A series of potent and selective, triazolylphenyl-based histone deacetylases inhibitors with activity against pancreatic cancer cells and Plasmodium falciparum

Article abstract of DOI:10.1021/jm701606b

The discovery of the rules governing the inhibition of the various HDAC isoforms is likely to be key to identifying improved therapeutics that act as epigenetic modulators of gene transcription. Herein we present results on the modification of the CAP reg

Full text of DOI:10.1021/jm701606b

J. Med. Chem. 2008, 51, 3437–3448
3437
A Series of Potent and Selective, Triazolylphenyl-Based Histone Deacetylases Inhibitors with
Activity against Pancreatic Cancer Cells and Plasmodium falciparum
Yufeng Chen,† Miriam Lopez-Sanchez,§ Doris N. Savoy,‡ Daniel D. Billadeau,‡ Geoffrey S. Dow,§ and Alan P. Kozikowski*,†
Drug DiscoVery Program, Department of Medicinal Chemistry and Pharmacognosy, UniVersity of Illinois at Chicago, 833 South Wood Street,
Chicago, Illinois 60612, DiVision of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant AVenue, SilVer
Springs, Maryland 20910, and Department of Immunology, DiVision of Oncology Research, Mayo Clinic College of Medicine, 13-42
Guggenheim, 200 First Street SW, Rochester, Minnesota 55905
ReceiVed December 20, 2007
The discovery of the rules governing the inhibition of the various HDAC isoforms is likely to be key to
identifying improved therapeutics that act as epigenetic modulators of gene transcription. Herein we present
results on the modification of the CAP region of a set of triazolylphenyl-based HDACIs, and show that the
nature of substitution on the phenyl ring plays a role in their selectivity for HDAC1 versus HDAC6, with
low to moderate selectivity (2-51-fold) being achieved. In light of the valuable selectivity and potency that
were identified for the triazolylphenyl ligand 6b in the inhibition of HDAC6 (IC50 ) 1.9 nM), this compound
represents a valuable research tool and a candidate for further chemical modifications. Lastly, these new
HDACIs were studied for both their anticancer and antimalarial activity, which serve to validate the superior
activity of the HDACI 10c.
Introduction
(histone deacetylase 1) and include HDAC4, 5, 6, 7, 9, and 10.8
This class is divided into class IIa, consisting of HDAC4, 5, 7,
and 9, and class IIb, consisting of HDAC6 and 10, which contain
two catalytic sites. All class I and II HDACs are zinc-dependent
enzymes. The active site of class I and II HDACs is found within
a highly conserved catalytic domain containing a divalent zinc
cation that is coordinated by both histidine and aspartate
residues. Deacetylation of the HDAC substrates occurs through
attack by a water molecule that is activated through interaction
with this zinc cation coupled with deprotonation through a
histidine-aspartate charge-relay system.9 Class III HDACs
(SIRT1-7) require NAD+ for their enzymatic activity.10 Class
IV HDAC is represented by HDAC11, which contains conserved
residues in the catalytic core region shared by both class I and
II enzymes.11
Acetylation and deacetylation of histones play an important
role in transcription regulation of eukaryotic cells.1 The steady
state of histone acetylation is established by the dynamic
equilibrium between competing histone deacetylases (HDACs)
and histone acetyltransferases (HATs). HATs add acetyl groups
to lysine residues, while HDACs remove the acetyl groups. In
general, hyperacetylation of histones facilitates gene expression,
whereas histone deacetylation is correlated with transcriptional
repression. The HDACs are able to control histone deacetylation,
which consequently promotes chromatin condensation. HDACa
inhibitors (HDACIs) selectively alter gene transcription, in part,
by permitting chromatin remodeling and by changing the
composition of multiprotein complexes bound to proximal
region of specific gene promoter.2 Further, the HDACs interact
with many nonhistone protein-substrates such as the hormone
receptors, chaperone proteins, and cytoskeletal proteins, which
regulate cell proliferation and cell death.3,4
In mammalian cells, the HDACs are divided into four classes
that depend on their sequence/structural homology to yeast
deacetylases, expression patterns, and catalytic mechanisms.5,6
Eighteen HDAC enzymes have been identified and classified3,7
based on homology to these yeast HDACs. Class I HDACs
include HDAC1, 2, 3, and 8, which are related to yeast RPD3
deacetylase and they share high homology in their catalytic sites.
Recent phylogenetic analyses suggest that this class can be
divided into classes Ia (HDAC1 and 2), Ib (HDAC3), and Ic
(HDAC8). The class II HDACs are related to yeast Hda1
The HDACIs currently in clinical trials and preclinical
development include pan-HDAC inhibitors (SAHA, 1a and 1b)
as well as those compounds showing some level of isozyme
selectivity (1c, 1d, 1e, and 1f (Apicidin)) in Chart 1.12 With
the approval of SAHA by the FDA for the treatment of
cutaneous T-cell lymphoma (CTCL) and with the ongoing
evaluation of HDACIs for various cancers,13–15 considerable
interest has been growing in the application of HDACIs to a
broader range of disease states, where chromatin remodeling
may play a role in the pathophysiology. An important question
that still needs to be adequately addressed concerns identification
of the individual HDAC or subset of HDACs that one might
best inhibit in order to achieve a desirable therapeutic outcome.
Genetic studies, knockout studies in yeast, and the use of
siRNAs in mammalian cells suggest that the class I HDACs
are essential to cell proliferation and survival.16,17 However,
no conclusive experimental evidence pointed to specific HDACs
as being selectively involved in cancer and other diseases.4
Furthermore, many of the HDACIs are cytotoxic agents and
induce cell-cycle arrest and apoptosis, which would, of course,
prevent their use in long-term therapy. Thus difficulties may
be anticipated in the application of this compound class to
inflammatory diseases, malaria, psychiatric disorders, and neu-
rodegenerative diseases. Moreover, it is not entirely clear how
* To whom correspondence should be addressed. Phone: 312-996-7577.
Fax: 312-996-7107. E-mail:kozikowa@uic.edu.
†
University of Illinois at Chicago.
Mayo Clinic College of Medicine.
Walter Reed Army Institute of Research.
‡
§
a Abbreviations: HDAC, histone deacetylase; HDACI, histone deacetylase
inhibitor; HAT, histone acetyltransferase; ZBG, zinc binding group; SAHA,
suberoylanilide hydroxamic acid; TSA, trichostatin A; CTCL, cutaneous
T-cell lymphoma; HDLP, HDAC-like protein; HDAH, HDAC-like ami-
dohydrolase; NOC, nitrile oxide cycloaddition; Rf, retention factor; TLC,
thin-layer chromatography; MTT, 3-[4,5-dimethylthiazol-2-yl]2,5-diphe-
nyltetrazolium bromide; HMEC, human microvascular endothelial cell.
10.1021/jm701606b CCC: $40.75
2008 American Chemical Society
Published on Web 05/22/2008

3438 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Chart 1. Examples of HDAC Inhibitors
Chen et al.
the HADCIs are working in vivo and whether histone or
nonhistone-mediated effects are the most important. The
development of inhibitors selective for the individual HDAC
isozymes and/or conditional knockout mice may resolve some
of the issues in this area.
HDACIs show inherent selectivity for HDAC6 versus HDAC1,
and we have suggested that such compounds may prove valuable
in the treatment of certain CNS disorders.22 We have also
suggested that certain polar or hydrophobic interactions between
the CAP recognition groups and the HDAC protein surface may
contribute to discrimination among the isoforms.23 In this paper,
we present our results obtained from the study of a number of
triazole-based CAP groups constructed by use of the Huisgen’s
versatile 1,3-dipolar cycloaddition chemistry.24,25 The present
study identifies the geometric pattern of substitution in the CAP
group as a possible structural determinant in achieving selectivity
for HDAC6 over HDAC1. These newly synthesized ligands
were screened against a panel of pancreatic cell lines and
Plasmodium falciparum strains, and these data are presented
as well.
Most HDAC inhibitors such as TSA and SAHA closely
resemble the aliphatic acetyl-lysine substrate [the so-called
acetylated histone tail] and deliver a hydroxamic acid or other
zinc binding group to the catalytic zinc ion at the bottom of a
narrow, active site pocket as seen from the cocrystal structures
of inhibited HDLP (HDAC-like protein from A. aeolicus),9
HDAH (HDAC-like amidohydrolase),18 and the human HDAC7
(PDB: 2PQO and 2PQP) and HDAC8 (PDB: 1VKG, 1T67,
1T64, 1T69, and 2V5X).19,20 These inhibitors all have the zinc
binding group and a CAP group connected to one another
through a linker comprised of a straight alkyl chain, or some
modification thereof that incorporates heteroatoms, olefinic, aryl,
or heteroaryl groups. Attempts to generate isozyme-specific
HDAC inhibitors generally focus on varying the CAP groups
to exploit the variability in the HDAC surface surrounding the
active site.21 Despite much effort, truly selective compounds
are few in number, and the precise structural determinants
required to achieve the selective inhibition of single HDAC
isozymes generally remain undefined. To date, we have made
concerted efforts to create structurally novel HDACIs with the
intent to elucidate the structural parameters relevant to achieving
isoform selectivity. Moreover, our plan was to study these new
compounds in parallel for their therapeutic potential in cancer,
stress-induced neuronal injury, and protozoan parasitic diseases.
To accomplish these objectives, we chose to systematically study
modifications of the CAP, linker, and zinc binding groups in
order to fine-tune for isoform selectivity. Along these lines, we
have shown previously that certain mercaptoacetamide-based
Results and Discussion
Chemistry. The synthesis of the triazole-based ligands
6a-6d, 10a-10d, 12a-12d, and 14a-14d was accomplished
according to the synthetic protocols shown in Schemes 1 and
2. The 1,3-dipolar cycloaddition reaction (“click-chemistry”) of
an azide with an alkyne catalyzed by a copper (I) salt26,27 or by
Cp*RuCl(PPh3)228 has recently been reported to generate either
a 1,4-disubstituted or a 1,5-disubstituted triazole, regioselec-
tively. This dipolar cycloaddition chemistry, much like our
previously reported applications of the nitrile oxide cycloaddition
(NOC) reaction,29 provides efficient and complementary syn-
thetic strategies to assemble diverse templates aimed at the
discovery and optimization of lead compounds. At the beginning
of this research, we first synthesized ligand 6a and screened it
against a panel of HDAC isoforms. The selectivity measured
for this ligand for HDAC6 versus HDAC1 (>20-fold) appeared
to be promising when compared with the pan-inhibition shown

HDACIs with ActiVity against Cancer Cells and P. falciparum
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3439
Scheme 1. Synthesis of 1,4-Disubstituted Triazole Ligands 6a-6d and 10a-10da
a Reagents and conditions: (a) Suberic acid monomethyl ester, POCL3, pyridine, 0 °C; (b) copper (II) sulfate pentahydrate, sodium ascorbate, azide 4a-4d,
rt; (c) NH2OH, KOH, rt.
