Synthesis of nordihydroguaiaretic acid derivatives and their bioactivities on S. pombe and K562 cell lines

Article abstract of DOI:10.1016/j.ejmech.2013.01.028

Nordihydroguaiaretic acid (NDGA) and its synthetic analogues are potentially useful in treating diseases related to cancers, diabetes, viral and bacterial infections, and inflammation. In this paper, we report the optimal synthetic methods and the bioactivity study of terameprocol 2, NDGA derivative 3, and its cyclized analogue 4. The IC₅₀ of these three compounds 2, 3 and 4 on the growth metabolism of Schizosacchromyces pombe and K562 cell lines were determined by microcalorimetry. The preliminary results showed that the compounds 2, 3 and 4 possessed good inhibition activities on S. pombe and K562 cell lines, and exhibited bidirectional biological effect and Hormesis effect. In particular, terameprocol 2 was found to possess the most potent inhibitory effect on K562 cell lines.

Full text of DOI:10.1016/j.ejmech.2013.01.028

European Journal of Medicinal Chemistry 62 (2013) 605e613
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of nordihydroguaiaretic acid derivatives and their
bioactivities on S. pombe and K562 cell lines
,
a
a
Xu Li a, Jian-Hong Jiang a, Qingqi Chen b , Sheng-Xiong Xiao , Chuan-Hua Li ,
***
**
Hui-Wen Gu c , Hui Zhang , Ji-Lin Hu , Fei-Hong Yao , Qiang-Guo Li
,
a
a
a
a,
*
a Hunan Provincial Key Laboratory of Xiangnan Rare-Precious Metals Compounds and Applications, Department of Chemistry and Life Science,
Xiangnan University, Chenzhou 423000, Hunan Province, PR China
b MedKoo Biosciences, Inc. P O Pox 16222, Chapel Hill, NC 27516-6222, USA
c State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University,
Changsha 410082, Hunan Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nordihydroguaiaretic acid (NDGA) and its synthetic analogues are potentially useful in treating diseases
related to cancers, diabetes, viral and bacterial infections, and inflammation. In this paper, we report the
optimal synthetic methods and the bioactivity study of terameprocol 2, NDGA derivative 3, and its
cyclized analogue 4. The IC50 of these three compounds 2, 3 and 4 on the growth metabolism of
Schizosacchromyces pombe and K562 cell lines were determined by microcalorimetry. The preliminary
results showed that the compounds 2, 3 and 4 possessed good inhibition activities on S. pombe and K562
cell lines, and exhibited bidirectional biological effect and Hormesis effect. In particular, terameprocol 2
was found to possess the most potent inhibitory effect on K562 cell lines.
Received 23 October 2012
Received in revised form
14 January 2013
Accepted 22 January 2013
Available online 4 February 2013
Keywords:
Nordihydroguaiaretic acid
NDGA derivatives
Microcalorimetry
Anticancer
Ó 2013 Elsevier Masson SAS. All rights reserved.
S. pombe
K562
1. Introduction
natural product. Remarkably, terameprocol 2, currently being
developed by Erimos Pharmaceuticals, is a promising anticancer
During the past century nordihydroguaiaretic acid (NDGA, 1)
(see Fig. 1), one of the naturally occurred lignins, has been an
attractive research topic due to its broad range of bioactivities and
potential medical applications [1e4] on the cardiovascular, im-
mune and neurological systems, cancer, tissue engineering, etc.
NDGA as a drug (generic name: masoprocol, trade name: ActinexÒ)
was approved by Food and Drug Administration (FDA, USA) in
September 1992. ActinexÒ is an antineoplastic drug used to treat
skin growths caused by sun exposure, which contains 10% maso-
procol or NDGA, is a topical cream product, developed by University
of Arizona Cancer Center. However, masoprocol was discontinued
in June 1996 due to its low market demand and minor side effects.
Terameprocol 2 (see Fig. 1), commonly known as tetra-O-methyl-
nordihydroguaiaretic acid, EM-1421 and M4N, is a semisynthetic
agent currently under Phase I/II clinical trials, which is the first
NDGA derivative in clinical trials. Although NDGA has been
extensively studied during the past decades, NDGA derivatives have
been less explored.
Biological microcalorimetry, providing a continuous measure-
ment of heat production, can be employed to directly determine the
biological activities of a living system. Heat flux is an expression of
overall metabolic flux, and the detection of small changes in heat
production to respond to toxic insult will be a sensitive indicator of
altered metabolism. Since microcalorimetry is a nondestructive
method with high accuracy and automaticity, it is now widely
applied in biological research [5] and pharmacological analysis [6].
In this paper we report the optimal synthetic methods and the
anticancer activities of DNGA derivative 3 (see Fig. 2), its cyclized
analogue 4 (see Fig. 2) and terameprocol 2. Their bioactivities were
determined by microcalorimetry [7e9], cytomorphology,
and fluorescence probe method. The results showed that ter-
ameprocol 2, NDGA derivative 3 and its cyclized analogue 4
exhibited good inhibitory activities on the growth metabolism of
Schizosacchromyces pombe and K562 cell lines. The values of IC50 of
* Corresponding author. Tel./fax: þ86 735 2653353.
** Corresponding author. Tel./fax: þ1 704 967 0446.
*** Corresponding author. Tel./fax: þ86 182 73153402.
E-mail addresses: qqchen@gmail.com (Q. Chen), gruyclewee@hnu.edu.cn
(H.-W. Gu), liqiangguo@163.com (Q.-G. Li).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.028

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X. Li et al. / European Journal of Medicinal Chemistry 62 (2013) 605e613
concentrations of terameprocol 2, NDGA derivative 3 and NDGA
derivative 4 were recorded on the TAM Air microcalorimeter at
32.00 ꢁC, respectively. All microcalorimetric experiments were
repeated three times. The results are illustrated in Fig. 4. From
Fig. 4, we can find that the thermogenic curves are similar to those
of S.pombe treated by different concentrations of three compounds.
