Dual inhibitors of inosine monophosphate dehydrogenase and histone deacetylase based on a cinnamic hydroxamic acid core structure

Article abstract of DOI:10.1016/j.bmc.2010.06.081

Small molecules that act on multiple biological targets have been proposed to combat the drug resistance commonly observed for cancer chemotherapy. By combining the structural features of known inhibitors of inosine monophosphate dehydrogense (IMPDH) and

Full text of DOI:10.1016/j.bmc.2010.06.081

Bioorganic & Medicinal Chemistry 18 (2010) 5950–5964
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dual inhibitors of inosine monophosphate dehydrogenase and histone
deacetylase based on a cinnamic hydroxamic acid core structure
a,d,
Liqiang Chen a, , Riccardo Petrelli
, Guangyao Gao a, Daniel J. Wilson a, Garrett T. McLean a,
*
Hiremagalur N. Jayaram b, Yuk Y. Sham a,c, Krzysztof W. Pankiewicz a
a Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware Street S.E., Minneapolis, MN 55455, USA
b Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and Richard Roudebush Veterans Affairs Medical Center,
1481 West Tenth Street, Indianapolis, IN 46202, USA
c Biomedical Informatics and Computational Biology Program, University of Minnesota, Minneapolis, MN 55455, USA
d School of Pharmacy, Medicinal Chemistry Unit, Università di Camerino, Via S. Agostino 1, 62032 Camerino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Small molecules that act on multiple biological targets have been proposed to combat the drug resistance
commonly observed for cancer chemotherapy. By combining the structural features of known inhibitors
of inosine monophosphate dehydrogense (IMPDH) and histone deacetylase (HDAC), dual inhibitors of
IMPDH and HDAC based on the scaffold of cinnamic hydroxamic acid (CHA) have been designed, synthe-
sized, and evaluated in biological assays. Key features, including the linker length, linker functionality,
substitution position, and interacting groups, have been explored. Their individual contribution to the
inhibitory activities against human IMPDH1 and IMPDH2 as well as HDAC has been assessed.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 6 May 2010
Revised 19 June 2010
Accepted 23 June 2010
Available online 1 July 2010
Keywords:
Inosine monophosphate dehydrogenase
Histone deacetylase
Dual inhibitor
Cinnamic hydroxamic acid
Cancer therapy
Drug resistance
1. Introduction
line therapy for chronic myelogenous leukemia (CML). The hall-
mark of CML is Philadelphia (Ph) chromosome derived from a chro-
Cancer cells have been characterized as those that can sustain
their growth, escape growth inhibition and evade apoptosis.1 This
lack of control has been attributed to genetic mutations and func-
tional alterations of proteins that are involved in signal transduc-
tions and cellular regulations. Extensive biological research has
delineated diverse signaling pathways that regulate the processes
of cell proliferation, differentiation, and apoptosis. Such informa-
tion has inspired and facilitated the design of a wide range of small
molecules and macromolecular biologics that target the precise
mechanisms causing and driving the pathological process of a par-
ticular type of cancer. A number of targeted anticancer therapeutics
have entered clinical trials and many have been approved for clin-
ical use, greatly improving the treatment of various cancers. How-
ever, resistance to targeted therapeutics usually occurs due to
gene amplification, amino acid point mutation, function redun-
dancy and pathway cross-talking, severely reducing the efficacy
of targeted therapy.2
mosomal translocation that fuses the genes of Bcr and Abl. The
resultant Bcr-Abl tyrosine kinase is constitutively active and is ex-
pressed in 95% of CML, representing an extremely attractive target
for CML therapy.3 Imatinib inhibits this aberrant Bcr-Abl chimeric
protein and interrupts the subsequent signaling cascades that
eventually lead to deregulated proliferation. Imatinib has revolu-
tionized the treatment of CML and established itself as a model
for future discovery and development of targeted anticancer ther-
apeutics. However, resistance to imatinib frequently stems from
amplification of Bcr-Abl gene. More significantly, a growing num-
ber of point mutations have emerged, either interrupting imati-
nib’s binding in the ATP binding pocket or preventing Bcr-Abl
from adopting the inactive conformation to which imatinib binds.4
The second generation of Bcr-Abl inhibitors such as nilotinib and
dasatinib are able to overcome a majority of Bcr-Abl mutations.
However, T315I, a critical mutation, still remains elusive.4
Consequently, there is an urgent need for new approaches to
combat the inevitable drug resistance.5 One strategy is to combine
a targeted therapy with conventional anticancer agents or another
targeted therapy. Another strategy is to design and develop a drug
that simultaneously inhibits a broad spectrum of biological targets.
For instance, multi-kinase inhibitors have been actively pursued
for the treatment of cancers.6 However, a broad spectrum inhibitor
The success of and challenges faced by targeted anticancer ther-
apeutics can be exemplified by imatinib mesylate, currently a first
* Corresponding author. Tel.: +1 612 624 2575; fax: +1 612 624 8154.
E-mail address: chenx462@umn.edu (L. Chen).
Current address.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.081

L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
5951
usually acts on protein targets in the same family or in closely re-
lated families. As a result, cross resistance is a potential drawback.
We report herein our design, synthesis and biological evalua-
tion of a new class of anticancer agents that inhibit both inosine
monophosphate dehydrogenase (IMPDH) and histone deacetylase
(HDAC). Blockage of both IMPDH and HDAC, two well-established
anticancer targets with significantly different mechanisms, could
reduce the probability of drug induced resistance and cross
resistance.
HDAC catalyzes the deactylation of the acetyl lysine residue on
histone tails. There are 18 members of human HDAC which are cat-
egorized into four classes. Class I, II, and IV HDACs require zinc me-
tal while Class III HDACs (SIRTs) are NAD-dependent. Together
with histone acetyltransferase (HAT), HDAC controls the histone
acetylation level and subsequent gene expression through chroma-
tin modification. It has been suggested that HDAC inhibitors allow
the expression of certain genes that are suppressed in cancer cells.7
In addition, a wide range of non-histone proteins have been discov-
ered as HDAC substrates, many of which have been suggested to be
important targets for cancer therapy.8,9 Suberoylanilide hydroxa-
mic acid (SAHA, vorinostat) (1, Fig. 1) and romidepsin (7, Fig. 1)
have been approved by the Food and Drug Administration (FDA),
validating HDAC inhibitors as anticancer therapeutics.
ing their activity against a second target. We anticipated that key
features present in individual IMPDH and HDAC inhibitors could
be combined or merged into one single molecule without compro-
mising the activity against either target. Our design was prompted
by an examination of known HDAC and IMPDH inhibitors, which
indicated that both groups of inhibitors exhibited considerable
chemical flexibility.
Known zinc-dependent HDAC inhibitors can be divided into
several classes, such as short-chain fatty acid, hydroxamic acid,
benzamide, and cyclic tetrapeptide (Fig. 1).13 A general pharmaco-
phore, which consists of a metal binding group, linker and cap re-
gion, can be proposed for the HDAC inhibitors. For the metal
binding group, a hydroxamic acid usually exhibits higher potency
than the corresponding benzamide. The linker region is fairly flex-
ible and allows for extensive structural modifications. A five to six
carbon member linear side chain, such as those in SAHA (1, Fig. 1),
fits very well in the active site of HDAC enzymes while aromatic
rings such as cinnamic acid derivatives (2–4, Fig. 1) are well
accommodated. The cap region tolerates a wide range of aromatic
and heterocyclic aromatic group and can be exploited for possible
selectivity among HDAC members.
IMPDH inhibitors can be divided into two categories based on
the active sites to which they bind: IMP site and NAD site.12 Among
those targeting the NAD binding site, tiazofurin (8, Fig. 2) needs an
initial conversion to mononucleotide and subsequent activation
through the formation of an NAD analogue. Mycophenolic acid
(9, MPA, Fig. 2) does not require activation and fits in the nicotin-
amide subsite of the NAD binding site. Importantly, structure-
based drug design based on the MPA binding mode has yielded ser-
ies of novel IMPDH inhibitors, as exemplified by VX-497 (10,
IMPDH, a nicotinamide adenine dinucleotide (NAD)-dependent
enzyme,10 catalyzes a rate-limiting step in the de novo synthesis of
guanine nucleotides, which are crucial for cell growth and prolifer-
ation. There are two forms of human IMPDH, type 1 and type 2. The
type 2 isoform (hIMPDH2) is selectively up-regulated in proliferat-
ing cells while the type 1 isoform (hIMPDH1) has been shown to
play a key role in angiogenesis, establishing IMPDH as an attractive
target for anticancer drug discovery.11,12
Fig. 2), based on
a N-[3-methoxy-4-(5-oxazolyl)phenyl]amino
(MOA) moiety which was believed to bind in a fashion analogous
to the substituted benzofuranone present in MPA.14 In addition
to those containing MOA, IMPDH inhibitors based on a N-(4-cya-
no-3-methoxyphenyl)amino (CMA) group have also been discov-
ered and developed, with VX-148 (12, Fig. 2) as a prominent
example.15 Further explorations of structural modifications have
identified additional warhead groups such as oxazolylindole,16
cyanoindole,17 4-pyridylindole, isoquinoline,18 and acridone.19
2. Results and discussion
2.1. Design
Our design of dual inhibitors was based on a premise that inhib-
itors targeting one enzyme can be structurally modified without
compromising their initial activity while simultaneously enhanc-
O
OH
R2
N
O
N
H
H
N
OH
N
H
O
R1
N
H
1, SAHA, vorinostat
2
, R1 = H, R2 = hydroxyethyl, LAQ824
3, R1 = Me, R2 = H, LBH589, panobinostat
O
O
O
O
O
N
H
NH2
S
OH
H
N
N
H
N
H
N
O
4, PXD-101, belinostat
5, MS-275/SNDX-275, entinostat
O
NH
HN
N
O
S
HN
O
S
N
N
H
NH2
H
N
NH
N
O
O
O
O
6, MGCD-0103, mocetinostat
7, FK-228, romidepsin
Figure 1. Examples of HDAC inhibitors.

