Novel chimeric histone deacetylase inhibitors: A series of lapatinib hybrides as potent inhibitors of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and histone deacetylase activity

Article abstract of DOI:10.1021/jm100665z

Reversible lysine-specific acetylation has been described as an important posttranslational modification, regulating chromatin structure and transcriptional activity in the case of core histone proteins. Histone deacetylases (HDAC) are considered as a promising target for anticancer drug development, with 2a as pan-HDAC inhibitor approved for cutanous T-cell lymphoma therapy and several other HDAC inhibitors currently in preclinical and clinical development. Protein kinases are a well-established target for cancer therapy with the EGFR/HER2 inhibitor 5 approved for treatment of advanced, HER2 positive breast cancer as a prominent example. In the present report, we present a novel strategy for cancer drug development by combination of EGFR/HER2 kinase and HDAC inhibitory activity in one molecule. By combining the structural features of 5 with an (E)-3-(aryl)-N-hydroxyacrylamide motif known from HDAC inhibitors like 1 or 3, we obtained selective inhibitors for both targets with potent cellular activity (target inhibition and cytotoxicity) of selected compounds 6a and 6c. By combining two distinct pharmacologically properties in one molecule, we postulate a broader activity spectrum and less likelihood of drug resistance in cancer patients.

Full text of DOI:10.1021/jm100665z

8546 J. Med. Chem. 2010, 53, 8546–8555
DOI: 10.1021/jm100665z
Novel Chimeric Histone Deacetylase Inhibitors: A Series of Lapatinib Hybrides as Potent Inhibitors
of Epidermal Growth Factor Receptor (EGFR), Human Epidermal Growth Factor Receptor 2 (HER2),
and Histone Deacetylase Activity
Siavosh Mahboobi,*,† Andreas Sellmer,† Matthias Winkler,† Emerich Eichhorn,† Herwig Pongratz,† Thomas Ciossek,‡
Thomas Baer,‡ Thomas Maier,‡ and Thomas Beckers*,§
†Department of Pharmaceutical Chemistry I, University of Regensburg, D-93040 Regensburg, Germany, ‡Nycomed GmbH, Therapeutic Area
Oncology, Byk-Gulden Strasse 2, D-78467 Konstanz, Germany, and §Oncotest GmbH, Institute for Experimental Oncology, Am Flughafen 12-14,
D-79108 Freiburg, Germany
Received June 18, 2010
Reversible lysine-specific acetylation has been described as an important posttranslational modifica-
tion, regulating chromatin structure and transcriptional activity in the case of core histone proteins.
Histone deacetylases (HDAC) are considered as a promising target for anticancer drug development,
with 2a as pan-HDAC inhibitor approved for cutanous T-cell lymphoma therapy and several other
HDAC inhibitors currently in preclinical and clinical development. Protein kinases are a well-
established target for cancer therapy with the EGFR/HER2 inhibitor 5 approved for treatment of
advanced, HER2 positive breast cancer as a prominent example. In the present report, we present a
novel strategy for cancer drug development by combination of EGFR/HER2 kinase and HDAC
inhibitory activity in one molecule. By combining the structural features of 5 with an (E)-3-(aryl)-N-
hydroxyacrylamide motif known from HDAC inhibitors like 1 or 3, we obtained selective inhibitors for
both targets with potent cellular activity (target inhibition and cytotoxicity) of selected compounds 6a
and 6c. By combining two distinct pharmacologically properties in one molecule, we postulate a broader
activity spectrum and less likelihood of drug resistance in cancer patients.
Introduction
The reversible lysine-specific acetylation of core histone pro-
teins has been well described as a mechanism to regulate
chromatin structure, transcription factor accessibility, and
finally transcriptional activity. Negatively charged DNA
binds to positively charged lysine residues in the amino-
terminal tails of histones, which is abrogated by lysine
N-terminal acetylation.7 HDAC isoenzymes are essential for
deacetylating histone proteins, allowing tight binding of DNA
and formation of a closed, transcriptionally inactive chroma-
tin conformation. Inhibitors of HDAC reactivate gene
expression but also affect acetylation of nonhistone targets
which have been described.8 On a cellular level, HDACi’s
reactivate tumor suppressor genes exhibit cell cycle arresting
and pro-apoptotic properties.9 Several HDACi’s exhibiting
the hydroxamic acid or benzamide motif as a Zn2þ complex-
ing headgroup are currently in preclinical and clinical devel-
opment (Figure 1, compounds 1-4) as represented by the
hydroxamic acid analogues “1 (PXD101)”,10 2a (phase II),
“2b (PCI(CRA)-24781)”11 (phase II), “3 (LBH589),”12or the
benzamide analogue “4 (SNDX/MS 275)” (phase II),9 to
name just a few.
Carcinogenesis and tumor suppression are controlled by
genetic as well as epigenetic events. Unlike genetic aberrations,
epigenetic changes in cancer cells such as DNA hypermethyla-
tion and histone deacetylation are largely reversible alterations.
Histone deacetylase (HDACa) class 1 and 2 enzymes with 11
isoforms are considered as promising targets for anticancer
drug development.1 Various inhibitors of HDAC class 1 and/or
class 2 enzymes (HDACi) are currently evaluated in clinical
phase 1 and 2 trials.2-6
Substrates of HDAC enzymes are core histone proteins
H2A/B, H3, and H4, highly conserved proteins complexed
with DNA and building up chromatin. Histone proteins are
post-translationally modified by various means, including
methylation and acetylation of N-terminal lysine residues.
*To whom correspondence should be addressed. For medicinal
chemistry (S.M.): phone, (þ49) (0) 941 943 4824; fax, (þ49) (0) 941-
9431737; E-mail, siavosh.mahboobi@chemie.uni-regensburg.de.
