Design of chimeric histone deacetylase- and tyrosine kinase-inhibitors: A series of Imatinib hybrides as potent Inhibitors of wild-type and mutant BCR-ABL, PDGF-Rβ, and histone deacetylases

Article abstract of DOI:10.1021/jm800988r

Inhibitors of histone deacetylases are a new class of cancer therapeutics with possibly broad applicability. Combinations of HDAC inhibitors with the kinase inhibitor 1 (Imatinib) in recent studies showed additive and synergistic effects. Here we present a new concept by combining inhibition of protein kinases and HDACs, two independent pharmacological activities, in one synthetic small molecule. In general, the HDAC inhibition profile, the potencies, and the probable binding modes to HDAC1 and HDAC6 were similar as for 6 (SAHA). Inhibition of Abl kinase in biochemical assays was maintained for most compounds, but in general the kinase selectivity profile differed from that of 1 with nearly equipotent inhibition of the wildtype and the Imatinib resistant Abl T315I mutant. A potent cellular inhibition of PDGFR and cytotoxicity toward EOL-1 cells, a model for idiopathic hypereosinophilic syndrome (HES), are restored or enhanced for selected analogues (12b, 14b, and 18b). Cytotoxicity was evaluated by using a broad panel of tumor cell lines, with selected analogues displaying mean IC₅₀ values between 3.6 and 7.1 μM.

Full text of DOI:10.1021/jm800988r

J. Med. Chem. 2009, 52, 2265–2279
2265
Design of Chimeric Histone Deacetylase- and Tyrosine Kinase-Inhibitors: A Series of Imatinib
Hybrides as Potent Inhibitors of Wild-Type and Mutant BCR-ABL, PDGF-Rꢀ, and Histone
Deacetylases†
Siavosh Mahboobi,*,‡ Stefan Dove,‡ Andreas Sellmer,‡ Matthias Winkler,‡ Emerich Eichhorn,‡ Herwig Pongratz,‡
Thomas Ciossek,§,3 Thomas Baer,§,4 Thomas Maier,§ and Thomas Beckers*,|
Institute of Pharmacy, UniVersity of Regensburg, D-93040 Regensburg, Germany, and Nycomed GmbH, Therapeutic Area Oncology,
Byk-Gulden Strasse 2, D-78467 Konstanz, Germany
ReceiVed August 5, 2008
Inhibitors of histone deacetylases are a new class of cancer therapeutics with possibly broad applicability.
Combinations of HDAC inhibitors with the kinase inhibitor 1 (Imatinib) in recent studies showed additive
and synergistic effects. Here we present a new concept by combining inhibition of protein kinases and
HDACs, two independent pharmacological activities, in one synthetic small molecule. In general, the HDAC
inhibition profile, the potencies, and the probable binding modes to HDAC1 and HDAC6 were similar as
for 6 (SAHA). Inhibition of Abl kinase in biochemical assays was maintained for most compounds, but in
general the kinase selectivity profile differed from that of 1 with nearly equipotent inhibition of the wild-
type and the Imatinib resistant Abl T315I mutant. A potent cellular inhibition of PDGFR and cytotoxicity
toward EOL-1 cells, a model for idiopathic hypereosinophilic syndrome (HES), are restored or enhanced
for selected analogues (12b, 14b, and 18b). Cytotoxicity was evaluated by using a broad panel of tumor
cell lines, with selected analogues displaying mean IC50 values between 3.6 and 7.1 µM.
Introduction
drugs: the abl/src dual kinase inhibitor N-(2-chloro-6-methylphe-
nyl)-2-(6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-ethylpyrimidin-4-
ylamino)thiazole-5-carboxamide, known as 2 (Dasatinib),10,4 and
the 1 analogue benzamide 3 (Nilotinib).11,12 Whereas 3 is a more
potent, close analogue of 1also binding to the inactive abl kinase,
2 as a 2-aminopyridinyl-thiazole analogue inhibits abl and src
kinases in the active as well as in the inactive conformation
with high potency. Both agents are effective toward most
clinically relevant Bcr-Abl mutants with the exception of the
T315I mutant.11,4
Selective inhibition of protein kinases is an important
therapeutic approach for the treatment of human cancers.1
However, as extensively documented for the Bcr-Abl oncogene
in chronic myeloid leukemia (CML)a patients treated with 1
(Imatinib, Figure 1A), clinical resistance caused by multiple
mutations affecting 1 binding to the Bcr-Abl kinase protein has
been observed.2,3 One frequent mutation found is T315I within
the ATP-binding site of Bcr-Abl, abrogating a direct hydrogen
bond of 1 with T315 of Bcr-Abl.2,4,5 In vitro studies of resistance
to 1 indicate additional mechanisms, including overexpression
of Bcr-Abl caused by gene amplification6-8 and increased efflux
mediated by the multidrug resistance P-glycoprotein (ABCB1,
Pgp).9 One strategy to circumvent drug resistance of CML
patients to therapy with 1 is exemplified by recently approved
A still preclinical strategy was exemplified by combination
of 1 or 2 with inhibitors of histone deacetylases (HDIs).
Cotreatment with 4 (LAQ 824),13 a close analogue of 5
(LBH589),14 or 6 (SAHA) as inhibitors of histone deacetylases
increased the potency of 1 and 2, inducing apoptosis of K562
and LAMA-84 blast crisis CML cell lines.15,16 The combination
showed additive or synergistic effects, which were explained
†
This paper is dedicated to Professor Gottfried Ma¨rkl on the occasion
by down-regulation of wild-type and mutant Bcr-Abl T315
protein.13
I
of his 80th birthday.
* Corresponding authors: S.M.: e-mail, siavosh.mahboobi@chemie.uni-
regensburg.de; telephone, (+49) (0) 941-9434824; fax, (+49) (0) 941-
9431737 (medicinal chemistry). T.B.: e-mail, Thomas.Beckers@oncotest.de;
telephone, (+49) (0) 761-5155916; fax, (+49) (0) 761 51559 55 (pharma-
cology).
4, 5, and 6 are broad spectrum inhibitors of HDAC class I
and II enzymes.17,18 Mammalian HDAC class I (isoenzymes
1-3, 8), class II (isoenzymes 4-7, 9, 10), and class IV (HDAC
11) isoenzymes are functionally related, Zn2+-containing ami-
dohydrolases inhibited by the natural compound 7 (Trichostatin
A, or TSA).19 HDAC enzymes catalyze the reversible deacety-
lation of acetylated lysine residues in respective substrates such
as core histone proteins H2A/B, H3, and H4.20 HDAC inhibitors,
by inducing histone hyperacetylation, cause a relaxation of
chromatin, correlating with a release from transcriptional
repression.20 Nevertheless, targets different from core histone
proteins exist. This might explain the complex transcriptional
signatures with up- and down-regulated genes seen in HDI
treated cancer cells.21,22 HDIs belonging to four different
chemical classes are currently in phase I to III clinical
development. 6 was approved for treatment of therapy resistant
cutaneous T-cell lymphoma (CTCL).23 5, 6, and 8 (MS-275),24
‡
University of Regensburg.
Nycomed GmbH.
Present address: Boehringer Ingelheim Pharma, Germany.
Present address: Dottikon AG, Switzerland.
§
3
4
|
Present address: Oncotest GmbH, Institute for Experimental Oncology,
Am Flughafen 12-14, D-79108 Freiburg, Germany.
a Abbreviations: APL, acute promyelocytic leukemia; AML, acute
myelogenous leukemia; AMC, 7-amino-4-methylcoumarin; ATCC, Ameri-
can tissue type collection; BOP, 1-benzotriazolyloxy-tris-(dimethylamino)-
phosphoniumhexafluorophosphate; CML, chronic myeloid leukemia; HDI,
histone deacetylase inhibitor; HDAC, histone deacetylase; HDLP, histone
deactetylase-like protein; HDAH, histone deacetylase-like amidohydrolase;
HES, idiopathic hypereosinophilic syndrome; NSCLC, non small cell lung
cancer; PDGFR, platelet derived growth factor receptor; PKA, protein kinase
A; rHDAC, recombinant HDAC; SAHA, suberoylanilide hydroxamic acid;
SDS, sodium dodecylsulfate; SDS-PAGE, SDS polyacrylamide gel elec-
trophoresis; TSA, trichostatin A; VEGFR, vascular endothelial growth factor
receptor.
10.1021/jm800988r CCC: $40.75
2009 American Chemical Society
Published on Web 03/20/2009