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L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
O
NH2
S
N
OH
O
OH
HO
O
O
O
OMe
OH OH
8, tiazofurin (TR)
9, mycophenolic aicd (MPA)
O
H
N
O
N
O
O
O
N
N
N
O
O
O
N
O
MeO
N
H
N
H
MeO
N
H
10, VX-497, merimepodib
11, BMS-337197
H
O
N
O
N
CN
O
H
NC
O
O
Me
N
N
MeO
OH
H
O
MeO
N
H
N
H
12, VX-148
13
Figure 2. Examples of IMPDH inhibitors.
In addition to the warhead moiety, the linker region can also
accommodate various structural modifications. VX-497, for exam-
ple, contains a urea linker. Extensive structure–activity relation-
ship (SAR) studies have revealed that the urea linker can be
replaced with amide,20 diamide21 and guanidine linkers.22 The
warhead can also be linked through heterocycles, such as tri-
azine,23 oxazole,24,25 quinolone,26 indole,27 quinazolinedione,28
and quinazolinethione.28
ate the individual contribution by key structural components to
the inhibitory activity of either IMPDH or HDAC. Four variations,
namely the length of linker (aminomethyl vs amino), the position
of substitution (para vs meta), the functionality of linker (urea vs
diamide) and the nature of IMPDH-interacting groups (MOA vs
CMA), are to be explored.
2.2. Chemistry
Given the structural flexibility in HDAC and IMPDH inhibitors,
the metal binding group such as hydroxamic acid in HDAC inhibi-
tors and the warhead in IMPDH inhibitors can be connected via a
linker that is tolerated for the inhibition of both IMPDH and HDAC.
Previously we have reported our design of dual inhibitors based on
either MPA or SAHA.29 Replacement of the carboxylic acid in MPA
with a hydroxamic acid led to MAHA (14, Fig. 3). On the other
hand, substitution of the phenyl group in SAHA with 5-oxazole
and methoxy groups yielded SMAHA (15, Fig. 3). Very recently, a
similar approach has been used to generate inhibitors simulta-
neously targeting HDAC and tyrosine kinases.30,31
In our current study we devised dual inhibitors based on a cin-
namic hydroxamic acid (CHA) core structure, which is present in
many HDAC inhibitors (Fig. 1). The target compounds shown in
Figure 4 were proposed based on a general pharmacophore model,
in which a hydroxamic acid interacts with the zinc atom in the ac-
tive site of HDAC. The cinnamic hydroxamic acid is connected via a
linker to MOA or CMA, a warhead which is expected to bind into
the NAD binding site of IMPDH. Two series of compounds (amino-
methyl CHA and amino CHA series) have been conceived to evalu-
Synthesis of the aminomethyl CHA series of dual inhibitors re-
quired key aminomethyl cinnamic esters 23a and 23b, whose
preparations are shown in Scheme 1. para-Substituted bromide
20a underwent a Heck reaction to give para-substituted aldehyde
21a,32,33 which was reduced to afford alcohol 22a. Conversion of
alcohol 22a to amine 23a was accomplished by initial mesylation,
replacement of the resultant mesylate with an azido group, and fi-
nal reduction of azide to amine. Amine 23a was isolated as a
hydrochloride form. The meta-substituted amine 23b was prepared
from meta-substituted bromide 20b in an identical sequence of
reactions as depicted in Scheme 1.
para-Substituted amine 23a and 3-methoxy-4-(5-oxazol-
yl)phenylamine (24)20 were combined to give urea 26a by the
action of triphosgene (Scheme 2). Removal of the tert-butyl group
was accomplished under acidic conditions to give carboxylic
acid 27a, which was in turn converted into a trityl-protected
hydroxamic acid 28a in the presence of 1-ethyl-3-(3-dimethylami-
nopropyl)carbodiimide hydrochloride (EDC) and 1-hydroxybenzo-
triazole (HOBt). Hydroxamic acid 16a was obtained after a facile
O
OH
O
H
N
N
O
OH
H
O
N
O
MeO
N
H
OH
OMe
O
14
15
Figure 3. Dual inhibitors of IMPDH and HDAC.

L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
5953
O
R
O
O
H
N
H
N
OH
H
N
OH
N
H
MeO
R
N
H
C
MeO
N
H
C
H2
H2
O
O
16a para, R = 5-oxazolyl
16b meta, R = 5-oxazolyl
16c para, R = CN
17a para, R = 5-oxazolyl
17b meta, R = 5-oxazolyl
16d meta, R = CN
O
R
O
O
H
O
OH
OH MeO
R
N
N
N
N
H
H
MeO
N
H
N
H
H
O
18a para, R = 5-oxazolyl
18b meta, R = 5-oxazolyl
18c para, R = CN
19a para, R = 5-oxazolyl
19b meta, R = 5-oxazolyl
18d meta, R = CN
Figure 4. Dual inhibitors of IMPDH and HDAC based on CHA core structure.
O
O
O
Br
OtBu
b
OtBu
OtBu
H2N
HCl
a
c
H
H
HO
C
C
H2
H2
O
O
20a para
20b meta
21a para
21b meta
22a para
22b meta
23a para
23b meta
Scheme 1. Reactions and conditions: (a) tert-butyl acrylate, Pd(OAc)2, P(O-tolyl)3, NaOAc, DMF, 130 °C, yield 69–78%; (b) NaBH4, EtOH, yield 83–86%; (c) (i) MsCl, Et3N, THF;
(ii) NaN3, DMF; (iii) Ph3P, THF/H2O; (iv) HCl, diethyl ether, yield 97–98%.
O
O
O
H
N
H
N
H
N
H
N
OtBu
MeO
R
b
OH
MeO
R
OtBu
C
a
H2N
HCl
C
C
H2
H2
O
O
H2
26a para, R = 5-oxazolyl
26b meta, R = 5-oxazolyl
26c para, R = CN
27a para, R = 5-oxazolyl
27b meta, R = 5-oxazolyl
27c para, R = CN
23a para
23b meta
26d meta, R = CN
27d meta, R = CN
O
O
H
H
N
H
N
H
OH
N
N
H
OTr
MeO
N
MeO
R
N
c
d
C
C
H
H2
H2
O
O
R
28a para, R = 5-oxazolyl
28b meta, R = 5-oxazolyl
28c para, R = CN
16a para, R = 5-oxazolyl
16b meta, R = 5-oxazolyl
16c para, R = CN
28d meta, R = CN
16d meta, R = CN
Scheme 2. Reactions and conditions: (a) aniline 24 or 25, triphosgene, Et3N, CH2Cl2, yield 26–72%; (b) TFA, CH2Cl2, yield 98–100%; (c) H2N–OTr, EDC, HOBt, DMF, yield 21–
62%; (d) TFA, Et3SiH, CH2Cl2, yield 57–97%.
removal of the trityl protecting group. meta-Substituted amine 23b
and aniline 24, after a series of chemical transformations, led to
hydroxamic acid 16b. Through similar reactions, 4-amino-2-
methoxybenzonitrile (25)34 was linked to amine 23a and 23b to
give hydroxamic acids 16c and 16d, respectively (Scheme 2).
A coupling of para-substituted amine 23a with 2-(3-methoxy-
4-(oxazol-5-yl)phenylamino)-2-oxoacetic acid (29)21 was medi-
ated by benzotriazol-1-yl-oxytripyrrolidinophosphonium hexa-
fluorophosphate (PyBOP) in the presence of N-methylmorpholine
(NMM), giving rise to diamide 30a (Scheme 3). Removal of the
tert-butyl group, formation of protected hydroxamate 32a, and
cleavage of the trityl protecting group afforded diamide-linked
hydroxamic acid 17a. When meta-substituted amine 23b and acid
29 were subjected to the same series of reactions, hydroxamic acid
17b was obtained.
As shown in Scheme 4, para-substituted aniline 33a and meta-
substituted 33b were subjected to chemical reactions similar to
those depicted in Scheme 2 except for the hydrolysis reaction that
was used to convert ethyl esters 34a–d to carboxylic acids 35a–d.
Accordingly, urea-linked amino CHA compounds 18a–d were ob-
tained. Preparation of diamide-linked amino CHA 19a and 19b is
depicted in Scheme 5 through transformations analogous to those

5954
L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
O
O
N
N
O
O
O
O
H
O
H
N
OtBu
OtBu
OH
a
b
H2N
HCl
N
C
MeO
N
H
C
MeO
N
H
C
H2
H2
H2
O
O
30a para
30b meta
31a para
31b meta
23a para
23b meta
O
O
N
N
O
O
O
O
H
N
C
H
N
OH
N
c
d
N
H
OTr
MeO
N
H
MeO
N
H
C
H
H2
H2
O
O
32a para
32b meta
17a para
17b meta
Scheme 3. Reactions and conditions: (a) compound 29, PyBOP, NMM, DMF, yield 42%; (b) TFA, CH2Cl2, yield 97–100%; (c) H2N–OTr, EDC, HOBt, DMF, yield 58–81%; (d) TFA,
Et3SiH, CH2Cl2, 93–94%.
O
R
O
R
O
O
O
a
b
OEt
OH
OEt
H2N
MeO
N
H
N
H
MeO
N
H
N
H
34a para, R = 5-oxazolyl
34b meta, R = 5-oxazolyl
34c para, R = CN
35a para, R = 5-oxazolyl
35b meta, R = 5-oxazolyl
35c para, R = CN
33a para
33b meta
34d meta, R = CN
35d meta, R = CN
R
MeO
O
R
O
O
O
OH
N
d
c
N
H
OTr
N
H
N
MeO
N
H
N
H
H
H
36a para, R = 5-oxazolyl
36b meta, R = 5-oxazolyl
36c para, R = CN
18a para, R = 5-oxazolyl
18b meta, R = 5-oxazolyl
18c para, R = CN
36d meta, R = CN
18d meta, R = CN
Scheme 4. Reactions and conditions: (a) aniline 24 or 25, triphosgene, Et3N, CH2Cl2, yield 27–97%; (b) NaOH, THF/H2O/MeOH, yield 66–98%; (c) H2N–OTr, EDC, HOBt, DMF,
yield 42–54%; (d) TFA, Et3SiH, CH2Cl2, yield 71–96%.
O
O
O
O
O
H
N
H
N
MeO
O
OEt
b
MeO
O
OH
a
OEt
N
H
N
H
H2N
O
O
N
N
O
38a para
38b meta
33a para
33b meta
37a para
37b meta
O
O
O
O
H
N
H
N
OH
N
MeO
N
H
OTr
MeO
O
c
d
N
H
N
H
H
O
N
O
N
39a para
39b meta
19a para
19b meta
Scheme 5. Reactions and conditions: (a) compound 29, PyBOP, NMM, DMF, yield 50–65%; (b) NaOH, THF/H2O/MeOH, 62–77%; (c) H2N–OTr, EDC, HOBt, DMF, yield 52–55%;
(d) TFA, Et3SiH, CH2Cl2, yield 22–25%.

