Modified cap group suberoylanilide hydroxamic acid histone deacetylase inhibitor derivatives reveal improved selective antileukemic activity

Article abstract of DOI:10.1021/jm901358y

A series of SAHA cap derivatives was designed and prepared in good-to-excellent yields that varied from 49% to 95%. These derivatives were evaluated for their antiproliferative activity in several human cancer cell lines. Antiproliferative activity was observed for concentrations varying from 0.12 to >100 μM, and a molecular modeling approach of selected SAHA derivatives, based on available structural information of human HDAC8 in complex with SAHA, was performed. Strikingly, two compounds displayed up to 10-fold improved antileukemic activity with respect to SAHA; however, these compounds displayed antiproliferative activity similar to SAHA when assayed against solid tumor-derived cell lines. A 10-fold improvement in the leukemic vs peripheral blood mononuclear cell therapeutic ratio, with no evident in vivo toxicity toward blood cells, was also observed. The herein-described compounds and method of synthesis will provide invaluable tools to investigate the molecular mechanism responsible for the reported selectively improved antileukemic activity.

Full text of DOI:10.1021/jm901358y

3038 J. Med. Chem. 2010, 53, 3038–3047
DOI: 10.1021/jm901358y
Modified Cap Group Suberoylanilide Hydroxamic Acid Histone Deacetylase Inhibitor Derivatives
Reveal Improved Selective Antileukemic Activity
†
†
‡
†
†
†
ꢀ
ꢀ
Chanaz Salmi-Smail, Aurelie Fabre, Franck Dequiedt, Audrey Restouin, Remy Castellano, Slaveia Garbit,
Philippe Roche,§ Xavier Morelli,§ Jean Michel Brunel,*, ,^ and Yves Collette†,^
†Uniteꢀ891 INSERM, Centre de Recherche en Canceꢀrologie de Marseille, 27 bd Lei Roure, 13009 Marseille 09, France, ‡Cellular and Molecular
Biology Unit, Fonds National de la Recherche Scientifique, FaculteꢀUniversitaire des Sciences Agronomiques de Gembloux, 13 Av. Mareꢀchal Juin,
B-5030 Gembloux, Belgium, §CNRS-IBSM, IMR Laboratory, 31 Chemin de Joseph Aiguier, 13402 Marseille 20, France, and Laboratoire
URMITE UMR 6236 CNRS, Faculteꢀ de Meꢀdecine et de Pharmacie, Universiteꢀ de la Meꢀditerraneꢀe, 27 bd Jean Moulin, 13385 Marseille 05,
France. ^J.M.B. and Y.C. share senior coauthorship.
Received September 16, 2009
A series of SAHA cap derivatives was designed and prepared in good-to-excellent yields that varied from
49% to 95%. These derivatives were evaluated for their antiproliferative activity in several human
cancer cell lines. Antiproliferative activity was observed for concentrations varying from 0.12 to >100
μM, and a molecular modeling approach of selected SAHA derivatives, based on available structural
information of human HDAC8 in complex with SAHA, was performed. Strikingly, two compounds
displayed up to 10-fold improved antileukemic activity with respect to SAHA; however, these
compounds displayed antiproliferative activity similar to SAHA when assayed against solid tumor-
derived cell lines. A 10-fold improvement in the leukemic vs peripheral blood mononuclear cell
therapeutic ratio, with no evident in vivo toxicity toward blood cells, was also observed. The herein-
described compounds and method of synthesis will provide invaluable tools to investigate the molecular
mechanism responsible for the reported selectively improved antileukemic activity.
Scheme 1. Structure of SAHA
Introduction
The reversible acetylation of lysine residues in histone tails
plays a critical role in transcriptional activation and repres-
sion.1,2 The regulation of these post-translational modifica-
tions is balanced by opposing histone acetyltransferase
(HATa) and histone deacetylase (HDAC) activities. In-
creased HDAC activity has generally been associated with
transcriptional repression, whereas increased HAT activity
(or HDAC inhibition) facilitates gene expression.1,2 HDACs
are also involved in reversible acetylation of non-histone
proteins.3-5 There are around 20 human HDACs that fall into
four classes6,7 based on their homology to yeast models: class I
and II are zinc-dependent metallohydrolases,6 whereas class III
HDACs or sirtuins are NADþ-dependent deacetylases.8 Class
IV, recently described (comprising only HDAC11), exhibits
properties of both class I and class II HDACs. Treatment of
tumor cells with general inhibitors of class I/II HDACs results in
growth arrest, differentiation, and apoptosis,1-11 promoting
these enzymes as potential cancer drug targets.12
tives are in both preclinical development and clinical trials.13
Among all of these inhibitors, suberoyl anilide hydroxamic
acid (SAHA, 1a)14 has emerged as an effective therapeutic
anticancer agent and recently gained FDA approval for the
treatment of advanced cutaneous T-cell lymphoma.15 Most
HDAC inhibitors synthesized to date closely resemble the
aliphatic acetyl-lysine substrate. These deliver an hydroxamic
acid or other zinc-binding group to the catalytic zinc ion at the
bottom of a narrow active site pocket, as seen in cocrystal
structuresofinhibitedHDLP (HDAC-like protein),16 HDAH
(HDAC-like amidohydrolase),17 and human HDAC8.18 In
addition to altering the metal-binding moiety toward a
HDAC inhibitor design, the hydrocarbon linker has been
diversified to focus on changing chain length, the creation of
unsaturation points along the chain, and the inclusion of an
arylcyclohexyl ring within the chain.19-22 Thus, distinguish-
ing characteristics of HDAC inhibitors like SAHA have
included a metal binding moiety, carbon linker, and capping
group (Scheme 1).