2.2.1.1. The growth rate constant (k) of S. pombe cells. As shown in
Fig. 4a, the poweretime curve of growth of S. pombe cells showed
that growth of S. pombe cells could be divided into four phases, that
is, a lag phase (AB), a log phase (BC), a stationary phase (CD) and
a decline phase (DE). During the log phase, the poweretime curves
obeyed the following equation:
Fig. 1. Structure of NDGA 1 and terameprocol 2.
terameprocol 2, NDGA derivative 3 and NDGA derivative 4 on the
growth metabolism of S. pombe cell lines were found to be 330.0,
290.0 and 350.0 mg Lꢀ1, respectively. The values of IC50 of ter-
ameprocol 2, NDGA derivative 3 and NDGA derivative 4 on the
growth metabolism of K562 cell lines were found to be 5.87, 8.57
and 11.23 mg Lꢀ1, respectively. The inhibitory activity of these three
compounds on the growth metabolism of the S. pombe has
been observed to decrease in the order of NDGA derivative
3 > terameprocol 2 > NDGA derivative 4. While their inhibitory
activity on K562 cell lines was found to decrease in the order of
terameprocol 2 > NDGA derivative 3 > NDGA derivative 4.
nt ¼ n0exp½kðt ꢀ t0Þꢂ
(1)
where t was the time after the start of exponential growth phase, t0
was the start time of exponential growth phase, nt and n0 were the
cell number at time t and t0, respectively. If the power output of
each S. pombe cell was one w, then
ntw ¼ n0wexp½kðt ꢀ t0Þꢂ
If pt ¼ ntw, p0 ¼ n0w, then
(2)
2. Results and discussions
2.1. Chemistry
pt ¼ p0exp½kðt ꢀ t0Þꢂ
or
(3)
NGDA derivatives 2e4 were synthesized using our developed
procedures based on literature reports [10e12], and were outlined
as in Fig. 3.
The key step to establish the stereochemistry of terameprocol 2
was the hydrogenation of tetra-substituted furan 4 to give the cor-
responding cis-tetrahydrofuran 9. The synthesis started with vera-
trole 5, which was acylated with propionyl chloride 6 using
chloroform as solvent to give a higher yield (94%) of the corre-
sponding ketone 7. Bromination of compound 7 in refluxing
ln pt ¼ lnp0 ꢀ kt0 þ kt
(4)
where Pt was the heat output power of the S. pombe cell at time t,
and k was the growth rate constant of the S. pombe cell at specified
conditions, whose size represented growth speed. Using this
equation, the growth rate constant k could be calculated and the
results are shown in Table 1. It could be seen from Fig. 4 and Table 1
that the compounds 2, 3 and 4 possessed the bidirectional bio-
logical effect and Hormesis effect, i.e., these three compounds
stimulated the growth of S. pombe at low concentration, but
inhibited the growth of S. pombe at high concentration.
chloroform then gave a-bromo-3,4-dimethoxypropiophenone 8 in
95% yield. Alkylation of 7 in liquid ammonia and sodium amide
(produced in situ via adding sodium) at 33 ꢁC with the bromoketone
8 gave racemic diketone 3 in 93%. The synthesis of compound 9 was
also reported by Faid et al. [12], who used sodium amide (in situ
produced), ferric chloride as catalysts and liquid ammonia as sol-
vent. Cyclodehydration of 9 in 1% HCleMeOH under reflux to give
the corresponding furan compound 4, followed by hydrogenation
created the cis-tetrahydrofuran 9, which was further converted to
terameprocol 2 via catalytic hydrogenation as shown in Fig. 3.
After preparing NDGA derivatives 3, 4 and terameprocol 2, we
next evaluate their bioactivities on S.pombe and K562 cell lines
(tumor cell lines) using micocalorimetry. The microcalorimetric
study was performed on a 3116-2/3239 TAM Air isothermal calo-
rimeter (Thermometric AB, Sweden).
2.2.1.2. Inhibition ratio (I) and half inhibition concentration (IC50).
The inhibition ratio of the growth metabolism of S. pombe cells
treated by drugs was defined as following:
I ¼ ðk ꢀ kcÞ=k0 ꢃ 100%
(5)
where k0 was the control rate constant (without any drug inhibi-
tion) of S. pombe and kc was the growth rate constant of S. pombe
treated by an inhibitor with a concentration of c. The values of (I)
are shown in Table 1. When the inhibition ratio was 50%, the drug
concentration was called as the half inhibition concentration (IC50).
The results showed that terameprocol 2, NDGA derivative 3 and
NDGA derivative 4 possessed significantly inhibitory effect on the
growth metabolism of S. pombe cell lines. The values of IC50 of
terameprocol 2, NDGA derivatives 3, and NDGA derivatives 4 on the
growth metabolism of S. pombe cell lines were 330.0, 290.0 and
350.0 mg Lꢀ1, respectively. The inhibitory ability of these three
compounds above on the growth of the S. pombe has been observed
to decrease according to the following order: NDGA derivative
3 > terameprocol 2 > NDGA derivative 4. The NDGA derivative 2, 3
and 4 possessed the bidirectional biological effect and Hormesis
effect. In other words, they stimulated the growth of the S.pombe at
low concentration, but inhibited the growth at high concentration.
The thermogenic curves for the growth metabolism of K562
cell lines treated by different concentrations of the compounds
2.2. Bioactivity
2.2.1. Microcalorimetry
The thermogenic curves (poweretime curves) for the growth
metabolism of S. pombe cells [13] treated by different
Fig. 2. Structure of NDGA derivative 3 and its cyclized analogue 4.

X. Li et al. / European Journal of Medicinal Chemistry 62 (2013) 605e613
607
Fig. 3. Synthesis of terameprocol 2 through hydrogenation.
terameprocol 2, NDGA derivative 3 and NDGA derivative 4 were
determined using the TAM Air calorimeter at 37.00 ꢁC, respectively.
All microcalorimetric experiments were repeated three times. The
results were illustrated in Fig. 5, in which the log phase of ther-
mogenic curves obeyed Eq. (4). Using this equation, the growth rate
constant k could be calculated and the results were shown in
Table 2. According to Eq. (5), the inhibition ratio (I) of the growth
metabolism of K562 cells treated by the terameprocol 2, NDGA
derivative 3 and NDGA derivative 4 could be calculated and the
results were shown in Table 2.