For pharmacology (T.B.): phone, (þ49) (0) 761 5155916; fax, (þ49)
(0) 761 5155955; E-mail, Thomas.Beckers@oncotest.de.
a Abbreviations: AMC, 7-amino-4-methylcoumarin; ATCC, American
Type Culture Collection; BOP, 1-benzotriazolyloxy-tris-(dimethyl-
amino)phosphonium hexafluorophosphate; CDK, cell division kinase;
CML, chronic myeloid leukemia; HDACi, histone deacetylase inhibitor;
HDAC, histone deacetylase; nd, not determined; InsR, insulin receptor;
NSCLC, nonsmall cell lung cancer; PDGFR, platelet derived growth
factor receptor; Plk1, polo-like kinase 1; PKA, protein kinase A; rHDAC,
recombinant HDAC; SAHA, suberoylanilide hydroxamic acid; SDS,
sodium dodecylsulfate; SDS-PAGE, SDS polyacrylamide gel electrophor-
esis; SMI, small molecule inhibitor; TSA, trichostatin A.
Inhibitors of protein tyrosine or serine/threonine kinases
are another well described drug class. The human epidermal
growth factor receptor (EGFR, HER1) and the family mem-
ber HER2 (c-erbB2) have been linked to various human
malignancies like breast, head and neck, pancreatic, colon,
gastric, and nonsmall cell lung cancer. Small synthetic inhibi-
tors (SMI) as well as monoclonal antibodies have been
developed for cancer therapy. The SMI 5 is one example
r
pubs.acs.org/jmc
Published on Web 11/16/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8547
Figure 1. Chemical structures of selected HDACi’s in preclinical and clinical developments (1-4), 5, and novel chimerically designed
compounds (6-8).
approved for treatment of HER2 positive advanced breast
cancer in combination with Capecitabine.13
catalytic reduction with hydrogen over sulfided platinum.16
The nitro-precursor 14 itself was prepared according to
Wallace et al.16 by SNAr-reaction of 2-chloro-1-fluoro-4-ni-
trobenzene and (3-fluorophenyl)methanol in DMF solution.
The desired furanyl- and thiophenyl- and phenyl-N-hy-
droxy-acrylamides 6a-6d (Scheme 2) were obtained from 16
by Suzuki coupling with the respective formylboronic acids
17a-17d analogous to Hosoya,17 Wittig olefination,18 and de-
protection of the resulting acrylic acid tert-butyl esters (19a-19d)
with trifluoroacetic acid. Amidation of the acrylic acids 20a-20d
with commercially available NH2OTHP (O-(tetrahydropyran-
2-yl)hydroxylamine) by use of BOP (1-benzotriazolyloxy-tris-
(dimethylamino)phosphoniumhexafluorophosphat) as cou-
pling reagent19 and cleavage of the tetrahydro-pyran-2-yl
protected acrylamides 21a-21d led to the N-hydroxy-acryl-
amides 6a-6d.
In this report, we present a novel strategy for cancer drug
development, combining the pharmacological activity of
EGFR/HER2 kinase inhibition with inhibition of HDAC
class 1/2 enzymes. As a scaffold, 5 was selected to include a
HDAC inhibitory headgroup (“warhead”) in order to com-
bine, in general, the antiproliferative activity of EGFR/HER2
inhibition with the pro-apoptotic, transcriptional reprogram-
ming activity of a HDACi. Of the various HDAC inhibitory
headgroups, we choose the hydroxamic acid and benzamide
motifs for combination with the [3-chloro-4-(3-fluorobenzyl-
oxy)phenyl]quinazolin-4-yl-amine core structure of 5 to ob-
tain chimerically designed compounds.
Chemistry
Analogously, the carboxylic acid (2-aminophenyl)amides 7a
and 7b as well as the carboxylic acid hydroxyamides 8a and 8b
were prepared as shown in Scheme 3 by amidation of the
carboxylic acids with NH2OTHP or the mono protected phe-
nylendiamine 25, followed by deprotection with trifluoroacetic
acid (for details, see Experimental Section). The mono protected
phenylendiamine 25 itself was easily accessible by reaction of
o-phenylendiamine (24) with BOC2O in THF solution.20
The (E)-N-(2-aminophenyl)-3-(furan-2-yl)acrylamide 7c as
well as the N-(2-aminophenyl)cinnamamides 7d and 7e were
The synthetic route to obtain the desired target compounds
is given in the following (Scheme 1): ring closure of 2-amino-5-
iodo benzoic acid (9) according to Ninishino14 formed the 1H-
quinazolin-4-one system 10. Chlorination with phosphorus
oxychloride and one-pot SN-reaction15 by 3-chloro-4-(3-
fluoro-benzyloxy)phenylamine (15), yielded the desired [3-
chloro-4-(3-fluorobenzyloxy)phenyl]-(6-iodo quinazolin-4-
yl)amine (16) as a central intermediate. The aniline derivative
15 was easily accessible from its nitro-precursor 1416 by

8548 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Mahboobi et al.
Scheme 1. Preparation of [3-Chloro-4-(3-fluorobenzyloxy)phenyl]-(6-iodoquinazolin-4-yl)amine (16) As a Central Intermediate
obtained in a similar manner by amidation19 of the acrylic
acids 20a-20d withthe mono protected phenylendiamine2520
and deprotection of the tert-butyl carbamates (28a, 28c, and
28d) with trifluoroacetic acid (Scheme 4). Selective catalytic
hydrogenation in presence of a benzyloxy- and a chloroaryl-
group by use of PtO2 yielded the N-(2-aminophenyl)-3-phe-
nylpropanamide 7f without additional cleavage.
(Table 2). The protein kinases PDGFRβ, InsR, Abl, CDK2,
PKA, and Plk1 were chosen to evaluate kinase target selectivity.
A change of the substitution patterns from meta (6c) to
para substitution drastically reduced HDAC inhibitory po-
tency, as seen for 6d and the bioisosteric built up thiophene
derivative 6b, while inhibition of EGFR and HER2 ki-
nases was not affected. In modifications bearing the N-(2-
aminophenyl)-benzamide substituent as a HDACi motif
taken from 4, the potent inhibition of EGFR and HER2
kinase activity and selectivity are conserved. However, no
significant HDAC inhibitory activity is evident for benz-
amide analogues 7a and 7b, structurally related to 8a and 8b.
