Synthesis, antiproliferation, and docking studies of N-phenyl-lipoamide and 8-mercapto-N-phenyloctanamide derivatives: Effects of C6 position thiol moiety

Article abstract of DOI:10.1007/s00044-011-9879-7

Some N-phenyl lipoamide and 8-mercapto- N-phenyloctanamide derivatives were synthesized and their in vitro antiproliferative activity was evaluated. The experimental results indicated that 8-mercapto-N-phenyloctanamides might be good histone deacetylase inhibitors rather than N-phenyl lipoamides, who had thiol moiety at C6 position. To verify the antiproliferation data on structural basis, in silico docking studies of the representative compounds into the crystal structure of histone deacetylase- like protein using AutoDock 4.0 program were performed. Furthermore, sulfur acetylated 8-mercapto-Nphenyloctanamide improved its in vitro antiproliferative activity, probably due to the increasing of its cell membrane permeability. While the identification of enzymatic target of N-phenyl lipoamides with dithiolane is still ongoing. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9879-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3312–3320
DOI 10.1007/s00044-011-9879-7
ORIGINAL RESEARCH
Synthesis, antiproliferation, and docking studies
of N-phenyl-lipoamide and 8-mercapto-N-phenyloctanamide
derivatives: effects of C6 position thiol moiety
•
Shi-Jie Zhang Wei-Xiao Hu
Received: 21 July 2011 / Accepted: 4 November 2011 / Published online: 16 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Some N-phenyl lipoamide and 8-mercapto-
N-phenyloctanamide derivatives were synthesized and
their in vitro antiproliferative activity was evaluated. The
experimental results indicated that 8-mercapto-N-pheny-
loctanamides might be good histone deacetylase inhibitors
rather than N-phenyl lipoamides, who had thiol moiety at
C6 position. To verify the antiproliferation data on struc-
tural basis, in silico docking studies of the representative
compounds into the crystal structure of histone deacetyl-
ase-like protein using AutoDock 4.0 program were
performed. Furthermore, sulfur acetylated 8-mercapto-N-
phenyloctanamide improved its in vitro antiproliferative
activity, probably due to the increasing of its cell mem-
brane permeability. While the identification of enzymatic
target of N-phenyl lipoamides with dithiolane is still
ongoing.
Administration (FDA) (Marks, 2007). As a promising lead
compound, vorinostat 1 has shown good in vitro anticancer
efficacy in a wide range of cancer cells (Curtin et al., 2002;
Gediya et al., 2005; Hanessian et al., 2007; Jones et al.,
2008). Crystallographic studies disclosed that the
hydroxamic acid group of vorinostat 1 coordinated to the
zinc ion in the active domain of A. aeolicus HDAC
homolog HDLP in a bidentate fashion and also formed
hydrogen bonds with Tyr 297, His 131, and His 132 resi-
dues (Finnin et al., 1999). The complex thereby blocked
substrate access, resulting in neoplastic cell cycle growth
arrest, terminal cell differentiation, and cell death
(Dokmanovic et al., 2007). However, hydroxamic acid
group was not the only coordinator that could bind to the
zinc ion in enzymes (Suzuki and Miyata, 2005b), and some
terminal sulfur-containing compounds were also found to
bind tightly to zinc-dependent enzymes (Brown et al.,
2000; Opie and Kowolik, 1995). Researchers discovered
that when the hydroxamic acid group of vorinostat 1 was
replaced by proper sulfur-containing moieties, for instance,
the corresponding a-mercaptoketone 2 and a-thioacet-
oxyketone 3 were observed to be even more potent HDAC
inhibitors than that of vorinostat 1 (Gu et al., 2006). The
HDAC inhibition of thiol-containing analogs was distinctly
dependent on chain length, such as x-mercapto alkyl amide
5 with chain length of n = 6 resulting better potent inhi-
bition than n = 5 (4) and n = 7 (6) (Suzuki et al., 2005c).
However, compounds with free thiol group might have low
in vitro or in vivo anticancer efficacy because of its poor
cell membrane permeability, and it would be enhanced by
hiding the sulfhydryl group with a disulfide bond or by
sulfur acylation (Gu et al., 2006; Suzuki et al., 2005c).