In this context, numerous programs involving short chain
fatty acids (butyrate and valproate derivatives), cyclic pep-
tides, depsipeptide (FK-228), and hydroxamic acid deriva-
*To whom correspondence should be addressed. Phone: (33) 4-86-13-
68-30. Fax: (33) 4 91 38 77 72. E-mail: bruneljm@yahoo.fr; yves.collette@
inserm.fr.
a Abbreviations: SAHA, suberoylanilide hydroxamic acid; HDAC,
histone deacetylase; HAT, histone acetyltransferase; HDLP, histone-
deacetylase-like protein; HDAH, histone-deacetylase-like amidohydro-
lase; BOP, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexa-
fluorophosphate.
Onthe basisof crystallographic analyses, the cappinggroup
is solvent-exposed and interacts with amino acids near the
entrance of the active site. In contrast, the metal-binding
moiety resides in the protein interior and complexes with the
r
pubs.acs.org/jmc
Published on Web 03/10/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3039
Scheme 2. General Pathway for SAHA Parent Derivative Synthesis
Table 1. Yields and log D of New Synthesized SAHA Analogues 1a-v
EC50 (μM)c
compd
yield (%)a
log D b
Skbr3
HT29
U937
JA16
0.87
HL60
1.2
K562
2.2
1a
1b
1c
1d
1e
1f
79
67
91
91
84
75
95
88
89
82
90
89
77
92
92
84
49
91
77
94
49
89
1.67
0.88
1.38
1.81
1.81
2.56
2.56
2.19
2.19
1.82
2.66
2.66
3.32
1.93
1.63
2.68
1.75
3.39
4.00
-0.93
-2.73
-0.92
2.1
8.5
11
2
18
0.82
6.4
15
6.4
0.5
1.9
3
1.4
4.2
6.4
1.4
2.1
1
0.64
1.25
2.4
1.8
1.2
26
1g
1h
1i
2.2
2.3
2
0.45
0.55
0.35
6.8
0.53
0.77
0.58
1.54
1.87
2.1
2,1
3.3
3.5
1j
17.5
1.1
8.8
73.5
4.2
58
17
1k
1l
0.95
42
0.12
7
0.24
0.85
1.3
1m
1n
1o
1p
1q
1r
1s
1t
27
14
1.7
3.15
76
22
1.8
>100
4
0.85
>100
2.5
1.4
32
0.35
26
0.4
1.16
3
0.6
0.3
0.43
0.18
1.13
0.35
3.5
0.8
1.2
27
23
>100
26.5
1u
1v
>100
40.5
>100
63
>100
16
>100
18
>100
>100
a Overall yield after two-step synthesis. b The log D value has been estimated using Marvin software. c EC50 (half maximal effective concentration)
refers to the concentration of a drug inducing a response halfway between the baseline and maximum after a specified exposure time.
metal ion involved in catalysis.16,18,32 However, the influence
of substitution of the phenyl SAHA capping group remains
unclear. This area of study remains relatively unexplored,
most likely because the paths of synthesis described thus far
have precluded such analogues from being easily produced.
To the best of our knowledge, the four SAHA synthetic
procedures described to date suffer from low yields and/or
long reaction times (up to 28 h for the first three).23-25 On the
other hand, Gediya et al.26 have reported an interesting
expeditious SAHA synthetic route. However, no extension
of such a procedure for the design of various SAHA deriva-
tives has been envisioned, and no evaluation of their potent
biological activities has been described. It is noteworthy that
several unpublished variations of the SAHA structure seem to

3040 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Salmi-Smail et al.
Figure 1. Dose-response antiproliferative activity of compounds 1a (SAHA) to 1v in breast-derived SkbR3 cells for the indicated range of
concentrations (0.1-100 μM). Results are expressed as the percent proliferative response after normalization to solvent (DMSO) treated cells.
Figure 2. Western blot analysis of acetylated H4 and p21 levels in SKBR3 cells following treatment with the indicated 1a-v analogues. Cells
were examined after 6, 12, and 24 h of incubation with SAHA derivatives (2 μM) or solvent (DMSO). β-Tubulin levels are shown to indicate
comparable loading of cell lysates.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3041
Figure 3. Structure-activity relationships of SAHA derivatives by correlation of their capping group hydrophobicity (log D) with respect to
their observed antiproliferative activity (EC50).
have been developed by Marks and Breslow27 as well as by
Meinke and co-workers from the Merck Research Labora-
tories.28 However, these were too toxic to be considered as
potential drug candidates.
In this paper, we report a mild two-step synthesis of a
library of new substituted SAHA parent derivatives from
commercially cheap available reagents (Scheme 2). This
high-yield synthesis varies the nature of the hydrophobic
capping group. While the presented chemistry might not be
considered particularly new, to the best of our knowledge, the
compounds and, more importantly, the influence of the
capping group substitution in the antitumoral selectivity of
Figure 4. Predicted binding of SAHA derivatives to HDAC8.
HDAC8 (PDB code 1T69) is represented as a solid surface and is
the corresponding SAHA derivatives remain unclear and a
few studies29-31 have examined the impact of this part of
molecule on its biological activities. Unexpectedly, the study
reveals selective improvement in the antileukemic activity of
some of these SAHA analogues.
oriented to illustrate the interaction with SAHA derivatives. The
color code corresponds to hydrophilicity. Ligands are shown as
sticks. FlexX 2.0 was used to predict the mode of binding of various
SAHA derivatives as described in the Experimental Section: (A) 1f
(orange) and 1g (blue); (B) 1s (orange), 1k (blue), SAHA (white).
activity of the considered compounds. The presence of a sub-
stituent in the ortho position reduced the compound’s anti-
proliferative activity with respect to meta- or para-substituted
derivatives. Nevertheless, in these two latter cases, the nature
of the substituents greatly influences the activity. In contrast
to iodo, naphthyl, or pyrene groups, electron-donating (e.g.,
NH2, NMe2, or OH) or electron-withdrawing (e.g., NO2 or
CN) groups did not improve the compound’s antiproliferative
activity. The results from these latter cases also suggest that
the steric hindrance generated and hydrophobic nature of
these groups constitute the most important factors to be taken
into consideration during the design of most potent SAHA
analogues.
