Synthesis and anticancer evaluation of α-lipoic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.03.112

α-Lipoic acid derivatives were synthesized and evaluated for their in vitro anticancer activities against NCI-460, HO-8910, KB, BEL-7402, and PC-3 cell lines. The results, for most compounds exhibited dose-dependent inhibitory property and several compounds had good inhibitions at the dose of 100 μg/mL. Compound 17m was further selected for in vivo evaluation against S180 xenograft in ICR mice, which had 24.7% tumor-weight inhibition through intragastric administration of 200 mg/kg of body weight. Moreover, the LD₅₀ in mice for 17m through ig exceeded 1000 mg/kg of body weight.

Full text of DOI:10.1016/j.bmcl.2010.03.112

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3078–3083
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anticancer evaluation of
a-lipoic acid derivatives
c
Shi-Jie Zhang a, Qiu-Fu Ge b, Dian-Wu Guo b, Wei-Xiao Hu a, , Hua-Zhang Liu
*
a College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, PR China
b Hangzhou Minsheng Pharmaceutical Group Co., Ltd, Hangzhou 310011, PR China
c College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310032, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
a
-Lipoic acid derivatives were synthesized and evaluated for their in vitro anticancer activities against
NCI-460, HO-8910, KB, BEL-7402, and PC-3 cell lines. The results, for most compounds exhibited dose-
dependent inhibitory property and several compounds had good inhibitions at the dose of 100 g/mL.
Received 26 January 2010
Accepted 27 March 2010
Available online 3 April 2010
l
Compound 17m was further selected for in vivo evaluation against S180 xenograft in ICR mice, which
had 24.7% tumor-weight inhibition through intragastric administration of 200 mg/kg of body weight.
Moreover, the LD50 in mice for 17m through ig exceeded 1000 mg/kg of body weight.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Lipoic acid
Synthesis
Anticancer
a
-Lipoic acid 1 is a naturally-occurring co-factor existed in a
Some lipoic acid derivatives also possessed notable anticancer
activities as illustrated in Figure 1. It was patented that bis acetyl
lipoate 3, bis benzoyl lipoate 4 and some other such sulfur-acylated
derivatives (6 and 7) exhibited positive activities to kill a wide
number of multi-enzyme complexes regulating metabolism and
has arisen significant interest from its fascinating biological prop-
erties.1
a-Lipoic acid 1 and its derivatives were proved to be effec-
tive against Type 2 diabetes,2,3 atherosclerosis,3 inflammatory skin
diseases,3 reperfusion arrhythmias,4,5 and Parkinson’s disease.6
Moreover, lipoic acid 1 has been widely reported to induce apopto-
sis in various cancer cell lines. It induced apoptosis in human hep-
atoma cells SMMC-7721 of 65% after 96 h with 5 mM dose of lipoic
acid 1,7 and human tumor cell lines FaDu and Jurkat were also ini-
tiated apoptosis following long-time exposure to lipoic acid in mM
concentrations, indicating a time- and dose-dependent fashion.8
Likewise, lipoic acid 1 was effective in suppressing growth on four
different HNSCC cell lines (FaDu, SCC9, SCC25, and Detroit-562) at
high concentrations up to 10 mM with more than 50% cell inhibi-
tion.9 Lipoic acid 1 and dihydrolipoic acid 2 induced apoptosis in
human colon cancer cells HT-29 with the corresponding EC50 val-
range of cancer cells at high concentrations between 60 lg/mL
and 800 lg/mL; however, bis carbamoyl lipoate 5 seemed not
active against these cell lines.13,14 Compounds 8 and 9 were effec-
tive on inhibiting the proliferation of human breast cancer cells
MCF-7 with IC50 at 15
colon cancer cells H-29 with IC50 at 10
l
M and 25
l
M, respectively; and of human
lM and 8
l
M.3 And treat-
ment of human skin cancer cells A-431 with quinazoline 10 related
to PD168393, in which the acrylamide group at the 6-position was
replaced by lipoic acid, induced a decrease of the cell proliferation
with IC50 at 22
derivatives (11, 12, 13 and 14) had an IC50 at 400, 240, 50, and
45 M on a human epithelial prostate cancer cell line (DU-145),
l
M.15 In addition, some thiolesters of lipoic acid
l
respectively.16 All these modifications suggested that the
variations on carboxyl group and disulfide bond of lipoic acid 1
with different substituents might enhance its anticancer efficacy.
