Identification of HDAC6-Selective Inhibitors of Low Cancer Cell Cytotoxicity

Article abstract of DOI:10.1002/cmdc.201500456

The histone deacetylases (HDACs) occur in 11 different isoforms, and these enzymes regulate the activity of a large number of proteins involved in cancer initiation and progression. The discovery of isoform-selective HDAC inhibitors (HDACIs) is desirable, as it is likely that such compounds would avoid some of the undesirable side effects found with the first-generation inhibitors. A series of HDACIs previously reported by us were found to display some selectivity for HDAC6 and to induce cell-cycle arrest and apoptosis in pancreatic cancer cells. In the present work, we show that structural modification of these isoxazole-based inhibitors leads to high potency and selectivity for HDAC6 over HDAC1-3 and HDAC10, while unexpectedly abolishing their ability to block cell growth. Three inhibitors with lower HDAC6 selectivity inhibit the growth of cell lines BxPC3 and L3.6pl, and they only induce apoptosis in L3.6pl cells. We conclude that HDAC6 inhibition alone is insufficient for disruption of cell growth, and that some degree of class 1 HDAC inhibition is required. Moreover, the highly selective HDAC6Is reported herein that are weakly cytotoxic may find use in cancer immune system reactivation. High or low selectivity: A new class of HDAC inhibitors bearing an isoxazole ring show high potency and selectivity for HDAC6 over HDAC1-3 and HDAC10, while unexpectedly showing little potency in blocking cell growth. These results suggest that HDAC6 inhibition alone is insufficient for disruption of cell growth, and that some degree of class 1 HDAC inhibition is required. The highly selective HDAC6 inhibitors reported herein that are weakly cytotoxic may find use in cancer immune system reactivation.

Full text of DOI:10.1002/cmdc.201500456

DOI: 10.1002/cmdc.201500456
Full Papers
Identification of HDAC6-Selective Inhibitors of Low Cancer
Cell Cytotoxicity
Irina N. Gaisina,[a, b] Werner Tueckmantel,[b] Andrey Ugolkov,[c] Sida Shen,[a] Jessica Hoffen,[c]
Oleksii Dubrovskyi,[c, d] Andrew Mazar,[c, d] Renee A. Schoon,[e] Daniel Billadeau,[e] and
Alan P. Kozikowski*[a]
The histone deacetylases (HDACs) occur in 11 different iso-
forms, and these enzymes regulate the activity of a large
number of proteins involved in cancer initiation and progres-
sion. The discovery of isoform-selective HDAC inhibitors
(HDACIs) is desirable, as it is likely that such compounds would
avoid some of the undesirable side effects found with the first-
generation inhibitors. A series of HDACIs previously reported
by us were found to display some selectivity for HDAC6 and to
induce cell-cycle arrest and apoptosis in pancreatic cancer
cells. In the present work, we show that structural modification
of these isoxazole-based inhibitors leads to high potency and
selectivity for HDAC6 over HDAC1–3 and HDAC10, while unex-
pectedly abolishing their ability to block cell growth. Three in-
hibitors with lower HDAC6 selectivity inhibit the growth of cell
lines BxPC3 and L3.6pl, and they only induce apoptosis in
L3.6pl cells. We conclude that HDAC6 inhibition alone is insuffi-
cient for disruption of cell growth, and that some degree of
class 1 HDAC inhibition is required. Moreover, the highly selec-
tive HDAC6Is reported herein that are weakly cytotoxic may
find use in cancer immune system reactivation.
Introduction
Histone acetylation is an important determinant of chromatin
organization and gene expression, with the hyperacetylation
of histones being associated with “open” euchromatic states of
chromatin, and hypoacetylation with a “closed” heterochro-
matic state.[1,2] Although the nuclear histones were identified
as the initial target of the HDACs, it was discovered that some
HDACs are also located in the cytoplasm, where they are in-
volved in the deacetylation of non-histone substrates including
PCAF, p300, MyoD, p53, STAT3, p65 (NFkB), Hsp90, and a-tubu-
lin.[3] There are four classes of HDACs composed of 18 different
enzymes with a highly conserved catalytic domain. Class I
HDACs are homologues of the yeast histone deacetylase RPD3
and are represented by HDAC1–3 and HDAC8; class II HDACs
(HDAC4–7, HDAC9, and HDAC10) share homology with the
yeast histone deacetylase HDAC1; class III HDACs are closely re-
lated to the NAD-dependent yeast Sir2 protein and are not af-
fected by inhibitors of other HDACs; and the most recently de-
scribed HDAC11 is the sole member of the class IV HDACs.[2,4]
HDACs exist in large multimolecular complexes and are recruit-
ed to promoter regions of genes in conjunction with other
proteins. The acetylation status of histone and non-histone
proteins has a wide range of effects on cellular function. In ad-
dition to influencing transcriptional activation and the DNA
binding affinity of several transcriptional factors, acetylation
also affects protein expression, stability, and degradation, and
protein-protein interactions within the cell or nucleus.[5] For ex-
ample, acetylation of a-tubulin results in the stabilization of
microtubules, an event not conducive to microtubule reorgani-
zation, and acetylation of Hsp90 inhibits its ATP binding and
chaperone association with its client proteins, including many
proteins that are required for maintenance of cellular transfor-
mation.[6] The aberrant activation of HDACs in cancer has been
suggested to be involved in the epigenetic silencing of tumor
suppressor genes.[1a,7] Yet it is clear that based on their regula-
tory roles for non-histone targets, the effect of HDACs in
cancer goes far beyond the modulation of histone acetylation
and gene expression. Consequently, there have been many ef-
forts to develop HDAC inhibitors (HDACIs) and to investigate
their effects on cancer cell growth. Several classes of HDACIs
have been found to have potent antitumor activities, with
some showing selectivity between cancer cells and normal
cells.[8] Most of the known HDACIs are pan-inhibitors, therefore,
the identification of compounds showing isoform selectivity is
crucial to developing better tools for studying underlying
mechanisms in different cancer cell lines. Previously, we have
[a] Dr. I. N. Gaisina, Dr. S. Shen, Prof. Dr. A. P. Kozikowski
Drug Discovery Program, Department of Medicinal Chemistry and Pharma-
cognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL
60612 (USA)
E-mail: kozikowa@uic.edu
[b] Dr. I. N. Gaisina, Dr. W. Tueckmantel
Psychogenics Inc., Tarrytown, NY 10591 (USA)
[c] Dr. A. Ugolkov, Dr. J. Hoffen, Dr. O. Dubrovskyi, Dr. A. Mazar
Center for Developmental Therapeutics, Feinberg School of Medicine, North-
western University, Evanston, IL 60208 (USA)
[d] Dr. O. Dubrovskyi, Dr. A. Mazar
Department of Pharmacology, Feinberg School of Medicine, Northwestern
University, Evanston, IL 60208 (USA)
[e] Dr. R. A. Schoon, Dr. D. Billadeau
Department of Immunology and Division of Oncology Research, College of
Medicine, Mayo Clinic, 13-42 Guggenheim, Rochester, MN 55905 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500456.
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compounds with consideration of the unique features of the
HDAC6 binding site.[13] The general structure of these inhibitors
consists of a hydroxamic acid function as a zinc binding group
(ZBG) attached to an isoxazole or benzene ring incorporated in
the linker, and a “cap” group, which in the present work is
a heteroaryl group bearing additional functionality. The choice
of the terminal motifs was based on HDAC inhibitors previous-
ly explored by us.[9b,c,13] The synthesis of compounds 1–5 is
outlined in Scheme 1. Employing a standard procedure,[9c] 4-
phenylthiazol-2-ylamine (19a) and its m- and p-nitrophenyl de-
rivatives (19b,c) were coupled with either 5-hexynoic acid
(20a) or 4-pentynoic acid (20b) to provide corresponding
amides 21. The acetylenic function in 21a,b was transformed
into isoxazole (compounds 23a,b) by 1,3-dipolar cycloaddition
with a nitrile oxide generated in situ from ethyl chlorooximi-
noacetate and a base.[14] The intermediates 21c,d were re-
duced to the aniline derivatives 22a,b, which after treatment
with ethyl chloroformate or di-tert-butyl dicarbonate to pro-
vide corresponding carbamates, were converted into the isoxa-
zoles 23c–e. Lastly, reaction of 23 with freshly prepared hy-
droxylamine[15] generated the desired hydroxamic acids 1–5.
The second series consisting of compounds 6–12 was pre-
pared according to Scheme 2. Coupling of 5-arylisoxazole-3-
carboxylic acids 24a–c with butynylamine provided acetylenes
25a–c,[9b] and the same procedure as outlined above was fol-
lowed to generate esters 26a–c. Deprotection and subsequent
acylation of the amino group of 27 with pivaloyl, cyclohexane-
carbonyl, and benzoyl chlorides, respectively, provided inter-
mediate esters 28a–c. Hydroxamate formation yielded the
target compounds 6–12. A small set of compounds containing
a benzene ring in the linker was synthesized as shown in
Scheme 3. The precursor acids 24a,b were coupled with
methyl 4-(aminomethyl)benzoate in the presence of PyBOP to
provide esters 29a,b, which were further transformed into hy-
droxamates 13 and 14. Alternatively, the tert-butoxycarbonyl
group was removed, and the resulting amines 30a,b were
Figure 1. Selected HDACIs with significant antiproliferative effects on pan-
creatic tumor cells.
generated several series of HDACIs (Figure 1).[9] Treatment of
pancreatic cancer cells with these compounds results in a G1
and/or G2 arrest and induction of apoptosis. In addition, our
data indicated that some of these HDACIs cause loss of the
DNA damage checkpoint kinase, Chk1, a known Hsp90 client
protein.[10] Our isozyme analysis indicates that some of these
inhibitors, such as compounds A–C, show low nanomolar po-
tency toward HDAC6 and HDAC3 in vitro. Our objective was to
increase the selectivity index in favor of HDAC6 by applying
some simple modifications to the scaffold of these lead com-
pounds that would not significantly affect their molecular
properties. Such isoform-selective agents should be valuable
tools in linking specific HDAC isoforms to tumorigenic activity,
especially considering the number of controversial reports on
the role of HDAC6-selective inhibitors in cancer.[11]
Results and Discussion
Synthesis of HDAC6-selective inhibitors
With the goal to improve selectivity of the previously synthe-
sized ligands[9a,c,12] toward HDAC6, we designed new series of
Scheme 1. Reagents and conditions: a) 1. EDCI, DMAP, CH2Cl2, or 2. POCl3, pyridine; b) Fe, NH4Cl, AcOH, EtOH/H2O; c) ethyl chlorooximinoacetate, Et3N, THF;
d) Boc2O, toluene, microwave, 1208C; e) ClCO2Et, Et3N, THF; f) NH2OH·HCl, KOH.
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Scheme 2. Reagents and conditions: a) but-3-ynylamine hydrochloride, PyBOP, Et3N, DMF; b) ethyl chlorooximinoacetate, Et3N, THF; c) CF3COOH, CH2Cl2; d) piv-
aloyl chloride, Et3N, CH2Cl2; e) cyclohexanecarbonyl chloride, Et3N, CH2Cl2; f) benzoyl chloride, Et3N, CH2Cl2; g) NH2OH·HCl, KOH, THF/MeOH.