Taking into consideration the log D parameter, which
reflects the true behavior and bioavailability of an ionizable
compoundinasolution at a given pH, a significant correlation
with antiproliferative activity was observed (Figure 3). In-
deed, derivatives displaying the highest logD values (e.g., the
most hydrophobic derivatives 1k, 1p, and 1s) also displayed
higher antiproliferative activity, in agreement with the pre-
viously mentioned results.
Discussion
The first step consists of a peptide coupling reaction using
various aniline derivatives and suberic acid monomethyl ester
with an efficient and versatile benzotriazol-1-yloxytris-
(dimethylamino)phosphonium hexafluorophosphate cou-
pling reagent (BOP).32 The obtained ester is successfully
transformed into its corresponding hydroxamic derivative
by using hydroxylamine (50% H2O) in refluxing methanol
for 2 h. All of the synthesized products were obtained in good-
to-excellent yields, varying from 49% to 95% (Table 1).
1a-v were first examined for their antiproliferative and
histone deacetylation activities in a cell-based assay. 1d, 1e,
1g-i, 1k, 1n, 1p, 1r, and 1s inhibited SKBR3-breast-derived
cell line proliferation, with EC50 values varying from 0.95 to 4
μM (Figure1, Table1). In contrast, 1b, 1c, 1q, 1m, 1o, 1f, 1l, 1j,
and 1v derivatives displayed the weakest antiproliferative
activity, with EC50 values ranging from 8.5 to 73.5 μM; 1u
and 1q proved inactive for concentrations up to 100 μM. As
expected, the antiproliferative activity of the various com-
pounds correlated well with their time-course ability to up-
regulate histone H4 acetylation and p21/WAF1 cell cycle
inhibitor accumulation in treated cell extracts (Figure 2).
The nature and presence of a substituent on the aryl moiety
of the capping group greatly influence the antiproliferative
We thus developed a molecular approach for modeling
selected SAHA derivatives based on available structural
information regarding human HDAC8 in complex with
SAHA (PDB code 1T69).18,33 For all the derivatives, the

3042 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Salmi-Smail et al.
Figure 5. Antiproliferative activity of SAHA derivatives in different cancer-derived cell lines. (A) Screening of SAHA analogues for their
antiproliferative activity in breast-derived SKBR3, colon-derived HT29, and leukemia-derived U937 tumor cell lines. The antiproliferative
activity profile was reported as the ratio between SAHA analogue and SAHA antiproliferative activities and calculated as the ratio R =
[1/EC50(x)]/[1/EC50(SAHA)]. These data were translated into a color code using MEV multiexperiment viewer (SAHA analogues with R > 1
are represented in red, and compounds with R < 1 are in green; a black color indicates molecules with R = 1). (B) Activity comparison for
SAHA analogues in U937, Jurkat JA16, HL60, and K562 leukemias, as described in (A). (C) Dose-response activity of 1s, 1k, and 1u
compared to SAHA in U937 cells for the indicated range of concentrations (0.1-100 μM). Results are expressed as relative light units (RLUs).
hydroxamate linker moiety adopted a very similar confor-
mation in the binding pocket. In contrast, the orientation of
the aromatic group varied, enabling the detection of two
populations correlated to biological activities. In all active
derivatives, the apolar head was found in the same orienta-
tion as SAHA in the X-ray structure; for inactive com-
pounds, however, a different orientation was observed
(Figures 4). The result was particularly striking with com-
pounds 1f and 1g, which correspond to the meta and para
isomers of the same compound (Figure 4A). The aromatic
moiety of biologically active SAHA derivatives was found to
be stabilized by direct interaction with the tyrosine Y306
residue.
The antiproliferative activity of the various SAHA deriva-
tives was further investigated in various tumor-derived cell
lines. Surprisingly, compared to SAHA, compounds 1d, 1e,
1g, 1h, 1i, 1k, 1n, 1p, 1r, 1s displayed more efficient antipro-
liferative activity toward the leukemia cell line U937. When
assayed against the SKBR3 and HT29 solid tumor-derived
cell lines, however, the antiproliferative activities of the same
compounds were comparable to that of SAHA (Figure 5A).
Interestingly, this more efficient antiproliferative activity
toward the leukemia cell line U937 was further verified on
other leukemia cell lines (Figure 5B).
Hence, the pyrene variant 1s (EC50 = 0.4 μM) and the iodo-
substituted SAHA derivative 1k (EC50 = 0.12 μM) proved 3-
and 10-fold more active than SAHA (EC50 = 1.2 μM),
respectively, when assayed against U937 cells. In contrast,
both compounds presented antiproliferative activity compar-
able to that of SAHA (EC50 ≈ 1 μM) when evaluated in
SKBR3 cells (Figure 5C). This differential activity of 1k and
1s correlated well with prolonged H4 acetylation and higher
induction of p21 levels in comparison to SAHA-treated cells
in U937 but not SKBR3 cells lines (Figure 6). Together, these
results indicate that the increased antiproliferative activity
displayed by 1k and 1s toward leukemia cell lines does not
result from increased broad toxicity. Indeed, when assayed on
cultures of peripheral blood mononuclear cells from normal
blood donors, 1k and 1s displayed cell toxicity comparable to
that of SAHA (therapeutic ratios of 15, 30, and 155 for 1s, 1k,
and SAHA, respectively) (Figure 7). In vivo toxicity was also
evaluated by monitoring the body weight, survival, and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3043
Figure 6. Western blot analysis of acetylated H4 and p21 levels in SKBR3 and U937 cells following treatment with SAHA analogues. Cells
were examined after 6, 12, and 24 h of incubation with SAHA derivatives (2 μM) or solvent (DMSO). Bands were quantified using ImageJ, and
results are presented after normalization of specific p21 and acetylated H4 signals to β-tubulin levels.
