on the nitrogen atoms of the urea
within the linker as a possible way to impart both selectivity and
potency to these compounds, and in particular for HDAC6 by
accessing a unique cavity on the surface of HDAC6. A summary
of the HDAC1 and HDAC6 inhibitory data obtained is
presented in Table 1.
The first round of analogues based on 1 maintained the same
2-methoxyphenyl cap group but contained varied substitutions
on the proximal nitrogen atom (5a−d). Introducing the
branching element at this position had a dramatic impact on
decreasing activity at HDAC1. Interestingly, inhibition at
HDAC6 was found to be dependent upon the nature of this
substitution. The dimethylamino substitution, as in 5a, and the
3-indolyl substitution, as in 5c, both proved detrimental to
HDAC6 inhibition, as they were over three times less potent
compared to 1. They did, however, maintain low micromolar
inhibitory activity at HDAC1. As the tertiary amine in 5a would
be protonated at physiological pH, it is possible that a positive
a
Scheme 1. Synthesis of Proximal Nitrogen Atom-Substituted Hydroxamic Acids
a
Reagents and conditions: (a) R1-NH2, NaCNBH3, rt, 5% AcOH/DCM, 16 h; (b) aryl-NCO, DCM, rt, 16 h; (c) 50 wt % NH2OH, NaOH, THF/
MeOH (1:1), 0 °C to rt, 30 min.
B
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a
Scheme 2. Synthesis of Distal Nitrogen Atom-Substituted Hydroxamic Acids
a
Reagents and conditions: (a) R1-NH2, CuI, K3PO4, ethylene glycol, iPrOH, 80 °C, 18 h; (b) i. triphosgene, sat. aq bicarbonate/DCM (1:1), 0 °C,
30 min; ii. methyl 4-(aminomethyl)benzoate-HCl, Et3N, DCM, rt, 16 h; (c) 50 wt % NH2OH, NaOH, THF/MeOH (1:1), 0 °C to rt, 30 min.
charge is unfavorable for proper target binding. Likewise, the
larger indole group of 5c may simply present too much steric
bulk to be properly accommodated by the active site. However,
the 3-hydroxypropyl derivative 5b and the 4-hydroxypheny-
lethyl derivative 5d displayed significant increases in the
inhibition of HDAC6. These substitutions had only a marginal
effect on HDAC1 activity. It is possible that the hydroxyl
groups of 5b and 5d are able to serve as H-bond acceptors or
donors and possess favorable interactions with key amino acid
residues on the HDAC6 surface, thus improving binding
affinity. Confirmation of this notion would require a high
quality X-ray structure of HDAC6, which is not yet available.
Concurrently, we queried the effect of masking the free
hydroxyl moiety of this branched linker design to examine the
influence of a H-bond acceptor. Capping the free hydroxyl with
a methyl group resulted in 5e, and this modification was found
to lead to a low nanomolar inhibitor with >400 fold selectivity
for HDAC6. Shortening this appendage to a hydroxyethyl
group as in 5f did not significantly alter HDAC6 inhibition but
did lead to slightly increased activity against HDAC1, thus
lowering the selectivity for our desired isozyme. The general
trend established was that a H-bond donor and a large aromatic
group detracted from the desired activity profile, whereas
smaller groups with H-bond acceptors favored HDAC6 activity.
Interestingly, the n-butyl-containing analogue 5g and phenethyl
analogue 5h were nanomolar potent HDAC6I. Furthermore, 5g
possessed excellent selectivity over HDAC1 (600-fold). These
data indicate that either a H-bond acceptor or lipophilic group
introduced as the branching element offers the best gains in
potency and selectivity for HDAC6.
Shifting the branching element to the distal nitrogen atom of
the urea relative to the ZBG resulted in analogues 8a−c. The
most potent of this series, 8a, possessed the same n-butyl
substitution as found in 5g. While 8a is a nanomolar HDAC6I,
it is five-times less potent and more importantly is less selective
than the proximally substituted analogue 5g. The methoxy
variant 8b suffered a dramatic decrease in potency toward
HDAC6. Decreasing the length of the alkyl chain as in 8c also
resulted in decreased HDAC6 inhibition. For compounds
possessing branched linkers originating from the urea, the most
significant gains in potency and selectivity for HDAC6 comes
from substitutions to the proximal nitrogen of the ZBG.
Lipophilic groups and H-bond acceptors are best accommo-
dated at this position, which we believe are most suitable to
access the side cavity at the HDAC6 surface.
Tubastatin A, an HDAC6I developed in our laboratory,9 reveals
that the inhibition of HDAC6 has been improved while
maintaining excellent selectivity relative to HDAC1. Nextura-
stat A also demonstrates comparable HDAC6 potency to
Trichostatin A (TSA) (Table 1). Additionally, we note that the
amino-benzamide ZBG has been incorporated into the design
of HDACIs, and its introduction reduces class 2 inhibition
resulting in class 1 selectivity; this is typified by MGCD0103,13
an HDACI that possesses antiproliferative activity and that has
recently entered clinical trials.14 Compared to MGCD0103,
Nexturastat A leads to a 30-fold reduction in activity at
HDAC1. Because the HDAC isozymes are highly homologous,
obtaining selectivity is critical to avoiding off-target effects and
is crucial to the anticipated development of HDAC6I.
Several pan-selective compounds have been approved by the
FDA, although only for use in cutaneous T-cell lymphoma.15
Avoiding cytotoxicity through isozyme selectivity would
ultimately prove advantageous and may open doors to a variety
of other therapeutic areas. Nexturastat A was thus screened
against all 11 isozymes (Table 2). In the similar class 1 and class
4 isozymes, Nexturastat A displayed low micromolar activity
compared to the low nanomolar activity against HDAC6.
Moreover, it also demonstrated high levels of selective
inhibition against members of the related class 2 HDAC
isozymes reaching >1000-fold selective in some cases. These
data establish Nexturastat A to be a potent and isozyme
selective HDAC6I. As more data emerge regarding the
biological activities of the other HDAC isozymes, maintaining
selectivity during inhibitor development will be paramount.
Next, we evaluated the ability of Nexturastat A to induce
hyperacetylation of α-tubulin, a hallmark of HDAC6 inhibition,
without elevating levels of acetylated histones. HDAC6
contains two catalytic domains. Its C-terminus domain is the
functional domain for both synthetic and physiological
substrates, whereas the N-terminal domain is devoid of
enzymatic activity.16 Low nanomolar treatment of Nexturastat
A on B16 murine melanoma cells led to a dose-dependent
increase of acetyl α-tubulin levels without a concomitant
elevation of histone H3 acetylation (Figure 2) indicating
binding to the second, enzymatically active catalytic domain.
Not until concentrations of 1 and 10 μM were used was an
observable increase in histone H3 acetylation found. This is not
surprising as the biochemical IC50 of Nexturastat A against the
class 1 HDACs, those responsible for histone acetylation, is in
the micromolar range. There is a clear preference for activity in
a cellular environment that corresponds to selective HDAC6
inhibition.
