Synthesis and biological evaluation of triazol-4-ylphenyl-bearing histone deacetylase inhibitors as anticancer agents

Article abstract of DOI:10.1021/jm901667k

Our triazole-based histone deacetylase inhibitor (HDACI), octanedioic acid hydroxyamide[3-(1-phenyl-1H-[1,2,3]triazol-4-yl)phenyl]amide (4a), suppresses pancreatic cancer cell growth in vitro with the lowest IC₅₀ value of 20nM against MiaPaca-2

Full text of DOI:10.1021/jm901667k

J. Med. Chem. 2010, 53, 1347–1356 1347
DOI: 10.1021/jm901667k
Synthesis and Biological Evaluation of Triazol-4-ylphenyl-Bearing Histone Deacetylase Inhibitors as
Anticancer Agents
Rong He,†,§ Yufeng Chen,†,§ Yihua Chen,† Andrei V. Ougolkov,‡, Jin-San Zhang,‡ Doris N. Savoy,‡ Daniel D. Billadeau,*,‡
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 and ‡Division of Oncology Research, Schulze Center for Novel Therapeutics, College of Medicine, Mayo Clinic, 19-254
Gonda, 200 First Street SW, Rochester, Minnesota 55905. These authors contributed equally. Present address: Department of Pathology,
Northwestern University, Chicago, Illinois 60611.
§
Received November 11, 2009
Our triazole-based histone deacetylase inhibitor (HDACI), octanedioic acid hydroxyamide[3-(1-
phenyl-1H-[1,2,3]triazol-4-yl)phenyl]amide (4a), suppresses pancreatic cancer cell growth in vitro with
the lowest IC50 value of 20 nM against MiaPaca-2 cell. In this study, we continued our efforts to develop
triazol-4-ylphenyl bearing hydroxamate analogues by embellishing the terminal phenyl ring of 4a with
different substituents. The isoform inhibitory profile of these hydroxamate analogues was similar to
those of 4a. All of these triazol-4-ylphenyl bearing hydroxamates are pan-HDACIs like SAHA.
Moreover, compounds 4h and 11a were found to be very effective inhibitors of cancer cell growth in
the HupT3 (IC50=50 nM) and MiaPaca-2 (IC50=40 nM) cancer cell lines, respectively. Compound 4a
was found to reactivate the expression of CDK inhibitor proteins and to suppress pancreatic cancer cell
growth in vivo. Taken together, these data further support the value of the triazol-4-ylphenyl bearing
hydroxamates in identifying potential pancreatic cancer therapies.
Introduction
of HDACIs such as (E)-(1S,4S,10S,21S)-7-[(Z)-ethylidene]-
4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-
[8.7.6]tricos-16-ene-3,6,9,19,22-pentone (FR901228)2 have
now reached clinical trials, and suberoylanilide hydroxamic
acid (SAHA, 1a) is marketed by Merck for use in cutaneous
T-cell lymphoma (CTCL).9 Published work on the use of
HDACIs in inflammatory disease,10 neurodegeneration,11
heart disease,12 and protozoan infections13 reflects the grow-
ing recognition that HDACIs might serve as therapeutic
interventions for diseases other than cancer.
There are 11 HDAC isoforms identified that operate
through zinc-dependent mechanisms.14 These include the
class I HDACs 1, 2, 3, and 8, class II that includes 4, 5, 6, 7,
9, and 10, and class IV that contains HDAC 11. Class III
enzymes are HDACs in yeast and include the SIRTs (sirtuins)
or Sir2- related proteins, which are NAD-dependent
HDACs.15 Most class I HDACs are subunits of multiprotein
nuclear complexes that are crucial for transcriptional repres-
sion and epigenetic landscaping. A variety of data suggest that
HDAC 1 plays an important role in tumorigenesis,16 and
therefore, class I inhibitors are being sought for use as anti-
cancer drugs. Class II HDACs regulate cytoplasmic processes
or function as signal transducers that shuttle between the
cytoplasm and the nucleus. The class IV enzyme HDAC 11 is
poorly understood compared to other HDAC isoforms, and
recently it has been found tonegativelyregulate the expression
of the gene encoding interleukin 10 (IL-10) in antigen-pre-
senting cells.17 HDACs of class I are expressed in most
cell types, whereas the expression pattern of class II HDACs
is more tissue-restricted. For example, HDACs 4 and 5
are highly expressed in cardiomyocytes and play a role in
Epigenetic alterations involve regulation of gene expression
and are critical to the pathogenesis of many diseases including
cancer and various neurodegenerative diseases.1,2 Histone
modification is one of the molecular mechanisms that mediate
epigenetic phenomena.3 Two types of enzymes, histone acet-
yltransferases (HATsa) and histone deacetylases (HDACs),
control the acetylation of histones. In general, HATs function
to acetylate lysine groups in nuclear histones, resulting in
neutralization of the charges on the histones and a more open,
transcriptionally active chromatin structure, while HDACs
function to deacetylate and suppress transcription. A shift in
the balance of acetylation on chromatin may result in changes
in the regulation of patterns of gene expression.4 HDAC
inhibitors (HDACIs) represent a class of molecularly tar-
geted agents that can modulate epigenetic changes to histone
proteins and thereby counteract aberrant gene expression.
HDACIs can be classified into structural classes, includ-
ing short chain fatty acids, small-molecule hydroxamates,5
cyclic peptides,2 benzamides,6 thiol-based compounds,7 ke-
tones,8 and other hybrid compounds (Figure 1). A number
*To whom correspondence should be addressed. For D.D.B.: phone,
507-266-4334; fax, 1-507-266-5146; e-mail, Billadeau.Daniel@
mayo.edu. For A.P.K.: phone, 312-996-7577; fax, 312-996-7107; e-mail,
kozikowa@uic.edu.
aAbbreviations: HDAC, histone deacetylase; HDACI, histone de-
acetylase inhibitor; HAT, histone acetyltransferase; ZBG, zinc binding
group; SAHA, suberoylanilide hydroxamic acid; TSA, trichostatin A;
CTCL, cutaneous T-cell lymphoma; NAD, nicotinamide adenine dinu-
cleotide; CDK, cyclin-dependent kinase; DNA, Deoxyribonucleic Acid;
TLC, thin-layer chromatography; MTT, 3-[4,5-dimethylthiazol-2-
yl]2,5-diphenyltetrazolium bromide.
r
2010 American Chemical Society
Published on Web 01/07/2010
pubs.acs.org/jmc

1348 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
He et al.
Figure 1. Classes of HDACIs.
robust neuroprotection in the homocysteic acid model of
oxidative stress.7,22 Moreover, one of these thiol-based HDA-
CIs was found toreduce neuronalcell lossina rodent model of
traumatic brain injury.23 We also have identified HDACIs
containing a phenylisoxazole as the cap group that possess
excellent selectivity and picomolar potency for HDAC 6.24 In
other studies, we have reported a series of trazolylphenyl-
based HDACIs for which we demonstrated that modifica-
tions to the cap region were able to alter selectivity for HDAC
1 versus HDAC 6.25 Preliminary studies of the anticancer and
antimalarial activity of these triazol-4-ylphenyl bearing hy-
droxamates (4a) suggested that these compounds were worth
further structural optimization (Figure 2). In this paper, we
present additional details of the structure-activity relation-
ship (SAR) studies of the parent compound 4a that focus
on steric and/or electronic effects on CAP region. The biolo-
gical studies presented here include HDAC isoform profiling
and cell-based antiproliferative screening toward pancrea-
tic cancer cell lines of all the newly synthesized triazolyl-
phenyl-based HDACIs, together with cell-cycle dysregula-
tion, induction of CDK inhibitor proteins, and in vivo xeno-
graft study of 4a.
Figure 2. Functional domains of HDACIs.
hypertrophy.18 HDACs 4, 8, and 9 are expressed to a greater
extent in tumor tissues.19 High expression level of HDAC 11
transcripts is limited to kidney, heart, brain, skeletal muscle,
and testis, while the class II enzyme, HDAC 6, is expressed in
most neurons.20 Understanding precise roles of HDAC iso-
forms will expand our understanding of the connections
between individual HDAC isoform and pathophysiology
as well as guide the development of selective HDACIs as
strategic therapeutic agents that elicit fewer undesirable side
effects.
Classical HDACIs have three chemical domains, each with
a specific function relating to the structure of the enzyme.