2266 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Mahboobi et al.
Figure 1. (A) Structures of Bcr-abl kinase inhibitors 1, 2, and 3 approved for treatment of CML, of HDAC inhibitors as the benzamide 8, and of
the hydroxamate class of compounds (4-7). (B) Concept of chimerically designed Imatinib-HDAC inhibitor hybrids by combining selected structural
features of a tyrosine kinase- and a HDAC-inhibitor exemplified by chimeric structures 10-19b.
examples for the prominent second generation HDAC inhibitors
exhibiting the hydroxamic acid and benzamide Zn2+-complexing
motif (headgroup), are shown in Figure 1a.
ester 25, respectively; alkaline cleavage afforded the corre-
sponding carboxylic acid 26. Amidation of 24 with O-(tetrahy-
dro-2H-pyran-2-yl)hydroxylamine (NH2OTHP) (27), or rather
of 26 with tert-butyl 2-aminophenylcarbamate (28),28 mediated
by BOP (1-benzotriazolyloxy-tris-(dimethylamino)phosphoni-
umhexafluorophosphate) as a coupling reagent,29 led to 29 and
30. After deprotection, the desired N-hydroxycinnamamide 10
and the N-(2-aminoaryl)benzamide 11 were obtained. The
terephthalamide 31 was obtained by monomethylation of the
amino intermediate 21a, affording 32, which was amidated with
4-(2-(tert-butoxycarbonylamino)phenylcarbamoyl)benzoic acid
(33). The resulting tert-butyl-2-benzamidocarbamate 34 was
deprotected with trifluoroacetic acid.
A closely related synthetic strategy (Scheme 2) was employed
for the syntheses of several compounds of the hydroxamate and
benzamide groups, exhibiting structural modifications also in
the B- and D-ring systems (Figure 1b). The N-(3-nitrophe-
nyl)thiazol-2-amines 35a and 35b, by catalytic reduction with
hydrogen, yielded the arylamines 36a, 36b. These intermediates
were used for amidation with the suitably protected and
substituted carboxylic acids 37-42. Amidation hereby was
performed either by transformation of the corresponding car-
boxylic acids 37-39 and 42 to their carboxylic acid chlorides
in a mixture of pyridine/thionyl chloride, followed by addition
of the primary arylamino compounds 21a, 21b and 36a, 36b,
respectively, or mediated by BOP.29 Deprotection of the tert-
butyl-2-benzamidocarbamates 43a-48b with trifluoroacetic
acid, respectively, of the N-(tetrahydro-2H-pyran-2-yloxy)cin-
namamides 49a-50b or N-(tetrahydro-2H-pyran-2-yloxy)ben-
zamides 53a, 53b with hydrochloric acid led to the desired N-(2-
Here we report on a novel tumor targeting strategy, combining
the structural features of the Abl, PDGFRꢀ, and Kit inhibitor
1, with respect to its bioisosteric thiazolyl derivative 9 (Figure
1b, exchange of the B-ring pyrimidine for a thiazole) with
HDAC inhibitory head groups to overcome resistance to 1 by
synergistic inhibition of two pharmacological targets. We
selected the hydroxamic acid and benzamide motif, respectively,
for combination with the N-(3-(4-(pyridin-3-yl)pyrimidin-2-
ylamino)phenyl)amide core structure of 1, or the N-(3-(4-
(pyridin-3-yl)thiazol-2-ylamino)phenyl)amide of its bioisosteric
analogue to obtain chimerically designed compounds. In early
reports the so-called “Flag-methyl”-group25,26 in the C-ring
system (Figure 1b) has been described to be responsible for
selectivity and enhancement of activity of the N-(3-(4-(pyridin-
3-yl)thiazol-2-ylamino)phenyl)amide pharmacophore25,26 con-
cerning Bcr-Abl and PDGFR activity. Therefore, the effect of
this substitution pattern has also been investigated.
Chemistry
The synthetic route to obtain the desired target compounds
(Scheme 1) is given in the following: Catalytic reduction with
hydrogen of the respective N-(3-nitrophenyl)-4-pyrimidin-2-
amines 20a and 20b (Scheme 1), prepared as described,25,27
led to the corresponding primary arylamines 21a, 21b, which
were used as central intermediates. Reductive amination with
(E)-3-(4-formylphenyl)acrylic acid (22) or methyl 4-formyl-
benzoate (23) led to the phenylacrylic acid 24 or to the methyl

Histone Deacetylase- and Tyrosine Kinase-Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2267
Scheme 1. Synthesis of Target Compounds 10, 11, and 31a
tion with n-BuLi, introduction of the carboxylic group, and
cleavage of the 1,3-dioxolane in one step,33 followed by
esterification of 6532 with methyl iodide and oxidation of 66.
The N-hydroxyacrylamide precursors 40 and 41 (Scheme 6)
were obtained from the aldehydes 69 and 66 by an aldol
condensation with malonic acid in a mixture of pyridine/
piperidine,34 amidation of the resulting acrylic acids 70,3571
with NH2OTHP 2736 by use of BOP as coupling reagent,29 and
alkaline cleavage of the methyl esters of 72 and 73, respectively.
Results and Discussion
The in vitro data for inhibition of recombinant HDAC 1 and
HDAC 6 (rHDAC1, rHDAC6) as well as inhibition of Abl
kinase by chimeric compounds in comparison to 1, 8, and 6 as
reference compounds are listed in Tables 1-3. In addition to
these biochemical data, the cellular efficacy of chimeric HDAC-
kinase inhibitors was quantified by induction of cellular histone
H3 K(Ac)9+14 hyperacetylation and cytotoxicity toward HeLa
and K562 cells. Combining the structural features of a 6-methyl-
N1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine (sub-
structure of 1 in Figure 1b) with a N-(2-aminophenyl)benzamide
or N-hydroxyacrylamide motif in general leads to potent
rHDAC1 inhibitors, with hydroxamate analogues 10 and 18b
having a potency quite similar to that of 6. Whereas hydrox-
amate analogues inhibit rHDAC1 and rHDAC6 as HDAC class
I and class II representatives with similar potency, benzamide
analogues display weak or no inhibition of rHDAC6. This
selectivity, which is illustrated by respective homologues in
Tables 2 and 3, was also described for other benzamides.37,38
For the pyrimidine analogues in Table 2, a minor trend toward
more potent inhibition of rHDAC1 and histone H3 hyperacety-
lation is seen from the pyridine derivative 13b, to the benzene,
12b, and thiophene derivative, 14b.
All rHDAC1 inhibiting compounds induce histone H3 hy-
peracetlyation in HeLa cells with a potency of EC50 around 2
µM, comparable to that of 6 or 8. Representative concentration-
effect curves for histone H3 hyperacetylation in HeLa cells are
shown in Figure 2. HeLa cervical carcinoma cells were treated
with compounds 12b, 11, 10, 18b, 16a, and 14b for 24 h before
fixation and staining with a H3(K)Ac9+14 specific antibody and
single cell image analysis. EC50 values were determined as 1.04
µM (12b), 4.8 µM (11), 1.5 µM (10), 4.2 µM (18b), 1.5 µM
(16a), and 2.6 µM (14b).
Different hydroxamate and benzamide derivatives, for ex-
ample, 10, 11, 12a/b, 18a/b, and 19a/b, inhibit constitutively
active Abl kinase in the biochemical assay with only up to 9-fold
lower potency than that of Imatinib. Compound 11 is most
active, with an IC50 of 2.0 µM. Changing the amine structure
to an amide (12b), a structural modification that led to the most
potent compounds in the parent Imatinib-series,26 is tolerated
but slightly reduces activity in this series. Also, introduction of
a heterocycle as a D-ring system (13b, 14b) or use of the
N-hydroxyacrylamide motif (10) decreases Abl kinase inhibition
significantly. Most important, the kinase selectivity profile as
summarized in Tables 4 and 5 differs from that of 1 with equal
inhibition of AblT315I mutant and VEGFR2/KDR. The cyto-
toxicity of these analogues toward K562 and HeLa CML cell
lines toward 1 (K562) or 6 and 8 (K562 and HeLa) is in the
low micromolar or sub-micromolar range, corresponding to the
range of the IC50 values. Nevertheless, the potency of 1 toward
K562 cells bearing the Bcr-Abl translocation with IC50 ) 0.049
µM is superior to that of all chimeric compounds.
a Conditions: (a) H2, Pd/C, 30 atm, 16 h; (b) paraformaldehyde, MeOH,
NaOMe, ∆, then NaBH4, ∆; (c) MeOH, HOAc, NaBH3CN, 20° C, 14 h;
(d) THF, HOAc, NaBH(OAc)3, 40° C, 16 h; (e) THF, NEt3, BOP, 20° C,
16 h or DMF, NEt3, EDC·HCl; (f) MeOH, H2O, LiOH, 20° C, 16 h; (g)
dioxane, HCl, 20° C, 4 h; (h) MeOH, H2O, HCl, 20° C, 16 h; (i) CF3COOH,
20° C, 1 h.
arylamino)arylcarboxamides 12a-17b and N-hydroxacryl-
amides 18a-19b, respectively, to the hydroxamic acids
54a-54b.
The N-(3-nitrophenyl)thiazol-2-amines 35a, 35b were pre-
pared analogously27 by reaction of 2-bromo-1-(pyridin-3-yl)-
ethanone hydrobromide30(59) and the pertinent phenylthiourea
derivatives 58a, 58b, which themselves were easily available
from 3-nitroaniline (56a) and 2-methyl-5-nitroaniline (56b) in
two steps by a modification of the excellent method described
by Rasmussen31 (Scheme 3).
The substituted carboxylic acids, 4-(2-(tert-butoxycarbony-
lamino)phenylcarbamoyl)benzoic acid (37) and 6-(2-(tert-bu-
toxycarbonylamino)phenylcarbamoyl)nicotinic acid (38) (Scheme
4), which were used for the central amidation step as described
in Schemes 1 and 2, were prepared from the corresponding
methoxycarbonyl arylic acids 59, 60 by reaction with tert-butyl
2-aminophenylcarbamate28 (28), followed by selective alkaline
cleavage of the methyl esters of 61 and 62 with LiOH (Scheme
4).
Following the same strategy for synthesis (Scheme 5), 5-(2-
(tert-butoxycarbonylamino)phenylcarbamoyl)thiophene-2-car-
boxylic acid (39) was prepared from 5-(methoxycarbonyl)th-
iophene-2-carboxylic acid32 (67) in two steps. Compound 67
hereby was obtained from thiophene-2-carbaldehyde (63) by
transformation to 2-(thiophen-2-yl)-1,3-dioxolane33(64), lithia-
The benzamide analogues with the bioisosteric (4-((pyridin-
3-yl)thiazol-2-ylamino)phenyl)amide substructure (Table 3)

