New aryldithiolethione derivatives as potent histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.05.011

A series of dithiolethione derivatives was synthesized and the in vitro HDAC inhibitory activity was tested. The most active compounds, 1 and 2, exhibited an IC₅₀ in nM range with a strong hyperacetylation of histone H4 in A549 cells. The HDAC

Full text of DOI:10.1016/j.bmc.2010.05.011

Bioorganic & Medicinal Chemistry 18 (2010) 4187–4194
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New aryldithiolethione derivatives as potent histone deacetylase inhibitors
Valerio Tazzari a, Graziella Cappelletti b, Manolo Casagrande c, Elena Perrino a, Luigi Renzi c,
Piero Del Soldato a, Anna Sparatore a,c,
*
a Sulfidris s.r.l., Viale Gran Sasso 17, 20131 Milan, Italy
b Department of Biology, University of Milan, Via Celoria 26, 20133 Milan, Italy
c Department of Pharmaceutical Science ‘Pietro Pratesi’, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dithiolethione derivatives was synthesized and the in vitro HDAC inhibitory activity was
tested. The most active compounds, 1 and 2, exhibited an IC50 in nM range with a strong hyperacetylation
of histone H4 in A549 cells. The HDAC inhibitory activity comparable to that of SAHA and the inhibition of
A549 cell proliferation suggest that these compounds are worthy of further studies as potential antican-
cer agents.
Received 5 March 2010
Revised 29 April 2010
Accepted 4 May 2010
Available online 7 May 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Histone deacetylase inhibitor
Dithiolethione derivatives
Anticancer agents
1. Introduction
activated the tumor suppressor protein phosphatase 2A (PP2A)
with antiproliferative effect in breast and lung cancer cell lines.12
Histone deacetylases (HDACs) are a family of enzymes which
regulate chromatin remodelling and gene transcription. HDAC
inhibitors represent a diverse group of compounds which interfere
with the functions of HDAC enzymes and are emerging as a prom-
ising class of anticancer agents.1,2 HDAC inhibitors have a potential
role in the regulation of gene expression, inhibit the proliferation
of tumor cells by induction of cell death, apoptosis etc. Several
HDAC inhibitors have been studied in phase I, II, III clinical trials
and two compounds, the hydroxamic acid SAHA (VorinostatÒ,
Merck)3 and the natural depsipeptide romidepsin (FK228, IstodaxÒ,
Celgene)4 have been approved for marketing by FDA in 2006 and
2009, respectively.5
One important aspect to be considered is related to cardiac tox-
icity (QTc prolongation) that is reported as a potential relevant side
effect of HDAC inhibitors.6
We have recently found that 5-(4-hydroxyphenyl)-3H-1,2-
dithiole-3-thione (ADTOH) and its valproic acid ester (ACS 2)
(Fig. 1) potently inhibited in vitro HDAC enzymatic activity as well
as A549 cell proliferation with strong hyperacetylation of histone
H4.7–9
In addition, there is evidence that dithiolethiones may exert
cardioprotection increasing human and rat cardiomyocytes resis-
tance to oxidative/electrophilic stress and doxorubicin toxicity13,14
or protecting rat aortic smooth muscle A10 cells against peroxyni-
trite or acrolein-induced toxicity.15,16 It has also been suggested by
some of us that ADTOH and its hybrids may have cardioprotective
activity due to the release of hydrogen sulfide (H2S) from dithiol-
ethione moiety.17 Indeed, it has been shown that incubation of AD-
TOH or dithiolethione hybrids with rat plasma or liver homogenate
caused a time-dependent release of H2S. Incubation of the same
compounds in phosphate buffer or boiled rat liver homogenate
released a much lesser, although still detectable amount of H2S,
suggesting that the majority of the gas released from the parent
molecule occurred as a result of a metabolic event. Increased
plasma H2S concentration was also observed when dithiolethione
derivatives were administered ip or iv in rats.18–20 It has been
shown that H2S may protect the heart mainly by activation of KATP
We showed that ACS 2 significantly inhibited A549 or NCI-
H1299 xenograft proliferation in nude mice and increased E-cad-
herin expression in NSCLC cells.10
Moreover, besides the chemopreventive effects by activation of
the KEAP1/Nrf2/ARE pathway;11 we found that dithiolethiones
* Corresponding author. Tel.: +39 02 50319365; fax: +39 02 50319359.
E-mail address: anna.sparatore@unimi.it (A. Sparatore).
Figure 1. Chemical structures of some HDAC inhibitors.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.011

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V. Tazzari et al. / Bioorg. Med. Chem. 18 (2010) 4187–4194
channels.21 NaHS (a H2S donor) in a myocardial ischemia-reperfu-
sion model of isolated rat heart, provided a concentration-depen-
dent reduction in myocardial infarct size.22
On this ground and with the aim to further explore the antican-
cer potentialities of dithiolethiones, we synthesized novel deriva-
tives taking the chemical structures of the well known HDAC
inhibitors SAHA and MS-275 (Fig. 1) as a model.23–25
As model inhibitors, the novel compounds conform to the phar-
macophore formed by a zinc binding motif, a spacer with a polar
cap group, which makes contacts with the rim of the tube-like
enzymatic active site.
It is well known that dithiolethiones are able to coordinate sev-
eral metal ions, including zinc.26,27 Indeed the potent HDAC inhib-
itory activity of ADTOH may be related to the strong nucleophilic
character acquired by the thione group as a consequence of the
delocalization of the negative charge of phenoxide anion as shown
in Figure 2.