Figure 2 shows the structure and functional domains of
SAHA (1a), which includes a zinc binding group (ZBG), a
linker region, and a surface recognition domain (cap). Under-
standing the mechanism of HDAC is beneficial for a study of
the interactions between HDACIs and the enzyme. The active
site of classes I, II, and IV HDACs is found within a highly
conserved catalytic domain containing a divalent zinc cation
that is coordinated to 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 histi-
dine-aspartate charge-relay system. To date, X-ray cocrys-
tallographic information is available for HDACs 4, 7, and 8,
in complex with several different inhibitors including SAHA
(1a), TSA (1b), and others.21 These crystal structures of the
HDAC enzymes and the derived homology models provide
valuable information in the design of selective HDACIs. We
have been able to obtain varying degrees of isozyme selectivity
for the HDACIs through chemical manipulations of the CAP
region in concert with the ZBG. For example, we have
reported that certain mercaptoacetamide-based HDACIs
show inherent selectivity for HDAC 6 versus HDAC 1 and
that some of these thiol-based agents are able to provide
Results and Discussion
Chemistry. The copper-catalyzed [3 þ 2]-cycloaddition of
acetylene 1 with azides 2a-l provided the 1,4-disubstituted
triazoles 3a-l, in analogy to work previously reported by
us.26 The terminal ester groups of compounds 3a-j and 3l
were then treated with KOH/NH2OH in methanol to afford
the final hydroxamates 4a-j and 4l (Scheme 1).
As shown in Scheme 2, the synthesis of the reversed 1,4-
disubstituted triazoles 11a and 11b started from the mono-
Boc protected 1,3-phenyldiamine 5. Compound 6 was pre-
pared by a coupling reaction between compound 5 and
suberic acid monomethyl ester in the presence of POCl3
and pyridine, which was further converted to compound 7
by deprotection of the Boc group in trifluoroacetic acid. The
diazotization of the resulting aniline 7 with NaN3 provided
the phenylazide 8. The cycloaddition of compound 8 with
phenylacetylene or cyclohexylacetylene provided the re-
versed triazoles 10a and 10b, respectively.26 Treating 10a
and 10b with KOH/NH2OH in methanol gave the desired
hydroxamates 11a and 11b (Scheme 2).
HDAC Isoform Inhibition Assay. The inhibitory effects of
compounds 4b-j, 4l, 11a,b, and 12a on HDAC activity were
determined by using a fluorescence-based assay as described
before.7 The data are presented as IC50 values in Table 1.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1349
Scheme 1. Synthesis of Ligands 4a-j and 4la
a Reagents and conditions: (a) copper sulfate pentahydrate, sodium ascorbate, azides 2a-j, room temp; (b) H2, 10% Pd-C, room temp, 4 h;
(c) NH2OH, KOH, room temp.
Scheme 2. Synthesis of Ligands 11a and 11ba
a Reagents and conditions: (a) suberic acid monomethyl ester, POCl3, pyridine, 0 °C; (b) TFA, 0 °C; (c) HOAc, NaNO2, 0 °C; (d) NaN3, 0 °C;
(e) copper sulfate pentahydrate, sodium ascorbate, phenylacetylene (9a) or cyclohexylacetylene (9b), room temp; (f) NH2OH, KOH, room temp.
Table 1. HDAC Isoform Inhibitory Activity (IC50, nM) of the Triazole-Based Ligands
IC50 [nM]a
compd
ClogP b
R
HDAC 1
HDAC 2
282
14
HDAC 3
HDAC 8
2290
1380
HDAC 10
HDAC 6
1a
1b
4a
4b
4c
1.44
2.23
2.39
2.59
3.55
3.35
2.79
1.47
0.96
2.37
3.44
1.47
2.39
3.44
3.85
96
4
17
2
72
5
14
1
Ph
4-F-Ph
4-I-Ph
18 ( 2
31.9 ( 2.9
8.0 ( 0.6
8.7 ( 0.3
5.3
63.9 ( 12.5
60 ( 10
24.6 ( 0.8
28.5 ( 1.0
NAc
7.2 ( 0.7
18.1 ( 1.9
3.0 ( 0.1
3.4 ( 0.2
1.4
2780 ( 470
12000 ( 800
>30000
5220 ( 390
NAc
27.7 ( 1.9
32.8 ( 5.0
10.7 ( 0.7
14.5 ( 0.6
NAc
9.6 ( 0.6
8.4 ( 1.8
2.2 ( 0.1
2.9 ( 0.1
1.7
4d
4e
4-CF3-Ph
3,5-F,F-Ph
4f
4-OHCH2-Ph
3,5-OHCH2-,OHCH2-Ph
4-F-,3-CH3O-Ph
cyclohexyl
4-NH2-Ph
3.04
5.7 ( 0.2
5.06
NAc
1.66
3.3 ( 0.2
3.39
NAc
NAc
7.6
1.9 ( 0.1
3.85
4g
4h
4j
27.8 ( 0.7
NAc
1320 ( 80
NAc
5.8 ( 0.1
NAc
8.6 ( 0.2
1.01
27.5 ( 1.0
NAc
3.7 ( 0.1
1.55
1190 ( 490
NAc
9.8 ( 0.1
NAc
3.3 ( 0.1
2.52
4l
11a
11b
12a
Ph
cyclohexyl
Ph
4.6 ( 0.1
12.1 ( 0.4
>30000
25.1 ( 2.0
48.2 ( 2.3
>30000
2.8 ( 0.2
5.3 ( 0.5
122 ( 9
4180 ( 390
2750 ( 120
>30000
6.8 ( 0.3
16.6 ( 0.5
>30000
3.1 ( 0.2
4.3 ( 0.5
>30000
a The isoform inhibition was tested at Nanosyn, http://www.nanosyn.com. b CLogP (KOWWIN) were calculated from http://146.107.217.178/lab/
alogps/start.html. c NA = not assayed.
TSA (1b) was used as a positive control. The inhibitory data
for SAHA (1a) are also presented for comparison.
The IC50 values of the newly synthesized hydroxamate
HDACIs are similar to those of the lead compound 4a. Most
of these analogues inhibit HDACs 1, 3, 10, and 6 in the
nanomolar range and are slightly less potent at HDAC 2
while showing only micromolar activity against HDAC 8.
No significant isozyme selectivity was observed in this set of
structures (4a-j, 4l, and 11a,b). Introduction of different
substituents on the terminal phenyl ring of 4a increased its

1350 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
He et al.
Table 2. Triazole-Based HDACIs Suppress Growth of Pancreatic
Cancer Cells
inhibition of pancreatic cancer cell lines (IC50, μM)
compd
BxPC-3 Hup T3 Mia Paca-2 Panc04.03 SU 86.86
SAHA (1a)
5
0.8
0.2
0.1
1.4
0.4
0.4
0.2
7
1.1
1.2
>0.5
0.2
1.3
0.8
0.6
7.7
1.5
1.7
1
4a
4b
4c
0.4
1
0.02
0.1
1.1
0.9
1.2
1
2.33
0.9
2.34
4d
4e
1.9
2.03
1
0.8
0.2
4f
4g
4h
4i
29
14
27
37
1
0.2
1.2
4
0.05
0.5
0.4
0.55
0.1
0.4
0.1
0.6
0.1
0.7
0.5
0.36
0.1
0.5
Figure 3. Effect of compound 4a on cell cycles. Panc04.03 cells
were treated with vehicle (DMSO) or compound 4a (5 μM) for 24 h.
Cells were subsequently harvested and stained with propidium
iodine, and cell cycle was analyzed using flow cytometry. The
percentage of cells in G1, S, and G2M is shown.
1.2
0.1
1.45
0.2
0.9
4j
0.1
4l
1.2
0.9
2
0.88
0.04
0.1
11a
11b
12a
>50
>50
>50
>50
32
IC50 values equal to or less than those of SAHA (1a) with the
exception of compounds 4g and 12a. Compound 4a was the
most active analogue against the MiaPaca-2 cells line with an
IC50 value of 20 nM. Generally, compound 4a was a more
effective inhibitor of cancer cell growth than compounds
4c-g, 4i-l, 11b, and 12a. Moreover, compounds 4h and 11a
were very effective inhibitors of cancer cell growth in the
HupT3 (IC50=50 nM) and MiaPaca-2 (IC50=40 nM) cancer
cell lines, respectively. On the other hand, compound 4g was
able to only moderately inhibit cell growth in the five
pancreatic cancer cell lines. The weak inhibitory activity of
compound 4g against the different pancreatic cancer cell
lines might be due to its polarity, as it has a lower ClogP value
than those of the other triazole analogues. Correlative
studies reporting aberrant expression and/or localization of
HDAC 3 in various tumors imply an important role of
HDAC 3 in carcinogenesis.30 HDAC 3 is also reported to
be a repressor of ULBPs expression in epithelial cancer
cells31 and to be linked to the cell cycle machinery of
MiaPaCa2 cells.32 While 12a exhibits HDAC 3 selectivity,
its poor growth inhibition may be due to its poorer HDAC
inhibitory activity.
To directly examine the effect on proliferation, we per-
formed cell cycle analysis on Panc04.03 cells, which were
treated for 24 h with either vehicle alone or compound 4a, the
best HDACI in this series. Compared to the vehicle-treated
Panc04.03 cancer cells, which show a cell cycle profile
consistent with rapidly proliferating cells, Panc04.03 cancer
cells treated with compound 4a demonstrate a loss of S-phase
cells and an increase in the percentage of cells in G1
(Figure 3). The increase in G2/M cells in the cells treated
with compound 4a may reflect additional effects on micro-
tubule stabilization, which is frequently associated with
increases in acetylated R-tubulin, which is deacetylated
by HDAC 6.