Figure 7. Dose-response activity of 1k, 1s, and 1u compared to SAHA in U937 cells and peripheral blood mononuclear cell (PBMC) cultures
for the indicated range of concentrations (0.1-100 μM). Results are expressed as relative light units (RLUs). EC50 values derived from these
curves were used to calculate a therapeutic ratio (TR = EC50(PBMC)/EC50(U937)) and are presented in the table in the lower-right corner.
hematological numeration of blood cells from mice treated
orally via forcedfeeding with50(mg/kg)/dayof1kandSAHA
for 2 weeks (Table 2). No deaths were observed among the
differentially treated groups during either the treatment per-
iod or in the month following interruption of the treatment.
Further, mice treated with 1k displayed indistinguishable

3044 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Table 2. Blood Analysis (Mean ( SEM)
Salmi-Smail et al.
HDAC representative) and HDAC6 (class II HDAC re-
presentative), showing comparable inhibition of both
HDAC1 and HDAC6 activity (Figure 8).
sample
CTL group
1a group
1k group
Number of Platelets (103/mm3)
J3
J5
n = 3
n = 3
n = 3
500 ( 33.5
454 ( 93
514 ( 42.8
428.0 ( 20.2
617.0 ( 29.7
538.0 ( 40.8
481.3 ( 27.4
478.3 ( 84.0
537 ( 28.3
Conclusion
J12
Potent SAHA derivatives with low micromolar range anti-
proliferative and histone hyperacetylation activities were
obtained by increasing the size of the hydrophobic region (1s)
or by substitution of the phenyl SAHA capping group in the
meta or para position (e.g., as in derivative 1k). In this context,
compounds displaying up to a 10-fold improvement in anti-
leukemic activity compared to SAHA have been identified. The
development of SAHA analogues like 1k with a peculiar profile
of inhibition toward leukemic cells not only raises the question
of the implication of distinct HDAC(s) and/or other molecular
determinants in this pathology but also may be useful for
dissecting their underlying biological functions. These com-
pounds and the related method of synthesis described will thus
provide invaluable tools to investigate further the underlying
molecular basis of improved cell-specific antiproliferative ac-
tivity. Such knowledge might prove useful in the future for
improving selectivity while reducing the side effects of HDAC
inhibitors developed in the clinic, particularly for the treatment
of leukemias. These compounds may further represent valuable
cancer therapy methods, for example, in association with other
proapoptotic drugs.
Mean Platelet Volume (μm3)
J3
J5
n = 3
n = 3
n = 3
8.2 ( 0.1
8.4 ( 0.5
8.8 ( 0.1
7.9 ( 0.3
7.7 ( 0.2
8.4 ( 0.1
8.2 ( 0.3
8.6 ( 0.6
8.7 ( 0.2
J12
Number of Red Blood Cells (106/mm3)
J3
J5
n = 3
n = 3
n = 3
8.6 ( 0.4
8.1 ( 0.2
7.7 ( 0.1
7.9 ( 0.05
8.3 ( 0.1
7.6 ( 0.1
7.9 ( 0.3
8.02 ( 0.4
6.9 ( 0.2
J12
Mean Cell Volume of red Cells (μm3)
J3
J5
n = 3
n = 3
n = 3
44 ( 0.3
43.7 ( 0.4
43.9 ( 0.5
44.4 ( 0.1
44.1 ( 0.2
44.5 ( 0.2
44.0 ( 0.05
43.9 ( 0.4
43.7 ( 0.5
J12
Number of White Blood Cells (103/mm3)
J3
J5
n = 3
n = 3
n = 3
7.4 ( 1.2
9.1 ( 1.2
8.1 ( 0.5
8.4 ( 1.4
5.9 ( 0.5
8.3 ( 0.6
8.5 ( 1.3
7.5 ( 0.7
7.5 ( 1.2
J12
Hematocrit (%)
J3
J5
n = 3
n = 3
n = 3
37.8 ( 2.2
35.3 ( 1.2
34.0 ( 0.8
35.0 ( 0.2
36.5 ( 0.8
33.7 ( 0.3
34.7 ( 1.5
35.2 ( 2.1
30.2 ( 0.7
J12
% of Weight at J3
Experimental Section
J3
J5
n = 3
n = 3
n = 3
100 ( 0
100 ( 0
100 ( 0
All solvents were purified according to reported procedures,
and the reagents used were commercially available. Methanol,
ethyl acetate, dichloromethane, and petroleum ether (35-60 ꢀC)
were purchased from SDS and used without further purifica-
tion. Column chromatography was performed on SDS silica gel
(70-230mesh). 1H NMRand13C NMRspectra wererecorded in
MeOD or DMSO-d6 on a Bruker AC 300 spectrometer working
at 300 and 75 MHz, respectively (the usual abbreviations are
used: s, singlet; d, doublet; t, triplet; q:, quadruplet; m, multiplet).
Tetramethylsilane was used as the internal standard. All chemi-
cal shifts are given in ppm. Purity (up to 95%) of all the
synthesized compounds has been established by HPLC analysis,
and mass spectroscopy analysis was performed by the Spectro-
96.7 ( 1.0
93.9 ( 1.7
94.9 ( 0.2
96.2 ( 1.4
99.7 ( 0.3
97.8 ( 0.8
J12
ꢀ
pole (Analytical Laboratory) of the University Paul Cezanne
(Marseille, France).