In more or less stable O-substituted derivatives the nucleophilic
character may be reduced with consequent decreased inhibitory
activity, as observed in valproic acid ester ACS 2.7 Nevertheless,
the exchange of valproic residue with more suitable (more ex-
tended and functionalized) substituents on the phenolic oxygen
may still give rise to compounds that will profitably occupy the
tube-like active site of HDAC.
Thus, a first subset of compounds is characterized by the
replacement of either the anilide cap group or the hydroxamic zinc
binding moiety of SAHA type inhibitors with a phenyldithiolethi-
one moiety, joined to the polymethylene spacer by means of an es-
ter or an ether linkage (3, 4 and 1, 2, 5, 6, respectively).
In compound 7 an acylated o-phenylenediamine moiety plays
the role of zinc binding functionality, as it is seen in MS-275 (Fig. 1),
CI 994 and other HDAC inhibitors.28 Moreover, we investigated
whether other kinds of dithiolethiones such as 8, 9 and 10
(Fig. 3) share the HDAC inhibitory activity of ADTOH, even if not
completely fulfilling the mentioned pharmacophore requirements.
On the basis that hydroxamic acids may bind to a wide range of
metals, and in an attempt to prevent untimely binding to other
metals outside the enzymatic active site, the hydroxamic acid
function of SAHA was protected by O-acylation with the two acids
8 and 9 to give compounds 11 and 12, respectively.
Finally, compound 13 is an hybrid between ADTOH and phen-
ylbutyric acid, which has been shown to be active against refrac-
tory solid tumor29 and whose weak HDAC inhibitory potency has
been strongly enhanced by tethering it with 4-aminobenzohydrox-
amic acid.30
The structures of the investigated compounds are depicted in
Figure 3; SAHA and ADTOH (Fig. 1) have been also tested as refer-
ence compounds.
2. Chemistry
Thesyntheses of the ether compounds 1, 2, 5, 6 and 7 were carried
out as indicated in Scheme 1, by coupling 5-[4-(3-thioxo-3H-1,2-
Figure 2. Resonance forms of deprotonated ADTOH.
Figure 3. Chemical structures of compounds investigated as HDAC inhibitors.

V. Tazzari et al. / Bioorg. Med. Chem. 18 (2010) 4187–4194
4189
Scheme 1. Reagents and conditions: (i) ADTOH, NaOH, EtOH, 80 °C; (ii) acetic acid, 50% H2SO4, 100 °C; (iii) TBDMSONH2, EDAC, CHCl3, rt; (iv) 1 N HCl; (v) aromatic amine,
EDAC, suitable solvent, rt.
Scheme 2. Reagents and conditions: (i) ADTOH, pyridine, THF; (ii) H2O; (iii) TBDMSONH2, EDAC, DMAP, CH2Cl2, 3 h, N2, rt; (iv) triethylsilane, TFA, CH2Cl2, 0 °C, 30 min.
dithiol-5-yl)phenoxy]pentanoic acid (16) or 7-[4-(3-thioxo-3H-1,2-
dithiol-5-yl)phenoxy]heptanoic acid (17) with the proper amine in
presence of EDAC and DMAP (or HOBt and TEA for 7). The starting
acids were prepared through the hydrolysis of the corresponding
ethyl esters (14 and 15) that were obtained by heating an ethanolic
solution of NaOH and ADTOH with ethyl 5-bromopentanoate or
ethyl 7-bromoheptanoate. To be noted that the yields of etherifica-
tion were very low (14%), due to the low reactivity of the phenolate
anion of ADTOH resonating with a thiolate form.7 The substitution
on the oxygen is proved by the identity of the UV–vis spectrum of
2 with that of anetholedithiolethione (ADT).31
silyl)hydroxylamine (TBDMSONH2) in presence of EDAC and DMAP.
In this case, deprotection of the silanated hydroxamic acid (19)
required stirring the compound with trifluoroacetic acid and trieth-
ylsilane for 30 min at 0 °C (Scheme 2).
Compound 4 was obtained coupling ADTOH with the monoani-
lide of the suberic acid (20), which was synthesized as previously
described32 (Scheme 3).
Compounds 11 and 12 were synthesized coupling SAHA with
1,3-dithiole-2-thioxo-4-carboxylic acid (8) and 4-(3-thioxo-3H-
1,2-dithiol-4-yl)benzoic acid (9), respectively, using EDAC and
HOBt as coupling reagents (Scheme 4).
The synthesis of compound 3 required first the preparation
of 8-oxo-8-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]octanoic acid
(18), obtained through the reaction of suberoyl chloride and ADTOH
in anhydrous THF, and then its coupling with O-(tert-butyldimethyl-
Compound 8 was synthesized as previously described by Dar-
tigues et al.33 and 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid
(9) was obtained by acidic hydrolysis of the corresponding methyl
ester prepared as described by Adelaere and Guemas.34
Scheme 3. Reagents and conditions: (i) acetic anhydride, reflux; (ii) aniline, THF, rt; (iii) ADTOH, EDAC, DMAP, CHCl3, rt.
Scheme 4. Reagents and conditions: (i) EDAC, HOBt, DMF, rt.

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V. Tazzari et al. / Bioorg. Med. Chem. 18 (2010) 4187–4194
[2-Methoxy-4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]acetic
acid (10) was obtained by acidic hydrolysis of the corresponding
methyl ester, which was synthesized as described by Lozac’h and
Mollier.35
Finally compound 13 was synthesized with good yields by
esterification of 4-phenylbutyric acid with ADTOH in presence of
DCC and DMAP as coupling reagents (Scheme 5).