2268 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Scheme 2. Synthesis of Target Compoundsa
Mahboobi et al.
a Conditions: (a) H2, Pd/C, 30 atm, 16 h; (b) SOCl2, pyridine, 20° C; (c) BOP, NEt3, THF or DMF, 20° C; (d) MeOH, H2O, LiOH, 20° C, 16 h; (e)
CF3COOH, 20° C, 1 h, then H2O, NH3, pH ) 9; (f) MeOH, H2O, HCl, 20° C, 16 h.
Scheme 3a
Scheme 4. Preparation of 4-(2-(tert-Butoxycarbonylamino)phe-
nylcarbamoyl)benzoic Acid (37) and 6-(2-(tert-Butoxycarbo-
nylamino)phenylcarbamoyl)nicotinic Acid (38)a
a Conditions: (a) NH4NCS, acetone, ∆, 15 min; (b) acetone, ∆, 30 min,
then 0° C, H2O; (c) LiOH, H2O, THF, ∆, 6 h; (d) EtOH, ∆, 2 h.
exhibited selective inhibition of rHDAC1 with IC50 values in
the range of 0.17-1.13 µM. The rHDAC1 inhibitory potency
a Conditions: (a) BOP, NEt3, DMF, 20° C; (b) LiOH, MeOH, H2O, then
0° C, H2O, HCl, pH ) 5.
decreases from the benzene derivatives (15a, 15b) to pyridine
(16a, 16b) and thiophene derivatives (17a, 17b). Cellular

Histone Deacetylase- and Tyrosine Kinase-Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2269
Scheme 5. Preparation of 5-(2-(tert-Butoxycarbonylamino)phe-
nylcarbamoyl)thiophene-2-carboxylic acid (39)a
significantly different from that of 1 (Table 5). The cytotoxicity
of these thiazolylamino analogues toward HeLa and K562 cells
correlates with histone H3 hyperacetylation, with K562 cells
being slightly more sensitive (Table 3).
Docking Studies of Compounds 10 and 11 on HDLP
and HDAH as well as on Abl and Abl-T315I. Docking studies
with the representative compounds 10 and 11 were performed
to suggest possible binding modes of the chimeras on HDAC1,
HDAC6, Abl, and Abl T315I (Figures 3 and 4). HDAC1 was
represented by the crystal structure of a histone deacetylase-
like protein (HDLP, PDB code 1c3s39) from the hyperthermo-
philic bacterium Aquifex aeolicus sharing 35% identity with
human HDAC1 over 375 residues. As a HDAC6 homologue, a
histone deacetylase-like amidohydrolase from Bordetella/Al-
caligenes strain FB188 was used (HDAH, PDB code 1zz1,40
35% identity over 291 amino acids with the second domain
HDAC6b of human HDAC6). Both structures are in complex
with 6. It was easily possible to dock compound 10 in an
energetically favorable conformation into both models. In the
minimized complexes, the N-hydroxyphenylacrylamide moiety
of 10 adopts the same position as the octanedioic acid
hydroxyamide chain of 6 (Figure 3A, B). In particular, the
interactions with eight amino acids of the active site, conserved
in all class I and II HDACs, are similar. The carbonyl and the
hydroxy oxygens of the hydroxamic acid are coordinated with
the zinc ion. The hydroxy group is hydrogen-bonded with an
imidazolyl nitrogen of H131 (HDLP) and H142 (HDAH), respec-
tively. Another H bond is formed between the carbonyl oxygen
and the OH group of Y297 (HDLP) or Y312 (HDAH). In both
models, the phenyl ring is parallelly aligned between two
phenylalanine residues (F141 and F198 in HDLP, F152 and F208 in
HDAH). The 4-((pyridin-3-yl)pyrimidin-2-ylamino)phenyl moi-
ety of 10 projects outward into the solvent (see also Figure 3D),
corresponding to the fact that the SARs of the hydroxamates
do not strongly depend on variations in this region. Some other
amino acids which are conserved between HDLP and HDAC1
or HDAH and HDAC6 lie within a sphere of 3 Å around
compound 10; examples for HDLP are Y91 (rHDAC1: E), E92
(D), and L265 (L), and those for HDAH are L21 (mHDAC6: L),
D98 (D), I100 (V), and E309 (E).
a Conditions: (a) Al2O3, CCl4, ∆, 48 h; (b) BuLi, THF, -78° C, then
CO2, followed by H2SO4 (10%); (c) CH3I, Na2CO3, DMF, 20° C, 48 h; (d)
Jones reagent; (e) BOP, NEt3, THF, 20° C, 24 h; (f) MeOH, H2O, LiOH,
24 h.
Scheme 6. Preparation of (E)-4-(3-Oxo-3-(tetrahydro-2H-pyran-
2-yloxyamino)prop-1-enyl)benzoic Acid (40) and (E)-5-(3-Oxo-
3-(tetrahydro-2H-pyran-2-yloxyamino)prop-1-enyl)thiophene-2-
carboxylic Acid (41)a
Compound 11 binds to HDLP in a similar mode as 10.
However, the 2-aminophenylbenzamide moiety must adopt
a deeper position in the binding pocket than the hydroxamate
group to enable coordination of the carbonyl oxygen with
the zinc ion (Figure 3C, D). Two hydrogen bonds of the
2-amino substituent with the backbone of G140 and of the
carbonyl function with the OH group of Y297 may contribute
to the tight fit. The phenyl ring of the benzamide moiety is
stacked between F141, H170, and F198. Additionally, compound
11 contacts the conserved amino acids M130 (rHDAC1: L),
C142 (C), and L265 (L). Just as in the case of the hydroxamate
10, the 4-((pyridin-3-yl)pyrimidin-2-ylamino)phenyl moiety
points outward (Figure 3D). The proposed binding mode of
11 may account for the HDAC1/class I selectivity of the
benzamide derivatives. Two significant mutations of glycines
strongly narrow the HDAH binding pocket for the 2-ami-
nophenyl group and for the corresponding aryl moieties of
the benzamides in Tables 2 and 3, leading to sterical
hindrance (Figure 3C). The HDLP residues G129 (rHDAC1:
G) and G294 (G) are mutated in HDAH into P140 (mHDAC6:
P) and E309 (E), respectively. In the aligned crystal structures,
the flexible side chain of M130 is in place of HDAH P140
clashing with compound 11.
a Conditions: (a) CH2(COOH)2, pyridine, piperidine, 80° C, 3 h; (b) BOP,
NEt3, DMF, 20° C; (c) LiOH, H2O, MeOH, then 0° C, HCl, pH ) 5.
potency as measured by histone H3 hyperacetylation is com-
parable with EC50 values down to 1.15 µM (15a). All com-
pounds showed only weak inhibition of Abl kinase. In addition
and as seen before, the kinase specificity profile changed
compared to that of 1 with a trend toward VEGFR2/KDR
inhibition for some analogues (Table 5). Exchange of the
benzamide headgroup by the hydroxamate moiety significantly
increased rHDAC1 inhibitory activity and led to highly potent
inhibition of rHDAC6 (Table 3). These analogues exhibited IC50
values for inhibition of rHDAC1 in the range of 0.08-0.21 µM.
The “methyl-flag” group at the phenyl C-ring system does not
affect rHDAC1 inhibition. Most potent compounds in this series
are 18a, 18b, and 19b, bearing the acrylamide motif. Although
inhibition of Abl kinase is retained, the selectivity profile is