General Procedure for the Synthesis of SAHA 1a and Its
Parent Analogues 1b-v. In a 50 mL two-necked round flask,
aniline (279mg, 310-3 mol) and methyl ester suberic acid (564 mg,
3 10-3 mol) were placed in anhydrous dichloromethane (15 mL)
at room temperature. The mixture was placed under stirring.
Diisopropylamine (1.37 mL, 1.06 ꢀ 10-2 mol) was added, fol-
lowed by the addition of the coupling reagent (BOP) (1.37 g,
3 ꢀ 10-3 mol) dissolved in 5 mL of CH2Cl2. The mixture was
stirred for 12 h at 20 ꢀC. The solvent was removed under vacuum
and the crude residue purified by chromatography on a silica gel
column using EtOAc/petroleum ether as eluent (1/1 to 1/0). This
procedure afforded the expected coupling product as a white solid
at 85% yield. 1H NMR (DMSO-d6): δ = 7.59-6.69 (m, 5H), 4.83
Figure 8. Effect of SAHA analogues (1 μM) on cell-derived
HDAC1 and HDAC6 deacetylase activity.
body weights and hematological counts compared to both
SAHA-treated and control mice.
(s, 1H), 3.63 (s, 3H), 2.38-2.27 (m, 3H), 1.70-1.29 (m, 7H). 13
C
NMR (DMSO-d6): δ = 176.44, 175.01, 140.33, 130.54, 130.28,
125.59, 121.70, 117.26, 52.55, 38.41, 35.21, 30.39, 30.33, 27.18,
26.30.
To a stirred solution of the previous derivative in methanol
(15 mL) was added a solution of hydroxylamine (50% in water).
The resulting solution was stirred for 3 h at 60 ꢀC. The solvent
was removed under vacuum and the crude residue purified by
chromatography on a silica gel column using EtOAc/MeOH
The reason for the difference in cellular activity among
these compounds is unclear, but it may be due to differences in
the cellular permeability or, more likely, HDAC expression
profile between leukemia cells and solid tumor-derived cell
lines. To gain preliminary insight into this possibility, 1d, 1e,
1g-i, 1k, 1n, 1p, 1r, and 1s were compared to SAHA in a
deacetylase activity assay using cell-derived HDAC1 (class I

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3045
as eluent (1/1 to 0/1). This afforded the expected derivative 1a as
a white solid at 92% yield: mp = 161 ꢀC. 1H NMR (DMSO-d6):
δ = 10.37 (s, 1H), 9.86 (s, 1H), 8.70 (s, 1H), 7.66-6.99 (m, 5H),
2.52-2.50 (m, 1H), 2.31-2.22 (m, 2H), 1.99-1.88 (m, 2H),
1.58-1.06 (m, 8H). 13C NMR (DMSO-d6): δ = 171.67, 169.58,
139.54, 129.00, 123.32, 119.42, 36.71, 32.59, 28.74, 25.38.
C14H20N2O3. MS (ESIþ) m/z 264.1474 (100%, (M þ Hþ)).
Compound 1b. Yellow oil crystallizing on standing: mp = 142 ꢀC.
1H NMR (DMSO-d6): δ = 9.43 (s, 1H), 7.21-7.18 (m, 2H),
6.50-6.47 (m, 2H), 2.51 (s, 1H), 2.22-1.92 (m, 4H), 1.48-1.26
(m, 10H). 13C NMR (DMSO-d6): δ = 171.18, 170.08, 145.42,
129.50, 121.80, 114.70, 37.07, 33.14, 30.27, 29.34, 29.30, 26.15,
25.95. C14H 21N3O3. MS (ESIþ) m/z 279.1583(100%, (M þ Hþ)).
Compound 1c. Yellow oil crystallizing on standing: mp =
29.30, 25.97. C15H19N3O3. MS (ESIþ) m/z 289.1426 (100%,
(M þ Hþ)).
Compound 1m. Yellow oil crystallizing on standing: mp =
172 ꢀC. 1H NMR (DMSO-d6): δ = 9.19 (s, 1H), 7.43-7.31
(m, 9H), 2.29-1.18 (m, 14H). 13C NMR (DMSO-d6): δ =
174.98, 171.91, 145.28, 140.04, 139.40, 135.26, 130.36, 129.10,
128.61, 127.11, 126.12, 117.12, 115.60, 32.60, 28.69, 25.40.
C20H24N2O3. MS (ESIþ) m/z 340.1787 (100%, (M þ Hþ)).
Compound 1n. Brown solid: mp = 128 ꢀC. 1H NMR (DMSO-
d6): δ = 9.52 (s, 1H), 7.37-7.34 (d, J = 12 Hz, 2H), 6.66-6.63
(d, J = 8 Hz, 2H), 2.80 (s, 6H), 2.52-1.23 (m, 14H). 13C NMR
(DMSO-d6): δ = 171.12, 169.80, 147.37, 125.37, 121.10, 112.99,
36.52, 32.57, 28.65, 25.50, 25.34. C16H25N3O3. MS (ESIþ) m/z
307.1896 (100%, (M þ Hþ)).
1
122 ꢀC. H NMR (DMSO-d6): δ=9.43 (s, 1H), 7.21-7.18 (m,
Compound 1o. White solid: mp = 110 ꢀC. 1H NMR (DMSO-
d6): δ = 7.96-7.93 (d, J = 12 Hz, 2H), 6.71-6.59 (m, 2H),
2.51-1.10 (m, 12H). 13C NMR (DMSO-d6): δ = 176.58, 169.58,
156.05, 136.98, 126.74, 112.74, 34.91, 32.57, 28.70, 25.37, 25.17.
