Histone deacetylase and microtubules as targets for the synthesis of releasable conjugate compounds

Article abstract of DOI:10.1016/j.bmcl.2009.09.075

Design and synthesis of an HDAC inhibitor and its merger with three tubulin binders to create releasable conjugate compounds is described. The biological evaluation includes: (a) in vitro reactivity with glutathione, (b) antiproliferative activity, (c) cell cycle analysis and (d) quantification of protein acetylation. The cellular pharmacology study indicated that the HDAC-inhibitor-drug conjugates retained antimitotic and proapoptotic activity with a reduced potency.

Full text of DOI:10.1016/j.bmcl.2009.09.075

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6358–6363
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Histone deacetylase and microtubules as targets for the synthesis of
releasable conjugate compounds
a
a
a
a
Daniele Passarella a, , Daniela Comi , Andrea Vanossi , Gianfranco Paganini , Francesco Colombo ,
*
Luca Ferrante a, Valentina Zuco b, Bruno Danieli a, Franco Zunino b,
*
a Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, Via Venezian 21, Italy
b Fondazione IRCCS Istituto Nazionale per lo Studio e la Cura dei Tumori, Via Venezian 1, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 June 2009
Revised 15 September 2009
Accepted 18 September 2009
Available online 30 September 2009
Design and synthesis of an HDAC inhibitor and its merger with three tubulin binders to create releasable
conjugate compounds is described. The biological evaluation includes: (a) in vitro reactivity with gluta-
thione, (b) antiproliferative activity, (c) cell cycle analysis and (d) quantification of protein acetylation.
The cellular pharmacology study indicated that the HDAC-inhibitor-drug conjugates retained antimitotic
and proapoptotic activity with a reduced potency.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
HDAC inhibitors
Tubulin binders
Releasable conjugate compounds
Disulfide bond
Anticancer compounds
The anticancer agents in clinical use suffer from severe dose-
limiting toxicities, but in any case chemotherapy continues to be
the primary systemic treatment of cancer. As a consequence there
is a need for innovative approaches to design anticancer drugs with
reduced toxicity1,2 and improved efficacy. Recently, the use of tar-
geted agents in combination with conventional cytotoxic treat-
ment3 has been proposed as a promising strategy for optimizing
antitumor therapies. In particular, histone deacetylase inhibitors
(HDAI) are potential antitumor agents (Fig. 1 reports two represen-
tative samples), because their biological effects include inhibition
of cell proliferation and induction of apoptosis. These effects result
in a significant antitumor activity in ‘in vivo’ models. HDAC inhib-
itors4 enhance histone acetylation, thus inducing chromatin relax-
ation and modulation of gene expression, and reversing the
epigenetic changes usually found in tumor cells.5 In addition, the
hydroxamic acid-based pan-HDAC inhibitors are known to modu-
On the base of this finding we thought to exploit the higher lev-
els of glutathione present in cancer cells (1 mM intracellular con-
centration compared to micromolar extracellular concentration)7
by designing novel disulfide containing hybrid compounds8 that
are activated selectively to release effective amounts of chemo-
therapeutic agents (HDAC inhibitor and tubulin binders) into can-
cer cells (Scheme 1). Reduced glutathione could secure reduction
of the disulfide bond, with subsequent release of the chemothera-
peutic agents by nucleophilic attack of the thiol group (thiolate an-
ion) at the ester function with formation of a thiolactone. As
tubulin-binder building blocks we selected thiocolchicine,9 paclit-
axel and cephalomannine in order to have agents representative of
both classes: inhibitors of tubulin polymerisation and stabilizers of
microtubules).10 Thiocolchicine (1) offers the possibility to remove
the acetyl group to obtain N-deacetylthiocolchicine 2 possessing an
amino group that can be easily acylated.11 Paclitaxel and cephalo-
manninne could be functionalized at the C20–OH group.12 4,40-
Dithiobutyric acid was selected as a proper linker because of the
possibility of enhancing the release of the therapeutic drugs by for-
mation of a five-membered thiolactone.13
late the acetylation status of non-histone proteins, including
a-
tubulin. Since tubulin acetylation may be implicated in regulation
of microtubule stability, the combination of microtubule-binding
agents with HDAC inhibitors may have a synergistic effect. In a re-
cent paper6 a combined treatment of Ark2 and KLE endometrial
cancer cells with TSA (5)/paclitaxel (3) (Fig. 1) is reported to cause
synergistic inhibition of cell growth and induction of apoptosis.
