Antiproliferative activities of a library of hybrids between indanones and HDAC inhibitor SAHA and MS-275 analogues

Article abstract of DOI:10.1016/j.bmcl.2007.09.041

New compounds derived from inhibitors of histone deacetylases (HDACs) have been synthesized and their antiproliferative activities towards non small lung cancer cell line H661 evaluated. Their design is based on hybrids between indanones to limit conformational mobility and other known HDAC inhibitors (SAHA, MS-275). The synthesis of these new derivatives was achieved by alkylation of appropriate indanones to introduce the side chain bearing a terminal ester group, the latter being a precursor of hydroxamic acid and aminobenzamide derivatives. These new analogues were found to be moderately active to inhibit H661 cell proliferation.

Full text of DOI:10.1016/j.bmcl.2007.09.041

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6142–6146
Antiproliferative activities of a library of hybrids between
indanones and HDAC inhibitor SAHA and MS-275 analogues
a
b
a
a,
*
´
Cedric Charrier, Joe¨lle Roche, Jean-Pierre Gesson and Philippe Bertrand
a
`
´ ´ ´
Synthese et Reactivite des Substances Naturelles, CNRS UMR 6514, Universite de Poitiers,
40 Avenue du Recteur Pineau, Poitiers F-86022, France
bInstitut de Physiologie et Biologie Cellulaires, CNRS UMR 6187, Universite´ de Poitiers,
40 Avenue du Recteur Pineau, Poitiers F-86022, France
Received 4 July 2007; revised 7 September 2007; accepted 8 September 2007
Available online 14 September 2007
Abstract—New compounds derived from inhibitors of histone deacetylases (HDACs) have been synthesized and their antiprolifer-
ative activities towards non small lung cancer cell line H661 evaluated. Their design is based on hybrids between indanones to limit
conformational mobility and other known HDAC inhibitors (SAHA, MS-275). The synthesis of these new derivatives was achieved
by alkylation of appropriate indanones to introduce the side chain bearing a terminal ester group, the latter being a precursor of
hydroxamic acid and aminobenzamide derivatives. These new analogues were found to be moderately active to inhibit H661 cell
proliferation.
Ó 2007 Elsevier Ltd. All rights reserved.
In eukaryotic cells, the reversible packing of DNA in the
chromatin structure provides the control of gene activa-
tion and repression. The fundamental element of the
chromatin structure is the nucleosome, assembled from
the highly conserved histone proteins H2A, H2B, H3,
and H4, forming an octameric core around which the
DNA is folded and sealed by histone H1.1 The regions
of chromatin that are involved in gene transcription,
DNA replication, and DNA repair are actively remod-
eled by regulation of histone acetylation, methylation,
phosphorylation, and ubiquitylation.2,3 Acetylation
and deacetylation of e-amino group of specific histone
lysines play a crucial role in the transcriptional process
and deregulation can lead to cancer.4–6 HATs (histone
acetyl transferases) are responsible for the acetylation
of lysines, while HDACs (histone deacetylases) activity
lead to deacetylation.7 Inhibition of HDACs elicits anti-
cancer effects in several tumors by inhibition of cell
growth and inducing cell differentiation.8 It is now well
established that a variety of proteins are also substrates
for HDACs and at least 50 non histone proteins of
known biological functions have been identified,9,10 with
functions beyond the alteration of chromatin struc-
tures.11 Therefore, inhibiting HDAC is an attractive tar-
get for anticancer therapy.12–14 Several programs for the
development of HDAC inhibitors (HDI) as anticancer
drugs have been initiated.11,15 Trichostatin A 116
(Fig. 1), a natural product isolated from Streptomyces
hygroscopicus, was later reported as HDI.17 Synthetic
analogues, like SAHA 2,18 MS-275 3, and NVP-
LAQ824 4, are in clinical trials.19 SAHA was recently
approved for treatment of cutaneous T-cell lym-
phoma.20 HDI activities requires (1) a functional group
O
O
OH
O
1 trichostatin A, TSA
2 SAHA
N
H
H
N
N
OH
O
N
O
H
N
N
H
H
NH2
O
N
3 MS-275
O
O
OH
HO
N
H
N
N
4 NVP-LAQ824
Keywords: Histone deacetylase; Cancer; Epigenetic; Trichostatin A;
SAHA; MS-275; Indanones; Hybrid.
H
*
Figure 1. Natural and synthetic HDI.
Corresponding author. E-mail: philippe.bertrand@univ-poitiers.fr
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.041