To investigate the effect of the system vinylogous to
benzamides, included in the HDAC-inhibitory structures
6a and 6c, we extended the study to the vinylogous benz-
amides 7c-7e as well as to benzamide 7f and the arylhydrox-
amates 8a and 8b. In the resulting benzamide and aryl-
hydroxamate series, EGFR and HER2 kinases were strongly
inhibited (Table 2). Regarding inhibition of HDAC en-
zymes, no activity is exhibited by benzamides 7a-7f, inde-
pendent from the substitution patterns (ortho or para), the
nature of the A-ring system (Figure.1) in the substructure of
5, or from the linker between the benzamide structure and
the A-ring system. This result is in contrast to our findings,
published earlier, where combination of a benzamide motif
with the imatinib pharmacophore led to chimeric HDAC
class I inhibitors.21 Weak inhibition of HeLa nuclear extract
HDAC, rHDAC 1, 3, and 8 class I enzymes activity with
stronger inhibition of rHDAC6 as a class 2 representative
was observed for the hydroxamate 8b. These compounds
were potent inhibitors of EGFR and HER2 with presumably
less selectivity (Table 2).
Results and Discussion
The synthesized chimeric compounds were characterized in
biochemical assays for HDAC as well as protein-kinase
inhibitory activity. In addition, cellular target specific activ-
ity was assessed in respective cancer cell lines. Finally, the
cytotoxic activity was determined in EGFR or HER2 over-
expressing cancer cell lines for selected analogues.
Pharmacological Profile in Biochemical and Cellular, Tar-
get-Specific Assays. The in vitro data for inhibition of HeLa
nuclear extract HDAC activity, a mixture of HDAC iso-
enzymes 1, 2, 3, 5, and 8, recombinant rHDAC 1, 3, and 6 as
well as cellular data for induction of histone H3 hyperacety-
lation are compiled in Table 1. As references, 5 as well as
2a and 4 are included as representatives for hydroxamate
and benzamide analogues. Combining the structural features
a [3-chloro-4-(3-fluorobenzyloxy)phenyl]quinazolin-4-yl-
amine (substructure of 5 in Figure.1) with an (E)-3-(aryl)-N-
hydroxyacrylamide motif taken from 1 (Figure 1; compounds
6a-6c) leads to inhibition of nuclear extract HDAC, rHDAC1
and rHDAC6 in the submicromolar (6a) or even nanomolar
range (6c) as well as cellular histone H3 hyperacetylation.
Compounds 6a to 6c also display very potent and specific
inhibition of EGFR and HER2 kinase activity in biochemical
assays, with 6c being only 2- to 5-fold less potent as Lapatinib

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8549
Scheme 2. Synthesis of N-Hydroxyacrylamides 6a-6d
Cytotoxic Properties toward EGFR and HER2 Expressing
Cancer Cell Lines. All compounds were tested for cytoxicity
toward cancer cell lines either with EGFR (A431, Cal27) or
HER2 (SKBR3, SKOV3) overexpression or of relevance for
HDAC inhibitor testing (HeLa, A549). The data are sum-
marized in Table 3, showing highly potent activity of 5 in the
Cal27, SKBR3, and SKOV3 cell lines with a bottom plateau
of the concentration-effect curve between 30% and 70%. In
the EGFR overexpressing cal27 head and neck cancer cell
line, 5 also strongly inhibited cellular EGF stimulated EGFR
phosphorylation (IC50 < 32 nM) with no effect on histone
H3 acetylation (Figure 2B). N-hydroxyacrylamide ana-
logues 6a and 6c were most interesting from the initial in vitro
profiling experiments. Whereas 6a potently inhibited EGF
breast cancer, leads to chimeric compounds with 6c acting as
truly pharmacologically chimeric compound. In biochemical
assays, this prototypic analogue shows selective and potent
inhibiton of EGFR/HER2 as well as HDAC enzymatic
activity. In cellular assays exemplified by the head and neck
cell line Cal27, EGFR phosphorylation and histone H3
acetylation are concentration dependently inhibited and in-
duced, respectively. Between 0.1 and 1 μM concentration,
maximal target inhibitory effects are visible, correlating with
an IC50 of 0.59 μM in the cytotoxicity assay. However, trans-
ferring the HDAC inhibitory benzamide motif so far resulted
in compounds with conserved kinase but no HDAC inhibi-
tory activity. By combination of two pharmacologically dis-
tinct properties in one molecule, we postulate a broader
activity spectrum with less likelihood of drug resistance. Thus,
chimeric HDAC-kinase inhibitors might constitute a new
class of experimental cancer drugs worth to be studied in
more detail.
stimulated EGFR phosphorylation in Cal27 cells (IC50
<
32nM), 6c was less potent (IC50 ≈ 0.5 μM) (Figure 2A).
Nevertheless, 6c was nicely inducing histone H3 hyperace-
tylation at 1 μM, behaving as a cellularly active, pharmaco-
logically bifunctional compound. In the cytotoxicity profile,
6c was broadly active with IC50 values e1 μM (Table 3) and a
complete cell kill at higher concentrations.
Experimental Section
Biological Methods. Biochemical HDAC Assays. For rHDAC1
and rHDAC6 expression, a clonal HEK293 (ATCC CRL1573)
human kidney cell line expressing the human rHDAC1 isoenzyme
bearing a C-terminal Flag epitope was provided by E. Verdin, The
Gladstone Institute/San Francisco, CA, USA. Human HDAC3-
Flag was coexpressed with the SMRT DAD domain in Sf21
insect cells, whereas human HDAC8-Flag was expressed
Conclusions
Transferring the (E)-3-(aryl)-N-hydroxyacrylamide motif
of 5, which is an EGFR/HER protein kinase inhibitor and a
drug approved for treatment of advanced, HER2 positive

8550 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Mahboobi et al.