For our purpose we designed the structure of compounds that
could be effective as HDAC inhibitor with the specific structural
characteristics that are required for the interaction with the en-
zyme active site.14
In particular we were guided by the need for an anchor point
suitable to introduce a spacer that warrants the obtainment of
the conjugate compounds. The general structure of the designed
* Corresponding authors.
E-mail address: Daniele.Passarella@unimi.it (D. Passarella).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.075

D. Passarella et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6358–6363
6359
O
O
MeO
MeO
H
N
Ph
R
NH
NH
AcO
OH
AcO
OH
O
H
O
H
O
O
OH
OH
H
Ph
Ph
OMe
2'
O
O
O
HO
HO
O
O
SCH3
OAc
OAc
OBz
OBz
1
2
R = COCH3 Thiocolchicine
R = H
3 Paclitaxel
4 Cephalomannine
O
R2
N
OH
O
O
N
OH
H
N
H
6a Panobinostat (LBH-589)
R1 = Me, R2 = H
Phase III (Novartis)
N
N
H
R1
5 TSA (Trichostatin)
6b LAQ-824
R1 = H, R2 = CH2CH2OH
Figure 1. Example of (a) tubulin-binders and (b) HDAC inhibitors.
Scheme 1. Design of novel molecules and their expected reactivity with glutathione.
HDAC inhibitors is reported in Figure 2 and was suggested by anal-
ogy with an established HDAC inhibitor, Panobinostat (LBH589,
Novartis) 6a, now in clinical trial.
In our first plan, we studied the use of LBH589 (6a) that we pre-
pared according the reported procedure but we had several prob-
lems in the formation of the dimeric structure due to the
presence of the indole nitrogen.
As consequence we designed the structure where the indole nu-
cleus is removed and replaced by a phenyl group. We picked out
compound 19 as our first goal. This was prepared in a five-step se-
quence using benzylamine and methyl ester 8 (Scheme 2) of 4-
formylcinnamic acid (7) as building blocks. The crucial reductive
amination step furnished compound 9 permitting the formation
of the required hydroxamic acid by subsequent manipulation of
the ester function.15 In particular this was realized by base-cata-
lyzed ester hydrolysis and condensation reaction with protected
hydroxylamine (NH2OTHP) in the presence of EDC and HOBt to
give compound 16 that was finally deprotected by camphorsulfo-
nic acid (CSA) to give 19. In a general attempt to obtain the desired
hybrids we planned to use the benzylic NH nitrogen for the intro-
duction of the dithiodiacyl spacers to take in the second scaffold
(tubulin binder unities). Surprisingly and without any plausible
explanation, acylation of 19 and 16 with 4,40-dithiobutyric acid
proved tricky and the corresponding amide could not be isolated.