C. Charrier et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6142–6146
6143
able to chelate the metal atom at the bottom of the ac-
O
O
Y= OH,
2-NH2-Ph
tive site, (2) a recognition moiety interacting with the
external surface of the protein, and (3) a spacer linking
these two moieties and fitting the tubular geometry of
this site.
Y
X
N
H
a : X=(CH2)3, R=Me
b : X=(CH2)4, R=Et
c : X=(CH2)5, R=Me
R
d : X=Ph, R=Et
e : X=thiazol, R=Et
Crystallographic data are available describing the bind-
ing mode to HDLP or HDAC8 for TSA 1, SAHA 2,21
MS-344 5,22 and the benzohydroxamate 623 (Fig. 2).
In each case, the aromatic ring of 1, 3, 5 and the pyridyl
moiety of 6 are located at the external surface of the ac-
tive site. Effective HDI with large recognition elements
such as 9 were also described.22 For TSA, SAHA, and
MS-344, the intermediate chain is stacked between two
phenylalanines (Phe141 and Phe198 in HDLP), the
hydroxamic acid function chelating the zinc. In com-
pound 6, the thiophenesulfonamide part behaves as
the aryl chain of SAHA.
O
i : R=H (commercial)
ii : R=F
iii : R=NMe2
COOR'
Br
X
R
10i-iii
11a-e
Figure 3. Aliphatic and aromatic indanones analogues.
(IC50 = 0.9 lM).26 Preparation of this library involves
the alkylation of indanones by convenient halogenated
esters. Hydroxamic acid and benzamide groups can then
be accessed from these esters. This strategy allows sepa-
rate preparation of the required indanones 10i–iii and
halogenated esters 11a–e (Fig. 3).
We previously described the synthesis of the racemic ri-
gid TSA analogue 7.24 This compound possesses anti-
proliferative activities (IC50 = 0.3 lM) on H661 cells
and HDI activity. The dimethylamino group present
on the aromatic ring was essential to achieve these high
activities, as the unsubstituted homologue was found 10-
fold less active.
Indanone 10ii was prepared according to known proce-
dure.27 Nucleophilic aromatic substitution of 10ii affor-
ded 10iii (Scheme 1) in 75% yield.28 Previous work
showed that a fluorine atom at the indanone aromatic
ring does not lead to an increase of activity. Thus the
intermediate 10ii (Fig. 3, R = F) was not used to prepare
fluorinated analogues. Bromoalkyl chains were commer-
cially available as ethyl bromoesters 11a, b, and c was
prepared by known procedure.29 The halogenated aro-
In the present work, we prepared hybrid compounds be-
tween known HDI like SAHA or MS-275 and the inda-
none moiety of compound 7 by replacement of the
butadien chain with aliphatic or aryl spacers. Hydroxa-
mic acid and benzamide groups were selected as Zn2+
chelating functions for each new derivative.
O
O
For aliphatic analogues we selected a chain length in the
range of four to six carbons, as in TSA and SAHA,
respectively (Fig. 3, X = (CH2)3–5). Aromatic spacers
were designed to keep the original arrangement of the
methylbutadienyl group and the insaturations of TSA
or 7. Thus a ring closure was envisioned between the
methyl group of the dien chain and the carbon adjacent
to the carbonyl group of the hydroxamic acid. This led
to 1,3 disubstituted aromatic rings. A benzyl group
was first selected (Fig. 3, X = Ph). Compounds like 10
being described as HDI,25 a methylthiazolyl group
(Fig. 3, X = thiazol) was also retained. Although our
aromatic spacers are 1,3 disubstituted, they are similar
to spacers of 6 and 9. Interestingly, unsubstituted
para-benzamide 8 (Fig. 2) without a methyl at C2 was
described with antiproliferative activity on HCT116 cells
i
N
F
10ii
10iii
Br
Br
O
Br
Br
O
O
O
O
ii
OR
+
Br
Br
OR
OR
OEt
iii
45%
O
12
11d
13
N
N
N
ii
OEt
OR
+
S
S
S
14
11e 30%
15
Scheme 1. Reagents: (i) DMSO:H2O, K2CO3, Me2NHÆHCl, 75%;
(ii) NBS, CCl4, BzO2; (iii) NEt3, (EtO)2PHO.
O
O
O
O
O
O
S
OH
OH
N
N
H
N
H
N
N
H
S
N
H
5 MS-344
O
O
6
N
OH
N
N
(+/-) 7
O
O
N
O
OH
NH2
O
N
S
N
OH
N
H
N
S N
O
H
H
N
H
9 CRA-19156
(+/-) 8
10
O
O
Figure 2. Examples of alkyl, aryl, and thiazolyl HDI.