Sulfur-acylated natural product largazole 7, isolated from
Floridian marine cyanobacterium Symploca sp. (Taori
et al., 2008), had exceptionally potent inhibition in a
Keywords Lipoic acid ꢀ
8-Mercapto-N-phenyloctanamide ꢀ Antiproliferation ꢀ
HDAC ꢀ Molecular docking
Introduction
Vorinostat 1 (suberoylanilide hydroxamic acid, SAHA) has
been approved for the clinical treatment of cutaneous
T-cell lymphoma (CTCL) by the U.S. Food and Drug
S.-J. Zhang (&)
Graduate School, Zhejiang Chinese Medical University,
Hangzhou 310053, People’s Republic of China
e-mail: shijie.zhang@163.com
W.-X. Hu
College of Pharmaceutical Science, Zhejiang University
of Technology, Hangzhou 310032, People’s Republic of China
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Med Chem Res (2012) 21:3312–3320
3313
number of cancer cell lines in vitro and by hydrolytic
removal of the octanoyl group, its largazole thiol 8, with
exposure of the free sulfhydryl residue as a Zn2?-binding
arm, exhibited an extraordinarily potent HDAC inhibitory
and antiproliferative activity (Bowers et al., 2008; Seiser
et al., 2008). Many other modifications of hydroxamic acid
group of HDAC inhibitors with sulfur-containing moieties
brought out more or less exciting conclusions (Anandan
et al., 2005; Chen et al., 2005; Dehmel et al., 2007; Suzuki
et al., 2004, 2005a).
be distinguished at around 2,500–3,000 cm-1 with weak
intensity. The free thiol group absorption of 15 is located at
2,557 cm-1, which neither appears in the spectra of the
corresponding disulfide 14 nor in bis acetyl lipoate 16. The
proton shifts on C6 (C–H) between lipoamide 14 and
1
dihydro lipoamide 15 are not identical in H NMR spectra
and the protons come into resonance at different frequen-
cies that in 14 it resonates about 0.6–0.7 ppm downfield 1H
chemical shift from that in 15. Moreover, additional proton
resonance signals in free thiols of 15 clearly show the
difference.
According to the SAR of the binding group of vorinostat
1, we conceived that the skeleton of a-lipoic acid (LA) 9
could be modified as LA-based vorinostat analogs. In our
previous article, we found that these analogs, mainly lip-
oamides exhibited dose-dependent inhibitory property and
functioned at high concentration of 100 lg/ml (Zhang
et al., 2010). This was quite agreeable with the results
made by Zachar’s group, who found that the threshold-
killing concentration of 12 was 60 lg/ml while the con-
centration of 11 even reached 600 lg/ml (Bingham and
Zachar, 2001). In this article, we detailed the synthesis and
antiproliferative activity of lipoamide analogs and com-
pared with 8-mercapto-N-phenyloctanamide derivatives
which have no thiol moiety at C6 position. In addition,
molecular docking studies revealed the effect of C6 posi-
tion thiol moiety on docking with HDAC.
Given that these compounds may target the HDAC’s
active-site zinc ion, which was positioned near the bottom
˚
of the 11 A deep tubular pocket of the enzyme, compounds
with C6 position mercapto group may have difficulty in
going through the pocket and binding the zinc ion with C8
position mercapto group. So compound 6 was synthesized
as shown in Scheme 2 and compared with compound 15 in
antiproliferative activity. Ethyl 8-bromooctanoate 21 was
prepared through Hunsdriecke reaction from 9-ethoxy-9-
oxononanoic acid 19, which was esterified from azelaic
acid 17 straightforward. Then compound 21 was converted
to 8-bromooctanoyl chloride 23 and then reacted with
aniline to get 8-bromo-N-phenyloctanamide 24, which was
treated with potassium thioacetate in ethanol to yield S-8-
oxo-8-(phenylamino)octyl ethanethioate 25 and hydrolyzed
by sodium hydroxide to obtain 8-mercapto-N- phenyloc-
tanamide 6.
Results and discussion
Antiproliferation studies
Chemistry
Table 1 shows the in vitro antiproliferative activity of the
representative compounds with or without the thiol side
chain at C6 position and the effect of sulfhydryl group
protection. In general, compounds 14, 15, 16, 6, 25 were
more active than LA 9 and dihydro LA 10, among which
compounds 14 and 25 were of most active, comparable
with or even better than vorinostat 1 at 100 lg/ml. It was
reported that compound 6 with free thiol has good HDAC
inhibitory activity with IC50 at 1.5 lM (Suzuki et al.,
2005c), but its antiproliferative activity against BEL-7402,
KB, NCI-460 shows weak at 100 lg/ml. This was due to its
N-Phenyl-6,8-dithioctamide derivatives were prepared
from 9 as shown in Scheme 1. Amidation of lipoyl chloride
13 and aniline in the presence of triethylamine as hydrogen
chloride capture in dichloromethane led to lipoamide 14.