Scheme 3. Synthesis of Carboxylic Acid (2-Aminophenyl)amides (7a, 7b) and Carboxylic Acid Hydroxyamides (8a, 8b)
Scheme 4. Synthesis of Phenylaminoacrylamides (7c-7e) and of N-(2-Aminophenyl)-3-(4-(4-(3-chloro-4-(3-fluorobenzyloxy)-
phenylamino)quinazolin-6-yl)phenyl)propanamide (7f)
without cofactor in Sf21 cells. The rHDAC proteins were
purified by M2-affinity gel chromatography according to the
manufacture protocol (Sigma no. A2220). Purified protein
samples were routinely analyzed by SDS-PAGE (12.5% or
10% Laemmli gels) followed by Coomassie stain and Western
blotting using a FLAG-specific antibody (anti M2-POX antibody,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8551
Table 1. IC50/EC50 Values for Chimeric Lapatinib-HDAC Inhibitors As Tested for Inhibition of HeLa Nuclear Extract HDAC Activity, Recombinant
HDAC Isoenzymes, and Induction of Cellular Histone H3 K9þ14 Hyperacetylation a
a In general, experiments were done in replicate and mean values are shown (data for 4 and 2a from ref 22).
Sigma no. A8592) followed by ECL-detection (GE Health-
care). In addition, protein batches were analyzed by Western
blotting for various HDAC isoenzymes including HDAC1, 3,
6, and 8.
The biochemical HDAC activity assay was essentially done as
described by Wegener et al.23
Biochemical Protein Kinase Assays. Active kinase proteins
were either obtained from commercial suppliers (ProQinase,

8552 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Mahboobi et al.
Table 2. Inhibition of Selected Recombinant Protein Tyrosine and Serine/Threonine Kinases in Biochemical Assays, Namely Epidermal Growth
Factor Receptor (EGFR), Human EGFR-Like Receptor 2 (HER2), Platelet-Derived Growth Factor Receptor β (PDGF-Rβ), Insulin Receptor (InsR),
Abl1, Cell Division Kinase 2 (CDK2), Protein Kinase A (PKA), and Polo-Like Kinase 1 (PlK1)a
EGFR IC50
[ μM]
HER2 IC50
[ μM]
PDGF-Rβ IC50
[ μM]
InsR IC50
[ μM]
Abl1 IC50
[ μM]
CDK2 IC50
[ μM]
PKA IC50
[ μM]
PlK1 IC50
[ μM]
no.
6a
6b
6c
6d
7a
7b
7c
7d
7e
7f
0.0025
0.0048
0.018
0.0041
0.0035
0.011
3.3
1.1
2.5
2.3
4.0
2.3
3.9
2.5
2.7
4.7
1.0
1.8
8.50
7.34
1.9
3.6
3.7
1.3
2.5
5.5
2.8
3.0
7.1
1.4
1.7
17.0
7.7
2.7
5.3
4.9
4.5
6.0
4.9
2.9
4.9
6.9
1.7
1.7
23.0
0.44
0.44
0.92
0.85
15.0
2.1
120
35
12.0
2.7
>100
>100
>100
>100
>100
>100
>100
>100
85
6.80
9.1
0.018
0.012
0.0029
0.0028
0.026
0.0057
0.0049
0.0087
0.014
3.9
5.6
1.9
8.5
5.5
0.049
0.029
0.015
0.85
1.2
0.015
6.2
0.0047
0.0020
0.0035
0.0045
1.4
27
8a
8b
5
0.0039
0.0022
0.0029
0.46
0.2
2.0
2.9
>100
>100
11.0
>100
a In general, experiments were done in replicate and mean IC50 values are shown.
Freiburg/Germany; InvitroGen/Panvera, Carlsbad, CA, USA)
Procedures and Analytical Data. 3-Chloro-4-(3-fluorobenzy-
or prepared in-house. Experimental details of the flash-plate
based radioactive enzyme assayhave been described previously.21
Cellular Histone H3 Hyperacetylation Assay. To assess the
cellular efficacy of a histone deacetylase inhibition, an assay was
set up for use on the Cellomics “ArrayScan II” platform for a
quantitative calculation of histone acetylation as described.24
Cellular Proliferation Assay. The antiproliferative activity of
selected compounds was evaluated using the tumor cell lines
HeLa (cervical carcinoma, ATCC CCL-2), A549 (NSCLC,
ATCC CCL-185), SKBR-3 (breast carcinoma, ATCC HTB-30),
SKOV-3 (ovarian carcinoma, ATCC HTB-77), Cal27 (tongue
carcinoma, ATCC CRL-2095), and A-431 (vulva carcinoma,
ATCC CRL-2592). For quantification of cellular proliferation/
cytotoxicity, the Alamar Blue (Resazurin) cell viability assay was
applied.25
Western Blot Analysis. For Western blot analysis, about
4 ꢀ 105 Cal27 cells/well in 6-well cell culture plates were treated
with the test compounds for 16 h. Next, cells were stimulated
with 100 ng/mL recombinant EGF for 5 min at room tempera-
ture before cell lysis. Cells were lysed in lysis buffer (50 mM Tris
HCl pH8, 150 mM NaCl, 1v/v NP-40, 0.5% w/v sodium
desoxycholate, 0.2% w/v disodium dodecylsulfate (SDS),
0.02% w/v NaN3, 1 mM sodium vanadate, 20 mM NaF,
100 μg/mL PMSF, 10 mM sodium pyrophosphate, protease
inhibitor mix/Roche and 50U/mL Benzonase) at 4 °C. Respec-
tive equal amounts of protein were separated by SDS-PAGE
before transfer to polyvinylidendifluoride (PVDF) membrane
(Biorad art. no. 162-0177) by semidry blotting. The following
antibodies were used: monoclonal mouse antibody specific for
β-actin (clone AC-12, Sigma art. no. A-5441), phosphorylated
EGFR (Y1068, Cell Signaling), EGFR (Upstate), acetylated
histone H3 (Cell Signaling), and histone H3 (Cell Signaling). As
secondary antibodies, goat antirabbit IgG-HRP conjugated
(Biorad: 170-6515), goat-antisheep (Santa Cruz) and goat-
antimouse IgG-HRP conjugated (Biorad: 170-6516) were used.