6144
C. Charrier et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6142–6146
matic counterparts were prepared by bromination. Ben-
zoate 12 afforded inseparable mono- and dibromo deriv-
atives 11d and 13. Mono debromination of the mixture
finally gave 11d in 45% yield.30 The same strategy ap-
plied to 14 gave the two separable mono- and dibromi-
nated compounds 11e and 15. Alkylation of 10i with
bromides 11d,e afforded aromatic esters 16d,e, while
bromides 11a–c were found unreactive (Scheme 2), even
when HMPA was used.
O
O
i
R
()4
10iii
17b
N
21 R=OEt
22 R=NHOH
ii
Scheme 3. Reagents and conditions: (i) NaHMDS, THF, À70 °C,
38%; (ii) EtOH, NH2OH 50% aq, 5 days, 45%.
hydroxamic acid 22 with a yield of 45%. Due to experi-
mental difficulties from this latter short strategy and the
modest overall yields, other alkylated derivatives of 10iii
were not prepared.
Conversion of 11a–c to the corresponding iodides 17a,31
17b,32 and 17c29 (Scheme 2), followed by alkylation,
finally gave the expected esters 16a–c in good yields.
Esters 16a–e were converted to acids 18a–e (Scheme 3)
and upon coupling with NH2-OTHP and subsequent
hydrolysis afforded hydroxamic acids 19a–e. Aminoben-
zamides 20a–e were obtained by coupling 18a–e with
1,2-diaminobenzene.
New derivatives were tested for tumor cell antiprolifera-
tive activity as this effect is well known for HDI and is
an important parameter for further development. After
48 h treatment of non small cell lung cancer H661 cells,
IC50 values (Table 1) were determined and compared to
TSA or SAHA (Figure 6, Supplementary data). In the
aromatic series, compounds 19d,e and 20d,e do not
show any activity at 25 lM (the highest tested concen-
tration). Some alkylated analogues showed a modest
activity, with compounds 19c and 20b having IC50 value
of 20 lM. Compounds 19a, 20a, and 20c are active but
In order to compare the activity of unsubstituted and
substituted indanone, attempts were made to alkylate
10iii with ester 17. As LDA alkylation protocol failed,
other bases were evaluated for the deprotonation
(NaH, THF, or NaH, dimethoxyethane), NaHMDS in
THF giving the best results despite the rather modest
yield (40%), but unreacted 10iii can be partially recov-
ered. Treatment of ester 21 in a mixture of DMF or
EtOH, and 50% aqueous NH2OH for 5 days gave
Table 1. Antiproliferative activities of 1, 2 and new TSA and SAHA
analogues 19a–e, 20a–e and 22 against H661 cells after 48 h treatment
Compound
IC50 lM
O
O
O
i
1 (TSA)
0.085
COOEt
COOEt
2 (SAHA)
5
11d
16d
16e
O
O
10i
Y
N
H
X
i
N
R
11e
S
R
X
Y
O
19a (IC4H)
19b (IC5H)
19c (IC6H)
H
H
H
(CH2)3
(CH2)4
(CH2)5
OH
OH
OH
>25
NA
20
O
iii
ii
OR
Br
( )n
11a-c
OR
I
( )n
17a-c
10i
19d (IBH)
19e (ITH)
H
H
OH
OH
NA
NA
O
O
a n=3, R=Me
b n=4, R=Et
c n=5, R=Me
N
S
( )n OR
16a-c
20a (IC4B)
20b (IC5B)
20c (IC6B)
H
H
H
(CH2)3
(CH2)4
(CH2)5
Ph–NH2 >25
Ph–NH2 20
Ph–NH2 P25
O
O
iv
X
OH
16a-e
18a-e
20d (IBB)
20e (ITB)
H
H
Ph–NH2 NA
v
vi
O
O
O
O
N
S
OH
Ph–NH2 NA
X
N
H
X
N
H
NH2
19a-e
20a-e
22 (Me2N-IC5H) Me2N (CH2)4
OH
NA
IC50 were determined from the curves of antiproliferative activities
(Figure 6, Supplementary data) at the concentration required for 50%
inhibition of cell proliferation. Experiments were done in triplicate.
NA, no activity detected at the maximum tested concentration
(25 lM).
Scheme 2. Reagents and conditions: (i) LDA, THF, À78 °C, 68–93%;
(ii) NaI, acetone, 98%; (iii) LDA, THF, À78 °C, HMPA, 88–95%;
(iv) LiOH, MeOH; 95% (v) a—H2N-OTHP, TBTU, DMF, NEt3;
b—CSA, CH2Cl2, MeOH, 36–60% (a + b); (vi) H2N–Ph–NH2, EDC,
THF, 73–85%.