3. Results and discussion
All synthesized compounds exhibited variable degrees of HDAC
inhibitory activity in vitro, and in some cases were comparable or
even more potent than SAHA. Nine compounds showed inhibition
of the proliferation of A549 cells with IC50 in the range from 3.4 to
66 lM and in most cases the antiproliferative activity was related
to the HDAC inhibition, since significant histone H4 hyperacetyla-
tion was observed in the same cells (Table 1 and Fig. 4).
Results are reported in Table 1.
Compounds 2 and 1 exhibit HDAC inhibitory activity higher
than SAHA, with IC50 = 0.01 lM and 0.08 lM versus 0.1 lM for
the reference drug, but the antiproliferative activity was 7–8 times
lower, suggesting that the determinants for the two activities are
different. In these molecules it is assumed that the hydroxamic
acid will prevail over the dithiolethione as zinc binding function.
However, the more extended oxyphenyldithiolethione moiety
may represent a better cap group than the anilide of SAHA, allow-
ing a more efficient accommodation and binding of the molecule
on the active site of HDAC with enhanced inhibitory potency. On
the other hand, the lower antiproliferative activity in respect to
SAHA, may be linked to a minor cellular uptake as well as to differ-
ent compound stability in the cell environment.
Compound 3 differs from 2 only due to the presence of an addi-
tional carbonyl group in the spacer, but exhibits a strongly reduced
Figure 4. Histone hyperacetylation in A549 cells exposed to 0.76
lM SAHA and
6.1 M compound 2 (ACS 63). Representative Western blot (A) and densitometric
l
analysis (B) showing the levels of hyperacetylated histone H4. Data are expressed as
means SE. **Significant difference versus control (P <0.001 according to ANOVA,
Dunnet post-hoc test).
HDAC inhibitory activity (IC50 = 10 lM) and a 10-fold lower anti-
proliferative activity. The observed lower activities in the two as-
says may be inherent to the molecule itself, whose dimensions
may exceed the optimal ones, but, they might also be due to the
products generated by hydrolysis of the ester linkage, which may
occur in different degrees in each assay, whose incubation time
were 1 and 48 h, respectively. Indeed, after 1 and 48 h of incuba-
tion increasing degrees of hydrolytic cleavage of ester 3 were ob-
served and a more accurate study of the kinetics of hydrolysis is
needed to explain the observed results of HDAC inhibition and
antiproliferation assays. Compounds 4, 5, 6 and 13, devoid of the
hydroxamic group, exhibited HDAC inhibitory activity that ranged
Scheme 5. Reagents and conditions: (i) DCC, DMAP, CH2Cl2, 3 h, rt.
Table 1
Effects of dithiolethione derivatives on inhibition of HDAC activity, inhibition of A549
cell proliferation and hyperacetylation of histone H4 in A549 cellsa
b
b
Compound
HDAC IC50
(lM)
A549 IC50
(lM)
Hyperac. H4 (A549)
SAHA
ADTOH
0.1 0.004
0.45 0.004
0.08 0.02
0.01 0.003
10 0.43
100 7.19
50 8.69
918.7 63.2
32 2.36
46.4 4.54
100 1.13
82.85 18
0.25 0.03
1.19 0.7
0.76 0.005
66 4.2
5.4 0.1
6.1 0.04
56 1.5
>1000
++
+
++
++
+
NT
NT
+
1 (ACS 91)
2 (ACS 63)
3 (ACS 60)
4 (ACS 66)
5 (ACS 69)
6 (ACS 72)
7 (ACS 88)
8 (ACS 5)
9 (ACS 48)
10 (ACS 50)
11 (ACS 90)
12 (ACS 58)
13 (ACS 68)
from moderate (IC50 = 50 and 100
respectively) to rather poor (IC50 = 179 and 918
l
M for compounds 5 and 4,
M for 13 and 6,
l
respectively). If for the esters 4 and 13 the activity could be, even
in part, related to the hydrolytic release of ADTOH, this possibility
does not apply to the best compound 5, whose activity must rely
on the whole molecule, supporting that the zinc binding role
may be accomplished by the dithiolethione moiety.
It is interesting to observe that compound 5 is 100-fold less po-
tent than ADTOH, but it is >200-fold more potent than the simple
methyl ether of the latter (ADT).7 Therefore the etherification of
the phenolic group of ADTOH may be exploited to form novel HDAC
inhibitors, provided that the attached moieties are able to occupy
the enzymatic channel and form both polar and hydrophobic bonds.
It is worth noting that, in this subset of compounds, the most (5)
and the least (6) active ones differ only for two methylene groups
in the spacer moiety. This confirms that the length of the spacer
should be optimized in relation to the dimensions of the cap and
>1000
60.2
31.7
3
6
+
>1000
ꢀ
102.9 8.6
879.8 63
3.4 0.8
1.68 0.23
31.8 0.007
+
NT
++
++
+
179.4 1.85
NT, not tested.
+ and ++, a significant increase (++, at least twofold) in the level of hyperacetylated
histone H4 was observed at concentrations equivalent to IC50 on cell proliferation.
ꢀ, no variation in the level of acetylated histone H4 were observed at concentra-
tions equivalent to IC50 on cell proliferation.
Values are means of at least three experiments.
Values represent IC50 standard error as resulting from sigmoidal dose–
response fitting.
a
b

V. Tazzari et al. / Bioorg. Med. Chem. 18 (2010) 4187–4194
4191
the zinc chelating groups. Indeed, while in this case the elongation
of the spacer has deleterious effects on activity, the opposite is ob-
served for the hydroxamic acids 2 and 1, previously discussed.