Scheme 3. Reagents and conditions: a) methyl 4-(aminomethyl)benzoate hydrochloride, PyBOP, Et3N, DMF; b) CF3COOH, CH2Cl2; c) cyclohexanecarbonyl chlo-
ride, Et3N, CH2Cl2; d) NH2OH·HCl, KOH, THF/MeOH; e) LiOH/NaOH, THF/H2O; f) ClCO2Et, NH2OH, N-methylmorpholine; g) 3-bromopropyne, NaH, DMF; h) ethyl
chlorooximinoacetate, Et3N, THF.
acylated with cyclohexanecarbonyl chloride. A two-step proce-
dure was employed to convert intermediate esters 28a,b into
the corresponding hydroxamic acids 15 and 16 (Scheme 3).
Carbazole (32) was alkylated with propargyl bromide, and the
standard reaction sequence was applied to afford compound
17 (Scheme 3). The carbazole-based analogue 18 with an elon-
gated linker was made by coupling an intermediate acid
formed from the ester 33 with methyl 4-(aminomethyl)ben-
zoate, followed by the aminolysis of the methyl ester with hy-
droxylamine.
Table 1. We first looked at effects of the linker length on iso-
zyme selectivity. In the phenylthiazole series, compound 2, ex-
hibiting an IC50 value at HDAC6 of 31 nm, was found to be
somewhat more potent than its longer-chain homologues,
such as compound 1 (IC50 81.8 nm) and the previously reported
compound A (IC50 67.7 nm). However, the shorter linker also re-
sulted in increased potency at HDAC3 and HDAC10. In light of
previously published results, the introduction of functional
groups, such as NH-Boc and NH-COOEt, either at the meta or
para position of the benzene ring, was hoped to increase the
selectivity of these compounds, possibly due to the formation
of additional hydrogen bonds with the enzyme. We therefore
synthesized ligands 3–12. The introduction of either of the
aforementioned groups in para position resulted in a twofold
improvement in potency (compounds 3 and 4, IC50 at HDAC6
HDAC isoform inhibition
All compounds were tested against both class I (1, 2, and 3)
and class II (6 and 10) HDACs, and their IC50 values are listed in
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Table 1. In vitro HDAC isozyme inhibition data for new hydroxamate derivatives.
Compd
HDAC isoform, IC50 [nm][a]
Class I
SI HDAC2/6
Class II
HDAC10
HDAC1
302
SI[b] HDAC1/6
HDAC2
HDAC3
29.6
SI HDAC3/6
HDAC6
SI HDAC10/6
A
B
C
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
TSA[d]
4.5
9.7
0.2
4.3
10
10
4.1
80
2817
429
146
6.3
25
0.3
15
0.4
1.8
1.7
11
10
11
7.2
32
3800
67.7
5.8
13.8
81.8Æ5.5
31Æ3.4
254
3.8
7.7
6.6
10
56.3
3.2
351
307
401
10.2
22.8
934
320
448
354
44.9
90.7
854
407
52.9
4.8
1220
1140
1140
834
1100
37
28
17
13
41.2Æ4.3
48.9Æ1.5
3.3Æ0.1
6.0Æ0.3
7.7Æ0.8
101.0Æ9.7
16.8Æ2.5
21.2Æ0.9
6.7Æ0.5
4.4Æ0.1
0.33Æ0.06
0.64Æ0.11
5.4Æ0.3
2.6Æ0.1
1510Æ65
0.61Æ0.14
0.78Æ0.23
1.0
6.6
82
201
266
323
271
333
107
16900
>50000
3910
6680
>50000
>50000
2930
436
444
350
212
38700
495
>50000[c]
>50000
41300
2360
22800
>50000
5190
1770
>50000
>50000
7050
135
789
215
669
44000
479
>50000
>50000
12500
5290
40100
>50000
4630
39
398
409
140
51
105
123
315
1910
>50000
>50000
10400
1900
666
1320
694
65
82
26
2360
5760
2160
461
1600
409
1230
40
257
29
1050
9580
4270
38
3160
2730
204
191
1380
2490
4300
1650
73
>50000
1370
>50000
22200
825
3.8
2280
798
9.3
37000
11.4
3.0
6.4
8.2
7.3
8.9
[a] Assays were conducted by the Reaction Biology Corp. (Malvern, PA, USA). All compounds were tested in duplicate in a 10-dose IC50 mode with threefold
serial dilution starting at 30 mm against HDAC6, and tested in singlet in a 10-dose IC50 mode with threefold serial dilution starting at 30 mm against
HDAC1,2,3,10. [b] Selectivity index. [c] No inhibition at 50 mm. [d] Trichostatin A.
41.2 and 48.9 nm, respectively). However, selectivity against
HDAC10 was diminished (HDAC10/6 selectivity ratio 1.0 and
6.6, respectively). On the contrary, the meta-(ethoxycarbonyl)-
amino substituent bestows significant selectivity on the inhibi-
tor 5. In the bis-isoxazole series, ligands 6 and 7 were equipo-
tent at HDAC6, with the meta-NH-Boc substituted analogue 7
being more selective against class 1. Surprisingly, replacement
of the Boc group by ethoxycarbonyl was found to be deleteri-
ous for activity (8 vs. 6).
Next, we chose to investigate the incorporation of other
bulky, electron-withdrawing substituents at an amino group in
meta position, and synthesized compounds 9–12 (Table 1). In
ligand 10, the single-bonded oxygen is deleted, resulting in an
amide rather than a urethane function. This replacement
caused little decrease in potency or selectivity (IC50 at HDAC6
21.2 nm, selectivity index (SI) at least 1900). Compound 9, bear-
ing a free amino group, was reasonably active at HDAC6, but
less selective over all other isoforms. Compound 11 containing
a (cyclohexanecarbonyl)amino group showed equal potency to
that of the NH-Boc analogue 7. Within this series, the benzoyl-
substituted analogue 12 was identified as the most potent
HDAC6 inhibitor with an acceptable selectivity profile.
and HDAC10, and at least 400-fold selectivity over HDAC1 and
HDAC3. These compounds have a higher cLogP value (2.66)
than their isoxazole analogues 6 and 7 (cLogP=1.35). Com-
pounds 15 and 16 were as potent as 6 and 7 at HDAC6, but
had relatively low SIs. Lastly, the effect of replacement of the
amidophenyl group by a more rigid and bulkier substituent,
such as carbazole, on the pattern of isozyme selectivity was
studied. The isoxazolylhydroxamate 17 was found to be
a weak inhibitor at all isozymes tested. On the other hand, the
elongated phenylhydroxamate 18 retained the HDAC6 potency
of its meta-BocNH substituted analogue 14, and showed an
improved selectivity over HDAC10.
The selectivity profile of HDACI 6 was more closely evaluat-
ed (Table S1, Supporting Information). This compound was
found to be highly selective for HDAC6 over all other isoforms.
Growth inhibition of pancreatic cancer cells by new HDACIs
Several HDACIs from different series, demonstrating varying
potencies and selectivity profiles in the isozyme assays, were
examined for their capacity to decrease pancreatic cancer cell
viability (Table 2). Previously, it has been shown that treatment
of BxPC3, Panc04.03, and MiaPaCa-2 cells with HDACIs A-C led
to a significant decrease in pancreatic cancer cell viability.[9b,c]
Some of our new HDACIs listed in Table 2, such as 1, 2, 4, and
5 showed antitumor activity similar to the reference com-
pound C when tested in two pancreatic cancer cell lines,
BxPC3 and L3.6pl. HDACIs 1, 2, 4, and 5 demonstrated reason-
able growth inhibition in at least one cancer cell line at low-
Because most of the hydroxamate-based HDACIs are very
polar (i.e., have low cLogP values, which could affect cell per-
meability), we decided to increase their lipophilicity by replac-
ing the heterocycle in the linker with a benzene ring. These
structural alterations resulted in ligands 13 and 14, which
stand out through their picomolar activity at HDAC6 and a se-
lectivity of more than three orders of magnitude over HDAC2
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Table 2. In vitro growth inhibition (GI) of pancreatic cancer cells by new
HDACIs.
Compd
BxPC3
GI50 [mm][a]
SE[b]
LogGI50 [mm]
L3.6pl
GI50 [mm][a]
SE[b]
LogGI50 [mm]
C
1
2
4
5
6
7
8
0.6
1.5
2.3
1.7
7.0
0.04
0.08
0.08
0.07
0.10
0.21
0.22
0.26
0.06
0.23
0.49
0.22
0.09
0.08
0.1
1.3
0.7
1.7
2.1
0.11
0.17
0.08
0.12
0.12
0.17
0.44
0.14
0.15
2.48
0.34
0.25
0.08
0.09
29
>50
>50
47
>50
>50
12
>50
>50
8.1
9
3.3
11
12
13
14
18
>50
>50
2
>50
1.3
7.8
2.3
[a] Pancreatic cancer cell lines BXPC3 and L3.6pl were obtained from the
American Type Culture Collection (ATCC, Manassas, VA, USA). The relative
number of viable cancer cells was determined 72 h post-treatment by
measuring the optical density (OD) using [3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt;
MTS] cell proliferation assay kit (Promega, Madison, WI, USA). OD values
were determined as the mean of five replicates per compound concentra-
tion in a 96-well plate. The GI50 value for each compound was calculated
with a nonlinear regression model of the standard slope using GraphPad
Prism 6.0 software. [b] Standard error.
Figure 2. HDAC inhibitors induce apoptosis in pancreatic cancer cells: L3.6pl
pancreatic cancer cells were treated with compounds 1, 2, or 4 at 10 mm for
24 h as indicated. Cells were collected and processed for Hoechst staining
(upper panel), and cell lysates were prepared for Western immunoblotting
(lower panel); 50 mg of the proteins were separated by SDS-PAGE, trans-
ferred to a PVDF membrane, and immunoblotted as indicated.
micromolar concentrations, whereas the majority of the more
selective HDAC6 inhibitors showed weak antiproliferative ef-
fects with GI50 values above 50 mm (Table 2). However, excep-
tions were observed with compound 13 that had GI50 values of
8 and 2 mm in BxPX3 and L3.6pl, and compound 18 that had
GI50 values of 2.3 and 1.3 mm in BxPX3 and L3.6pl, respectively
(Table 2).
ty.[17] To gain a better understanding of the utility and selectivi-
ty of HDAC6 inhibitors, PANC1 pancreatic cancer cells were
treated with a panel of the new HDAC6-selective inhibitors,
and with the non-selective inhibitors B and C for comparison.