With the knowledge of the anticancer activities of lipoic acid
and its derivatives, we conceived that the skeleton of lipoic acid
1 could be modified, and such compounds may have potent
anticancer activities with low toxicity. Therefore, in this Letter,
ues around 250
about 27% and 13% diminution of human lung epithelial cells
H-460 growth after 24 h by 100 M of lipoic acid 1 and dihydroli-
l
M for both compounds.10 Besides, there was an
l
poic acid 2, respectively, and it seemed that the effect of lipoic acid
1 was more pronounced than dihydrolipoic acid 2.11 As expected,
lipoic acid 1 was inactive at low
l
M concentrations,12 and the
concentrations required to induce effects were considerably high
in mM ranges.2 As an endogenous agent widely used as a dietary
supplement, lipoic acid 1 is safe to normal cells without adverse
side effects caused by many chemotherapeutic agents and the rat
LD50 of lipoic acid 1 was assumed to exceed 2000 mg/kg of body
weight.1
we are endeavored to synthesize these
and evaluate their anticancer activities.
a-lipoic acid derivatives
Compound 3 was synthesized in three steps from 1 by sodium
borohydride (NaBH4) reduction to dihydrolipoic acid 2, followed
by treatment with acetyl chloride (3 equiv) in dichloromethane
(DCM) to form anhydride 23 and selectively hydrolyzed in isopro-
panol/H2O as illustrated in Scheme 1 and compound 4 was ob-
tained by benzoyl chloride (3 equiv) to form anhydride 24 and
selectively hydrolyzed in dioxane/H2O.13
* Corresponding author. Tel./fax: +86 571 88320557.
E-mail address: huyang@mail.hz.zj.cn (W.-X. Hu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.112

S.-J. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3078–3083
3079
O
O
O
O
S
O
S
R3
R2
N
S
R1
OH
R1
NH
OH
S
R3
S
R2
S S
O
1
entry
O
entry
R3
MCF-7 H-29
H
O
2
3
S
R5
R4
8
15
10
S-S bond
S
R4
R4
O
NH3Cl
9
25
8
O
entry
R5
DU-145
400
4
5
6
7
O
S
S
11
O
O
Br
O
H2N
A-431
10 22
N
N
12
13
240
73
O
O
O
O
HN
N
H
N
S
O
N
O
O
O
O
N
H
H
14
N
50
S S
S
Figure 1. Structures of lipoic acid and related derivatives with anticancer property. Values refer to IC50
(lM).
O
O
O
O
O
a
b
c
Ac
Bz
HS
S
S
S
S
OH
OH
Ac
Bz
O
O
Ac
Bz
OH
OH
S S
SH
S
S
S
S
Ac
Bz
Ac
Bz
d
1
2
23
24
3
4
O
e
Scheme 1. Reagents and conditions: (a) NaOH, NaBH4, H2O, 2 N HCl, rt, 2–3 h, 47%; (b) acetyl chloride, triethylamine, DCM, 0 °C to rt, 2 h; (c) isopropanol, H2O, 40 °C, 4.5 h,
30% from 2; (d) benzoyl chloride, triethylamine, DCM, 0 °C to rt, 9 h; (e) dioxane, H2O, rt, 60 h, 24% from 2.
O
O
O
O
a
c
b
OH
OH
Cl
HS
O
O
N
H
N
H
Cl
S S
S S
SH
15
21
22
16
Scheme 2. Reagents and conditions: (a) Na2S, S, TBAB, toluene, reflux, 5 h; (b) hydroxylamine hydrochloride, KOH, methanol, rt, 4–6 h, 32%; (c) NaBH4, THF, 0 °C, 3 h, 63%.