Reduction of disulfide bond by sodium borohydride gave
the corresponding dihydro lipoamide 15, which was fol-
lowed by acylation with acetyl chloride to afford 16.
The presence of thiol (S–H) absorption in an IR spec-
trum is diagnostic evidence for the sodium borohydride
reduction of S–S bond that S–H stretching frequency can
Scheme 1 Reagents and
conditions. a Thionyl chloride,
benzene, 0°C, 3 h. b Aniline,
triethylamine, dichloromethane,
0°C, 4 h. c Sodium borohydride,
tetrahydrofuran, 0°C, 7 h.
d Acetyl chloride, triethylamine,
dichloromethane, 0°C to rt, 2 h
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Med Chem Res (2012) 21:3312–3320
Scheme 2 Reagents and conditions. a Ethanol, p-methyl benzene-
sulfonic acid, toluene, reflux, 8 h. b Sodium hydroxide, ethanol, rt,
1 h, then 4 N hydrochloric acid. c Silver nitrate, potassium hydroxide,
H2O, rt, 15 min. d Bromine, carbon tetrachloride, 1.5 h. e Sodium
hydroxide, H2O, 45°C, 2 h, then 4 N hydrochloric acid. f Thionyl
chloride, reflux, 1 h. g Aniline, triethylamine, dichloromethane, 0°C
to rt, 4 h. h Potassium thioacetate, ethanol, rt, 20 h; i sodium
hydroxide, H2O, ethanol, rt, 48 h
analogs as the active species (Suzuki et al., 2005c), such as
FK228, FR901375, Spiruchostatin (Bowers et al., 2008),
SCOP (Nishino et al., 2008), largazole 7 (Bowers et al.,
2008), and 9 (Novotny et al., 2008). Thus, it can be inferred
that the antiproliferative activity of disulfide 14, which can
be reduced to bis sulfhydryl 15, would be no better than
that of 15 under the hypothesis of HDAC inhibitory action.
In fact, the antiproliferative activity of 14 exhibits 1-fold
higher against NCI-460, 3-fold higher against KB, and
7-fold higher against BEL-7402 than 15, and the expla-
nation might be that 14 is functioning through other
mechanism such as mammalian pyruvate dehydrogenase
complex (PDC) inhibition rather than HDAC inhibition.
Table 1 Antiproliferative activity of compounds against cancer cell
lines at 100 lg/ml
Compound
BEL–7402
KB
NCI–460
14
60.57 1.74
7.67 4.96
14.13 3.23
Na
65.32 2.30
15.78 4.23
22.95 1.54
18.59 10.30
77.01 2.75
10.96 4.47
16.34 0.34
64.60 1.61
88.83 0.54
47.59 5.38
25.42 2.59
44.77 2.57
32.39 7.35
82.26 1.43
5.72 6.86
7.84 1.02
61.06 2.89
97.00 0.46
15
16
6
25
66.31 6.76
8.17 2.14
0.59 2.07
65.74 2.28
86.20 0.79
9
10
Vorinostat 1
Cisplatin
Values presented are means S.D.
In silico molecular docking
na not active
The docking studies of the compounds were performed
with AutoDock 4.0 program, based on a Lamarckian
genetic algorithm (LGA) method (Morris et al., 1998;
Huey et al., 2007). Basically, this program calculates total
interaction energies between random pairs of ligands and
various selected portions of protein to determine docking
poses. The AutoDock 4.0 program with the following
parameters: AutoDockTools 1.4.5 (ADT) is prepared for
ligands and receptor. The AutoDock search parameters
select Genetic Algorithm. The number of GA-Run is 100,
the population size 200, and the maximum number of Evals
is 25,000,000 generations. Other parameters are default
values. The generate files are calculated by AutoGrid 4.0
and AutoDock 4.0 (Figs. 1, 2).