As can be seen from Fig. 5 and Table 2 that the compound ter-
ameprocol 2, NDGA derivative 3 and NDGA derivative 4 possessed
the bidirectional biological effect and Hormesis effect, that is, they
stimulated the growth of K562 at low concentration, but inhibited
the growth of K562 at high concentration. More importantly, it was
found that terameprocol 2, NDGA derivative 3 and NDGA derivative
0.0018
0.0018
0.0016
(b)
D
(a)
C
0.0016
0.0014
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000
-0.0002
1
4
0.0014
c( C
H
O ) / m g · m L
2
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000
-0.0002
1
2
3
4
5
6
7
8
0. 00
0. 02
0. 04
0. 08
0. 16
0. 24
0. 32
0. 38
5
3
6
p
7
B
8
A
0
E
0
50000
100000
150000
200000
100000
200000
t
/ s ec
t/Sec
0.0018
0.0016
0.0014
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000
-0.0002
c( C
H O ) / mg· m L
1
2
3
0.0016
0.0014
0.0012
0.0010
0.0008
0.0006
0.0004
0.0002
0.0000
-0.0002
1
2
3
4
5
6
7
8
0. 00
0. 02
0. 04
0. 08
0. 16
0. 24
0. 32
0. 40
(c)
(d)
3
2
1
4
4
c( C
H
O ) / mg· m L
1
2
3
4
5
6
7
8
0. 00
0. 02
0. 04
0. 08
0. 16
0. 24
0. 32
0. 40
5
5
6
p
p
6
7
7
8
8
0
50000
100000
150000
200000
0
50000
100000
150000
200000
t / s ec
t / s ec
Fig. 4. Metabolism thermogenic curves of S. pombe cells affected by the compounds at 32.00 ꢁC. (a): For control; (b): For terameprocol 2; (c): For NDGA derivative 3; (d): For NDGA
derivative 4.

608
X. Li et al. / European Journal of Medicinal Chemistry 62 (2013) 605e613
Table 1
Thermokinetic parameters of the growth of S. pombe affected by inhibitors at different concentrations at 32.00 ꢁC.
Inhibitors
ca/mg ,Lꢀ1
kb/minꢀ1
Rc
Id
IC50e/mg ,Lꢀ1
Terameprocol 2
0.00
0.00443 ꢄ 3.7723 ꢃ 10ꢀ6f
0.00401 ꢄ 2.391 ꢃ 10ꢀ6
0.00407 ꢄ 2.7923 ꢃ 10ꢀ6
0.00425 ꢄ 2.1328 ꢃ 10ꢀ6
0.00392 ꢄ 1.4008 ꢃ 10ꢀ6
0.00322 ꢄ 1.2102 ꢃ 10ꢀ6
0.00245 ꢄ 1.2343 ꢃ 10ꢀ6
0.00143 ꢄ 4.8528 ꢃ 10ꢀ7
0.9987
0.9995
0.9991
0.9995
0.9997
0.9997
0.9993
0.9997
0.00
9.48
8.13
4.06
11.51
27.31
44.70
67.72
330.0
20.00
40.00
80.00
160.0
240.0
320.0
380.0
NDGA derivative 3
0.00
0.00444 ꢄ 4.7937 ꢃ 10ꢀ6
0.00479 ꢄ 5.1510 ꢃ 10ꢀ6
0.00462 ꢄ 6.3506 ꢃ 10ꢀ6
0.00460 ꢄ 4.7560 ꢃ 10ꢀ6
0.00421 ꢄ 3.0529 ꢃ 10ꢀ6
0.00341 ꢄ 2.0422 ꢃ 10ꢀ6
0.00163 ꢄ 8.4427 ꢃ 10ꢀ7
0.00075 ꢄ 3.6659 ꢃ 10ꢀ7
0.9980
0.9980
0.9973
0.9977
0.9988
0.9991
0.9991
0.9990
0.00
ꢀ7.88
ꢀ4.05
ꢀ3.60
5.18
23.20
63.29
83.00
290.0
20.00
40.00
80.00
160.0
240.0
320.0
400.0
NDGA derivative 4
0.00
0.00422 ꢄ 1.3357 ꢃ 10ꢀ6
0.00427 ꢄ 1.7166 ꢃ 10ꢀ6
0.00429 ꢄ 1.8595 ꢃ 10ꢀ6
0.00431 ꢄ 2.1920 ꢃ 10ꢀ6
0.00430 ꢄ 2.3583 ꢃ 10ꢀ6
0.00346 ꢄ 2.1549 ꢃ 10ꢀ6
0.00273 ꢄ 2.7096 ꢃ 10ꢀ6
0.00113 ꢄ 5.7325 ꢃ 10ꢀ6
0.9998
0.9996
0.9996
0.9994
0.9994
0.9991
0.9983
0.9990
0.00
ꢀ1.18
ꢀ1.66
ꢀ2.13
ꢀ1.90
18.00
35.31
73.22
350.0
20.00
40.00
80.00
160.0
240.0
320.0
400.0
a
The concentration.
The growth rate constant of K562.
The correlation coefficient.
The inhibitive ratio.
b
c
d
e
The half inhibition concentration.
f
Mean ꢄ S.D.; n ¼ 3.
4 possessed better inhibitory activities on the growth metabolism
of K562 than S. pombe cell lines. That is because the values of IC50 of
terameprocol 2, NDGA derivative 3 and NDGA derivative 4 on the
growth metabolism of K562 cell lines were found to be 5.87, 8.57
and 11.23 mg Lꢀ1, respectively, which is much smaller than those of
terameprocol 2, NDGA derivative 3 and NDGA derivative 4 on the
growth metabolism of S. pombe cell lines. The inhibitory ability of
these three compounds above on the growth of K562 has been
0.00009
1
0.00008
0.00009
0.00008
0.00007
0.00007
3
0.00006
0.00005
D
0.00006
0.00005
0.00004
0.00003
0.00002
0.00001
C
4
0.00004
2
E
6
A
B
0.00003
0.00002
7
5
0.00001
0
5 0 00 0
1 0 0 0 0 0
1 5 0 00 0
2 00 0 0 0
2 5 0 0 0 0
0
50000 100000
200000 250000
150000
t/sec
t/sec
(a)
(b)
0.00008
0.00007
0.00006
0.00005
0.00004
0.00003
0.00002
0.00001
0.00005
0.00004
0.00003
0.00002
0.00001
0.00000
1
1
2
3
7
4
3
6
6
5
7
2
4
5
0
50000 100000 150000 200000 250000
0
50000
100000 150000
200000 250000
(c)
(d)
Fig. 5. Metabolism thermogenic curves of K562 cells affected by the compounds at 37.00 ꢁC. (a): For control; (b): For terameprocol 2; (c): For NDGA derivative 3; (d): For NDGA
derivative 4.

X. Li et al. / European Journal of Medicinal Chemistry 62 (2013) 605e613
609
Table 2
Thermokinetic parameters of the growth of K562 affected by inhibitors at different concentrations at 37.00 ꢁC.