Chemical Procedures. General. NMR spectra were recorded
with a Bruker Avance 300 MHz spectrometer at 300 K, using
TMS as an internal standard. IR spectra (KBr or pure solid)
were measured with a Bruker Tensor 27 spectrometer. Melting
loxy)phenylamine (15). Preparation analogous lit.16 as follows:
2-chloro-1-(3-fluorobenzyloxy)-4-nitrobenzene (14) (20.0 g;
70.9 mmol) was dissolved in THF (300 mL), dry sulfided plati-
num on carbon (Aldrich) (4.00 g) were added and the mixture
was stirred under a hydrogen atmosphere (10 atm) overnight.
The platinum was filtered off over celite, washed with THF, the
solvent removed under reduced pressure and the residue treated
with light petrol to afford the title compound as an analytically
pure solid. Yield (17.49 g; 98%). 1H NMR (DMSO-d6) δ (ppm)
4.98 (s, 2H, exchangeable), 5.04 (s, 2H), 6.54 (dd, 1H, J = 8.8 Hz,
4J = 2.6 Hz), 6.74 (d, 1H, 4J = 2.6 Hz), 6.92 (d, 1H, J = 8.8 Hz),
7.09-7.15 (m, 1H), 7.25-7.30 (m, 2H), 7.37-7.45 (m, 1H).
[3-Chloro-4-(3-fluorobenzyloxy)phenyl]-(6-iodoquinazolin-
4-yl)amine (16).26 Preparation analogous to lit.15 as follows:
To a mixture of 6-iodo-1H-quinazolin-4-one (10) (6.80 g;
25.0 mmol), toluene (5.0 mL), and POCl3 (27.5 mmol; 2.60 mL),
triethylamine (27.5 mmol; 3.81 mL) was added carefully. The
mixture was heated to 80 °C for 2 h, cooled to room tempera-
ture, a solution of 3-chloro-4-(3-fluorobenzyloxy)phenylamine (15)
(27.50 mmol; 6.92 g) in 2-butanone (20.0 mL) added, and the
mixture stirred at 80 °C for another hour. The mixture was cooled
to 0 °C, and the yellow precipitate was filtered off and added to a
NaOH solution (1N; 150 mL) by stirring. After 30 min, the yellow
solid was filtered off, washed with water and a small amount of
acetone, and dried in vacuo. Yield (8.38 g; 66%) analytical pure
1
sample. H NMR (DMSO-d6) δ (ppm) 5.26 (s, 2H), 7.15-7.22
(m, 1H), 7.27 (d, 1H, J = 9.1 Hz), 7.29-7.35 (m, 2H), 7.43-7.51
(m, 1H), 7.56 (d, 1H, J = 8.8 Hz), 7.74 (dd, 1H, J = 9.1 Hz, 4J =
2.5 Hz), 8.02 (d, 1H, J = 2.5 Hz), 8.12 (dd; 1H, J = 8.8 Hz,
4
4
4J = 1.7 Hz), 8.62 (s, 1H), 8.96 (d, J = 1.7 Hz), 9.90 (s, 1H,
exchangeable).
General Procedure for Suzuki Coupling. Preparation ana-
logous to Hosoya.17 A mixture of 16 (5.00 g; 9.88 mmol), the re-
spective arylboronic acid (17a, 17b, 22b Aldrich; 17c, 17d Alfa
Aesar; 22a Rare Chemicals) (13.1 mmol), (PPh3)2PdCl2 (0.35 g;
0.5 mmol), DME (30 mL), ethanol (20 mL), and 2 M aqueous
Na2CO3 (30 mL) was heated at 60 °C for 3 h. After cooling to
room temperature, the crude product precipitated as a yellow
solid. It was removed by filtration, washed with water, and dried
in vacuo overnight. Crystallization from acetone afforded the
title compound in sufficient purity for further synthesis. An
analytical sample was obtained by column chromatography
(SiO2; CH2Cl2/ethyl acetate = 4:1).
€
points were determined with a Buchi B-545. MS spectra were
measured with a Finnigan MAT 95 (EI, 70 eV), or with a
Finnigan Thermo Quest TSQ 7000 (ESI) (DCM/MeOH þ
10 mmol/L NH4Ac), respectively. All reactions were carried
out under nitrogen atmosphere. Elemental analyses were per-
formed by the Analytical Laboratory of the University of
Regensburg and are in a range of (0.4% of the calculated
values if not given otherwise, ensuring a purity g95%. Chemical
names were created using ChemBioDraw Ulta 11.0 software.
Furane- and Benzene-carbaldehydes (18a, 18c). 5-{4-[3-Chloro-
4-(3-fluorobenzyloxy)phenylamino]quinazolin-6-yl}furan-2-car-
1
baldehyde (18a).26 Yield (3.65 g, 78%); mp 229.8-234.1 °C. H
NMR (DMSO-d6) δ (ppm) 5.26 (s, 2H), 7.17 (dt, 1H, J = 2.5 Hz,
J = 8.5), 7.26-7.34 (m, 3H), 7.40 (d, 1H, J = 3.6 Hz), 7.43-7.50

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8553
Table 3. Cytotoxicity of the Chimeric HDAC Inhibitors Towards Selected Cancer Cell Lines, Namely A549 NSCL and HeLa Cervical Cancer (Source
for HDAC Nuclear Activity), A431 Epidermoid Carcinoma, and Cal27 Head and Neck Cancer (EGFR Overexpression) and SKOV3 Ovarian and
SKBR3 Breast Cancer (HER2 Overexpression)a
a In general, experiments were done in replicate and mean IC50 values are shown (data for 2a from Beckers et al., 200722).