poor cell membrane permeability resulted from the highly
polar character of the compound with free thiol. Remark-
ably, masking the sulfhydryl group of compound 6 as thi-
oester enhanced its cancer cell growth inhibition rapidly. It
stands to reason that the thioester 25 increased the possi-
bility of permeating the membrane of cancer cells and then
hydrolyzed under physiological conditions to release the
free thiol compound 6 to target the active site of HDAC
(Gu et al., 2006). However, bis acetyl compound 16 failed
to exhibit an admiring growth inhibitory effect as com-
pound 25, only relatively potent compared with bis sulf-
hydryl compound 15. This result suggests that even though
16 can get through cell membrane as efficiently as 25 and
convert to thiol 15 within the cell, hardly can it squeeze
into pocket-shaped HDAC tunnel because of the block of
the thiol side chain at C6 position. It is widely believed that
in cellular environment, compounds with disulfide bond or
sulfur-acylated can be reduced, releasing the free thiol
As shown in Fig. 3a, b, compound 6 bound by
inserting its unbranched long alkyl chain into the HDLP
pocket snugly, making multiple contacts to the tube-like
hydrophobic portion of the pocket. The thiol moiety at
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Med Chem Res (2012) 21:3312–3320
3315
Fig. 1 Structures of known
HDAC inhibitors and others
related. IC50 refers to the
inhibition of HDAC1, unless
otherwise stated
Fig. 2 Illustration of proposed
pathway of compounds against
cancer cell lines
one end of the chain reached the polar bottom of the
pocket, where it coordinated the zinc ion and also con-
tacted active-site residue Tyr 297 with a distance of
where the distance of C8 thiol moiety to Tyr 297 was
˚
2.7 A and unbonded to zinc, as shown in Fig. 3c, d. For
compound 14 in Fig. 3e, f, the dithiolane was almost
sandwiched between the phenyl groups of Phe 141 and
Phe 198 and could not reach the active site.
˚
2.5 A. While the phenyl-amino ketone group contacted
residues at the rim of the pocket and in an adjacent sur-
face groove formed by Pro 22, Tyr 91, and Phe 141. Due
to the existence of C6 thiol moiety, compound 15 could
not get into the pocket and was blocked at the tightest
point of the pocket shaped by Phe 141 and Phe 198,
As shown in Table 2, compound 6 displayed free
binding energy value of -7.36 kcal/mol, indicating that the
affinity of compound 6 for HDLP was stronger than com-
pounds 15 or 14.
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Med Chem Res (2012) 21:3312–3320
Fig. 3 a, b Docking interaction of compound 6 with the target HDLP. c, d Docking interaction of compound 15 with the target HDLP. e, f
Docking pose of compound 14 with the target HDLP
Experimental
spectrometer operating at 400 MHz using tetramethyl
silane (TMS) as an internal standard in CDCl3 and chem-
ical shifts were expressed in d (ppm) values downfield
from TMS, and J values were in Hz (The splitting pattern
abbreviations are as follows: s, singlet; d, doublet; t, triplet;
m, multiplet and br, broad peak). Mass spectra (MS) were
run on an HP 5989B instrument at an ionizing voltage of
70 eV. High resolution mass spectra (HRMS) were
Chemistry-general aspects
Melting points were taken on an XRC-1 apparatus and are
uncorrected. Infrared spectra (IR) were obtained on a
1
Thermo Nicolet Avatar 370 FT-IR spectrophotometer. H
NMR spectra were recorded on a Brucker AC-80
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Med Chem Res (2012) 21:3312–3320
3317
1H NMR (CDCl3, 400 MHz) d: 7.52 (d, J = 8.0 Hz, 2H,
Ar), 7.33 (t, J = 8.0 Hz, 2H, Ar), 7.16 (br, 1H, NH), 7.11
(t, J = 7.2 Hz, 1H, Ar), 3.59 (m, J = 6.9 Hz, 1H, –S–CH–
), 3.22–3.09 (m, 2H, –S–CH2–), 2.38 (t, J = 7.4 Hz, 2H,
–CH2–CO–), 1.97–1.49 (m, 8H, –CH2–CH–CH2–CH2–
CH2–); EI-MS m/z(%): 281 (M?, 97), 248 (16), 216 (17),
155 (30), 148 (27), 135 (51), 93 (100), 77 (18); UV
(EtOH): kmax (nm, log e) = 202 (4.83), 243 (4.57).