L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
5955
shown in Scheme 3 except that ethyl esters 37a and 37b were
hydrolyzed to give the corresponding acids 38a and 38b.
to those with a cyano group as indicated by comparing 16a and
16b with 16c and 16d.
2.3. Biological evaluations
2.3.2. Amino CHA series
In the amino CHA series 18a–d, para-substituted ureas gener-
ally showed higher activity against both IMPDH1 and IMPDH2 than
the corresponding meta-substituted compounds even though para-
substituted urea 18a was slightly less potent than meta-substi-
tuted urea 18b. This trend is in contrast to the observation that
meta-substitution generally enhanced the inhibition of human
IMPDHs in the aminomethyl CHA series. Nevertheless, para-substi-
tuted diamide 19a was less potent against IMPDH1 and IMPDH2
than meta-substituted diamide 19b. As far as the linker is con-
cerned, para-substituted urea 18a was an IMPDH inhibitor much
more active than its diamide counterpart 19a. However, meta-
substituted urea 18b and meta-substituted diamide 19b showed
similar inhibitory activity.
Compounds containing a cyano group generally possessed
weaker potency against either IMPDH1 or IMPDH2 than their 5-
oxazole counterparts. For example, para-substituted urea 18a and
18b showed significantly higher activity against IMPDH1 than
18c and 18d. A similar enhancement of activity was also observed
for compound 18b against IMPDH2 in relation to compound
18d. However, 5-oxazole-containing compound 18a exhibited
inhibition of IMPDH2 that was nearly identical to that of com-
pound 18c.
Mirroring the trend observed for the urea-linked aminomethyl
CHA series, the urea-linked para-substituted compounds were sig-
nificantly more potent inhibitors of HDAC than the corresponding
meta-substituted counterparts, regardless of whether MOA or CMA
was incorporated. However, the trend was reversed for diamide-
linked compounds as HDAC inhibitors, in which meta-substituted
19b is nearly 10-fold more potent than para-substituted 19a. This
observation indicates that a meta-substitution seems to favor
HDAC inhibition for the diamide-linked compounds, contradicting
the observation for the urea-linked compounds.
When the linker is considered, diamide-linked compounds such
as 19a and 19b were weaker HDAC inhibitors than those with
urea-linked compounds 18a and 18b, a trend consistent with that
observed in the aminomethyl CHA series. Compounds with CMA
were as potent inhibitors as those with MOA, a phenomenon that
was also observed in the aminomethyl CHA series.
The aminomethyl CHA 16a–17b and amino CHA 18a–19b were
tested for their activity against human IMPDH1 and IMPDH2, as
well as a nuclear extract of HDAC enzymes. Compounds 16a–19b
were also evaluated for their anti-proliferative activity against
the CML cancer cell line K562. The resulting biological data is sum-
marized in Table 1. These dual inhibitors were weaker than VX-497
or VX-148 (Fig. 2),15,35 generally inhibiting IMPDHs at high nano-
molar to low micromolar levels. However, these compounds
showed high potency against HDAC and the best compounds dis-
played low nanomolar activity that is comparable to that of
LAQ824 (Fig. 1).36 In order to uncover potential SAR trends, the
aminometyl CHA and amino CHA series were examined individu-
ally. In each series, the influence of substitution patterns, linkers
and IMPDH-interacting groups was evaluated. Furthermore, a com-
parison between the aminomethyl CHA and amino CHA series was
also investigated.
2.3.1. Aminomethyl CHA series
In the aminomethyl CHA series 16a–d and 17a–b, meta-substi-
tuted inhibitors exhibited higher activity against IMPDH1 than the
corresponding para-substituted compounds. A similar trend was
observed against IMPDH2, even though diamide 17a was as potent
as 17b. Inhibitors with a urea linker appeared to possess higher
inhibitory activity against IMPDH1 than those with a diamide lin-
ker, as indicated by comparing ureas 16a and 16b with diamides
17a and 17b. As observed in the urea series 16a–d, replacement
of 5-oxazole with a cyano group greatly diminished inhibition
against both IMPDH 1 and IMPDH2.
In contrast to the trend observed for the inhibition of IMPDH1
and IMPDH2, para-substituted ureas 16a and 16c displayed drasti-
cally enhanced inhibitory ability against HDAC than the corre-
sponding meta-substituted ureas 16b and 16d. Para-substituted
diamide 17a was approximately threefold more potent than
meta-substituted diamide 17b. Urea-linked compound 16a was
more active than diamide-linked analogue 17a whereas com-
pounds 16b and 17b showed similar activity. In addition, hydroxa-
mic acids containing a 5-oxazole displayed HDAC inhibition similar
Table 1
Biological evaluations of dual inhibitors of IMPDH and HDAC
O
O
R
H
N
H
N
OH
O
H
N
N
H
OH
MeO
R
N
H
X
N
H
X
MeO
O
O
urea linker
diamide linker
IMPDH1 Kai pp
(lM)
IMPDH2 Kai pp
(lM)
Compound
X
Position
Linker
R
HDAC nuclear extract
IC50 M)
K562 cell proliferation
50 (lM)
(l
16a
16b
16c
16d
17a
17b
18a
18b
18c
18d
19a
19b
CH2
CH2
CH2
CH2
CH2
CH2
None
None
None
None
None
None
para
meta
para
meta
para
meta
para
meta
para
meta
para
meta
Urea
Urea
Urea
Urea
Diamide
Diamide
Urea
Urea
Urea
5-Oxazolyl
5-Oxazolyl
CN
7.8 0.8
2.3 0.1
>100
44 18
17 10
4.4 0.5
0.30 0.05
0.58 0.06
1.2 0.3
8.2
1.2 0.2
0.34 0.06
0.026 0.006
0.83 0.39
0.036 0.020
0.64 0.56
0.23 0.10
0.89 0.28
0.55 0.14
3.0 0.7
3.5
18
11
1
NDa
28
CN
2.6 0.3
1.4 0.6
1.5 0.1
0.25 0.12
0.12 0.01
0.23 0.10
1.2
5-Oxazolyl
5-Oxazolyl
5-Oxazolyl
5-Oxazolyl
CN
CN
5-Oxazolyl
5-Oxazolyl
5.3
>100
4.9
>100
4.0
38
0.63 0.46
3.4 0.7
17 12
Urea
Diamide
Diamide
7.1 0.6
0.46 0.03
1.8 0.3
0.20 0.02
72
28
1.8 0.5
a
ND, not determined due to low aqueous solubility.

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L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
2.3.3. Aminomethyl CHA series versus amino CHA series
When the aminomethyl CHA and amino CHA series were com-
pared, it was clear that compounds included in the amino CHA ser-
ies exhibited higher potency against human IMPDH1 and IMPDH2
with the exception of compound 19a, which displayed approxi-
mately the same potency against IMPDH2 as its aminomethyl
counterpart compound 17a. However, when the inhibition of
HDAC was under consideration, aminomethyl CHA series consis-
tently showed higher activity than the corresponding the amino
CHA series.
From our SAR studies as discussed above, it can be concluded
that in the aminomethyl series a meta-substitution pattern is gen-
erally preferred for the inhibition of IMPDHs whereas a para-sub-
stitution leads to potent inhibition of HDAC enzymes. In other
words, structural modifications which increased the inhibitory
activity towards IMPDH decreased that against HDAC. Compounds
in the aminomethyl series generally inhibited human IMPDHs at
micromolar levels whereas they possessed anti-HDAC activity at
sub-micromolar levels. Worth noting are compounds 16a and
16c, which displayed anti-HDAC IC50’s of approximately 30 nM.
In the amino CHA series, the SAR trend observed for compounds
connected via a urea linker contradicts that drawn from com-
pounds with a diamide linker. A meta-substitution is necessary
for increased potency for compounds with a diamide linker. In con-
trast, in the urea-linked compounds, a para-substitution is gener-
ally preferred for higher activity against both human IMPDH and
HDAC. These convergent SAR trends allowed for simultaneous
enhancement of activity against IMPDH and HDAC, a task which
was not feasible in the aminomethyl CHA series. Several com-
pounds in the amino CHA series, such as compound 18a and 18c,
exhibited potent and comparable activity against IMPDH and
HDAC.
2.3.4. Anti-proliferation assay in K562 cells
Compounds 16a–19b showed modest to weak inhibition of
K562 as indicated by IC50’s above micromolar levels. The IC50’s
did not appear to correlate with compounds’ ability to inhibit
either IMPDH or HDAC. The low inhibitory activity and erratic
SAR trend might be attributed to the poor aqueous solubility of
these compounds, a phenomenon that was observed in our syn-
thetic efforts.
2.4. Computational modeling
A HDAC1 homology model was constructed based on the solved
X-ray crystallographic structure of histone deacetylase-like protein
(HDLP) in complex with SAHA.37 The initial sequence alignment of
HDAC1-11 and HDLP showed HDLP has the highest sequence sim-
ilarity to HDAC1 with 59% sequence identity in the catalytic site.
Compound 16a, which is the most potent HDAC inhibitor in the
current series of dual inhibitor, and SAHA were docked into the
HDAC1 homology model (Fig. 5). Compound 16a, with a para-sub-
stitution pattern on the cinnamic ring, fits snugly into the narrow
tube-like catalytic cavity in a manner similar to SAHA, a prototype
of HDAC inhibitors. For both compounds, the hydroxamic acid,
along with residues Asp264, His178, and Asp176, coordinates with
the zinc ion at the bottom of the cavity, accounting for a significant
fraction of the total energy contributions. Additionally, for com-
Figure 5. Comparison between compound 16a (A) and SAHA (B) within HDAC1
homology model binding site. All key amino acid residues are shown. (C) Per
residue interaction energy in kcal/mol of compound 16a (A) and SAHA (B) within
the HDAC1 binding site.
stitution were consistently more potent than ones that are meta-
substituted. Since the MOA or CMA binds outside the cavity and
is engaged in similar interactions, compounds with MOA displayed
virtually identical activity against HDAC when compared to similar
compounds with CMA.
Modeling of compounds 18a and 18b into the NAD binding
pocket of the solved X-ray crystallographic structure of hIMPDH2
was based on the binding mode of VX-497 (Fig. 6) as previously re-
ported.14 Both compounds engage in an energetically favorable
stacking interaction with IMP, the enzymatic reaction substrate.
The urea functionality forms hydrogen bonds with Asp274, a key
interaction observed for VX-497 and its analogues. For compound
18a with its para-substitution on the cinnamic phenyl moiety,
the hydroxamic acid side chain is extended into the adenosine sub-
site of the NAD binding site. For compound 18b, the hydroxamic
pound 16a the phenyl ring of CHA is involved in p–p stacking with
Phe150 and Phe205. The MOA moiety binds in the cap region in a
fashion very similar to that of SAHA, engaging in a favorable inter-
action with Pro29. Judged by the binding mode of compound 16a
in HDAC1, a para-substitution is clearly preferred in order to fit
in the tube-like catalytic cavity whereas a meta-substitution pat-
tern elicits an unfavorable clash with the residues that form the
wall. Therefore, it is no surprise that compounds with a para-sub-

L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
5957
evaluate a new type of dual inhibitors of IMPDH and HDAC based
on a CHA core structure. Two series of compounds, namely the
aminomethyl CHA and amino CHA series, have been conceived.
Variations of key components in either series allowed us to as-
sess the individual contribution of these elements. In the amino-
methyl CHA series, a meta-substitution pattern is desired for
improved inhibition of IMPDHs whereas a para-substitution is
generally preferred for inhibition of IMPDH in the urea-linked
amino CHA compounds. Nevertheless, a para-substitution pattern
leads to higher activity against HDAC in both series except for
diamide-linked amino CHA compounds. While the SAR trends
for the enhancement of IMPDH and HDAC inhibition are diver-
gent in the aminomethyl CHA series, the convergent SAR ob-
served in the amino CHA series has permitted us to prepare
compounds with comparable activity against both IMPDH and
HDAC. In summary, we have successfully designed CHA-based
dual inhibitors of IMPDH and HDAC by exploiting structural flex-
ibility, a strategy which can be applied to devise new classes of
dual inhibitors. Our SAR study and molecular modeling have also
revealed a general approach for the design of balanced dual
inhibitors. All these inhibitors may prove valuable to combat
the inevitable drug resistance emerging from targeted cancer
chemotherapies.
4. Experimental
4.1. General methods
All commercial reagents (Sigma–Aldrich, Acros) were used as
provided unless otherwise indicated. An anhydrous solvent dis-
pensing system (J. C. Meyer) using two packed columns of neutral
alumina was used for drying THF, Et2O, and CH2Cl2, while two
packed columns of molecular sieves were used to dry DMF. Sol-
vents were dispensed under argon. Flash chromatography was per-
formed with Ultra Pure silica gel (Silicycle) with the indicated
solvent system. Melting points were determined on a Mel-Temp
apparatus and were uncorrected. Nuclear magnetic resonance
spectra were recorded on a Varian 600 MHz with Me4Si or signals
from residual solvent as the internal standard for 1H or 13C. Chem-
ical shifts are reported in ppm, and signals are described as s (sin-
glet), d (doublet), t (triplet), q (quartet), m (multiplet), bs (broad
singlet), and dd (double doublet). Values given for coupling con-
stants are first order. High resolution mass spectra were recorded
on an Agilent TOF II TOF/MS instrument equipped with either an
ESI or APCI interface.
Figure 6. Comparison between compound 18a (A) and 18b (B) within Human
IMPDH Type II binding site. All key amino acid residues are shown. (C) Per residue
interaction energy in kcal/mol of compound 18a and 18b within the hIMPDH2 NAD
binding site.
4.2. Chemical synthesis
4.2.1. (E)-tert-Butyl 3-(4-formylphenyl)acrylate (21a)
acid is projected into a channel that is perpendicular to the NAD
binding site, emulating the bind mode of VX-497. The comparison
between compounds 18a and 18b indicates that either substitution
pattern is tolerated and does not significantly alter a compound’s
ability to inhibit IMPDH, at least in the urea series.
Our molecular modeling and SAR study clearly suggest a gen-
eral approach for the design of compounds with balanced activities
against both IMPDH and HDAC. In these compounds a desired
para-substitution pattern would confer an extended and linear
molecular shape like that of compound 18a, a structural feature
accommodated by HDAC as well as IMPDH.
A solution of 4-bromobenzaldehyde (20a, 9.33 g, 50.4 mmol),
tert-butyl acrylate (10.9 mL, 75.1 mmol), Pd(OAc)2 (226 mg,
1.01 mmol), P(O-tolyl)3 (928 mg, 3.05 mmol), and NaOAc (8.49 g,
103 mmol) in dry DMF (100 mL) in a sealed flask was evacuated
and then back-filled with argon five times. The mixture was heated
at 130 °C for 24 h. After cooling to rt, the mixture was filtered
through a pad of Celite and washed with EtOAc. After the filtrate
was concentrated, the residue was dissolved in EtOAc (500 mL)
and washed with water (2 Â 200 mL) and brine (2 Â 200 mL). The
organic layer was dried over Na2SO4 and filtered. The filtrate was
concentrated and the residue was triturated with hot hexanes
(100 mL). After filtration, aldehyde 21a was obtained as a white so-
lid (8.08 g, 69%). 1H NMR (CDCl3, 600 MHz) d 10.02 (s, 1H), 7.89 (d,
J = 7.8 Hz, 2H), 7.66 (d, J = 7.8 Hz, 2H), 7.61 (d, J = 16.2 Hz, 1H), 6.48
(d, J = 16.2 Hz, 1H), 1.54 (s, 9H). HRMS calcd for C14H17O3 233.1178
(M+H)+, found 233.1179.
3. Conclusions
By combining structural features from known inhibitors of
IMPDH and HDAC, we have been able to design, synthesize and