Compound 13 shows close structural analogies with the previ-
ously studied7 valproic ester of ADTOH (ACS 2, Fig. 1) and, indeed,
they exhibit comparable activities as enzyme and cell proliferation
inhibitor. Compound 7 is 29 times more potent than 6 but 3200
times less than 2, from which it differs, respectively, for an addi-
tional ortho-amino group and for the replacement of the NHOH
with an o-phenylenediamine moiety. The increase of potency pro-
duced by the insertion of an amino group in the anilide cap of 6
clearly indicates that the formed acylated o-phenylenediamine be-
came a better zinc chelating motif than the dithiolethione moiety.
On the other hand, the striking difference of potency between 2
and 7 may not be attributed exclusively to a better metal chelating
capability of the hydroxamic function in respect to the acylated o-
phenylenediamine, since the larger molecular size of 7 might also
exceed the enzyme channel capacity.
Antiproliferative activity, with IC50 in the low
l
M range, was
observed in nine compounds. Generally this activity is related to
the HDAC inhibition, since increased levels of hyperacetylated his-
tone H4 was observed in A549 cells.
Due to their potency as HDAC inhibitors and antiproliferative
agents, compounds 2 and 1 deserve further investigation, particu-
larly to explore if the peculiar biological characteristics of the sulf-
urated moieties are able to improve the safety profile of SAHA and
its analogs. This is the object of an ongoing study.
These results support our hypothesis that the dithiolethione
moiety is a suitable scaffold to build new potential anticancer
agents.
5. Experimental
5.1. General
All commercially available solvents and reagents were used
without further purification, unless otherwise stated. CC = flash
column chromatography. Mps: Büchi apparatus, uncorrected. 1H
NMR spectra: Varian Mercury 300VX spectrometer; CDCl3 or
DMSO-d6; d in ppm, J in hertz. High resolution mass spectra
(HRMS) on a LTQ ORBITRAPÒ XL mass spectrometer in positive
electro spray ionization (ESI; Source: FINNIGAN ION MAX). UV–
vis spectra: Varian DMS 80 spectrophotometer.
The dithiolethione carboxylic acids 8, 9 and 10 resulted en-
dowed with moderate HDAC inhibitory activity (IC50 in the range
from 46 to 100 lM), despite not fulfilling the requirement of the
pharmacophore model for HDAC inhibition.
Even less potent than ADTOH, these acids present some interest
for either the generation of extended molecules potentially more
potent, or as protective group for the sensitive hydroxamic func-
tion of several HDAC inhibitors.
5.2. Ethyl 5-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]pentanoate
(14)
Thus, coupling the hydroxamic function of SAHA with the dith-
iolethione carboxylic acids 8 and 9 the N,O-diacylated hydroxyl-
amines 11 and 12 were obtained. These compounds were
5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione (890 mg; 3.93
mmol) was added to a solution of NaOH (157 mg; 3.93 mmol) in
ethanol (4 ml) and the mixture was stirred for 15 min, thereafter
ethyl 5-bromopentanoate 98% (838 mg; 0.63 ml) and KI (65 mg;
0.1 equiv) were added and the mixture was refluxed for 4 h. The
solvent was evaporated and the crude product was chromato-
graphed on silica gel (cyclohexane/ethyl acetate; 85:15) and after
washing with petrol ether, ethyl 5-(4-(3-thioxo-3H-1,2-dithiol-5-
yl)phenoxy)pentanoate as an orange solid was obtained. Mp 81–
82 °C. Yield: 14%. 1H NMR (DMSO-d6): d = 7.86 (d, J = 8.80 Hz,
2H); 7.75 (s, 1H); 7.05 (d, J = 9.09 Hz, 2H); 4.08–3.99 (m, 4H);
2.35 (t, J = 6.94 Hz, 2H); 1.73–1.66 (m, 4H); 1.16 (t, J = 7.03 Hz,
3H). HRMS (ESI) m/z calcd for C16H19O3S3 [M+H]+: 355.04908;
found: 355.04896.
endowed with strong HDAC inhibitory activity (IC50 = 0.25
lM
and 1.19 M, respectively) that was however 2.5-fold and 12-fold
l
lower than that of SAHA. This decrease of activity may be related
either to an intrinsic weaker zinc ion binding capacity or to the
exceeding molecular dimension in respect to the enzyme active
site channel. Indeed, the more extended 12 is about fivefold less
potent than 11. The observed activity could be also related to the
hydrolytic release of SAHA and the difference between 12 and 11
may depend from the different rates of their hydrolytic cleavage.
Additional studies, that are beyond the scope of the present
work, will be needed to explore the effects of masking the hydroxa-
mic function of SAHA on its ADME characteristics, as well as on its
safety profile.
As it is shown in Table 1, the inhibition of A549 cell proliferation
paralleled, in most cases, the HDAC inhibitory activity, and signif-
icant histone H4 hyperacetylation was observed on the same cells.
However, for some compounds such a parallelism does not hold,
since moderate enzyme inhibitors (8, 5, 10 and 4) are practically de-
void of any antiproliferative activity, while, on the contrary, weaker
HDAC inhibitors (9, 13 and 6) still display a fair antiproliferative
5.3. Ethyl 7-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]heptanoate
(15)
5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione (400 mg; 1.76
mmol) was added to a solution of NaOH (70 mg, 1.76 mmol) in eth-
anol (2 ml) and the mixture was stirred at 45 °C for 10 min, then
ethyl 7-bromo-eptanoate (0.34 ml; 1.76 mmol) was added and
the mixture was stirred under nitrogen at 80 °C for 6 h. After evap-
oration of the solvent, the crude product was purified by CC (silica
gel; cyclohexane/CH2Cl2; 6:4) as a red solid. Mp 70.3–70.9 °C.