C14H19N3O5. MS (ESIþ) m/z 309.3215 (100%, (M þ Hþ)).
Compound 1p. White solid: mp = 124 ꢀC. 1H NMR (DMSO-
d6): δ = 10.12 (s, 1H), 8.31 (s, 1H), 7.85-7.35 (m, 7H), 2.52-1.22
(m, 13H). 13C NMR (DMSO-d6): δ = 172.00, 169.61, 137,25,
133.81, 129.98, 128.61, 127.77, 127.57, 126.71, 124.79, 120.35,
115.38, 36.79, 32.61, 28.76, 25.40. C18H22N2O3. MS (ESIþ) m/z
314.1630 (100%, (M þ Hþ)).
2H), 6.50-6.47 (m, 2H), 2.51 (s, 1H), 2.22-1.92 (m, 4H), 1.48-
1.26 (m, 10H). 13C NMR (DMSO-d6): δ = 175.03, 172.87, 155.43,
131.03, 119.06, 116.37, 37.48, 32.12, 24.82, 24.75, 23.61. C14H20-
N2O 4. MS (ESIþ) m/z 280.1423 (100%, (M þ Hþ)).
Compound 1d. Yellow oil crystallizing on standing: mp =
154 ꢀC. 1H NMR (DMSO-d6): δ = 10.37 (s, 1H), 10.08 (s, 1H),
7.63-6.90 (m, 4H), 2.52-2.23 (m, 3H), 2.02-1.86 (m, 2H),
1.59-1.26 (m, 8H). 13C NMR (DMSO-d6): δ = 172.04, 169.58,
162.45, 141.40, 130.51, 115.04, 1109.63, 106.12, 36.73, 32.58,
28.71, 25.37, 25.24. C14H19FN O3. MS (ESIþ) m/z 282.1383
2
Compound 1q. Pale viscous yellow oil. 1H NMR (DMSO-d6):
δ = 7.74-7.27 (m, 7H), 2.28-1.31 (m, 14H). 13C NMR (DMSO-
d6): δ = 179.80, 172.54, 143.65, 137.14, 128.18, 127.30, 124.82,
118.07, 111.67, 35.97, 32.95, 29.29, 29.09. C17H21N3O3. MS
(ESIþ) m/z 315.1583 (100%, (M þ Hþ)).
(100%, (M þ Hþ)).
Compound 1e. Pale viscous yellow oil: 1H NMR (DMSO-d6):
δ = 9.95 (s, 1H), 7.61-7.08 (m, 4H), 2.51-1.26 (m, 14H). 13
C
NMR (DMSO-d6): δ = 173.86, 171.67, 135.96, 121.28, 121.15,
115.66, 115.36, 36.53, 33.55, 28.69, 28.51, 25.34, 24.62.
C14H19FN2O3. MS (ESIþ) m/z 282.1383 (100%, (M þ Hþ)).
Compound 1f. Yellow oil crystallizing on standing: mp = 52
ꢀC. 1H NMR (DMSO-d6): δ = 9.60 (s, 1H), 7.38-7.34 (m, 2H),
6.99-6.44 (m, 4H), 2.52-2.50 (m, 1H), 2.25-1.93 (m, 4H),
1.58-1.22 (m, 7H). 13C NMR (DMSO-d6): δ = 170.97, 169.60,
153.47, 148.62, 140.92, 131.31, 121.28, 115.90, 115.34, 36.55,
32.59, 28.73, 25.39. C15H19F3N2O3. MS (ESIþ) m/z 332.1348
(100%, (M þ Hþ)).
Compound 1r. Yellow oil crystallizing on standing: mp = 132
ꢀC. 1H NMR (DMSO-d6): δ = 9.97 (s, 1H), 7.95-7.25 (m, 9H),
3.89 (s, 2H), 2.52-1.29 (m, 12H). 13C NMR (DMSO-d6): δ =
171.66, 168.58, 144.06, 143.11, 141.38, 138.73, 136.46, 127.07,
126.38, 125.35, 120.38, 119.72, 118.25, 116.28, 36.84, 32.60,
28.76, 25.45, 25.40. C21H24N2O3. MS (ESIþ) m/z 352.1787
(100%, (M þ Hþ)).
Compound 1s. Gray solid: mp = 158 ꢀC. 1H NMR (DMSO-
d6):δ=10.31 (s, 1H), 8.33-7.38 (m, 10H), 6.35 (s, 1H), 2.62-1.40
(m, 12H). 13C NMR (DMSO-d6): δ = 170.23, 167.29, 142.44,
130.10, 130.06, 129.75, 128.90, 128.56, 126.19, 125.77, 125.29,
124.41, 123.93, 122.92, 120.98, 120.43, 119.82, 112.78, 111.19,
34.03, 30.38, 26.62, 26.54, 23.37, 23.19. C24H24N2O3. MS (ESIþ)
m/z 388.1787 (100%, (M þ Hþ)).
Compound 1g. Yellow oil crystallizing on standing: mp = 64
ꢀC. 1H NMR (DMSO-d6): δ = 10.26 (s, 1H), 7.79-7.62 (m, 4H),
2.51-1.26 (m, 14H). 13C NMR (DMSO-d6): δ = 172.36, 169.59,
143.17, 126.27, 119.22, 36.74, 32.57, 28.69, 25.35, 24.62.
C15H19F3N2O3. MS (ESIþ) m/z 332.1348 (100%, (M þ Hþ)).
Compound 1h. White solid: mp = 146 ꢀC. 1H NMR (DMSO-
d6): δ = 10.01 (s, 1H), 7.64-7.32 (m, 4H), 2.51-1.27 (m, 14H).