Inhibitors
ca/mg Lꢀ1
kb/minꢀ1
Rc
Id
IC50e/mg Lꢀ1
Terameprocol 2
0.00
2.00
3.00
4.00
5.00
6.00
8.00
4.1881 ꢃ 10ꢀ4 ꢄ 1.00210 ꢃ 10ꢀ6f
4.7239 ꢃ 10ꢀ4 ꢄ 7.1697 ꢃ 10ꢀ7
4.4342 ꢃ 10ꢀ4 ꢄ 7.5337 ꢃ 10ꢀ7
3.9762 ꢃ 10ꢀ4 ꢄ 6.0102 ꢃ 10ꢀ7
2.7992 ꢃ 10ꢀ4 ꢄ 3.0152 ꢃ 10ꢀ7
1.9712 ꢃ 10ꢀ4 ꢄ 3.2544 ꢃ 10ꢀ7
8.5275 ꢃ 10ꢀ5 ꢄ 4.3549 ꢃ 10ꢀ7
0.9932
0.9952
0.9954
0.9961
0.9967
0.9934
0.9375
0.00
5.87
ꢀ12.79
ꢀ5.88
5.06
33.16
52.93
79.64
NDGA derivative 3
0.00
4.00
6.00
7.00
8.00
9.00
11.00
3.9397 ꢃ 10ꢀ4 ꢄ 7.3096 ꢃ 10ꢀ7
4.4893 ꢃ 10ꢀ4 ꢄ 4.9357 ꢃ 10ꢀ7
3.9703 ꢃ 10ꢀ4 ꢄ 7.2831 ꢃ 10ꢀ7
3.6261 ꢃ 10ꢀ4 ꢄ 9.4969 ꢃ 10ꢀ7
2.2866 ꢃ 10ꢀ4 ꢄ 4.9898 ꢃ 10ꢀ7
1.7435 ꢃ 10ꢀ4 ꢄ 8.4754 ꢃ 10ꢀ6
1.6810 ꢃ 10ꢀ4 ꢄ 5.3231 ꢃ 10ꢀ7
0.9911
0.9945
0.9922
0.9948
0.9953
0.8884
0.9442
0.00
ꢀ13.95
ꢀ0.78
7.96
41.96
55.75
57.33
8.57
NDGA derivative 4
0.00
2.00
4.00
6.00
8.00
3.8465 ꢃ 10ꢀ4 ꢄ 7.4564 ꢃ 10ꢀ7
3.9155 ꢃ 10ꢀ4 ꢄ 3.7194 ꢃ 10ꢀ7
4.3527 ꢃ 10ꢀ4 ꢄ 8.7860 ꢃ 10ꢀ7
3.1202 ꢃ 10ꢀ4 ꢄ 4.4618 ꢃ 10ꢀ7
3.0855 ꢃ 10ꢀ4 ꢄ 4.9220 ꢃ 10ꢀ7
1.6500 ꢃ 10ꢀ4 ꢄ 4.4866 ꢃ 10ꢀ7
7.6784 ꢃ 10ꢀ5 ꢄ 5.8538 ꢃ 10ꢀ7
0.9946
0.9941
0.9938
0.9972
0.9959
0.9806
0.8605
0.00
ꢀ1.79
ꢀ13.16
18.88
19.78
57.10
80.04
11.23
12.00
14.00
a
The concentration.
b
The growth rate constant of K562.
The correlation coefficient.
The inhibitive ratio.
c
d
e
f
The half inhibition concentration.
Mean ꢄ S.D.; n ¼ 3.
observed to decrease in the order of terameprocol 2 > NDGA
derivative 3 > NDGA derivative 4.
necrosis and rupture of membrane happened in partial cell. Cell
membrane structure disappeared, and cytoplasm filled by a large
number of lipid droplets. Cell surface extended long protrusions.
All of these observations confirmed that there were late apoptotic
changes in cell membrane structure. Furthermore, the treated cells
stained using PI (propidium iodide) (Fig. 6c) showed that the cell
nucleus changed red, which demonstrated the late apoptotic
changes.
2.2.2. Pharmacological mechanisms
The results of the stained K562 cells were illustrated in Fig. 6.
The experimental results showed that cells of control group, i.e.
normal cells without any drugs inhibition (Fig. 6a) were round,
varied in size and had some big spherical protrusions and a small
amount number of microvilli on the surface. At the same time,
there existed some notches on their nuclear surface. In addition,
intracytoplasmic mitochondrial showed some small tubes with
thick crest and abundant free mitochondrial ribosomes. After K562
cells were treated by the terameprocol 2 (Fig. 6b), it was found that
the cell membrane structure changed. It was further found that,
the nuclear size became smaller, the chromatin gathered under
nuclear envelope, and the nuclear membrane structure was clear.
Moreover, cell membrane structure showed early apoptotic, and
The results of mitochondrial transmembrane potential of K562
cells were shown in Table 3.
The above experimental results showed that the mitochondrial
transmembrane potential of K562 cells treated by drugs decreased
significantly comparing with the control group. The potentials of
high concentration groups were smaller than those of low con-
centration groups, and were found to be decreased significantly as
time went on. The difference between groups had statistical sig-
nificance by t-test.
Fig. 6. Morphological change of K562 cells: (a) Control group; (b) treated by terameprocol 2; (c) the PI staining of the cells treated by terameprocol 2.

610
X. Li et al. / European Journal of Medicinal Chemistry 62 (2013) 605e613
Table 3
4-necked round bottom flask (equipped with N2 inlet, overhead
magnetic stir drive, addition funnel, thermocouple, and condenser)
was added aluminum chloride (349 g, 2.61 mol) followed by CH2Cl2
(870 mL). The suspension was cooled to ꢀ10 ꢁC using a dry ice/
acetone bath. Propionyl chloride (138 g, 1.49 mol) in CH2Cl2
(145 mL) was added in portions via addition funnel over 15 min.
Keeping the mixture between ꢀ2 ꢁC and 2 ꢁC, the mixture was
stirred for an additional 10 min. Veratrole (170 g, 1.23 mol) in
CH2Cl2 (100 mL) was added via the addition funnel over 20 min, the
reaction mixture was kept under the temperature between ꢀ4 ꢁC
and 1 ꢁC. After 5 min, TLC showed complete consumption of
starting material (SiO2, 1:1 EtOAceheptane, UV, veratrole Rf ¼ 0.54,
propiophenone Rf ¼ 0.42). The reaction was cooled to ꢀ10 ꢁC and
hydrochloric acid (3 M, 2 L) was slowly and cautiously added over
25 min while keeping the reaction mixture between ꢀ1 ꢁC and
16 ꢁC. The phases were separated and the aqueous phase was
extracted once with CH2Cl2 (500 mL). The combined CH2Cl2 was
washed with NaOH (3 M, 1 L), dried over MgSO4 (34.5 g), and then
concentrated in vacuo to give a viscous oil. The oil was dissolved in
hot MeOH (300 mL) and the solution was held at 0e5 ꢁC for 16 h.