Yield: 14%. 1H NMR (DMSO-d6): d = 7.85 (d, J = 8.50 Hz, 2H); 7.75
(s, 1H); 7.05 (d, J = 8.80 Hz, 2H); 4.06–3.98 (m, 4H); 2.27 (t,
J = 7.33 Hz, 2H); 1.72–1.30 (m, 8H); 1.15 (t, J = 7.03 Hz, 3H). HRMS
(ESI) m/z calcd for C18H23O3S3 [M+H]+: 383.08038; found:
383.08029.
activity. For instance, compound 13 exhibited a IC50 = 31.8
antiproliferative activity, while its IC50 for HDAC inhibition is as high
as 179 M.
lM for
l
Therefore, in these cases additional mechanisms may play a role
in the inhibition of A549 cell proliferation.
4. Conclusions
A series of dithiolethione derivatives was synthesized and
found to display variable degrees of HDAC inhibitory activity, that
in some cases is comparable or even higher than that of SAHA, used
as reference drug.
5.4. 5-[4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy]pentanoic acid
The dithiolethione moiety may be either directly responsible for
the enzyme inhibition, acting as zinc ion chelating group, or play a
role as a more appropriate cap group (in comparison to the anilide
group of SAHA), when a more effective chelating group is also pres-
ent in the molecule.
(16)
Ethyl 5-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]pentanoate
(155 mg; 0.474 mmol) was suspended in a mixture of acetic acid
(5.9 ml) and 50% sulfuric acid (0.9 ml) and heated, under stirring,

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V. Tazzari et al. / Bioorg. Med. Chem. 18 (2010) 4187–4194
at 100 °C for 1.5 h. After cooling, the mixture was diluted with
water and extracted with CH2Cl2. The organic phase was dried on
anhydrous sodium sulfate, filtered and evaporated to dryness.
The orange product was washed with ethyl ether. Mp 114.5–
117 °C (dec.). Yield: 89% 1H NMR (DMSO-d6): d = 12.05 (s,1H, col-
lapses with D2O); 7.85 (d, J = 8.80 Hz, 2H); 7.75 (s, 1H); 7.05 (d,
J = 8.80 Hz, 2H); 4.06 (t, J = 5.23 Hz, 2H); 2.27 (t, J = 7.43 Hz, 2H);
1.75–1.60 (m, 4H). HRMS (ESI) m/z calcd for C14H15O3S3 [M+H]+:
327.01778; found: 327.01793.
5.8. N-Phenyl-5-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]
pentanamide (5)
A solution of 5-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]pen-
tanoic acid (155 mg, 0.474 mmol), EDAC (109 mg, 0.57 mmol)
and DMAP (6 mg) in 7 ml of CH2Cl2 was cooled on an ice bath
and stirred under nitrogen for 20 min. A solution of aniline
(44.1 mg, 0.474 mmol) in 3 ml of CH2Cl2 was added dropwise, then
the ice bath was removed and the mixture was stirred under nitro-
gen for 2 h. The mixture was diluted with 20 ml of CH2Cl2 and ex-
tracted with cold 1 N HCl and then with cold water. The organic
phase was dried on anhydrous sodium sulfate and evaporated to
dryness under reduced pressure. The crude product was chromato-
graphed on silica gel (CH2Cl2/methanol; 99.8:0.2) and the obtained
orange product was washed with ethyl ether. Mp 199.5–201.5 °C.
Yield: 76%. 1H NMR (DMSO-d6): d = 9.88 (s, 1H); 7.85 (d,
J = 8.53 Hz, 2H); 7.75 (s, 1H); 7.57 (d, J = 8.50 Hz, 2H); 7.26 (t,
J = 7.15 Hz, 2H); 7.07–7.00 (m, 3H); 4.09 (br s, 2H); 2.36 (br s,
2H); 1.76 (br s, 4H). HRMS (ESI) m/z calcd for C20H20NO2S3
[M+H]+: 402.06507; found: 402.06480.
5.5. 7-[4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy]heptanoic acid
(17)
Ethyl 7-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]heptanoate
(89 mg; 0.23 mmol) was suspended in a mixture of acetic acid
(3 ml) and 50% sulfuric acid (0.48 ml) and the mixture was stirred
at 100 °C, for 3 h. Water was added after cooling and the product
was extracted with CH2Cl2. The organic solution was dried on
anhydrous sodium sulfate and evaporated to dryness and the res-
idue was washed with ethyl ether. A red solid was obtained. Mp
114.8–115.6 °C. Yield: 94%. 1H NMR (DMSO-d6): d = 12.06 (s, 1H,
collapses with D2O); 7.92 (d, J = 8.50 Hz, 2H); 7.83 (s, 1H); 7.13
(d, J = 8.80 Hz, 2H); 4.12 (t, J = 6.15 Hz, 2H); 2.27 (t, J = 7.03 Hz,
2H); 1.81–1.29 (m, 8H). HRMS (ESI) m/z calcd for C16H19O3S3
[M+H]+: 355.04908; found: 355.04914.