Lipoic acid-based hydroxamates 15 and 16 were obtained as
outlined in Scheme 2. The esterification of 1 with ethanol under re-
flux was not feasible in a general way, as the procedure suffered
from the formation of polymerized by-product. Therefore, to access
ethyl lipoate 22, ethyl 6,8-dichlorooctanoate 21, a precursor in
preparation of 1, was used to react with Na2S and elemental sulfur
in presence of tetrabutylammonium bromide (TBAB) in toluene
under reflux to form the heterocyclic disulfide circle with quantita-
tive yield.17 Hydroxamate 15 was obtained by treating with freshly
prepared hydroxylamine and reduction of disulfide bond of 15 by
NaBH4 in tetrahydrofuran (THF) gave 16.
The major series of lipoic acid derivatives as amides were pre-
pared from 1 as shown in Scheme 3. Amidation of lipoyl chloride
25 with amines in presence of triethylamine as hydrogen chloride
capture in DCM led to amides 17a–o. Reduction of disulfide bond
gave the corresponding dihydrolipoamides 18a–o, several of which
were followed by acylation with acetyl chloride and benzoyl chlo-
ride to afford 19a–d and 20a–d, respectively.
1 (½a 2D5
ꢀ
ꢁ117.8 (c 1.0, EtOH)) was obtained with S-(ꢁ)-
a-methyl-
benzylamine (S-(ꢁ)-FEA) under the same procedure. The obtained
enantiomers were applied to synthesize amides with 4-nitro-3-(tri-
fluoromethyl)aniline, respectively, as illustrated in Scheme 4.
In addition, some lipoamide analogues were synthesized with-
out mercapto group at C6 position as shown in Scheme 5. Com-
pound 32 was prepared and compared with compound 18m in
anticancer activities. 8-Bromooctanoic acid 28 was converted to
8-bromooctanoyl chloride 29 and reacted with 5-chloropyridin-
2-amine to get 8-bromo-N-(5-chloropyridin-2-yl)octanamide 30,
which was treated by a novel method we developed with potas-
sium O-ethylxanthate in alkaline condition to have S-8-(5-chloro-
pyridin-2-ylamino)-8-oxooctyl O-ethyl carbonodithioate 31 and
acidified by hydrochloric acid to obtain N-(5-chloropyridin-2-yl)-
8-mercaptooctanamide 32 one pot. Besides, S-8-oxo-8-(phenyla-
mino)octyl ethanethioate 34 and 8-mercapto-N-phenyloctanamide
35 were synthesized to confirm the effect of C6 position mercapto
group compared with 18a and 19a.
In order to probe the effect of chirality in the anticancer
All the compounds were tested in vitro at different concentra-
tions against human lung NCI-460, ovarian HO-8910, nasopharynx
KB, hepatoma BEL-7402 cancer cell lines, in comparison with
Vorinostat and Cisplatin. Some selected compounds were also tested
against human prostate PC-3. By analysis of the data reported in
Table 1, we can draw the following structure–activity relationships
(SAR).
activities of lipoamides, we prepared enantiopure R-(+)-1 (½a D25
ꢀ
+119.7 (c 1.0, EtOH), equals to ee >99%) according to a literature
method.18 Racemic lipoic acid 1 was treated with the optically
active base R-(+)-a-methylbenzylamine (R-(+)-FEA) to obtain the
diastereoisomeric salt, followed by repeatedly crystallization and
finally scission of the salt with citric acid to get R-(+)-1 and S-(ꢁ)-

3080
S.-J. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3078–3083
O
O
O
O
O
R6
R6
R6
R6
a
b
c
d
HS
S
S
1
Cl
N
H
N
H
Ac
Bz
N
H
S S
S S
Cl
SH
18a-18o
S
25
Ac
17a-17o
19a-19d
e
c
e
a
b
d
f
N
H
Cl
CF3
S
R6 =
Bz
Cl
F
20a-20d
j
g
o
h
i
l
m
n
k
CF3
NO2
CH3
N
N
N
OCF3
OCH3
NO2
Br
Cl
Scheme 3. Reagents and conditions: (a) thionyl chloride, benzene, 0 °C, 3–4 h; (b) R6NH2, triethylamine, DCM, 0 °C, 2–6 h, 19–76% from 1; (c) NaBH4, THF, 0 °C, 7 h, 22–87%;
(d) acetyl chloride, triethylamine, DCM, 0 °C to rt, 2 h, 40–77%; (e) benzoyl chloride, triethylamine, DCM, 0 °C to rt, 9 h, 57–91%.