For this reason we realised the opportunity to move the acylation
point out from the encumbered nitrogen and far from the disulfide
moiety. Compounds 18 and 20 were therefore synthesized accord-
ing to Scheme 2. Compound 10 was prepared from 7 by NaBH4
reduction in the presence of Dowex1-x8,16 formation of the mesyl
derivative 15 and final reaction with 2-benzylamino ethanol in the
presence of DIPEA. Manipulation of the carboxylic function re-
quired: (a) ester hydrolysis and successively (b) protection of the
hydroxyl group as acetate to give compound 13. Reaction of 13
with NH2OTHP in the presence of EDC and HOBt furnished com-
pound 17, that was converted into compound 18 by potassium car-
bonate hydrolysis of the acetate ester. The need to have at hand
compound 20 for biological evaluation drove us to submit com-
pound 18 to reaction with CSA in MeOH. In this case the critical
purification step was carried out by filtration on SPE ISOLUTE PE-
AX (quaternary amine functionalized silica). We used compound
11 as starting material for the preparation of reference compounds
16a and 19a. We explored different strategies for the preparation
of the disulfide hybrid. After many unsuccessful trials we realized
that only the THP-protected hydroxylamides 16 and 18, could be
used for a simple and efficient synthesis of the desired compounds.
For the preparation of the N-deacetylthiocolchicine heterodimer
derivative 24 (Scheme 3), reaction of N-deacetylthiocolchicine (2)
N
H
X
N
OH
O
Anchor point
Figure 2. General structure of designed HDAC inhibitors.

6360
D. Passarella et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6358–6363
O
N
R
H
d
N
R
i
H
N
OR
OR'
OTHP
O
O
O
7 R = H
8 R = Me
a
16 R = H
16a R = Boc
17 R = CH2CH2OAc
18 R = CH2CH2OH
9
R = H
R' = Me
f
11 R = H
11a R = Boc
R' = H
R' = H
g
l
10 R = CH2CH2OH R' = Me
12 R = CH2CH2OH R' = H
13 R = CH2CH2OAc R' = H
f
b
h
m
N
R
RO
e
H
N
NH
OH
OMe
OH
O
O
19 R = H
14 R = H
15 R = Ms
c
19a R = Boc
20 R = CH2CH2OH
Scheme 2. Reagents: (a) SOCl2, MeOH; (b) NaBH4, Dowex-8, THF; (c) MsCl, Et3N, DMAP, THF; (d) PhCH2NH2, NaBH3CN, CH3COOH, MeOH; (e) DIPEA, CH2Cl2; (f) KOH, MeOH;
(g) (Boc)2O, K2CO3; (h) CH3COCl, TEA, DMAP, THF; (i) NH2OTHP, EDC, HOBt, DMF; (l) K2CO3, MeOH, H2O; (m) CSA (Amberlite IR120 for 16a), MeOH.
Scheme 3. Reagents: (a) HOCH2CH2Br, DMAP, DCC, CH2Cl2; (b) 16, K2CO3, CH3CN; (c) CSA, MeOH; (d) 4,40-dithiobutyric acid, DMAP, DCC, CH2Cl2.
with 4,40-dithiobutyric acid furnished the monoamide derivative
21 that was subsequently reacted with 2-bromoethanol to give
compound 22. The last compound gave rise to N-alkylation of
intermediate 16 and the hybrid structure 23 was obtained after
20 h at 60 °C. Finally, removal of the THP protective group from
the hydroxamic acid function by means of camphorsulfonic acid
(CSA) furnished the target compound 24. Purification of 24 was
possible by scavenging of CSA with Amberlist A-21 (basic resin).