5.9. N-Phenyl-7-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)
heptanamide (6)
A solution of 7-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]hep-
tanoic acid (150 mg; 0.423 mmol), EDAC (97 mg; 0.5 mmol), DMAP
(5.2 mg) in 8 ml of CHCl3 was cooled on an ice bath and stirred un-
der nitrogen for 200. A solution of aniline (39.4 mg; 0.423 mmol), in
2 ml of CHCl3, was added dropwise, then the ice bath was removed
and the mixture was stirred under nitrogen for 3 h. The mixture
was diluted with 20 ml of CH2Cl2 and extracted first with cold
HCl 1 N and then with cold water. The organic phase was dried
on anhydrous sodium sulfate and evaporated to dryness under re-
duced pressure. The product, after addition of CH2Cl2 crystallized.
Mp 149–150 °C. Yield: 33%. 1H NMR (DMSO-d6): d = 9.83 (s, 1H);
7.84 (d, J = 8.53 Hz, 2H); 7.74 (s, 1H); 7.56 (d, J = 8.25 Hz, 2H);
7.26 (t, J = 7.42 Hz, 2H); 7.06–6.97 (m, 3H); 4.05 (t, J = 6.32 Hz,
2H); 2.29 (t, J = 7.15 Hz, 2H); 1.73–1.39 (m, 8H). HRMS (ESI) m/z
calcd for C22H24NO2S3 [M+H]+: 430.09637; found: 430.09600.
5.6. N-Hydroxy-5-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)
pentanamide (1)
5-[4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy]pentanoic acid
(125 mg; 0.38 mmol), EDAC (87 mg; 0.45 mmol) and a catalytic
quantity of DMAP were dissolved in 6 ml of CHCl3 and the solution
was stirred under nitrogen for 20 min on an ice bath. Thereafter a
solution of O-(tert-butyldimethylsilyl)hydroxylamine (59 mg,
0.38 mmol) in CHCl3 was added and the reaction was stirred at
room temperature for 3 h. The solution was diluted with chloro-
form and shaken first with 1 N HCl and then with water. The or-
ganic phase was dried on anhydrous sodium sulfate, filtered and
evaporated to dryness. The solid orange product was washed with
ethyl ether. Mp 101.5–103.5 °C. Yield: 65% 1H NMR (DMSO-d6):
d = 10.37 (s, 1H, collapses with D2O); 8.69 (s, 1H, collapses with
D2O); 7.85 (d, J = 8.79 Hz, 2H, 2H); 7.75 (s, 1H,); 7.05 (d,
J = 8.21 Hz, 2H); 4.05 (t, J = 5.91 Hz, 2H); 2.00 (t, J = 6.16 Hz, 2H);
1.75–1.57 (m, 4H). HRMS (ESI) m/z calcd for C14H15NO3S3Na
[M+Na]+: 364.01063; found: 364.01035.
5.10. N-(2-Aminophenyl)-7-(4-(3-thioxo-3H-1,2-dithiol-5-yl)
phenoxy)heptanamide (7)
o-Phenylenediamine (39.65 mg, 0.37 mmol) was added to a
solution of 7-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]heptanoic
acid (130 mg, 0.36 mmol), EDAC (83 mg, 0.43 mmol), HOBt
(66 mg, 0.43 mmol) and triethylamine (TEA, 0.05 ml) in anhydrous
DMF (3 ml) and the mixture was stirred under nitrogen, at room
temperature for 24 h. At the end of the reaction DMF was evapo-
rated and the residue was taken up with CH2Cl2. The organic phase
was extracted with water, dried on anhydrous sodium sulfate and
evaporated to dryness. The crude product was purified by CC (silica
gel; CH2Cl2/methanol; 99:1). The orange solid product was washed
with ethyl ether. Mp 144–146 °C. Yield: 44%. 1H NMR (DMSO-d6):
d = 9.07 (s, 1H); 7.84 (d, J = 8.50 Hz, 2H,); 7.74 (s, 1H); 7.12 (d,
J = 7.33 Hz, 1H); 7.05 (d, J = 8.80 Hz, 2H); 6.87 (t, J = 7.33 Hz, 1H);
6.70 (d, J = 7.62 Hz, 1H); 6.51 (t, J = 7.33 Hz, 1H); 4.79 (s, 2H, col-
lapses with D2O); 4.06 (t, J = 6.45 Hz, 2H); 2.30 (t, J = 7.04 Hz,
2H); 1.75–1.36 (m,8H). HRMS (ESI) m/z calcd for C22H25NO2S3
[M+H]+: 445.10727; found: 445.10682.
5.7. N-Hydroxy-7-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)
heptanamide (2)
A mixture of 7-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]hep-
tanoic acid (66.7 mg; 0.19 mmol), EDAC (1.2 equiv 0.245 mmol;
46.8 mg) and DMAP (2.5 mg) in CHCl3 (6 ml) was cooled on an
ice bath, stirring under nitrogen for 20 min. A solution of O-(tert-
butyldimethylsilyl)hydroxylamine (30 mg; 0.19 mmol) in CHCl3
(1.2 ml) was added dropwise, then the ice bath was removed and
the mixture was stirred for 3 h at rt. After addition of CH2Cl2, the
mixture was shaken with 1 N HCl and then with cold water. The or-
ganic phase was dried on anhydrous sodium sulfate and evapo-
rated to dryness. The product crystallized with CH2Cl2. Mp
110.6–111.9 °C. Yield: 69.5%. 1H NMR (DMSO-d6): d = 10.32 (s,1H,
collapses with D2O); 8.65 (s,1H, collapses with D2O); 7.85 (d,
J = 8.20 Hz, 2H); 7.75 (s, 1H); 7.05 (d, J = 8.80 Hz, 2H); 4.04 (t,
J = 6.32 Hz, 2H); 1.93 (t, J = 7.03 Hz, 2H); 1.70–1.27 (m, 8H). HRMS
(ESI) m/z calcd for C16H19NO3S3 [M+H]+: 392.04193; found:
5.11. 8-Oxo-8-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]octanoic
acid (18)
392.04271. UV–vis (EtOH), kmax, nm (log
e
): 236 (4.10), 265 sh
5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH, 226
(3.82), 348 (4.31), 428 (4.06); kmin: 288, 389.