Evaluation of this lead against other HDACI developed by
others and us reveals 5g, termed Nexturastat A, is in fact a
potent and selective HDAC6I. Comparing Nexturastat A to
C
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a
Table 1. Initial HDAC Inhibition Screen of Substituted Urea Compounds
a
IC50 values displayed are the mean of two experiments standard deviation obtained from curve-fitting of a 10-point enzyme assay starting from 30
b
c
μM of the HDACI with 3-fold serial dilution. Values are extracted from fitting dose−response curves to the data points; Reference 9. Trichostatin
A.
D
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polarity, as its cLogP = 0.54 (ChemDraw 12.0), and thus the
compound fails to efficiently permeate the cell membrane.
Comparing the most active compounds 5d and 5f−h in this
whole-cell assay reveals that the most selective HDAC6I have
the greatest efficacy in inhibiting cell growth. It should also be
noted that they also have higher cLogP values, possibly
contributing to improved cell permeability. As the cLogP is
adjusted to more optimal levels, as exemplified for Nexturastat
A (cLogP = 2.20), cellular efficacy is restored, demonstrating
that a proper balance of physiochemical parameters must be
maintained.
Compared to the pan-selective HDACI LBH589, Nexturastat
A is approximately 100-fold less potent in inducing murine B16
melanoma cell death. This decreased efficacy is unlikely due to
poor cell permeability, for as shown above, the treatment of
B16 cells with nanomolar doses of Nexturastat A results in
increased acetyl-tubulin levels (Figure 2). Additionally, both
compounds possess similar cLogP values (2.64 vs 2.20 for
LBH589 and Nexturastat A, respectively). Rather, the effects of
nonselective HDAC inhibition with LBH589 treatment are
likely contributing to its increased potency, and in particular, its
class 1 activity. It is also of interest to note that Nexturastat A
has increased potency against the B16 cell line in comparison to
Tubastatin A (Table 3). While a definitive explanation for this
difference in cellular activity is lacking, it is possible that this is
due to the improved HDAC6 activity of Nexturastat A. While
HDAC6-selective inhibitors have not played a prominent role
in cancer therapy to date, the present data suggest that further
explorations in these directions may be valuable to pursue.
Table 2. Inhibitory Profile of Nexturastat A (5g) against
HDAC1−11
a
isoform
IC50 (μM)
fold selective for HDAC6
HDAC1
HDAC2
HDAC3
HDAC4
HDAC5
HDAC6
HDAC7
HDAC8
HDAC9
HDAC10
HDAC11
3.02 1.04
6.92 0.763
6.68 1.75
600
1380
1330
1870
2330
−
9.39 0.863
11.7 0.141
0.00502 0.00060
4.46 0.665
0.954 0.0799
6.72 1.15
888
190
1340
1510
1020
7.57 0.481
5.14 0.686
a
IC50 displayed is the mean of two experiments standard deviation
obtained from curve fitting of 10-point enzyme assay starting from 30
μM analogue with 3-fold serial dilution. Values are extracted from
fitting dose−response curves to the data points.
CONCLUSIONS
■
This study has investigated the use of a branched linker
introduced into the canonical HDACI platform. Introduction of
this branching element, specifically to the nitrogen atom
proximal to the zinc binding group, has led to the discovery of
potent inhibitors that show excellent selectivity for HDAC6
versus the full panel of HDACs. The SAR data indicate that the
branched urea scaffold imparts substantial gains in the desired
biochemical activity. These compounds were evaluated in a
cellular context, and the best compound, Nexturastat A, was
found to be capable of increasing acetylated α-tubulin levels.
Moreover, Nexturastat A inhibited the growth of B16
melanoma cells, albeit with lower potency than LBH589. The
work thus demonstrates that this design-platform for selective
HDAC6Is, typified in Nexturastat A, leads to agents useful not
only as chemical tools but also of possible value in oncology.
Figure 2. Comparison of acetylation status of tubulin and histone H3
demonstrates HDAC6-substrate selectivity of Nexturastat A.
Antiproliferative Activity in Melanoma Cells. The
above compounds were then evaluated in an MTS assay to
determine the ability of selective HDAC6I to exert an
antiproliferative effect on B16 murine melanoma cells.
Treatment with the above compounds for 48 h resulted in
dose-dependent growth inhibition of the melanoma cells as
summarized in Table 3. The general trend for inhibiting cell
growth correlates with potency for HDAC6. However, the
biochemically potent and selective 5b performed very poorly in
the cellular assay. This may possibly be attributed to its high
Table 3. Antiproliferative Activity against B16 Murine
Melanoma Cells
EXPERIMENTAL SECTION
■
1
General Information. H and 13C spectra were obtained on a
compound
GI50 melanoma cells (μM)
Bruker spectrometer with TMS as an internal standard. The following
abbreviations for multiplicity were used: s = singlet, d = doublet, t =
triplet, m = multiplet, dd = double doublet, br = broad. Reactions were
monitored by TLC using precoated silica gel plates (Merck silica gel
60 F254, 250 μm thickness) and visualized under UV light. LRMS
experiments were carried out using an Agilent 1946A LC-MSD with
MeCN and H2O spiked with 0.1% formic acid as mobile phase. HRMS
determinations were done with a Shimadzu IT-TOF instrument with
MeCN and H2O spiked with 0.1% formic acid as mobile phase. Flash
chromatography was accomplished using the automated CombiFlash
Rf system from Teledyne ISCO and prepacked silica gel cartridges
according to the recommended loading capacity. Preparatory HPLC
was used in purification of all final compounds using a Shimadzu
preparative liquid chromatograph with the following specifications:
Column: ACE 5AQ (150 × 21.2 mm) with 5 μm particle size. Method
5a
5b
5c
5d
5e
5f
75.3 1.23
>100
30.4 1.32
18.4 1.23
22.2 1.41
19.1 1.19
14.3 1.15
15.4 1.20
65.8 1.19
>100
5g “Nexturastat A”
5h
8a
8b
8c
>100
Tubastatin
LBH589
40.5 1.21
0.150 0.00115
E
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1: 25−100% MeOH/H2O, 30 min; 100% MeOH, 5 min; 100−25%
MeOH/H2O, 4 min. Method 2: 8−100% MeOH/H2O, 30 min; 100%
MeOH, 5 min; 100−8% MeOH/H2O, 4 min. Method 3: 0% MeOH, 5
min; 0−100% MeOH/H2O, 25 min; 100% MeOH, 5 min; 100−0%
MeOH/H2O, 4 min. Flow rate = 17 mL/min with monitoring at 254
and 280 nm. Both solvents were spiked with 0.05% TFA. Analytical
HPLC was carried out using an Agilent 1100 series instrument with
the following specifications: column: Luna 5 C18(2) 100A (150 ×
4.60 mm) 5 μm particle size; gradient 10−100% MeOH/H2O, 18 min,
100% MeOH, 3 min; 100−10% MeOH/H2O, 3 min; 10% MeOH/
H2O, 5 min. Both solvents were spiked with 0.05% TFA. The purity of
all tested compounds was >95%, as determined by analytical HPLC.