We demonstrated that treatment with either group leads to an
accumulation of acetylated a-tubulin, a well-known target of
HDAC6 (Figure 3A).[18] Pancreatic cancer cell migration was
measured using the scratch-wound assay. Importantly, we
found that compounds B and C demonstrated a significant de-
crease in PANC1 cell migration in this assay, relative to diluent-
treated control cells (Figure 3B and Table 3). On the other
hand, the HDAC6-selective inhibitors 6, 9–13, and 18 had only
a minor effect on pancreatic cancer cell migration, in contrast
to what has previously been reported for tubacin.[19]
Moreover, compounds 1, 2, and 4 effectively induced apop-
tosis as determined by Hoechst staining and PARP cleavage,
a marker of apoptosis, in HDACI-treated L3.6pl pancreatic
cancer cells 24 h after the initiation of treatment (Figure 2). On
the other hand, none of compounds 1, 2, or 4 induced apop-
tosis in BxPC3 pancreatic cancer cells (data not shown). It is
worth noting that the nonselective HDACI, panobinostat, has
recently been found to promote apoptosis and decrease
tumor growth in the BxPC3 subcutaneous xenograft mouse
model.[16] L3.6pl is characterized as a poorly differentiated, fast-
growing pancreatic cell line with high tumorigenicity, while
BxPC3 is characterized as more differentiated, slow-growing
cell line with low tumorigenicity. Our results suggest that selec-
tion of HDACIs as effective pancreatic cancer therapies will
likely depend on the precise histopathological features of the
tumor.
In summary, we have demonstrated that simple structural al-
terations, such as the incorporation of a rigid aromatic ring
into the linker and the introduction of a functional group in
the cap residue, can be used to improve selectivity toward the
HDAC6 isoform in this series of compounds. We were able to
generate compounds possessing low-nanomolar to sub-nano-
molar potency and high HDAC6 selectivity. Among those
tested, the most selective compounds demonstrated only low
antiproliferative activity against pancreatic cancer cell lines as
compared with their non-selective analogues. Treatment of
pancreatic cancer cells with the newly synthesized HDACIs
leads to an accumulation of acetylated a-tubulin, but does not
affect cell migration.
Selective HDAC6 inhibitors do not suppress pancreatic
cancer cell migration
Previous studies suggested that levels of HDAC6 but not its
deacetylase activity affect both cell migration and cytotoxici-
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were run in a Biotage Initiator microwave synthesizer. Unless
1
stated otherwise, H and 13C NMR spectra were recorded on Bruker
DPX-400 or AVANCE-400 spectrometers at 400 and 100 MHz, re-
spectively, with TMS as an internal standard. Standard abbrevia-
tions indicating multiplicity were used as follows: s=singlet, d=
doublet, t=triplet, q=quadruplet, m=multiplet, and br=broad.
Mass spectra were measured in ESI mode at an ionization potential
of 70 eV with an LC–MS instrument containing a Hewlett–Packard
MSD. Analytical TLC was performed on Merck 60 F254 silica gel glass
plates, layer thickness: 250 mm. Preparative TLC was performed on
Analtech silica gel GF plates, layer thickness: 1 mm. For flash chro-
matography, silica gel of 230–400 mesh particle size was used. Ana-
lytical HPLC was carried out on an ACE 3AQ C18 column [150”
4.6 mm, particle size: 3 mm; flow rate: 2.0 mLminÀ1; from 10%
CH3CN in H2O to 50% in 10 min and to 100% CH3CN in 5 min,
both solvents containing 0.05% TFA (method A), or from 30%
CH3CN in H2O to 100% CH3CN in 15 min, both solvents containing
0.05% TFA (method B)].
General procedures for amide coupling
Figure 3. Evaluation of the ability of novel HDAC6 inhibitors to induce tubu-
lin acetylation and to arrest cancer cell migration. A) PANC1 cells were treat-
ed with DMSO or HDACIs at 1 mm for 24 h. Cells were harvested and lysed,
and equivalent amounts of protein were loaded per lane and immunoblot-
ted as indicated. B) PANC1 cells were treated with the indicated HDACIs, and
a scratch was made through a confluent layer of cells. Images of the cells
were taken at 24 and 48 h post wounding.
Method A: To a solution of an aminothiazole (1 equiv) and a car-
boxylic acid (1.1 equiv) in dry CH2Cl2 (10 mLmmolÀ1) at ambient
temperature were added EDCI (1.5 equiv) and DMAP (0.1 equiv),
and the resulting reaction mixture was stirred for 12 h. Upon reac-
tion completion as indicated by TLC, the reaction mixture was di-
luted with 50 mL of CH2Cl2, and then washed with saturated aque-
ous NaHCO3 (50 mL) and brine (50 mL), dried over anhydrous
Na2SO4, and concentrated under vacuum. The residue was purified
by flash chromatography to afford the product.
Table 3. Inhibition of cell migration.[a]
Method B: To a mixture of 4-(3- or 4-nitrophenyl)thiazol-2-ylamine
(1 equiv) and 5-hexynoic acid (1 equiv) in anhydrous pyridine
Compd
24 h
48 h
Compd
24 h
48 h
(4 mLmmolÀ1
)
at À158C was added phosphorus oxychloride
DMSO
–
+ + +
+ + +
+
–
+ +
+ +
–
DMSO
10
11
12
13
–
–
+
–
–
+
–
–
–
–
–
–
(1 equiv) dropwise with vigorous stirring. After 45 min of stirring at
À158C, the reaction mixture was allowed to warm to ambient tem-
perature, heated for 1 h at 608C, and stirred overnight at ambient
temperature. The reaction was quenched by addition of ice-water,
and the mixture was extracted with EtOAc (3”50 mL). The organic
layer was washed with brine (50 mL), dried over Na2SO4, and con-
centrated under vacuum. The residue was purified by CombiFlash
chromatography (EtOAc/hexane; 25–40%) to provide the amide.
B
C
6
7
9
+
+
+
–
18
[a] Level of inhibition (high to low): + + +, + +, or +; no inhibition: –.
Method C: To a solution of the carboxylic acid (1 equiv) in DMF
(0.5 mLmmolÀ1) was added PyBOP (1 equiv), and the mixture was
stirred for 20 min at ambient temperature. But-3-ynylamine hydro-
chloride (1.05 equiv) and Et3N (3 equiv) were then added, and the
reaction mixture was stirred for 1 h. Upon completion of the reac-
tion as ascertained by TLC, Et3N was evaporated, and the residue
was purification by HPLC.
Conclusions
In summary, while inhibition of class 1 HDACs has been proven
to be essential for decreasing the viability of pancreatic cancer
cells, the contribution of HDAC6 to pancreatic cancer cell via-
bility remains to be investigated. However, the low growth in-
hibition induced by some of these compounds may make
them more valuable as adjuvants in reactivation of the
immune system through control of the expression levels of
PD-L1.[20] This possibility is currently under study.
General procedure for 1,3-dipolar cycloaddition with a nitrile
oxide (Method D)
To a stirred solution of an acetylene (1 equiv) and Et3N (15 equiv)
in dry THF (15 mLmmolÀ1) at ambient temperature was added
dropwise over 36 h by syringe pump a solution of ethyl chlorooxi-
minoacetate (15 equiv) in 20 mL of THF. The white solids formed
were separated by filtration and washed with EtOAc (200 mL). The
combined organic phases were concentrated under vacuum. The
residue was purified by column chromatography.
Experimental Section
All reactions were conducted under an argon atmosphere and
stirred magnetically. Starting materials, reagents, and solvents were
purchased from commercial suppliers and used without further pu-
rification unless otherwise stated. Microwave-assisted reactions
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(t, J=8.0 Hz, 2H), 1.96–2.03 (t, J=8.0 Hz, 3H), 2.93 (m, 2H), 2.21 (t,
J=7.5 Hz, 2H), 2.71 (t, J=7.5 Hz, 2H), 4.46 (q, J=8.0 Hz, 2H), 6.30
(s, 1H), 7.34 (s, 1H), 7.36–7.47 (m, 3H), 7.84 (d, J=8.4 Hz, 2H),
10.62 ppm (s, 1H).
General procedure for hydroxamic acid formation (Method E)
To a stirred solution of the ester (0.1 g, 1 equiv) in CH2Cl2
(15 mLmmolÀ1) was added at 08C a freshly prepared solution of
NH2OH. After 15 min, the reaction mixture was diluted with 30 mL
of CH2Cl2, and the product was extracted into H2O. The aqueous
phase was acidified with 1n HCl to pH~4. The precipitate was fil-
tered off and washed with H2O and hexane to give the hydroxamic
acid, which was purified by HPLC.
5-[3-[N-(4-Phenylthiazol-2-yl)carbamoyl]propyl]isoxazole-3-car-
bohydroxamic acid (1): The title compound was synthesized from
the ester 23a (0.10 g, 0.27 mmol) according to Method E and puri-
fied by HPLC (method A). Yield: 0.053 g, 54%. 1H NMR (400 MHz,
[D6]DMSO): d=2.02 (m, 2H), 2.54 (t, J=7.4 Hz, 2H), 2.86 (t, J=
7.4 Hz, 3H), 3.30 (brs, 1H), 6.58 (s, 1H), 7.30–7.45 (m, 3H), 7.88 (d,
J=7.4 Hz, 2H), 9.35 (s, 1H), 12.28 ppm (s, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=22.8, 25.7, 34.3, 102.3, 108.3, 124.6, 126.0, 128.1,
129.1, 134.7, 149.1, 158.2, 171.2, 171.5 ppm; HPLC purity: 98.2%;
HRMS-FAB: m/z [M+H]+ calcd for C17H15N4O4S: 373.0965, found:
373.0978.
Preparation of NH2OH: To a stirring solution of NH2OH·HCl (1.0 g,
10 wt. equiv) in MeOH (10 mL) was added portionwise at 08C a so-
lution of KOH (1.0 g) in MeOH (4.0 mL). The resulting mixture was
filtered, and the obtained solution of NH2OH in MeOH was used in
a reaction with an ester.
N-[4-(4-Nitrophenyl)thiazol-2-yl]hex-5-ynamide (21c): Method B
was used to couple 4-(4-nitrophenyl)thiazol-2-ylamine (19b)
(0.50 g, 2.26 mmol) and 5-hexynoic acid (20a) (0.25 g, 2.26 mmol).
The residue was purified by flash chromatography (EtOAc/hexane;
25–40%) to provide the acetylene 21c (0.61 g, 84%).
General procedure for the reduction of nitro groups (Method
F)
A mixture of the nitro compound (1 equiv) and NH4Cl (2 equiv) in
H2O/EtOH (3:5, 17 mLmmolÀ1) was heated at reflux, and iron filings
(9 equiv) and AcOH (1 mLmmolÀ1) were added subsequently. The
reaction mixture was held at reflux for 2 h, and then allowed to
cool to ambient temperature. It was diluted with EtOAc (100 mL),
washed with H2O (40 mL) and brine (40 mL), dried over Na2SO4,
and evaporated. The residue was purified by CombiFlash chroma-
tography.
N-[4-(4-Aminophenyl)thiazol-2-yl]hex-5-ynamide (22a): The title
compound was obtained from the nitro compound 21c (0.61 g,
1.90 mmol) and NH4Cl (0.20 g, 3.8 mmol) according to Method F
and purified by flash chromatography (EtOAc/hexane; 30–50%).