HDAC inhibitory activity, although these modifications
failed to influence the levels of HDAC isozyme selectivity.
For example, compound 4d with an electron withdrawing
p-trifluoro group on the terminal phenyl ring was 2- to 3-fold
more potent than compound 4a against HDACs 1, 2, 3, 10,
and 6 while it was 1.9-fold less potent than compound 4a in
HDAC 8. Comparison of inhibitors 4l and 4a shows that the
addition of a p-amino group on the terminal phenyl ring of 4a
also leads to an increase in inhibitory activity against HDAC
1 (18-fold), HDAC 3 (5-fold), and HDAC 6 (4-fold). Repla-
cement of the terminal phenyl ring of 4a with a cyclohexyl
group as in 4j led to a 2- to 3-fold increase in potency against
all tested HDACs (HDACs 1, 2, 3, 8, 10, and 6). The reversed
1,4-disubstituted triazole 11a was more potent than com-
pound 4a against HDACs 1, 2, 3, 10, and 6. The other
reversed 1,4-disubstituted triazole 11b was slightly less po-
tent than compound 4j in all tested HDACs. Compound 12a
has previously been reported by us, and it represents a
benzamide-based HDACI that selectively inhibits HDAC 3
over the other tested HDAC isoforms.27
Triazole-Based HDACIs Reactivate Expression of CDK
Inhibitors and Suppress Growth of Pancreatic Cancer Cells.
Pancreatic cancer is the fourth leading cause of cancer death
in the U.S. and essentially remains an incurable disease with
the incidence nearly equaling the mortality. Altered HDAC
activity is seen to be associated with many cancers, validating
HDACs as promising targets for cancer therapy. In fact,
treatment of pancreatic cancer cells with the broad spectrum
class I/II HDACI, SAHA (1a), leads to up-regulation of p21,
growth inhibition, and an increase in gemcitabine-induction
of apoptosis.28 Furthermore, SAHA (1a) was found to
enhance the antiproliferative effect of a DNA methyl-
ation inhibitor, 5-aza-20-deoxycytidine, on pancreatic cancer
cells.29
In continuation of our efforts to develop HDACIs as
antipancreatic cancer therapeutics, we assayed these newly
synthesized triazoles (4b-j, 4l, 11a,b, and 12a) against a
panel of pancreatic cancer cell lines, which consisted of
BxPC-3, HupT3, MiaPaca-2, Pan04.03, and SU86.86 cell
lines. The effect of the tested analogues on cancer cell growth
was measured by MTT assay, and the IC50 values obtained
against these cell lines are summarized in Table 2. The data
for SAHA (1a) and compound 4a are provided for compari-
son purposes. As is apparent from the data in Table 2, the
majority of our HDACIs (4a-f, 4h-j, 4l, and 11a,b) have
One of the key features of HDACIs is that they have the
capability of reactivating tumor suppressor genes in cancer
cells that can inhibit proliferation and survival of the cancer
cells. The CDK inhibitor protein p21 is frequently epigene-
tically silenced in tumor cells, and reactivation of this protein
can cause cell cycle arrest and induce senescence and apo-
ptosis in cancer cells. We therefore examined whether treat-
ment of the BXPC-3 cell line with compound 4a would lead
to the expression of p21 (Figure 4). Indeed, although p21
protein levels are nearly undetectable in the BXPC-3 cell
line treated with vehicle (DMSO) alone, the addition of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1351
m = multiplet, and br = broad. HRMS experiment was per-
formed on a Q-TOF-2TM (Micromass) instrument. TLC was
performed with Merck 250 mm 60F254 silica gel plates. Pre-
parative TLC was performed with Analtech 1000 mm silica gel
GF plates. Column chromatography was performed using
Merck silica gel (40-60 mesh). HPLC was carried out on
Agilent 1100 HPLC system with a Synergi 4 μm Hydro-RP
80A column, with detection at 254 on a variable wavelength
detector G1314A. Method 1 consisted of the following: flow
rate = 1.4 mL/min; gradient eluation over 20 min from 30%
CH3OH-H2O to 100% CH3OH with 0.05% TFA. Method 2
consisted of the following: flow rate = 1.4 mL/min; gradient
eluation over 20 min, from 10% CH3OH-H2O to 100%
CH3OH with 0.05% TFA. Method 3 consisted of the following:
flow rate = 1.4 mL/min; gradient eluation over 20 min from
100% H2O to 100% CH3OH with 0.05% TFA.
Figure 4. HDACI 4a reactivates expression of the CDK inhibitor
p21 in BXPC3 pancreatic cancer cells. The pancreatic cancer cell line
BXPC3 was treated with vehicle (DMSO) or compound 4a (5 μM)
for 18 h. Cell lysates were prepared, separated by SDS-PAGE,
transferred to PVDF membrane, and immunoblotted as indicated.
7-{3-[1-(4-Fluoro-phenyl)-1H-[1,2,3]triazol-4-yl]phenylcarba-
moyl}heptanoic Acid Methyl Ester (3b). In a mixture of 1 (100
mg, 0.34 mmol) and 1-azido-4-fluorobenzene (2b, 143 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 copper33 sulfate penta-
hydrate (17 mg, 0.07 mmol, dissolved in 1 mL of water). The
heterogeneous mixture was stirred vigorously overnight at room
temperature. The reaction mixture was diluted with EtOAc and
washed thoroughly with brine, dried over Na2SO4, filtered, and
concentrated. The residue was purified by column chromato-
graphy on silica gel (hexanes/EtOAc, 1:1) to afford 77 mg (52%)
of 3b. Rf=0.47 (1:1 hexane/EtOAc). 1H NMR (DMSO-d6, 400
MHz): δ=1.30 (br s, 4H), 1.53 (t, J=8.0 Hz, 2H), 1.58 (t, J=8.0
Hz, 2H), 2.28-2.34 (m, 4H), 3.57 (s, 3H), 7.40 (t, J=8.0 Hz, 1H),
7.49 (t, J=8.0 Hz, 2H), 7.55 (d, J=8.0 Hz, 1H), 7.59 (d, J=8.0
Hz, 1H), 8.01-8.04 (m, 2H), 8.26 (s, 1H), 9.24 (s, 1H), 10.02 (s,
1H). 13C NMR (DMSO-d6, 100 MHz): δ=24.7, 25.3, 28.6, 28.7,
33.6, 36.7, 51.6, 116.3, 117.0, 117.3, 119.3, 120.3, 120.6, 122.7,
122.8, 129.7, 131.0, 133.6, 140.3, 147.7, 171.8, 173.7.
7-{3-[1-(4-Iodophenyl)-1H-[1,2,3]triazol-4-yl]phenylcarba-
moyl}heptanoic Acid Methyl Ester (3c). Compound 3c (yield
38%) was prepared from 1 and 1-azido-4-iodobenzene (2c)
according to the methodology described for the preparation of
compound 3b. Rf=0.71 (1:1 hexane/EtOAc). 1H NMR (DMSO-
d6, 300 MHz): δ=1.28 (br s, 4H), 1.51 (t, J=6.8 Hz, 2H), 1.58 (t, J
=6.8 Hz, 2H), 2.25-2.33 (m, 4H), 3.55 (s, 3H), 7.38 (t, J=7.9
Hz, 1H), 7.53 (d, J=7.5 Hz, 1H), 7.57 (d, J=7.9 Hz, 1H), 7.79 (d,
J=8.2 Hz, 2H), 7.97 (d, J=8.2 Hz, 2H), 8.25 (s, 1H), 9.27 (s, 1H),
10.01 (s, 1H). 13C NMR (DMSO-d6, 75 MHz): δ=25.1, 25.7,
29.0, 29.1, 34.0, 37.1, 51.9, 116.6, 119.7, 120.3, 121.0, 122.6,
130.1, 131.2, 137.0, 139.4, 140.7, 148.2, 172.1.
Figure 5. CDK inhibitors are re-expressed in SU86.86 and
Panc04.03 mouse xenograft tumors upon treatment with HDACI
4a. (A) Female athymic nude mice (8-10 weeks old) were inoculated
subcutaneously with 3 ꢀ 106 Su86.86 (left flank) and Panc04.03
(right flank) pancreatic cancer cells mixed with Matrigel (BD
Biosciences). Two weeks after injection, tumors were size-matched
and mice were randomized into three treatment groups: (a) control
DMSO, four mice; (b) compound 4a (10 mg/kg), four mice;
(c) compound 4a (100 mg/kg), four mice. Established SU86.86
and Panc04.03 xenografts (tumor volume 150-200 mm3) were
treated by ip injections with diluent (50 μL of DMSO) or compound
4a (10 and 100 mg/kg; every 12 h for 48 h). (B) Tumor proteins were
extracted from fresh tumor tissues taken from mouse, separated by
SDS-PAGE (50 μg/well), transferred to polyvinylidene difluoride
membrane, and probed with the indicated antibodies.
compound 4a resulted in an increased expression of p21. As
shown in Figure 5, expression of CDK inhibitors p15, p16,
p21, p27, and p57 was reactivated in mouse xenograft tumors
upon treatment with compound 4a.