Methyl 4-(((3-(Dimethylamino)propyl)amino)methyl)-
benzoate (3a). The synthesis of 3a is representative, general
procedure A. A round-bottom flask charged with methyl 4-
formylbenzoate (328 mg, 2 mmol) and 3-(dimethylamino)-
propylamine (0.252 mL, 2 mmol) was taken up in a solution of 5%
AcOH in DCM (10 mL). After 5 min, NaCNBH3 (126 mg, 2 mmol)
was added in portions and the resulting mixture was stirred at room
temperature under an atmosphere of Ar overnight. The reaction was
quenched with 1 N NaOH (10 mL) and the aqueous layer extracted
with DCM (3 × 10 mL). The combined organic extracts were washed
with brine, dried over sodium sulfate, concentrated in vacuo, and
purified via flash chromatography, affording the product as a waxy solid
NMR (400 MHz, CDCl3) δ 7.99 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0
Hz, 2H), 3.91 (s, 3H), 3.84 (s, 2H), 2.62 (t, J = 7.2 Hz, 2H), 1.49 (m,
2H), 1.34 (m, 3H), 0.91 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 167.00, 145.89, 129.65, 128.70, 127.87, 53.63, 51.95, 49.14,
32.14, 20.38, 13.93. LRMS ESI: [M + H]+ = 222.1.
Methyl 4-((Phenethylamino)methyl)benzoate (3h). Made
according to general procedure A, affording a colorless oil (203 mg,
1
75%). H NMR (400 MHz, CDCl3) δ 8.01 (m, 2H), 7.35 (m, 4H),
7.22 (m, 3H), 3.93 (s, 3H), 3.88 (s, 2H), 2.89 (m, 4H). 13C NMR
(100 MHz, CDCl3) δ 166.97, 145.66, 139.81, 129.65, 128.65, 128.43,
127.82, 126.15, 53.38, 51.96, 50.46, 36.29. LRMS ESI: [M + H]+ =
270.1.
Methyl 4-((1-(3-(Dimethylamino)propyl)-3-(2-
methoxyphenyl)ureido)methyl)benzoate (4a). The synthesis of
4a is representative, general procedure B. A solution of 3a (99 mg,
0.395 mmol) in DCM (5 mL) was added the appropriate isocyanate
(0.053 mL, 0.395 mmol) at room temperature under an atmosphere of
Ar, and the resulting solution was stirred overnight. The reaction was
quenched with saturated bicarbonate (10 mL) and extracted with
DCM (3 × 10 mL). The combined organics were washed with brine
(15 mL), dried over sodium sulfate, concentrated in vacuo, and
purified via flash chromatography, affording the urea ester as a waxy
solid (156 mg, 98%). 1H NMR (400 MHz, CDCl3) δ 8.64 (br s, 1H),
8.18 (d, J = 7.2 Hz, 1H), 7.99 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz,
2H), 6.97−6.94 (m, 2H), 6.84 (d, J = 7.2 Hz, 1H), 4.64 (s, 2H), 3.91
(s, 3H), 3.81 (s, 3H), 3.42 (t, J = 6.0 Hz, 2H), 2.34 (t, J = 6.0 Hz, 2H),
2.20 (s, 6H), 1.74 (quint, J = 6.0 Hz, 2H). 13C NMR (100 MHz,
CDCl3) δ 166.85, 156.64, 148.86, 143.75, 129.77, 129.43, 129.02,
127.53, 122.25, 120.94, 120.36, 109.92, 55.52, 54.60, 51.96, 49.19,
44.64, 44.18, 24.98. LRMS ESI: [M + H]+ = 400.2.
1
(313 mg, 63%). H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 8.0 Hz,
2H), 7.38 (d, J = 8.0 Hz, 2H), 3.89 (s, 3H), 3.84 (s, 2H), 2.79 (br s,
1H), 2.68 (t, J = 6.8 Hz, 2H), 2.35 (t, J = 6.8 Hz, 2H), 2.23 (s, 6H),
1.70 (quint, J = 6.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 166.96,
145.27, 129.71, 128.87, 127.95, 58.04, 53.42, 51.99, 47.87, 45.36,
27.41. LRMS ESI: [M + H]+ = 251.1
Methyl 4-(((3-Hydroxypropyl)amino)methyl)benzoate (3b).
Methyl 4-((1-(3-Hydroxypropyl)-3-(2-methoxyphenyl)-
Made according to general procedure A, affording a waxy solid (89 mg,
ureido)methyl)benzoate (4b). Made according to general proce-
1
29%). H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 8.0 Hz, 2H), 7.39
1
dure B, affording a waxy solid (113 mg, 74%). H NMR (400 MHz,
(d, J = 8.0 Hz, 2H), 3.91 (s, 3H), 3.90 (s, 2H), 3.78 (broad, 4H), 2.93
(t, J = 5.6 Hz, 2H), 1.78−1.73 (m, 2H). 13C NMR (100 MHz, CDCl3)
δ 166.79, 143.16, 129.77, 129.44, 128.26, 63.55, 53.14, 52.10, 48.88,
30.18. LRMS ESI: [M + H]+ = 224.2.
CDCl3) δ 8.07−8.03 (m, 3H), 7.39 (d, J = 8.0 Hz, 2H), 7.20 (br s,
1H), 6.93−6.90 (m, 2H), 6.77−6.47 (m, 1H), 4.59 (s, 2H), 3.91 (s,
3H), 3.67−3.62 (m, 7H), 1.80−1.77 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 166.65, 156.31, 147.85, 142.24, 130.14, 129.70, 128.43,
126.77, 122.44, 121.04, 119.48, 109.88, 58.25, 55.53, 52.12, 50.41,
44.00, 30.32. LRMS ESI: [M + H]+ = 373.2.
Methyl 4-(((2-(1H-Indol-3-yl)ethyl)amino)methyl)benzoate
(3c). Made according to general procedure A, affording a waxy solid
1
(446 mg, 72%). H NMR (400 MHz, CDCl3) δ 8.28 (br s, 1H), 7.98
Methyl 4-((1-(2-(1H-Indol-3-yl)ethyl)-3-(2-methoxyphenyl)-
ureido)methyl)benzoate (4c). Made according to general proce-
dure B, affording a solid (200 mg, 95%). 1H NMR (400 MHz, CDCl3)
δ 8.23−8.21 (m, 1H), 8.15 (br s, 1H), 7.99 (d, J = 8.0 Hz, 2H), 7.60
(d, J = 7.6 Hz, 1H), 7.36−7.34 (m, 3H), 7.22−7.10 (m, 3H), 7.02 (s,
1H), 6.97−6.94 (m, 2H), 6.81−6.79 (m, 1H), 4.59 (s, 2H), 3.91 (s,
3H), 3.70−3.67 (m, 5H), 3.12 (t, J = 7.6 Hz, 2H). 13C NMR (100
MHz, CDCl3) δ 166.83, 155.17, 147.60, 143.28, 136.29, 129.98,
129.34, 128.79, 127.30, 127.13, 122.18, 122.08, 121.15, 119.51, 118.96,
118.46, 112.53, 111.29, 109.29, 55.56, 52.10, 50.89, 48.98. LRMS ESI:
[M + H]+ = 458.2.