2270 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Mahboobi et al.
Table 1. Inhibition of rHDAC1, rHDAC6, and c-Abl Kinase, Induction of Histone H3 Hyperacetylation and Cytotoxicity toward HeLa and K562 Cells
Mediated by Chimerically Designed 2-Aminophenylpyrimidines as well as 1, 6, and 8 as Reference Compoundsa
a Mean IC50 values were calculated from respective concentration-effect curves done in replicate using GraphPad Prism statistical analysis software.
Table 2. Inhibition of rHDAC1, rHDAC6, and c-Abl Kinase, Induction of Histone H3 Hyperacetylation and Cytotoxicity toward HeLa and K562 Cells
Mediated by Chimerically Designed 2-Aminophenylpyrimidinesa
a Mean IC50 values were calculated from respective concentration-effect curves done in replicate using GraphPad Prism statistical analysis software. (n.d.
) not determined).
With respect to Abl and Abl T315I, the docking modes of
structure of Abl in complex with 1 (PDB code 1iep41), poses
compounds 10 and 11 are rather ambiguous. Based on the crystal
strongly consistent with the binding mode of the cocrystallized

Histone Deacetylase- and Tyrosine Kinase-Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2271
Table 3. Inhibition of rHDAC1, rHDAC6, and c-Abl Kinase, Induction of Histone H3 Hyperacetylation and Cytotoxicity toward HeLa Cells Mediated
by Chimerically Designed 2-Aminophenythiazolylamines with a Benazmide or Hydroxamate Head Groupa
a Mean IC50 values were calculated from respective concentration-effect curves done in replicate using GraphPad Prism statistical analysis software.
Table 4. Inhibition of Wild-Type (wt) Abl and Mutant Abl T315I,
PDGF-Rꢀ, VEGF-R2/KDR, and PKA by 2-Aminophenylpyrimidines
and 1 as Referencea
Abl (wt) ABL T315
example IC50 (µM) IC50 (µM) IC50 (µM) IC50 (µM) IC50 (µM)
I
PDGF-Rꢀ VEGF-R2
PKA
11
10
31
12b
13b
14b
12a
1
2.0
9.2
1.12
6.9
2.7
4.6
7.3
2.6
7.8
5.2
1.1
0.24
7.8
9.2
6.1
9.8
8.6
11
1.5
25.8
>100
>100
38
>100
>100
>100
>100
31
5.3
32
25
7.8
1.1
34
4.1
31
22
9.8
>100
87.5
a Mean IC50 values were calculated from respective concentration-effect
curves done in replicate using GraphPad Prism statistical analysis software.
Figure 2. Induction of histone H3 hyperacetylation in HeLa cells.
inhibitor may be suggested (Figure 4A and B). There are exactly
the same interactions with Abl if only the corresponding
substructures of 1 and 10 are considered. In particular, the
hydrogen bonds of the pyridyl nitrogen with the backbone of
M318 (hinge), of the pyrimidin-2-ylamino group with the side
chain of T315 (hinge), and of the phenylamino NH with the
carboxylate of E286 (RC) are reproduced. The N-hydroxyacry-
lamide and the 2-aminophenyl moieties of 10 and 11, respec-