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L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
4.2.2. (E)-tert-Butyl 3-(3-formylphenyl)acrylate (21b)
2H), 1.53 (s, 9H). HRMS calcd for C14H20NO2 234.1488 (M+H)+,
found 234.1506.
Following procedures similar to those described for aldehyde
21a, 3-bromobenzaldehyde (20b, 5.90 mL, 50.4 mmol) was con-
verted into aldehyde 21b as yellowish syrup (9.17 g, 78%). 1H
NMR (CDCl3, 600 MHz) d 10.01 (s, 1H), 7.98 (s, 1H), 7.84 (d,
J = 7.8 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 16.2 Hz, 1H),
7.53 (d, J = 7.5 Hz, 1H), 6.44 (d, J = 16.2 Hz, 1H), 1.51 (s, 9H). HRMS
calcd for C14H17O3 233.1178 (M+H)+, found 233.1185.
4.2.7. tert-Butyl ester 26a
To a solution of 3-methoxy-4-(oxazol-5-yl)aniline20 (456 mg,
2.40 mmol) in dry CH2Cl2 (25 mL) were added triphosgene
(242 mg, 0.82 mmol) and Et3N (0.34 mL, 2.44 mmol). The resulting
mixture was heated at reflux for 1 h. After cooling to rt, additional
Et3N (1.0 mL, 7.2 mmol) was added, followed by an addition of
(E)-tert-butyl 3-(4-(aminomethyl)phenyl)acrylate hydrochloride
(777 mg, 2.88 mmol). The mixture was allowed to stir at rt for
24 h and then concentrated. The residue was dissolved in MeOH
(15 mL) and slowly added to ice/water (150 mL plus 20 mL of 1 N
HCl). The solid precipitate was washed with water, collected and
purified by silica gel column chromatography to give tert-butyl ester
26a as a light-yellow solid (414 mg, 38%). 1H NMR (CD3OD,
600 MHz) d 8.14 (s, 1H), 7.62 (d, J = 9.0 Hz, 1H), 7.58–7.52 (m, 3H),
7.44 (d, J = 1.8 Hz, 1H), 7.40–7.35 (m, 3H), 6.92 (dd, J = 8.2, 1.8 Hz,
1H), 6.40 (d, J = 15.6 Hz, 1H), 4.42 (s, 2H), 3.92 (s, 3H), 1.52 (s, 9H).
HRMS calcd for C25H27N3O5Na 472.1842 (M+Na)+, found 472.1810.
4.2.3. (E)-tert-Butyl 3-(4-(hydroxymethyl)phenyl)acrylate (22a)
To a solution of aldehyde 21a (3.66 g, 15.8 mmol) in EtOH
(100 mL) was added NaBH4 (2.38 g, 62.9 mmol) in portions within
30 min. The resulting mixture was allowed to stir at rt for 1 h and
concentrated. The residue was dissolved in water (100 mL) and
acidified to pH ꢀ 5. The solid formed was filtered, washed with
water and then dried to give alcohol 22a as a white solid (3.05 g,
83%). 1H NMR (CDCl3, 600 MHz) d 7.54 (d, J = 16.2 Hz, 1H), 7.47
(d, J = 6.0 Hz, 2H), 7.34 (d, J = 6.0 Hz, 2H), 6.33 (d, J = 15.6 Hz, 1H),
4.68 (s, 2H), 1.81 (br s, 1H), 1.54 (s, 9H). HRMS calcd for
C
14H19O3 235.1334 (M+H)+, found 235.1329.
4.2.4. (E)-tert-Butyl 3-(3-(hydroxymethyl)phenyl)acrylate (22b)
Following procedures similar to those described for alcohol 22a,
aldehyde 21b (4.83 g, 20.8 mmol) was reduced with NaBH4 (3.14 g,
83.0 mmol) to give alcohol 22b as a yellowish syrup (4.20 g, 86%).
1H NMR (CDCl3, 600 MHz) d 7.54 (d, J = 15.6 Hz, 1H), 7.48 (s, 1H),
7.39 (t, J = 4.2 Hz, 1H), 7.34–7.30 (m, 2H), 6.34 (d, J = 16.2 Hz,
1H), 4.68 (s, 2H), 1.50 (s, 9H). HRMS calcd for C14H19O3 235.1334
(M+H)+, found 235.1337.
4.2.8. tert-Butyl ester 26b
Following procedures similar to those described for tert-butyl
ester 26a, (E)-tert-butyl 3-(3-(aminomethyl)phenyl)acrylate
hydrochloride (776 mg, 2.88 mmol) was converted into tert-butyl
ester 26b as a light-yellow solid (284 mg, 26%). 1H NMR (CD3OD,
600 MHz)
d 8.14 (s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.57 (d,
J = 16.2 Hz, 1H), 7.55 (s, 1H), 7.49–7.45 (m, 1H), 7.44 (d,
J = 1.8 Hz, 1H), 7.40–7.36 (m, 3H), 6.92 (dd, J = 8.1, 2.1 Hz, 1H),
6.44 (d, J = 15.6 Hz, 1H), 4.42 (s, 2H), 3.94 (s, 3H), 1.52 (s, 9H).
HRMS calcd for C25H27N3O5Na 472.1842 (M+Na)+, found 472.1802.
4.2.5. (E)-tert-Butyl 3-(4-(aminomethyl)phenyl)acrylate
hydrochloride (23a)
To a solution of alcohol 22a (2.98 g, 12.7 mmol) and Et3N
(3.60 mL, 25.8 mmol) in anhydrous THF (80 mL) at 0 °C was added
dropwise MsCl (1.50 mL, 19.3 mmol). The mixture was allowed to
stir at 0 °C for 1 h, warm to rt and stir at rt for 1 h. After the reaction
mixture was diluted with EtOAc (400 mL), the resulting solution
was washed with water (100 mL), satd NaHCO3 (100 mL), water
(100 mL) and brine (200 mL). The organic layer was dried over
Na2SO4 and filtered. The filtrate was concentrated and re-dissolved
in anhydrous DMF (60 mL). After NaN3 (1.84 g, 28.3 mmol) was
added at rt, the resulting mixture was heated at 50 °C for 30 min
and cooled to rt. The mixture was then diluted with EtOAc
(300 mL) and washed with water (3 Â 60 mL) and brine
(2 Â 100 mL). The organic layer was dried over Na2SO4 and filtered.
The filtrate was concentrated and re-dissolved in a mixture of THF
(60 mL) and water (6 mL). After PPh3 (4.34 g, 16.6 mmol) was
added at rt, the reaction mixture was allowed to stir at rt overnight
and then concentrated. The residue was dissolved in Et2O (15 mL),
and hexanes (200 mL) was added. The solid precipitated was fil-
tered and washed with Et2O/hexanes. The filtrate was concen-
trated and the residue was dissolved in hexanes (600 mL). After
an addition of a solution of HCl in Et2O (1.0 M, 30 mL), the solid
precipitated was filtered, washed and dried to give amine hydro-
chloride 23a as a white solid (3.37 g, 98%). 1H NMR (CD3OD,
600 MHz) d 7.66 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 16.2 Hz, 1H), 7.51
(d, J = 8.4 Hz, 2H), 6.48 (d, J = 16.2 Hz, 1H), 4.15 (s, 2H), 1.53 (s,
9H). HRMS calcd for C14H20NO2 234.1488 (M+H)+, found 234.1495.
4.2.9. tert-Butyl ester 26c
Following procedures similar to those described for tert-butyl
ester 26a, (E)-tert-butyl 3-(4-(aminomethyl)phenyl)acrylate
hydrochloride (23a, 850 mg, 3.15 mmol) and 4-amino-2-methoxy-
benzonitrile34 (25, 443 mg, 2.40 mmol) were coupled to give tert-
butyl ester 26c as a yellowish solid (705 mg, yield 72%). 1H NMR
(CDCl3, 600 MHz) d 7.76 (s, 1H), 7.59 (s, 1H), 7.45 (d, J = 15.6 Hz,
1H), 7.34–7.30 (m, 3H), 7.28–7.24 (m, 2H), 6.63 (dd, J = 8.4,
1.2 Hz, 1H), 6.24 (d, J = 16.2 Hz, 1H), 5.86 (t, J = 5.7 Hz, 1H), 4.43
(d, J = 5.4 Hz, 2H), 3.84 (s, 3H), 1.54 (s, 9H). HRMS calcd for
C
23H26N3O4 408.1923 (M+H)+, found 408.1915.
4.2.10. tert-Butyl ester 26d
Following procedures similar to those described for tert-butyl
ester 26a, (E)-tert-butyl 3-(3-(aminomethyl)phenyl)acrylate
hydrochloride (23b, 850 mg, 3.15 mmol) and 4-amino-2-methoxy-
benzonitrile (25, 443 mg, 2.40 mmol) were coupled to give tert-bu-
tyl ester 26d as an off-white solid (623 mg, yield 64%) 1H NMR
(CDCl3, 600 MHz) d 7.64 (s, 1H), 7.57 (d, J = 1.2 Hz, 1H),7.46 (d,
J = 16.2 Hz, 1H), 7.36–7.24 (m, 6H), 6.62 (dd, J = 9.0, 1.2 Hz, 1H),
6.30 (d, J = 15.6 Hz, 1H), 5.77 (t, J = 5.7 Hz, 1H), 4.40 (d, J = 6.0 Hz,
2H), 3.84 (s, 3H), 1.52 (s, 9H). HRMS calcd for C23H25N3O4Na
430.1737 (M+Na)+, found 430.1715.
4.2.11. Carboxylic acid 27a
A solution of tert-butyl ester 26a in dry CH2Cl2 (6 mL) and TFA
(6 mL) was allowed to stir at rt overnight and then concentrated.
After co-evaporation with toluene, carboxylic acid 27a was ob-
tained as a light-yellow solid (292 mg, 99%). 1H NMR (DMSO-d6,
600 MHz) d 8.92 (s, 1H), 8.32 (s, 1H), 7.67–7.58 (m, 3H), 7.56 (d,
J = 16.2 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.43 (s, 1H), 7.36 (s, 1H),
7.33 (d, J = 7.8 Hz, 2H), 7.01 (d, J = 7.8 Hz, 1H), 6.82 (t, J = 5.7 Hz,
1H), 6.49 (d, J = 15.6 Hz, 1H), 4.33 (d, J = 6.0 Hz, 2H), 3.87 (s, 3H).
HRMS calcd for C21H20N3O5 394.1397 (M+H)+, found 394.1415.
4.2.6. (E)-tert-Butyl 3-(3-(aminomethyl)phenyl)acrylate hydro-
chloride (23b)
Following procedures similar to those described for amine 23a,
alcohol 22b (3.86 g, 16.5 mmol) was converted into amine hydro-
chloride 23b as a white solid (4.33 g, 97%). 1H NMR (CD3OD,
600 MHz) d 7.71 (s, 1H), 7.60 (d, J = 16.2 Hz, 1H), 7.56 (td, J = 7.5,
3.0 Hz, 1H), 7.53–7.47 (m, 2H), 6.52 (d, J = 16.2 Hz, 1H), 4.16 (s,