Methyl 4-((1-(4-Hydroxyphenethyl)-3-(2-methoxyphenyl)-
ureido)methyl)benzoate (4d). Made according to general proce-
dure B, affording a solid (178 mg, 90%). 1H NMR (400 MHz, CDCl3)
δ 8.15 (d, J = 4.8 Hz, 1H), 8.01 (d, J = 7.6 Hz, 2H), 7.35 (d, J = 8.0
Hz, 2H), 7.11 (s, 1H), 7.02 (d, J = 8.0 Hz, 2H), 6.95−6.93 (m, 2H),
6.80−6.77 (m, 3H), 6.43 (br s, 1H), 4.53 (s, 2H), 3.91 (s, 3H), 3.73
(s, 3H), 3.56 (t, J = 6.8 Hz, 2H), 2.87 (t, J = 6.8 Hz, 2H). 13C NMR
(100 MHz, CDCl3) δ 166.89, 155.25, 154.99, 147.69, 142.93, 130.04,
129.83, 129.76, 129.40, 128.50, 127.21, 122.35, 121.18, 119.10, 115.67,
109.78, 55.60, 52.15, 50.97, 50.33, 33.88. LRMS: [M + H]+ = 435.2.
Methyl 4-((1-(3-Methoxypropyl)-3-phenylureido)methyl)-
benzoate (4e). Made according to general procedure B, affording a
colorless oil (70 mg, 73%). 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J =
8.4 Hz, 2H), 7.84 (s, 1H), 7.45 (d, J = 8.4. Hz, 2H), 7.40 (d, J = 8.0
Hz, 2H), 7.30 (m, 2H), 7.02 (t, J = 7.6 Hz, 1H), 4.64 (s, 2H), 3.92 (s,
3H), 3.49 (t, J = 5.2 Hz, 2H), 3.44 (s, 3H), 3.415 (t, J = 6.4 Hz, 2H),
1.77 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 166.87, 156.36, 143.76,
139.85, 129.88, 129.18, 128.81, 127.72, 122.45, 119.17, 68.16, 58.63,
52.05, 49.41, 43.05, 27.55. LRMS ESI: [M + H]+ = 358.2.
(d, J = 8.0 Hz, 2H), 7.62 (d, J = 7.6 Hz, 1H), 7.36−7.34 (m, 3H), 7.21
(t, J = 7.6 Hz, 1H), 7.13 (t, J = 7.6 Hz, 1H), 7.00 (s, 1H), 3.92 (s, 3H),
3.87 (s, 2H), 3.02−2.98 (m, 4H), 1.68 (br s, 1H). 13C NMR (100
MHz, CDCl3) δ 167.06, 145.66, 136.34, 129.60, 128.62, 127.85,
127.31, 122.02, 121.89, 119.13, 118.74, 113.47, 111.15, 53.36, 51.97,
49.28, 25.64. LRMS ESI: [M + H]+ = 309.1.
Methyl 4-(((4-Hydroxyphenethyl)amino)methyl)benzoate
(3d). Made according to general procedure A, affording a waxy solid
1
(130 mg, 46%). H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.0 Hz,
2H), 7.32 (d, J = 8.0 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.71 (d, J = 8.4
Hz, 2H), 3.90 (s, 3H), 3.86 (s, 2H), 2.86 (t, J = 6.8 Hz, 2H), 2.76 (t, J
= 6.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 167.07, 154.60,
145.02, 131.05129.78, 129.75, 128.91, 128.04, 115.55, 53.29, 52.08,
50.37, 35.02. LRMS ESI: [M + H]+ = 268.1.
Methyl 4-(((3-Methoxypropyl)amino)methyl)benzoate (3e).
Made according to general procedure A, affording a colorless oil (127
1
mg, 54%). H NMR (400 MHz, CDCl3) δ 7.94 (d, J = 8.0 Hz, 2H),
7.35 (d, J = 8.0 Hz, 2H), 3.86 (s, 3H), 3.80 (s, 2H), 3.41 (t, J = 6.4 Hz,
2H), 3.28 (s, 3H), 2.67 (t, J = 6.4 Hz, 2H), 1.72 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ 166.88, 145.78, 129.54, 128.58, 127.76, 71.17,
58.48, 53.50, 51.85, 46.72, 29.83. LRMS ESI: [M + H]+ = 238.2.
Methyl 4-(((2-Methoxyethyl)amino)methyl)benzoate (3f).
Made according to general procedure A, affording a colorless oil (29
1
mg, 13%). H NMR (400 MHz, CDCl3) δ 7.97 (d, J = 8.0 Hz, 2H),
7.38 (d, J = 8.0 Hz, 2H), 3.88 (s, 3H), 3.84 (s, 2H), 3.49 (t, J = 5.2 Hz,
2H), 3.33 (s, 3H), 2.77 (t, J = 5.2 Hz, 2H), 1.88 (br, 1H). 13C NMR
(100 MHz, CDCl3) δ 166.98, 145.58, 129.65, 128.74, 127.90, 71.85,
58.74, 53.47, 51.94, 48.69. LRMS ESI: [M + H]+ = 224.2.
Methyl 4-((Butylamino)methyl)benzoate (3g). Made according
1
to general procedure A, affording a colorless oil (150 mg, 68%). H
F
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Methyl 4-((1-(2-Methoxyethyl)-3-phenylureido)methyl)-
N - H y d r o x y - 4 - ( ( 1 - ( 4 - h y d r o x y p h e n e t h y l ) - 3 - ( 2 -
methoxyphenyl)ureido)methyl)benzamide (5d). Made accord-
ing to general procedure C and purified via method 2, affording the
benzoate (4f). Made according to general procedure B, affording a
1
colorless oil (40 mg, 91%). H NMR (400 MHz, CDCl3) δ 8.33 (br,
1
title compound (63 mg, 63%). H NMR (400 MHz, DMSO-d6) δ
1H), 8.02 (d, J = 8.0 Hz, 2H), 7.35 (m, 6H), 7.02 (t, J = 7.2 Hz, 1H),
4.68 (s, 2H), 3.93 (s, 3H), 3.50 (s, 3H), 3.46 (s, 4H). 13C NMR (100
MHz, CDCl3) δ 166.88, 157.07, 143.81, 139.37, 129.92, 129.28,
128.81, 127.73, 122.34, 119.15, 72.59, 59.28, 52.07, 50.90, 48.44.
LRMS ESI: [M + H]+ = 343.2.
11.20 (br s, 1H), 9.20 (br s, 1H), 7.82 (d, J = 8.0 Hz, 2H), 7.74 (d, J =
8.0 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.37 (s, 1H), 7.04 (d, J = 8.4 Hz,
2H), 6.98 (d, J = 8.0 Hz, 2H), 6.89−6.85 (m, 1H), 6.69 (d, J = 8.4 Hz,
2H), 4.57 (s, 2H), 3.75 (s, 3H), 3.49 (t, J = 7.6 Hz, 2H), 2.75 (t, J =
7.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 164.00, 155.76,
154.62, 148.97, 141.88, 131.63, 129.63, 128.83, 127.12, 122.72, 120.44,
120.33, 115.22, 110.69, 55.69, 49.68, 49.49, 33.21. HRMS ESI: calcd
for C24H25N3O5 [M + H]+ m/z = 436.1867; found 436.1858.