Scheme 2. Synthesis of 1,3-Disubstituted Triazole Ligands 12a-12d and 14a-14da
a Reagents and conditions: (a) Cp*RuCl(PPh3)2, azide 4a, 4b, and 4d, THF, reflux; (b) NH2OH, KOH, rt; (c) 4c, toluene, reflux.
by SAHA (Table 1) Thus, we chose to expand upon this lead
scaffold possessing the triazole core in order to characterize the
structural determinants relevant to achieving higher levels of
isoform selectivity. Three azides were chosen for the first round
of optimization of ligand 6a, and modifications were made to
probe functional group tolerability on the terminal phenyl ring
(6b), presence of a spacer between the terminal phenyl ring and
the triazole ring (6c), and presence of an additional hydrogen
bond donor (6d). Moreover, the regioisomeric triazoles
10a-10d were prepared from the meta-substituted aniline 7 in
order to examine the effects of geometry on isoform selectivity.
As shown in Scheme 1, the POCl3-pyridine-mediated coupling
of suberic acid monomethyl ester with 4-ethynylaniline (1)
yielded amide 3, which underwent cycloaddition with azide 4a
catalyzed by CuSO4 and sodium ascorbate30 to give the 1,4-
disubstituted triazole 5a in good yield and regioselectivity.
Displacement of the methyl ester moiety in 5a by hydroxylamine
afforded the final hydroxamic acid 6a. Following a similar
sequence of reactions, ligands 6b-6d and 10a-10d were
prepared in good yield.
Scheme 2 outlines the synthesis of the 1,5-disubstituted
triazoles (12a-12d and 14a-14d). The 1,3-Dipolar cycload-
dition between 3 and 4a was carried out in refluxing THF under
argon to afford the 1,5-disubstituted triazole 11a regioselectively.
This intermediate was subsequently converted to the hydroxamic
acid 12a using the same conditions as described above.
Hydroxamic acids 12b and 12d were synthesized readily using
the same reaction conditions. However, the synthesis of hy-
droxamic acids 12c and 14c from phenylazide 4c and the
acetylene 3 or 8 with ruthenium catalyst failed to provide the
desired products, even after prolonged reaction times at higher
temperatures. Finally, we switched to the standard Huisgen
reaction conditions. Thus, on heating an excess of the pheny-
lazide and acetylene 3 in refluxing toluene for 2 days, we

3440 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Chen et al.
Table 1. HDAC Isoform in Vitro Inhibition Profiles for Prepared Triazole Ligands
a Taken from reference 32. b The isoform inhibition was tested at Amphora Discovery Corporation (http://www.amphoracorp.com/). c Not determined.

HDACIs with ActiVity against Cancer Cells and P. falciparum
Chart 2. Compound 6a and Tubacin
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3441
obtained the 1,4- and 1,5- disubstituted triazoles 5c and 11c.
The same reaction of the phenylazide and acetylene 8 resulted
in 1,4- and 1,5- disubstituted triazoles 9c and 13c. Structures
of 11c and 13c have been elucidated based upon comparison
of retention factor (Rf) on thin-layer chromatography (TLC) and
the NMRs between 11c vs 5c and 13c vs 9c, which were
obtained from the copper catalyzed cycloaddition reactions in
Scheme 1. Treatment of 11c and 13c with hydroxylamine and
KOH gave the corresponding hydroxamic acids 12c and 14c.
HDAC Isoform Inhibition Assay. The new HDACIs de-
scribed above were screened both in vitro for enzyme-inhibitory
activity and then for their ability to block cancer cell growth
and P. falciparum growth. The inhibitory effects of these
compounds on histone deacetylase (HDAC) activity were
determined using a fluorescence-based assay with electrophoretic
separation of substrate and product carried out using a microf-
luidic system followed by quantitation of fluorescence intensity
in the substrate and product peaks. The assays were performed
using isolated HDAC isoforms that had been expressed as 6×
His-tagged fusion proteins in a baculovirus expression system
in Sf9 cells. HDACs 1, 2, 3, 6, and 8 were expressed as full
length fusion proteins. The HDAC10 fusion protein was
expressed as a carboxy-terminal deletion of 38 amino acids
(residues 632-669). HDAC3 was coexpressed with a fragment
of the SMRT gene (residues 395-489) to generate enzymati-
cally active protein. The data are presented as IC50 values in
Table 1. TSA was used as a positive control. The recently
published inhibitory data for SAHA against a panel of recom-
binant HDACs are also presented for comparison.
selective inhibitors has been identified, and in this class of
ligands, the selectivity between deacetylation of histone H4 and
R-tubulin could be modified by varying the CAP region
functional groups.38,39 In the present research, the newly
identified HDAC6 selective ligand 6a shares the same linker
and zinc binding group as found in tubacin, which was identified
through combinatorial synthesis and cell-based screening of a
>7000-compound library.21 The structural differences between
tubacin and 6a are easily discerned. Tubacin structure is a
T-shaped molecule, while 6a is a linear molecule (Chart 2).
Consistent with the isoform inhibitory profile of 6a, the
p-fluorinated compound 6b shows even better selectivity for
HDAC6 over HDAC1 (51-fold). For compounds 6c and 6d,
the inhibition of HDAC1 increased slightly, but the selectivity
between HDAC6 and HDAC1 is similar to that of 6a (21-fold
for 6c and 16-fold for 7d). On the other hand, a decreased level
of selectivity between HDAC6 and HDAC1 was found for
ligands 10a-10d (2-8-fold). Within this small library, ligand
10c shows the lowest level of selectivity between HDAC6 and
HDAC1 (only 2-fold). It is apparent that this subclass of ligands
10a-10d shows better HDAC1 inhibition than their regioiso-
mers 6a-6d, thus accounting for their lower selectivity index.
On the basis of the SAR shown here, it is likely that further
levels of HDAC6 selectivity could be achieved through further
modifications of the terminal phenyl ring of 6a and 6b.
A similar but slightly decreased level of selectivity variation
in the inhibition of HDAC1 versus HDAC6 was found by the
compounds belonging to the two subclasses of 1,5-disubstituted
triazoles, namely 12a-12d and 14a-14d (7-22-fold versus
4-8-fold). In general, structural variations in the triazole ring
functionality do not play a significant role in the selectivity for
inhibition of HDAC6 over HDAC1 compared with the substitu-
tion pattern of the central phenyl ring (para versus meta). This
finding may be a consequence of the location of the central
phenyl group nearer to the entrance of the enzyme’s catalytic
pocket.9,19,20 Consistent with our previous paper concerning
p-substituted biphenyl bearing HDAC inhibitors,23 as well as
the tubacin-like molecules,35–37 this 1,4-substituted phenyl CAP
might thus contribute to the preferable inhibition of HDAC6
over HDAC1, although further chemistry biology studies will
be needed to confirm this hypothesis.
In addition to the subclass selectivity for HDAC6 versus
HDAC1, minor changes in structure and geometrical orientation
of the CAP region recognition groups was found to lead to
differences in inhibitory activity against some of the other
isozymes. For example, the benzyl triazole 6a is more than 10-
fold less active in inhibiting HDAC2 compared to its phenyl
counterpart 6c (IC50: 47.5 nM for 6c and 679 nM for 6a). The
inhibition of HDAC10 by 10a increases almost 10-fold com-
pared with that of its regioisomer 6a (IC50: 18.5 nM for 10a
Results and Discussion
The results of the HDAC isoform assays are shown in Table
1. As noted above, the present study evolved as a consequence
of our finding that compound 6a showed some degree of
selectivity for HDAC6 versus HDAC1. Compared to other
members of this zinc-dependent HDAC family, HDAC6 deacetyl-
ates tubulin31 and HSP90.32 HDAC6 has a specific ubiquitin-
binding domain at the C-terminus that associates with protein
ubiquitination and inhibition of proteasome.33 Recently, HDAC6
was shown to be required for the autophagic degradation of
aggregated mutant HTT in mouse neuroblastoma cells owing
to its function in the transportation of autophagosomes and
lysosomes.34 Tubacin, a specific inhibitor of HDAC6 that
inhibits the deacetylation of R-tubulin but not of histones,35–37
and RNA interference techniques have been used as research
tools in elucidating the cellular function of HDAC6 and its
substrates. Because of the important role of HDAC6 in human
biology, more selective small molecule inhibitors of HDAC6
or even substrate-selective HDAC6 inhibitors are in great
demand. Recently a new class of thiol- and lysine-based HDAC6

3442 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Chen et al.
Table 2. In Vitro Inhibition against Pancreatic Cancer Cell Lines by the
Triazole-Based HDAC Inhibitors
Table 4. In Vitro Growth Inhibition of P. falciparum Strains by the
Triazole-Based HDAC Inhibitors
inhibition of pancreatic cancer cell lines (IC50, µM)
inhibition of P. falciparum (IC50, nM)
compd BxPC-3 Hup T3 Mia Paca-2 Panc 04.03 SU 86.86
compd
D6
W2
TM90C235 TM90C2A
SAHA
6a
6b
5
9
22
3
0.8
5
4
1.1
<1
<0.5
0.4
1.2
2
3
1.3
10
11
1
chloroquinea 14 ( 6
405 ( 206
5.9 ( 2.1
161
138 ( 71
42 ( 23
310
189 ( 46
46 ( 22
110
mefloquinea
SAHA
6a
11 ( 5.6
247
210
6c
1
1
530
300
160
6d
18
3
1
12
1
1
0.2
11
5
4
10
46
2
0.6
<0.5
0.3
0.02
0.8
2
8
8
37
1
2
3
11
10
2
1
<0.5
14
2
2
8
44
4
9
12
2
1
0.8
12
9
10
11
42
7
8
4
6d
10a
80
140
180
390
160
350
110
220
280
190
190
110
10a
10b
10c
10d
12a
12b
12c
12d
14a
14b
14c
14d
10b
0.4
10cb
10d
17 [17-18] 32 [20-45] 35 [31-39] 17 [15-19]
11
11
11
13
25
12
11
10
43
110
400
240
350
230
210
300
320
180
560
>1200
460
620
>950
600
590
730
380
260
520
290
470
390
370
460
500
390
110
300
120
170
230
150
210
330
120
12a
12b
12c
12d
14a
14b
14c
14d
5
8
42
2
25
23
a Data represent average IC50 +/- STDEV for last 20 assays. b Values
represent mean of two experiments with range in brackets.