L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
5959
4.2.12. Carboxylic acid 27b
6.45 (d, J = 14.4 Hz, 1H), 4.29 (s, 2H), 3.82 (s, 3H). HRMS calcd for
C
Following procedures similar to those described for carboxylic
acid 27a, tert-butyl ester 26b (257 mg, 0.57 mmol) was converted
into carboxylic acid 27b as a light-yellow solid (226 mg, 100%).
1H NMR (DMSO-d6, 600 MHz) d 8.89 (s, 1H), 8.31 (s, 1H), 7.62–
7.58 (m, 2H), 7.57–7.51 (m, 3H), 7.43 (s, 1H), 7.40–7.33 (m, 3H),
7.01 (dd, J = 8.4, 1.8 Hz, 1H), 6.80 (t, J = 5.7 Hz, 1H), 6.50 (d,
J = 15.6 Hz, 1H), 4.33 (d, J = 5.4 Hz, 2H), 3.87 (s, 3H). HRMS calcd
for C21H20N3O5 394.1397 (M+H)+, found 394.1413.
38H31N4O4 607.2345 (MÀH)À, found 607.2340.
4.2.18. Protected hydroxamate 28d
Following procedures similar to those described for protected
hydroxamate 28a, carboxylic acid 27b (459 mg, 1.31 mmol) was
converted into protected hydroxamate 28d as
a pale solid
(491 mg, 62%). 1H NMR (DMSO-d6, 600 MHz) d 10.44 (s, 1H), 9.16
(s, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.45 (s, 1H), 7.42–7.18 (m, 20H),
6.97 (d, J = 6.6 Hz, 1H), 6.88 (s, 1H), 6.48 (d, J = 15.6 Hz, 1H), 4.28
(d, J = 4.8 Hz, 2H), 3.82 (s, 3H). HRMS calcd for C38H31N4O4
607.2345 (MÀH)À, found 607.2341.
4.2.13. Carboxylic acid 27c
Following procedures similar to those described for carboxylic
acid 27a, tert-butyl ester 26c (658 mg, 1.62 mmol) was converted
into carboxylic acid 27c as a pale solid (570 mg, 100%). 1H NMR
(DMSO-d6, 600 MHz) d 12.36 (br s, 1H), 9.25 (s, 1H), 7.64 (d,
J = 8.4 Hz, 2H), 7.56 (d, J = 15.6 Hz, 1H), 7.51 (d, J = 9.0 Hz, 1H),
7.46 (d, J = 1.2 Hz, 1H), 7.32 (d, J = 7.8 Hz, 2H), 7.02–6.94 (m, 2H),
6.49 (d, J = 15.6 Hz, 1H), 4.32 (d, J = 6.0 Hz, 2H), 3.83 (s, 3H). HRMS
calcd for C19H18N3O4 352.1291 (M+H)+, found 352.1285.
4.2.19. Hydroxamic acid 16a
To
a suspension of protected hydroxamate 28a (256 mg,
0.39 mmol) in anhydrous CH2Cl2 (10 mL) was added TFA
(0.50 mL). The resulting yellow solution was treated with Et3SiH
till there was no change of color. After stirring for additional
40 min, the solid precipitate was filtered, washed with CH2Cl2
and collected to give hydroxamic acid 16a as a yellowish solid
(151 mg, 94%). 1H NMR (DMSO-d6, 600 MHz) d 8.88 (s, 1H), 8.32
(s, 1H), 7.56–7.48 (m, 3H), 7.45–7.39 (m, 2H), 7.36 (s, 1H), 7.33
(d, J = 7.8 Hz, 2H), 7.01 (dd, J = 9.0, 1.2 Hz, 1H), 6.76 (br s, 1H),
6.43 (d, J = 16.2 Hz, 1H), 4.32 (d, J = 4.2 Hz, 2H), 3.87 (s, 3H). HRMS
calcd for C21H21N4O5 409.1506 (M+H)+, found 409.1500.
4.2.14. Carboxylic acid 27d
Following procedures similar to those described for carboxylic
acid 27a, tert-butyl ester 26d (567 mg, 1.39 mmol) was converted
into carboxylic acid 27d as a pale solid (480 mg, 98%). 1H NMR
(DMSO-d6, 600 MHz) d 9.21 (s, 1H), 7.59 (d, J = 6.0 Hz, 1H), 7.55
(d, J = 5.4 Hz, 1H), 7.51 (d, J = 9.0 Hz, 1H), 7.46 (s, 1H), 7.38 (t,
J = 7.5 Hz, 1H), 7.34 (d, J = 7.2 Hz, 1H), 6.98 (dd, J = 9.0, 1.2 Hz,
1H), 6.94 (t, J = 5.7 Hz, 1H), 6.50 (d, J = 16.2 Hz, 1H), 4.32 (d,
J = 5.4 Hz, 2H), 3.83 (s, 3H). HRMS calcd for C19H18N3O4 352.1291
(M+H)+, found 352.1280.
4.2.20. Hydroxamic acid 16b
Following procedures similar to those described for hydroxamic
acid 16a, protected hydroxamate 28b (56 mg, 0.086 mmol) was
converted into hydroxamic acid 16b as a pale solid (34 mg, 97%)
1H NMR (DMSO-d6, 600 MHz) d 8.86 (s, 1H), 8.32 (s, 1H), 7.54 (d,
J = 8.4 Hz, 1H), 7.48 (s, 1H), 7.46–7.40 (m, 3H), 7.38 (d, J = 7.8 Hz,
1H), 7.36 (s, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H),
6.77 (br s, 1H), 6.45 (d, J = 15.6 Hz, 1H), 4.32 (d, J = 4.2 Hz, 2H),
3.87 (s, 3H). HRMS calcd for C21H21N4O5 409.1506 (M+H)+, found
409.1506.
4.2.15. Protected hydroxamate 28a
A solution of carboxylic acid 27a (272 mg, 0.69 mmol), 1-ethyl-
3-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC,
410 mg, 2.07 mmol), 1-hydroxy-benzotriazole (HOBt, 189 mg,
1.40 mmol) and O-tritylhydroxylamine (380 mg, 1.38 mmol) in
anhydrous DMF (10 mL) was stirred at rt for 4 d. After concentra-
tion, the residue was diluted with EtOAc (60 mL) and washed with
water (10 mL), satd NaHCO3 (10 mL), water (10 mL) and brine
(30 mL). The organic layer was dried over Na2SO4 and filtered.
The filtrate was concentrated and the residue was purified by silica
gel column chromatography (0–4% MeOH/CH2Cl2) to give pro-
tected hydroxamate 28a as a yellowish solid (281 mg, 62%). 1H
NMR (DMSO-d6, 600 MHz) d 10.37 (s, 1H), 8.84 (s, 1H), 8.32 (s,
1H), 7.53 (d, J = 8.4 Hz, 1H), 7.48–7.40 (m, 3H), 7.40–7.18 (m,
18H), 7.00 (d, J = 8.4 Hz, 1H), 6.71 (t, J = 5.7 Hz, 1H), 6.44 (d,
J = 16.2 Hz, 1H), 4.29 (d, J = 5.4 Hz, 2H), 3.86 (s, 3H). HRMS calcd
for C40H34N4O5Na 673.2421 (M+Na)+, found 673.2421.
4.2.21. Hydroxamic acid 16c
Following procedures similar to those described for hydroxamic
acid 16a, protected hydroxamate 28c (300 mg, 0.49 mmol) was
converted into hydroxamic acid 16c as a pale solid (150 mg,
83%). 1H NMR (DMSO-d6, 600 MHz) d 9.22 (s, 1H), 7.55–7.48 (m,
3H), 7.46 (s, 1H), 7.42 (d, J = 15.6 Hz, 1H), 7.32 (d, J = 7.2 Hz, 2H),
6.98 (d, J = 8.4 Hz, 1H), 6.93 (s, 1H), 6.42 (d, J = 15.6 Hz, 1H), 4.31
(d, J = 4.2 Hz, 2H), 3.83 (s, 3H). HRMS calcd for C19H19N4O4
367.1406 (M+H)+, found 367.1387.
4.2.22. Hydroxamic acid 16d
Following procedures similar to those described for hydroxamic
acid 16a, protected hydroxamate 28d (300 mg, 0.49 mmol) was
converted into hydroxamic acid 16d as a pale solid (103 mg,
57%). 1H NMR (DMSO-d6, 600 MHz) d 9.21 (s, 1H), 7.51 (d,
J = 9.0 Hz, 1H), 7.48–7.40 (m, 4H), 7.37 (t, J = 7.5 Hz, 1H), 7.30 (d,
J = 7.2 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.94 (t, J = 6.0 Hz, 1H), 6.44
(d, J = 15.6 Hz, 1H), 4.32 (d, J = 5.4 Hz, 2H), 3.83 (s, 3H). HRMS calcd
for C19H19N4O4 367.1406 (M+H)+, found 367.1380.
4.2.16. Protected hydroxamate 28b
Following procedures similar to those described for protected
hydroxamate 28a, carboxylic acid 27b (206 mg, 0.52 mmol) was
converted into protected hydroxamate 28b as a yellowish solid
(71 mg, 21%). 1H NMR (DMSO-d6, 600 MHz) d 10.42 (s, 1H), 8.81
(s, 1H), 8.32 (s, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.44–7.18 (m, 21H),
7.00 (d, J = 8.4 Hz, 1H), 6.71 (t, J = 5.7 Hz, 1H), 6.47 (d, J = 15.6 Hz,
1H), 4.29 (d, J = 6.0 Hz, 2H), 3.86 (s, 3H). HRMS calcd for
4.2.23. tert-Butyl ester 30a
C
40H34N4O5Na 673.2421 (M+Na)+, found 673.2422.
A solution of (E)-tert-butyl 3-(4-(aminomethyl)phenyl)acrylate
hydrochloride (405 mg, 1.50 mmol), 2-(3-methoxy-4-(oxazol-5-
yl)phenylamino)-2-oxoacetic acid21 (472 mg, 1.80 mmol), PyBOP
(1.17 g, 2.25 mmol) and NMM (0.66 mL, 6.00 mmol) in anhydrous
DMF (15 mL) was stirred at rt for 48 h. The reaction mixture was
slowly added to ice/water (150 mL plus 20 mL of 1 N HCl). The so-
lid precipitate was washed with water, collected and purified by
silica gel column chromatography (30–50% EtOAc/hexanes) to give
4.2.17. Protected hydroxamate 28c
Following procedures similar to those described for protected
hydroxamate 28a, carboxylic acid 27c (538 mg, 1.53 mmol) was
converted into protected hydroxamate 28c as
a pale solid
(505 mg, 54%). 1H NMR (DMSO-d6, 600 MHz) d 10.39 (s, 1H), 9.20
(s, 1H), 7.56–7.16 (m, 21H), 6.98 (d, J = 6.0 Hz, 1H), 6.89 (s, 1H),