Table 2 Binding energy data of compounds 6, 15, and 14
Compound
Estimated free
binding
energya
(kcal/mol)
6
-7.36
-5.86
-6.01
15
14
a
Free binding energy calculations were determined by docking
studies using AutoDock 4.0 program
6,8-Dimercapto-N-phenyloctanamide (15)
To a solution of 14 (0.84 g, 3 mmol) in tetrahydrofuran
(30 ml) at 0°C was added a solution of sodium borohydride
(0.23 g, 6 mmol in 5 ml H2O) dropwise. The mixture was
allowed to stir at the same temperature for an additional
7 h to reach completion. 1 N hydrochloric acid (10 ml) was
added, and the solvent was removed under vacuum. The
residue was extracted with dichloromethane (3 9 10 ml).
The organic layer was washed with brine (3 9 10 ml),
dried over anhydrous magnesium sulfate, and concentrated
under vacuum to obtain crude product, which was chro-
matographed over silica gel (PE:EtOAc = 2:1) to yield 15
as white solid, 0.74 g (87.0%); mp: 43–44°C; IR mmax
(KBr)/cm-1: 3310, 2929, 2557, 1653, 1599, 1534, 1498,
measured on Agilent 6210 TOF LC/MS instrument with
ESI source. UV absorption spectra were recorded on UV-
1800 UV–vis spectrophotometer from Shimadzu. Column
chromatography was carried out on silica gel (200–300
mesh) eluting with solvents as indicated. Reaction progress
was monitored by thin layer chromatography (TLC) on
precoated silica gel GF254 plates with fluorescent indica-
tor. All the chemicals and solvents were of analytical
reagent and used as received, unless otherwise stated.
Petroleum ether (PE) referred to a mixture of alkanes with
the boiling range from 60 to 90°C. a-LA was racemic and
used before recrystallization (cyclohexane:ethyl acetate
(EtOAc) = 15:1).
1
1442, 759, 692, 501; H NMR (CDCl3, 400 MHz) d: 7.53
(d, J = 7.6 Hz, 3H, Ar ? NH), 7.31 (t, J = 7.8 Hz, 2H,
Ar), 7.11 (t, J = 7.4 Hz, 1H, Ar), 2.96–2.88 (m, 1H, –S–
CH–), 2.78–2.62 (m, 2H, –S–CH2–), 2.37 (t, J = 7.4 Hz,
2H, –CH2–CO–), 1.94–1.45 (m, 8H, –CH2–CH–CH2–
CH2–CH2–), 1.36 (t, J = 8.4 Hz, 1H, SH–CH2–), 1.31 (d,
J = 7.2 Hz, 1H, SH–CH); ESI-HRMS: [M ? H]?
284.1157 for C14H21NOS2 ? H (Calcd 284.1143),
[M ? Na]? 306.0976 for C14H21NOS2 ? Na (Calcd
306.0962); UV (EtOH): kmax (nm, log e) = 202 (4.77), 243
(4.50).
Lipoyl chloride (13) in dichloromethane
To a stirred solution of thionyl chloride (0.89 g, 7.5 mmol)
in benzene (10 ml, calcium chloride dried) at 0°C was
added a solution of lipoic acid (1.03 g, 5 mmol) in dried
benzene (20 ml) dropwise over 1 h. The mixture was
allowed to stir for an additional 3 h at the same tempera-
ture. After completion of the reaction, the solvent was
removed under vacuum to get lipoyl chloride 13 almost
quantitatively. The residue without further purification was
diluted in dichloromethane (20 ml) for the next step.
S,S0-(8-Anilino-8-oxo octane-1,3-diyl)diethanethioate (16)
5-(1,2-Dithiolan-3-yl)-N-phenylpentanamide (14)
To a solution of 15 (0.28 g, 1 mmol) and triethylamine
(0.25 g, 2.5 mmol) in dichloromethane (20 ml) at 0°C was
cautiously added a solution of acetyl chloride (0.20 g,
2.5 mmol) in dichloromethane (5 ml). The mixture was
stirred in an ice bath for additional 2 h to reach completion.
Dichloromethane (15 ml) was added to dilute the mixture
and washed with 1% citric acid (3 9 10 ml), brine
(3 9 10 ml), and dried over anhydrous magnesium sulfate.