Table 3. In Vitro Inhibition against Nontransformed Human Cells by
In the development of HDAC inhibitors as anticancer
therapeutics, it will be invaluable to elucidate the interplay of
the structural parameters relevant to isoform inhibitory activity,
antiproliferative activity, and toxicity to normal cells. It is still
unclear, for example, as to whether pan-selective HDACIs, such
as SAHA and 1b, are superior to class I selective inhibitors
such as 1c and 1d.41 In this work, these new triazole-based
ligands were tested for their antiproliferative activity against a
panel of pancreatic cancer cell lines, which consisted of BxPC-
3, Hup T3, Mia Paca-2, Pan 04.03, and SU 86.86 cells. Cell
growth inhibition of the tested analogues was measured by MTT
assay, and the IC50 values obtained against these cell lines are
summarized in Table 2. Comparison data are provided for
SAHA. All compounds in this library demonstrated reasonably
good antiproliferative activity against all cancer cell types
examined, especially for the Mia Paca-2 line. This finding is
consistent with an earlier report showing that the cyclopeptide-
based HDAC inhibitor 1e was most active against Mia Paca-2
when tested against a panel of five pancreatic cancer lines.42 In
our triazole library, 10c gave the best inhibition against all
pancreatic cancer cell lines, with the lowest IC50 value being
shown against the Mia Paca-2 cell (20 nM). For comparison
we note that the structurally related regioisomers 6c, 12c, and
14c showed different inhibitory profiles. In general, the 1,4-
disubstituted triazoles showed better cell inhibitory activity than
the 1,5-disubstituted triazoles. Another trend from this small
library of compounds is that the meta-substituted phenyl ring
compounds 10a-10d are more potent than their para-substituted
counterparts 6a-6d. The previous experience on the biphenyl-
based HDAC inhibitors indicated that the meta-substituted
biphenyl HDACIs were more potent in cellular assays than the
p-biphenyl counterparts.43 But this trend is not obvious in the
subclass of vicinal disubstituted triazole ligands 12a-12d and
14a-14d. It is valuable to note that the subclass of ligands
10a-10d has the lowest selectivity between HDAC1 and
HDAC6 in this library, and yet these compounds show better
antiproliferative activity than their regioisomers possessing
higher isozyme selectivity, 6a-6d. These data may further
substantiate the importance of potency in the inhibition of the
nuclear HDAC1 in the induction of cancer cell apoptosis, as
this particular HDAC is involved in the regulation of the c-jun
promoter.44
the Triazole-Based HDAC Inhibitors
inhibition of nontransformed cells (IC50, µM)
compd
HMEC
HPDE6c7
SAHA
6c
10b
10c
1.3
1
<1
<1
1.3
1
0.5
<0.2
and 188 nM for 6a). In general, the selectivity of these inhibitors
for HDAC1 and HDAC10 vary less than the selectivity
measured between HDAC1 and HDAC6. The majority of these
compounds fail to provide any remarkable levels of isoform
selectivity, and all of them show only weak activity for HDAC8.
Although the present findings are of interest and can be
analyzed in a number of ways, it is well appreciated that the
isozyme selectivity shown by an HDACI can vary within
the context of the whole cell in light of the fact that, inter
alia, the HDACs form complexes involving both other
HDACs as well as corepressor proteins (e.g., mSin3A-HDAC
complex). Moreover, some of these HDACs may work together
in a complementary fashion, whereas others may oppose one
another in terms of their ultimate effects on gene transcription.
As such, both cell-based screening methods and diverse disease
models together with appropriate ADMET studies remain
relevant to evaluating the true value of any new HDACIs. Below
we present the preliminary results of both the growth inhibition
of these triazole-based HDACIs against a panel of pancreatic
cancer lines, nontransformed cells, and P. falciparum strains
(Tables 2, 3, and 4).
Pancreatic Cancer Lines Proliferation Assay. Pancreatic
cancer is the fourth leading cause of cancer death in the United
States and essentially remains an incurable disease with a five-
year survival of less than 5%. There is increasing evidence that
signaling and transcriptional pathways that control cell prolifera-
tion, differentiation, and apoptosis are dysregulated in pancreatic
cancer. Recently, SAHA had been tested against six pancreatic
cancer cell lines and showed induction of apoptosis, G2 cell
cycle arrest, and differentiation. Also, the combination of SAHA
and an inhibitor of DNA methylation, 5-aza-2′-deoxycytidine,
had an enhanced antiproliferative effect toward pancreatic cancer
cells.40 At present, there exists no effective therapy against
pancreatic cancer.
We further measured the cytotoxicity the three most active
ligands, 6c, 10b, and 10c, against two human normal cells,

HDACIs with ActiVity against Cancer Cells and P. falciparum
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3443
namely HMEC (human microvascular endothelial cell) and
HPDE6c7 (an immortalized nontransformed pancreatic epithelial
cell line), and SAHA was chosen as control (Table 3). A similar
extent of inhibition was found out between the tested pancreatic
cancer cells and nontransformed cells, including SAHA.
will be required to qualify this as a real drug lead. Additionally,
efforts are being made to obtain measurements of IC50 values
against the missing HDAC isoforms, which will be reported in
due course.
Experimental Section
Activity against P. falciparum. Falciparum malaria is
responsible for more than a million deaths a year, mostly in
sub-Saharan Africa.45 There is a general need to find new
antimalarial drugs with novel modes of action, given that there
is only one class of antimalarial drugs (artemisinin derivatives)
effective against all drug-resistant malaria. Initial studies suggest
that HDAC may be an attractive target in P. falciparum. The
potential antimalarial activity of HDAC inhibitors was reported
by Darkin-Rattray et al. in 1996.46 1f, a fungal metabolite, was
found to exhibit a minimum inhibitory concentration of 125
ng/mL against P. falciparum and exhibited a suppressive effect
on blood-stage parasites when administered orally at a dose rate
of 50 mg/kg. Functional studies suggested that the target of 1f
was indeed malarial parasite HDAC. The compound did not
appear to have much selectivity for malaria because its IC50
against proliferating mammalian cells was 50-100 nM. Sub-
sequently, Joshi et al. subcloned an HDAC directly from P.
falciparum, showed that it localized in the parasite nucleus, and
was expressed primarily in mature asexual blood stages and in
gametocytes.47 A subsequent study also demonstrated that other
putative HDAC inhibitors were active in vitro and in vivo.48
These studies suggest that HDAC is a promising target in
malaria but have not yielded a promising lead candidate.
1
Chemistry. H NMR and 13C NMR spectra were recorded on
Bruker spectrometer at 300/400 MHz and 75/100 MHz, respec-
tively, with TMS as an internal standard. HRMS experiment was
performed on Q-TOF-2TM (Micromass). TLC was performed with
Merck 60 F254 silica gel plates. Preparative TLC was performed
with Analtech 1000 µm silica gel GF plates. Column chromatog-
raphy was performed using Merck silica gel (40-60 mesh). HPLC
was carried out on an ACE AQ columns (100 mm × 4.6 mm and
250 mm × 10 mm), with detection at 210, 240, 254, 280, and 300
nm on a Shimadzu SPD-10A VP detector; flow rate ) 2.0-3.5
mL/min; from 10% acetonitrile in water to 100% acetonitrile with
0.05% TFA.
7-(4-Ethynylphenylcarbamoyl)heptanoic Acid Methyl Ester (3).
To a solution of 4-ethynylaniline (1, 2.00 g, 7 mmol) and suberic
acid monomethyl ester (2, 3.21 g, 17 mmol) in 30 mL of dry
pyridine at 0 °C was added 3.14 g (21 mmol) of POCl3 dropwise.
This solution was stirred at this temperature for 30 min, then diluted
with EtOAc and washed thoroughly with saturated aqueous KHSO4
and brine, dried over Na2SO4, filtered, and concentrated. Silica gel
chromatography (hexanes/EtOAc 3:1) of the crude mixture afforded
2.16 g (44%) of 3. Rf ) 0.35 (2:1 hexane:EtOAc). 1H NMR
(DMSO-d6, 400 MHz) δ (ppm): 10.01 (s, 1H), 7.59 (d, J ) 8.4
Hz, 2H), 7.38 (d, J ) 8.0 Hz, 2H), 4.05 (s, 1H), 3.56 (s, 3H),
2.31-2.26 (m, 4H), 1.58-1.49 (m, 4H), 1.22 (br s, 4H). 13C NMR
(DMSO-d6, 100 MHz) δ (ppm): 173.7, 171.9, 140.3, 132.7, 119.2,
116.2, 84.0, 80.1, 51.6, 36.8, 33.6, 28.7, 28.6, 25.2, 24.7.
7-[4-(1-Benzyl-1H-[1,2,3]triazol-4-yl)phenylcarbamoyl]heptano-
ic Acid Methyl Ester (5a). In a mixture of 3 (100 mg, 0.34 mmol)
and benzyl azide (4a, 124 mg, 1.04 mmol) in water and ethyl
alcohol (v/v ) 1:1, 10 mL) sodium ascorbate (28 mg, 0.14 mmol,
dissolved in 1 mL of water) was added, followed by the addition
of copper(II) sulfate pentahydrate (18 mg, 0.07 mmol, dissolved
in 1 mL of water). The heterogeneous mixture was stirred
vigorously overnight at room temperature. TLC analysis indicated
complete consumption of the 3 in 12 h. The reaction mixture was
diluted with EtOAc and washed thoroughly with brine, dried over
Na2SO4, filtered, and concentrated. Silica gel chromatography
(hexanes/EtOAc 1:1) of the crude mixture afforded 97 mg (66%)
Some of the triazole analogs described here display interesting
antimalarial activity. The 50% inhibitory concentrations (IC50s)
against a number of drug resistant strains of P. falciparum are
presented in Table 4. Most of the analogs exhibit IC50s > 100
nM against all strains tested, whereas mefloquine has an IC50
of 5.9 nM against the sensitive W2 strain while chloroquine
has an IC50 of 14 nM against the sensitive D6 strain. In contrast,
compound 10c is an order of magnitude more potent than most
of the other compounds tested and is more active than
mefloquine and chloroquine against the multiple drug-resistant
strains C235 and C2A. Compound 10c exhibits potency
equivalent to TSA48 and 1f47 and is more active by orders of
magnitude than other HDAC inhibitors evaluated by Andrews
et al.48 The selectivity indices of compound 10c relative to P.
falciparum C235 were 0.62, 5.7, 11.4, and 22.8 against the Mia
Paca-2, Hup T3, Bx-PC-3, and SU 86.86 cell lines, respectively.