(m, 1H), 7.68-7.74 (m, 2H), 7.84 (d, 1H, J = 8.5 Hz), 7.98 (d, 1H,
J = 2.5 Hz), 8.28 (dd, 1H, J = 1.1 Hz, J = 8.8 Hz), 8.58 (s, 1H),
8.95 (d, 1H, J = 0.8 Hz), 9.66 (s, 1H), 10.10 (s, 1H, exchangeable).
J = 8.7 Hz), 7.97 (td, 1H, J = 1.2 Hz, J = 7.6 Hz), 8.01 (d, 1H,
J = 2.6 Hz), 8.21 (m, 2H), 8.38 (t, 1H, J = 1.6 Hz), 8.60 (s, 1H),
8.85 (d, 1H, J = 1.6 Hz), 9.94 (s, 1H, exchangeable), 10.14
(s, 1H). EI-þMS (70 eV) m/z (%): 483 (18) [Mþ 3 ], 374 (100)
[M-C7H6F3 ] . IR (KBr): 3380, 2729, 1696 cm-1. Anal. (C28-
þp ESI m/z (%): 476 [M þ Hþ]þ (37); 474 [M þ H]þ (100). -p
3
ESI m/z (%): 474 [M - Hþ]- (37), 472 [M - H]- (100). IR (KBr):
3399, 1673 cm-1
.
H19ClFN3O2 1/5H2O): C, H, N.
3
3-{4-[3-Chloro-4-(3-fluorobenzyloxy)phenylamino]quinazolin-
6-yl}benzaldehyde (18c). Yield (2.73 g, 54%) yellow crystals; mp
Acrylic Acid tert-Butyl Esters (19a-d). A mixture of the
respective aryl-2-carbaldehydes 18a-18d (1.0 mmol), (tert-bu-
toxycarbonylmethyl)triphenylphosphonium chloride (0.56 g;
1.02 mmol), NaOH (0.08 g; 2.04 mmol), and NEt3 (0.3 g;
3.06 mmol) in CH2Cl2 (100 mL) and water (2.04 mL) was stirred
at room temperature overnight. The organic layer was separated,
1
243.2-247.5 °C. H NMR (DMSO-d6) δ (ppm) 5.25 (s, 2H),
7.18 (dt, 1H, J = 2.2 Hz, J = 8.3 Hz), 7.28 (d, 1H, J = 9.1 Hz),
7.33 (m, 2H), 7.47 (dt, 1H, J = 6.1 Hz, J = 8.0 Hz), 7.74 (dd, 1H,
J = 2.2 Hz, J = 6.7 Hz), 7.78 (t, 1H, J = 5.4 Hz), 7.86 (d, 1H,

8554 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Mahboobi et al.
Figure 2. Western blot analysis of Cal27 head and neck cancer cells treated for 16 h with compounds 5 (A) and 6c or 6a (B). As a control, cells
treated with 1 μM 5, 6a, or 6c were documented by phase contrast microscopy in (C).
subsequently washed with satd NH4Cl solution (3 ꢀ 50 mL),
dried (Na2SO4), filtered, and concentrated under reduced pres-
sure. The crude product was purified by column chromato-
graphy (DCM/EE = 1:1) and precipitated by addition of light
petrol.
7.41-7.50 (m, 1H), 7.46 (d, 1H, Jtrans = 15.9 Hz), 7.73 (dd, 1H,
J = 2.5 Hz, J = 8.9 Hz), 7.82 (d, 1H, J = 8.8 Hz), 8.00 (d, 1H, J =
2.5 Hz), 8.27 (dd, 1H, J = 1.6 Hz, J = 8.8 Hz), 8.57 (s, 1H), 8.86
(d, 1H, J = 1.4 Hz), 9.96 (s, 1H, exchangeable), 12.45 (s, 1H,
exchangeable). þp ESI m/z (%): 518 [M þ Hþ] (37), 516 [M þ Hþ]
(100). -p ESI m/z (%): 516 [M - Hþ]- (40), 514 [M þ Hþ]þ (100).
IR (KBr): 3406, 1673 cm-1. Anal. (C28H19ClFN3O4) C, H, N.
3-{3-[4-(3-Chloro-4-(3-fluorobenzyloxy)phenylamino]quinazolin-
6-yl}-phenyl)acrylic Acid (20c). Yield (0.78 g, 99%) orange crystals;
mp 275.1-275.2 °C. 1H NMR (DMSO-d6) δ (ppm) 5.29 (s, 2H),
6.73 (d, 1H, J = 16.0 Hz), 7.19 (dt, 1H, J = 2.2 Hz, J = 8.3 Hz),
7.33 (m, 3H), 7.48 (m, 1H), 7.62 (t, 1H, J = 7.7 Hz), 7.71 (m, 2H),
7.80 (d, 1H, J = 7.8 Hz), 7.92 (m, 2H), 7.96 (d, 1H, J = 2.5 Hz),
8.19 (s, 1H), 8.42 (dd, 1H, J = 1.6 Hz, J = 8.8 Hz), 8.83 (s, 1H),
8.94 (d, 1H, J = 1.2 Hz), 10.88 (s, 1H, exchangeable), 12.58 (s, 1H,
exchangeable). ES-MS (DCM/MeOH þ 10 mmol/L NH4Ac)
3-{5-[4-(3-Chloro-4-(3-fluorobenzyloxy)phenylamino]quinazolin-
6-yl}furan-2-yl)acrylic Acid tert-Butyl Ester (19a). Yield (0.43 g,
1
76%) yellow crystals; mp 201.3-201.4 °C. H NMR (DMSO-
d6) δ (ppm) 1.50 (s, 9H), 5.28 (s, 2H), 6.50 (d, 1H, Jtrans
=
15.6 Hz), 7.13 (d, 1H, J = 3.6 Hz), 7.19 (dt, 1H, J = 2.4 Hz, J =
8.6 Hz), 7.28-7.36 (m, 4H), 7.44 (d, 1H, Jtrans = 15.6 Hz),
7.45-7.52 (m, 1H), 7.72 (dd, 1H, J = 2.5 Hz, J = 8.8 Hz), 7.82
(d, 1H, J = 8.8 Hz), 7.99 (d, 1H, J = 2.5 Hz), 8.29 (dd, 1H, J =
1.5 Hz, J = 8.6 Hz), 8.57 (s, 1H), 8.87 (d, 1H, J = 1.1 Hz), 9.97
(s, 1H, eþxchangeable). þp ESI m/z (%): 574 [M þ Hþ] (39), 572
[M þ H ] (100%). -p ESI m/z (%): 572 [M - Hþ]- (40), 570
[M - Hþ]-. IR (KBr): 2977, 1697 cm-1. Anal. (C32H27ClFN3-
O4): C, H, N.