Conclusion
7-{3-[1-(4-Trifluoromethylphenyl)-1H-[1,2,3]triazol-4-yl]phe-
nylcarbamoyl}heptanoic Acid Methyl Ester (3d). Compound 3d
(yield 51%) was prepared from 1 and 1-azido-4-trifluoromethyl-
benzene (2d) according to the methodology described for the
preparation of compound 3b. Rf=0.54 (1:1 hexane/EtOAc). 1H
NMR (DMSO-d6, 400 MHz): δ=1.31(br s, 4H), 1.55 (t, J=8.0
Hz, 2H), 1.61 (t, J=8.0 Hz, 2H), 2.35-2.28 (m, 4H), 3.57 (s, 3H),
7.42 (t, J=8.0 Hz, 1H), 7.58 (t, J=8.0 Hz, 2H), 8.03 (d, J=8.0
Hz, 2H), 8.24 (d, J=8.0 Hz, 2H), 8.30 (s, 1H), 9.42 (s, 1H), 10.03
(s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ=24.7, 25.3, 28.6,
28.7, 33.6, 36.7, 51.6, 116.3, 119.5, 120.2, 120.7, 120.8, 125.6,
127.6, 127.7, 128.9, 129.2, 129.8, 130.7, 139.8, 140.4, 148.0,
171.8, 173.7.
7-{3-[1-(3,5-Difluorophenyl)-1H-[1,2,3]triazol-4-yl]phenylcar-
bamoyl}heptanoic Acid Methyl Ester (3e). Compound 3e (yield
59%) was prepared from 1 and 1-azido-3,5-difluorobenzene (2e)
according to the methodology described for the preparation of
compound 3b. Rf=0.55 (1:1 hexane/EtOAc). 1H NMR (DMSO-
d6, 400 MHz): δ=1.32 (br s, 4H), 1.55 (t, J=8.0 Hz, 2H), 1.60
(t, J = 8.0 Hz, 2H), 2.28-2.35 (m, 4H), 3.57 (s, 3H), 7.48-
7.40 (m, 2H), 7.53-7.59 (m, 2H), 7.85 (d, J=8.0 Hz, 2H), 8.29
(s, 1H), 9.36 (s, 1H), 10.03 (s, 1H). 13C NMR (DMSO-d6,
Substituted 1-aryl-1H-[1,2,3]triazolylphenyl-based ana-
logues of compound 4a were designed, synthesized, and
profiled for their HDAC isoform selectivity and antiprolifera-
tive activities against pancreatic cell lines. The preliminary in
vivo evaluation of compound 4a on established pancreatic
tumor cell line xenografts was also determined. We found that
compound 4a could reactivate expression of CDK inhibitor
proteins as well as suppress pancreatic cancer cell growth in
vivo. The fluorinated analogues 4b, 4e, 4h of compound 4a
may have potential in PET imaging studies. Taken together,
our current findings strongly suggest that compound 4a and
its related compounds may ultimately be useful in various
combination treatment strategies for pancreatic cancer.
Experimental Section
Chemistry. 1H NMR and 13C NMR spectra were recorded on
a Bruker spectrometer at 300/400 MHz and 75/100 MHz,
respectively, with TMS as an internal standard. Standard
abbreviation indicating multiplicity was used as follows:
s=singlet, d=doublet, t=triplet, q=quartet, quin=quintuplet,

1352 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
He et al.
100 MHz): δ = 24.7, 25.3, 28.6, 28.7, 33.6, 36.7, 51.6, 104.1,
104.4, 116.3, 119.5, 120.6, 120.6, 125.8, 129.8, 130.6, 140.4,
148.0, 161.9, 162.1, 171.8, 173.8.
44%) was prepared from 1 and 1-azido-4-nitrobenzene (2k)
according to the methodology described for the preparation of
compound 3b. Rf=0.60 (1:1 hexane/EtOAc). 1H NMR (DMSO-
d6, 500 MHz): δ=1.31 (br s, 4H), 1.52-1.55 (m, 2H), 1.59-1.62
(m, 2H), 2.29-2.35 (m, 4H), 3.58 (s, 3H), 7.42 (t, J=7.5 Hz, 1H),
7.59 (d, J=6.5 Hz, 2H), 7.93 (t, J=8.0 Hz, 1H), 8.31 (s, 1H), 8.35
(d, J=8.5 Hz, 1H), 8.49 (d, J=8.5 Hz, 1H), 8.82 (s, 1H), 9.53 (s,
1H), 10.03 (s, 1H). 13C NMR (DMSO-d6, 125 MHz): δ=24.3,
24.9, 28.2, 28.3, 33.2, 36.3, 51.1, 114.6, 115.9, 119.1, 120.0, 120.2,
123.0, 125.9, 129.3, 130.2, 131.5, 137.2, 139.9, 147.6, 148.5,
171.3, 173.3.
7-{3-[1-(4-Aminophenyl)-1H-[1,2,3]triazol-4-yl]phenylcarba-
moyl}heptanoic Acid Methyl Ester (3l). A suspension of com-
pound 3l (0.080 g, 0.17 mmol) and Pd/C (10 wt %, 20 mg) in
EtOAc (20 mL) was stirred under hydrogen atmosphere at room
temperature for 5 h. The catalyst was removed by filtration
through a pad of Celite and washed thoroughly with MeOH.
The solvent was evaporated. The residue was purified by column
chromatography on silica gel (EtOAc/hexane, 2:1) to give
compound 3l (0.054 g, 72%). Rf = 0.30 (1:1 hexane/EtOAc).
1H NMR (CD3OD, 500 MHz): δ=1.40 (br s, 4H), 1.61-1.67 (m,
2H), 1.71-1.75 (m, 2H), 2.34 (t, J=7.5 Hz, 2H), 2.41 (t, J=8.0
Hz, 2H), 3.65 (s, 3H), 4.61 (br s, 2H), 6.81 (d, J=8.0 Hz, 1H),
7.10 (d, J=8.0 Hz, 1H), 7.21 (s, 1H), 7.27 (t, J=8.0 Hz, 1H), 7.41
(t, J=8.0 Hz, 1H), 7.59 (d, J=7.5 Hz, 1H), 7.64 (d, J=7.5 Hz,
1H), 8.11 (s, 1H), 8.74 (s, 1H). 13C NMR (CD3OD, 125 MHz):
δ=25.8, 26.7, 29.9, 30.0, 34.7, 37.9, 52.0, 107.5, 109.9, 116.4,
118.4, 120.4, 121.2, 122.5, 130.5, 131.4, 132.1, 139.2, 140.5,
149.0, 150.9, 174.7, 176.0.
7-{3-[1-(4-Hydroxymethylphenyl)-1H-[1,2,3]triazol-4-yl]phe-
nylcarbamoyl}heptanoic Acid Methyl Ester (3f). Compound
3f (yield 69%) was prepared from 1 and (4-azidophe-
nyl)methanol (2f) according to the methodology described for
the preparation of compound 3b. Rf=0.57 (EtOAc). 1H NMR
(DMSO-d6, 400 MHz): δ=1.32 (br s, 4H), 1.53 (t, J=8.0 Hz,
2H), 1.60 (t, J=8.0 Hz, 2H), 2.28-2.35 (m, 4H), 3.57 (s, 3H),
4.59 (s, 2H), 5.37 (br s, 1H), 7.40 (t, J=8.0 Hz, 1H), 7.54-7.61
(m, 4H), 7.93 (d, J=8.0 Hz, 2H), 8.26 (s, 1H), 9.23 (s, 1H), 10.01
(s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ = 24.7, 25.3,
28.6, 28.7, 33.6, 36.7, 51.6, 62.6, 116.3, 119.3, 119.9, 120.2,
120.6, 125.8, 128.0, 129.7, 131.1, 135.6, 140.3, 143.7, 147.6,
171.8, 173.8.
7-{3-[1-(3,5-Bis-hydroxymethylphenyl)-1H-[1,2,3]triazol-4-yl]-
phenylcarbamoyl}heptanoic Acid Methyl Ester (3g). Compound
3g (yield 40%) was prepared from 1 and (3-azido-5-hydroxy-
methylphenyl)methanol (2g) according to the methodology
described for the preparation of compound 3b. Rf = 0.26
(EtOAc). 1H NMR (DMSO-d6, 400 MHz): δ=1.32 (br s, 4H),
1.54 (t, J=8.0 Hz, 2H), 1.61 (t, J=8.0 Hz, 2H), 2.28-2.35 (m,
4H), 3.58 (s, 3H), 4.62 (s, 4H), 7.38-7.41 (m, 2H), 7.59 (d, J=8.0
Hz, 2H), 7.79 (s, 2H), 8.29 (s, 1H), 9.27 (s, 1H), 10.00 (s, 1H). 13
C
NMR (DMSO-d6, 100 MHz): δ=24.7, 25.3, 28.6, 28.7, 33.6,
36.7, 51.6, 62.8, 116.3, 116.4, 119.3, 119.9, 120.7, 124.6, 129.7,
131.1, 136.9, 140.3, 145.0, 147.6, 171.8, 173.8.