N-Hydroxy-4-((1-(3-methoxypropyl)-3-phenylureido)-
methyl)benzamide (5e). Made according to general procedure C
and purified via method 2, affording the title compound (48 mg, 76%).
1H NMR (400 MHz, DMSO-d6) δ 11.19 (br, 1H), 8.36 (s, 1H), 7.72
(d, J = 8.4 Hz, 2H), 7.45 (d, J = 7.6 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H),
7.23 (m, 2H), 6.94 (t, J = 7.2 Hz, 1H), 4.61 (s, 2H), 3.34 (m, 4H),
3.21 (s, 3H), 1.74 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ164.04,
155.30, 142.25, 140.42, 131.49, 128.31, 127.06, 121.87, 119.88, 69.16,
57.88, 49.08, 43.56, 27.81. HRMS ESI: calcd for C19H23N3O4 [M +
H]+ m/z = 358.1761; found 358.1785.
N-Hydroxy-4-((1-(2-methoxyethyl)-3-phenylureido)methyl)-
benzamide (5f). Made according to general procedure C and purified
method 2, affording the title compound (20 mg, 50%). 1H NMR (400
MHz, DMSO-d6) δ 11.17 (s, 1H), 9.01 (br, 1H), 8.44 (s, 1H), 7.72 (d,
J = 8.0 Hz, 2H), 7.42 (d, J = 7.6 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H),
7.23 (t, J = 7.6 Hz, 2H)H, 6.94 (t, J = 7.2 Hz, 1H), 4.64 (s, 2H), 3.49
(s, 4H), 3.28 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 164.06,
155.46, 142.17, 140.30, 131.43, 128.32, 126.97, 121.83, 119.64, 70.88,
58.31, 49.82, 46.35. HRMS ESI: calcd for C18H21N3O4 [M + H]+ m/z
= 344.1605; found 344.1601.
Methyl 4-((1-Butyl-3-phenylureido)methyl)benzoate (4g).
Made according to general procedure B, affording colorless oil (59
1
mg, 98%). H NMR (400 MHz, CDCl3) δ8.04 (d, J = 8.0 Hz, 2H),
7.39 (d, J = 8.0 Hz, 2H), 7.27 (m, 4H), 7.03 (t, J = 7.2 Hz, 1H), 6.32
(s, 1H), 4.65 (s, 2H), 3.93 (s, 3H), 3.36 (t, J = 7.2 Hz, 2H), 1.64 (m,
2H), 1.37 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 166.74, 155.30, 143.11, 138.83, 130.14, 129.50, 128.85,
127.04, 123.16, 119.90, 52.12, 50.49, 47.74, 30.52, 20.18, 13.81. LRMS
ESI: [M + H]+ = 341.1
Methyl 4-((1-Phenethyl-3-phenylureido)methyl)benzoate
(4h). Made according to general procedure B, affording a colorless
oil (113 mg, 92%). 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 8.4 Hz,
2H), 7.36 (m, 4H), 7.30 (d, J = 7.2 Hz, 1H), 7.21 (m, 4H), 7.08 (d, J =
8.0 Hz, 2H), 6.99 (t, J = 7.2 Hz, 1H), 6.00 (s, 1H), 4.58 (s, 2H), 3.91
(s, 3H), 3.59 (t, J = 6.8 Hz, 2H), 2.90 (t, J = 6.8 Hz, 2H). 13C NMR
(100 MHz, CDCl3) δ 166.71, 155.61, 142.99, 138.86, 138.72, 130.06,
129.43, 129.00, 128.86, 128.65, 127.28, 126.92, 122.95, 119.83, 52.08,
50.38, 49.94, 34.74. LRMS ESI: [M + H]+ = 389.2.
4-((1-(3-(Dimethylamino)propyl)-3-(2-methoxyphenyl)-
ureido)methyl)-N-hydroxybenzamide (5a). The synthesis of 5a is
representative, general procedure C. Solid NaOH (125 mg, 3.12
mmol) was dissolved in an aqueous solution (50 wt %, 1 mL) at 0 °C.
Then a solution of 4a (156 mg, 0.390 mmol) in THF/MeOH (1:1, 6
mL total) was added dropwise where the biphasic solution became
homogeneous upon compete addition. The resulting solution was
stirred 30 min at room temperature. The reaction was quenched with
AcOH (0.223 mL, 3.90 mmol) and concentrated in vacuo, and the
crude product was purified via HPLC method 2 and neutralized with
4-((1-Butyl-3-phenylureido)methyl)-N-hydroxybenzamide
(5g). Made according to general procedure C and purified by method
1
2, affording the title compound (40 mg, 68%). H NMR (400 MHz,
DMSO-d6) δ 11.17 (br, 1H), 8.36 (s,1H), 7.72 (d, J = 8.0 Hz, 2H),
7.46 (d, J = 7.6 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.22 (t, J = 7.6 Hz,
2H), 6.94 (t, J = 7.2 Hz, 1H), 4.62 (s, 2H), 3.30 (m, 1H), 1.48 (m,
2H), 1.27 (m, 2H), 0.86 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 164.01, 155.22, 142.30, 140.45, 131.42, 128.19, 127.00,
126.96, 121.80, 120.04, 49.01, 46.11, 29.64, 19.43, 13.77. HRMS ESI:
calcd for C19H23N3O3 [M + H]+ m/z = 342.1812; found 342.1802.
N-Hydroxy-4-((1-phenethyl-3-phenylureido)methyl)-
benzamide (5h). Made according to general procedure C and
purified by method 2, affording the title compound (71 mg, 63%). 1H
NMR (400 MHz, DMSO-d6) δ 11.15 (br, 1H), 7.72 (d, J = 8.0 Hz,
2H), 7.45 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 7.24 (m, 7H),
6.95 (t, J = 7.2 Hz, 1H), 4.60 (s, 2H), 3.54 (t, J = 7.6 Hz, 2H), 2.82 (t,
J = 7.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 164.04, 155.12,
142.12, 140.35, 139.04, 131.50, 128.77, 128.31, 128.20, 127.09, 127.05,
126.16, 121.91, 120.13, 49.20, 48.04, 33.91. HRMS ESI: calcd for
C23H23N3O3 [M + H]+ m/z = 390.1812; found 390.1793.