The evaporation of solvent left crude product, which was
purified with chromatography (PE: EtOAc = 2: 1) to
obtain 16 as viscous liquid, 0.28 g (77.1%); IR mmax (KBr)/
cm-1: 3311, 2933, 1690, 1600, 1541, 1499, 1442, 1134,
953, 757, 694, 630, 507; 1H NMR (CDCl3, 400 MHz)
d: 7.67 (br, 1H, NH), 7.54 (d, J = 7.6 Hz, 2H, Ar), 7.31
(t, J = 7.8 Hz, 2H, Ar), 7.09 (t, J = 7.2 Hz, 1H, Ar), 3.56
To an ice-cooled solution of aniline (0.56 g, 6 mmol) and
triethylamine (0.61 g, 6 mmol) in dichloromethane (20 ml)
was cautiously added the prepared solution of 13 in
dichloromethane dropwise. The reaction mixture was stir-
red in an ice bath for 4 h to reach completion, monitored by
TLC. The mixture was washed by saturated sodium
bicarbonate (3 9 10 ml), 1 N hydrochloric acid
(3 9 10 ml), brine (3 9 10 ml) and dried over anhydrous
magnesium sulfate, and the solvent was then removed
under reduced pressure. The crude product was purified
with chromatography (PE:EtOAc = 1:1) to get 14 as yel-
low solid, 0.97 g (68.9%); mp: 67–69°C; IR mmax (KBr)/
cm-1: 3296, 2920. 1658, 1599, 1537, 1498, 1444, 691, 502;
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Med Chem Res (2012) 21:3312–3320
(m, J = 6.8 Hz, 1H, –S–CH–), 3.01–2.94 (m, 1H, –S–
CHH–), 2.87–2.80 (m, 1H, –S–CHH–), 2.35 (t, J = 7.6 Hz,
2H, –CH2–CO–), 2.32 (s, 6H, 2 9 CH3), 1.92–1.37 (m,
8H, –CH2–CH–CH2–CH2–CH2–); ESI-HRMS: [M ? H]?
368.1381 for C18H25NO3S2 ? H (Calcd 368.1354),
[M ? Na]? 390.1202 for C18H25NO3S2 ? Na (Calcd
390.1174).
8-bromooctanoic acid 22 as pale yellow crystal, 3.3 g
(97.7%); low mp; IR mmax (KBr)/cm-1: 2933, 2852, 1701,
1467, 1410, 1252, 929, 726, 678.
The stirred mixture of 8-bromooctanoic acid 22 (3.2 g,
15 mmol) and thionyl chloride (20 ml) was refluxed for
1 h. Evaporation of excess thionyl chloride left 8-bromo-
octanoyl chloride 23 quantitatively.
8-Bromo-N-phenyloctanamide 24 was prepared from
aniline (0.93 g, 9.9 mmol) and 8-bromooctanoyl chloride
23 using the procedure described for 14: yellow solid, 1.1 g
(44.6%); low mp; IR mmax (KBr)/cm-1: 3308, 2933, 2856,
1661, 1600, 1540, 1499, 1443, 1384, 1251, 1180, 757, 693.
To a suspension of 8-bromo-N-phenyloctanamide 24
(0.80 g, 2.68 mmol) obtained above in ethanol (10 ml) was
added potassium thioacetate (1.1 g, 9.73 mmol), and the
mixture was stirred at room temperature for 20 h. The
reaction mixture was diluted with ethyl acetate (50 ml) and
tetrahydrofuran (50 ml), washed with H2O (3 9 20 ml)
and brine (3 9 20 ml), and dried over anhydrous sodium
sulfate. Filtration and concentration in vacuo and purifi-
cation by silica gel chromatography (PE:EtOAc = 1:1)
gave 25 as brown oil, 142 mg (18.0%); IR mmax (KBr)/
cm-1: 3312, 2929, 2856, 1690, 1667, 1600, 1543, 1499,
1443, 1384, 756, 693, 507; 1H NMR (CDCl3, 400 MHz) d:
7.52 (d, J = 7.6 Hz, 2H, Ar), 7.32 (t, J = 7.6 Hz, 2H, Ar),
7.25 (brs, 1H, NH), 7.10 (t, J = 7.4 Hz, 1H, Ar), 2.86 (t,
2H, J = 7.4 Hz, –CH2–S–), 2.35 (t, J = 6.8 Hz, 2H,
–CH2–CO–), 2.32 (s, 3H, CH3–CO–), 1.77–1.72 (m, 2H,
–CH2–CH2–S–), 1.60–1.53 (m, 2H, –CH2–CH2–CO–),
1.41–1.36 (m, 6H, –CH2–CH2–CH2–CH2–CH2–S–); EI-MS
m/z(%): 293 (M?, 5), 253 (30), 218 (19), 204 (11), 135
(90), 120 (18), 93 (100), 77 (38).