2272 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Mahboobi et al.
Table 5. Inhibition of Wild-Type (wt) Abl and Mutant Abl T315I,
PDGF-Rꢀ, VEGF-R2/KDR, and PKAa
F382 (DFG motif). An H bond of the pyridine nitrogen with the
protonated amino group of K271 is possible.
Abl (wt) ABL T315
example IC50 (µM) IC50 (µM) IC50 (µM) IC50 (µM) IC50 (µM)
I
PDGF-Rꢀ VEGF-R2
PKA
This “inverse binding mode” of 10 on an active-like Abl wild-
type conformation can be almost completely transferred to the
structure of the Abl T315I mutant in complex with 75, also
representing an overall active state.42 Comparing the minimized
wild-type and mutant complexes (parts C and D, respectively,
of Figure 4), the rms deviation of the CR atoms amounts to
only 1.04 Å. The only significant differences arise from the
alignment of the 4-(pyridin-3-yl)pyrimidin-2-ylamino moiety of
10 with the 2-methoxyphenyl group of 75, leading to interac-
tions, especially with Y253 (P-loop), V256 (ꢀ2), K271 (ꢀ3), N368
(ꢀ7), and D381 (DFG motif). Again, an H bond of the pyridine
nitrogen with the protonated amino group of K271 can be
suggested.
Since the similar “inverse binding modes” of 10 on active-
like conformations of Abl and Abl T315I correspond to the almost
equal inhibitory potencies of the present compounds at both
Abl species, it could be speculated that indeed active-like states
are stabilized in both cases. However, the generation of other
ligand-specific conformations not represented by the available
crystal structures cannot be ruled out. Therefore, the given
binding modes are rather working hypotheses which must be
investigated by further experiments.
Benzamides
15a
15b
16a
16b
17a
17b
39
34
24
35
24
24
20
14
10
12
9.0
8.5
71
8.7
13
47
28
11
24
46
4.4
9.0
12
4.8
>100
>100
>100
>100
>100
>100
Hxdroxamates
40
54a
54b
18a
18b
19a
19b
17
9
4.5
2.7
3.8
2.6
19
4
43
14
8.7
11
8.6
6.7
>100
>100
>100
>100
>100
>100
8.8
2.1
13
3.9
11
6.2
1.8
2.0
1.7
a Mean IC50 values were calculated from respective concentration-effect
curves done in replicate using GraphPad Prism statistical analysis software.
tively, are aligned with a mainly hydrophobic pocket between
the N- and the C-terminal domain and contact V289 (RC), I293
(RC), L354 (RE), and F359 (RE-ꢀ7 loop). The N-hydroxy group
of 10 may form an H bond with the backbone oxygen of I360
(RE-ꢀ7 loop). Figure 4B indicates that even longer substituents
projecting into the solvent are possible.
Selected compounds of all series were pharmacologically
characterized in more detail.
However, these “Imatinib modes” do not answer the question
why the present compounds are nearly equiactive as inhibitors
of Abl and Abl T315I, since 1 itself does not bind to the mutant.
The problem is complicated by the flexibility of, in particular,
the nucleotide binding (P-loop) and the activation loop and thus
by the induction of ligand-specific Abl conformations, repre-
sented by different crystal structures of the wild-type [PDB code
1m52,41 complex with 6-(2,6-dichlorophenyl)-8-methyl-2-
(3-(methylthio)phenylamino)pyrido[2,3-d]pyrimidin-7(8H)-
one (74) (PD173955)]41 and of the Abl T315I mutant [PDB codes
2z6042 and 2v7a,43 complexes with (5-[3-(2-methoxyphenyl)-
1H-pyrrolo[2,3-b]pyridin-5-yl]-N,N-dimethylpyridine-3-carboxa-
mide) (75) (PPY-A)42 and N-[5-(2-methoxy-2-phenylacetyl)-
1,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl]-4-(4-methylpiperazin-
1-yl)benzamide (76) (PHA739358),44 respectively].
In the “Imatinib mode”, the pyrimidinyl ring of 10 is parallelly
aligned with F382 (DFG motif). This interaction, essential for
binding of 1, requires an inactive, autoinhibiting conformation
of the activation loop.41,45 By contrast, the complex of Abl with
74 represents a conformation of the activation loop like in active
kinases.41 It is impossible to dock compound 10 on this Abl
structure in a reasonable conformation. Since the amide
analogues 18a and 18b (Table 3) are even slightly more potent
than 10, both phenyl rings must be nearly coplanar, which
markedly restricts the degrees of conformational freedom.
However, compound 10 can be docked into PDB 1m52 (active-
like conformation) in an “inverse mode” where the phenylami-
nomethylphenyl moiety is aligned with the hinge region (Figure
4C). As in the case of the aminopyrimidine group of 74,41 an
H bond with the backbone oxygen of M318 is formed. L248 (P-
loop) and L370 (ꢀ7) cover the “inner” phenyl ring from both
sides. The N-hydroxyacrylamide moiety penetrates into the
solvent, which again explains the low variability of the potency
within the series. Also corresponding to the SAR, the “methyl-
flag” is not involved in direct interactions. The 4-(pyridin-3-
yl)pyrimidin-2-ylamino moiety adopts the position of the
dichlorophenyl group of 74, mainly interacting with Y253
(P-loop), V256 (ꢀ2), K271 (ꢀ3), I313 (ꢀ5), T315 (hinge), D381, and
The inhibition of PDGF stimulated PDGFRꢀ autophospho-
rylation in Swiss 3T3 cells and the constitutive Bcr-Abl
phosphorylation in K562 cells were studied, using the benzamide
analogues 12b and 12a (with and without the “methyl-flag”),
the hydroxamate/benzamide homologues 10 and 11, and the
aminothiazolyl hydroxamate derivatives 18b and 18a (with and
without the “methyl-flag”). As shown in Figure 5, all analogues
showed no or only marginal inhibition of cellular Bcr-Abl
activity in K562 cells at 1 and 10 µM concentration. This might
be explained by a reduced binding to the inactive Bcr-abl protein
in cells, in contrast to 1.
In contrast to the inefficacy of Bcr-abl kinase inhibition in
cells, ligand induced PDGFRꢀ autophosphorylation was potently
inhibited by 12b at 1 and 10 µM, but not by the analog 12a. A
similar result was obtained with 18b at 10 µM. For both
examples, only the analogues with the “methyl-flag” were active
cellular PDGFRꢀ inhibitors. These results are substantiated by
a high sensitivity of the eosinophilic leukemia cell line EOL1,
bearing the fusion protein FIP1L1-PDGFRR, which causes a
constitutive activation of the PDGF-R pathway.46,47 12b and
18b, in addition to 14b, are highly cytotoxic toward EOL1 cells
with IC50 values of 7 and 6 nM, respectively (Table 6). The
EOL1 cell line has been described as an in vitro model for
chronic eosinophilic leukemia, with idiopathic HES as an
indication for Imatinib therapy.47 Finally, the cytotoxicity profile
for the selected compounds of the present series as well as for
1, 6, and 8 as reference compounds was evaluated using a panel
of 22 to 24 different tumor cell lines emanating from 16 different
tumor entities/histotypes.
As summarized in Table 6 and highlighted for 1, 10, and 11
(Figure 6), the chimeric compounds are broadly active with a
higher potency toward leukemic cell lines K562, EOL1, and
CCRF-CEM. The mean inhibitory potency ranges from 3.6 µM
for 10 to 7.1 µM for 14b, quite comparable to that of 6 or 8
with IC50 values of 2.6 and 2.8 µM, respectively.
The antiproliferative, cytotoxic activity profiles of 11 and 10
as structural homologues with the benzamide and hydroxamate
headgroups in comparison to 1 are shown in Figure 6. The

Histone Deacetylase- and Tyrosine Kinase-Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2273
Figure 3. Docking of compounds. 10 and 11 at HDLP (HDAC1 homologue, derived from PDB 1c3s) and HDAH (HDAC6 homologue, derived
from PDB 1zz1). Colors of atoms, unless otherwise indicated: O, red; N, blue; S, yellow; Zn2+, magenta. C atoms of the eight conserved amino
acids of the binding site, green; C atoms of other residues within a sphere of 3 Å around the ligands, cyan; C and some essential H atoms of 10
and 11, gray; C atoms of 6, yellow. The backbones are represented as cyan ribbon (ꢀ-sheets) and tube models, and the R-helices are shown as
cylinders. (A) Compounds 10 and 6 complexed with HDLP. (B) Compounds 10 and 6 complexed with HDAH. (C) Compound 11 complexed with
HDLP. The red CPK models indicate the positions of P140 (below) and E309 (above) in HDAH (clash with the 2-aminophenyl moiety). (D) CPK
model of HDLP with compounds 10 (C atoms yellow) and 11 (C atoms orange) represented as sticks. The eight conserved amino acids of the
binding site are green-colored.
deviation from the mean IC50 value (shown in parentheses) is
given as lgM. For details about the different cell lines and
individual IC50 values, see the Experimental Part and the
Supporting Information, respectively.
The HDAC inhibition profile of most chimeras remained
conserved in biochemical and cellular assays. Their potency was
comparable to that of 6 or 8 as reference HDIs. Inhibition of
active Abl kinase in biochemical assays was also conserved in
most cases, although inhibition of Bcr-Abl autophosphorylation
in K562 CML cells was neclectable and the biochemical
selectivity profile was altered. This might be explained by a
dimished activity toward the inactive Bcr-abl kinase. Neverthe-
less, potent cellular inhibition of PDGFR and cytotoxicity toward
EOL-1 cells as one feature of 1 is restored in selected analogues,
namely 12b, 14b, and 18b. The kinase selectivity profile differed
from that of 1 with potent inhibition of the Abl T315I mutant,
being resistant to 1. Docking studies argue in favor of a binding
mode “inverse” to that of 1 on an autoinhibiting Abl conforma-
tion. Finally, the cytotoxicity toward a broad panel of tumor
cell lines emanating from 16 different tumor histotypes was
evaluated for selected analogues. In contrast to 1, all chimeric
compounds displayed a broad cytotoxicity with mean IC50 values
from 3.6 to 7.1 µM, which is comparable to 6, with a mean
IC50 of 2.6 µM. Few compounds showed a significantly higher
Conclusions
In recent studies, combinations of HDIs with the kinase
inhibitor 1 showed additive and synergistic effects based on
down-regulation of wild-type or mutant Bcr-Abl kinase in blast
crisis CML. The chimeric HDAC-kinase inhibitory compounds
of the present study are based on the Imatinib scaffold, bearing
benzamide or hydroxamate head groups which transfer the
HDAC inhibitory property and which determine the selectivity
profile, discriminating between hydroxamate-based unselective,
pan HDIs and benzamide-based, presumably class I selective
HDIs. We could show that the concept of chimeric compounds
bearing the pharmacological activities of a HDI as well as a
protein kinase inhibitor is feasible. Chimeric compounds based
on the structure of 1 with a hydroxamate or benzamide
headgroup are potent, broadly active cellular HDAC inhibitors.