1
Yield: 0.39 g (71%). H NMR (300 MHz, [D6]DMSO): d=1.75 (m, 2H),
2.20 (m, 2H), 2.56 (m, 2H), 2.82 (s, 1H), 5.23 (brs, 2H), 6.56 (d, J=
9.0 Hz, 2H), 7.16 (s, 1H), 7.53 (d, J=9.0 Hz, 2H), 12.15 ppm (s, 1H).
N-(4-Phenylthiazol-2-yl)pent-4-ynamide (21b): Method A was
used to couple 2-amino-4-phenylthiazole (19a) (1.01 g, 5.67 mmol)
and 4-pentynoic acid (20b) (0.61 g, 6.24 mmol). The residue was
purified by column chromatography (EtOAc/hexane; 30%) to
afford 21b (1.24 g, 85%). 1H NMR (400 MHz, CDCl3): d=2.20 (m,
2H), 2.40 (m, 2H), 7.18 (s, 1H), 7.38–7.47 (m, 3H), 7.85 (m, 2H),
11.24 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=14.0, 34.4, 69.4,
82.0, 108.0, 126.3, 128.3, 128.9, 134.2, 149.7, 169.2 ppm.
5-[3-[N-[4-[4-(tert-Butoxycarbonylamino)phenyl]thiazol-2-yl]car-
bamoyl]propyl]isoxazole-3-carboxylic acid ethyl ester (23c):
(a) Synthesis of N-[4-[2-(hex-5-ynoylamino)thiazol-4-yl)phenyl]carba-
mic acid tert-butyl ester. A mixture of the amine 22a (0.20 g,
0.70 mmol) and Boc2O (0.20 g, 0.91 mmol) in 2 mL of toluene was
placed in a microwave oven and heated for 20 min at 1208C. The
solvent was evaporated, and the residue was purified by flash
chromatography (EtOAc/hexane; 30–50%) to provide the inter-
5-[2-[N-(4-Phenylthiazol-2-yl)carbamoyl]ethyl]isoxazole-3-carbox-
ylic acid ethyl ester (23b): The title compound was synthesized
from 21b (0.56 g, 2.18 mmol) and ethyl chlorooximinoacetate
(4.92 g, 32.7 mmol) according to Method D. The residue was puri-
fied by column chromatography (EtOAc/hexane; 25–50%). Crystal-
lization from 10% EtOAc/hexane provided the pure ester 23b
1
mediate urethane (0.21 g, 79%). H NMR (300 MHz, CDCl3): d=1.25
(m, 9H), 1.99 (m, 2H), 2.30 (m, 2H), 2.01 (s, 1H), 2.58 (m, 2H), 7.05
(s, 1H), 7.40 (m, 2H), 7.74 ppm (d, J=9.0 Hz, 2H).
(b) The title compound 23c was synthesized from the above inter-
mediate (0.21 g, 0.54 mmol) and ethyl chlorooximinoacetate
(1.23 g, 8.17 mmol) according to Method D and purified by flash
chromatography (EtOAc/hexane; 25–70%). Yield: 0.12 g (44%).
1H NMR (400 MHz CDCl3): d=1.41 (t, J=7.1 Hz, 3H), 1.52 (s, 9H),
1.98 (m, 2H), 2.21 (m, 2H), 2.69 (m, 2H), 4.44 (q, J=7.1 Hz, 2H),
6.37 (s, 1H), 7.04 (s, 1H), 7.33 (m, 2H), 7.64 (m, 2H), 11.50 ppm
(brs, 1H).
1
(0.41 g, 50%). H NMR (400 MHz, CDCl3): d=1.44 (t, J=8.0 Hz, 2H),
2.21 (t, J=8.0 Hz, 3H), 2.93 (t, J=8.0 Hz, 2H), 4.45 (q, J=8.0 Hz,
2H), 6.14 (s, 1H), 7.17 (s, 1H), 7.28–7.43 (m, 3H), 7.49 (d, J=8 Hz,
2H) (m, 2H), 12.18 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=
14.1, 21.4, 32.3, 62.1, 101.9, 108.3, 126.4, 128.6, 129.0, 134.2, 149.5,
156.2, 159.9, 150.0, 168.9, 172.9 ppm.
5-[2-[N-(4-Phenylthiazol-2-yl)carbamoyl]ethyl]isoxazole-3-carbo-
hydroxyamic acid (2): The title compound was synthesized from
the ester 23b (0.10 g, 0.27 mmol) according to Method E. Yield:
0.05 g, 50%. A sample for biological testing was purified by HPLC.
1H NMR (400 MHz, [D6]DMSO): d=2.92 (t, J=7.2 Hz, 2H), 3.16 (t,
J=7.2 Hz, 3H), 6.58 (s, 1H), 7.31–7.45 (m, 3H), 7.90 (d, J=7.4 Hz,
2H), 9.35 (s, 1H), 11.49 (s, 1H), 12.38 ppm (s, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=21.8, 31.1, 32.5, 191.1, 108.4, 126.0, 128.2,
129.1, 134.7, 149.2, 156.6, 157.9, 158.1, 170.1, 173.8 ppm; HPLC
purity: 97.4%; HRMS-FAB: m/z [M+H]+ calcd for C16H14N4O4S:
359.0808, found: 359.0820.
N-[4-[2-[4-[3-(N-Hydroxycarbamoyl)isoxazol-5-yl]butyrylamino]-
thiazol-4-yl]phenyl]carbamic acid tert-butyl ester (3): The title
compound was synthesized from the ester 23c (0.12 g, 0.24 mmol)
according to Method E. Yield: 0.025 g (24.5%). 1H NMR (400 MHz,
[D6]DMSO): d=1.41 (s, 9H), 1.93 (m, 2H), 2.44 (m, 2H), 2.79 (m,
2H), 6.54 (s, 1H), 7.37–7.44 (m 3H), 7.89 (d, J=5.1 Hz, 2H), 9.37 (s,
1H), 11.40 (brs, 1H), 12.19 ppm (s, 1H); 13C NMR (100 MHz
[D6]DMSO): d=22.8, 25.6, 28.5, 34.3, 79.6, 101.1, 106.6, 118.5, 126.4,
128.7, 139.5, 149.1, 153.1, 156.6, 157.8, 158.1, 171.1, 174.3 ppm;
HPLC purity: 97.9%; HRMS-FAB: m/z [M+H]+ calcd for
C22H25N5O6S: 488.1598, found: 488.1618.
5-[2-[N-(4-Phenylthiazol-2-yl)carbamoyl]propyl]isoxazole-3-car-
boxylic acid ethyl ester (23a): The title compound was synthe-
sized from 2-amino-4-phenylthiazole according to Methods A and
5-[3-[N-[4-[4-(Ethoxycarbonylamino)phenyl]thiazol-2-yl]carba-
moyl]propyl]isoxazole-3-carboxylic acid ethyl ester (23d):
(a) Synthesis of N-[4-[2-(hex-5-ynoylamino)thiazol-4-yl]phenyl]carba-
1
D in 32% yield over two steps. H NMR (400 MHz, CDCl3): d=1.41
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mic acid ethyl ester. To a mixture of the amine 22a (0.18 g,
0.64 mmol) and Et3N (0.17 mL, 1.3 mmol) in THF (5 mL) was added
ethyl chloroformate (0.08 g, 0.77 mmol) at ambient temperature.
The resulting mixture was stirred for 1 h. Upon completion of the
reaction as ascertained by TLC, EtOAc (20 mL) was added. The or-
ganic phase was washed with H2O (20 mL) and brine (20 mL), dried
over Na2SO4, and evaporated. The residue was purified by flash
chromatography (EtOAc/hexane; 25–100%) to provide the inter-
N-[3-[2-[3-[3-(N-Hydroxycarbamoyl)isoxazol-5-yl]propionylami-
no]thiazol-4-yl]phenyl]carbamic acid ethyl ester (5): The title
compound was synthesized from the ester 23e (0.072 g,
1
0.163 mmol) according to Method E. Yield: 0.035 g (50%). H NMR
(400 MHz, [D6]DMSO): d=1.30 (t, J=7.0 Hz, 3H), 2.95 (t, J=7.3 Hz,
2H), 3.25 (t, J=7.3 Hz, 2H), 4.19 (q, J=7.0 Hz, 2H), 6.60 (s, 1H),
7.28–7.32 (m, 3H), 7.55 (d, J=7.0 Hz, 1H), 8.07 (s, 1H), 9.27 ppm (s,
1H); 13C NMR (100 MHz, [D6]DMSO): d=14.9, 21.9, 32.5, 60.6, 102.3,
108.5, 116.1, 118.2, 120.2, 129.4, 135.2, 140.0, 149.3, 153.9, 157.4,
158.0, 161.3, 170.1, 174.6 ppm; HPLC purity: 97.6%; HRMS-FAB:
m/z [M+H]+ calcd for C19H19N5O6S: 446.1128, found: 446.1141.
1
mediate urethane (0.23 g, 99%). H NMR (400 MHz, [D6]DMSO): d=
1.23 (t, J=7.1 Hz, 3H), 1.81 (m, 2H), 2.22 (m, 2H), 2.55 (m, 2H),
2.82 (m, 1H), 4.14 (q, J=7.1 Hz, 2H), 7.44 (s, 1H), 7.52 (d, J=8.5 Hz,
2H), 7.77 (d, J=8.5 Hz, 2H), 9.72 (s, 1H), 12.2 ppm (s, 1H); 13C NMR
(400 MHz, [D6]DMSO): d=14.9, 17.7, 23.9, 34.1, 60.6, 72.2, 84.2,
106.7, 118.5, 126.5, 129.0, 139.2, 149.0, 153.9, 158.1, 171.3 ppm.
N-[4-[3-(N-But-3-ynylcarbamoyl)isoxazol-5-yl]phenyl]carbamic
acid tert-butyl ester (25a): Method C was used to couple the acid
24a (0.60 g, 1.97 mmol)) and but-3-ynylamine hydrochloride
(0.22 g, 2.08 mmol). The crude product was purified by HPLC to
(b) The title compound 23d was synthesized from the above inter-
mediate (0.23 g, 0.64 mmol) and ethyl chlorooximinoacetate
(0.97 g, 6.43 mmol) according to Method D and purified by flash
chromatography (EtOAc/hexane; 30–50%). Yield: (0.20 g, 65%).
1H NMR (400 MHz, [D6]DMSO): d=1.26 (m, 3H), 2.01 (m, 2H), 2.52
(m, 2H), 2.89 (m, 2H), 4.33 (m, 2H), 6.73 (s, 1H), 7.45 (s, 1H), 7.49
(d, J=8.5 Hz, 2H), 7.77 (d, J=8.5 Hz, 2H), 9.55 (s, 1H), 9.72 (s, 1H),
12.2 (s, 1H), 13.2 ppm (s, 1H).