7-{3-[1-(3-Fluoro-4-methoxyphenyl)-1H-[1,2,3]triazol-4-yl]-
phenylcarbamoyl}heptanoic Acid Methyl Ester (3h). Compound
3h (yield 70%) was prepared from 1 and 4-azido-2-fluoro-1-
methoxybenzene (2h) according to the methodology described
for the preparation of compound 3b. Rf = 0.25 (1:1 hexane/
Octanedioic Acid {3-[1-(4-Fluorophenyl)-1H-[1,2,3]triazol-4-
yl]phenyl}amide Hydroxyamide (4b). To a solution of hydro-
xylamine hydrochloride (1.96 g, 28 mmol) in 20 mL of MeOH,
KOH (1.58 g, 28 mmol) was added at 40 °C for 10 min. The
reaction mixture was cooled to 0 °C and filtered. Compound 3b
(60 mg, 0.14 mmol) was added to the filtrate followed by KOH
(0.158 g, 2.8 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 residue was
purified by preparative HPLC to give compound 4b (31 mg,
1
EtOAc). H NMR (DMSO-d6, 400 MHz): δ=1.32 (br s, 4H),
1.54 (t, J=8.0 Hz, 2H), 1.60 (t, J=8.0 Hz, 2H), 2.28-2.34 (m,
4H), 3.57 (s, 3H), 3.93 (s, 1H), 7.38-7.44 (m, 2H), 7.53 (d, J=8.0
Hz, 1H), 7.58 (d, J=4.0 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.93
(dd, J=12.0 and 4.0 Hz, 1H), 8.25 (s, 1H), 9.20 (s, 1H), 10.01 (s,
1H). 13C NMR (DMSO-d6, 100 MHz): δ=24.7, 25.3, 28.6, 28.7,
33.6, 36.7, 51.6, 56.8, 109.1, 109.3, 114.9, 115.0, 116.2, 116.8,
119.3, 120.1, 120.6, 129.7, 130.0, 130.1, 131.0, 140.3, 147.6,
147.7, 150.5, 152.9, 171.8, 173.7.
7-{3-[1-(4-tert-Butylphenyl)-1H-[1,2,3]triazol-4-yl]phenylcar-
bamoyl}heptanoic Acid Methyl Ester (3i). Compound 3i (yield
58%) was prepared from 1 and 1-azido-4-tert-butylbenzene
(2i) according to the methodology described for the prepara-
tion of compound 3b. Rf = 0.47 (1:1 hexane/EtOAc). 1H
NMR (DMSO-d6, 400 MHz): δ=1.35-1.40 (m, 13H), 1.51 (t,
J=8.0 Hz, 2H), 1.60 (t, J=8.0 Hz, 2H), 2.28-2.35 (m, 4H), 3.57
(s, 3H), 7.40 (t, J = 8.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H),
7.60-7.64 (m, 3H), 7.88 (d, J=8.0 Hz, 2H), 8.25 (s, 1H), 9.20 (s,
1H), 10.01 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ=24.7,
25.3, 28.6, 28.7, 31.4, 33.6, 34.9, 36.7, 51.6, 116.3, 119.3, 119.9,
120.1, 120.7, 125.8, 127.0, 129.7, 131.1, 134.7, 140.3, 147.6,
151.7, 171.8, 173.7.
7-[3-(1-Cyclohexyl-1H-[1,2,3]triazol-4-yl)phenylcarbamoyl]-
heptanoic Acid Methyl Ester (3j). Compound 3j (yield 45%) was
prepared from 1 and azidocyclohexane (2j) according to the
methodology described for the preparation of compound 3b.
Rf=0.30 (1:1 hexane/EtOAc). 1H NMR (DMSO-d6, 400 MHz):
δ=1.20-1.32 (m, 5H), 1.40-1.60 (m, 6H), 1.68 (d, J=12.0 Hz,
1H), 1.79-1.85 (m, 4H), 2.11 (d, J=12.0 Hz, 2H), 2.27-2.32
(m, 4H), 3.57 (s, 3H), 4.50 (t, J = 8.0 Hz, 1H), 7.33 (t,
J=8.0 Hz, 1H), 7.45 (d, J=8.0 Hz, 1H), 7.54 (d, J=8.0 Hz,
1H), 8.14 (s, 1H), 8.56 (s, 1H), 9.95 (s, 1H). 13C NMR (DMSO-
d6, 100 MHz): δ=24.7, 25.0, 25.1, 25.3, 28.6, 28.7, 33.2, 33.6,
36.7, 51.6, 59.5, 116.1, 118.8, 119.9, 120.4, 129.6, 131.8, 140.2,
146.4, 171.7, 173.7.
1
51%). HPLC purity: 14.1 min, 95.6% (method 2). H NMR
(DMSO-d6, 400 MHz): δ=1.29 (br s, 4H), 1.51 (t, J=8.0 Hz,
2H), 1.60 (t, J=8.0 Hz, 2H), 1.94 (t, J=8.0 Hz, 2H), 2.32 (t,
J=8.0 Hz, 2H), 7.40 (t, J=8.0 Hz, 1H), 7.49 (t, J=8.0 Hz, 2H),
7.55 (d, J=8.0 Hz, 1H), 7.59 (d, J=8.0 Hz, 1H), 8.01-8.04 (m,
2H), 8.26 (s, 1H), 8.65 (s, 1H), 9.24 (s, 1H), 10.02 (s, 1H), 10.33
(s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ=25.4, 28.8, 32.6,
36.8, 116.3, 117.0, 117.3, 171.8, 119.4, 120.3, 120.6, 122.7, 122.8,
129.7, 131.0, 133.6, 140.3, 147.7, 169.5. ESI-HRMS calculated
for [C22H24FN5O3 - H]-: 424.1790. Found: 424.1790.
Octanedioic Acid Hydroxyamide {3-[1-(4-Iodophenyl)-1H-
[1,2,3]triazol-4-yl]phenyl}amide (4c). Compound 4c (yield
27%) was prepared according to the methodology described
for the preparation of compound 4b. HPLC purity: 15.6 min,
95.2% (method 2). 1H NMR (DMSO-d6, 400 MHz): δ=1.29
(br s, 4H), 1.50 (t, J=8.0 Hz, 2H), 1.60 (t, J=8.0 Hz, 2H), 1.94
(t, J=8.0 Hz, 2H), 2.33 (t, J=8.0 Hz, 2H), 7.40 (t, J=8.0 Hz,
1H), 7.54-7.60 (m, 2H), 7.81 (d, J=8.0 Hz, 2H), 7.99 (d, J=8.0
Hz, 2H), 8.26 (s, 1H), 8.65 (s, 1H), 9.29 (s, 1H), 10.01 (s, 1H),
10.33 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ = 172.2,
169.8, 148.2, 140.7, 139.4, 137.0, 131.2, 130.1, 122.6, 121.0,
120.3, 119.8, 116.6, 37.1, 33.0, 29.2, 25.8. ESI-HRMS calcu-
lated for [C22H24IN5O3 - H]-: 532.0851. Found: 532.0849.
Octanedioic Acid Hydroxyamide {3-[1-(4-Trifluoromethyl-
phenyl)-1H-[1,2,3]triazol-4-yl]phenyl}amide (4d). Compound 4d
(yield 39%) was prepared according to the methodo-
logy describedforthe preparationofcompound4b. HPLCpurity:
1
15.6 min, 98.6% (method 2). H NMR (DMSO-d6, 400 MHz):
δ=1.29 (br s, 4H), 1.50 (t, J=8.0 Hz, 2H), 1.60 (t, J=8.0 Hz, 2H),
1.95 (t, J=8.0 Hz, 2H), 2.33 (t, J=8.0 Hz, 2H), 7.42 (t, J=8.0 Hz,
7-{3-[1-(4-Nitrophenyl)-1H-[1,2,3]triazol-4-yl]phenylcarba-
moyl}heptanoic Acid Methyl Ester (3k). Compound 3k (yield

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1353
1H), 7.59 (t, J=8.0 Hz, 2H), 8.03 (d, J=8.0 Hz, 2H), 8.25 (d, J=
8.0 Hz, 2H), 8.66 (s, 1H), 8.30 (s, 1H), 9.43 (s, 1H), 10.03 (s, 1H),
10.33 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ=25.4, 28.8,
32.6, 36.8, 116.3, 119.5, 120.2, 120.7, 120.8, 125.8, 127.6, 127.7,
128.9, 129.8, 130.7, 139.8, 140.4, 148.0, 169.5, 171.8. ESI-HRMS
calculated for [C23H24F3N5O3 - H]-: 474.1759. Found: 474.1757.