S-8-Oxo-8-(phenylamino)octyl ethanethioate (25)
A mixture of azelaic acid 17 (75.3 g, 400 mmol), excess
ethanol (180 ml), toluene (100 ml), and catalytic amount
of p-methyl benzenesulfonic acid (6.0 g, 35 mmol) was
stirred at reflux for 8 h to get diethyl ester 18, one ethyl
ester moiety of which was hydrolyzed by sodium hydrox-
ide (17.5 g, 440 mol) in ethanol (400 ml) for 1 h and then
adjusted to pH 2 by 4 N hydrochloric acid. The aqueous
phase was extracted by ethyl ether (3 9 100 ml) and dried
over anhydrous magnesium sulfate to get azelaic acid
monoethyl ester 19 as yellowish clear oil, 73.8 g (85.3%);
IR mmax (KBr)/cm-1: 3446, 2934, 2858, 1736, 1710, 1384,
1182, 1096, 1035.
To a stirring solution of azelaic acid monoethyl ester 19
(21.6 g, 100 mmol) and potassium hydroxide (5.6 g,
100 mmol) in H2O (240 ml) was added a solution of silver
nitrate (17.0 g, 100 mmol) in H2O (200 ml) in 5 min. The
reaction continued for 15 min before filtration of the gray
precipitation. The precipitated silver salt 20 was washed
with water (60 ml) and methanol (60 ml) and then dried
thoroughly under an oil pump at 110°C and powdered
finely to get 19.4 g (60.1%).
To a mixture of silver salt 20 (19.4 g, 60 mmol) and
carbon tetrachloride (70 ml, phosphorus pentoxide dried)
was added bromine (8.8 g, 55 mmol, sulfuric acid dried)
over 40 min in an ice bath with generation of carbon
dioxide. After completion, the violet mixture was heated to
reflux in a 110°C oil bath for 1.5 h. The mixture was fil-
tered and the left silver bromide was washed with warm
carbon tetrachloride. The filtrate was washed with 10%
sodium carbonate (60 ml) and dried over anhydrous mag-
nesium sulfate to obtain ethyl 8-bromooctanoate 21 as pale
yellow oil, 8.9 g (64.4%); IR mmax (KBr)/cm-1: 2933,
2857, 1734, 1463, 1384, 1250, 1177, 1096, 1035, 782, 726,
644, 562; EI-MS m/z(%): 171 ([M-79Br]?, 100), 152 (32),
125 (17), 97 (50), 83 (28), 69 (31).
8-Mercapto-N-phenyloctanamide (6)
To a solution of S-8-oxo-8-(phenylamino)octyl ethanethi-
oate 25 (37.9 mg, 0.13 mmol) in ethanol (5 ml) was added
sodium hydroxide (17.1 mg, 0.43 mmol) in H2O (1 ml).
The mixture was stirred at room temperature for 48 h
before H2O (10 ml) was added to dilute the mixture and
neutralized with 2 N hydrochloric acid to pH 4–5 with
cooling in an ice-water bath. The clear yellow solution was
extracted with ethyl acetate (20 ml) and washed with H2O
(3 9 5 ml), brine (3 9 5 ml), and dried over anhydrous
sodium sulfate. Filtration and concentration in vacuo gave
6 as light yellow solid, 21.8 mg (67.1%); low mp; IR mmax
(KBr)/cm-1: 2961, 2926, 2855, 2549, 1663, 1600, 1545,
1499, 1443, 1384, 757, 693, 509; 1H NMR (CDCl3,
400 MHz) d: 7.51 (d, J = 8.0 Hz, 2H, Ar), 7.31 (t,
J = 7.4 Hz, 2H, Ar), 7.09 (t, J = 7.0 Hz, 1H, Ar), 2.73–
2.65 (m, 2H, HS–CH2–), 2.35 (t, J = 7.6 Hz, 2H, –CH2–
CO–), 1.81–1.43 (m, 10H, –CH2–CH2–CH2–CH2–CH2–
CH2–HS), 1.36 (t, J = 7.6 Hz, 1H, SH–CH2–); EI-MS
The mixture of ethyl 8-bromooctanoate 21 (3.8 g,
15 mmol) and sodium hydroxide (0.7 g, 16.5 mmol) in
H2O (60 ml) was heated to 45°C for 2 h and then more
water (180 ml) was added and washed with carbon tetra-
chloride (3 9 30 ml). The aqueous phase was acidified by
4 N hydrochloric acid to pH 2. The solution was extracted
by ethyl acetate (3 9 80 ml) and washed with brine
and dried over anhydrous magnesium sulfate to get
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Med Chem Res (2012) 21:3312–3320
3319
m/z(%): 219 ([M–SH ? H]?, 4), 205 (6), 191 (7), 179 (9),
163 (10), 149 (17), 137 (16), 123 (25), 93 (51).