13C NMR (DMSO-d6): δ = 171.75, 169.46, 138.64, 128.89,
126.78, 120.89, 36.70, 32.59, 28.74, 25.38, 25.29, 24.79.
C14H19ClN2O3. MS (ESIþ) m/z 298.1084 (100%, (M þ Hþ)).
Compound 1i. Yellow solid: mp = 140 ꢀC. 1H NMR (DMSO-
d6): δ = 10.08 (s, 1H), 7.80 (s, 1H), 7.40-7.26 (m, 2H), 7.07-
7.03 (m, 1H), 2.31-2.26 (m, 2H), 1.98-1.93 (m, 2H), 1.57-
1.22 (m, 10H). 13C NMR (DMSO-d6): δ = 172.24, 169.77,
140.97, 133.37, 130.66, 123.05, 118.91, 117.79, 36.67, 32.56,
28.64, 25.33. C14H19ClN2O3. MS (ESIþ) m/z 298.1084 (100%,
(M þ Hþ)).
Compound 1t. White solid: mp = 164 ꢀC. 1H NMR (DMSO-
d6): δ = 2.51(s, 1H), 1.96-1.91 (m, 5H), 1.47-1.19 (m, 11H). 13
C
NMR (DMSO-d6): δ = 169.78, 32.54,28.54, 25.32. C8H16-
N2O4. MS (ESIþ) m/z 204.1110 (100%, (M þ Hþ)).
Compound 1u. White solid: mp = 126 ꢀC. 1H NMR (DMSO-
d6): δ = 3.16 (s, 2H), 2.03-1.92 (m, 4H), 1.43-1.27 (m, 9H). 13
C
NMR (DMSO-d6): δ = 180.63, 170.40, 48.90, 37.01, 32.50,
29.16, 28.91, 28.59, 25.88, 25.32. C8H15NO4. MS (ESIþ) m/z
189.1001 (100%, (M þ Hþ)).
Compound 1v. Yellow oil crystallizing on standing: mp =
62 ꢀC. 1H NMR (DMSO-d6): δ = 7.71-7.14 (m, 4H), 2.62-1.20
(m, 12H). 13C NMR (DMSO-d6): δ = 176.91, 167.79, 141.38,
125.57, 121.43, 121.26, 116.53, 109.73, 47.05, 30.65, 26.69, 23.44.
C12H17FN4O4. MS (ESIþ) m/z 300.2932 (100%, (M þ Hþ)).
Cell Culture Conditions and Proliferation Assay. The human
breast cancer cell line SKBR3 and colon adenocarcinoma cell
line HT29 were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) and
0.1% sodium pyruvate. The human U937 and HL60 myeloid
leukemia cell lines, K562 chronic erythromyeloid leukemia
cells, and clone Jurkat leukemia JA16 cells were maintained in
RPMI-1640 medium supplemented with 10% FBS at 37 ꢀC
with 5% CO2. Peripheral blood mononuclear cells (PBMCs)
were obtained from donors whole blood cells and cultured in
RPMI-1640 medium supplemented with 10% FBS at 37 ꢀC with
5% CO2.
Compound 1j. Brown solid: mp = 68 ꢀC. 1H NMR (DMSO-
d6): δ = 9.41 (s, 1H), 7.67-7.11 (m, 4H), 2.56-1.27(m, 14H). 13
C
NMR (DMSO-d6): δ = 171.81, 169.49, 136.76, 132.94, 128.27,
127.31, 118.57, 35.99, 32.62, 28.73, 25.45. C14H19BrN2O3. MS
(ESIþ) m/z 342.0579 (100%, (M þ Hþ)).
Compound 1k. White solid: mp = 150 ꢀC. 1H NMR (DMSO-
d6): δ = 9.99 (s, 1H), 7.62-7.41 (m, 4H), 2.52-2.51 (m, 2H),
2.30-2.25 (m, 2H), 1.96-1.92 (m, 2H), 1.58-1.25 (m, 8H). 13
C
NMR (DMSO-d6): δ = 171.92, 169.61, 139.43, 137.64, 121.66,
86.62, 36.73, 32.57, 28.69, 25.35, 26.26. C14H19IN2O3. MS
(ESIþ) m/z 390.0440 (100%, (M þ Hþ)).
1
Compound 1l. Pale-yellow oil: H NMR (DMSO-d6): δ =
9.780 (s, 1H), 7.38-6.94 (m, 4H), 2.52-2.50 (m, 1H), 2.25-1.22
(m, 13H). 13C NMR (DMSO-d6): δ = 180.28, 170.60, 152.71,
150.46, 127.36, 126.85, 121.50, 119.64, 114.27, 37.34, 33.20,

3046 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Salmi-Smail et al.
Stock solutions of synthesized and commercial (Merck and
Co., Inc.) suberoyl anilide hydroxamic acid (SAHA) as well
as the synthesized SAHA derivatives (10 mM) dissolved in
dimethyl sulfoxide (DMSO) were aliquotted and stored at
-20 ꢀC.
the Cscore Sybyl module34 for docking experiments with FlexX
2.0:35 FlexXscore, Gscore, Dscore, PMFscore, and Chemscore
(http://www.biosolveit.de). We also rescored each docking with
our in-house scoring function, GFscore (http://gfscore.cnrs-
mrs.fr).36
The Tripos Sybyl “Structure Preparation Tool” was used to
optimize the protein structure before use by FlexX 2.0. Hydro-
gen atoms were added, bumps and side chain amides corrected,
and missing residues added and minimized. The newly obtained
PDB file was then injected as such into FlexX 2.0.