Octanedioic Acid {3-[1-(3,5-Difluorophenyl)-1H-[1,2,3]tria-
zol-4-yl]phenyl}amide Hydroxyamide (4e). Compound 4e (yield
41%) was prepared according to the methodology described for
the preparation of compound 4b. HPLC purity: 15.0 min, 96%
(method 2). 1H NMR (DMSO-d6, 400 MHz): δ=1.29 (br s, 4H),
1.49 (t, J=8.0 Hz, 2H), 1.60 (t, J=8.0 Hz, 2H), 1.94 (t, J=8.0 Hz,
2H), 2.33 (t, J=8.0 Hz, 2H), 7.40-7.48 (m, 2H), 7.53-7.59 (m,
2H), 7.86 (d, J=8.0 Hz, 2H), 8.29 (s, 1H), 8.65 (s, 1H), 9.36 (s,
1H), 10.03 (s, 1H), 10.33 (s, 1H). 13C NMR (DMSO-d6, 100
MHz): δ=25.4, 28.8, 32.6, 36.8, 104.1, 104.3, 104.4, 104.6, 116.3,
119.5, 120.3, 120.6, 129.8, 130.6, 138.8, 138.9, 140.4, 148.0,
161.9, 162.1, 164.4, 164.5, 169.5, 171.8. ESI-HRMS calculated
for [C22H23F2N5O3 - H]-: 442.1696. Found: 442.1696.
Octanedioic Acid Hydroxyamide {3-[1-(4-Hydroxymethyl-
phenyl)-1H-[1,2,3]triazol-4-yl]phenyl}amide (4f). Compound 4f
(yield 45%) was prepared according to the methodology des-
cribed for the preparation of compound 4b. HPLC purity: 12.2
min, 95.1% (method 2). 1H NMR(DMSO-d6, 400 MHz): δ=1.29
(br s, 4H), 1.50 (t, J=8.0 Hz, 2H), 1.60 (t, J=8.0 Hz, 2H), 1.94 (t,
J=8.0 Hz, 2H), 2.33 (t, J=8.0 Hz, 2H), 4.59 (d, J=4.0 Hz, 2H),
5.36 (t, J=8.0 Hz, 1H), 7.40 (t, J=8.0Hz, 1H), 7.54-7.61 (m,4H),
7.93 (d, J=8.0 Hz, 2H), 8.26 (s, 1H), 8.65 (s, 1H), 9.24 (s, 1H),
10.02 (s, 1H), 10.33 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ=
25.4, 28.8, 32.6, 36.8, 62.6, 116.3, 119.3, 119.9, 120.2, 120.6, 125.8,
128.0, 129.7, 131.1, 135.6, 140.3, 143.7, 147.6, 169.5, 171.8. ESI-
HRMS calculated for [C23H27N5O4- H]-: 436.1990. Found:
436.1989.
(d, J=8.0 Hz, 2H), 8.25 (s, 1H), 8.65 (s, 1H), 9.20 (s, 1H), 10.02
(s, 1H), 10.34 (s, 1H). 13C NMR (DMSO-d6, 100 MHz):
δ=25.4, 28.8, 31.4, 32.6, 34.9, 36.8, 116.3, 119.3, 119.9, 120.1,
120.7, 127.0, 129.7, 131.1, 134.7, 140.3, 147.6, 151.7, 169.5, 171.8.
ESI-HRMS calculated for [C26H33N5O3 þ H]þ: 464.2656.
Found: 464.2653.
Octanedioic Acid [3-(1-Cyclohexyl-1H-[1,2,3]triazol-4-yl)phe-
nyl]amide Hydroxyamide (4j). Compound 4j (yield 27%) was
prepared according to the methodology described for the pre-
paration of compound 4b. HPLC purity: 7.9 min, 97.4%
1
(method 1). H NMR (DMSO-d6, 400 MHz): δ = 1.20-1.35
(m, 5H), 1.38-1.53 (m, 4H), 1.59 (t, J=8.0 Hz, 2H), 1.67 (d, J=
12.0 Hz, 1H), 1.78-1.84 (m, 4H), 1.94 (d, J=8.0 Hz, 2H), 2.09
(d, J=12.0 Hz, 2H), 2.31 (t, J=8.0 Hz, 2H), 4.46-4.52 (m, 1H),
7.34 (t, J=8.0 Hz, 1H), 7.46 (d, J=8.0 Hz, 1H), 7.55 (d, J=8.0
Hz, 1H), 8.16 (s, 1H), 8.56 (s, 1H), 9.96 (s, 1H), 10.35 (s, 1H). 13
C
NMR (DMSO-d6, 100 MHz): δ=25.0, 25.1, 25.4, 28.8, 32.6,
33.2, 36.8, 59.6, 116.1, 118.8, 119.8, 120.4, 129.6, 131.8, 140.2,
146.4, 169.5, 171.7. ESI-HRMS calculated for [C22H31N5O3 -
H]-: 412.2354. Found: 412.2352.
Octanedioic Acid {3-[1-(3-Aminophenyl)-1H-[1,2,3]triazol-4-
yl]phenyl}amide Hydroxyamide (4l). Compound 4l (yield 26%)
was prepared according to the methodology described for the
preparation of compound 4b. HPLC purity: 10.6 min, 97.1%
(method 3). 1H NMR (DMSO-d6, 400 MHz): δ=1.28 (br s, 4H),
1.49-1.70 (m, 4H), 1.94 (t, J=8.0 Hz, 2H), 2.32 (t, J=8.0 Hz,
2H), 5.58 (br s, 2H), 6.66 (d, J=8.0 Hz, 1H), 7.00 (d, J=8.0 Hz,
1H), 7.15 (s, 1H), 7.21 (dd, J=8.0, 8.0 Hz, 1H), 7.38 (dd, J=8.0,
8.0 Hz, 1H), 7.54-7.72 (m, 2H), 8.25 (s, 1H), 8.66 (br s, 1H), 9.09
(s, 1H), 10.00 (s, 1H), 10.33 (s, 1H). 13C NMR (DMSO-d6, 100
MHz): δ=24.7, 28.1, 31.9, 36.0, 104.5, 106.5, 113.6, 115.5, 118.4,
119.0, 119.9, 128.9, 129.8, 130.4, 137.1, 139.5, 146.7, 149.7,
168.7, 171.0.
7-(3-tert-Butoxycarbonylaminophenylcarbamoyl)heptanoic
Acid Methyl Ester (6). To a solution of 5 (1.00 g, 4.8 mmol) and
suberic acid monomethyl ester (0.903 g, 4.8 mmol) in 15 mL of
dry pyridine at 0 °C was added 0.883 g (5.7 mmol) of POCl3
dropwise. This solution was stirred at 0 °C for 1 h, then diluted
with EtOAc and washed thoroughly with saturated aqueous
KHSO4 and brine, dried over Na2SO4, filtered, and concen-
trated. The residue was purified by column chromatography on
silica gel (EtOAc/hexane, 1:3) to give compound 6 (0.847 g,
46%). Rf=0.55 (1:1 hexane/EtOAc). 1H NMR (DMSO-d6, 400
MHz): δ=1.28 (br s, 4H), 1.46-1.55 (m, 13H), 2.24-2.30 (m,
4H), 3.57 (s, 3H), 7.00 (d, J=8.0 Hz, 1H), 7.11 (t, J=8.0 Hz, 1H),
7.28 (d, J=8.0 Hz, 1H), 7.76 (s, 1H), 9.30 (s, 1H), 9.79 (s, 1H).
13C NMR (DMSO-d6, 100 MHz): δ=24.7, 25.4, 28.5, 28.6, 28.7,
33.6, 36.7, 79.3, 109.5, 113.5, 129.0, 140.0, 140.1, 153.1, 171.5,
173.7.
7-(3-Aminophenylcarbamoyl)heptanoic Acid Methyl Ester (7).
To a solution of compound 6 (0.800 g, 2.1 mmol) in CH2Cl2 (15
mL) at 0 °C, TFA (5 mL) was added. After 1 h, the reaction
mixture was concentrated in vacuo. The crude product 7 (0.49 g,
83%) was used for the next reaction directly. The analytical
sample was purified by preparative thin-layer chromatography
(hexanes/EtOAc, 1:1). 1H NMR (DMSO-d6, 400 MHz): δ =
1.37-1.38 (m, 4H), 1.63-1.67 (m, 2H), 1.68-1.73 (m, 2H), 2.31
(t, J=7.2 Hz, 4H), 3.67 (s, 3H), 6.43 (d, J=7.2 Hz, 1H), 6.67 (d, J
=7.2 Hz, 1H), 7.07 (t, J=7.6 Hz, 1H), 7.20 (s, 1H). 13C NMR
(DMSO-d6, 100 MHz): δ = 24.6, 25.3, 28.7, 33.9, 37.6, 51.5,
106.6, 109.6, 111.0, 129.6, 171.2, 174.2.