Conclusion
In summary, 8-mercapto-N-phenyloctanamide derivatives
were successfully synthesized and their antiproliferative
activity was evaluated and compared with N-phenyl-lipo-
amide analogs. The experimental results showed that in
vitro antiproliferative activity of sulfur acetylated 8-mer-
capto-N-phenyloctanamide was much higher than that of
8-mercapto-N-phenyloctanamide, indicating the sulfur
protected thioctamide increased its cell membrane perme-
ability and would be deacetylated to become 8-mercapto-
N-phenyloctanamide with free thiol moiety as HDAC
inhibitor. In addition, docking studies of molecules with or
without C6 thiol moiety were performed to determine their
effectiveness as HDAC inhibitors. Based on their docking
results and scoring, compounds with C6 thiol moiety or
dithiolane were unable to reach the active site of HDLP,
while 8-mercapto-N-phenyloctanamide without C6 thiol
moiety successfully targeted HDAC with its C8 thiol
moiety as zinc chelating group. N-Phenyl lipoamides with
dithiolane also showed good antiproliferative activity, but
it could not be docked with HDAC, the indentification of
enzymatic target of which is still ongoing. In follow-up
studies, these experimental and predictive results described
may give rise to design new and more potent HDAC
inhibitors with proper thiol moiety.
Antiproliferation assay
The human cancer cell lines (BEL-7402, KB and NCI-460
derived from Shanghai Institutes for Biological Science,
Chinese Academy of Sciences) were cultivated at 37°C,
5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM,
purchased from Gibco) supplemented with 800 (U/v)
penicillin, 0.1% (w/v) streptomycin, 10% (v/v) fetal
bovine serum for 3–5 days. Human cancer cells, treated
with Trypsin–EDTA solution, were seeded into 96-well flat
bottom plates at 106 cells/well and incubated in a 5% CO2
incubator at 37°C for 24 h. Cultures were treated with
compounds prepared in 100 lg/ml. Mitochondrial metab-
olism was measured as a marker for cell growth by adding
10 ll/well MTT (5 mg/ml in medium, Sigma) with 3 h of
incubation at 37°C. Crystals formed were dissolved in
150 ll of DMSO. The absorbance was determined using a
microplate reader at 490 nm. The absorbance data were
converted into a cell proliferation percentage, compared to
DMSO treated cells, to determine growth inhibition. Each
assay was performed in triplicate.
In silico molecular docking
Acknowledgments The authors are thankful to Hangzhou Minsh-
eng Pharmaceutical Group Co., Ltd. for antiproliferative evaluation.
Partial financial support from Hangzhou Minsheng Pharmaceutical
Group Co., Ltd. are also gratefully acknowledged.
The synthesized compounds with R conformation were
taken for prediction of 3D structures and energy was
minimized for flexible docking. Each molecule was added
Gasteiger charges and nonpolar hydrogens were merged to
carbon atoms by AutoDockTools (ADT 1.4.5). The 3D
structure of HDLP (1C3R) was obtained from Protein Data
Bank at the website: http://www.pdb.org/. HDLP is similar
to human HDAC1 with 35.2% identity and specially, all of
the polar residues in the active site and the hydrophobic
residues that make up the walls of the pocket are identical
(Finnin et al., 1999). Trichostatin A (TSA) molecule and
nonreceptor atoms such as water, ions were removed from
1C3R, and Kollmann charges were assigned and converted
to pdbqt format as receptor file. The grid maps defining the
search region and representing the protein in the docking
process were calculated with AutoGrid and had dimensions
Conflict of interest The authors declare that they have no conflict
of interest regarding the work reported in this manuscript.
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