Spectroscopic data reported in the Experimental section (Supple-
mentary data) fully support the complex structure. For the prepa-
ration of target compounds 28 and 30 (Scheme 4) we preferred to
exploit the higher reactivity of the hydroxyl group at position 20 of
the taxanes building blocks (3 and 4, Fig. 1). The easily obtained 20-
dithiodibutyroyl-paclitaxel 25 and cephalomannine 26 were con-
densed with the N-hydroxyethyl 18 in the presence of DCC and
DMPA. Removal of the THP protective group by CSA furnished
the target compounds that were purified by Amberlist A-21 treat-
ment and characterized. We first investigated from a chemical
point of view if glutathione is really able to cleave the disulfide
bond of the conjugate compounds 24, 28 and 30. Solution of these
(1.8
(1.8
l
l
M, acetone/water 1:1) were treated with
M) for 6 h and the mixtures was directly analysed by HRES-
L-glutathione
I-FT-MS. The experiment is far from the idea to reproduce the cel-
lular medium but the detected ionic species offer a qualitative
evidence of the release of the drugs (Table 1). The antiproliferative
activity of the tested antimicrotubule agents (thiocolchicine 1, pac-
litaxel 3 and cephalomannine 4), HDAC inhibitor moieties 19 and
20, N-Boc derivatives 16a and 19a and conjugate compounds was
evaluated under the same conditions following 72 h-exposure in
ovarian carcinoma cells (IGROV-1) (Table 2). All tested compounds
exhibited a significant antiproliferative activity in a submicromolar
range apart from N-Boc derivative 16a and 19a. However, all con-
jugated compounds were characterized by a substantial reduction
of activity, as compared with free antimicrotubule agent most evi-
dent in the paclitaxel conjugate (28). Among the tested conjugates,
the thiocolchicine analogue (24) was the most effective and there-
fore used for additional cellular and biochemical pharmacological
studies. Again, the compound 24 exhibited a reduction of potency
as compared to thiocolchicine (1) and its deacetyl derivative (2). A
comparable reduction of potency was found with the thiocolchi-

D. Passarella et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6358–6363
6361
Scheme 4. Reagents: (a) 4,40-dithiobutyric acid, DMAP, DCC, CH2Cl2; (b) 18, DMAP, DCC, CH2Cl2; (c) CSA, MeOH.
Table 1
HRESI-FT-MS detection of ionic species after treatment with glutathione
Detected species
24
HDACIn-H
Tio-CO(CH2)3-SS-
(CH2)2CO-Tio
Tio-CO(CH2)3-SS-G
G-SS-G
O
O
MeO
MeO
Ph
H
N
NH
O
AcO
OH
NH
O
O
H
AcO
OH
O
O
H
OH
O
OH
H
Ph
Ph
MeO
O
O
28
30
HDACIn-H
Pacli-H
G-SS-G
O
O
O
Tio
OAc
OAc
SCH3
OBz
OBz
Cepha
Pacli
O
O
O
HDACIn-CO(CH2)3-SS-
G
Cepha-H
Cepha-CO(CH2)3-SS-G
G-SS-G
HO
NH
O
HN OH
O
G
H
HADCIn
N
NH2
N
OH
Ph
O
HDACIn-H
cine dimmer. In contrast, no reduction of the cytotoxic potency
was observed with the compound 21. The cell cycle analysis of
cells treated with thiocolchicine or its conjugate (24) at equitoxic
concentrations (IC80) indicated similar pattern of perturbation that
was consistent with a G2/M arrest as expected for antimicrotubule
agents (Fig. 3a–c). The comparable effects of thiocolchicine and the
derivative 24 on cell cycle examined at 24 h reflected the antimi-
crotubule activity of these compounds, because under the same
conditions the HDAC inhibitor 20 induced only marginal perturba-
tions. The drug ability to induce mitotic arrest was also docu-
mented by morphological evidence of mitotic cells (Fig. 3d).
Treated cells also exhibited large sub-G1 peaks, which were consis-
tent with the presence of dead cells. Indeed, at equitoxic concen-
trations (IC80) used in these experiments, thiocolchicine and its
conjugate (24) exhibited a comparable ability to induce apoptosis
(52 2% and 45 3%, respectively, as detected by the TUNEL as-
say). Western blot analysis performed in ovarian carcinoma cells
Table 2
Antiproliferative activity
Compounds
IC50
(
l
g/ml)
IC50
(
l
M)
1
2
3
4
19
20
16a
19a
21
24
28
30
21a
0.005 0.002
0.012 0.005
0.023 0.002
0.06 0.01
0.30
0.48 0.03
0.018 0.03
0.53
0.05 0.01
0.250
0.14 0.01
0.06 0.01
1.4
41
19.1
0.019 0.001
0.078 0.015
0.72
0.74
0.050 0.001
0.07 0.014
1.0
1.0

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D. Passarella et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6358–6363
Figure 3. Cell cycle analysis.