2274 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Mahboobi et al.
Figure 4. Docking of compounds. 10 and 11 at Abl and Abl-T315I. Colors of atoms, unless otherwise indicated: O, red; N, blue; S, yellow. The
Abl backbones are represented as light cyan ribbon (ꢀ-sheets) and tube models, and the R-helices are shown as cylinders. Important regions are
highlighted: nucleotide binding loop (P-loop), yellow; hinge region, green; catalytic loop, red; activation loop, orange. Drawn are amino acids
within a sphere of 3 Å around the ligands. For the C atoms, the same coloring scheme as for the backbone was applied. (A) Compound 10 docked
at the Abl structure PDB 1iep (complex with 1). (B) MOLCAD surface of Abl PDB 1iep (wt abl, inactive conformation) with compounds 10
(C and H atoms magenta) and 11 (C and H atoms yellow). Mapped is the lipophilic potential (color ramp from blue, polar, to brown, hydrophobic).
(C) Compound 10 docked on the Abl structure PDB 1m52 (complex with 74). (D) Compound 10 docked on the Abl-T315I structure PDB 2z60
(complex with 75).
Figure 5. Analysis of Bcr-Abl and PDGFRꢀ phosphophorylation in K562 and Swiss 3T3 cells. K562 CML (A) and PDGF stimulated Swiss 3T3
cells (B) were treated with chimeric compounds at 1 and 10 µM concentration, as indicated for 1-3 h before cell lysis and Western blot analysis,
detecting c-abl phosphoY245, c-Abl, and phosphorylated PDGFRꢀ proteins. As a loading control, membranes were reprobed with an antibody
specific for ꢀ-Actin. Cells treated with 1 µM of 1 or 0.5 vol % DMSO were included as a positive and negative control, respectively.
activity toward the EOL1 chronic eosinophilic leukemia cell
with the potent inhibition of ligand activated PDGFR in 3T3
line, a model for HES treated clinically with 1. This correlated
fibroblast cells by chimeric compounds 12b and 18b. Thus, these

Histone Deacetylase- and Tyrosine Kinase-Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2275
Chart 1. Chemical Structures of 74, 75, and 76
Table 6. Anti-proliferative, Cytotoxic Activity of Selected Compounds
Evaluated Using a Panel of 22 to 25 Different Human Tumor Cell Lines
(Number of Individual Tumor Cell Lines in Parenthesis)a
cytotox
mean cytotox CCRF-CEM
cytotox
K562
cytotox
EOL-1
cytotox
A549
example IC50 (µM)
IC50 (µM) IC50 (µM) IC50 (µM) IC50 (µM)
6
8
1
2.6 (22)
2.8 (22)
27.6 (25)
5.2 (24)
7.1 (25)
3.6 (24)
6.1 (24)
4.7 (25)
5.2 (25)
0.8
0.71
36
2.0
1.4
0.73
2.0
1.8
1.3
0.67
0.72
0.049
0.76
0.59
0.74
0.49
0.74
0.9
1.0
2.2
3.3
15.5
0.053
0.0002
0.007
0.003
0.11
0.10
0.12
0.006
12b (B)
14b (B)
10 (H)
11 (B)
16a (B)
18b (H)
3.57
2.61
3.42
2.60
3.74
6.62
a Mean IC50 values are given in the first column with the sensitivity of
selected cell lines CCRF-CEM (ALL), K562 (CML), EOL1 (HES), and
A549 (NSCLC) shown for comparison. For details regarding cell lines, see
the Experimental Part. (B, benzamide analog; H, hyxdroxamate analog.)
Cytotoxicity on selected cell lines is shown by mean IC50 values, calculated
from respective concentration-effect curves done in replicate using GraphPad
Prism statistical analysis software.
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, Sigma
#A8592) followed by ECL-detection (GE Healthcare). In addition,
protein batches were analyzed by Western blotting for various
HDAC isoenzymes including HDAC1 and HDAC6.
The biochemical HDAC activity assay was essentially done as
described by Wegener et al.48
For further details, see the Supporting Information.
Biochemical Protein Kinase Assays. Active kinase proteins
were either obtained from commercial suppliers (Abl, PDGF-Rꢀ
ProQinase, Freiburg/Germany; AblT315I: Millipore/Upstate, Bil-
lerica/USA; protein kinase A (PKA): InvitroGen/Panvera, Carlsbad/
USA) or prepared in-house (KDR, c-terminal kinase domain
AA790-1356). As substrates, poly-AGKY for Abl, AblT315I, and
PDGF-Rꢀ (#P-1152 Sigma, Munich/Germany), poly-GT for KDR
(#P-0275, Sigma, Munich/Germany) or a PKA substrate peptide
for PKA (#12-394, Millipore/Upstate, Billerica/USA) were used.
Into each well of a 96-well flashplate (#SMP-200 in the case of
Abl, AblT315I, PDGF-Rꢀ, and KDR, #SMP-103 in the case of PKA;
Perkin-Elmer, Waltham/USA) kinase protein was diluted into kinase
assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 3 mM MnCl2,
1 mM DTT, 3 µM sodium orthovanadate, 5 µg/mL PEG 8000)
containing the appropriate substrate (final volume 89 µL). DMSO
vehicle or compounds are added as 1 µL/well prior to the initiation
of the kinase reaction by 10 µL 1 mM [33P]-γATP (4 × 105 cpm).
Reactions were allowed to proceed for a time predetermined to
show a linear dependence on a time versus phosphorylation plot
(30 min for PKA, otherwise 80 min) and terminated with the
addition of an equal volume of phosphoric acid (2% H3PO4 for 5
min). Plates were washed three times with 0.9% w/v NaCl and
quantitated by liquid scintillation counting.
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.49
Cellular Proliferation Assay. The antiproliferative activity of
selected compounds was evaluated using numerous tumor cell
lines.37,50 For quantification of cellular proliferation/cytotoxicity,
the Alamar Blue (Resazurin) cell viability assay was applied.51 For
further details, see the Supporting Information.
Figure 6. Cytotoxicity profile of selected compounds.
Western Blot Analysis. For Western blot analysis, 1.9 × 106/
well K562 or 1 × 106/well NIH 3T3 cells in six well cell culture
plates were treated with the test compounds for 3 or 2 h,
respectively. Next, NIH3T3 cells were stimulated with 50 ng/mL
of human recombinant PDGF-BB for 10 min at room temperature
before cell lysis. Cells were lysed in 200 or 300 µL lysis buffer
(50 mM Tris HCl pH 8, 150 mM NaCl, 1v/v NP-40, 0.5% w/v
sodium desoxycholate, 0.2% w/v sodiumdodecylsulfate (SDS),
0.02% w/v NaN3, 1 mM sodium vanadate, 20 mM NaF, 100 µg/
mL PMSF, 10 mM sodiumpyrophosphate, protease inhibitor mix/
Roche and 50 U/ml Benzonase) at 4 °C. Respective 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:
analogues most likely bear one feature of 1, conferring selective
cytotoxicity toward PDGFR driven diseases such as HES. We
conclude that chimeric HDAC-kinase inhibitory compounds are
a new approach for the combination therapy of cancer.
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. The rHDAC1 and rHDAC6
proteins were purified by M2-affinity gel chromatography according
to the manufacturer’s protocol (Sigma #A2220). Purified protein