1
afford acetylene 25a (0.126 g, 18%). H NMR (400 MHz, CDCl3): d=
1.45 (s, 9H), 1.98 (t, J=2.6 Hz, 1H), 2.45 (m, 2H), 3.55 (m, 2H), 6.58
(s, 1H), 6.79 (s, 1H), 7.07 (m, 1H), 7.18 (s, 1H), 7.42 (d, J=8.6 Hz,
2H), 7.63 ppm (d, J=8.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=
19.3, 28.2, 30.9, 38.0, 70.4, 80.89, 81.2, 98.1, 118.3, 121.3, 126.8,
140.6, 12.2, 158.9, 159.0, 171.4 ppm.
5-[2-[[5-[4-(tert-Butoxycarbonylamino)phenyl]isoxazole-3-carbon-
yl]amino]ethyl]isoxazole-3-carboxylic acid ethyl ester (26a): The
title compound was synthesized from the acetylene 25a (0.126 g,
0.36 mmol) and ethyl chlorooximinoacetate (1.00 g, 6.60 mmol) ac-
cording to Method D and purified by flash chromatography
(EtOAc/hexane; 25–70%). Yield: 0.105 g (63%). 1H NMR (400 MHz,
CDCl3): d=1.34 (t, J=7.2 Hz, 3H), 1.47 (s, 9H), 3.14 (m, 2H), 3.75 (t,
J=6.8 Hz, 2H), 4.37 (q, J=7.2 Hz, 2H), 6.51 (s, 1H), 6.80 (s, 1H),
7.46 (d, J=8.5 Hz, 2H), 7.63 ppm (d, J=8.5 Hz, 2H); 13C NMR
(100 MHz, CDCl3): d=13.9, 26.7, 28.1, 37.1, 62.1, 97.8, 102.5, 118.3,
120.7, 126.7, 141.1, 152.7, 156.4, 158.6, 159.6, 159.9, 171.5,
172.1 ppm.
N-[4-[2-[4-[3-(N-Hydroxycarbamoyl)isoxazol-5-yl]butyrylamino]-
thiazol-4-yl]phenyl]carbamic acid ethyl ester (4): The title com-
pound was synthesized from the ester 23d (0.20 g, 0.42 mmol) ac-
cording to Method E. Yield: 0.075 g (38%). 1H NMR (400 MHz,
[D6]DMSO): d=1.25 (t, J=7.1 Hz, 3H), 2.02 (m, 2H), 2.54 (m, 2H),
2.86 (m, 2H), 4.14 (d, J=7.1 Hz, 3H), 6.61 (s, 1H), 7.46 (s, 1H), 7.50
(d, J=7.1 Hz, 2H), 7.78 (d, J=7.1, 2H), 9.72 (s, 1H), 11.48 (s, 1H),
12.27 ppm (s, 1H); 13C NMR (100 MHz, [D6]DMSO): d=14.9, 22.8,
25.7, 31.1, 34.2, 60.6, 101.1, 106.8, 118.5, 126.5, 129.0, 139.2, 149.0,
153.9, 157.9, 158.1, 171.1, 174.3 ppm; HPLC purity: 98.1%; HRMS-
FAB: m/z [M+H]+ calcd for C20H21N5O6S: 460.1285, found:
460.1300.
N-[4-[3-[N-[2-[3-(N-Hydroxycarbamoyl)isoxazol-5-yl]ethyl]carba-
moyl]isoxazol-5-yl]phenyl]carbamic acid tert-butyl ester (6): The
title compound was synthesized from the ester 26a (0.105 g,
0.223 mmol) according to Method E and purified by HPLC (meth-
od A). Yield: 0.025 g (24.5%). 1H NMR (400 MHz, [D6]DMSO): d=
1.42 (s, 9H), 3.04 (m, 2H), 3.52 (m, 2H), 6.58 (s, 1H), 7.11 (s, 1H),
7.56 (d, J=8.4 Hz, 2H), 7.65 (d, J=8.4 Hz, 2H), 8.92 (m, 1H), 9.28
(brs, 1H), 9.63 (s, 1H), 11.4 ppm (s, 1H); 3C NMR (100 MHz,
[D6]DMSO): d=26.4, 28.4, 37.2, 80.0, 98.8, 101.6, 118.5, 120.3, 127.0,
142.3, 153.0, 156.6, 157.8, 159.1, 159.7, 170.9, 172.5 ppm; HPLC
purity: 97.6%; HRMS-FAB: m/z [M+H]+ calcd for C21H23N5O7S:
458.1670, found: 458.1669.
5-[2-[N-[4-[3-(Ethoxycarbonylamino]phenyl)thiazol-2-yl]carba-
moyl]ethyl]isoxazole-3-carboxylic acid ethyl ester (23e): (a) Syn-
thesis of N-[3-[2-(pent-4-ynoylamino)thiazol-4-yl]phenyl]carbamic
acid ethyl ester. To a solution of the amine 22b (0.16 g, 0.60 mmol)
and Et3N (0.24 mL, 1.80 mmol) in THF (3 mL) was added dropwise
at 08C a solution of ethyl chloroformate (0.07 mL, 0.56 mmol) in
THF (1 mL). The reaction mixture was stirred for 20 min, diluted
with EtOAc (25 mL), washed with H2O (20 mL), aqueous NaHCO3
(20 mL), and brine (20 mL), dried over Na2SO4, and evaporated. The
residue was purified by preparative TLC (EtOAc/hexane; 30%) to
provide the intermediate urethane (0.10 g, 48%). 1H NMR
(400 MHz, [D6]DMSO): d=1.42 (t, J=7.0 Hz, 3H), 2.84 (t, J=7.2 Hz,
2H), 2.98 (m, 1H), 4.31 (q, J=7.0 Hz, 2H), 7.47 (m, 2H), 7.66 (m,
2H), 8.28 (s, 1H), 9.84 ppm (s, 1H); 13C NMR (100 MHz, [D6]DMSO):
d=18.9, 19.7, 39.1, 52.6, 53.3, 65.3, 76.8, 88.4, 113.2, 120.8, 122.9,
125.0, 134.1, 140.0, 144.7, 144.8, 154.0, 158.7, 162.8, 174.9 ppm.
N-[3-[3-(N-But-3-ynylcarbamoyl)isoxazol-5-yl]phenyl]carbamic
acid tert-butyl ester (25b): Method C was used to couple the acid
24b (1.30 g, 4.30 mmol) and but-3-ynylamine hydrochloride
(0.50 g, 4.73 mmol). The crude product was purified by HPLC to
1
afford acetylene 25b (0.96 g, 63%). H NMR (400 MHz, CDCl3): d=
1.54 (s, 9H), 2.07 (t, J=2.6 Hz, 1H), 2.54 (m, 2H), 3.67 (dd, J=6.4,
12.8 Hz, 2H), 6.67 (s, 1H), 6.98 (s, 1H), 7.19 (m, 1H), 7.40–7.47 (m,
3H), 7.88 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=19.0, 27.9,
30.5, 37.6, 70.0, 80.5, 80.7, 99.0, 99.4, 115.2, 120.0, 120.1, 127.0,
129.4, 138.8, 152.1, 158.5, 171.0 ppm.
(b) The title compound 23e was synthesized from the above inter-
mediate (0.08 g, 0.23 mmol) and ethyl chlorooximinoacetate
(0.35 g, 2.32 mmol) according to Method D and purified by prepa-
rative TLC (EtOAc/hexane; 20%). Yield: 0.072 g (65%). 1H NMR
(400 MHz, CDCl3): d=1.41 (t, J=8.0 Hz, 3H), 2.65 (t, J=7.3 Hz, 2H),
3.10 (t, J=7.3 Hz, 2H), 4.23 (m, 2H), 4.44 (q, J=8.0 Hz, 2H), 6.33 (s,
1H), 7.12 (m, 2H), 7.29 (m, 2H), 7.43 (m, 1H), 7.87 (brs, 1H),
11.64 ppm (brs, 1H); 13C NMR (100 MHz, CDCl3): d=14.1, 21.6, 32.6,
62.1, 102.0, 108.5, 121.1, 129.5, 134.8, 138.6, 149.0, 153.9, 156.3,
159.2, 160.0, 169.1, 173.2 ppm.
5-[2-[[5-[3-(tert-Butoxycarbonylamino)phenyl]isoxazole-3-carbon-
yl]amino]ethyl]isoxazole-3-carboxylic acid ethyl ester (26b): The
title compound was synthesized from the acetylene 25b (0.96 g,
2.70 mmol) and ethyl chlorooximinoacetate (4.0 g, 27.0 mmol) ac-
cording to Method D and purified by flash chromatography
(EtOAc/hexane; 25–40%). Yield: 0.93 g (73%). 1H NMR (300 MHz,
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CDCl3): d=1.29 (t, J=7.1 Hz, 3H), 1.55 (s, 9H), 3.21 (t, J=6.8 Hz,
2H), 3.86 (q, J=6.8 Hz, 2H), 4.43 (q, J=7.1 Hz, 2H), 6.55 (s, 1H),
6.69 (s, 1H), 6.99 (s, 1H), 7.13 (m, 1H), 7.43 (m, 3H), 7.89 ppm (s,
1H).
thesized from the ester 27 (0.06 g, 0.16 mmol) according to Meth-
od E and purified by HPLC (method B). Yield: 0.02 g (34%). H NMR
1
(400 MHz, [D6]DMSO): d=3.04 (m, 2H), 3.53 (m, 2H), 5.36 (brs, 2H),
6.58 (s, 1H), 6.65 (d, J=8.0 Hz, 1H), 6.98 (m, 2H), 7.06 (s, 1H), 7.10
(m, 2H), 8.95 (t, J=5.4 Hz), 9.28 (s, 1H), 11.42 ppm (s, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=26.4, 37.2, 99.4, 101.6, 110.6, 113.9, 116.7,
127.1, 130.2, 149.6, 156.6, 157.8, 159.1, 159.6, 171.7, 172.5 ppm;
HPLC purity: 97.1%; HRMS-FAB: m/z [M+H]+ calcd for C16H15N5O5:
358.1146, found: 358.1156.
N-[3-[3-[N-[2-[3-(N-Hydroxycarbamoyl)isoxazol-5-yl]ethyl]carba-
moyl]]isoxazol-5-yl]phenyl]carbamic acid tert-butyl ester (7): The
title compound was synthesized from the ester 26b (0.10 g,
0.21 mmol) according to Method E and purified by HPLC (meth-
1
od A). Yield: 0.02 g (20%). H NMR (400 MHz, [D6]DMSO): d=1.42 (s,
9H), 3.04 (m, 2H), 3.54 (m, 2H), 6.59 (s, 1H), 7.17 (s, 1H), 7.35 (d,
J=7.8 Hz, 2H), 7.45 (m, 2H), 8.01 (s, 1H), 8.99 (t, J=5.5 Hz, 1H),
9.28 (s, 1H), 9.54 (s, 1H), 11.42 ppm (s, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=26.4, 28.5, 31.1, 37.2, 79.9, 100.1, 101.6, 114.9, 120.2,
120.8, 127.0, 130.2, 140.8, 153.2, 156.6, 157.8, 158.9, 159.8, 170.9,
172.5 ppm; HPLC purity: 97.8%; HRMS-FAB: m/z [M+Na]+ calcd
for C21H23N5NaO7: 480.1490, found: 480.1505.