5960
L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
tert-butyl ester 30a as a yellow solid (298 mg, 42%). 1H NMR
(CDCl3, 600 MHz) d 9.38 (s, 1H), 7.92 (t, J = 5.7 Hz, 1H), 7.90 (s,
1H), 7.75 (d, J = 7.8 Hz, 1H), 7.60–7.54 (m, 2H), 7.53 (s, 1H), 7.50
(d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.17 (dd, J = 8.4, 1.8 Hz,
1H), 6.36 (d, J = 15.6 Hz, 1H), 4.58 (d, J = 6.0 Hz, 2H), 3.98 (s, 3H),
1.54 (s, 9H). HRMS calcd for C26H27N3O6Na 500.1792 (M+Na)+,
found 500.1793.
4.2.29. Hydroxamic acid 17a
To solution of protected hydroxamate 32a (250 mg,
a
0.37 mmol) in anhydrous CH2Cl2 (10 mL) was added TFA
(0.50 mL). The resulting yellow solution was treated with Et3SiH
till there was no change of color. After stirring for additional
30 min, the solid precipitate was filtered, washed with CH2Cl2
and collected to give hydroxamic acid 17a as a yellowish solid
(151 mg, 94%). 1H NMR (DMSO-d6, 600 MHz) d 10.79 (s, 1H), 9.58
(t, J = 6.3 Hz, 1H), 8.39 (s, 1H), 7.75 (s, 1H), 7.68–7.61 (m, 2H),
7.52 (d, J = 7.8 Hz, 2H), 7.48 (s, 1H), 7.42 (d, J = 16.2 Hz, 1H), 7.33
(d, J = 7.8 Hz, 2H), 6.43 (d, J = 15.6 Hz, 1H), 4.41 (d, J = 6.6 Hz, 2H),
3.90 (s, 3H). HRMS calcd for C22H21N4O6 437.1455 (M+H)+, found
437.1451.
4.2.24. tert-Butyl ester 30b
Following procedures similar to those described for tert-butyl
ester 30a, (E)-tert-butyl 3-(3-(aminomethyl)phenyl)acrylate hydro-
chloride (405 mg, 1.50 mmol) was converted into tert-butyl ester
30b as a yellow solid (305 mg, 42%). 1H NMR (CDCl3, 600 MHz) d
9.38 (s, 1H), 7.92 (t, J = 5.7 Hz, 1H), 7.90 (s, 1H), 7.76 (d, J = 8.4 Hz,
1H), 7.60–7.52 (m, 3H), 7.48–7.42 (m, 2H), 7.37 (t, J = 7.5 Hz, 1H),
7.31 (d, J = 7.8 Hz, 2H), 7.18 (dd, J = 8.4, 1.8 Hz, 1H), 6.38 (d,
J = 16.2 Hz, 1H), 4.58 (d, J = 6.0 Hz, 2H), 3.98 (s, 3H), 1.53 (s, 9H).
HRMS calcd for C26H27N3O6Na 500.1792 (M+Na)+, found 500.1797.
4.2.30. Hydroxamic acid 17b
Following procedures similar to those described for hydroxamic
acid 17a, protected hydroxamate 32b (175 mg, 0.26 mmol) was
converted into hydroxamic acid 17b as a pale solid (105 mg,
93%). 1H NMR (DMSO-d6, 600 MHz) d 10.79 (s, 1H), 9.58 (t,
J = 6.0 Hz, 1H), 8.38 (s, 1H), 7.76 (s, 1H), 7.69–7.61 (m, 2H), 7.50
(s, 1H), 7.48 (s, 1H), 7.46–7.40 (m, 2H), 7.37 (t, J = 7.5 Hz, 1H),
7.31 (d, J = 7.2 Hz, 1H), 6.45 (d, J = 15.6 Hz, 1H), 4.42 (d, J = 6.6 Hz,
2H), 3.91 (s, 3H). HRMS calcd for C22H21N4O6 437.1455 (M+H)+,
found 437.1450.
4.2.25. Carboxylic acid 31a
A solution of tert-butyl ester 30a in dry CH2Cl2 (6 mL) and TFA
(6 mL) was allowed to stir at rt overnight and then concentrated.
After co-evaporation with toluene, carboxylic acid 31a was ob-
tained as a yellow solid (240 mg, 100%). 1H NMR (DMSO-d6,
600 MHz) d 10.79 (s, 1H), 9.59 (t, J = 6.6 Hz, 1H), 8.38 (s, 1H),
7.75 (s, 1H), 7.68–7.61 (m, 4H), 7.56 (d, J = 15.6 Hz, 1H), 7.47 (s,
1H), 7.34 (d, J = 8.4 Hz, 2H), 6.49 (d, J = 16.2 Hz, 1H), 4.42 (d,
J = 6.0 Hz, 2H), 3.90 (s, 3H). HRMS calcd for C22H20N3O6 422.1346
(M+H)+, found 422.1350.
4.2.31. Ethyl ester 34a
To a solution of 3-methoxy-4-(oxazol-5-yl) aniline (570 mg,
3.0 mmol) and triphosgene (296 mg, 1.0 mmol) in CH2Cl2 (10 mL)
was added Et3N (0.50 mL, 5.0 mmol) at 5 °C and the mixture was
refluxed for 2.5 h. The mixture was cooled to room temperature
and a solution of ethyl 4-aminocinnamate hydrochloride (33a,
555 mg, 2.0 mmol) in CH2Cl2 was added. Additional Et3N
(0.25 mL) was added and the mixture was stirred at rt for 2.5 h
and then poured into water. The product was extracted with EtOAc
and purified on silica gel column (Hexanes/EtOAc/MeOH = 6:3:1)
to obtain ethyl ester 34a (410 mg, 50%). 1H NMR (DMSO-d6,
600 MHz): d 9.91 (s, 1H), 8.99 (s, 1H), 8.36 (s, 1H), 7.65 (d,
J = 8.4 Hz, 2H), 7.61 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 16.2 Hz, 1H),
7.52 (d, J = 8.4 Hz, 2H), 7.46 (s, 1H), 7.42 (s, 1H), 7.08 (dd, J = 8.4,
1.2 Hz, 1H), 6.48 (d, J = 16.2 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 3.93
(s, 3H), 1.25 (t, J = 7.2 Hz, 1H). 13C NMR (DMSO-d6): d 167.1,
156.5, 152.8, 150.9, 147.9, 144.8, 142.4, 141.7, 130.0, 128.4,
126.6, 124.1, 118.8, 116.3, 111.1, 110.8, 101.8, 102.1, 60.5, 56.1,
14.9. HRMS calcd for C22H21N3O5Na 430.1373 (M+Na)+, found
430.1385.
4.2.26. Carboxylic acid 31b
Following procedures similar to those described for carboxylic
acid 31a, tert-butyl ester 30b (280 mg, 0.59 mmol) was converted
into carboxylic acid 31b as a yellow solid (240 mg, 97%). 1H NMR
(DMSO-d6, 600 MHz) d 10.79 (s, 1H), 9.56 (t, J = 6.3 Hz, 1H), 8.38
(s, 1H), 7.76 (s, 1H), 7.69–7.60 (m, 3H), 7.60–7.53 (m, 2H), 7.47
(s, 1H), 7.40–7.32 (m 2H), 6.50 (d, J = 16.2 Hz, 1H), 4.43 (d,
J = 6.0 Hz, 2H), 3.90 (s, 3H). HRMS calcd for C22H20N3O6 422.1346
(M+H)+, found 422.1346.
4.2.27. Protected hydroxamate 32a
A solution of carboxylic acid 31a (215 mg, 0.51 mmol), EDC
(310 mg, 1.57 mmol), HOBt (139 mg, 1.03 mmol) and O-tritylhydr-
oxylamine (281 mg, 1.02 mmol) in anhydrous DMF (15 mL) was
stirred at rt for 4 d. After concentration, the residue was dissolved
in MeOH (15 mL) and slowly added to ice/water (150 mL). The so-
lid precipitate was washed with water, collected and purified by
silica gel column chromatography (0–4% MeOH/CH2Cl2) to give
protected hydroxamate 32a as a light-yellow solid (281 mg, 81%).
1H NMR (CDCl3, 600 MHz) d 9.38 (s, 1H), 7.94–7.86 (m, 2H), 7.75
(d, J = 7.8 Hz, 1H), 7.57 (d, J = 1.2 Hz, 1H), 7.53 (s, 1H), 7.43 (br s,
5H), 7.36–7.31 (m, 7H), 7.31–7.26 (m, 4H), 7.24 (d, J = 7.8 Hz,
2H), 7.18 (dd, J = 8.7, 1.5 Hz, 1H), 6.09 (d, J = 16.2 Hz, 1H), 4.54 (d,
4.2.32. Ethyl ester 34b
Following procedures similar to those described for ethyl ester
34a, 3-methoxy-4-(oxazol-5-yl) aniline (24, 570 mg, 3.0 mmol)
and ethyl 3-aminocinnamate (33b, 382 mg, 2.0 mmol) were com-
bined to give ethyl ester 34b (220 mg, 27%). 1H NMR (DMSO-d6,
600 MHz): d 8.13 (s, 1H), 7.71 (s, 1H), 7.57 (d, J = 8.4 Hz, 2H),
7.56 (d, J = 15.6 Hz, 1H), 7.44 (s, 1H), 7.40 (d, J = 7.2 Hz, 1H), 7.34
(d, J = 1.2 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.21 (d, J = 7.8 Hz, 1H),
6.97 (d, J = 8.4 Hz, 1H), 6.43 (d, J = 15.6 Hz, 1H), 4.14 (q, J = 7.2 Hz,
2H), 3.89 (s, 3H), 1.22 (t, J = 7.2 Hz, 3H). 13C NMR (DMSO-d6): d
166.8, 156.6, 153.1, 150.3, 148.2, 144.7, 141.5, 140.3, 135.3,
129.7, 126.3, 123.5, 122.5, 120.9, 118.6, 118.1, 110.9, 110.8,
101.8, 60.5, 55.3, 13.9. HRMS calcd for C22H22N3O5 408.1553
(M+H)+, found 408.1561.
J = 6.6 Hz, 2H), 3.98 (s, 3H). HRMS calcd for
C41H34N4O6Na
701.2370 (M+Na)+, found 701.2366.
4.2.28. Protected hydroxamate 32b
Following procedures similar to those described for protected
hydroxamate 32a, carboxylic acid 31b (215 mg, 0.51 mmol) was
converted into protected hydroxamate 32b as a light-yellow solid
(200 mg, 58%). 1H NMR (CDCl3, 600 MHz) d 9.35 (s, 1H), 7.93–
7.84 (m, 2H), 7.76 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 1.8 Hz, 1H), 7.53
(s, 1H), 7.43 (br s, 5H), 7.36–7.31 (m, 7H), 7.30–7.23 (m, 6H),
7.16 (dd, J = 8.1, 1.5 Hz, 1H), 6.10 (d, J = 15.6 Hz, 1H), 4.53 (d,
4.2.33. Ethyl ester 34c
Following procedures similar to those described for ethyl ester
34a, 4-cyano-3-methoxy aniline hydrochloride (25, 554 mg,
3.0 mmol) and ethyl 4-aminocinnamate hydrochloride (33a,
555 mg, 2.0 mmol) were combined to give ethyl ester 34c
J = 6.0 Hz, 2H), 3.97 (s, 3H). HRMS calcd for
C41H34N4O6Na
701.2370 (M+Na)+, found 701.2369.