m/z (%): 526 (100) [M þ Hþ]þ. IR (KBr): 3442, 2934, 1671 cm-1
.
Anal. (C30H21ClFN3O3 3/4TFA) C, H, N.
3
3-{-[4-(3-Chloro-4-(3-fluorobenzyloxy)phenylamino]quinazolin-
6-yl)phenyl)acrylic Acid tert-Butyl Ester (19c). Yield (2.30 g,
76%) colorless crystals; mp 202.9-203.0 °C. 1H NMR (DMSO-
d6) δ (ppm) 1.51 (s, 9H), 5.27 (s, 2H), 6.73 (d, 1H, J = 16.0 Hz),
7.19 (dt, 1H, J = 2.2 Hz, J = 8.2 Hz), 7.32 (m, 3H), 7.48 (m, 1H),
7.60 (t, 1H, J = 7.7 Hz), 7.69 (d, 1H, J = 16.0 Hz), 7.76 (m, 2H),
7.87 (d, 1H, J = 8.7 Hz), 7.94 (d, 1H, J = 7.9 Hz), 8.03 (d, 1H,
J = 2.6 Hz), 8.20 (m, 1H), 8.29 (dd, 1H, J = 1.7 Hz, J = 8.7 Hz),
8.60 (s, 1H), 8.83 (d, 1H, J = 1.5 Hz), 9.90 (s, 1H, exchangeable).
EI-MS (70 eV) m/z (%): 581 (18) [Mþ 3], 236 (100) [M-C11H14F3]þ.
IR (KBr): 3379, 2982, 1676 cm-1. Anal. (C34H29ClFN3O3) C, H, N.
Synthesis of Acrylic Acids (20a-20d) by Cleavage of the Acrylic
Acid tert-Butyl Esters. The respective acrylic acid tert-butyl ester
(3.50 mmol) was dissolved in 10 mL TFA. The mixture was
stirred at 20 °C for 30 min, added to water (50 mL) by stirring,
and the yellow precipitate filtered off, washed with water, and
dried in vacuo.
General Procedure for Amidation by Use of BOP as Coupl-
ing Reagent: Preparation of compounds 21a-21d, 26a-26b and
27a-27b. A mixture of the respective carboxylic acid (1.15 mmol),
BOP (0.53 g; 1.20 mmol), the respective amine (2-aminophenyl)-
carbamic acid tert-butyl ester20 (25) or O-tetrahydropyran-2-
yl)hydroxylamine (Aldrich)) (1.15 mmol), and NEt3 (0.24 g; 2.40
mmol) in DMF (10 mL) was stirred at room temperature for 12 h.
The solution was added to water (50 mL) by stirring, the pre-
cipitating product filtered off, washed with water, and dried in
vacuo. Crystallization from the respective solvent given afforded
the pure compound as yellow crystals.
(E)-3-(5-(4-(3-Chloro-4-(3-fluorobenzyloxy)phenylamino)quina-
zolin-6-yl)furan-2-yl)-N-(tetrahydro-2H-pyran-2-yloxy)acrylamide
(21a). Crystallization from acetone. Yield: (0.55 g, 78%); mp
232.8-232.9 °C. 1H NMR (DMSO-d6) δ (ppm) 1.54 (s, br., 3H),
1.70 (s, br, 3H), 3.54 (d, br., 1H, J = 11.0 Hz), 3.92-4.02 (m, br,
1H), 4.93 (s, 1H), 5.28 (s, 2H), 6.55 (d, 1H, Jtrans = 15.6 Hz), 7.04
(d, 1H, J = 3.3 Hz), 7.16-7.52 (m, 7H), 7.74 (dd, 1H, J = 8.5 Hz,
J = 1.4 Hz), 7.84 (d, 1H, J = 8.8 Hz), 8.01 (d, 1H, J = 2.5 Hz),
8.22 (dd, 1H, J = 8.8 Hz, J = 1.4 Hz), 8.58 (s, 1H), 8.86 (s, 1H),
9.98 (s, 1H,exchangeable), 11.30 (s, 1H, exchangeable). þpESIm/z
(%): 617 [M þ Hþ] (39), 615 [M þ Hþ] (100). -p ESI m/z (%): 615
3-{5-[4-(3-Chloro-4-(3-fluorobenzyloxy)phenylamino]quinazolin-
6-yl}furan-2-yl)acrylic Acid (20a). Yield (0.18 g, 99%) yellow
crystals; mp 268.8-268.9 °C. 1H NMR (DMSO-d6) δ (ppm) 5.27
(s, 2H), 6.53 (d, 1H, Jtrans = 15.9 Hz), 7.11 (d, 1H, J = 0 3.6 Hz).
7.18 (dt, 1H, J = 2.0 Hz, J = 8.6 Hz), 7.26-7.35 (m, 4H, aromat),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8555
[M - Hþ]- (37), 613 [M - Hþ]- (100). IR (KBr): 3295, 2950, 1651
(2) Minucci, S.; Pelicci, P. G. Histone deacetylase inhibitors and the
promise of epigenetic (and more) treatments for cancer. Nat. Rev.