mg, 1 mmol) and anhydrous pyridine (79 mg; 1 mmol) were added

V. Tazzari et al. / Bioorg. Med. Chem. 18 (2010) 4187–4194
4193
to a solution of suberoyl chloride (211 mg; 1 mmol) in anhydrous
tetrahydrofuran (THF). After stirring for 1 h at rt, THF was evapo-
rated to dryness and the residue dissolved in CH2Cl2. The dichloro-
methane solution was thoroughly washed with cold water, then
was dried on anhydrous sodium sulfate and evaporated to dryness.
After chromatographic separation (silica; CH2Cl2/methanol; 99:1)
an orange solid was obtained which was characterized only by
1H NMR and used for the preparation of 3 without any further
characterization. Yield: 40%. 1H NMR (DMSO-d6): d = 12.00 (s, 1H,
collapses with D2O); 7.95 (d, J = 8.53 Hz, 2H); 7.80 (s, 1H); 7.30
(d, J = 8.53 Hz, 2H); 2.57 (t, J = 6.87 Hz, 2H); 2.20 (t, J = 6.87 Hz,
2H); 1.60–1.28 (m, 8H).
CH2Cl2/methanol (9:1). The organic phase was dried on anhydrous
sodium sulfate and evaporated to dryness and the residue was
washed with ether and CH2Cl2 to obtain a yellow-orange solid.
Mp 240–245 °C. Yield: 93%. 1H NMR (DMSO-d6): d = 13.03 (s, 1H,
collapses with D2O); 9.23 (s, 1H); 7.97 (d, J = 7.63 Hz, 2H); 7.69
(d, J = 7.63 Hz, 2H).
5.15. [2-Methoxy-4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]
acetic acid (10)
Methyl [2-methoxy-4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]
acetate35 was hydrolyzed as above described for the preparation of
compound 9. Mp 198–200 °C. Yield: 92%. 1H NMR (DMSO-d6):
d = 7.86 (s, 1H); 7.45–7.42 (m, 2H); 6.96 (d, J = 9.09 Hz, 1H); 4.78
(s, 2H); 3.86 (s, 3H).
5.12. 4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenyl 8-(hydroxyamino)-
8-oxooctanoate (3)
A
dichloromethane solution of O-(tert-butyldimethylsi-
5.16. N1-Phenyl-N8-(2-thioxo-1,3-dithiole-4-carbonyloxy)
lyl)hydroxylamine (TBDMSONH2, 49.3 mg; 0.335 mmol) was added
dropwise to a solution of 8-oxo-8-[4-(3-thioxo-3H-1,2-dithiol-5-
yl)phenoxy]octanoic acid (18) (130 mg; 0.335 mmol), 1-ethyl-3-
(30-dimethylaminopropyl)carbodiimide hydrochloride (EDAC;
77 mg; 0.40 mmol) and 4-(dimethylamino)pyridine (DMAP; 4 mg)
dissolved in CH2Cl2 (10 ml, previously filtered through basic alu-
mina). The reaction mixture was stirred at rt, under nitrogen for
3 h. The solution, after washing with 1 N HCl and cold water, was
dried on anhydrous sodium sulfate and evaporated to dryness.
The crude compound was chromatographed on silica gel CC
(CH2Cl2/CH3OH; 99:1). The obtained compound (19; yield: 62.5%)
still contain the silyl protective group.
A part of the obtained compound (50 mg; 0.097 mmol) was dis-
solved in CH2Cl2 (2 ml), and after cooling at 0 °C, 0.02 ml of triethyl-
silane and 0.4 ml of trifluoroacetic acid were added. The reaction
mixture was stirred for 30 min at 0 °C, then the solvent was evapo-
rated and after addition of few drops of CH2Cl2 the product, kept in
refrigerator overnight, crystallized. Crystals were washed with a
CH2Cl2/ether (1:1) mixture. Mp 93–97 °C. Yield: 63.6%. 1H NMR
(DMSO-d6): d = 10.32 (s, 1H, collapses with D2O); 8.66 (s,1H, col-
lapses with D2O); 7.95 (d, J = 8.53 Hz, 2H); 7.75 (s, 1H); 7.30 (d,
J = 8.53 Hz, 2H); 2.59 (t, J = 6.87 Hz, 2H); 1.91 (t, J = 6.87 Hz, 2H);
1.53–1.28 (m, 8H). HRMS (ESI) m/z calcd for C17H20NO4S3 [M+H]+:
398.05490; found: 398.05479.
octanediamide (11)
A
solution of N8-hydroxy-N1-phenyloctanediamide (SAHA,
100 mg; 0.38 mmol) in anhydrous DMF (1 ml) was added to a solu-
tion of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDAC,
86.8 mg, 0.45 mmol), HOBt (69.5; 0.45 mmol) and 2-thioxo-1,3-
dithiole-4-carboxylic acid33 (8, 60 mg; 0.38 mmol) in anhydrous
DMF (2 ml) and the mixture was stirred under nitrogen, at room
temperature, for 24 h. At the end of the reaction, DMF was evapo-
rated and the residue dissolved in ethyl acetate. The organic phase
was washed with water, dried on anhydrous sodium sulfate and
evaporated to dryness. The product was washed with a little of ethyl
acetate and then with ether and crystallized from THF. Mp 138–
141 °C (dec.). Yield: 31%. 1H NMR (DMSO-d6): d = 12.15 (s, 1H, col-
lapses with D2O); 9.82 (s, 1H, collapses with D2O); 8.69 (s, 1H);
7.60 (d, J = 7.91 Hz, 2H); 7.26 (t, J = 7.92 Hz, 2H); 6.99 (t,
J = 7.32 Hz, 1H); 2.27 (t, J = 7.33 Hz, 2H); 2.16 (t, J = 7.33 Hz, 2H).