CF3
NO2
O
O
O
O
O
d, e
d, e
c
c
OH
OH
N
H
O H3N
S S
S S
S S
S S
S S
S S
a
R-(+)-1
26
R-(+)-17h
CF3
1
NO2
b
O
N
H
O H3N
27
S-(-)-1
S-(-)-17h
Scheme 4. Reagents and conditions: (a) R-(+)-FEA, toluene, 38–39 °C, recrystallization repeated 5 times; (b) S-(ꢁ)-FEA, toluene, 38–39 °C, recrystallization repeated 5 times;
(c) citric acid, methanol, toluene, 1 h; (d) thionyl chloride, benzene, 0 °C, 3–4 h; (e) 4-nitro-3-(trifluoromethyl)aniline, triethylamine, DCM, 0 °C, 2–6 h, 70% for R-(+)-17h from
R-(+)-1, 66% for S-(ꢁ)-17h from S-(ꢁ)-1.
Cl
O
O
b
N O
a
Br
Cl
Br
Br
Cl
HO
N
H
28
29
30
Cl
N
O
N O
d
c
S
O
SH
N
H
N
H
S
31
32
O
O
O
b
e
f
SH
Br
S
29
N
H
N
N
H
H
O
35
33
34
Scheme 5. Reagents and conditions: (a) thionyl chloride, reflux, 1 h; (b) 5-chloropyridin-2-amine, triethylamine, DCM, 0 °C to rt, 3–4 h, 44% from 28; (c) potassium O-
ethylxanthate (freshly prepared from CS2, EtOH, KOH in H2O), NaOH, H2O, overnight, without separation; (d) 1 N HCl, 80% from 30; (e) AcSK, EtOH, rt, 20 h, 18%; (f) NaOH,
H2O, EtOH, rt, 48 h, 67%.
6,8-Bis(benzoylthio)octanoic acid 4 was most effective among
compounds 1, 2, and 3 on the inhibition of NCI-460, HO-8910,
KB, and BEL-7402 cancer cells. The results quite agreed with Za-
char, Z.’s study that the threshold-killing concentration of 4 was
decreased the inhibitory activities. On the whole, these compounds
exhibited dose-dependent anticancer property, most of which
showed excellent inhibitory activities at the dose of 100 lg/mL.
Enantioselectivity was not a rare event in the screening of anti-
cancer drugs. In our results, the anticancer activities of S-enantio-
mer of lipoamide (S-(ꢁ)-17h) was about twofold stronger than the
racemic 17h against NCI-460, HO-8910 and KB cancer cell lines
and fourfold stronger against BEL-7402 cells at the concentration
60 lg/mL while the concentration of 3 reached 600 lg/mL in kill-
ing a series of cancer cell lines.13 However, comparison with
17a–20a, 17b–20b, 17c–20c, and 17d–20d, bis(benzoylthio)
amides of lipoic acid (20a–d) retained little activities. In addition,
it was difficult to draw a conclusion whether oxidized or reduced
form of the corresponding lipoamides was preferable from the re-
sults of in vitro evaluation. It seemed that the amide group might
play a more important role in anticancer efficacy. Comparing the
compounds 17a–k, it had the trends that the electron-withdrawing
groups such as NO2, CF3, Cl on the aryl ring enhanced the inhibitory
activities, and the electron-donating groups such as H, OCH3
of 100 lg/mL. The anticancer activities of its R-antipode (R-(+)-
17h) showed no improvement than racemic 17h, but possessed
twofold stronger inhibitory activity than the racemic 17h against
BEL-7402. These data underlined the different stereochemical pref-
erence in a series of cancer cell lines.