N-Butylaniline (6a). Synthesized in a manner analogous to that
previously reported.12 Briefly, CuI (19 mg, 0.1 mmol) and freshly
ground K3PO4 (849 mg, 4 mmol) were placed in a sealed tube
followed by sequential addition of 2-propanol (2 mL), ethylene glycol
(0.222 mL, 4.0 mL), phenyl iodide (0.224 mL, 2.0 mmol), and n-
butylamine (0.237 mL, 2.4 mmol). The tube was then sealed, and
stirring commenced at 80 °C for 18 h. After cooling to room
temperature, the reaction was diluted with water:ethyl ether (1:1, 10
mL). The aqueous layer was extracted with ether (3 × 5 mL), washed
with brine (15 mL), dried over sodium sulfate, and concentrated in
vacuo. Purification via flash chromatography afforded the title
compound as a yellow oil (235 mg, 79%). 1H NMR (400 MHz,
CDCl3) δ 7.19 (m, 2H), 6.70 (t, J = 7.2 Hz, 1H), 6.61 (d, J = 8.4 Hz,
2H), 3.60 (br, 1H), 3.12 (t, J = 7.2 Hz, 2H), 1.62 (m, 2H), 1.44 (m,
2H), 0.98 (t, J = 7.2 Hz, 3H). Spectra matches that reported in the
literature.17
1
bicarbonate wash, affording the title compound (20 mg, 13%). H
NMR (400 MHz, DMSO-d6) δ 11.22 (br s, 1H), 9.02 (br s, 1H),
7.80−7.76 (m, 3H), 7.39 (d, J = 8.4 Hz, 2H), 7.00−6.94 (m, 2H),
6.88−6.84 (m, 2H), 4.62 (s, 2H), 3.72 (s, 3H), 3.43−3.40 (m, 2H),
2.82 (br s, 2H), 2.56 (s, 6H), 1.91−1.86 (m, 2H). 13C NMR (100
MHz, DMSO-d6) δ 155.35, 149.62, 141.56, 131.69, 128.60, 127.24,
127.15, 127.02, 123.14, 121.40, 120.23, 110.78, 55.62, 54.33, 49.32,
44.24, 42.76, 23.45. HRMS ESI: calcd for C21H28N4O4 [M + H]+ m/z
= 401.2183; found 401.2164.
N-Hydroxy-4-((1-(3-hydroxypropyl)-3-(2-methoxyphenyl)-
ureido)methyl)benzamide (5b). Made according to general
procedure C and purified via method 3 affording the title compound
1
(95 mg, 84%). H NMR (400 MHz, DMSO-d6) δ 11.19 (br s, 1H),
7.82 (d, J = 7.6 Hz, 1H), 7.76−7.71 (m, 3H), 7.37 (d, J = 8.0 Hz, 2H),
6.95 (d, J = 4.0 Hz, 2H), 6.88−06.85 (m, 1H), 4. 58 (s, 2H), 3.74 (s,
3H), 3.48 (t, J = 5.6 Hz, 2H), 3.40 (t, J = 6.8 Hz, 2H), 1.73−1.69 (m,
2H). 13C NMR (100 MHz, DMSO-d6) δ 164.10, 155.18, 149.08,
141.99, 131.56, 128.91, 127.18, 127.10, 126.93, 122.61, 120.49, 120.29,
110.76, 57.59, 55.69, 49.28, 43.97, 30.61. HRMS ESI: calcd for
C19H23N3O5 [M + H]+ m/z = 374.1710; found 374.1693.
4-((1-(2-(1H-Indol-3-yl)ethyl)-3-(2-methoxyphenyl)ureido)-
methyl)-N-hydroxybenzamide (5c). Made according to general
procedure C and purified via method 1, affording the title compound
1
(62 mg, 57%). H NMR (400 MHz, DMSO-d6) δ 11.21 (br s, 1H),
10.86 (s, 1H), 10.12 (br s, 1H), 7.86 (t, J = 7.6 Hz, 1H), 7.75 (d, J =
8.0 Hz, 2H), 7.56 (d, J = 8.0 Hz, 1H), 7.42−7.40 (m, 3H), 7.34 (d, J =
8.0 Hz, 1H), 7.18 (s, 1H), 7.07 (t, J = 7.2 Hz, 1H), 7.01−6.96 (m,
3H), 6.90−6.86 (m, 1H), 4.64 (s, 2H), 3.71 (s, 3H), 3.62 (t, J = 7.6
Hz, 2H), 3.00 (t, J = 7.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ
163.97, 158.37, 154.72, 148.88, 141.90, 136.21, 131.65, 128.70, 127.12,
122.98, 122.65, 120.97, 120.35, 118.31, 118.18, 111.41, 111.11, 110.66,
55.66, 49.77, 48.47, 23.89. HRMS ESI: calcd for C26H26N4O4 [M +
H]+ m/z = 459.2027; found 459.2030.
N-(3-Methoxypropyl)aniline (6b). Made following the same
procedure for 6a affording a light yellow oil (282 mg, 85%). 1H NMR
G
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(400 MHz, CDCl3) δ 7.18 (m, 2H), 6.69 (t, J = 7.2 Hz, 1H), 6.61 (d, J
= 8.4 Hz, 2H), 3.92 (br, 1H), 3.52 (t J = 6.0 Hz, 2H), 3.60 (s, 3H),
3.23 (t, J = 6.8 Hz, 2H), 1.90 (m, 2H). Spectra matches that reported
in the literature.18
(100 MHz, DMSO-d6) δ 164.21, 156.34, 144.54, 142.00, 130.92,
129.57, 128.32, 126.75, 126.67, 43.70, 43.39, 13.76. HRMS ESI: calcd
for C17H19N3O3 [M + H]+ m/z = 314.1499; found 314.1489.
HDAC Inhibition Assays. HDAC inhibition assays were
performed by Reaction Biology Corp. (Malvern, PA) using isolated
human, recombinant full-length HDAC1 and -6 from a baculovirus
expression system in Sf9 cells. An acetylated fluorogenic peptide,
RHKKAc, derived from residues 379−382 of p53 was used as substrate.
The reaction buffer was made up of 50 mM Tris-HCl pH 8.0, 127 mM
NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mg/mL BSA, and a final
concentration of 1% DMSO. Compounds were delivered in DMSO
and delivered to enzyme mixture with preincubation of 5−10 min
followed by substrate addition and incubation for 2 h at 30 °C.
Trichostatin A and a developer were added to quench the reaction and
generate fluorescence, respectively. Dose−response curves were
generated starting at 30 μM compound with 3-fold serial dilutions
to generate a 10-dose plot. IC50 values were then generated from the
resulting plots, and the values expressed are the average of duplicate
trials standard deviation.
Tubulin and Histone Acetylation Western Blot Assay. B16
melanoma cells were plated at 105 cells/well in 12-well plates and
allowed to adhere overnight. A 50 mM stock of compound was then
added by serial dilutions in complete medium to the indicated
concentrations. Cells were incubated for 24 h under humidified
conditions (37 °C, 5% CO2). Wells were then washed with cold PBS,
and cells were lysed in a buffer containing 10 mM Tris-HCl pH 8.0,
10% SDS, 4 mM urea, 100 mM DTT, and 1x protease inhibitor
(Roche). Cells were lysed for 30 min on ice and then sonicated for 8
min (8 cycles of 30 s on/30 s rest). Cells were then boiled for 10 min
with 6x gel loading buffer and resolved on 4−15% gradient gels and
subsequently transferred onto nitrocellulose membranes. Membranes
were blocked with 5% milk in PBS-T, and specific antigens were
detected using antibodies against acetyl-H3 and H3 (Cell Signaling)
and acetyl-α-tubulin and α-tubulin (Sigma). Bands were detected by
scanning blots with an LI-COR Odyssey imaging system using both
700 and 800 channels.
Methyl 4-((3-Butyl-3-phenylureido)methyl)benzoate (7a).