In antiproliferative assays, compounds were assayed for their
growth-inhibiting activity toward the described cancer cell lines
or PBMCs using the CellTiter-Glo luminescent cell viability
assay as described by the manufacturer (Promega Corp.).
Briefly, for adherent cells, 104 cells were plated onto 96-well
plates (white with clear bottom (3610, Corning Costar)) in
100 μL of media per well and were allowed to grow overnight
before the assay. For cells growing in suspension, 104 cells were
plated onto 96-well plates immediately before the assay. Com-
pounds were added at different concentrations (varying from
100 to 0.1 μM) to each well, and cell cultures were incubated for
48 h. Vehicle (DMSO) was used as a control, and all compounds
were tested in a constant percentage of DMSO (1%). After
addition of 50 μL of CellTiter GLO, luminescence was measured
using a Centro luminometer (Berthold). EC50 values were
determined as the dose of compound required to reduce lumi-
nescent values to 50% of the signal obtained for untreated cell
cultures.
SDS-PAGE and Western Blot Analysis. SKBR3 and U937
cells (0.5 ꢀ 106) were plated onto six-well cell culture dishes
(Corning Costar) and cultured for 6, 12, and 24 h in 2 mL of
media with the tested compounds (2 μM final concentration). At
each time point, proteins were prepared from the total cell
lysates obtained using 50 μL of SDS-PAGE sample buffer
(80 mM Tris-HCl (pH 6.8, 2% SDS, 100 mM DTT, 10%
glycerol, 0.1% bromophenol blue). Proteins were resolved by
SDS-PAGE (15% acrylamide) and transferred to nitrocellu-
lose membranes. After blocking with 5% BSA in TBS with
0.05% Tween (TBST buffer) for 1 h at room temperature,
membranes were probed overnight at 4 ꢀC with antiacetylated
H4 (Upstate) and anti-p21/WAF1 (Abcam) antibodies and for
1 h with an antitubulin β antibody (Sigma). Following incuba-
tion with the primary antibody, blots were washed three times
for 10 min in TBST buffer, followed by incubation for 1 h at
room temperature with secondary horseradish peroxidase-con-
jugated antimouse or antirabbit antibodies. After washing,
luminescence was developed using WestPico reagent, as recom-
mended by the manufacturer (Pierce). Films were scanned, and
densitometric analysis was performed using ImageJ.
For each docking, the “active site” used for docking always
comprises a pocket containing all atoms of human HDAC8 no
˚
farther than 8 A from the SAHA crystalline position with a core-
˚
subpocket of 3 A around the SAHA residue. The best para-
meters were evaluated for each scoring function according to
their ability to mimic the crystallographic pose of the SAHA
ligand (we formerly corrected the SAHA structure and atom
valences according to the NCBI Pubchem structural data server
(http://pubchem.ncbi.nlm.nih.gov/)).
Docking with FlexX 2.0. FlexX 2.0 is an incremental con-
struction-docking algorithm involving three steps. FlexX cuts
ligand into fragments, places the best one into the binding site,
and then makes an incremental construction of the whole ligand.
Conformational flexibility of the ligand is taken into account by
considering both torsion angle flexibility and conformational
flexibility of ring systems.37 FlexX by itself does not recognize
receptor flexibility; rather, it sees proteins as rigid elements
(bodies). Default FlexX parameters were used as supplied in
the Tripos Sybyl 7.3.1 package to carry out flexible docking with
30 conformations for each molecule, using the place particle
option as defined by Rarey et al.38 We also adjusted the
protonation state of “Receptor Descriptor File” residues to
both protonated lysine and arginine residues as well as ionized
aspartic and glutamic acids residues.
In Vitro HDAC Activity. The relative HDAC-inhibiting
potential of each compound was tested against purified HDAC1
and HDAC6. Recombinant FLAG-tagged HDAC1 and
HDAC6 were immunopurified from stable expressing cell lines
according to previously described procedures.39 For inhibition
studies, the immunoprecipitated HDACs were washed twice
with HDAC buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl,
10% glycerol) and preincubated with each inhibitor in HDAC
buffer for 30 min at 4 ꢀC. Beads were resuspended in 30 μL of
HDAC buffer containing 20 000 cpm of a peracetylated H4
peptide. HDAC activity was determined after incubation for 2 h
at 37 ꢀC. The reaction was stopped by adding 0.04 M acetic acid and
250 mM HCl. The mixture was extracted with ethyl acetate, and the
released [3H]acetic acid was quantified by scintillation counting.
In Vivo Toxicity. The compounds were dissolved in a 50%
Cremophor EL (Sigma)/50% ethanol mixture followed by
heating (up to 60 ꢀC) and sonication for 30 min. Once the
compound was in solution, water was added to generate the
working solution.
Fvb mice were purchased from CERJ (Centre d’Elevage
Roger Janvier, France). Mice were maintained under specific
pathogen-free conditions in individual ventilated cages with
acidified water. All animal procedures were performed in ac-
cordance with protocols approved by the local Committee for
Animal Experimentation. The 8-week-old mice were randomly
assigned to receive either compound 1a or 1k (n = 3 mice per
group) at 50 mg/kg of body weight or vehicle (solvent-only
control, n = 3 mice per group) by oral gavage five times per week
for 2 weeks. Differential blood counts were assessed by retro-
orbital nonlethal eyebleeds before study initiation (J-5), during
the study (J5), and at study end points (J12). Heparinized blood
samples were immediately processed on a Medonic CA620
analyzer (Boule Medical AB) to determine a full blood picture
profile (white cell count, hematocrit, red cell count, and platelet
count).
Note Added after ASAP Publication. This paper was pub-
lished on March 10, 2010 with an incorrect version of Figure 7.
The revised version was published on March 22, 2010.
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