The resulting white solids were broken up with spatula and vac-
uum filtered. The filter cake was washed with heptane (125 mL)
and dried on the funnel (179.9 g). A second crop of solids was
obtained by concentrating the mother liquor, diluting with MeOH
and holding at 0e5 ꢁC for 3 h (15 g). The crop 1 and 2 solids were
combined and dried in a vacuum oven (30 ꢁC, 18 h) to give pro-
piophenone 7 as a white solid (192 g, 80.5% yield, purity >98% as
Mitochondrial transmembrane potential of K562 cells affected by terameprocol 2 at
different concentrations ðx ꢄ s; n ¼ 3Þ.
Drug
concentration mg/L
R h123(%)
12 h
18 h
24 h
Control
4.0
6.0
668.2 ꢄ 0.8
597.2 ꢄ 0.6
477.8 ꢄ 2.8a
788.8 ꢄ 1.0
615.3 ꢄ 1.5a
331.5 ꢄ 2.9a
277.9 ꢄ 1.4
168.0 ꢄ 2.3a
170.7 ꢄ 1.2a
a
Compared with control group p < 0.01.
Mitochondria are one of the most important organelles for
cells. It is well known that mitochondria are apoptosis receptors
and amplifier of cell survival in many physiological or patho-
logical situations. Thus, mitochondria play a very important role
in cell survival [14]. The permeability transition pores of mito-
chondria located in the junction of outer and inner membranes
of mitochondria, which is formed by a variety of protein com-
ponents. It is because of the opening of the permeability tran-
sition pores that a series of phenomenon such as the decrease or
disappearance of the potential of the mitochondrial membrane,
the swelling of mitochondrial, the rupturing of outer membrane,
the activation of caspases, and the releasing of cytochrome
occurred. It is indicated that mitochondria participated in the
apoptosis of cells and some important events in the apoptotic
cascade [15].
determined by HPLC). 1H NMR (CDCl3, 400 MHz)
d: 1.21 (t, 3H,
3. Conclusions
J ¼ 7.3 Hz), 2.96 (q, 2H, J ¼ 7.3 Hz), 3.93 (s, 3H), 3.94 (s, 3H), 6.88 (d,
In this paper, we report the synthesis and characterization of
terameprocol 2, NDGA derivative 3 and its cyclized analogue 4.
Their bioactivities on S. pombe and K562 cell lines have been also
determined by microcalorimetry. The preliminary results showed
that the values of IC50 of terameprocol 2, NDGA derivative 3 and
NDGA derivative 4 on the growth metabolism of S. pombe cell lines
were 330.0, 290.0 and 350.0 mg Lꢀ1, respectively; the values of IC50
of terameprocol 2, NDGA derivative 3 and NDGA derivative 4 on the
growth metabolism of K562 cell lines were 5.87, 8.57 and
11.23 mg Lꢀ1, respectively. The inhibitory activity of these com-
pounds on the growth of the S. pombe has been observed to
decrease in the order of NDGA derivative 3 > terameprocol
2 > NDGA derivative 4. While their inhibitory activity on K562 cell
lines was found to decrease according to the order: terameprocol
2 > NDGA derivative 3 > NDGA derivative 4. The compounds 2, 3
and 4 possessed the bidirectional biological effect and Hormesis
effect. They stimulated the growth of the S. pombe and K562 at low
concentration, but inhibited the growth of S. pombe and K562 at
high concentration.
The experimental has substantiated elementarily that ter-
ameprocol 2 possessed the action of inducing cancer cell-apoptosis,
reducing mRNA levels and subsequent protein production of the
cyclin-dependent kinase CDC2, resulting in the inactivation of the
CDC2/cyclin B complex (maturation promoting factor) [16]. The
antitumor activities of terameprocol 2 may be based on selective
inhibition of specificity protein 1 (Sp1)-regulated proteins, includ-
ing VEGF, survivin and cdc2 that control cell cycle and apoptosis
[17e19].
1H, J ¼ 8.3 Hz), 7.54 (d, 1H, J ¼ 1.6 Hz), 7.58 (dd, 1H, J ¼ 8.3, 1.6 Hz).
13C NMR (CDCl3, 100 MHz)
d: 8.27, 30.98, 55.64, 55.73, 109.74,
109.83, 122.24, 129.87, 148.69, 152.79, 199.10. LC/MS (m/z ¼ 194.8).
4.1.2. 2-Bromo-3,4-dimethoxypropiophenone (8)
This compound was prepared by a modified procedure based on
the method of Perry et al. [10], as outlined in Fig. 3. A 3 L, 3-neck
round bottom flask was fitted with an addition funnel, overhead
magnetic stir, thermocouple, N2 inlet and condenser. The con-
denser was vented into a base trap containing NaOH (50.2 g,
1.26 mol) in 1.8 L deionized water. The vessel was charged with
propiophenone 7 (241.55 g,1.25 mol) and chloroform (900 mL). The
mixture was heated to 62e64 ꢁC. To the refluxing solution was
added a solution of bromine (203.9 g, 1.27 mol, in 300 mL chloro-
form) via the addition funnel over 35 min. The reaction mixture
was vigorously stirring during addition. After addition, the mixture
was vigorously stirred for 20 min and cooled to 20 ꢁC. The solvent
was removed in vacuo and the resulting solids were dissolved in
CHCl3 (250 mL) and MeOH (625 mL). The solution was con-
centrated until solids formed and the slurry was cooled to 0e5 ꢁC
hand held for 10 min. The slurry was vacuum filtered on a 2 L fritted
funnel (medium frit), and the filter cake was washed with cold
MeOH (2 ꢃ 50 mL). The solids were dried in a vacuum oven (35 ꢁC,
15 h) to give 228 g
obtained by concentrating the mother liquor to a solid, and crys-
tallizing from hot MeOH (300 mL) to give an additional 58 g
bromoketone 8 (17.3% yield). 1H NMR (CDCl3, 400 MHz)
: 1.90 (d,
a-bromoketone 8 (67% yield). A second crop was
a
-
d
3H, J ¼ 6.7 Hz), 3.95 (s, 3H), 3.96 (s, 3H), 5.29 (q, 1H, J ¼ 6.7 Hz), 6.91
(d, 1H, J ¼ 8.4 Hz), 7.59 (d, 1H, J ¼ 2.0 Hz), 7.66 (dd, 1H, J ¼ 8.4,
4. Experiments
J ¼ 2.0 Hz). 13C NMR (CDCl3, 100 MHz)
d: 20.23, 41.13, 55.90, 109.96,
111.03, 123.35, 126.85, 149.10, 153.69, 191.94. LC/MS (m/z ¼ 274.8).