Thus, for three of the four mammalian cell lines, 10c displayed
more selectivity for P. falciparum than was reported for 1f47 in
earlier studies. Consequently, it may represent a useful lead
structure for the synthesis of new HDAC inhibitors with
selectivity for malaria. ADME studies are ongoing in our
laboratory at WRAIR to determine the viability of 10c as a lead
candidate.
1
of 5a. Rf ) 0.62 (1:2 hexane:EtOAc). H NMR (DMSO-d6, 400
MHz) δ (ppm): 9.93 (s, 1H), 8.52 (s, 1H), 7.75 (d, J ) 7.6 Hz,
2H), 7.64 (d, J ) 8.0 Hz, 2H), 7.38-7.33 (m, 5H), 5.62 (s, 2H),
3.57 (s, 3H), 2.29 (t, J ) 7.2 Hz, 4H), 1.57-1.52 (m, 4H), 1.29
(br s, 4H). 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 171.1, 147.0,
139.4, 136.4, 129.2, 128.5, 128.3, 126.0, 125.7, 121.3, 119.6, 53.4,
51.6, 36.7, 33.6, 28.7, 28.6, 25.3, 24.7.
7-{4-[1-(4-Fluorobenzyl)-1H-[1,2,3]triazol-4-yl]phenylcarbamoyl}-
heptanoic Acid Methyl Ester (5b). Compound 5b (yield 80%) was
prepared according to the methodology described for the preparation
of compound 5a by substituting 4a with 4-fluorobenzyl azide (4b).
Rf ) 0.45 (1:2 hexane:EtOAc). 1H NMR (DMSO-d6, 400 MHz) δ
(ppm): 9.93 (s, 1H), 8.51 (s, 1H), 7.74 (d, J ) 8.4 Hz, 2H), 7.64
(d, J ) 8.4 Hz, 2H), 7.43-7.40 (m, 2H), 7.24-7.19 (m, 2H), 5.61
(s, 2H), 3.56 (s, 3H), 2.29 (t, J ) 6.8 Hz, 4H), 1.59-1.52 (m, 4H),
1.29 (br s, 4H). 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 173.7,
171.7, 163.5, 161.1, 147.0, 139.4, 132.7, 130.7, 130.6, 126.0, 125.7,
121.2, 119.6, 116.1, 115.9, 52.6, 51.6, 36.7, 33.6, 28.7, 28.6, 25.3,
24.7.
Summary. The present studies further add to the ever
growing database of structural information that is relevant to
the design of isozyme selective HDACIs. Modification of the
CAP region of the present set of triazolylphenyl-based HDACIs
reveals that the nature of substitution of the phenyl ring plays
a role in their selectivity for HDAC1 versus HDAC6, with low
to moderate selectivity (2-51-fold) being achieved. In light of
the valuable selectivity and potency shown by ligand 6b in the
inhibition of HDAC6, this compound represents a useful
research tool and would appear to represent a candidate for
further chemical modifications. Moreover, the anticancer and
antimalarial screening studies validate the superior activity of
the HDACI 10c, however, both in vivo and ADMET studies
7-[4-(1-Phenyl-1H-[1,2,3]triazol-4-yl)phenylcarbamoyl]heptano-
ic Acid Methyl Ester (5c). Compound 5c (yield 57%) was prepared
according to the methodology described for the preparation of
compound 5a, by substituting 4a with phenyl azide (4c). Rf ) 0.70
1
(1:2 hexane:EtOAc). H NMR (CDCl3, 400 MHz) δ (ppm): 8.19
(s, 1H), 7.88 (d, J ) 8.4 Hz, 2H), 7.81 (d, J ) 7.6 Hz, 2H), 7.66
(d, J ) 8.0 Hz, 2H), 7.57 (t, J ) 7.6 Hz, 1H), 7.54 (d, J ) 5.6 Hz,

3444 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Chen et al.
1H), 7.48 (d, J ) 7.6 Hz, 1H), 7.00 (s, 1H), 3.69 (s, 3H), 2.40 (t,
J ) 7.2 Hz, 2H), 2.34 (t, J ) 7.2 Hz, 2H), 1.77 (t, J ) 6.8 Hz,
2H), 1.66 (t, J ) 7.2 Hz, 2H), 1.41 (br s, 4H). 13C NMR (CDCl3,
100 MHz) δ (ppm): 173.8, 171.0, 151.1, 147.6, 137.8, 136.6, 135.3,
129.3, 128.3, 127.8, 126.1, 125.6, 125.1, 120.1, 119.6, 116.9, 51.1,
37.2, 33.8, 33.5, 29.9, 28.3, 24.9, 24.2, 20.7.
(t, J ) 7.3 Hz, 2H), 1.94 (t, J ) 7.4 Hz, 2H), 1.59 (t, J ) 6.7 Hz,
2H), 1.50 (t, J ) 6.4 Hz, 2H), 1.29 (br s, 4H). 13C NMR (DMSO-
d6, 100 MHz) δ (ppm): 171.3, 169.1, 144.9, 130.5, 128.9, 125.5,
125.2, 121.6, 121.5, 119.3, 70.7, 56.4, 32.3, 28.4, 25.0. ESI-HRMS
calculated for [C24H28Br1N5O4 + Na]+: 552.1217; found: 552.1210.
7-(3-Ethynylphenylcarbamoyl)heptanoic Acid Methyl Ester (8).
Compound 8 (yield 37%) was prepared according to the methodol-
ogy described for the preparation of compound 3, by substituting
4-ethynylaniline (1) with 3-ethynylaniline (7). Rf ) 0.40 (1:1
hexane:EtOAc). 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 9.94 (s,
1H), 7.78 (s, 1H), 7.54 (d, J ) 8.0 Hz, 1H), 7.29 (t, J ) 7.6 Hz,
1H), 7.12 (d, J ) 7.6 Hz, 1H), 4.14 (s, 1H), 3.56 (s, 3H), 2.28 (t,
J ) 7.6 Hz, 4H), 1.58-1.50 (m, 4H), 1.28 (br s, 4H). 13C NMR
(DMSO-d6, 100 MHz) δ (ppm): 173.7, 171.9, 139.5, 129.5, 126.6,
125.8, 122.3, 122.2, 120.1, 83.8, 80.6, 51.5, 36.7, 33.6, 28.7, 28.6,
25.2, 24.7.
7-(4-Phenylcarbamoyl)heptanoic Acid Methyl Ester (5d). Com-
pound 5d (yield 77%) was prepared according to the methodology
described for the preparation of compound 5a by substituting 4a
with 2-azido-1-(3-bromophenyl)ethanol (4d). Rf ) 0.43 (1:2 hexane:
1
EtOAc). H NMR (DMSO-d6, 400 MHz) δ (ppm): 9.94 (s, 1H),
8.39 (s, 1H), 7.74 (d, J ) 8.4 Hz, 2H), 7.66 (d, J ) 8.4 Hz, 2H),
7.60 (s, 1H), 7.48 (d, J ) 8.0 Hz, 1H), 7.38 (d, J ) 8.0 Hz, 1H),
7.31 (t, J ) 7.6 Hz, 1H), 5.99 (d, J ) 4.8 Hz, 1H), 5.06-5.03 (m,
1H), 4.61-4.56 (m, 1H), 4.51-4.46 (m, 1H), 3.57 (s, 3H),
2.32-2.27 (m, 4H), 1.60-1.51 (m, 4H), 1.29 (br s, 4H). 13C NMR
(DMSO-d6, 100 MHz) δ (ppm): 173.7, 171.6, 146.3, 145.2, 139.3,
130.8, 129.3, 126.0, 125.9, 125.6, 122.0, 121.9, 119.7, 71.1, 56.8,
51.6, 36.7, 33.6, 28.7, 28.6, 25.3, 24.7.
7-[3-(1-Benzyl-1H-[1,2,3]triazol-4-yl)phenylcarbamoyl]heptano-
ic Acid Methyl Ester (9a). Compound 9a (yield 58%) was prepared
according to the methodology described for the preparation of
compound 5a by substituting 3 with 8. Rf ) 0.64 (1:2 hexane:
Octanedioic Acid [4-(1-Benzyl-1H-[1,2,3]triazol-4-yl)phenyl]-
amide Hydroxyamide (6a). To a solution of hydroxylamine
hydrochloride (2.64 g, 38 mmol) in 20 mL of MeOH, KOH (2.13
g, 38 mmol) was added at 40 °C for 10 min. The reaction mixture
was cooled to 0 °C and filtered. Compound 5a (80 mg, 0.19 mmol)
was added to the filtrate followed by KOH (250 mg, 4.45 mmol)
at room temperature for 30 min. The reaction mixture was extracted
with EtOAc. The organic layer was washed with saturated NH4Cl
aqueous solution and brine, dried over Na2SO4, filtered, and
concentrated. The crude solid was purified by preparative HPLC
to give compound 6a (35 mg, 43%). 1H NMR (DMSO-d6, 400
MHz) δ (ppm): 10.34 (s, 1H), 9.96 (s, 1H), 8.54 (s, 1H), 7.76 (d,
J ) 8.5 Hz, 2H), 7.66 (d, J ) 8.5 Hz, 2H), 7.42-7.34 (m, 5H),
6.38 (s, 2H), 2.30 (t, J ) 7.5 Hz, 2H), 1.94 (t, J ) 7.3 Hz, 2H),
1.58 (t, J ) 6.9 Hz, 2H), 1.49 (t, J ) 6.7 Hz, 2H), 1.27 (br s, 2H).
13C NMR (DMSO-d6, 100 MHz) δ (ppm): 171.3, 169.1, 146.6,
139.1, 136.1, 128.8, 128.2, 127.9, 125.6, 125.3, 120.9, 119.2, 53.0,
36.4, 32.3, 28.4, 25.0. ESI-HRMS calculated for [C23H27N5O3 +
H]+: 422.2186; found: 422.2180.