Octanedioic Acid {3-[1-(3,5-Bis(hydroxymethyl)phenyl)-1H-
[1,2,3]-triazol-4-yl]phenyl}amide Hydroxyamide (4g). Com-
pound 4g (yield 43%) was prepared according to the methodol-
ogy described for the preparation of compound 4b. HPLC
purity: 11.4 min, 95.2% (method 2). 1H NMR (DMSO-d6, 400
MHz): δ = 1.29 (br s, 4H), 1.50 (t, J = 6.8 Hz, 2H), 1.60 (t,
J=6.4 Hz, 2H), 1.94 (t, J=7.2 Hz, 2H), 2.32 (t, J=7.6 Hz, 2H),
4.62 (d, J=5.6 Hz, 4H), 5.41 (t, J=6.0 Hz, 2H), 7.38-7.42 (m,
2H), 7.58 (dd, J=8.0 and 4.0 Hz, 2H), 7.79 (s, 2H), 8.29 (s, 1H),
8.65 (d, J=4.0 Hz, 1H), 9.27 (s, 1H), 10.01 (s, 1H), 10.33 (s, 1H).
13C NMR (DMSO-d6, 100 MHz): δ=25.4, 28.8, 32.6, 36.8, 62.8,
116.3, 116.4, 119.3, 119.9, 120.7, 124.6, 125.8, 129.7, 131.1,
136.9, 140.3, 145.0, 147.6, 169.5, 171.8. ESI-HRMS calculated
for [C24H29N5O5 - H]-: 466.2096. Found: 466.2095.
Octanedioic Acid {3-[1-(3-Fluoro-4-methoxyphenyl)-1H-
[1,2,3]-triazol-4-yl]phenyl}amide Hydroxyamide (4h). Com-
pound 4h (yield 41%) was prepared according to the methodol-
ogy described for the preparation of compound 4b. HPLC
1
purity: 14.1 min, 96% (method 2). H NMR (DMSO-d6, 400
MHz): δ=1.29 (br s, 4H), 1.50 (t, J=8.0 Hz, 2H), 1.60 (t, J=8.0
Hz, 2H), 1.94 (t, J=8.0 Hz, 2H), 2.33 (t, J=8.0 Hz, 2H), 3.93 (s,
3H), 7.38-7.44 (m, 2H), 7.53 (d, J=8.0 Hz, 1H), 7.58 (d, J=8.0
Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.92 (d, J=12.0 Hz, 1H), 8.26
(s, 1H), 8.65 (s, 1H), 9.20 (s, 1H), 10.02 (s, 1H), 10.33 (s, 1H). 13C
NMR (DMSO-d6, 100 MHz): δ=25.0, 25.1, 28.4, 32.3, 36.4,
56.4, 108.7, 108.9, 114.6, 115.9, 116.4, 118.9, 119.7, 120.2, 129.4,
129.6, 130.6, 140.0, 147.3, 150.1, 152.5, 169.1, 171.4. ESI-
HRMS calculated for [C23H26FN5O4 þ H]þ: 456.2042. Found:
456.2034.
Octanedioic Acid {3-[1-(4-tert-Butylphenyl)-1H-[1,2,3]triazol-
4-yl]phenyl}amide Hydroxyamide (4i). Compound 4i (yield
33%) was prepared according to the methodology described
for the preparation of compound 4b. HPLC purity: 16.3 min,
95.5% (method 2). 1H NMR (DMSO-d6, 400 MHz): δ=1.30 (br
s, 4H), 1.33 (s, 9H), 1.50 (t, J=8.0 Hz, 2H), 1.60 (t, J=8.0 Hz,
2H), 1.95 (t, J=8.0 Hz, 2H), 2.33 (t, J=8.0 Hz, 2H), 7.40 (t, J=
8.0 Hz, 1H), 7.56 (d, J=8.0 Hz, 1H), 7.57-7.64 (m, 3H), 7.88
7-(3-Azidophenylcarbamoyl)heptanoic Acid Methyl Ester (8).
To a solution of 7 (0.450 g, 1.6 mmol) in a 1:3 mixture of acetic
acid and water (15 mL) was added NaNO2 (0.446 g, 6.4 mmol) at
0 °C, and the mixture was stirred for 30 min. To this was added
NaN3 (0.420 g, 6.4 mmol) at the same temperature, and stirring
was continued for 1 h. To this was added saturated aqueous
NaHCO3 solution followed by NaHCO3 powder until the
mixture reached about pH 7. The mixture was extracted with
EtOAc, and the combined organic extracts were washed with

1354 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
He et al.
brine, dried over Na2SO4, filtered, and concentrated. The
residue was purified by column chromatography on silica gel
(EtOAc/hexane, 1:4) to give compound 8 (0.28 g, 56%). Rf =
mmol) in a mixture of MeOH (10 mL) and water (10 mL) was
added LiOH H2O (0.412 g, 9.84 mmol), and the mixture was
3
stirred at room temperature for 1 h. The reaction mixture was
acidfied with 1 N HCl dropwise to pH 5 and extracted with
EtOAc. The organic layer was washed with water and brine,
dried over Na2SO4, and then filtered. The solvent was evapo-
rated to give compound 13 (0.172 g, 89%). 1H NMR (DMSO-
d6, 400 MHz): δ=1.32 (br s, 4H), 1.51 (t, J=8.0 Hz, 2H), 1.61 (t,
J=8.0 Hz, 2H), 2.20 (d, J=8.0 Hz, 2H), 2.31 (t, J=8.0 Hz, 2H),
7.40 (t, J=8.0 Hz, 1H), 7.52-7.65 (m, 5H), 7.98 (d, J=8.0 Hz,
1
0.69 (1:1 hexane/EtOAc). H NMR (CD3OD, 400 MHz): δ=
1.38-1.40 (m, 4H), 1.65 (t, J=6.8 Hz, 2H), 1.70 (t, J=6.4 Hz,
2H), 2.32-2.39 (m, 4H), 3.65 (s, 3H), 6.79 (d, J=8.0 Hz, 1H),
7.25-7.33 (m, 2H), 7.50 (d, J=1.6 Hz, 1H), 9.88 (br s, 1H). 13C
NMR (CD3OD, 100 MHz): δ=24.1, 25.1, 28.4, 33.2, 36.4, 50.5,
109.9, 113.9, 115.9, 125.8, 129.7, 140.1, 140.5, 173.3, 174.5.
7-[3-(4-Phenyl[1,2,3]triazol-1-yl)phenylcarbamoyl]heptanoic
Acid Methyl Ester (10a). In a mixture of 8 (0.12 g, 0.39 mmol)
and phenylacetylene (0.06 g, 0.59 mmol) in water and ethyl
alcohol (v/v=1:1, 10 mL), sodium ascorbate (31 mg, 0.15 mmol,
dissolved in 1 mL of water) was added, followed by the addition
of copper33 sulfate pentahydrate (19 mg, 0.08 mmol, dissolved in
1 mL of water). The heterogeneous mixture was stirred vigor-
ously overnight at room temperature. The reaction mixture was
diluted with EtOAc and washed thoroughly with brine, dried
over Na2SO4, filtered, and concentrated. The residue was
purified by column chromatography on silica gel (hexane/
EtOAc, 1:1) to afford 0.078 g (48%) of 10a. Rf = 0.31 (1:1
hexane/EtOAc). 1H NMR (400 MHz, CD3OD): δ=1.31 (br s,
4H), 1.53 (t, J=8.0 Hz, 2H), 1.60 (t, J=8.0 Hz, 2H), 2.30 (t, J=
8.0 Hz, 2H), 2.35 (t, J=8.0 Hz, 2H), 3.57 (s, 3H), 7.39 (t, J=8.0
Hz, 1H), 7.50 (t, J=8.0 Hz, 2H), 7.54-7.56 (m, 2H), 7.64 (d, J=
8.0 Hz, 1H), 7.97 (d, J=8.0 Hz, 2H), 8.36 (s, 1H), 9.26 (s, 1H),
10.22 (s, 1H). 13C NMR (DMSO-d6, 90 MHz): δ=172.5, 141.4,
137.6, 131.0, 129.8, 129.0, 126.1, 120.4, 119.7, 115.1, 111.2, 52.0,
37.2, 34.0, 29.1, 29.0, 25.6, 25.1.
7-[3-(4-Cyclohexyl[1,2,3]triazol-1-yl)phenylcarbamoyl]hepta-
noic Acid Methyl Ester (10b). Compound 10b (yield 40%) was
prepared from 8 and cyclohexylacetylene according to the
methodology described for the preparation of compound 10a.
Rf=0.41 (1:1 hexane/EtOAc). 1H NMR (400 MHz, CD3OD): δ
=1.40-1.59 (m, 8H), 1.61-1.69 (m, 2H), 1.71-1.80 (m, 3H),
1.86-1.89 (m, 2H), 2.10 (d, J=12.0 Hz, 2H), 2.34 (t, J=8.0 Hz,
2H), 2.42 (t, J=8.0 Hz, 2H), 2.80-2.90 (m, 1H), 3.65 (s, 3H),
7.50-7.60 (m, 3H), 8.22 (s, 1H), 8,26 (s, 1H), 10.07 (br s, 1H).