5-[2-[[5-[3-[(2,2-Dimethylpropionyl)amino]phenyl]isoxazole-3-
carbonyl]amino]ethyl]isoxazole-3-carboxylic acid ethyl ester
(28a): To a mixture of the amine 27 (0.06 g, 0.16 mmol) and Et3N
(0.05 mL, 0.36 mmol) in CH2Cl2 (1 mL) was added pivaloyl chloride
(22 mL, 0.18 mmol) at 08C. The resulting mixture was warmed to
ambient temperature and stirred for 3 h. The solvent was evaporat-
ed, and the residue was purified by flash chromatography (EtOAc/
1
hexane; 40–100%) to provide the product (0.054 g, 73%). H NMR
[4-[3-(But-3-ynylcarbamoyl)isoxazol-5-yl]phenyl]carbamic
acid
(400 MHz, CDCl3): d=1.42 (t, J=7.0 Hz, 3H), 3.22 (m, 2H), 3.86 (m,
1H), 4.45 q, J=7.0 Hz, 2H), 6.56 (s, 1H), 7.45–7.54 (m, 3H), 7.66 (d,
J=8.0 Hz, 1H), 8.04 ppm (s, 1H); 13C NMR (100 MHz, CDCl3): d=
14.1, 27.0, 27.5, 37.2, 39.7, 62.1, 99.4, 102.7, 117.1, 121.5, 122.0,
127.3, 129.8, 138.8, 158.6, 159.1, 159.8, 171.3, 171.8, 176.8 ppm.
ethyl ester (25c): Method C was used to couple the acid 24c
(0.8 g, 2.9 mmol) and but-3-ynylamine hydrochloride (0.34 g,
3.19 mmol). The crude product was purified by flash chromatogra-
phy to afford acetylene 25c (0.94 g, 99%).
5-[2-[[5-[4-(Ethoxycarbonylamino)phenyl]isoxazole-3-carbonyl]-
amino]ethyl]isoxazole-3-carboxylic acid ethyl ester (26c): The
title compound was synthesized from the acetylene 25c (0.94 g,
2.87 mmol) and ethyl chlorooximinoacetate (4.35 g, 28.7 mmol) ac-
cording to Method D and purified by flash chromatography
(EtOAc/hexane; 40–100%). Yield: 0.36 g (28%). 1H NMR (400 MHz,
CDCl3): d=1.29 (t, J=6.5 Hz, 3H), 1.43 (t, J=7.1 Hz, 3H), 3.21 (t, J=
6.5 Hz, 2H), 3.86 (q, J=7.1 Hz, 2H), 4.42 (q, J=7.1 Hz, 2H), 6.56 (s,
1H), 6.78 (s, 1H), 6.90 (d, J=4.4 Hz, 1H), 7.07 (t, J=5.6 Hz, 1H),
7.52 (d, J=8.4 Hz, 1H), 7.65 ppm (d, J=8.4 Hz, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=14.4, 27.0, 37.2, 61.6, 62.1, 98.1, 102.7,
118.5, 121.5, 126.9, 140.3, 153.1, 156.5, 158.6, 159.3, 159.8, 171.4,
171.8 ppm.
5-[2-[[5-[3-[(2,2-Dimethylpropionyl)amino]phenyl]isoxazole-3-
carbonyl]amino]ethyl]isoxazole-3-carbohydroxyamic acid (10):
The title compound was synthesized from the ester 28a (0.054 g,
0.118 mmol) according to Method E and purified by HPLC (meth-
1
od A). Yield: 0.022 g (42%). H NMR (400 MHz, [D6]DMSO): d=1.18
(s, 9H), 3.05 (m, 2H), 3.54 (m, 2H), 6.59 (s, 1H), 7.21 (s, 1H), 7.40 (t,
J=7.8 Hz, 1H), 7.56 (d, J=7.8 Hz, 1H), 7.76 (d, J=7.8 Hz, 1H), 8.19
(s, 1H), 9.01 (t, J=5.0 Hz, 1H), 9.28 (s, 1H), 9.37 (s, 1H), 11.42 ppm
(s, 1H); 13C NMR (100 MHz, [D6]DMSO): d=26.4, 27.5, 37.2, 100.1,
101.6, 117.2, 121.2, 122.7, 126.8, 130.0, 140.6, 156.6, 157.8, 158.9,
159.8, 170.9, 172.5, 177.2 ppm; HPLC purity: 99.9%; HRMS-FAB:
m/z [M+H]+ calcd for C21H23N5O6: 442.1721, found: 442.1738.
N-[4-[3-[N-[2-[3-(N-Hydroxycarbamoyl)isoxazol-5-yl]ethyl]carba-
moyl]isoxazol-5-yl]phenyl]carbamic acid ethyl ester (8): The title
compound was synthesized from the ester 26c (0.15 g, 0.34 mmol)
according to Method E and purified by HPLC (method A). Yield:
5-[2-[[5-[3-[(Cyclohexanecarbonyl)amino]phenyl]isoxazole-3-car-
bonyl]amino]ethyl]isoxazole-3-carboxylic acid ethyl ester (28b):
To a mixture of the amine 27 (0.06 g, 0.16 mmol) and Et3N
(0.06 mL, 0.48 mmol) in CH2Cl2 (1 mL) was added cyclohexanecar-
bonyl chloride (0.026 mL, 0.19 mmol) at 08C. After stirring 3 h at
08C, H2O (2 mL) was added, and the mixture was extracted with
CH2Cl2 (3”5 mL). The combined organic layers were dried over
Na2SO4 and evaporated. The residue (0.08 g) was used in next step
1
0.065 g (44%). H NMR (400 MHz, [D6]DMSO): d=1.17 (t, J=7.1 Hz,
3H), 3.03 (m, 2H), 3.53 (m, 2H), 4.09 (q, J=6.5 Hz, 2H), 6.58 (s, 1H),
7.12 (s, 1H), 7.54 (d, J=8.7 Hz, 2H), 7.76 (d, J=8.7 Hz, 2H), 8.93 (t,
J=5.7 Hz, 1H), 9.27 (s, 1H), 9.90 (s, 1H), 11.4 ppm (s, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=14.8, 26.4, 37.2, 60.9, 98.9, 101.6, 118.6,
120.6, 127.1, 142.0, 153.8, 156.6, 157.8, 159.1, 159.7, 170.9,
172.5 ppm; HPLC purity: 97.6%; HRMS-FAB: m/z [M+H]+ calcd for
C19H19N5O7: 430.1357, found: 430.1374.
1
without additional purification. H NMR (400 MHz, CDCl3): d=0.97–
1.47 (m, 8H), 1.47–1.84 (m, 5H), 2.35 (m, 1H), 3.14 (m, 2H), 3.62 (m,
2H), 4.38 (m, 2H), 6.80 (s, 1H), 7.26 (s, 1H), 7.47 (t, J=7.7 Hz, 1H),
7.60 (d, J=7.7 Hz, 1H), 7.69 (d, J=7.7 Hz, 1H), 8.25 (s, 1H), 9.07 (m,
1H), 10.05 ppm (s, 1H).
5-[2-[[5-(3-Aminophenyl)isoxazole-3-carbonyl]amino]ethyl]isoxa-
zole-3-carboxylic acid ethyl ester (27): A solution of tert-butyl
ester 26b (0.30 g, 0.64 mmol) in a mixture of CH2Cl2 and trifluoro-
acetic acid (3:1, 4 mL) was stirred at ambient temperature for 48 h.
The solvents were evaporated, and the residue was purified by
flash chromatography (EtOAc/hexane; 50–100%) to provide the
5-[3-[(Cyclohexanecarbonyl)amino]phenyl]-N-[2-[3-(N-hydroxy-
carbamoyl)isoxazol-5-yl]ethyl]isoxazole-3-carboxamide (11): The
title compound was synthesized from the ester 26b (0.08 g,
0.17 mmol) according to Method E and purified by HPLC (meth-
1
1
od A). Yield: 0.022 g (25%). H NMR (400 MHz, [D6]DMSO): d=1.11–
amine 27 (0.12 g, 50%). H NMR (400 MHz, [D6]DMSO): d=1.30 (t,
1.44 (m, 5H), 1.58 (m, 2H), 1.74 (m, 2H), 2.28 (m, 1H), 3.05 (m, 2H),
3.54 (m, 2H), 6.59 (s, 1H), 7.20 (m, 1H), 7.39 (t, J=7.6 Hz, 1H), 7.54
(d, J=7.6 Hz, 1H), 7.60 (d, J=7.6 Hz, 1H), 8.18 (s, 1H), 9.00 (m, 1H),
9.98 (s, 1H), 11.41 ppm (s, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
25.6, 25.8, 26.4, 29.5, 37.2, 45.3, 100.2, 101.6, 116.0, 121.0, 126.9,
130.2, 140.7, 156.6, 157.8, 158.9, 159.8, 170.8, 172.5, 175.1 ppm;
HPLC purity: 98.4%; HRMS-FAB: m/z [M+H]+ calcd for C23H25N5O6:
468.1877, found: 468.1873.
J=7.0 Hz, 3H), 3.13 (m, 2H), 3.61 (m, 2H), 4.34 (q, J=7.0 Hz, 2H),
6.70 (dd, J=5.5 Hz, 8.0 Hz, 1H), 6.78 (s, 1H), 7.04–7.19 (m, 4H),
9.02 ppm (t, J=5.5 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
14.3, 26.5, 37.1, 62.1, 99.4, 102.8, 110.6, 113.9, 116.8, 127.1, 130.2,
149.5, 156.4, 159.1, 159.6, 159.9, 171.7, 173.7 ppm.
5-(3-Aminophenyl)-N-[2-[3-(N-hydroxycarbamoyl)isoxazol-5-yl]-
ethyl]isoxazole-3-carboxamide (9). The title compound was syn-
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5-[2-[[5-[3-(Benzoylamino)phenyl]isoxazole-3-carbonyl]amino]-
ethyl]isoxazole-3-carboxylic acid ethyl ester (28c): To a mixture
of the amine 27 (0.056 g, 0.15 mmol) and Et3N (0.04 mL,
0.33 mmol) in CH2Cl2 (1 mL) was added benzoyl chloride (19 mL,
0.16 mmol) at 08C. After stirring for 1 h at 08C, H2O (2 mL) was
added, and the mixture was extracted with CH2Cl2 (3”5 mL). The
organic phase was dried over Na2SO4 and evaporated, and the resi-
due was purified by preparative TLC (EtOAc/hexane, 50%) to pro-
pound was synthesized from the acid 24b (1.00 g, 3.29 mmol),
methyl
4-(aminomethyl)benzoate
hydrochloride
(0.54 g,
3.29 mmol), PyBOP (1.88 g, 3.62 mmol), and N,N-diisopropylethyl-
amine (0.96 mL, 7.23 mmol) using the same procedure as for com-
1
pound 29a. Yield: 0.77 g (50%). H NMR (400 MHz, CDCl3): d=1.48
(s, 9H), 4.58 (d, J=5.2 Hz, 2H), 4.94 (s, 1H), 6.93 (s, 1H), 7.31–7.40
(m, 10H), 7.61 (d, J=6.3 Hz, 2H), 7.70 (m, 1H), 7.80 (s, 1H),
7.95 ppm (m, 1H).