1
EtOAc). H NMR (DMSO-d6, 400 MHz) δ (ppm): 9.95 (s, 1H),
8.58 (s, 1H), 8.14 (s, 1H), 7.54 (d, J ) 8.0 Hz, 1H), 7.45 (d, J )
8.0 Hz, 1H), 7.41-7.31 (m, 6H), 5.63 (s, 2H), 3.56 (s, 3H),
2.32-2.27 (m, 4H), 1.60-1.50 (m, 4H), 1.30 (br s, 4H). 13C NMR
(DMSO-d6, 100 MHz) δ (ppm): 171.7, 147.0, 140.2, 136.4, 131.4,
129.6, 129.2, 128.6, 128.3, 125.9, 125.8, 121.9, 120.4, 118.9, 116.0,
53.4, 51.6, 36.7, 33.6, 28.7, 28.6, 25.3, 24.7.
7-{3-[1-(4-Fluorobenzyl)-1H-[1,2,3]triazol-4-yl]phenylcarbamoyl}-
heptanoic Acid Methyl Ester (9b). Compound 9b (yield 37%) was
prepared according to the methodology described for the preparation
of compound 5b, by substituting 3 with 8. Rf ) 0.61 (1:2 hexane:
1
EtOAc). H NMR (DMSO-d6, 400 MHz) δ (ppm): 9.94 (s, 1H),
8.57 (s, 1H), 8.13 (s, 1H), 7.53 (d, J ) 7.6 Hz, 1H), 7.45-7.41
(m, 3H), 7.33 (t, J ) 8.0 Hz, 1H), 7.24-7.19 (m, 2H), 5.62 (s,
2H), 3.56 (s, 3H), 2.31-2.27 (m, 4H), 1.59-1.48 (m, 4H), 1.25
(br s, 4H). 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 173.7, 171.7,
147.0, 140.2, 131.4, 130.7, 129.6, 121.9, 120.4, 118.9, 116.1, 115.9,
52.6, 51.6, 36.7, 33.6, 28.7, 28.6, 25.3, 24.7.
Octanedioic Acid {4-[1-(4-Fluorobenzyl)-1H-[1,2,3]triazol-4-
yl]phenyl}amide Hydroxyamide (6b). Compound 6b (yield 67%)
was prepared according to the methodology described for the
7-[3-(1-Phenyl-1H-[1,2,3]triazol-4-yl)phenylcarbamoyl]heptano-
ic Acid Methyl Ester (9c). Compound 9c (yield 56%) was prepared
according to the methodology described for the preparation of
compound 5c by substituting 3 with 8. Rf ) 0.78 (1:2 hexane:
1
preparation of compound 6a. H NMR (DMSO-d6, 400 MHz) δ
1
(ppm): 10.33 (s, 1H), 9.96 (s, 1H), 8.66 (s, 1H), 8.53 (s, 1H), 7.75
(d, J ) 8.0 Hz, 2H), 7.65 (d, J ) 8.4 Hz, 2H), 7.42-7.41 (m, 2H),
7.25-7.21 (m, 2H), 5.62 (s, 2H), 2.30 (t, J ) 6.8 Hz, 2H), 1.94 (t,
J ) 7.6 Hz, 2H), 1.58 (t, J ) 6.0 Hz, 2H), 1.49 (br s, 2H), 1.28 (br
s, 4H). 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 171.3, 169.1,
163.1, 160.7, 146.6, 139.1, 132.3, 130.3, 130.2, 125.6, 125.3, 120.8,
119.3, 115.8, 115.5, 52.2, 36.4, 32.3, 28.4, 25.0. ESI-HRMS
calculated for [C23H26F1N5O3 + H]+: 440.2092; found: 440.2086.
Octanedioic Acid Hydroxyamide [4-(1-Phenyl-1H-[1,2,3]triazol-
4-yl)phenyl]amide (6c). Compound 6c (yield 58%) was prepared
according to the methodology described for the preparation of
compound 6a. 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.35 (s,
1H), 10.02 (s, 1H), 9.21 (s, 1H), 8.67 (s, 1H), 7.94 (d, J ) 7.2 Hz,
2H), 7.86 (d, J ) 7.6 Hz, 2H), 7.71 (d, J ) 7.6 Hz, 2H), 7.63 (t,
J ) 7.6 Hz, 2H), 7.51 (t, J ) 7.2 Hz, 1H), 2.32 (t, J ) 6.4 Hz,
2H), 1.94 (t, J ) 6.8 Hz, 2H), 1.59 (br s, 2H), 1.49 (br s, 2H), 1.28
(br s, 4H). 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 171.4, 169.1,
147.2, 139.4, 136.7, 130.0, 128.7, 125.8, 124.9, 120.0, 119.3, 118.9,
35.8, 32.3, 28.4, 25.1, 25.0. ESI-HRMS calculated for [C22H25N5O3
+ H]+: 408.2030; found: 408.2025.
EtOAc). H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.00 (s, 1H),
9.25 (s, 1H), 8.25 (s, 1H), 7.97 (d, J ) 7.6 Hz, 2H), 7.65-7.49
(m, 5H), 7.40 (t, J ) 7.6 Hz, 1H), 3.57 (s, 3H), 2.34-2.28 (m,
4H), 1.60 (t, J ) 6.8 Hz, 2H), 1.53 (t, J ) 7.2 Hz, 2H), 1.31 (br s,
4H). 13C NMR (DMSO-d6, 100 MHz) δ (ppm): 171.8, 147.7, 140.3,
137.0, 131.0, 130.3, 129.7, 129.1, 125.8, 120.7, 120.4, 120.0, 119.3,
116.3, 51.6, 36.7, 33.6, 28.7, 28.6, 25.3, 24.7.
7-(3-{1-[2-(3-Bromophenyl)-2-hydroxyethyl]-1H-[1,2,3]triazol-
4-yl}phenylcarbamoyl)heptanoic Acid Methyl Ester (9d). Com-
pound 9d (yield 71%) was prepared according to the methodology
described for the preparation of compound 5d by substituting 3
with 8. Rf ) 0.52 (1:2 hexane:EtOAc). 1H NMR (CD3OD, 400
MHz) δ (ppm): 8.24 (s, 1H), 7.99 (s, 1H), 7.60 (s, 1H), 7.57 (dd,
J ) 8.0 and 0.8 Hz, 1H), 7.53 (d, J ) 8.0 Hz, 1H), 7.46 (d, J )
8.0 Hz, 1H), 7.38 (t, J ) 8.0 Hz, 2H), 7.27 (t, J ) 8.0 Hz, 1H),
5.11 (dd, J ) 8.0 and 4.0 Hz, 1H), 4.67-4.64 (m, 1H), 4.61-4.57
(m, 1H), 3.65 (s, 3H), 2.40 (t, J ) 7.6 Hz, 2H), 2.34 (t, J ) 7.2
Hz, 2H), 1.72 (t, J ) 7.2 Hz, 2H), 1.64 (t, J ) 7.2 Hz, 2H), 1.41
(br s, 4H). 13C NMR (CD3OD, 100 MHz) δ (ppm): 173.2, 143.8,
139.1, 130.9, 130.6, 129.9, 129.0, 128.7, 124.5, 122.1, 122.0, 120.9,
119.5, 116.8, 71.4, 56.8, 50.5, 36.4, 33.2, 28.5, 28.4, 25.2, 24.4.
Octanedioic Acid [3-(1-Benzyl-1H-[1,2,3]triazol-4-yl)phenyl]-
amide Hydroxyamide (10a). Compound 10a (yield 36%) was
prepared according to the methodology described for the preparation
of compound 6a. 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.33
(s, 1H), 9.96 (s, 1H), 8.59 (s, 1H), 8.15 (s, 1H), 7.55 (d, J ) 8.0
Hz, 1H), 7.46 (d, J ) 8.0 Hz, 1H), 7.41-7.32 (m, 5H), 5.64 (s,
2H), 2.31 (t, J ) 8.0 Hz, 2H), 1.94 (t, J ) 8.0 Hz, 2H), 1.58 (t, J
) 8.0 Hz, 2H), 1.49 (t, J ) 8.0 Hz, 2H), 1.28 (br s, 4H). 13C NMR
Octanedioic Acid (4-{1-[2-(3-bromophenyl)-2-hydroxyethyl]-
1H-[1,2,3]triazol-4-yl}phenyl)amide Hydroxyamide (6d). Com-
pound 6d (yield 41%) was prepared according to the methodology
described for the preparation of compound 6a. 1H NMR (DMSO-
d6, 400 MHz) δ (ppm): 10.34 (s, 1H), 9.96 (s, 1H), 8.66 (s, 1H),
8.41 (s, 1H), 7.76 (d, J ) 8.5 Hz, 2H), 7.67 (d, J ) 8.1 Hz, 2H),
7.61 (s, 1H), 7.49 (d, J ) 8.3 Hz, 1H), 7.40 (d, J ) 7.5 Hz, 1H),
7.32 (t, J ) 7.5 Hz, 1H), 6.00 (d, J ) 4.7 Hz, 1H), 5.05 (dd, J )
7.8 and 3.7 Hz, 1H), 4.62-4.57 (m, 1H), 4.52-4.47 (m, 1H), 2.31

HDACIs with ActiVity against Cancer Cells and P. falciparum
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3445
(DMSO-d6, 100 MHz) δ (ppm): 171.8, 169.5, 147.0, 140.2, 136.4,
131.4, 129.6, 129.2, 128.6, 128.3, 125.8, 121.9, 120.4, 118.9, 116.1,
53.4, 36.8, 32.6, 28.8, 25.4. ESI-HRMS calculated for [C23H27N5O3
+ H]+: 422.2186; found: 422.2180.
7-[4-(3-Phenyl-3H-[1,2,3]triazol-4-yl)phenylcarbamoyl]heptanoic Acid
Methyl Ester (11c). A mixture of phenylazide (4c, 1.65 g, 13.9
mmol) and phenylacetylene 3 (200 mg, 0.69 mmol) in 20 mL of
toluene was heated to refluxed for 2 days. The toluene was removed
under vacuum, and the crude mixture was purified by preparative
TLC to give compound 11c (65 mg, yield 23%, Rf ) 0.53,
hexane:EtOAc 1:2) and compound 5c (57 mg, yield 20%, Rf )
0.70, hexane:EtOAc 1:2). Compound 11c: 1H NMR (CD3OD,
400 MHz) δ (ppm): 7.96 (s, 1H), 7.58 (d, J ) 8.8 Hz, 2H),
7.53-7.50 (m, 3H), 7.40-7.38 (m, 2H), 7.21 (d, J ) 8.8 Hz, 2H),
3.63 (s, 3H), 2.36 (t, J ) 7.2 Hz, 2H), 2.31 (t, J ) 7.6 Hz, 2H),
1.66 (t, J ) 7.2 Hz, 2H), 1.61 (t, J ) 7.2 Hz, 2H), 1.37 (br s, 4H).