HRMS (ESI) m/z calcd for C18H21N2O4S3 [M+H]+: 425.06580; found:
425.06540.
5.17. N1-Phenyl-N8-(4-(3-thioxo-3H-1,2-dithiol-4-yl)benzoyloxy)
octanediamide (12)
A
solution of N8-hydroxy-N1-phenyloctanediamide (SAHA)
(100 mg; 0.38 mmol) in 1 ml of dimethylformamide (DMF) was
added to a solution of 9 (96 mg; 0.38 mmol), 1-hydroxybenzotria-
zole hydrate (HOBt, 64 mg; 0.418 mmol), EDAC (80 mg; 0.418
mmol) in 2 ml of anhydrous DMF. The reaction was stirred under
nitrogen at room temperature for 24 h. At the end of the reaction
DMF was evaporated and the crude product was taken up in water,
filtered and washed with CH2Cl2 and then with THF. The obtained
product has a melting point of 139.5–140 °C. Yield: 66%. 1H NMR
(DMSO-d6): d = 11.90 (s, 1H, collapses with D2O); 9.85 (s, 1H); 9.25
(s, 1H); 8.05 (d, J = 7.91 Hz, 2H); 7.76 (d, J = 8.21 Hz, 2H); 7.55 (d,
J = 7.62 Hz, 2H); 7.25 (t, J = 7.91 Hz, 2H); 6.99 (t, J = 6.88 Hz, 1H);
2.28 (t, J = 7.03 Hz, 2H); 2.18 (t, J = 6.74 Hz, 2H); 1.60–1.25 (m, 8H).
HRMS (ESI) m/z calcd for C24H25N2O4S3 [M+H]+: 501.09710; found:
501.09666.
5.13. 4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenyl 8-oxo-8-(phenylamino)
octanoate (4)
A mixture of the monoanilide of suberoyl acid prepared as previ-
ous described32 (199.5 mg; 0.8 mmol), EDAC (193.6 mg 1.0 mmol)
and DMAP (10.3 mg) in 12 ml of CHCl3 were stirred for 20 min on
an ice bath under nitrogen. 5-(4-Hydroxyphenyl)-3H-1,2-dithiole-
3-thione (ADTOH, 181 mg; 0.8 mmol) was added and the reaction
mixture was stirred at rt for 2 h. At the end of the reaction, the organ-
ic phase was extracted with water and then with a solution of NaH-
CO3, dried on anhydrous sodium sulfate and evaporated to dryness.
The obtained residue was washed with CH2Cl2. Mp 177.5–179.5 °C.
Yield: 66%. 1H NMR (DMSO-d6): d = 9.85 (s, 1H); 7.94 (d, J = 8.53 Hz,
2H); 7.80 (s, 1H); 7.57 (d, J = 7.43 Hz, 2H); 7.30–7.23 (m, 4H); 7.00 (t,
J = 6.87 Hz, 1H); 2.60 (t, J = 7.15 Hz, 2H); 2.29 (t, J = 6.88 Hz, 2H);
1.64–1.35 (m, 8H). HRMS (ESI) m/z calcd for C23H24NO3S3 [M+H]+:
458.09128; found: 458.09096.
5.18. 4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenyl 4-phenylbutanoate
(13)
A dichloromethane solution of dicyclohexylcarbodiimide (DCC)
(755 mg; 1.2 equiv) was added dropwise at room temperature to a
suspension of 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (680
mg; 3 mmol), 4-phenylbutyric acid (500 mg; 3 mmol) and DMAP
(18.5 mg) in CH2Cl2 (25 ml). The reaction was stirred for 3 h at rt.
At the end of the reaction the dicyclohexylurea (DCU) was filtered
and the solution was chromatographed on SiO2 (cyclohexane/
5.14. 4-(3-Thioxo-3H-1,2-dithiol-4-yl)benzoic acid (9)
A suspension of methyl 4-(3-thioxo-3H-1,2-dithiol-4-yl)benzo-
ate34 (100 mg, 0.35 mmol) in 4.5 ml of acetic acid and 0.72 ml of
50% (v/v) H2SO4 was stirred at 100 °C for 4 h. After cooling, the
solution was diluted with water and extracted with a mixture of

4194
V. Tazzari et al. / Bioorg. Med. Chem. 18 (2010) 4187–4194
CH2Cl2; 7:3). The product crystallized with ethyl ether. Mp 82.5–
83 °C. Yield: 80%. 1H NMR (DMSO-d6): d = 7.95 (d, J = 8.50 Hz, 2H);
7.81 (s, 1H); 7.31–7.16 (m, 6H); 2.69–2.58 (m, 4H); 1.98–1.88 (m,
2H). HRMS (ESI) m/z calcd for C19H17O2S3 [M+H]+: 373.03852;
found: 373.03822.
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acetylated fluorogenic substrate (100
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l
l
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Acknowledgment
This work was supported in part by grants from the Italian Min-
istry of University and Research (M.U.R.)