Figure 2 showed the in vitro anticancer activity of the represen-
tative compounds with or without the thiol side chain at C6

Table 1
Anticancer activities against NCI-460, HO-8910, KB, BEL-7402, and PC-3 cell linesa
Compds
Molecular
weight
Inhibition rate of NCI-460 (%)
Concentration ( g/mL)
Inhibition rate of HO-8910 (%)
Concentration ( g/mL)
Inhibition rate of KB (%)
Concentration ( g/mL)
Inhibition rate of BEL-7402 (%)
Concentration ( g/mL)
Inhibition rate of PC-3 (%)
Concentration ( g/mL)
l
l
l
l
l
100
10
1
0.1
100
10
1
0.1
100
10
1
0.1
100
10
1
0.1
100
10
1
0.1
1
2
3
4
15
16
206.3
208.3
292.4
416.6
221.3
223.4
281.4
283.5
367.5
491.7
315.9
317.9
402.0
526.1
315.9
317.9
402.0
526.1
315.9
317.9
402.0
526.1
299.4
301.4
349.4
351.5
365.4
367.5
394.4
396.4
295.5
297.5
311.5
313.5
326.4
328.5
282.4
284.4
316.9
318.9
361.3
363.3
331.5
333.5
394.4
394.4
264.3
300.1
5.72
7.84
2.39
5.02
6.04
na
4.65
12.25
9.89
0.77
0.64
1.17
5.86
4.30
13.07
5.93
10.14
8.19
13.19
14.40
8.85
7.20
0.31
9.48
10.90
6.76
15.40
9.80
11.44
6.84
4.39
8.26
12.16
19.12.
19.07
6.29
7.82
9.43
0.51
na
8.71
9.25
9.96
na
5.11
1.17
na
86.71
na
49.30
57.76
13.68
22.27
na
na
na
na
na
68.28
na
38.15
na
74.76
21.27
58.76
na
78.13
22.25
74.83
34.78
84.53
66.75
47.65
92.67
62.72
na
43.76
12.55
64.52
51.21
51.89
14.17
68.55
71.16
32.12
56.84
na
na
na
1.61
na
na
26.24
4.71
5.49
3.20
na
4.66
na
na
1.33
na
na
na
na
0.75
na
1.47
na
3.89
6.81
5.99
8.86
8.57
6.11
na
1.05
3.93
na
0.29
na
3.39
na
na
na
na
10.96
16.34
8.03
89.81
12.44
58.00
65.32
15.78
22.95
na
0.72
na
6.93
na
76.54
8.04
48.79
na
83.12
44.11
71.95
5.78
85.38
28.21
79.95
30.49
90.11
73.33
49.51
95.52
67.95
17.49
48.32
22.21
71.31
41.06
25.07
18.53
74.45
76.55
42.32
59.24
na
7.18
3.38
8.88
2.68
0.89
41.52
10.24
8.62
2.50
2.67
na
8.60
8.79
12.39
0.68
2.57
10.56
6.06
11.60
1.68
na
na
na
8.21
3.05
na
0.14
12.00
7.24
na
7.92
7.62
10.92
9.49
7.24
1.52
5.11
na
8.17
0.59
8.35
4.77
5.59
9.22
2.76
11.76
28.24
2.27
3.72
na
na
na
na
3.09
6.63
4.15
3.22
na
2.52
6.98
12.82
na
7.39
9.12
3.28
1.32
2.84
na
5.33
3.08
5.01
7.37
1.11
1.14
0.56
na
0.64
0.27
3.07
9.62
1.79
na
na
nt
nt
nt
91.24
nt
nt
59.24
nt
nt
nt
nt
nt
nt
nt
79.46
nt
nt
nt
41.17
nt
nt
nt
80.79
nt
88.74
nt
88.55
75.45
90.25
79.88
75.14
nt
nt
nt
79.58
nt
nt
nt
nt
nt
7.43
nt
nt
12.98
nt
nt
nt
nt
nt
nt
nt
13.85
nt
nt
nt
14.67
nt
nt
nt
13.85
nt
38.50
nt
38.17
42.46
na
45.52
9.57
nt
nt
nt
6.67
nt
nt
nt
nt
nt
9.86
nt
nt
13.62
nt
nt