13C NMR (CD3OD, 100 MHz) δ (ppm): 174.0, 172.9, 139.4, 137.7,
136.1, 131.9, 129.0, 128.8, 128.5, 124.9, 120.9, 119.1, 50.1, 36.1,
32.8, 28.1, 28.0, 24.7, 24.0.
7-(4-{3-[2-(3-Bromo-phenyl)-2-hydroxyethyl]-3H-[1,2,3]triazol-
4-yl}phenylcarbamoyl)heptanoic Acid Methyl Ester (11d). Com-
pound 11d (yield 40%) was prepared according to the methodology
described for the preparation of compound 11a by substituting 4a
with 2-azido-1-(3-bromophenyl)ethanol (4d). Rf ) 0.30 (1:2 hexane:
EtOAc). 1H NMR (CDCl3, 400 MHz) δ (ppm): 8.60 (s, 1H), 7.67
(d, J ) 8.4 Hz, 2H), 7.52 (s, 1H), 7.40 (s, 1H), 7.34 (d, J ) 6.8
Hz, 1H), 7.23 (d, J ) 8.0 Hz, 2H), 7.12-7.10 (m, 3H), 5.29 (br s,
1H), 4.98 (br s, 1H), 4.45-4.40 (m, 2H), 3.63 (s, 3H), 2.37 (t, J )
7.2 Hz, 2H), 2.28 (t, J ) 7.2 Hz, 2H), 1.69 (t, J ) 6.4 Hz, 2H),
1.59 (t, J ) 6.8 Hz, 2H), 1.34 (br s, 4H). 13C NMR (CDCl3, 100
MHz) δ (ppm): 174.0, 171.8, 142.5, 139.3, 138.5, 131.9, 130.7,
129.7, 129.3, 128.5, 124.2, 122.3, 121.0, 119.6, 71.6, 54.3, 51.1,
36.0, 33.5, 28.3, 24.8, 24.2.
Octanedioic Acid {3-[1-(4-Fluorobenzyl)-1H-[1,2,3]triazol-4-
yl]phenyl}amide Hydroxyamide (10b). Compound 10d (yield 34%)
was prepared according to the methodology described for the
preparation of compound 6a. 1H NMR (CD3OD, 400 MHz) δ
(ppm): 8.31 (s, 1H), 8.02 (s, 1H), 7.54 (d, J ) 7.6 Hz, 2H), 7.44
(dd, J ) 8.4 and 5.2 Hz, 2H), 7.38 (t, J ) 8.0 Hz, 1H), 7.14 (t, J
) 8.8 Hz, 2H), 5.64 (s, 2H), 2.40 (t, J ) 7.2 Hz, 2H), 2.11 (t, J )
7.6 Hz, 2H), 1.74-1.63 (m, 4H), 1.42 (br s, 4H). 13C NMR
(CD3OD, 100 MHz) δ (ppm): 172.9, 171.1, 163.5, 161.2, 147.2,
138.7, 131.0, 130.4, 129.5, 128.6, 120.5, 119.2, 116.5, 115.1, 114.8,
99.5, 52.4, 36.0, 31.8, 28.0, 24.8, 24.7. ESI-HRMS calculated for
[C23H26F1N5O3 + H]+: 440.2092; found: 440.2088.
Octanedioic Acid Hydroxyamide [3-(1-Phenyl-1H-[1,2,3]triazol-
4-yl)phenyl]amide (10c). Compound 10c (yield 47%) was prepared
according to the methodology described for the preparation of
compound 6a. 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.34 (s,
1H), 10.03 (s, 1H), 9.27 (s, 1H), 8.66 (s, 1H), 8.27 (s, 1H), 7.98
(d, J ) 7.6 Hz, 2H), 7.66-7.50 (m, 5H), 7.41 (t, J ) 7.6 Hz, 1H),
2.33 (t, J ) 7.2 Hz, 2H), 1.95 (t, J ) 7.2 Hz, 2H), 1.61 (t, J ) 6.4
Hz, 2H), 1.50 (t, J ) 6.8 Hz, 2H), 1.30 (br s, 4H). 13C NMR
(DMSO-d6, 100 MHz) δ (ppm): 171.4, 169.1, 147.3, 140.0, 136.7,
130.7, 129.9, 129.4, 128.7, 120.3, 120.0, 119.6, 119.0, 115.9, 99.5,
36.4, 32.3, 28.4, 25.0. ESI-HRMS calculated for [C22H25N5O3 +
H]+: 408.2030; found: 408.2033.
Octanedioic Acid [4-(3-Benzyl-3H-[1,2,3]triazol-4-yl)phenyl]-
amide Hydroxyamide (12a). Compound 12a (yield 57%) was
prepared according to the methodology described for the preparation
Octanedioic Acid (3-{1-[2-(3-Bromophenyl)-2-hydroxyethyl]-
1H-[1,2,3]triazol-4-yl}phenyl)amide Hydroxyamide (10d). Com-
pound 10d (yield 56%) was prepared according to the methodology
described for the preparation of compound 6a. 1H NMR (DMSO-
d6, 400 MHz) δ (ppm): 10.34 (s, 1H), 9.98 (s, 1H), 8.45 (s, 1H),
8.16 (s, 1H), 7.62 (s, 1H), 7.55 (d, J ) 7.6 Hz, 1H), 7.50-7.45
(m, 3H), 7.39 (t, J ) 6.8 Hz, 1H), 7.37-7.31 (m, 2H), 5.07 (br s,
1H), 4.59-4.52 (m, 2H), 2.32 (t, J ) 6.4 Hz, 2H), 1.95 (t, J ) 7.2
Hz, 2H), 1.60 (br s, 2H), 1.50 (br s, 2H), 1.29 (br s, 4H). 13C NMR
(DMSO-d6, 100 MHz) δ (ppm): 171.4, 169.1, 146.0, 144.9, 139.9,
131.2, 130.5, 129.3, 128.9, 125.2, 122.1, 121.6, 119.9, 118.4, 115.6,
70.7, 56.4, 36.4, 32.3, 28.4, 25.1. ESI-HRMS calculated for
[C24H28Br1N5O4 + H]+: 530.1397; found: 530.1388.
7-[4-(3-Benzyl-3H-[1,2,3]triazol-4-yl)phenylcarbamoyl]heptanoic Acid
Methyl Ester (11a). A mixture of benzyl azide (4a, 139 mg, 1.04
mmol), phenylacetylene 3 (100 mg, 0.35 mmol), and Cp*RuCl-
(PPh3)2 (83 mg, 0.10 mmol) was refluxed in 20 mL of THF for 8 h
under argon. The progress of reaction was monitored by TLC, and
phenylacetylene 3 was consumed completely at the end of reaction.
The solvent was removed under vacuum, and the crude solid was
purified by preparative TLC to give compound 11a (89 mg, 61%).
Rf ) 0.45 (1:2 hexane:EtOAc). 1H NMR (CDCl3, 400 MHz) δ
(ppm): 8.62 (s, 1H), 7.71 (s, 1H), 7.69 (d, J ) 8.4 Hz, 2H),
7.27-7.26 (m, 3H), 7.19 (d, J ) 8.4 Hz, 2H), 7.08-7.06 (m, 2H),
5.53 (s, 2H), 3.64 (s, 3H), 2.40 (t, J ) 7.2 Hz, 2H), 2.29 (d, J )
7.6 Hz, 2H), 1.72 (t, J ) 6.8 Hz, 2H), 1.61 (t, J ) 7.2 Hz, 2H),
1.36 (br s, 4H). 13C NMR (CDCl3, 100 MHz) δ (ppm): 173.8, 171.7,
139.4, 137.7, 135.0, 132.5, 129.0, 128.4, 128.2, 127.8, 126.6, 121.2,
119.5, 51.3, 51.1, 36.9, 33.5, 28.3, 24.8, 24.2.
1
of compound 6a. H NMR (CD3OD, 400 MHz) δ (ppm): 7.82 (s,
1H), 7.67 (d, J ) 8.4 Hz, 2H), 7.30 (d, J ) 8.4 Hz, 2H), 7.27-7.25
(m, 3H), 7.04-7.02 (m, 2H), 5.64 (s, 2H), 2.39 (t, J ) 7.2 Hz,
2H), 2.10 (t, J ) 7.2 Hz, 2H), 1.70 (t, J ) 7.2 Hz, 2H), 1.63 (t, J
) 6.8 Hz, 2H), 1.39 (br s, 4H). 13C NMR(CD3OD, 75 MHz) δ
(ppm): 173.0, 171.1, 139.6, 137.9, 135.1, 131.9, 128.7, 128.0, 127.4,
126.3, 120.9, 119.3, 51.2, 36.1, 31.8, 28.1, 28.0, 24.7. ESI-HRMS
calculated for [C23H27N5O3 + Na]+: 444.2006; found: 444.2007.
Octanedioic Acid {4-[3-(4-Fluorobenzyl)-3H-[1,2,3]triazol-4-
yl]phenyl}amide Hydroxyamide (12b). Compound 12b (yield 66%)
was prepared according to the methodology described for the
preparation of compound 6a. 1H NMR (CD3OD, 400 MHz) δ
(ppm): 9.97 (s, 1H), 7.80 (s, 1H), 7.70-7.67 (m, 2H), 7.32 (d, J )
8.4 Hz, 2H), 7.09-7.05 (m, 2H), 7.03-6.98 (m, 2H), 5.64 (s, 2H),
2.40 (t, J ) 7.6 Hz, 2H), 2.10 (t, J ) 7.2 Hz, 2H), 1.71 (t, J ) 6.8
Hz, 2H), 1.64 (t, J ) 7.2 Hz, 2H), 1.41 (br s, 4H). 13C NMR
(CD3OD, 100 MHz) δ (ppm): 173.3, 171.5, 163.8, 161.2, 140.0,
138.1, 132.5, 131.5, 129.1, 129.0, 128.9, 125.8, 121.4, 119.7, 115.2,
115.0, 50.8, 36.4, 32.2, 28.4, 28.3, 25.1. ESI-HRMS calculated for
[C23H26F1N5O3 + H]+: 440.2092; found: 440.2087.