1
vide the ester 28c (0.05 g, 69%). H NMR (400 MHz, CDCl3): d=1.32
N-[3-[3-[N-[4-(N-Hydroxycarbamoyl)benzyl]carbamoyl]isoxazol-5-
yl]phenyl]carbamic acid tert-butyl ester (14): The title compound
was synthesized from the ester 29b (0.35 g, 0.78 mmol) using the
same procedure as for compound 13. The crude product was puri-
fied by HPLC (method A) to give the title compound (0.06 g, 17%).
1H NMR (400 MHz, [D6]DMSO): d=4.43 (d, J=5.9 Hz, 2H), 7.21 (s,
1H), 7.34 (m, 3H), 7.38 (m, 2H), 7.66 (d, J=8.1 Hz, 2H), 8.03 (s, 1H),
9.38 (t, J=6.1 Hz, 1H), 9.55 (s, 1H), 11.12 ppm (s, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=28.5, 42.5, 79.9, 100.2, 114.9, 120.2, 120.7,
127.0, 127.4, 127.6, 130.2, 131.9, 140.8, 142.5, 153.2, 159.0, 159.9,
164.5, 170.9 ppm; HPLC purity: 96.4%; HRMS-FAB: m/z [M+H]+
calcd for C23H24N4O6: 453.1774, found: 453.1781.
(m, 3H), 3.13 (m, 2H), 3.75 (m, 2H), 4.35 (m, 2H), 6.50 (s, 1H), 6.92
(s, 1H), 7.28 (s, 1H), 7.34–7.50 (m, 5H), 7.70 (m, 1H), 7.87 (m, 2H),
7.98 (d, J=5.0 Hz, 1H), 8.03 (s, 1H), 9.14 ppm (s, 1H).
5-[3-(Benzoylamino)phenyl]-N-[2-[3-(N-hydroxycarbamoyl)isoxa-
zol-5-yl]ethyl]isoxazole-3-carboxamide (12): The title compound
was synthesized from the ester 28c (0.05 g, 0.10 mmol) according
to Method E and purified by HPLC (method A). Yield: 0.019 g
(39%). 1H NMR (400 MHz, [D6]DMSO): d=3.11 (t, J=6.6 Hz, 2H),
3.63 (dd, J=6.6 Hz, 12.7 Hz, 2H), 6.66 (s, 1H), 7.30 (s, 1H), 7.52–
7.64 (m, 5H), 7.67 (d, J=7.9 Hz, 1H), 7.94 (dd, J=1.2 Hz, 6.6 Hz,
1H), 7.98 (m, 2H), 8.39 (m, 1H), 9.08 (t, J=5.8 Hz, 1H), 9.35 (brs,
1H), 10.48 (s, 1H), 11.49 ppm (s, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=26.1, 36.9, 99.5, 99.9, 101.2, 117.0, 121.4, 122.5,
126.6, 127.7, 128.5, 129.8, 131.8, 134.6, 140.0, 156.3, 157.5, 158.6,
159.5, 165.8, 170.4, 172.2 ppm; HPLC purity: 97.3%; HRMS-FAB:
m/z [M+H]+ calcd for C23H19N5O6: 462.1408, found: 462.1416.
4-[[[5-[3-[(Cyclohexanecarbonyl)amino]phenyl]isoxazole-3-car-
bonyl]amino]methyl]benzoic acid (31b): (a) Synthesis of 4-[[[5-(3-
aminophenyl)isoxazole-3-carbonyl]amino]methyl]benzoic
acid
methyl ester. A solution of tert-butyl carbamate 29b (0.76 g,
1.68 mmol) in 12 mL of a mixture of CH2Cl2 and trifluoroacetic acid
(1:1) was stirred overnight at ambient temperature. The solvents
were evaporated, and the residue was recrystallized from EtOAc/
hexane (10%) to provide the intermediate amine (0.50 g) which
was used directly in next step.
4-[[[5-[4-[(tert-Butoxycarbonyl)amino]phenyl]isoxazole-3-carbon-
yl]amino]methyl]benzoic acid Methyl Ester (29a): To a solution of
the acid 24a (0.50 g, 1.64 mmol) in DMF (3.0 mL) was added
PyBOP (0.94 g, 1.80 mmol), and the mixture was stirred for 15 min
at ambient temperature. Methyl 4-(aminomethyl)benzoate hydro-
chloride (0.35 g, 2.16 mmol) and Et3N (0.65 mL, 4.93 mmol) were
added subsequently, and the mixture was heated in a microwave
reactor for 20 min at 658C. H2O (20 mL) was added, and the mix-
ture was extracted with EtOAc (3”25 mL). The combined organic
phases were washed with brine (40 mL), dried over Na2SO4, and
evaporated. The residue was purified by flash chromatography
(EtOAc/hexane; 40–100%) to provide the ester 29a (0.60 g, 81%).
1H NMR (400 MHz, [D6]DMSO): d=1.48 (s, 9H), 4.01 (s, 3H), 4.54 (d,
J=6.0 Hz, 2H), 7.14 (s, 1H), 7.45 (d, J=8.1 Hz, 2H), 7.61 (d, J=
8.5 Hz, 2H), 7.81 (d, J=8.1 Hz, 2H), 7.95 (d, J=8.6 Hz, 2H), 9.42 (t,
J=6.0 Hz, 1H), 9.7 ppm (s, 1H).
(b) Synthesis of 4[[[5-[3-[(cyclohexanecarbonyl]amino]phenyl]isoxa-
zole-3-carbonyl]amino]methyl]benzoic acid methyl ester (30b). To
a solution of the amine (0.50 g, 1.42 mmol) in 20 mL of a mixture
of CH2Cl2 and THF (1:1) were added Et3N (0.37 mL, 2.84 mmol) and
cyclohexanecarbonyl chloride (0.38 mL, 2.84 mmol) at ambient
temperature. After 15 min, H2O (15 mL) was added, and the mix-
ture was extracted with CH2Cl2 (3”25 mL). The combined organic
phases were washed with brine (40 mL), dried over Na2SO4, and
evaporated. The residue was purified by flash chromatography
(CH2Cl2/MeOH; 5%) to provide the acylation product (0.54 g, 82%),
which was used directly in next step.
(c) To a stirred solution of intermediate 30b (0.54 g, 1.17 mmol) in
THF (20 mL) was added a freshly prepared solution of LiOH (0.12 g,
4.28 mmol) in H2O (10 mL) at ambient temperature. After 24 h,
NaOH (0.10 g, 2.1 mmol) was added. The mixture was stirred for
30 min, acidified with 1n HCl to pH~2, diluted with EtOAc
(25 mL), washed with H2O (15 mL) and brine (15 mL), dried over
Na2SO4, and concentrated under vacuum to provide the acid 31b
(0.26 g, 50%). 1H NMR (400 MHz, [D6]DMSO): d=1.25–2.33 (m,
10H), 2.33 (t, J=11.0 Hz, 1H), 4.53 (d, J=5.7 Hz, 2H), 7.31 (s, 1H),
7.37 (d, J=8.0 Hz, 2H), 7.46 (t, J=7.9 Hz, 1H), 7.61 (d, J=7.9 Hz,
1H), 7.72 (d, J=7.9 Hz, 1H), 7.90 (d, J=8.0 Hz, 2H), 8.28 (s, 1H),
9.46 (t, J=6.0 Hz, 1H), 10.14 ppm (s, 1H).
N-[4-[3-[N-[4-(N-Hydroxycarbamoyl)benzyl]carbamoyl]isoxazol-5-
yl]phenyl]carbamic acid tert-butyl ester (13): To a solution of the
methyl ester 29a (0.20 g, 0.44 mmol) and hydroxylamine hydro-
chloride (0.18 g, 2.65 mmol) in a mixture of MeOH and THF (2:1,
15 mL) was added MeONa (0.81 mL, 3.54 mmol, as a 21% solution
in MeOH) at ambient temperature. The mixture was stirred over-
night, and then 1n HCl was added to adjust the solution to pH~4.
The mixture was diluted with EtOAc (50 mL), washed with brine
(25 mL), dried over Na2SO4, and concentrated under vacuum. The
residue was purified by HPLC (method A) to give the title com-
1
pound. Yield: 0.02 g (10%). H NMR (400 MHz, [D6]DMSO): d=1.43
(s, 9H), 4.43 (d, J=5.9 Hz, 2H), 7.15 (s, 1H), 7.31 (d, J=8.0 Hz, 2H),
7.55 (d, J=8.6 Hz, 2H), 7.64 (d, J=8.0 Hz, 2H), 7.75 (d, J=8.6 Hz,
2H), 9.33 (t, J=5.9 Hz, 1H), 9.64 (s, 1H), 11.11 ppm (s, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=28.1, 30.7, 42.1, 79.7, 98.5, 99.5, 118.2,
120.0, 126.6, 127.0, 127.2, 131.5, 142.0, 142.2, 152.6, 158.8, 159.4,
164.1, 170.6 ppm; HPLC purity: 97.6%; HRMS-FAB: m/z [M-H]À
calcd for C23H24N4O6: 451.1623, found: 451.1639.
5-[3-[(Cyclohexanecarbonyl)amino]phenyl]-N-[4-(N-hydroxycar-
bamoyl)benzyl]isoxazole-3-carboxamide (16): To a solution of the
acid 31b (0.12 g, 0.26 mmol) and N-methylmorpholine (0.032 g,
0.32 mmol) in THF (8 mL) was added ethyl chloroformate
(0.030 mL, 0.32 mmol) at 08C, and the mixture was stirred for
30 min. The precipitate was filtered off, and the filtrate was added
to a mixture of hydroxylamine hydrochloride (0.037 g, 0.53 mmol)
and Et3N (0.07 mL, 0.53 mmol) in DMF (2 mL). The reaction mixture
was stirred for 30 min at 08C. 1n HCl was added to bring the solu-
4-[[[5-[3-[(tert-Butoxycarbonyl)amino]phenyl]isoxazole-3-carbon-
yl]amino]methyl]benzoic acid Methyl Ester (29b): The title com-
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tion to pH~4, and the mixture was extracted with EtOAc (3”
25 mL). The combined organic phases were washed with brine
(40 mL), dried over Na2SO4, and evaporated. The residue was puri-
fied by HPLC (method B) to give the hydroxamic acid. Yield: 0.01 g
(8%). 1H NMR (400 MHz, [D6]DMSO): d=1.18–1.84 (m, 10H), 2.33
(m, 1H), 4.51 (d, J=6.0 Hz, 2H), 7.30 (s, 1H), 7.36 (d, J=8.2 Hz, 2H),
7.47 (t, J=7.9 Hz, 1H), 7.61 (d, J=7.9 Hz, 1H), 7.71 (d, J=7.9 Hz,
1H), 7.73 (d, J=8.2 Hz, 2H), 8.26 (s, 1H), 9.00 (brs, 1H), 9.46 (t, J=
6.0 Hz, 1H), 10.06 (s, 1H), 11.18 ppm (s, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=25.6, 25.8, 29.5, 42.5, 45.3, 100.2, 116.0, 121.0, 121.6,
127.0, 127.4, 127.6, 130.2, 131.9, 140.7, 142.5, 159.0, 159.9, 164.5,
170.8, 175.1 ppm; HPLC purity: 97.2%; HRMS-FAB: m/z [M+H]+
calcd for C25H26N4O6: 463.1976, found: 463.1988.