nt
nt
nt
nt
nt
8.44
nt
nt
nt
13.66
nt
nt
nt
14.49
nt
8.44
nt
10.84
12.58
na
1.08
9.98
nt
nt
nt
19.87
nt
nt
nt
nt
nt
9.54
nt
nt
5.50
nt
nt
nt
nt
nt
nt
nt
17.06
nt
nt
nt
18.89
nt
nt
nt
15.64
nt
6.27
nt
6.69
14.94
5.37
8.06
2.13
nt
nt
nt
13.88
nt
nt
nt
2.22
na
nt
nt
10.40
10.30
1.09
10.40
5.96
3.22
0.85
na
10.97
72.80
na
43.74
47.59
25.42
44.77
na
80.97
12.48
47.52
60.57
7.67
14.13
na
4.74
na
3.90
5.70
9.48
5.50
15.39
0.60
8.00
6.36
16.58
15.13
6.08
9.78
na
15.64
12.97
na
9.89
9.36
15.64
5.64
na
11.95
7.05
17.25
18.81
3.20
7.97
9.34
3.82
5.69
11.93
5.69
2.61
5.01
3.93
na
17.73
0.75
8.37
14.24
11.44
14.64
18.22
11.43
12.10
10.14
na
2.09
9.12
18.32
21.95
14.85
12.56
18.79
18.05
6.77
11.44
20.32
29.23
11.98
0.97
4.15
7.67
9.82
1.43
22.68
13.38
na
na
na
17a
18a
19a
20a
17b
18b
19b
20b
17c
18c
19c
20c
17d
18d
19d
20d
17e
18e
17f
10.95
6.38
10.63
3.79
7.74
7.72
na
2.35
na
na
11.16
7.37
8.33
3.12
8.17
6.11
2.48
na
na
na
0.27
9.02
na
1.04
1.81
3.68
6.59
8.11
5.00
10.77
5.53
na
na
3.38
6.83
5.11
6.61
5.72
0.33
3.09
3.72
1.70
3.37
1.06
9.96
9.20
na
na
na
2.85
na
na
5.04
6.54
9.88
10.05
5.48
5.35
na
1.88
0.89
na
12.07
15.24
na
65.23
24.83
42.81
na
68.71
40.91
47.34
na
64.50
41.60
68.75
32.06
82.95
65.14
51.65
95.85
52.90
23.07
45.73
23.49
56.67
39.11
39.27
11.71
58.55
55.78
52.00
55.88
na
na
na
na
na
6.55
2.53
7.88
na
7.05
3.62
2.85
na
68.25
16.11
20.76
na
76.39
25.53
45.50
na
74.45
11.65
61.90
25.31
78.41
49.69
23.93
91.67
49.93
7.08
38.73
22.50
52.63
33.35
37.64
3.62
na
na
3.38
na
na
0.04
12.66
18.64
13.35
14.79
11.71
4.05
8.84
9.50
na
1.54
6.00
na
10.19
5.20
3.31
5.03
2.49
na
0.93
na
na
0.76
na
4.33
2.51
8.37
na
5.72
10.69
16.36
9.11
11.44
16.95
11.27
6.78
0.33
8.41
1.97
7.27
4.50
na
4.29
5.75
4.00
na
0.22
na
na
na
na
na
na
na
na
na
na
na
22.93
3.51
16.56
21.72
18.22
18.08
10.62
4.44
13.26
10.86
1.97
8.49
14.99
na
4.04
4.13
7.23
2.94
8.93
14.36
13.68
na
0.28
28.58
21.59
35.12
34.06
na
na
na
na
57.71
96.28
na
7.01
1.16
5.78
4.87
1.92
6.14
4.43
1.21
1.24
14.16
na
2.81
0.35
1.29
5.82
12.21
4.76
13.71
10.02
0.56
0.96
1.17
4.54
2.76
3.54
1.79
0.33
0.76
na
na
2.10
na
4.27
2.51
6.95
na
na
na
56.90
66.59
na
18f
1.66
0.29
7.32
7.46
0.68
10.31
11.13
7.93
3.96
na
1.73
na
na
7.44
2.34
13.08
4.71
9.55
0.69
na
17g
18g
17h
18h
17i
18i
17j
18j
na
na
na
na
6.34
4.14
0.61
8.96
na
1.95
3.68
4.92
1.64
na
na
na
1.14
na
na
2.65
na
na
0.34
0.76
na
1.06
18.06
35.23
na
9.01
14.07
6.32
10.43
4.83
14.44
5.77
na
16.06
17.82