Octanedioic Acid Hydroxyamide [3-(3-Phenyl-3H-[1,2,3]triazol-
4-yl)phenyl]amide (12c). Compound 12c (yield 57%) was prepared
according to the methodology described for the preparation of
compound 6a. 1H NMR (CD3OD, 400 MHz) δ (ppm): 7.97 (s, 1H),
7.58 (d, J ) 8.4 Hz, 2H), 7.54-7.51 (m, 3H), 7.41-7.38 (m, 2H),
7.22 (d, J ) 8.4 Hz, 2H), 2.37 (t, J ) 7.2 Hz, 2H), 2.09 (t, J ) 7.6
Hz, 2H), 1.69-1.61 (m, 4H), 1.39 (br s, 4H). 13C NMR (CD3OD,
100 MHz) δ (ppm): 173.3, 171.5, 139.8, 138.1, 136.4, 132.2, 129.4,
129.2, 128.9, 125.8, 125.3, 121.3, 119.5, 36.4, 32.2, 28.4, 28.4,
25.1. ESI-HRMS calculated for [C22H25N5O3 + H]+: 408.2030;
found: 408.2026.
Octanedioic Acid (4-{3-[2-(3-Bromophenyl)-2-hydroxy-ethyl]-
3H-[1,2,3]triazol-4-yl}phenyl)amide Hydroxyamide (12d). Com-
pound 12d (yield 41%) was prepared according to the methodology
described for the preparation of compound 6a. 1H NMR (DMSO-
d6, 400 MHz) δ (ppm): 10.32 (s, 1H), 10.06 (s, 1H), 8.64 (br s,
1H), 7.75 (s, 1H), 7.71 (d, J ) 8.8 Hz, 2H), 7.43 (d, J ) 7.6 Hz,
1H), 7.38 (d, J ) 8.8 Hz, 2H), 7.34 (s, 1H), 7.23 (t, J ) 7.6 Hz,
1H), 7.13 (d, J ) 8.0 Hz, 1H), 5.93 (d, J ) 4.0 Hz, 1H), 4.97 (br
7-{4-[3-(4-Fluorobenzyl)-3H-[1,2,3]triazol-4-yl]phenylcarbamoyl}-
heptanoic Acid Methyl Ester (11b). Compound 11b (yield 50%) was
prepared according to the methodology described for the preparation
of compound 11a by substituting 4a with 4-fluorobenzyl azide (4b).
Rf ) 0.45 (1:2 hexane:EtOAc). 1H NMR (CDCl3, 400 MHz) δ
(ppm): 8.38 (s, 1H), 7.70 (s, 1H), 7.68 (d, J ) 8.4 Hz, 2H), 7.19
(d, J ) 8.4 Hz, 2H), 7.08-7.04 (m, 2H), 6.97-6.93 (m, 2H), 5.50
(s, 2H), 3.65 (s, 3H), 2.40 (t, J ) 7.6 Hz, 2H), 2.30 (t, J ) 7.6 Hz,
2H), 1.73 (t, J ) 6.8 Hz, 2H), 1.62 (t, J ) 7.2 Hz, 2H), 1.37 (br s,
4H). 13C NMR (CDCl3, 100 MHz) δ (ppm): 173.8, 171.6, 163.3,
160.8, 139.4, 137.5, 132.7, 130.7, 129.0, 128.7, 128.6, 121.2, 119.4,
115.5, 115.3, 51.1, 50.7, 37.0, 33.5, 28.3, 28.2, 24.8, 24.2.

3446 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Chen et al.
s, 1H), 4.49-4.46 (m, 2H), 2.32 (t, J ) 7.2 Hz, 2H), 1.93 (t, J )
7.2 Hz, 2H), 1.58 (t, J ) 6.4 Hz, 2H), 1.49 (t, J ) 6.8 Hz, 2H),
1.28 (br s, 4H). 13C NMR (100 MHz, DMSO-d6): 172.0, 169.5,
145.1, 140.6, 138.4, 132.6, 130.8, 129.6, 129.0, 125.4, 122.0, 121.4,
119.4, 71.2, 54.7, 36.8, 32.6, 28.8, 25.4. ESI-HRMS calculated for
[C24H28Br1N5O4 + H]+: 530.1397; found: 530.1391.
7-[3-(3-Benzyl-3H-[1,2,3]triazol-4-yl)phenylcarbamoyl]heptano-
ic Acid Methyl Ester (13a). Compound 13a (yield 38%) was
prepared according to the methodology described for the preparation
of compound 11a by substituting 3 with 8. Rf ) 0.55 (1:2 hexane:
EtOAc). 1H NMR (CDCl3, 300 MHz) δ (ppm): 8.56 (s, 1H),
7.72-7.68 (m, 2H), 7.63 (d, J ) 8.2 Hz, 1H), 7.30 (t, J ) 7.8 Hz,
1H), 7.24-7.21 (m, 3H), 7.06-7.03 (m, 2H), 6.92 (d, J ) 7.5 Hz,
1H), 5.56 (s, 2H), 3.64 (s, 3H), 2.38 (t, J ) 7.3 Hz, 2H), 2.31 (t,
J ) 6.6 Hz, 2H), 1.68 (t, J ) 6.9 Hz, 2H), 1.60 (t, J ) 6.7 Hz,
2H), 1.35 (br s, 4H). 13C NMR (CDCl3, 75 MHz) δ (ppm): 174.7,
172.5, 139.5, 138.5, 135.6, 133.5, 129.9, 129.1, 128.5, 127.7, 127.5,
124.3, 121.1, 120.5, 52.3, 37.7, 34.3, 29.1, 25.6, 25.0.
7-{3-[3-(4-Fluorobenzyl)-3H-[1,2,3]triazol-4-yl]phenylcarbamoyl}-
heptanoic Acid Methyl Ester (13b). Compound 13b (yield 55%) was
prepared according to the methodology described for the preparation
of compound 11a, by substituting 3 with 8. Rf ) 0.55 (1:2 hexane:
EtOAc). 1H NMR (CDCl3, 300 MHz) δ (ppm): 8.71 (s, 1H), 7.75
(s, 1H), 7.68 (s, 1H), 7.56 (d, J ) 8.0 Hz, 1H), 7.30 (t, J ) 7.9 Hz,
1H), 7.05-7.00 (m, 2H), 6.92-6.86 (m, 3H), 5.52 (s, 2H), 3.62
(s, 3H), 2.38 (t, J ) 7.3 Hz, 2H), 2.27 (t, J ) 7.3 Hz, 2H), 1.68 (t,
J ) 6.8 Hz, 2H), 1.59 (t, J ) 6.7 Hz, 2H), 1.33 (br s, 4H). 13C
NMR (CDCl3, 75 MHz) δ (ppm): 174.7, 172.6, 161.1, 139.6, 138.4,
133.5, 131.3, 129.9, 129.8, 129.7, 127.4, 124.3, 121.1, 120.5, 116.2,
115.9, 51.9, 51.6, 37.6, 34.3, 29.1, 25.6, 25.0.
Octanedioic Acid {4-[3-(4-Fluorobenzyl)-3H-[1,2,3]triazol-4-
yl]phenyl}amide Hydroxyamide (14b). Compound 14b (yield 44%)
was prepared according to the methodology described for the
preparation of compound 6a. 1H NMR (CD3OD, 400 MHz) δ
(ppm): 10.32 (s, 1H), 10.02 (s, 1H), 7.90 (s, 1H), 7.75 (s, 1H),
7.61 (d, J ) 8.0 Hz, 1H), 7.39 (t, J ) 8.0 Hz, 1H), 7.12-7.04 (m,
5H), 5.65 (s, 2H), 2.29 (t, J ) 7.2 Hz, 2H), 1.93 (t, J ) 7.2 Hz,
2H), 1.57 (t, J ) 6.8 Hz, 2H), 1.48 (t, J ) 7.2 Hz, 2H), 1.27 (br s,
4H). 13C NMR (CD3OD, 100 MHz) δ (ppm): ESI-HRMS calculated
for [C23H26F1N5O3 + H]+: 462.1911; found: 462.1912.
Octanedioic Acid Hydroxyamide [4-(3-Phenyl-3H-[1,2,3]triazol-
4-yl)phenyl]amide (14c). Compound 14c (yield 42%) was prepared
according to the methodology described for the preparation of
compound 6a. 1H NMR (DMSO-d6, 400 MHz) δ (ppm): 10.31 (s,
1H), 9.96 (s, 1H), 8.64 (s, 1H), 8.05 (s, 1H), 7.64 (s, 1H), 7.59 (d,
J ) 8.4 Hz, 1H), 7.53-7.51 (m, 3H), 7.40-7.39 (m, 2H), 7.27 (t,
J ) 7.6 Hz, 1H), 6.84 (d, J ) 7.6 Hz, 1H), 2.25 (t, J ) 7.2 Hz,
2H), 1.92 (t, J ) 7.2 Hz, 2H), 1.53-1.47 (m, 4H), 1.24 (br s, 4H).
13C NMR (DMSO-d6, 100 MHz) δ (ppm): 171.8, 169.5, 140.2,
138.0, 136.6, 133.5, 130.0, 129.9, 129.6, 127.2, 125.8, 123.4, 120.0,
119.3, 36.7, 32.6, 28.8, 28.7, 25.4, 25.3. ESI-HRMS calculated for
[C22H25N5O3 + Na]+: 430.1849; found: 430.1845.