4 h
24 h
20
19
20
19
dose
(μM)
0
3.5 0.35 0.87 0.06 3.5 0.35 0.87 0.06
acetylated
histone H4
acetylated
histone H4
acetylated
tubulin
acetylated
tubulin
β-tubulin
actin
Figure 4. Western blot analysis of protein acetylation in IGROV-1 cells heated with
HDAC inhibitors. Analysis was performed after 24 h exposure. b-Tubulin is shown
as control of protein loading.
Figure 6. Western blot analysis of protein acetylation in IGROV-1 cells treated with
21, 21a and SAHA (Suberoylanilide hydroxamic acid).
24
and able to act synergistically when released simultaneously inside
the tumor cells. All the tested conjugates, prepared using various
antimicrotubule agents, exhibited an appreciable reduction of the
cytotoxic potency. The cellular/molecular basis of the disappoint-
ing result is unclear, because in vitro glutathione was able to pro-
mote the release of the drug moieties. A plausible explanation of
this behaviour could be an inefficient reduction of S–S bond and
slow release of the HDAC inhibitor as suggested by a delayed in-
crease of the tubulin acetylation in cells treated with 24 (Fig. 3).
In addition, since the acetylation of protein substrates by the HDAC
inhibitory moiety is dose-dependent and an effective HDAC inhibi-
24h
48h
0
0.33 0.1
0
0.33 0.1
acetylated
tubulin
β-tubulin
Figure 5.
a-Tubulin acetylation in IGROV-1 cells following 24-h exposure to
conjugate 24. b-Tubulin is shown as control of protein loading.
tion could be achieved at higher concentration (ꢀ1
l
M) than that
M),
indicated that HDAC inhibitor moiety (19 and 20) used for conju-
gation was able to enhance acetylation of both histone H4 and a-
released by the conjugate at the tested concentration (ꢀ0.3
l
it is likely that the intracellular level of the HDAC inhibitor is inad-
equate to produce the expected synergistic interaction. Alterna-
tively, and perhaps concomitantly, a reduced tubulin interaction
of the conjugate itself or of the released mercapto-acylated thi-
ocolchicine 21 could account for the lower potency as compared
with free drug. This interpretation is consistent with the marked
reduction (ꢀ10-fold) of the potency of the paclitaxel conjugate
28. Indeed, substituents at the 20-position are expected to impair
drug-microtubule interactions. Finally, it should be noted that
the large molecular size of the conjugates may impair their ability
to penetrate cell membrane. Thus a low membrane permeability
could contribute to the observed reduction of potency. If this is
tubulin (Fig. 4). This early event, already evident at 4 h, was clearly
dose-dependent. In cells treated with the conjugate 24, tubulin
acetylation was a delayed event, detectable only at 48 h (Fig. 5),
thus suggesting a sluggish release of the HDAC inhibitor. We can-
not rule out the possibility that the thiocolchicine derivative con-
taining the mercapto-acylated chain may have HDAC inhibitory
activity. However, the marginal effects of thiocolchicine dimer
21a, which is expected to generate the thiol-containing monomer,
do not support this interpretation (Fig. 6). The rationale for the
synthesis of the novel prodrugs described in the present study
stems from the combination of two moieties, antimicrotubule
agent and HDAC inhibitor, each affecting different cellular targets

D. Passarella et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6358–6363
6363
5. Haberland, M.; Montgomery, R. L.; Olson, E. N. Nat. Rev. Genet. 2009, 10, 32.
the case, antimicrotubule agents with lower molecular weight may
be more appropriate in this approach. In spite of the reduction of
the cytotoxic potency, we retain that the dynamics of the release
of the conjugate compounds described in the present study have
to be investigated in order to take out any possible suggestion that
could be useful in the design of new anticancer compounds. At the
same time, the therapeutic advantages of these conjugates should
be documented by in vivo models.