24.46
23.17
6.27
na
17k
18k
17l
7.45
4.64
0.81
2.31
6.26
na
13.07
10.69
4.15
na
18l
na
na
nt
nt
nt
17m
18m
17n
18n
17o
18o
R-(+)-17h
S-(ꢁ)-17h
Vorinostat
DDP
33.69
27.38
38.01
39.25
na
na
na
na
54.93
84.24
27.47
21.90
29.64
29.42
na
na
na
na
63.08
97.33
48.46
50.67
45.51
41.15
7.22
na
na
na
na
3.40
na
na
na
10.75
25.47
42.33
32.37
nt
nt
nt
17.12
10.76
nt
nt
nt
2.00
0.89
nt
nt
nt
nt
na
na
9.00
6.40
2.36
na
na
na
nt
nt
na
na
2.18
na
na
na
nt
nt
na
na
35.44
92.20
61.06
87.55
na
na
40.85
45.54
39.24
92.27
69.59
94.99
49.15
95.77
64.60
95.43
na
na
51.23
71.59
43.35
94.04
65.74
86.56
na
na
54.83
88.67
59.00
79.83
IC50 6.5
92.56
na
38.75
65.84
15.12
17.99
l
Mb
81.89
52.92
na
a
Values presented are means of three experiments; values in bold are comparable to Vorinostat or DDP at the concentration; na = not active; nt = not tested.
Data taken from the literature.19
b

3082
S.-J. Zhang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3078–3083
100
90
80
70
60
50
40
30
20
10
0
thioester 34 increased the possibility of permeating the membrane
inhibition rate (%)
of cancer cells. However, bis acetyl compound 19a failed to exhibit
an admiring growth inhibitory effect as compound 34, only similar
to bis sulfhydryl compound 18a. With the hypothesis that 19a
could get through the cell membrane as efficiently as 34, the only
explanation was that the thiol side chain at C6 position impaired its
anticancer activities. Hydrolysis of compound 34 under cellular
physiological conditions could release free thiol compound 35,
which was considered as a good HDAC inhibitor with IC50 at
1.5 l
M.20,21 This might give explanation why compound 34 was
of most active against these cancer cell lines. It could be inferred
that the thiol side chain at C6 position might prohibit compounds
19a or 18a from squeezing into the pocket-shaped HDAC tunnel.22
It is worth noting that the anticancer activities of 17a exhibited
onefold higher against NCI-460, threefold higher against KB and
sevenfold higher against BEL-7402 than 18a, indicating lipoamides
with intact dithiolane ring might function through other mecha-
nisms rather than HDAC.9 As far as we know, the exact mecha-
nisms by which lipoic acid or its derivatives induce cancer cell
inhibition are still unclear.1,7,9,13,15,16,23
NCI-460
17a
18a
KB
BEL-7402
cell lines
19a
compds
35
34
32
Figure 2. Comparative profiling of compounds against cancer cell lines at 100 lg/
mL.