2276 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Mahboobi et al.
monoclonal mouse antibody specific for ꢀ-Actin (clone AC-12,
Sigma Art. No. A-5441), polyclonal rabbit antibody specific for
c-abl kinase (Cell Signaling #2862), polyclonal rabbit antibody
specific for phospho c-abl Y245 (Cell Signaling #2861), polyclonal
rabbit antibody specific for PDGF-Rꢀ (Santa Cruz #sc-432), mouse
monoclonal antibody clone 4G10 specific for posphotyrosine
(Upstate #05-321), goat antirabbit IgG-HRP conjugated (Biorad:
170-6515), and goat-antimouse IgG-HRP conjugated (Biorad: 170-
6516).
ethyl acetate (3 × 20 mL), cooled to 0 °C, and acidified with diluted
acetic acid to pH ) 5-6. The precipitating product was removed
by filtration and dried in vacuo.
4-(2-(tert-Butoxycarbonylamino)phenylcarbamoyl)benzoic Acid
(37).53 1H NMR (DMSO-d6): δ (ppm) ) 1.44 (s, 9H), 7.15 (dt,
1H, J ) 1.6 Hz, J ) 7.6 Hz), 7.22 (dt, 1H, J ) 1.8 Hz, J ) 7.7
Hz), 7.55 (dt, 2H, J ) 1.5 Hz, J ) 7.8 Hz), 8.07 (m, 4H), 8.73 (s,
1H), 9.97 (s, 1H), 13.33 (s, 1H).
5-(2-(tert-Butoxycarbonylamino)phenylcarbamoyl)thiophene-
2-carboxylic Acid (39).54 1H NMR (DMSO-d6): δ (ppm) ) 1.45
(s, 9H), 7.16 (m, 2H), 7.26 (d, 1H, J ) 3.8 Hz), 7.51 (m, 2H), 7.77
(d, 1H, J ) 3.8 Hz), 8.75 (s, 1H), 9.87 (s, 1H).
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 points were determined with a Bu¨chi
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 + 10 mmol/L NH4Ac), respectively. All reactions were
carried out under nitrogen. Elemental analyses were performed by
the Analytical Laboratory of the University of Regensburg. Chemi-
cal names were created using ChemDraw Ultra 10.0 software.
N-(2-Methyl-5-nitrophenyl)-4-(pyridin-3-yl)pyrimidin-2-
amine (20b) was prepared as described.27
(E)-4-(3-Oxo-3-(tetrahydro-2H-pyran-2-yloxyamino)prop-1-
enyl)benzoic Acid (40). 1H NMR (DMSO-d6): δ (ppm) ) 1.54 (s,
3H), 1.70 (s, 3H), 3.54 (dd, 1H, J ) 4.6 Hz, J ) 6.9 Hz), 3.98 (m,
1H), 4.93 (s, 1H), 6.62 (d, 1H, J ) 15.8 Hz), 7.54 (d, 1H, J ) 15.7
Hz), 7.69 (d, 2H, J ) 8.1 Hz), 7.96 (d, 2H, J ) 8.3 Hz), 11.34 (s,
1H). Anal. (C15H17NO5 ·3/2H2O) C, H, N.
tert-Butyl 2-(4-(4-Methyl-3-(4-(pyridin-3-yl) pyrimidin-2-
ylamino) phenylcarbamoyl) benzamido)phenylcarbamate (43b).
4-(2-(tert-Butoxycarbonylamino)phenylcarbamoyl)benzoic acid (37)53
(0.68 g, 1.91 mmol) was dissolved in dry THF (20.0 mL) and BOP
(benzotriazolyloxy-tris-(dimethylamino)phosphonium-
hexafluorophosphate) (2.00 mmol, 0.88 g), and NEt3 (0.60 mL, 4.19
mmol) and 6-methyl-N1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-
diamine (21b)26 (0.50 g, 1.91 mmol) were added. After stirring at room
temperature for 24 h, the mixture was poured into water with stirring,
and the precipitating product was filtered off, dried in vacuo, and
purified by column chromatography (CH2Cl2/MeOH ) 8/1).
1H NMR (DMSO-d6): δ (ppm) ) 1.45 (s, 9H), 2.25 (s, 3H),
7.22 (m, 3H), 7.43 (d, 1H, J ) 5.6 Hz), 7.53 (m, 4H), 8.11 (m,
5H), 8.49 (m, 2H), 8.68 (m, 2H), 8.96 (s, 1H, exchangeable), 9.28
(s, 1H), 9.95 (s, 1H, exchangeable), 10.40 (s, 1H, exchangeable).
Anal. (C35H33N7O4 ·1/2H2O) C, H, N.
Preparation of Carbamic Acid tert-Butyl Esters 44b, 45b,
46a, 46b, 47a, 47b, 48a, and 48b by Amidation of the Respec-
tive Carboxylic Acids. The respective carboxylic acid was dis-
solved in 10 mL of dry pyridine, and 1.1 equiv of SOCl2 was added.
The mixture was stirred at room temperature for half an hour, and
1.0 equiv of the respective arylamine was added. After stirring at
room temperature for 24 h, the mixture was poured into water with
stirring, and the precipitating product was removed by filtration,
dried in vacuo, and purified by column chromatography (DCM/
MeOH ) 10/1).
6-Methyl-N1-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-di-
amine (21b).26 1H NMR (DMSO-d6): δ (ppm) ) 2.09 (s, 3 H),
4.85 (bs, 2 H), 6.35 (dd, 1 H, J ) 2.4 Hz, J ) 8.1 Hz), 6.80 (m,
2 H), 7.35 (m, 1 H), 7.51 (dd, 1 H, J ) 5.0 Hz, J ) 8.0 Hz), 8.46
(m, 1 H), 8.69 (m, 2 H), 9.24 (d, 1 H, J ) 2.4 Hz).
Preparation of (E)-3-(4-(Methoxycarbonyl)phenyl)acrylic Acid
(70) and (E)-3-(5-(Methoxycarbonyl)thiophen-2-yl)acrylic Acid
(71). A mixture of the respective aldehyde (69 or 66, 15.0 mmol),
malonic acid (30.0 mmol), and pyridine/piperidine (2:1; 3.75 mL)
was heated to 80 °C for 3 h. The hot mixture was poured into water
(20 mL) under stirring and acidified with diluted HCl. The mixture
was cooled in an ice bath, and the precipitating crystals were
removed by filtration, washed with diluted HCl, and dried in vacuo.
(E)-3-(4-(Methoxycarbonyl)phenyl)acrylic Acid (70).35 1H
NMR (DMSO-d6): δ (ppm) ) 3.87 (s, 3H), 6.66 (d, 1H, J ) 16.0
Hz), 7.64 (d, 1H, J ) 16.1 Hz), 7.83 (d, 2H, J ) 8.3 Hz), 7.97 (d,
2H, J ) 8.3 Hz), 12.57 (s, 1H).
Preparation of Carboxylic Acid Methyl Esters (61, 62, 68,
72, and 73) by Amidation with 28 or NH2OTHP (O-(Tetrahy-
dro-2H-pyran-2-yl)hydroxylamine) (27). The respective meth-
oxycarbonyl aroylic acid (10.0 mmol) (59, 60, 70, 71, or 52a and
52b) was dissolved in dry THF (50.0 mL) or DMF (20.0 mL), and
1.1 equiv of BOP (benzotriazolyloxy-tris-(dimethylamino)phos-
phoniumhexafluorophosphate), 2.2 equiv of NEt3, and 1.1 equiv
of the respective amine (2828 or NH2OTHP 2736) were added. After
stirring at room temperature for 24 h, the mixture was poured into
water with stirring, and the precipitating product was filtered off,
dried in vacuo, and purified by column chromatography (CH2Cl2/
MeOH ) 10/1).
tert-Butyl-2-(5-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylami-
1
no)phenylcarbamoyl)picolinamido)phenylcarbamate (44b). H
NMR (DMSO-d6): δ (ppm) ) 1.51 (s, 9H), 2.26 (s, 3H), 7.19 (m,
1H), 7.28 (m, 3H), 7.46 (d, 1H, J ) 5.2 Hz), 7.53 (m, 2H), 8.02 (d,
1H, J ) 8.1 Hz), 8.13 (d, 1H, J ) 1.9 Hz), 8.32 (d, 1H, J ) 8.2 Hz),
8.50 (m, 1H), 8.54 (d, 1H, J ) 5.2 Hz), 8.58 (dd, 1H, J ) 2.1 Hz, J
) 8.2 Hz), 8.70 (dd, 1H, J ) 1.5 Hz, J ) 4.7 Hz), 9.03 (s, 1H), 9.12
(d, 1H, J ) 1.6 Hz), 9.18 (s, 1H), 9.30 (d, 1H, J ) 1.8 Hz), 10.57 (s,
1H), 10.62 (s, 1H). Anal. (C34H32N8O4 ·1/2H2O) C, H, N.
tert-Butyl 2-(5-(4-Methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylami-
no)phenylcarbamoyl)thiophene-2-carboxamido)phenylcarbam-
ate (45b). 1H NMR (DMSO-d6): δ (ppm) ) 1.46 (s, 9H), 2.24 (s,
3H), 7.15 (dt, 1H, J ) 1.6 Hz, J ) 7.6 Hz), 7.23 (m, 2H), 7.46 (m,
1H), 7.50 (dd, 2H, J ) 1.2 Hz, J ) 7.8 Hz), 7.55 (m, 2H), 7.94 (d,
1H, J ) 4.0 Hz), 8.07 (m, 2H), 8.50 (m, 1H), 8.53 (d, 1H, J ) 5.2
Hz), 8.70 (dd, 1H, J ) 1.6 Hz, J ) 4.8 Hz), 8.77 (s, 1H), 9.01 (s,
1H), 9.29 (d, 1H, J ) 1.7 Hz), 9.97 (s, 1H), 10.38 (s, 1H). Anal.
(C33H31N7O4S·3/4H2O) C, H, N.
Preparation of 12b, 13b, 14b, 15a, 16a, 17a, 15b, 16b, and
17b by Cleavage of the tert-Butyl Phenylcarbamate Group.
General procedure: The respective carbamic acid tert-butyl-esters
30, 44b, 45b, 46a, 46b, 47a, 47b, 48a, and 48b were dissolved in
TFA (5.0 mL for 0.5 mmol ester) and stirred at room temperature
for 2 h. The solution was poured into water (100 mL) under stirring,
and the mixture was alkalized with NH3 (pH ) 9). The precipitating
product was filtered off, washed with H2O, and dried in vacuo.
Methyl 4-(2-(tert-Butoxycarbonylamino)phenylcarbamoyl)ben-
zoate (61).52 1H NMR (DMSO-d6): δ (ppm) ) 1.44 (s, 9H), 3.91
(s, 3H), 7.22 (dt, 1H, J ) 1.8 Hz, J ) 7.7 Hz), 7.15 (dt, 1H, J )
1.7 Hz, J ) 7.6 Hz), 7.55 (m, 2H), 8.10 (m, 4H), 8.71 (s, 1H),
9.98 (s, 1H).
(E)-Methyl 4-(3-Oxo-3-(tetrahydro-2H-pyran-2-yloxyamino)-
prop-1-enyl)benzoate (72). 1H NMR (DMSO-d6): δ (ppm) ) 1.54
(s, 3H), 1.70 (s, 3H), 3.54 (dd, 1H, J ) 4.6 Hz, J ) 6.9 Hz), 3.86
(s, 3H), 4.02 (m, 1H), 4.93 (s, 1H), 6.63 (d, 1H, J ) 15.9 Hz),
7.55 (d, 1H, J ) 15.8 Hz), 7.72 (d, 2H, J ) 8.2 Hz), 7.98 (d, 2H,
J ) 8.3 Hz), 11.34 (s, 1H). Anal. (C16H19NO5) C, H, N.
Preparation of Suitably Protected and Substituted Carboxy-
lic Acids 37, 38, 39, 40, and 41 by Alkaline Cleavage of the
Corresponding Carboxylic Acid Methyl Esters. The respective
methyl esters 61, 62, 68, 72, and 73 (5.0 mmol) were dissolved in
MeOH (50 mL), and 2 equiv of LiOH in H2O (50 mL) was added.
After stirring at room temperature overnight, MeOH was removed
under reduced pressure, and the aqueous layer was extracted with