L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
5961
(710 mg, 97%). 1H NMR (DMSO-d6, 600 MHz): d 9.29 (s, 1H), 9.11 (s,
1H), 7.66 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 15.6 Hz, 1H), 7.58 (d,
J = 7.2 Hz, 1H), 7.52 (d, J = 9.0 Hz, 2H), 7.49 (s, 1H), 7.04 (d,
J = 8.4 Hz, 1H), 6.50 (d, J = 15.6 Hz, 1H), 4.17 (q, J = 7.2 Hz, 2H),
3.88 (s, 3H), 1.25 (t, J = 7.2 Hz, 1H). 13C NMR (DMSO-d6): d 167.1,
162.4, 152.5, 146.4, 144.8, 141.9, 134.8, 130.0, 128.7, 119.0,
117.6, 116.6, 111.1, 101.5, 93.5, 60.5, 56.6, 14.9. HRMS calcd for
to give carboxylic acid 35d (250 mg, 74%). 1H NMR (DMSO-d6,
600 MHz): d 6.99 (s, 1H), 6.83 (d, J = 15.6 Hz, 1H), 6.79 (s, 1H),
6.72 (t, J = 10.2, 8.4 Hz, 2H), 6.58 (t, J = 7.8 Hz, 1H), 6.51 (d,
J = 7.2 Hz, 1H), 6.23 (d, J = 10.2 Hz, 1H), 5.70 (d, J = 15.6 Hz, 1H),
4.16 (q, J = 7.2 Hz, 2H), 3.78 (s, 3H). 13C NMR (DMSO-d6): d 168.8,
162.7, 153.1, 146.4, 144.8, 140.1, 135.7, 134.4, 129.8, 123.0,
121.2, 119.5, 118.5, 117.2, 110.9, 101.4, 94.1, 55.9. HRMS calcd
for C18H15N3O4Na 360.0954 (M+Na)+, found 360.0963.
C
20H19N3O4Na 388.1267 (M+Na)+, found 388.1281.
4.2.34. Ethyl ester 34d
4.2.39. Hydroxamic acid 18a
Following procedures similar to those described for ethyl ester
34a, 4-cyano-3-methoxy aniline hydrochloride (25, 369 mg,
2.0 mmol) and ethyl 3-aminocinnamate (33b, 573 mg, 3.0 mmol)
were combined to give ethyl ester 34d (485 mg, 66%). 1H NMR
(DMSO-d6, 600 MHz): d 9.29 (s, 1H), 8.90 (s, 1H), 7.74 (s, 1H),
7.60 (d, J = 15.6 Hz, 1H), 7.57 (d, J = 9.0 Hz, 1H), 7.50 (s, 1H), 7.48
(d, J = 6.6 Hz, 1H), 7.38 (d, J = 7.2 Hz, 1H), 7.35 (t, J = 6.8 Hz, 1H),
7.01 (dd, J = 7.8, 1.2 Hz, 1H), 6.52 (d, J = 16.2 Hz, 1H), 4.16 (q,
J = 7.2 Hz, 2H), 3.87 (s, 3H), 1.25 (t, J = 7.2 Hz, 1H). 13C NMR
(DMSO-d6): d 166.0, 161.7, 152.1, 145.9, 144.3, 139.6, 134.6,
134.1, 129.4, 122.2, 120.7, 118.4, 118.3, 116.9, 110.3, 100.7, 92.7,
To a solution of carboxylic acid 35a (100 mg, 0.26 mmol) in
dried DMF (5 mL) were added O-tritylhydroxylamine (109 mg,
1.5 mmol), EDC (101 mg, 0.52 mmol) and 1-hydroxybenzotriazole
(53 mg, 0.40 mmol). The mixture was stirred at room temperature
overnight. Solvent was removed and the residue was purified on
silica gel column (CHCl3: MeOH = 9: 1) to give protected hydroxa-
mate 36a (70 mg, 42%). To a portion of 36a (64 mg, 0.10 mmol) in
CH2Cl2 (5 mL) was added TFA (0.25 mL) and triethylsilane
(0.25 mL). The mixture was allowed to stir overnight. The precipi-
tate was filtered, collected and dried to give hydroxamic acid 18a
(38 mg, 96%). 1H NMR (DMSO-d6, 600 MHz): d 9.05 (s, 1H), 9.00
(s, 1H), 8.36 (s, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.48–7.53 (m, 5H),
7.42 (d, J = 12.0 Hz, 1H), 7.39 (d, J = 10.2 Hz, 1H), 7.07 (d,
J = 8.4 Hz, 1H), 6.35 (d, J = 15.6 Hz, 1H), 3.92 (s, 3H). 13C NMR
(DMSO-d6): d 213.6, 180.2, 156.4, 152.8, 146.7, 141.8, 129.0,
126.6, 118.9, 113.3, 111.0, 110.8, 102.2, 56.1. HRMS calcd for
60.1, 55.9, 14.1. HRMS calcd for
C20H19N3O4Na 388.1267
(M+Na)+, found 388.1284.
4.2.35. Carboxylic acid 35a
To a suspension of ethyl ester 34a (300 mg, 0.74 mmol) in THF
(5 mL) were added methanol (2.5 mL) and NaOH (590 mg,
15 mmol in 5 mL of water). The mixture was stirred at room tem-
perature overnight. After solvents were removed, the residue was
diluted with water and pH was adjusted with aqueous HCl to 1–
2. The precipitate of the product was filtered, collected and dried
to give carboxylic acid 35a (245 mg, 87%). 1H NMR (DMSO-d6,
600 MHz): d 9.01 (br s, 2H), 8.34 (s, 1H), 7.53–7.61 (m, 5H), 7.45
(d, J = 16.2 Hz, 1H), 7.48–7.56 (m, 2H), 7.08 (d, J = 6.6 Hz, 1H),
6.40 (d, J = 16.2 Hz, 1H), 3.92 (s, 3H). 13C NMR (DMSO-d6): d
168.5, 156.5, 152.9, 150.9, 147.9, 146.7, 144.5, 142.2, 141.8,
129.9, 128.6, 126.6, 124.1, 118.8, 117.4, 113.4, 111.1, 110.9,
102.1, 56.2. HRMS calcd for C20H18N3O5 380.1240 (M+H)+, found
380.1233.
C
20H19N4O5 395.1349 (M+H)+, found 395.1331.
4.2.40. Hydroxamic acid 18b
Following procedures similar to those described for hydroxamic
acid 18a, carboxylic acid 35b (100 mg, 0.26 mmol) was converted
into protected hydroxamate 36b (90 mg, 54%). A portion of 36b
(64 mg, 0.10 mmol) was deprotected to give hydroxamic acid
18b (37 mg, 93%). 1H NMR (DMSO-d6, 600 MHz): d 9.02 (s, 1H),
8.87 (s, 1H), 8.35 (s, 1H), 7.78 (s, 1H), 7.61 (d, J = 9.0 Hz, 1H),
7.38–7.45 (m, 4H), 7.32 (t, J = 7.8 Hz, 2H), 7.17 (d, J = 7.8 Hz, 1H),
7.08 (d, J = 7.8 Hz, 1H), 6.43 (d, J = 16.2 Hz, 1H), 3.92 (s, 3H). 13C
NMR (DMSO-d6): d 162.6, 158.5, 152.4, 150.2, 147.2, 141.2, 140.0,
138.4, 135.4, 129.4, 125.9, 123.3, 121.8, 119.6, 116.5, 110.3,
110.0, 101.3, 55.4. HRMS calcd for C20H19N4O5 395.1349 (M+H)+,
found 395.1339.
4.2.36. Carboxylic acid 35b
Following procedures similar to those described for carboxylic
acid 35a, ethyl ester 34b (580 mg, 1.43 mmol) underwent hydroly-
sis to give carboxylic acid 35b (360 mg, 66%). 1H NMR (DMSO-d6,
600 MHz): d 9.91 (s, 1H), 9.80 (s, 1H), 8.34 (s, 1H), 7.76 (s, 1H),
7.58 (d, J = 7.2 Hz, 1H), 7.56 (d, J = 14.4 Hz, 1H), 7.48–7.56 (m,
2H), 7.40 (s,1H), 7.29–7.31 (m, 2H), 7.05 (d, J = 6.8 Hz, 1H), 6.43
(d, J = 15.6 Hz, 1H), 3.91 (s, 3H). 13C NMR (DMSO-d6): d 166.9,
155.3, 152.2, 149.7, 146.8, 143.5, 141.0, 139.8, 134.2, 128.9,
125.4, 122.7, 121.3, 119.4, 118.7, 116.6, 109.6, 109.3, 100.4, 54.9.
HRMS calcd for C20H18N3O5 380.1240 (M+H)+, found 380.1233.
4.2.41. Hydroxamic acid 18c
Following procedures similar to those described for hydroxamic
acid 18a, carboxylic acid 35c (100 mg, 0.30 mmol) was converted
into protected hydroxamate 36c (85 mg, 48%). A portion of 36c
(60 mg, 0.10 mmol) was deprotected to give hydroxamic acid 18c
(25 mg, 71%). 1H NMR (DMSO-d6, 600 MHz): d 10.7 (s, 1H), 9.31
(s, 1H), 9.10 (s, 1H), 8.98 (s, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.50 (br
s, 5H), 7.40 (d, J = 15.6 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.35 (d,
J = 16.2 Hz, 1H), 3.88 (s, 3H). 13C NMR (DMSO-d6): d 162.4, 152.6,
146.7, 146.5, 141.0, 138.7, 134.8, 129.6, 129.0, 119.2, 117.6,
113.3, 111.0, 101.4, 93.4, 56.6. HRMS calcd for C18H16N4O4Na
375.1063 (M+Na)+, found 375.1065.
4.2.37. Carboxylic acid 35c
Following procedures similar to those described for carboxylic
acid 35a, ethyl ester 34c (365 mg, 1.0 mmol) underwent hydrolysis
to give carboxylic acid 35c as a yellow solid (330 mg, 98%). 1H NMR
(DMSO-d6, 600 MHz): d 12.2 (br s, 1H), 10.1 (br s, 1H), 9.91 (br s, 1H),
7.62 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.4 Hz, 1H), 7.49–7.53 (m, 4H),
7.02 (dd, J = 8.4, 1.2 Hz, 1H), 6.40 (d, J = 16.2 Hz, 1H), 3.88 (s, 3H).
13C NMR (DMSO-d6): d 168.5, 162.4, 152.8, 146.6, 144.4, 141.9,
134.8, 129.9, 118.7, 117.7, 117.5, 113.4, 110.8, 101.1, 93.3, 56.6.
HRMS calcd for C18H15N3O4Na 360.0954 (M+Na)+, found 360.0965.
4.2.42. Hydroxamic acid 18d
Following procedures similar to those described for hydroxamic
acid 18a, carboxylic acid 35d (100 mg, 0.30 mmol) was converted
into protected hydroxamate 36d.
A portion of 36d (60 mg,
0.10 mmol) was deprotected to give hydroxamic acid 18d
(32 mg, 90%). 1H NMR (DMSO-d6, 600 MHz): d 9.30 (s, 1H), 8.99
(s, 1H), 7.76 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.48 (s, 1H), 7.41 (d,
J = 15.6 Hz, 1H), 7.39 (d, J = 11.4 Hz, 1H), 7.33 (t, J = 7.8, 7.2 Hz,
1H), 7.19 (d, J = 7.8 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.43 (d,
J = 15.6 Hz, 1H), 3.88 (s, 3H). 13C NMR (DMSO-d6): d 161.7, 152.1,
145.9, 139.6, 138.3, 135.4, 134.1, 129.4, 122.1, 119.8, 119.2,
4.2.38. Carboxylic acid 35d
Following procedures similar to those described for carboxylic
acid 35a, ethyl ester 34d (365 mg, 1.0 mmol) underwent hydrolysis