4.1. Synthesis
4.1.3. 1,4-Bis(3,4-dimethoxyphenyl)-2,3-dimethylbutane-1,4-dione (3)
As outlined in Fig. 3, to a dry 5 L, 3-necked round bottom flask
(equipped with an overhead magnetic stir drive, addition funnel,
thermocouple and N2 inlet and outlet) was added solid 97% t-BuOK
4.1.1. 3,4-Dimethoxypropiophenone (7)
This compound was prepared by a modified procedure based on
the method of Perry et al. [10] as outlined in Fig. 3. To a 5 L,

X. Li et al. / European Journal of Medicinal Chemistry 62 (2013) 605e613
611
(103 g, 898.5 mmol, corrected for purity), followed by THF (615 mL).
The solution was cooled to 0e1 ꢁC with an ice-water bath. A solu-
tion of propiophenone 7 (170 g, 876.3 mmol) in THF (340 mL) was
added in portions over 15 min while keeping the internal temper-
ature <7 ꢁC giving a white/yellow-white slurry. After 15 min the
mixture was warmed to 18 ꢁC and DMF (850 mL) was added via the
addition funnel over 2 min giving a clear yellow/orange solution.
After 15 min, the reaction mixture was cooled to ꢀ70 ꢁC using a dry
completely dried (no weight loss between drying turns), which
gave 2194 g of furan 4 (91% yield, purity>98% as determined by
HPLC). 1H NMR (CDCl3, 400 MHz)
d: 2.22 (s, 6H), 3.92 (s, 6H), 3.95 (s,
6H), 6.94 (d, 2H, J ¼ 6.9 Hz), 7.24e719 (m, 4H). 13C NMR (CDCl3,
100 MHz) d: 9.82, 55.87, 55.90, 109.14, 111.25, 117.77, 118.36, 125.06,
146.86, 148.00, 148.92. LC/MS (m/z ¼ 368.8).
4.1.5. Terameprocol (2)
ice/acetone bath. With vigorous stirring, a solution of
a
-bromoke-
Terameprocol 2 is anticancer drug candidate currently in clinical
trials. Large quantity of terameprocol 2 may be needed in the
future. We were interested in exploring synthetic process which
would allow us to effectively produce terameprocol 2 at kilograms
per batch since methods reported in the literature [10] was not
scalable in our hand. By using different catalysts, solvents, reaction
temperatures, reaction times, and hydrogen pressures, we were
able to develop three optimized procedures (methods A, B and C),
which were scalable, reproducible and could produce terameprocol
2 at relatively large quantity.
tone 8 (240 g, 876.3 mmol) in 2:1 THF-DMF (510 mL) was added in
portions over 25 min while maintaining an internal temperature
between ꢀ60 ꢁC and ꢀ55 ꢁC. After an additional 15 min at ꢀ60 ꢁC,
the reaction was complete as determined by LC/MS analysis. The
reaction was quenched at ꢀ60 ꢁC with water (900 mL) containing
70 mL 1 M HCl and the reaction was warmed to 18e20 ꢁC over 1 h.
The bulk of THF was removed in vacuo (1415 mL solvent removed)
and the resulting mixture was extracted with CH2Cl2 (1.5 L). The
organic layer was separated and the aqueous layer (pH 2e3) was
back extracted twice with CH2Cl2 (2 ꢃ 400 mL). The combined
CH2Cl2 was washed with water (425 mL). The bulk of the CH2Cl2
(1550 mL) was removed in vacuo to give crude product 3, which was
further purified from methanol to give the expected product 321.7 g
(95% yield, purity>98% as determined by HPLC). 1H NMR (CDCl3,
4.1.5.1. Method A. To an 8 L hydrogenator, equipped with an over-
head magnetic stir drive and heating mantle, were charged a finely
divided mixture of 78.65 g (32.6 mmol) 10% Pd-C catalyst (10R39,
55.9% wet) catalyst (available through JohnsoneMatthey Inc) and
11.55 g (5.4 mmol) Degussa E101 10% Pd/C (50% wet) catalyst
(available from SigmaeAldrich Inc.). To the vessel was charged n-
propyl acetate (3.74 L) and the vessel was pressurized to 800 psi
with H2. The mixture was stirred at the maximum stir speed at
room temperature for 30 min to 1 h. The vessel was vented to at-
mospheric pressure, the lid opened under N2 atmosphere and
slurry of furan compound 4 (400 g, 108 mmol) in n-propyl acetate
(1.86 L was charged to the vessel). The mixture was heated to
100 ꢁC under 1000 psi H2 pressure at maximum stir speed. The
reaction was monitored by HPLC until all furan 4 is consumed and
less than 2% THF intermediate 9 is present. The reaction mixture
was cooled to 20e25 ꢁC and the vessel was vented to atmospheric
pressure. The reactor lid was removed and the reaction mixture was
sparged with N2. Immediately, the reaction mixture was filtered
through a bed of CeliteÒ 545 filter material (800 g) and the CeliteÒ
filter material cake was washed with n-propyl acetate (4 L). The
combined n-propyl acetate filtrate was washed with water (2 L),
5 wt % aqueous potassium carbonate solution (2 L) and brine (2 L).
The organic stream was dried over Na2SO4 (400 g), filtered, and
then solvent was removed in vacuo at 50 ꢁC. The resulting residue
was diluted with heptane (2 L) and solvent was removed in vacuo at
50 ꢁC. The resulting solids were suspended in 15% (v/v) IPAe
heptane (1.6 L), heated to 50e60 ꢁC and cooled to 20 ꢁC over 1 h.