Histone Deacetylase- and Tyrosine Kinase-Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2277
N1-(2-Aminophenyl)-N4-(4-methyl-3-(4-(pyridin-3-yl)pyrimi-
din-2-ylamino)phenyl)terephthalamide (12b). 1H NMR (DMSO-
d6): δ (ppm) ) 2.24 (s, 3H), 4.95 (s, 2H), 6.58-6.64 (m, 1H),
6.80 (dd, 1H, J ) 8.0 Hz, J ) 1.1 Hz), 6.96-7.02 (m, 1H), 7.19
(d, 1H, J ) 7.7 Hz), 7.23 (d, 1H, J ) 8.2 Hz), 7.44 (d, 1H, J ) 5.2
Hz), 7.50-7.55 (m, 2H), 8.06-8.15 (m, 5H), 8.47-8.51 (m, 1H),
8.52 (d, 1H, J ) 5.2 Hz), 8.69 (dd, 1H, J ) 4.8 Hz, J ) 1.5 Hz),
8.99 (s, 1H), 9.28 (d, 1H, J ) 1.6 Hz), 9.91 (s, 1H), 10.36 (s, 1H).
Anal. (C30H25N7O2 ·1/2H2O) C, H, N.
Gasteiger-Hueckel charges. Each complex was refined in a
stepwise approach. First, minimization (100 cycles of steepest
descent, then Powell conjugate gradient) with fixed ligand (AM-
BER_FF99 force field,55 dielectric constant of 4) until a root-mean-
square (rms) force of 0.1 kcal mol-1 Å-1 was approached, second,
minimization of the ligand and the surrounding (distance up to 6
Å) protein residues (Tripos force field,56 dielectric constant of 1,
Powell), and, third, again minimization with fixed ligand (AM-
BER_FF99 force field, dielectric constant of 4, Powell). The second
and the third steps were stopped at an rms force of 0.05 kcal mol-1
Å-1. Molecular surfaces and lipophilic potentials (protein variant
with the new Crippen parameter table57,58) were calculated and
visualized by the program MOLCAD (J. Brickmann et al., Technical
University of Darmstadt, Germany) contained within SYBYL.
N2-(2-Aminophenyl)-N5-(4-methyl-3-(4-(pyridin-3-yl)pyrimi-
din-2-ylamino)phenyl)thiophene-2,5-dicarboxamide (14b). 1H
NMR (DMSO-d6): δ (ppm) ) 2.24 (s, 3H), 6.63 (dt, 1H, J ) 1.0
Hz, J ) 7.7 Hz), 6.81 (dd, 1H, J ) 1.0 Hz, J ) 8.0 Hz), 7.01 (dt,
1H, J ) 1.3 Hz, J ) 8.0 Hz), 7.15 (dd, 1H, J ) 1.0 Hz, J ) 7.7
Hz), 7.24 (d, 1H, J ) 8.4 Hz), 7.46 (m, 2H), 7.54 (dd, 1H, J ) 4.8
Hz, J ) 7.9 Hz), 8.02 (m, 2H), 8.08 (d, 1H, J ) 1.8 Hz), 8.52 (m,
2H), 8.70 (dd, 1H, J ) 1.4 Hz, J ) 4.7 Hz), 9.01 (s, 1H), 9.29 (d,
1H, J ) 1.7 Hz), 9.88 (s, 1H), 10.35 (s, 1H). Anal. (C28H23N7O2S)
C, H, N.
Preparation of N-(Tetrahydro-2H-pyran-2-yloxy)amides 49a,
49b and 50a, 50b by Amidation of the Respective Carboxylic
Acids 40 and 41. The respective carboxylic acid was dissolved in
the necessary amount of dry DMF, and 1.1 equiv of BOP, 2.0 equiv
of NEt3, and 1.1 equiv of the respective arylamine were added.
After stirring at room temperature for 24 h, the mixture was poured
into water under stirring, and the precipitating product was removed
by filtration, dried in Vacuo, and purified by column chromatography
(DCM/MeOH ) 10/1).
Acknowledgment. We thank E. Verdin, Gladstone Institute
for Virology and Immunology, San Francisco, CA, for providing
rHDAC1 and rHDAC6 expressing Hek293 cell lines. We also
thank colleagues at ALTANAsNycomed Discovery Research,
in particular U. Bosch and G. Quintini, for providing rHDAC
and kinase protein preparations as well as C. Burkhardt, H.
Wieland, C. Engesser, and H. Julius for excellent technical
assistance.
Supporting Information Available: Full experimental details
for synthetic preparations, analytical data of all compounds, and
biological test systems are available free of charge via the Internet
at http://pubs.acs.org.
(E)-N-(4-Methyl-3-(4-(pyridin-3-yl)thiazol-2-ylamino)phenyl)-4-
(3-oxo-3-(tetrahydro-2H-pyran-2-yloxyamino)prop-1-enyl)benza-
1
References
mide (49b). H NMR (DMSO-d6): δ (ppm) ) 1.55 (s, 3H), 1.71
(s, 3H), 2.28 (s, 3H), 3.55 (m, 1H), 3.97 (m, 1H), 4.94 (s, 1H),
6.64 (d, 1H, J ) 15.9 Hz), 7.20 (d, 1H, J ) 8.5 Hz), 7.38 (dd, 1H,
J ) 2.1 Hz, J ) 8.2 Hz), 7.43 (ddd, 1H, J ) 0.6 Hz, J ) 4.9 Hz,
J ) 8.0 Hz), 7.49 (s, 1H), 7.57 (d, 1H, J ) 15.9 Hz), 7.75 (d, 2H,
J ) 8.2 Hz), 8.01 (d, 2H, J ) 8.4 Hz), 8.33 (m, 1H), 8.49 (dd, 1H,
J ) 1.6 Hz, J ) 4.7 Hz), 8.66 (d, 1H, J ) 2.0 Hz), 9.17 (d, 1H, J
) 1.7 Hz), 9.48 (s, 1H), 10.30 (s, 1H), 11.33 (s, 1H). Anal.
(C30H29N5O4S·1/2H2O) C, H, N.
Preparation of N-Hydroxyamides 10, 18a, 18b, 19a, 19b,
and 54a, 54b by Cleavage of the Respective N-(Tetrahydro-
2H-pyran-2-yloxy)amides (49a, 49b, 50a, 50b, and 53a, 53b).
The respective N-(tetrahydro-2H-pyran-2-yloxy)amide was dis-
solved in the necessary amount of MeOH, 1 N HCl (half of the
amount of MeOH) was added, and the mixture was stirred at room
temperature overnight. The organic solvent was removed in Vacuo,
and the product was filtered off, washed with a small amount of
MeOH, and dried in vacuo.
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