15 min, the reaction mixture was acidified with 1n HCl (pH ~4)
and diluted with EtOAc. The resulting mixture was washed with
H2O and brine, and the organic phase was dried over Na2SO4 and
evaporated. The solid residue was purified by HPLC (method A) to
give the hydroxamic acid as an off-white solid (0.150 g, 47%).
1H NMR (400 MHz, [D6]DMSO): d=5.86 (s, 2H), 6.60 (s, 1H), 7.18 (t,
J=7.5 Hz, 2H), 7.40 (t, J=7.5 Hz, 2H), 7.68 (d, J=7.5 Hz, 2H), 8.09
(d, J=7.5 Hz, 2H), 9.27 (s, 1H), 11.36 ppm (s, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=37.7, 102.2, 109.5, 119.6, 120.4, 122.4,
126.0, 139.7, 155.8, 157.4, 169.6 ppm; HRMS-FAB: m/z [M+H]+
calcd for C17H13N3O3: 308.1029, found: 308.1036.
5-(9H-Carbazol-9-ylmethyl)-N-[4-(hydroxycarbamoyl)benzyl]isox-
azole-3-carboxamide (18). (a) Synthesis of 5-(9H-carbazol-9-ylme-
thyl)isoxazole-3-carboxylic acid. To a stirred solution of the ester 33
(0.83 g, 2.58 mmol) in 5.0 mL of THF was added a freshly prepared
solution of LiOH (0.124 g, 5.17 mmol) in 5.0 mL of H2O at ambient
temperature. The mixture was stirred overnight, NaOH (0.103 g,
2.59 mmol) was added, and the reaction mixture was stirred for
30 min. The reaction mixture was acidified with 1n HCl (pH~4), di-
luted with EtOAc, and washed with H2O and brine. The organic
phase was dried over Na2SO4 and evaporated to give the carboxyl-
ic acid as an off-white solid (0.63 g, 83%).
5-[4-[(Cyclohexanecarbonyl)amino]phenyl]-N-[4-(N-hydroxycar-
bamoyl)benzyl]isoxazole-3-carboxamide (15): (a) Synthesis of 4-
[[[5-[3-[(cyclohexanecarbonyl)amino]phenyl]isoxazole-3-carbonyl]-
amino]methyl]benzoic acid (31a). Intermediate 29a was subjected
to the same three-step sequence as employed in the transforma-
tion of intermediate 29b to 31b. Compound 31a: 1H NMR
([D6]DMSO): d=1.16–1.76 (m, 10H), 2.28 (t, J=11.0 Hz, 1H), 4.47 (d,
J=6.2 Hz, 2H), 7.17 (s, 1H), 7.37 (d, J=8.2 Hz, 2H), 7.72 (d, J=
8.8 Hz, 2H), 7.80 (d, J=8.8 Hz, 2H), 7.85 (d, J=8.2 Hz, 2H), 9.35 (t,
J=6.0 Hz, 1H), 10.03 (s, 1H), 12.79 (brs, 1H).
(b) Synthesis of 4-[[5-(9H-carbazol-9-ylmethyl)isoxazole-3-carboxa-
mido]methyl]benzoic acid methyl ester. To a solution of the carbox-
ylic acid (0.33 g, 1.13 mmol) in DMF (3 mL) was added PyBOP
(0.64 g, 1.24 mmol), and the mixture was stirred for 15 min at am-
bient temperature. Methyl 4-(aminomethyl)benzoate hydrochloride
(0.24 g, 1.5 mmol) and Et3N (0.45 mL, 3.4 mmol) were added. The
resulting mixture was heated in a microwave oven at 658C for
20 min. H2O was added, and the mixture was extracted with
EtOAc. The combined organic phases were washed with NaCl,
dried over Na2SO4, and evaporated. The residue was purified by
CombiFlash chromatography (EtOAc/hexane; 40% then 100%) to
provide the ester intermediate (1.0 g, 66%). 1H NMR (400 MHz,
[D6]DMSO): d=3.81 (s, 3H), 4.43 (d, J=6.1 Hz, 2H), 5.94 (s, 2H),
6.65 (s, 1H), 7.24 (t, J=7.4 Hz, 2H), 7.35 (d, J=8.3 Hz, 2H), 7.48 (t,
J=7.4 Hz, 2H), 7.75 (d, J=8.3 Hz, 2H), 7.88 (t, J=8.3 Hz, 2H), 8.16
(d, J=7.4 Hz, 2H), 9.31 (t, J=6.1 Hz, 2H), 11.7 ppm (s, 1H);
13C NMR (100 MHz, [D6]DMSO): d=28.2, 43.1, 77.0, 77.9, 99.5, 126.8,
127.1, 128.3, 128.9, 130.7, 136.0, 144.0, 155.8 ppm.
(b) Compound 15 was obtained from the preceding intermediate
in the same manner as described for the synthesis of compound
1
16 from its precursor 31b. H NMR (400 MHz, [D6]DMSO): d=1.11–
1.76 (m, 10H), 2.32 (m, 1H), 4.44 (d, J=6.0 Hz, 2H), 7.16 (s, 1H),
7.33 (d, J=8.1 Hz, 2H), 7.66 (d, J=8.1 Hz, 2H), 7.72 (d, J=8.6 Hz,
2H), 7.80 (d, J=8.6 Hz, 2H), 8.94 (brs, 1H), 9.35 (t, J=6.0 Hz, 1H),
10.05 (s, 1H), 11.12 ppm (s, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
25.6, 25.7, 29.4, 42.5, 45.3, 99.1, 119.6, 121.1, 127.0, 127.4, 127.5,
131.8, 142.1, 142.5, 159.1, 159.8, 164.5, 170.8, 175.2 ppm; HPLC
purity: 95.3%; HRMS-FAB: m/z [M+H]+ calcd for C25H26N4O6:
463.1976, found: 463.1988.
5-(Carbazol-9-ylmethyl)isoxazole-3-carboxylic acid ethyl ester
(33): (a) Synthesis of 9-prop-2-ynyl-9H-carbazole. To a solution of
carbazole (1.00 g, 6.00 mmol) in dry DMF (5 mL) was added NaH
(0.31 g, 7.8 mmol) as a 60% suspension in mineral oil at 08C. The
mixture was stirred for 15 min, then propargyl bromide (0.73 mL,
6.6 mmol) was added, and the reaction mixture was allowed to
warm to ambient temperature. The reaction mixture was poured
into ice-water, and the solution was extracted with EtOAc. The or-
ganic phase was washed with H2O and brine, dried over anhydrous
Na2SO4, and concentrated under vacuum. The crude product was
purified by combiflash chromatography (EtOAc/hexane; 5–70%) to
(c) To a solution of the methyl ester (0.33 g, 0.75 mmol) and hy-
droxylamine hydrochloride (0.31 g, 4.5 mmol) in MeOH-THF (20 mL,
1:1) was added MeONa (1.3 mL, 6.0 mmol, as a 21% solution in
MeOH) at ambient temperature. The mixture was stirred at ambi-
ent temperature for 60 h, acidified with 1n HCl (pH ~4), and ex-
tracted with EtOAc. The combined organic phases were washed
with NaCl, dried over Na2SO4, and evaporated. The residue was pu-
rified by HPLC (method A) to give the hydroxamic acid as a white
1
give 0.72 g (58%) of the carbazole intermediate. H NMR (300 MHz,
CDCl3): d=2.07 (t, J=0.4 Hz, 1H), 2.84 (m, 2H), 3.43 (m, 2H), 7.41
(t, J=8.3 Hz, 2H), 7.50 (t, J=8.3 Hz, 2H), 8.02 (d, J=8.3 Hz, 2H),
8.26 ppm (d, J=8.3 Hz, 2H).
1
solid (0.022 g, 6.6%). H NMR (400 MHz, [D6]DMSO): d=4.32 (d, J=
5.9 Hz, 2H), 5.88 (s, 2H), 7.18 (m, 4H), 7.42 (t, J=7.2 Hz, 2H), 7.58
(d, J=8.2 Hz, 2H), 7.69 (d, J=8.2 Hz, 2H), 8.10 (d, J=7.2 Hz, 2H),
8.92 (s, 1H), 9.21 (t, J=5.9 Hz, 1H), 11.07 ppm (s, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=37.8, 42.0, 102.4, 109.5, 119.6, 120.4,
122.5, 126.0, 126.9, 127.1, 131.4, 139.7, 142.0, 158.3, 158.7, 164.1,
170.0 ppm; HRMS-FAB: m/z [M+H]+ calcd for C25H20N4O4:
441.1557, found: 441.1577.
(b) To a solution of 9-prop-2-ynyl-9H-carbazole (0.72 g, 3.50 mmol)
and Et3N (9.8 mL, 70 mmol) in 20 mL of dry THF under an Ar at-
mosphere stirred at ambient temperature, was added dropwise
over 24 h by syringe pump a solution of ethyl chlorooximinoace-
tate (5.32 g, 35.0 mmol) in 20 mL of THF. The reaction mixture was
filtered and washed with EtOAc, and the filtrate was evaporated.
The residue was purified by CombiFlash chromatography (EtOAc/
hexane; 5% then 40%) to provide a mixture of the ester 33 and
the dimer formed from the reagent (2.35 g).
Biological assays
5-(Carbazol-9-ylmethyl)isoxazole-3-carbohydroxamic acid (17).
To a stirred solution of the ester 33 (0.33 g, 1.03 mmol) in 5 mL of
THF was added at 08C a freshly prepared solution of NH2OH. After
HDAC inhibition assays: HDAC inhibition assays were performed
by the Reaction Biology Corporation (Malvern, PA, USA) using
human, full-length recombinant HDAC1 and 6 isolated from a bacu-
ChemMedChem 2016, 11, 81 – 92
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This work was supported by the US National Institutes of Health
(R43 CA 133985 to W.T.) and the Cancer Center Support Grant
2 P30 (CA060553-19 to A.M. and A.U.) at the Robert H. Lurie Com-
prehensive Cancer Center, Northwestern University. We thank Dr.
Arsen Gaisin for some technical assistance in compound purifica-
tion.
Keywords: HDAC6 selectivity
·
histone deacetylases
·
hydroxamic acids · immune activation · inhibitors · pancreatic
cancer
Received: October 6, 2015
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J. M. Denu, C. D. Allis, Annu. Rev. Biochem. 2001, 70, 81–120.
Published online on November 23, 2015
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