The slurry was agitated for 1 h at 20 ꢁC and vacuum filter (up to
18 h). The crude solids were transferred (289 g) to a 2 L vessel,
equipped with an overhead stir drive, condenser and heating
mantle, and 15% (v/v) IPAeheptane (578 mL) was added. The
mixture was heated to 65 ꢁC until the slurry thinned (about 5 min)
and the mixture was allowed to cool to 15 ꢁC over 3.5 h. The slurry
was vacuumed and the cake was washed with chilled (5e10 ꢁC) 15%
(v/v) IPAeheptane (300 mL). The solids were dried in vacuo to
constant weight (217 g, 55.9% yield, purity > 99% as determined by
400 MHz)
d
: 1.31 (d, J ¼ 7.1 Hz, 2CH3, 6H), 3.55 (m, 2H), 3.85 (s,
4OCH3, 12H), 6.98 (d, 2H, J ¼ 6.9 Hz), 7.24e719 (m, 4H). 13C NMR
(CDCl3, 100 MHz) d: 11.82, 42.11, 58.87, 110.14,110.23, 131.33, 149.44,
150.11, 198.22. LC/MS (m/z ¼ 386.4).
4.1.4. 2,5-Bis(3,4-dimethoxyphenyl)-3,4-dimethylfuran (4)
To a dry 50 L, 3-necked round bottom flask (equipped with
a mechanical stirrer, addition funnel, thermocouple and N2 inlet
and outlet) was added solid 97% t-BuOK (774.8 g, 6.56 mol, cor-
rected for purity), followed by THF (4.60 L). The solution was cooled
to 0e4 ꢁC with an ice-water bath. A solution of propiophenone 7
(1.243 kg, 6.40 mol) in THF (2.50 L) was added in portions over
60 min while keeping the internal temperature <7 ꢁC giving
a white/yellow-white slurry. After 15 min the mixture was warmed
to 10 ꢁC and DMF (6.20 L) was added via the addition funnel over
15 min giving a clear yellow/orange solution. After 15 min, the re-
action mixture was cooled to ꢀ70 ꢁC using a dry ice/acetone bath.
With vigorous stirring, a solution of
a-bromoketone 8 (1.748 kg,
6.4 mol) in a solution of 2:1 THF-DMF (THF: 2.5 L; DMF: 1.25 L) was
added in portions over 90 min while maintaining an internal
temperature between ꢀ60 ꢁC and ꢀ55 ꢁC. After an additional
15 min at ꢀ60 ꢁC, the reaction was complete as determined by LC/
MS analysis. The reaction was quenched at ꢀ60 ꢁC with water
(8.60 L) containing 1M HCl 510 mL and the reaction were warmed
to 18e20 ꢁC over 1 h. The bulk of THF was removed in vacuo
(9000 mL solvent removed) and the resulting mixture was extrac-
ted with CH2Cl2 (5.6 L). The organic layer was separated and the
aqueous layer (pH 2e3) was back extracted twice with CH2Cl2
(2 ꢃ 7.6 L). The combined CH2Cl2 was washed with water (3.5 L).
The bulk of the CH2Cl2 (8000 mL) was removed in vacuo and was
transferred to a 3-neck round bottom flask equipped with me-
chanical stirrer, addition funnel and condenser. The resulting so-
lution was heated to reflux (44 ꢁC) and a solution of 3% HCl in
MeOH (prepared by adding 460 mL acetyl chloride to 8000 mL
methanol) was added in a steady stream over a period of 90 min.
Solids precipitated within 15e20 min. Reflux was continued (54 ꢁC)
for 5 h and the mixture was cooled to 0e2 ꢁC over 2 h. The solids
were vacuum filtered and the filter cake was washed with MeOH
(2920 mL), then heptane (2920 mL). The white solids were dried in
a vacuum oven (20 h, 50 ꢁC), then the solid was stirred and crushed
to break down larger pieces. The solid was dried in a vacuum oven
(20 h, 50 ꢁC). The procedure was repeated 3 times till solid was
GC). 1H NMR (CDCl3, 400 MHz)
d
: 0.85 (d, 6H, J ¼ 6.6 Hz), 1.83e1.92
(m, 2H), 2.30 (dd, 2H, J ¼ 9.3, 13.5 Hz), 2.76 (dd, 2H, J ¼ 5.0, 13.5 Hz),
3.85 (s, 6H), 3.86 (s, 6H), 6.65 (d, 2H, J ¼ 2.0 Hz), 6.70 (dd, 2H, J ¼ 8.0,
2.0 Hz), 6.79 (d, 2H, J ¼ 8.0 Hz). 13C NMR (CDCl3, 200 MHz)
d: 16.19,
38.80, 39.14, 55.76, 55.86, 110.99, 112.22, 120.90, 134.42, 147.02,
148.68. LCMS (m/z ¼ 358.9).
4.1.5.2. Method B. Furan compound 4 (400 g) was hydrogenated in
n-propyl acetate using 10R39 catalyst (2.5 mol%) and E101 NE/W
GG (0.5 mol%) at 100 ꢁC. After pre-reducing the catalyst mixture at

612
X. Li et al. / European Journal of Medicinal Chemistry 62 (2013) 605e613
Table 4
Results of hydrogenation of furan 4 under various pressure and time.
No.
Time (h)
T (ꢁC)
P (psi)
Furan 4 (%)
Compound 9 (%)
Terameprocol 2 (%)
Other impurity (%)
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
100
100
100
100
100
23
23
100
100
960
980
980
980
980
980e600
600
950
840
NA
18.2
0.0
0.0
0.0
NA
NA
NA
29.5
24.7
17.1
11.8
38.4
55.4
61.2
64.7
13.9
19.9
21.7
23.5
6e18
18
21
22
Held without stirring, water added at t ¼ 18 h
0.0
0.0
0.0
0.0
9.5
4.3
2.8
1.2
64.1
67.9
67.1
98.2
26.4
27.8
30.1
0.6
Isolated product (46.6% yield)
room temperature, the reactor lid was opened and substrate was
added as a solid. The mixture was heated under H2 pressure and
monitored by HPLC. The reaction scheme is shown in Fig. 3 and the
reaction profile is shown in Table 4. Within 3 h, the furan 4 was
completely converted to THF intermediate 9 and products (Table 4,
entry 3). The amount of THF intermediate 9 steadily decreased over
the next 2 h (Table 4, entries 4e5), and the mixture was cooled to
room temperature and held for 12 h (Table 4, entry 6). Water (10%
by volume of n-PrOAc) was then added and the mixture was heated
under H2 pressure. After an additional 4 h of heating under H2
pressure, HPLC analysis showed low levels of THF intermediate 9
(Table 4, entry 9).
monitored to ensure consistent pressure (1000 psi) during the
course of the reaction. The results for this run, using 3.0 mol%
10R39 catalyst and 0.5 mol% Degussa E101 catalyst, monitored by
HPLC, are shown in Table 5. After 3 h, complete consumption of
furan 4 was observed by HPLC (Table 5, entry 1). Within the next
5 h, the THF intermediate 9 was steadily converted to products
(Table 5, entries 2e4). After 8 h, the reaction mixture was cooled to
room temperature and immediately filtered through CeliteÒ filter
material. The organic stream was held overnight at room
temperature.