5962
L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
116.9, 116.8, 110.3, 100.7, 92.7, 55.9. HRMS calcd for C18H16N4O4-
Na 375.1063 (M+Na)+, found 375.1055.
143.5, 141.0, 139.8, 134.2, 128.9, 125.4, 122.7, 121.3, 119.4,
118.7, 116.6, 109.6, 109.3, 100.4, 54.9. HRMS calcd for
C
21H18N3O6 408.1051 (M+H)+, found 408.1053.
4.2.43. Ethyl ester 37a
To a solution of 2-(3-methoxy-4-(oxazol-5-yl)phenylamino)-2-
oxoacetic acid (29, 600 mg, 2.29 mmol) in anhydrous DMF
(15 mL) were added benzotriazol-1-yloxy-tripyrrolidinophospho-
nium hexafluorophosphate (PyBOP) (2.38 g, 4.56 mmol), 4-methyl-
morpholine (NMM) (1.10 mL, 9.16 mmol) and (E)-ethyl 3-(4-
aminophenyl)acrylate (33a, 525 mg, 2.75 mmol). The mixture
was stirred at rt for 48 h. After concentration, the resulting residue
was diluted with EtOAC (50 mL) and subsequently washed with
H2O (2 Â 10 mL) and brine (2 Â 10 mL). The organic layer was
dried over Na2SO4 and filtered. The filtrate was concentrated and
the residue was purified by flash chromatography column (0–3%
MeOH/CH2Cl2) to give ethyl ester 37a as a light-yellow solid
(500 mg, 50%). Mp = 187.6–188.1 °C. 1H NMR (DMSO-d6,
600 MHz): d 11.04 (s, 1H), 11.01 (s, 1H), 8.38 (s, 1H), 7.91 (d,
J = 8.51 Hz, 1H), 7.82 (s, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.67 (d,
J = 8.5 Hz, 2H), 7.63 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 16.1 Hz, 1H),
7.48 (s, 1H), 6.58 (d, J = 15.8 Hz, 1H), 4.17 (q, J = 6.7 Hz, 2H), 3.91
(s, 3H), 1.23 (t, J = 6.3 Hz, 3H). HRMS calcd for C23H22N3O6
436.2054 (M+H)+, found 436.2058.
4.2.47. Protected hydroxamate 39a
A solution of carboxylic acid 38a (250 mg, 0.61 mmol), O-trit-
ylhydroxylamine (277 mg, 1.22 mmol), EDC (295 mg, 1.53 mmol),
and HOBt (166 mg, 1.22 mmol) in anhydrous DMF (20 mL) was
stirred at rt for 24 h. After concentration, the residue was diluted
with EtOAc (25 mL) and washed with water (2 Â 15 mL) and brine
(2 Â 20 mL). The organic layer was dried over Na2SO4 and filtered.
The filtrate was concentrated and the residue was purified by flash
column chromatography (0–5% MeOH/CH2Cl2) to give protected
hydroxamate 39a as a white solid (210 mg, 52%). Mp = 209.5–
211.1 °C. 1H NMR (DMSO-d6, 600 MHz): d 11.09 (s, 1H), 11.05 (s,
1H), 10.61 (s, 1H), 8.38 (s, 1H), 7.82 (s, 1H), 7.67 (s, 1H), 7.66 (d,
J = 8.4 Hz, 2H), 7.64 (s, 1H), 7.61 (d, J = 7.1 Hz, 2H), 7.48 (s, 1H),
7.43 (d, J = 15.7 Hz, 1H), 7.38–7.23 (m, 15H), 6.44 (d, J = 15.5 Hz,
1H), 3.95 (s, 3H). HRMS calcd for C40H33N4O6 665.2642 (M+H)+,
found 665.2649.
4.2.48. Protected hydroxamate 39b
Following procedures similar to those described for protected
hydroxamate 39a, carboxylic acid 38b (120 mg, 0.29 mmol) in
anhydrous DMF (10 mL) was treated with H2N-OTr (135 mg,
0.59 mmol), EDC (142 mg, 0.73 mmol), and HOBt (80 mg,
0.59 mmol) to give protected hydroxamate 39b as a white solid
(70 mg, 55%). Mp = 218.5–219.2 °C. 1H NMR (DMSO-d6, 600 MHz):
d 10.94 (s, 1H), 10.89 (s, 1H), 10.46 (s, 1H), 8.36 (s, 1H), 7.98 (s,
1H), 7.92 (s, 1H), 7.79–7.77 (m, 2H), 7.67 (d, J = 8.5 Hz, 1H), 7.62
(d, J = 8.8 Hz, 1H), 7.48 (s, 1H), 7.41 (d, J = 16.4 Hz, 1H), 7.39–7.18
(m, 15H), 6.44 (d, J = 15.5 Hz, 1H), 3.91 (s, 3H). HRMS calcd for
4.2.44. Ethyl ester 37b
Following procedures similar to those described for ethyl ester
37a, oxoacetic acid 29 (200 mg, 0.763 mmol) in anhydrous DMF
(4 mL) was treated with PyBOP (790 mg, 1.52 mmol), NMM
(0.33 mL, 3.05 mmol) and (E)-ethyl 3-(3-aminophenyl)acrylate
(33b, 175 mg, 0.92 mmol) to give ethyl ester 37b as a pale solid
(210 mg, 65%). Mp = 185.7–187.1 °C. 1H NMR (DMSO-d6,
600 MHz): d 10.98 (s, 1H), 10.91 (s, 1H), 8.38 (s, 1H), 8.10 (s, 1H),
7.92 (d, J = 6.7 Hz, 1H), 7.81 (s, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7.66
(d, J = 7.6 Hz, 1H), 7.59 (d, J = 16.1 Hz, 1H), 7.51 (d, J = 7.1 Hz, 1H),
7.47 (s, 1H), 6.53 (d, J = 15.8 Hz, 1H), 4.17 (q, J = 6.7 Hz, 2H), 3.91
(s, 3H), 1.24 (t, J = 6.3 Hz, 3H). 13C NMR (DMSO-d6, 150 MHz): d
166.8, 165.3, 156.6, 153.1, 150.3, 148.2, 144.7, 141.5, 140.3,
135.3, 129.7, 126.3, 123.5, 122.5, 120.9, 118.6, 118.1, 110.9,
C
40H33N4O6 665.2332 (M+H)+, found 665.2337.
4.2.49. Hydroxamic acid 19a
To a solution of protected hydroxamate 39a (70 mg, 0.10 mmol)
in anhydrous CH2Cl2 (6 mL) was added TFA (0.15 mL). The resulting
yellow solution was treated with Et3SiH till it was colorless. After
being stirring for an additional 30 min, the solid formed was filtered,
washed with anhydrous CH2Cl2 and dried under high vacuum to
give hydroxamic acid 19a as a light-yellow solid (9.3 mg, 22%).
Mp = 174.3–175.9 °C. 1H NMR (DMSO-d6, 600 MHz): d 11.01 (s,
1H), 10.96 (s, 1H), 10.83 (s, 1H), 8.39 (s, 1H), 8.05 (s, 1H), 7.87 (d,
J = 7.9 Hz, 1H), 7.83 (s, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.62 (d,
J = 8.2 Hz, 2H), 7.49 (s,1H), 7.43 (d, J = 15.7 Hz, 1H), 7.37 (d,
J = 7.7 Hz, 1H), 6.39 (d, J = 15.9 Hz, 1H), 3.92 (s, 3H). HRMS calcd
for C21H19N4O6 423.1292 (M+H)+, found 423.1310.
110.8, 101.8, 60.5, 55.3, 13.9. HRMS calcd for
C23H22N3O6
436.1433 (M+H)+, found 436.1439.
4.2.45. Carboxylic acid 38a
To a suspension of ethyl ester 37a (450 mg, 1.03 mmol) in THF
(18 mL) were added methanol (6 mL) and NaOH (910 mg in 18 mL
water). The mixture was stirred at room temperature overnight.
Solvents were removed and the residue was diluted with water
and 1 N HCl was added until pH ꢀ 1–2. The solid that formed
was collected by filtration and dried to carboxylic acid 38a as a
white solid (260 mg, 62%). Mp = 184.2–185.7 °C. 1H NMR (DMSO-
d6, 600 MHz): d 12.07 (br s, 1H), 10.85 (s, 1H), 10.82 (s, 1H), 8.37
(s, 1H), 7.81 (d, J = 8.2 Hz, 1H), 7.66 (d, J = 4.4 Hz, 2H), 7.64 (s,
1H), 7.62 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.5 Hz, 1H), 7.51 (d,
J = 15.8 Hz, 1H), 7.45 (s, 1H), 6.44 (d, J = 16.1 Hz, 1H), 3.91 (s, 3H).
HRMS calcd for C21H18N3O6 408.1233 (M+H)+, found 408.1236.
4.2.50. Hydroxamic acid 19b
Following procedures similar to those described for hydroxamic
acid 19a, protected hydroxamate 39b (50 mg, 0.075 mmol) in
anhydrous CH2Cl2 (6 mL) was treated with TFA (0.1 mL) to give
hydroxamic acid 19b as a white solid (7.5 mg, 25%). Mp = 173.5–
174.8 °C. 1H NMR (DMSO-d6, 600 MHz): d 11.04 (s, 1H), 10.97 (s,
1H), 10.83 (s, 1H), 8.39 (s, 1H), 8.05 (s, 1H), 7.84 (d, J = 7.9 Hz,
1H), 7.81 (s, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H),
7.48 (s, 1H), 7.43 (s, 1H), 7.41 (d, J = 15.5 Hz, 1H), 7.34 (d,
J = 7.6 Hz, 1H), 6.41 (d, J = 15.8 Hz, 1H), 3.92 (s, 3H), 1.21 (s, 1H).
HRMS calcd for C21H19N4O6 423.1642 (M+H)+, found 423.1645.
4.2.46. Carboxylic acid 38b
Following procedures similar to those described for carboxylic
acid 38a, ethyl ester 37b (210 mg, 0.48 mmol) in THF (6 mL) was
treated with methanol (3 mL) and NaOH (0.29 g in 6 mL water)
to give carboxylic acid 38b as a light-yellow solid (150 mg, 77%).
Mp = 188.5–189.3 °C. 1H NMR (DMSO-d6, 600 MHz): d 12.44 (br s,
1H), 10.99 (s, 1H), 10.91 (s, 1H), 8.37 (s, 1H), 8.09 (s, 1H), 7.89 (d,
J = 7.9 Hz, 1H), 7.81 (s, 1H), 7.67 (d, J = 7.9 Hz, 2H), 7.65 (d,
J = 7.6 Hz, 1H), 7.53 (d, J = 16.4 Hz, 1H), 7.47 (s, 1H), 7.42 (d,
J = 7.3 Hz, 1H), 6.43 (d, J = 15.8 Hz, 1H), 3.95 (s, 3H). 13C NMR
(DMSO-d6, 150 MHz): d 166.9, 165.2, 155.3, 152.2, 149.7, 146.8,
4.3. Biological evaluation
4.3.1. IMPDH inhibition
IMPDH inhibition assays were performed as previously de-
scribed.38 Briefly, assays were set up in duplicate using IMPDH type
1 (150 nM) and type 2 (50 nM) and varying concentrations of

L. Chen et al. / Bioorg. Med. Chem. 18 (2010) 5950–5964
5963
inhibitor. IMPDH and inhibitors were added to 100
fer (50 mM Tris, pH 8.0, 100 mM KCl, 1 mM DTT, 100
100 M NAD) at 25 °C, mixed gently and the production of NADH
was monitored by following changes in absorbance at 340 nm on
a Molecular Devices M5e multimode plate reader. Steady state
velocities were used to determine IC50 and Kai pp values by fitting
the velocities versus inhibitor concentration to the sigmoidal con-
centration-response curve (variable slope) and the Morrison equa-
tion,39 respectively, using GraphPad Prism.
l
L reaction buf-
thank Dr. Courtney Aldrich for his help with the biological assays.
We also thank Dr. Yanli Xu for her help with HRMS characteriza-
tion of several compounds. The University of Minnesota Supercom-
puting Institute provided all the computational resources.
lM IMP,
l
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.081.
4.3.2. HDAC inhibition
References and notes
HDAC inhibition assays were performed using the HDAC Activ-
ity/Inhibitor Screening Assay Kit (Cayman Chemical) per the man-
ufacturer’s instructions. Inhibitors were suspended in either DMSO
or methanol. End point readings were used to determine IC50 val-
ues by fitting the fractional fluorescence versus inhibitor concen-
tration to the sigmoidal concentration-response curve (variable
slope) using GraphPad Prism. Curves were corrected for Auto-fluo-
rescence by making a standard concentration curve, substituting
compound for solvent using the background well conditions.
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l
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4.4. Computational modeling
All modeling was carried out using the Schrodinger modeling
package.40 The structure of HDAC1 was homology modeled based
on the solved X-ray crystallographic structure of histone deacetyl-
ase-like protein (HDLP) in complex with SAHA (PDB: 1C3S).37 The
initial sequence alignment of HDAC1-11 and HDLP using clustalW
showed HDAC2-3, 8 and HDLP have the highest sequence similar-
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(see Supplementary Fig. S1). The homology model of HDAC1 was
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and 18b into the NAD binding pocket of the solved X-ray crystallo-
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viously reported mode of binding of Vertex compound VX-497 in
H93A Cricetulus griseus IMPDH.14 Distance restraints between
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