Methyl 4-(aminomethyl)benzoate hydrochloride (101 mg, 0.5
mmol) was taken up in a biphasic solution of DCM:sat. bicarbonate
(1:1, 4 mL), and triphosgene (49 mg, 0.17 mmol) was added at 0 °C.
After 30 min, the aqueous layer was extracted with DCM (3 × 5 mL),
washed with brine (15 mL), and concentrated in vacuo. The crude
isocyanate was taken up in DCM (2 mL), 6a (75 mg, 0.5 mmol) and
Et3N (0.209 mL, 1.5 mmol) were added, and the resulting solution
was stirred overnight at room temperature. The reaction was quenched
with with sat. bicarbonate (5 mL) and extracted with DCM (3 × 5
mL). The combined organics were washed with brine (15 mL), dried
over sodium sulfate, and concentrated in vacuo. The crude material
was purified via flash chromatography, affording the title compound as
an off-white waxy solid (93 mg, 55%). 1H NMR (400 MHz, CDCl3) δ
7.95 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 7.6 Hz, 2H), 7.32 (m, 1H), 7.25
(m, 4H), 4.45 (t, J = 5.6 Hz, 1H), 4.41 (d, J = 6.0 Hz, 2H), 3.89 (s,
3H), 3.70 (t, J = 7.6 Hz, 2H), 1.48 (m, 2H), 1.31 (m, 2H), 0.89 (t, 7.2
Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 166.89, 156.87, 145.14,
141.61, 130.08, 129.78, 128.85, 128.73, 127.81, 126.96, 52.01, 49.21,
44.25, 30.68, 19.92, 13.82. LRMS ESI: [M + H]+ = 341.1.
Methyl 4-((3-(3-Methoxypropyl)-3-phenylureido)methyl)-
benzoate (7b). Made according to that of 7a except using 6b as
the secondary amine, affording the title compound as an off-white
waxy solid (65 mg, 36%). 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J =
8.0 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.33 (m, 1H), 7.27 (m, 4H),
4.69 (br, 1H), 4.42 (6.0 Hz, 2H), 3.90 (s, 3H), 3.80 (t, J = 7.2 Hz,
2H), 3.43 (t, J = 6.4 Hz, 2H), 3.27 (s, 3H), 1.83 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ 166.88, 156.95, 145.05, 141.63, 130.10, 129.80,
128.90, 127.79, 127.00, 70.23, 58.54, 52.03, 46.86, 44.29, 28.82. LRMS
ESI: [M + H]+ = 357.1.
Methyl 4-((3-Ethyl-3-phenylureido)methyl)benzoate (7c).
Made according to that of 7a except using commercially available N-
ethylaniline as the secondary amine, affording the title compound as an
1
B16 Melanoma Cell Growth Inhibition Assay. B16 murine
melanoma cells were plated at 5 × 103/well in 96-well flat-bottom
plates. The following day, medium was changed to that containing
various concentrations of HDACI or matched DMSO vehicle
concentrations diluted in complete medium performed in triplicate.
Cells were incubated for 48 h at 37 °C and 5% CO2. Density of viable,
metabolically active cells was quantified using a standard MTS assay
(CellTiter 96 AQueous One, Promega, Madison, WI) as per
manufacturer’s instructions. Briefly, 20 μL of reagent was added per
well and incubated at 37 °C for 3 h. Absorbances at 490 nM were
measured spectrophotometrically with background subtraction at 690
nM. All values were then normalized and expressed as a percentage of
medium control (100%).
off-white waxy solid (197 mg, 63%). H NMR (400 MHz, CDCl3) δ
7.94 (d, J = 8.4 Hz, 2H), 7.24 (t, J = 7.6 Hz, 2H), 7.32 (t, J = 7.2 Hz,
1H), 7.26 (m, 4H), 4.57 (br, 1H), 4.41 (d, J = 5.6 Hz, 1H), 3.88 (s,
3H), 3.76 (dd, J = 14, 7.2 Hz, 2H), 1.12 (t, J = 7.2 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 166.82, 156.64, 145.09, 141.22, 130.03, 129.72,
128.79, 127.82, 126.92, 51.95, 44.17, 44.09, 13.82. LRMS ESI: [M +
H]+ = 313.1.
4-((3-Butyl-3-phenylureido)methyl)-N-hydroxybenzamide
(8a). Made according to general procedure C and purified by method
2, affording the title compound as an off-white solid (74 mg, 80%). 1H
NMR (400 MHz, DMSO-d6) δ 11.13 (br, 1H), 8.72 (br, 1H), 7.66 (d,
J = 8.0 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.27 (m, 5H), 6.23 (t, J = 5.6
Hz, 1H), 4.20 (d, J = 5.6 Hz, 2H), 3.57 (t, J = 6.8 Hz, 2H), 1.35 (m,
2H), 1.23 (m, 2H), 0.82 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 164.17, 156.48, 144.50, 142.12, 130.129.55, 128.22,
126.67, 126.61, 48.44, 43.38, 30.21, 19.35, 13.72. HRMS ESI: calcd for
C19H23N3O3 [M + H]+ m/z = 342.1812; found 342.1825.
N-Hydroxy-4-((3-(3-methoxypropyl)-3-phenylureido)-
methyl)benzamide (8a). Made according to general procedure C
and purified by method 2, affording an off-white solid (59 mg, 91%).
1H NMR (400 MHz, DMSO-d6) δ 11.14 (br, 1H), 7.66 (d, J = 8.0 Hz,
2H), 7.43 (t, J = 7.6 Hz, 2H), 7.27 (m, 5H), 6.27 (t, J = 6.0 Hz, 1H),
4.21 (d, J = 5.8 Hz, 2H), 3.62 (t, J = 7.2 Hz, 2H), 3.28 (t, J = 6.4 Hz,
2H), 3.14 (s, 3H), 1.63 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ
164.13, 156.51, 144.44, 142.19, 130.89, 129.57, 128.18, 126.70, 126.64,
69.49, 57.80, 46.35, 43.39, 28.32. HRMS ESI: calcd for C19H23N3O4
[M + H]+ m/z = 358.1761; found 358.1749.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: kozikowa@uic.edu. Tel: 312-996-7577.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants provided by the NIH
(CA134807 to E.M.S. and A.P.K.) and the International Rett
Syndrome Foundation (IRSF).
4-((3-Ethyl-3-phenylureido)methyl)-N-hydroxybenzamide
(8c). Made according to general procedure C and purified by method
1
2, affording an off-white solid (91 mg, 96%). H NMR (400 MHz,
ABBREVIATIONS USED
■
DMSO-d6) δ 11.13 (br, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 7.6
Hz, 2H), 7.26 (m, 5H), 6.30 (t, J = 6.0 Hz, 1H), 4.21 (d, J = 5.6 Hz,
2H), 3.61 (dd, J = 14, 7.2 Hz, 2H), 0.99 (t, J = 7.2 Hz, 3H). 13C NMR
HDAC, histone/protein decetylase; HDACI, histone deacety-
lase inhibitor; TSA, Trichostatin A; ZBG, zinc-binding group
H
dx.doi.org/10.1021/jm301098e | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
active histone deacetylase inhibitor. J. Med. Chem. 2008, 51 (14),
4072−4075.