Table 2
Antitumor efficacy of selected compound 17m against S180 xenograft in micea
We also evaluated the in vivo antitumor activity of lipoamide
against mice S180 xenograft model. Compound 17m was selected
for the in vivo evaluation in comparison with cyclophosphamide
and Vorinostat and 0.9% saline solution was used as blank control.
All the drugs with different doses were administered once daily
through intragastric (ig) administration route for consecutive se-
ven days before animals were euthanized and the tumors were ex-
cised and weighted. The results presented in Table 2 showed that
compound 17m through ig administration of 200 mg/kg of body
weight exhibited moderate experimental therapeutic efficacy
in vivo against S180 xenograft in ICR mice by 24.7% compared with
vehicle alone. This result was lower than that of cyclophosphamide
at 74.5% but higher than Vorinostat, which showed no significant
suppression of S180 tumor growth. In general, the in vitro evalua-
tion against S180 revealed that the activity of 17m was weaker
than that of Vorinostat as shown in Table 3.
Groups
Dose
(mg/kg)
ARb
No.
TWd (g)
TWIe (%)
animalc
Control
17m
17m
Vorinostat
Vorinostat
Cyclophosphamide
/
Ig
Ig
Ig
Ig
Ig
Ig
8
6
6
6
6
6
1.53 0.56
1.53 0.59
1.15 0.18
1.61 0.29
1.53 0.40
0.39 0.15
/
/
100
200
100
200
100
24.7
/
/
*
74.5
a
Values presented are means SD. Statistical analysis: student t-test, *p <0.05
compared to control.
b
Administration route.
Number of mice tested in the group.
Average tumor-weight after drug treatment.
Percentage of tumor-weight inhibition versus control group.
c
d
e
Table 3
Compound 17m was further profiled for its acute toxicity to
mice as shown in Table 4. It should be noted that all the mice given
a single injection of 17m at the dose concentration of 1000 mg/kg
of body weight through ig administration route showed no gross
pathological changes and the mortality rate was zero, implying
the normal growth of mice and the LD50 in mice was even higher
than 1000 mg/kg. In preliminary studies, the maximum tolerated
dose (MTD) of compound 17m was determined following intra-
peritoneal (ip) dosed at 100 mg/kg per day for consecutive four
days, as defined by no morality and body weight loss less than
10% on day eight. However, when the dose was increased to
200 mg/kg per day from day nine, none were alive on day 11.
Anticancer activity against S180 cell linesa
Compds
Molecular weight
Inhibition rate of S180 (%)
Concentration ( g/mL)
l
100
10
1
0.1
17m
Vorinostat
DDP
316.9
264.3
300.1
74.28
71.70
98.30
24.51
81.68
62.25
13.03
11.91
18.49
5.92
6.34
0.52
a
Values presented are means of three experiments.
Table 4
In conclusion, these a-lipoic acid derivatives are quite promis-
ing for their low toxicity and in vivo efficacy and worth further
studying as cancer preventive or therapeutic agents.
Acute toxicity of selected compound 17m on mice
Group
Dose
ARa No.
animalb
Sex
Gross
Mortality
pathological
findings
Acknowledgments
Experimental 1000
Ig
Ig
6
Female Normal
Female Normal
0
0
mg/kg
Control
/
6
The authors thank Center of Analysis & Measurement of
Zhejiang University and Zhejiang University of Technology for
characterization of the compounds and partial financial support.
We are also grateful to Zhejiang Provincial Top Key Discipline of
Biochemical Engineering and Hangzhou Minsheng Pharmaceutical
Group Co., Ltd for financial support.
a
Administration route.
Number of mice tested in the group.
b
position and the effect of sulfhydryl group protection. Compounds
18a, 35 and 32 showed weak anticancer activities against BEL-
7402, KB and NCI-460 at 100 lg/mL. This might due to their poor
Supplementary data
cell membrane permeability resulted from the highly polar charac-
ter of the compounds with free thiols. Remarkably, masking the
sulfhydryl group of compound 35 as thioester 34 enhanced its
cancer cell growth inhibition rapidly. It stood to reason that the
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.112.

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