6. (a) Dowdy, S. C.; Jiang, S.; Zhou, X. C.; Hou, X.; Jin, F.; Podratz, K. C.; Jiang, S.-W.
Mol. Cancer Ther. 2006, 5, 2767; (b) Itoh, Y.; Suzuki, T.; Kouketsu, A.; Suzuki, N.;
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7. Saito, G.; Swanson, J. A.; Lee, K. D. Adv. Drug Delivery Rev. 2003, 55, 199.
8. Previous preparation of disulfide containing bivalent compounds: Danieli, B.;
Giardini, A.; Lesma, G.; Passarella, D.; Peretto, B.; Sacchetti, S.; Silvani, A.;
Pratesi, G. J. Org. Chem. 2006, 71, 2848.
9. Shi, Q.; Verdier-Pinard, P.; Brossi, A.; Hamel, E.; McPhail, A. T.; Lee, K.-H. J. Med.
Chem. 1997, 40, 961.
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Acknowledgments
11. Shi, Q.; Verdier-Pinard, P.; Brossi, A.; Harmel, E.; Lee, K.-H. Bioorg. Med. Chem.
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12. Danieli, B.; Giardini, A.; Lesma, G.; Passarella, D.; Silvani, A.; Appendino, G.;
Noncovich, A.; Fontana, G.; Bombardalli, E.; Sterner, O. Chem. Biodiv. 2004, 1,
327.
13. For other example of exploitation of disulfide bond in releasable conjugate
compounds see: Dubikovskaya, E. A.; Thorne, S. H.; Pillow, T. H.; Contag, C. H.;
Wender, P. A. PNAS 2008, 105, 12128.
This research has been developed under the umbrella of CM
0602 COST Action ‘Inhibitors of Angiogenesis: design, synthesis
and biological exploitation’. This work was partially supported by
Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR)
PRIN 2007—Program ‘Sviluppo e caratterizzazione di nuovi inibito-
ri di tirosine chinasi cellulari con attività antiproliferativa e antian-
giogenica nei confronti di differenti tumori’, by Ministero della
Salute and by Fondazione Italo Monzino, Milan, Italy.
14. Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51, 1505.
15. Preparation of hydroxamic acids. From hydroxylamine and ester: (a) Mordini,
A.; Reginato, G.; Russo, F.; Taddei, M. Synthesis 2007, 20, 3201; (b) Hauser, C. R.;
Renfrow, W. B., Jr.. In Organic Synthesis; Wiley: New York, 1943; Collect. Vol. II;
Cleavage of an ester on a solid support: (c) Thouin, E.; Lubell, W. Tetrahedron
Lett. 2000, 41, 457; From carboxylic acids and amine using coupling reagents:
(d) De Luca, L.; Giacomelli, G.; Taddei, M. J. Org. Chem. 2001, 66, 2534; (e)
Giacomelli, G.; Porcheddu, A.; Salaris, M. Org. Lett. 2005, 5, 2715; (f) Ech-
Chahad, A.; Minassi, A.; Berton, L.; Appendino, G. Tetrahedron Lett. 2005, 46,
5113; (g) Katritzky, A. R.; Kirichenko, N.; Rogovoy, B. V. Synthesis 2003, 18,
2777; (h) Bailèn, M. A.; Chinchilla, R.; Dodsworth, D. J.; Nàjera, C. Tetrahedron
Lett. 2001, 42, 5013; (i) Sekar Reddy, A.; Suresh Kumar, M.; Ravindra Reddy, G.
Tetrahedron Lett. 2000, 41, 6285; From N-acyloxazolidinones: (l) Sibi, M. P.;
Hasegawa, H.; Ghorpade, S. R. Org. Lett. 2002, 4, 3343; From esters and O–Bn
hydroxylamine: (m) Pirrung, M. C.; Chau, G. H. L. J. Org. Chem. 1995, 60, 8084;
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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.2009.09.075.
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