(15) Kelly, W. K.; Marks, P. A. Drug Insight: histone deacetylase
inhibitors - development of the new targeted anticancer agent
suberoylanilide hydroxamic acid. Nat. Clin. Pract. Oncol. 2005, 2 (3),
150−157.
REFERENCES
■
(1) Zhang, Y.; Kwon, S.; Yamaguchi, T.; Cubizolles, F.; Rousseaux,
S.; Kneissel, M.; Cao, C.; Li, N.; Cheng, H. L.; Chua, K.; Lombard, D.;
Mizeracki, A.; Matthias, G.; Alt, F. W.; Khochbin, S.; Matthias, P. Mice
lacking histone deacetylase 6 have hyperacetylated tubulin but are
viable and develop normally. Mol. Cell. Biol. 2008, 28 (5), 1688−1701.
(2) Santo, L.; Hideshima, T.; Kung, A. L.; Tseng, J. C.; Tamang, D.;
Yang, M.; Jarpe, M.; van Duzer, J. H.; Mazitschek, R.; Ogier, W. C.;
Cirstea, D.; Rodig, S.; Eda, H.; Scullen, T.; Canavese, M.; Bradner, J.;
Anderson, K. C.; Jones, S. S.; Raje, N. Preclinical activity,
pharmacodynamic, and pharmacokinetic properties of a selective
HDAC6 inhibitor, ACY-1215, in combination with bortezomib in
multiple myeloma. Blood 2012, 119 (11), 2579−2589.
(3) Peltonen, K.; Kiviharju, T. M.; Jarvinen, P. M.; Ra, R.; Laiho, M.
Melanoma cell lines are susceptible to histone deacetylase inhibitor
TSA provoked cell cycle arrest and apoptosis. Pigm. Cell Research
2005, 18 (3), 196−202. Facchetti, F.; Previdi, S.; Ballarini, M.;
Minucci, S.; Perego, P.; La Porta, C. A. M. Modulation of pro- and
anti-apoptotic factors in human melanoma cells exposed to histone
deacetylase inhibitors. Apoptosis 2004, 9 (5), 573−582.
(4) Wagner, J. M.; Hackanson, B.; Lubbert, M.; Jung, M Histone
deacetylase (HDAC) inhibitors in recent clinical trials for cancer
therapy. Clin. Epigenet. 2010, 1 (3−4), 117−136.
(16) Zou, H.; Wu, Y. Q.; Navre, M.; Sang, B. C. Characterization of
the two catalytic domains in histone deacetylase 6. Biochem. Biophys.
Res. Commun. 2006, 341 (1), 45−50.
(17) Okano, K.; Tokuyama, H.; Fukuyama, T. Synthesis of secondary
arylamines through copper-mediated intermolecular aryl amination.
Org. Lett. 2003, 5 (26), 4987−4990.
(18) Guo, D. L.; Huang, H.; Xu, J. Y.; Jiang, H. L.; Liu, H. Efficient
Iron-Catalyzed N-Arylation of Aryl Halides with Amines. Org. Lett.
2008, 10 (20), 4513−4516.
NOTE ADDED AFTER ASAP PUBLICATION
■
After this paper was published on the Web October 23, 2012, a
correction was made to Table 1. The revised version was
reposted October 26, 2012.
(5) Gao, L.; Cueto, M. A.; Asselbergs, F.; Atadja, P. Cloning and
functional characterization of HDAC11, a novel member of the human
histone deacetylase family. J. Biol. Chem. 2002, 277 (28), 25748−
25755.
(6) Villagra, A.; Cheng, F.; Wang, H. W.; Suarez, I.; Glozak, M.;
Maurin, M.; Nguyen, D.; Wright, K. L.; Atadja, P. W.; Bhalla, K.;
Pinilla-Ibarz, J.; Seto, E.; Sotomayor, E. M. The histone deacetylase
HDAC11 regulates the expression of interleukin 10 and immune
tolerance. Nat. Immunol. 2009, 10 (1), 92−100.
(7) Wang, H.; Cheng, F.; Woan, K.; Sahakian, E.; Merino, O.; Rock-
Klotz, J.; Vicente-Suarez, I.; Pinilla-Ibarz, J.; Wright, K. L.; Seto, E.;
Bhalla, K.; Villagra, A.; Sotomayor, E. M. Histone Deacetylase
Inhibitor LAQ824 Augments Inflammatory Responses in Macro-
phages through Transcriptional Regulation of IL-10. J. Immunol. 2011,
186 (7), 3986−3996.
(8) Parmigiani, R. B.; Xu, W. S.; Venta-Perez, G.; Erdjument-
Bromage, H.; Yaneva, M.; Tempst, P.; Marks, P. A. HDAC6 is a
specific deacetylase of peroxiredoxins and is involved in redox
regulation. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (28), 9633−9638.
(9) 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 (31), 10842−10846.
(10) Kalin, J. H.; Butler, K. V.; Akimova, T.; Hancock, W. W.;
Kozikowski, A. P. Second-Generation Histone Deacetylase 6 Inhibitors
Enhance the Immunosuppressive Effects of Foxp3+ T-Regulatory
Cells. J. Med. Chem. 2012, 55 (2), 639−651.
(11) Vickers, C. J.; Olsen, C. A.; Leman, L. J.; Ghadiri, M. R.
Discovery of HDAC Inhibitors That Lack an Active Site Zn2+-Binding
Functional Group. ACS Med. Chem. Lett. 2012, 3 (6), 505−508.
(12) Kwong, F. Y.; Klapars, A.; Buchwald, S. L. Copper-catalyzed
coupling of alkylamines and aryl iodides: An efficient system even in
an air atmosphere. Org. Lett. 2002, 4 (4), 581−584.
(13) Fournel, M.; Bonfils, C.; Hou, Y.; Yan, P. T.; Trachy-Bourget,
M.-C.; Kalita, A.; Liu, J.; Lu, A.-H.; Zhou, N. Z.; Robert, M.-F.;
Gillespie, J.; Wang, J. J.; Ste-Croix, H.; Rahil, J.; Lefebvre, S.; Moradei,
O.; Delorme, D.; MacLeod, A. R.; Besterman, J. M.; Li, Z.
MGCD0103, a novel isotype-selective histone deacetylase inhibitor,
has broad spectrum antitumor activity in vitro and in vivo. Mol. Cancer
Ther. 2008, 7 (4), 759−768.
(14) Zhou, N.; Moradei, O.; Raeppel, S.; Leit, S.; Frechette, S.;
Gaudette, F.; Paquin, I.; Bernstein, N.; Bouchain, G.; Vaisburg, A.; Jin,
Z.; Gillespie, J.; Wang, J.; Fournel, M.; Yan, P. T.; Trachy-Bourget, M.
C.; Kalita, A.; Lu, A.; Rahil, J.; MacLeod, A. R.; Li, Z.; Besterman, J.
M.; Delorme, D. Discovery of N-(2-aminophenyl)-4-[(4-pyridin-3-
ylpyrimidin-2-ylamino)methyl]benzamide (MGCD0103), an orally
I
dx.doi.org/10.1021/jm301098e | J. Med. Chem. XXXX, XXX, XXX−XXX