Octanedioic Acid (4-{3-[2-(3-Bromophenyl)-2-hydroxyethyl]-
3H-[1,2,3]triazol-4-yl}phenyl)amide Hydroxyamide (14d). Com-
pound 12b (yield 49%) was prepared according to the methodology
described for the preparation of compound 6a. 1H NMR (CD3OD,
400 MHz) δ (ppm): 7.71 (s, 1H), 7.67 (s, 1H), 7.58 (d, J ) 8.4
Hz, 1H), 7.41 (t, J ) 8.0 Hz, 1H), 7.36 (d, J ) 8.0 Hz, 1H), 7.30
(s, 1H), 7.12 (t, J ) 7.6 Hz, 1H), 7.06 (t, J ) 7.2 Hz, 2H), 5.05 (t,
J ) 6.8 Hz, 1H), 4.69-4.64 (m, 1H), 4.59-4.54 (m, 1H), 2.41 (t,
J ) 7.6 Hz, 2H), 2.10 (t, J ) 7.2 Hz, 2H), 1.73 (t, J ) 6.8 Hz,
2H), 1.66 (t, J ) 7.6 Hz, 2H), 1.42 (br s, 4H). 13C NMR (CD3OD,
100 MHz) δ (ppm): 173.3, 171.5, 143.6, 139.1, 139.0, 131.9, 130.6,
129.7, 129.1, 128.7, 126.8, 124.5, 124.0, 122.0, 120.5, 120.1, 71.5,
54.4, 36.4, 32.2, 28.5, 28.4, 25.2, 25.1. ESI-HRMS calculated for
[C24H28Br1N5O4 + H]+: 530.1397; found: 530.1392.
7-[3-(3-Phenyl-3H-[1,2,3]triazol-4-yl)phenylcarbamoyl]heptano-
ic Acid Methyl Ester (13c). Compound 13c (yield 16%, Rf ) 0.59,
hexane:EtOAc 1:2) was prepared according to the methodology
described for the preparation of compound 11c, together with its
regioisomer 9c (yield 12%, Rf ) 0.78, hexane:EtOAc 1:2).
1
Compound 13c: H NMR (CDCl3, 400 MHz) δ (ppm): 7.84 (s,
1H), 7.61 (d, J ) 8.0 Hz, 1H), 7.58 (s, 1H), 7.44-7.41 (m, 3H),
7.38-7.36 (m, 2H), 7.25 (t, J ) 8.0 Hz, 1H), 6.83 (d, J ) 7.6 Hz,
1H), 3.66 (s, 3H), 2.35 (t, J ) 7.2 Hz, 2H), 2.31 (t, J ) 7.6 Hz,
2H), 1.71 (t, J ) 6.8 Hz, 2H), 1.62 (t, J ) 6.8 Hz, 2H), 1.35 (br s,
4H). 13C NMR (CD3OD, 100 MHz) δ (ppm): 173.8, 171.2, 138.5,
137.1, 136.0, 133.1, 129.0, 128.9, 126.9, 124.7, 123.6, 120.0, 119.2,
51.1, 37.0, 33.5, 28.2, 24.8, 24.2.
7-(3-{3-[2-(3-Bromophenyl)-2-hydroxyethyl]-3H-[1,2,3]triazol-
4-yl}phenylcarbamoyl)heptanoic Acid Methyl Ester (13d). Com-
pound 13d (yield 43%) was prepared according to the methodology
described for the preparation of compound 11a, by substituting 3
with 8. Rf ) 0.35 (1:2 hexane:EtOAc). 1H NMR (CD3OD, 400
MHz) δ (ppm): 7.71 (s, 1H), 7.62 (s, 1H), 7.58 (d, J ) 8.0 Hz,
1H), 7.42 (t, J ) 8.0 Hz, 1H), 7.36 (d, J ) 8.0 Hz, 1H), 7.13 (t, J
) 7.6 Hz, 1H), 7.06 (t, J ) 7.2 Hz, 2H), 5.05 (t, J ) 6.4 Hz, 1H),
4.70-4.65 (m, 1H), 4.60-4.55 (m, 1H), 3.64 (s, 3H), 2.41 (t, J )
7.2 Hz, 2H), 2.33 (t, J ) 7.6 Hz, 2H), 1.74 (t, J ) 6.8 Hz, 2H),
1.64 (t, J ) 7.2 Hz, 2H), 1.42 (br s, 4H). 13C NMR (CDCl3, 100
MHz) δ (ppm): 173.9, 171.7, 142.3, 138.5, 138.4, 132.1, 131.6,
131.5, 131.0, 129.7, 129.2, 128.6, 128.3, 128.1, 126.5, 124.2, 123.9,
122.2, 120.3, 119.8, 71.7, 54.5, 51.1, 36.9, 33.5, 28.4, 28.3, 24.8,
24.2.
Octanedioic Acid [3-(3-Benzyl-3H-[1,2,3]triazol-4-yl)phenyl]-
amide Hydroxyamide (14a). Compound 14a (yield 66%) was
prepared according to the methodology described for the preparation
of compound 6a. 1H NMR (CD3OD, 400 MHz) δ (ppm): 10.32 (s,
1H), 10.01 (s, 1H), 8.65 (br s, 1H), 7.90 (s, 1H), 7.77 (s, 1H), 7.61
(d, J ) 8.0 Hz, 1H), 7.38 (t, J ) 8.0 Hz, 1H), 7.29-7.22 (m, 5H),
7.09 (d, J ) 7.6 Hz, 1H), 7.01 (d, J ) 6.4 Hz, 2H), 5.66 (s, 2H),
2.29 (t, J ) 7.2 Hz, 2H), 1.93 (t, J ) 7.2 Hz, 2H), 1.57 (t, J ) 6.4
Hz, 2H), 1.48 (t, J ) 6.4 Hz, 2H), 1.27 (br s, 4H). 13C NMR
(CD3OD, 100 MHz) δ (ppm): 171.9, 171.8, 140.2, 140.1, 138.0,
136.3, 133.3, 129.9, 129.0, 128.2, 127.4, 127.2, 125.8, 123.3, 120.2,
119.4, 119.3, 51.6, 36.8, 32.6, 28.8, 25.4, 25.3. ESI-HRMS
calculated for [C23H27N5O3 + H]+: 422.2186; found: 422.2181.
Biological Methods. HDAC Inhibition Assay. The inhibitory
effects of compounds on histone deacetylase (HDAC) activity were
determined using a fluorescence-based assay with electrophoretic
separation of substrate and product carried out using a microfluidic
system followed by quantitation of fluorescence intensity in the
substrate and product peaks. The assays were performed using
isolated HDAC isoforms that had been expressed as 6× His-tagged
fusion proteins in a baculovirus expression system in Sf9 cells.
HDACs 1, 2, 3, 6, and 8 were expressed as full length fusion
proteins. The HDAC10 fusion protein was expressed as a carboxy-
terminal deletion of 38 amino acids (residues 632-669). HDAC3
was coexpressed with a fragment of the SMRT gene (residues
395-489) to generate enzymatically active protein. Purified proteins
were incubated with 1 µM carboxyfluorescein (FAM)-labeled
acetylated peptide substrate and test compound for 17 h at 25 °C
in HDAC assay buffer containing 100 mM HEPES (pH 7.5), 25
mM KCl, 0.1% BSA, and 0.01% Triton X-100. Reactions were
terminated by the addition of buffer containing 0.078% SDS for a
final SDS concentration of 0.05%. Substrate and product were
separated electrophoretically using a Caliper LabChip 3000 system
with blue laser excitation and green fluorescence detection (CCD2).
The fluorescence intensity in the substrate and product peaks was
determined using the Well Analyzer software on the Caliper system.
The reactions were performed in duplicate for each sample. IC50
values were automatically calculated using the IDBS XLFit version
4.2.1 plug-in for Microsoft Excel and the XLFit 4 Parameter
Logistic Model (Sigmoidal Dose-Response Model): ((A + ((B -
A)/1 + ((C/x)^D))), where x is compound concentration, A is the
estimated minimum and B is the estimated maximum of %
inhibition, C is the inflection point, and D is the Hill slope of the
sigmoidal curve. The standard errors of the IC50s were automatically
calculated using the IDBS XLFit version 4.2.1 plug-in for Microsoft
Excel and the formula xf4_FitResultStdError().
Cytotoxicity Assays. The pancreatic cancer cell lines BxPc-3,
HupT3, MiaPaCa-2, Panc 04.03, and SU86.86 were obtained from

HDACIs with ActiVity against Cancer Cells and P. falciparum
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3447
(10) Frye, R. A. Phylogenetic classification of prokaryotic and eukaryotic
Sir2-like proteins. Biochem. Biophy. Res. Commun. 2000, 273, 793–
798.
(11) 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, 25748–25755.
(12) Glaser, K. B. HDAC inhibitors: Clinical update and mechanism-based
potential. Biochem. Pharmacol. 2007, 74, 659–671, and references
cited therein.
(13) Garber, K. HDAC inhibitors overcome first hurdle. Nat. Biotechnol.
2007, 25, 17–19.
(14) Marks, P. A. Discovery and development of SAHA as an anticancer
agent. Oncogene 2007, 26, 1351–1356.
ATCC (Rockville, MD) and were grown in medium (DMEM or
RPMI) containing 10% fetal calf serum and L-glutamine. Pancreatic
cancer cells were plated out in duplicate into 6 wells of a 96-well
microtiter plate at 2.5-4 × 103 cells per well. Four-hours post
plating, individual wells were treated with diluent (DMSO) or
varying concentrations of SAHA or the indicated HDACIs from a
concentration of 1 nm to 50 µM. Cytotoxicity was measured at
time “0” and 72 h post treatment using the colorimetric MTS assay
according to the manufacturer’s suggestions (Promega, Madison,
WI). The IC50s were calculated using XLfit (IDBS Limited,
Guildford, UK).
Parasite Growth Inhibition Assay. The in vitro activities of
HDAC inhibitors against P. falciparum strains W2, D6, TM91C235
and TM90C2A were evaluated using the method the labeled
hypoxanthine assay of Desjardins et al.,49 as modified by Milhous
et al.50 and described in one of our earlier papers.51 W2 is
chloroquine resistant and mefloquine sensitive, D6 is chloroquine
sensitive but naturally less susceptible to mefloquine, TM91C235
is resistant to mefloquine, chloroquine, and pyrimethamine as is
TM90C2A, however, this latter parasite is a two pfmdr1 copy strain.
Data are reported as a single IC50 value for each compound in Table
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Acknowledgment. We are indebted to the ADDF/Elan (grant
no. 271210) for their support of these studies. We thank Dr.
Rong He for assisting in the preparation of the manuscript,
and Dr. Arsen Gaysin for providing technical support. This
manuscript was reviewed by the WRAIR and the US Army
Medical and Material Research Command and there is no
objection to its publication or dissemination. The opinions
expressed herein are those of the authors and do not reflect
the views of the Department of the Army or the Department
of Defense.
Supporting Information Available: The HPLC reports for the
purity check of the final compounds measured in two different
mobile phases. This material is available free of charge via the
Internet at http://pubs.acs.org.
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