C. Charrier et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6142–6146
6145
compared to the structure of HDI obtained from crys-
tallographic data (Figure 7, Supplementary data) and
then docked in the active site of HDAC8.33 The key
parameters for comparison studies were the overlapping
of the hydroxamic acids, the fitting of the aliphatic
chains and carbonyl group of indanone for 19b,c with
SAHA, and the overlapping of the aromatic ring for
19d and 19e with 6 or 9.
Conformers of 19b compared to SAHA (Figure 8, Sup-
plementary data) gave interactions with the surface
while conformer c6c of 19c did not (Fig. 5A). The chain
of SAHA presents a particular folding shortening the
distance between the carbonyl of the amide group and
the hydroxamic acid. Conformers c5a and c6c were
remodeled according to this folding, although not giving
the most stable conformers (Fig. 5B and C). Similarly to
the optimized conformers, the C2 methyl group in 19b
gave interaction with the surface while 19c did not.
19a is probably too short to fold as SAHA, with a
methyl group close to the entry of the site and is not pla-
nar enough to compete with TSA or 7. These results ap-
peared consistent with the observed activities for 19c
compared to 19a,b. The activity of hybrid 19c is proba-
bly due to the flexible nature of the aliphatic chain. De-
spite the sterics of the methyl group at C2, the length of
this chain appears to be optimal. Compound 20b is ac-
tive (25 lM), while hydroxamate 19b is not, suggesting
that in this case the benzamide group compensates the
shorter chain length.
Figure 4. Possible orientations of spacers linked to the indanone
moiety and TSA bound to HDAC8 in conformation A.
IC50 values could not be determined because at the high-
est concentration (25 lM), inhibition was less than 50%.
The expected improvement of activity for compound 22,
compared to its unsubstituted homologue 19b, was not
obtained. Observed activities were then analyzed in rela-
tion with the structure of SAHA and compounds 6 and
9 bound to HDAC8. Thus compounds 19b–e were iter-
atively submitted to molecular dynamics (MM2 meth-
od) and then optimized (AM1 level) until stable
conformers were obtained.
Three orientations A, B, and C of the spacer relative to
the indanone were considered (Fig. 4), A being adopted
by the chain of TSA and 7. 3D structures of para-ben-
zohydroxamates being only accessible, the para equiva-
lent of 19d was modeled for comparative purposes.
Several stable conformers were obtained in each case.
Conformers of 19d and for the para derivatives of 19d
gave analoguous conformations, the position of the
hydroxamic acid function on the phenyl ring having lit-
tle impact on the stability. All conformers were first
In the case of 19d compared to 6 or 9 conformation A is
obtained for conformers mb2 and mb3, with different
orientations of the hydroxamic acid (Figures 9 and 10,
Supplementary data). Results for the para derivative of
19d are almost identical. Conformer mb3 of 19d gave
conformational similarity to TSA (Fig. 5D) validating
our initial hypothesis for the design of 1,3 disubstituted
spacers. Despite this, the orientation of the phenyl ring,
compared to the butadien system of TSA or 7, gave
interactions in the tubular access. In addition, the inter-
Figure 5. Comparison of the binding modes in the active site of HDAC8. (A) SAHA (CPK) and conformer c6c of 19c (pink); (B and C) remodeled
conformers c5a (B) and c6c (C) according to the folding of SAHA; (D) TSA (CPK) and conformer mb3 of 19d (pink). Grey areas show interactions
with the surface of the site.

6146
C. Charrier et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6142–6146
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In conclusion, hybrid molecules designed between inda-
nones and MS-275 or SAHA derivatives were prepared.
Only hybrid analogues of SAHA showed antiprolifera-
tive activities. In contrast to para-benzohydroxamate
like MS-275 or 6, our derivatives based on meta-ben-
zohydroxamate and thiazolyl derivatives do not show
substantial activity. In this study, we measured antipro-
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pounds. With this approach, we could determine the
parameters for the interactions of our indanones’ deriv-
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spacers were used to link the chelating function to the
indanone, major interactions were obtained. For 1,3
disubstituted aromatic spacers, the link between this
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C2 in both the aliphatic and aromatic groups of ana-
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19c is a promising candidate for further modifications
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Acknowledgments
We thank MENRT, CNRS, and La Ligue Contre le
´
Cancer, Comite de Charente-Maritime for financial
support.
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Patent 2005234033 A1 20051020.
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.09.041.
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