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cm-1. Anal. (C33H28ClFN4O5 1/2H2O) C, H, N.
3
(E)-3-(3-(4-(3-Chloro-4-(3-fluorobenzyloxy)phenylamino)quina-
zolin-6-yl)phenyl)-N-(tetrahydro-2H-pyran-2-yloxy)acrylamide
(21c). Yield (0.24 g, 67%) colorless crystals. Purification by cc
(SiO2, ethyl acetate, CH2Cl2 1:1) and crystallization from ethyl
acetate. 1H NMR (DMSO-d6) δ (ppm) 1.55 (s, 3H), 1.71 (s, 3H),
3.54 (m, 1H), 3.96 (m, 1H), 4.94 (s, 1H), 5.27 (s, 2H), 6.68 (d, 1H,
J = 15.8 Hz), 7.19 (dt, 1H, J = 2.2 Hz, J = 8.2 Hz), 7.32 (m,
3H), 7.48 (m, 1H), 7.64 (m, 3H), 7.76 (dd, 1H, J = 2.5 Hz, J =
8.9 Hz), 7.88 (d, 1H, J = 8.7 Hz), 7.92 (m, 1H), 8.03 (d, 1H, J =
2.5 Hz), 8.07 (s, 1H), 8.25 (dd, 1H, J = 1.7 Hz, J = 8.7 Hz), 8.61
(s, 1H), 8.85 (d, 1H, J = 1.1 Hz), 9.95 (s, 1H, exchangeable),
11.29 (s, 1H, exchangeable). ES-MS (DCM/MeOH þ 10 mmol/L
NH4Ac) m/z (%): 625 (100) [M þ Hþ]. IR (KBr): 3444, 2872,
1668 cm-1. Anal. (C35H30ClFN4O4) C, H, N.
Synthesis of N-Hydroxyacrylamides (6a-6d, 8a, and 8b from
the Respective (Tetrahydropyran-2-yloxy)amide: Precursors. To
a stirred solution of the corresponding (tetrahydropyran-2-
yloxy)amide (0.5 mmol) in MeOH (50 mL) was added 1N HCl
(50 mL). The mixture was stirred at room temperature over-
night. Half of the solvent was removed under reduced pressure,
and the precipitating product was filtered off, crystallized from
MeOH, and dried in vacuo.
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3-{5-[4-(3-Chloro-4-(3-fluorobenzyloxy)phenylamino]quina-
zolin-6-yl}furan-2-yl)-N-hydroxy-acrylamide Hydrochloride Monohy-
drate (6a). Yield: (0.20 g, 60%) yellow crystals; mp 190.9-192.0 °C.
1HNMR(DMSO-d6) δ(ppm) 5.32 (s, 2H), 6.62 (d, 1H, Jtrans = 15.6
Hz), 7.01 (d, 1H, J = 3.6 Hz), 7.19 (dt, 1H, J = 2.2 Hz, J = 8.9 Hz),
7.20-7.32 (m, 4H), 7.45-7.53 (m, 2H), 7.72 (dd, 1H, J = 2.5 Hz,
J = 8.8 Hz), 7.95 (d, 1H, J = 2.5 Hz), 7.98 (d, 1H, J = 8.8 Hz), 8.40
(d, 1H, J = 0.8 Hz, J = 9.1 Hz), 8.91 (s, 1H), 9.40 (d, 1H, 0.8 Hz),
10.84 (s, 1H, exchangeable), 11,98 (s, 1H, exchangeable). þp ESI
m/z (%): 533 [M þ Hþ] (38), [M þ Hþ] (531). -p ESI m/z (%): 589
[M þ Ac-]- (91), 565 [M þ Cl-]- (51), 592 [M - Hþ]- (100). IR
(KBr): 3418, 2855, 1660 cm-1. Anal. (C28H20ClFN4O4 H2O HCl)
3
3
C, H, N, Cl.
(E)- 3-{3-[4-(3-Chloro-4-(3-fluorobenzyloxy)phenylamino)quina-
zolin-6-yl)phenyl)-N-hydroxyacrylamide (6c). Yield (0.13 g, 88%)
yellow crystals. NMR (DMSO-d6) δ (ppm) 5.32 (s, 2H), 6.70
(d, 1H, J = 15.8 Hz), 7.20 (dt, 1H, J = 2.1 Hz, J = 8.3 Hz), 7.35
(m, 3H), 7.49 (dt, 1H, J = 6.1 Hz, J = 8.0 Hz), 7.61 (dd, 2H, J =
7.8 Hz, J = 15.5 Hz), 7.70 (m, 2H), 7.97 (m, 2H), 8.04 (d, 1H, J =
8.7 Hz), 8.15 (s, 1H), 8.51 (dd, 1H, J = 1.1 Hz, J = 8.8 Hz), 8.97
(s, 1H), 9.28 (d, 1H, J = 0.9 Hz, exchangeable), 10.85 (s, 1H,
exchangeable), 11.89 (s, 1H, exchangeable). ES-MS (DCM/
MeOH þ 10 mmol/L NH4Ac) m/z (%): 541 (100) [M þ Hþ].
IR (KBr): 3385, 2860, 1662 cm-1. Anal. (C30H22ClFN4O3 HCl
IV.
N-[Oxytris(dimethylamino)phosphonium]benzotriazole
3
3
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2H2O) C, H, N.
Acknowledgment. We thank E. Verdin, Gladstone Institute
for Virology and Immunology, San Francisco, California,
USA, for providing rHDAC1 and rHDAC6 expressing
Hek293 cell lines. We also thank colleagues at ALTANA-
Nycomed Discovery Research, in particular U. Bosch and
G. Quintini, for providing rHDAC and kinase protein prep-
arations, and C. Burkhardt, H. Wieland, C. Engesser, and H.
Julius for excellent technical assistance.
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Supporting Information Available: Full experimental details
for synthetically preparations, analytical data, and biological
test systems. This material is available free of charge via the
Internet at http://pubs.acs.org.
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