13C NMR (100 MHz, CD3OD): δ=24.7, 25.2, 26.0, 26.1, 28.6,
28.7, 32.8, 33.6, 35.0, 36.7, 51.6, 110.8, 114.6, 118.9, 119.3, 173.7,
130.5, 137.5, 140.9, 153.7, 172.0.
2H), 8.26 (s, 1H), 9.26 (s, 1H), 10.01 (s, 1H), 11.92 (br s, 1H). 13
C
NMR (DMSO-d6, 100 MHz): δ=24.8, 25.4, 28.7, 28.8, 34.0,
36.8, 116.3, 119.3, 120.4, 120.7, 129.7, 130.3, 131.0, 137.0, 140.3,
171.7, 174.9. ESI-HRMS calculated for [C22H24N4O3 - H]-:
391.1776. Found: 391.1774.
Octanedioic Acid (2-Aminophenyl)amide [3-(1-phenyl-1H-
[1,2,3]triazol-4-yl)phenyl]amide (14). To a stirred solution of
compound 13 (0.1 g, 0.25 mmol) and 1,2-phenyldiamine
(0.275 g, 2.54 mmol) in dry DMF (10 mL) at room temperature,
HOAt (0.113 g, 0.76 mmol), triethylamine (0.35 mL, 2.54
mmol), and DMAP (0.031 g, 0.25 mmol) were added sequen-
tially. Stirring was continued overnight. The reaction mixture
was diluted with ethyl acetate, washed with water, saturated
NaHCO3 solution, saturated NH4Cl solution, and brine, dried
over Na2SO4, filtered, and concentrated. The crude material was
purified by preparative HPLC to give compound 14 (0.042 g,
1
34%). H NMR (DMSO-d6, 400 MHz): δ = 1.36 (br s, 4H),
1.61-1.63 (m, 4H), 2.29-2.36 (m, 4H), 4.81 (br s, 1H), 6.53 (t,
J=8.0 Hz, 1H), 6.71 (d, J=8.0 Hz, 1H), 6.88 (t, J=8.0 Hz, 1H),
7.14 (d, J=4.0 Hz, 1H), 7.41 (t, J=8.0 Hz, 1H), 7.50-7.65 (m,
5H), 7.98 (d, J=8.0 Hz, 2H), 8.27 (s, 1H), 9.09 (s, 1H), 9.26 (s,
1H), 10.03 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ=25.5,
25.6, 28.9, 36.1, 36.8, 116.3, 116.6, 119.3, 120.4, 120.7, 124.0,
125.7, 125.8, 126.1, 129.1, 129.7, 130.3, 131.0, 137.0, 140.3,
142.3, 147.7, 171.5, 171.8. ESI-HRMS calculated for [C28H30-
N6O2 þ H]þ: 483.2503. Found: 483.2502.
Biological Methods. HDACs Inhibition Assay. Purified
HDACs 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. Reac-
tions 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 four-parameter
logistic model (sigmoidal dose-response model): A þ [(B - A)/
1 þ (C/x)D], where x is compound concentration, A is the esti-
mated minimum, 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 IC50 were automatically
calculated using the IDBS XLFit, version 4.2.1, plug-in for
Microsoft Excel and the formula xf4_FitResultStdError().
MTS Cell Proliferation Assay. All the pancreatic cancer cell
lines were obtained from ATCC (Rockville, MD) and were
maintained in a humidified environment at 37 °C with 5% CO2.
BxPc-3 and HupT3 cells were grown in RPMI 1640 (Mediatech,
Hercules, CA) containing 10% fetal calf serum (FBS).
Panc04.03 was grown in RPMI 1640 medium with 15% FBS.
MiaPaca-2 was grown in DMEM (Mediatech, Hercules, CA)
with 10% FBS. For cytotoxicity assay, cultured cells were
detached with trypsin, washed, and counted. An aliquot
((3-5) ꢀ 103 cells) was placed in triplicate into 6 wells of a
96-well microtiter plate in a total volume of 150 μL. Four hours
after plating, 50 μL of the culture medium containing either
Octanedioic Acid Hydroxyamide [3-(4-Phenyl[1,2,3]triazol-1-
yl)phenyl]amide (11a). Compound 11a (yield 35%) was pre-
pared according to the methodology described for the prepara-
tion of compound 4a. HPLC purity: 13.7 min, 99% (method 2).
1H NMR (DMSO-d6, 400 MHz): δ=1.29 (br s, 4H), 1.50 (t, J=
8.0 Hz, 2H), 1.61 (t, J=8.0 Hz, 2H), 1.94 (t, J=8.0 Hz, 2H), 2.35
(t, J=8.0 Hz, 2H), 7.39 (t, J=4.0 Hz, 1H), 7.48-7.54 (m, 3H),
7.56 (s, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.97 (d, J=8.0 Hz, 2H),
8.36 (s, 1H), 8.65 (s, 1H), 9.26 (s, 1H), 10.22 (s, 1H), 10.33 (s,
1H). 13C NMR (DMSO-d6, 100 MHz): δ = 25.3, 25.4, 28.8,
32.6, 36.8, 110.9, 114.8, 119.3, 120.0, 125.8, 128.6, 129.4, 130.6,
137.3, 147.0, 147.7, 169.5, 172.1. ESI-HRMS calculated for
[C22H25N5O3 þ H]þ: 408.2030. Found: 408.2030.
Octanedioic Acid [3-(4-Cyclohexyl-[1,2,3]triazol-1-yl)phenyl]-
amide Hydroxyamide (11b). Compound 11b (yield 30%) was
prepared according to the methodology described for the pre-
paration of compound 4a. HPLC purity: 9.1 min, 96.1%
1
(method 1). H NMR (DMSO-d6, 400 MHz): δ= 1.29 (br s,
4H), 1.40-1.49 (m, 5H), 1.59 (t, J=8.0 Hz, 2H), 1.68 (d, J=12.0
Hz, 1H), 1.76 (d, J=12.0 Hz, 2H), 1.94 (t, J=8.0 Hz, 2H), 2.01
(d, J=12.0 Hz, 2H), 2.33 (t, J=8.0 Hz, 2H), 2.74 (t, J=8.0 Hz,
1H), 7.47 (d, J=4.0 Hz, 2H), 7.58 (d, J=4.0 Hz, 1H), 8.25 (s,
1H), 8.47 (s, 1H), 10.16 (s, 1H), 10.32 (s, 1H). 13C NMR
(DMSO-d6, 100 MHz): δ = 25.3, 25.4, 26.0, 28.8, 32.6, 32.8,
35.0, 36.8, 110.8, 114.6, 118.9, 119.3, 130.5, 137.5, 140.9, 153.7,
169.5, 172.1. ESI-HRMS calculated for [C22H31N5O3 - H]-:
412.2354. Found: 412.2357.
7-[3-(1-Phenyl-1H-[1,2,3]triazol-4-yl)phenylcarbamoyl]hepta-
noic Acid (13). To a solution of compound 12 (0.200 g, 0.49

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1355
diluent (DMSO) or varying concentrations of the indicated
HDACIs from a concentration of 1 nM to 50 μM was added
to each well. Cytotoxicity was measured at time 0 and 72 h after
treatment using the CellTiter 96 Aqueous Non-Radioactive Cell
Proliferation Assay kit (Promega, Madison, WI) according to
the manufacturer’s instructions. The absorbance of the product
formazan, which is considered to be directly proportional to the
number of living cells in the culture, was measured at 490 nm
using a SpectraMax M2 microplate reader (Molecular Devices,
Sunnyvale, CA). The IC50 values were calculated using XLfit
(IDBS Ltd., Guildford, U.K.).
Xenograft Tumor Model. Female athymic nude mice (8-10
weeks old) were inoculated subcutaneously with 3 ꢀ 106 Su86.86
(left flank) and Panc04.03 (right flank) pancreatic cancer cells
mixed with Matrigel (BD Biosciences). Two weeks after injec-
tion, tumors were size-matched and mice were randomized
into three treatment groups: (a) control DMSO, four mice;
(b) compound 4a (10 mg/kg), four mice; (c) compound 4a
(100 mg/kg), four mice. Established SU86.86 and Panc04.03
xenografts (tumor volume 150-200 mm3) were treated by ip
injections with diluent (50 μL DMSO) or compound 4a (10 and
100 mg/kg every 12 h for 48 h). After cessation of therapy,
tumors were dissected from sacrificed animals. Tumor proteins
were extracted from fresh tumor tissues taken from mouse,
separated by SDS-PAGE (50 μg/well), transferred to poly-
vinylidene difluoride membrane, and probed with antibodies.
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Acknowledgment. This work was supported in part by gift
funds from an anonymous donor, by Grant No. 271210 from
the ADDF/Elan, and by the Mayo Foundation and the
Pancreatic Cancer SPORE P50 CA102701.
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