After the overnight hold, the organic stream was concentrated
under vacuum till dryness to give waxy solids, which were then
slurried in 15% IPAeheptane (4 mL/g input) at 55 ꢁC for 15 min and
the resulting uniform slurry was cooled to 20 ꢁC over 1 h. After an
additional 1 h of stirring at 20 ꢁC, the slurry was filtered. Instead of
immediately washing the cake with heptane, the cake was
allowed to dry on the vacuum funnel. The resulting crude cake
(289.58 g), was transferred to a vessel (2 L) and suspended in 15%
IPAeheptane (2 mL/g). The suspension was heated to 65 ꢁC and
held until the slurry thinned (5 min). The slurry was cooled to
15 ꢁC over 4 h, vacuum filtered, then washed once with chilled 15%
IPAeheptane (1 mL/g, cooled to 5e10 ꢁC). After drying in vacuo at
70 ꢁC, terameprocol was isolated in 55.9% yield, with 99.0% purity
(by HPLC). Analytical data were identical with that shown in
Method A.
After filtering the mixture through CeliteÒ filter material and
washing the filter cake with additional n-propyl acetate, the
product stream underwent aqueous work-up. The solvent was
evaporated and the residual n-propyl acetate was chased with
heptane. Terameprocol was then crystallized from heptane. The
resulting sticky solids were difficult to manipulate and a spatula
was used to free the solids from the walls of the vessel. After fil-
tration, three heptane washes were used to remove the residual
cyclized impurity 48 from terameprocol. After drying in vacuo at
50 ꢁC, terameprocol was isolated in 46.6% yield, with 98.2% purity
(by HPLC) (Table 4, entry 10). Analytical data were identical with
that shown in Method A.
4.1.5.3. Method C. In this reaction, several modifications based on
the results in entries 6e9 were implemented in order to optimize
the yield and purity of terameprocol 2. First, to avoid a slower
conversion, consistent H2 pressure was maintained at 1000 psi
during the reaction. Second, to improve conversion of starting
material to products, the loading of 10R39 catalyst was increased
from 2.5 mol% to 3 mol%. Third, in order to avoid unnecessary losses
due to either product absorption onto carbon or decomposition
under acidic conditions, the reaction mixture was filtered through
CeliteÒ filter material immediately upon reaction completion.
Finally, to avoid losses during product isolation the product was
initially isolated from 15% IPAeheptane, then reslurried with IPAe
heptane instead of washing several times with heptane.
4.2. Determination of bioactivity
4.2.1. Cell lines and culture conditions
S. pombe (ACCC 20047) was provided by Agricultural Culture
Collection of China. The Edinburgh minimal medium (YES) culture
composition was 5 g/L yeast extract, 30 g/L glucose, 225 mg/L
adenine, histidine, leucine, uracil and lysine hydrochloride. The
yeast extract medium composition was dissolved by 100 mL dis-
tilled water and constant volume to 1000 mL (natural pH).
K562 (3115CNCB00237) was provided by China Center for Type
Culture Collection. The complete growth medium: RPMI 1640
medium with 2 mM
L-glutamine adjusted to contain 1.5 g/L sodium
After pre-reducing the catalyst, the vessel was opened and
a slurry of the furan compound 4 (400 g) in n-propyl acetate was
added. The mixture was heated and the pressure was carefully
bicarbonate, 4.5 g/L glucose, 10 mM HEPES and 1.0 mM sodium
pyruvate 90%, fetal bovine serum 10%, 100
100 g/mL Penicillin.
mg/mL streptomycin,
m
Table 5
Results of hydrogenation of furan 4 under various reaction times.
No.
Time (h)
T (ꢁC)
P (psi)
Furan 4 (%)
Compound 9 (%)
Terameprocol 2 (%)
Other impurity (%)
1
2
3
4
5
3
5
7
8
100
100
100
100
1000
1000
1000
1000
0.0
0.0
0.0
0.0
0.0
21.4
5.6
2.8
1.2
0.0
58.5
69.1
70.2
70.6
99.0
20.1
25.3
26.9
28.2
0.5
Isolated product (55.9% yield)

X. Li et al. / European Journal of Medicinal Chemistry 62 (2013) 605e613
613
Fig. 7. TAM air isothermal calorimeter. (a) The outside drawing. (b) The schematic diagram. 1: Air thermostat; 2: calorimeter unit; 3: fan; 4: temperature sensor; 5: Peltier
temperature control module.
4.2.2. Microcalorimeter
added Rhodamine (Rh123) (SigmaeAldrich Co.), then incubated for
45 min in the dark. The dye was washed with PBS. Mitochondrial
transmembrane potentials were assessed using a fluorescence
spectrophotometer (F-4600 Hitachi Co., Japan) [21]. Date are given
as mean ꢄ S.D. at least 3 individual experiments. The results were
analyzed by t-test using SPSS 19.0 software.
An eight-channel heat conduction isothermal microcalorimeter
(Fig. 7, 3116-2/3239 TAM air, Thermometric AB Company, Sweden)
was employed in this research. This eight-channel heat conduction
isothermal microcalorimeter for heat flow measurements in the
milliwatt range under isothermal conditions was held together in
a single removable block. The block was placed in an air thermostat,
which kept the temperature within 0.02 ꢁC indicating that tem-
perature gradient is very low. All calorimetric channels were of twin
type, consisting of a sample and a reference vessel. Each vessel was
connected to the surrounding heat sink by a Peltier module, and
when heat was produced or consumed due to any process, the
temperature of the sample vessel was to be changed. The temper-
ature of the surrounding was constant and thus a temperature
gradient across the Peltier module was developed. This would
generate a measurable voltage and the voltage was proportional to
the heat flow across the Peltier module and to the rate of the pro-
cesses taking place in the sample vessel. Such voltage signal was
recorded continuously and in real-time through an eight channel
data acquisition system connected to a computer. The software
supplied to TAM air was used to monitor and record the heat flow
Acknowledgments
This research was financially supported by the National Natural
Science Foundation of China (No. 20973145 and No. 21273190), the
Hunan Provincial Key Laboratory Special Foundation of China (No.
2011TP4016-1), the Hunan Provincial Educational Ministry Foun-
dation of China (No. 11A112), and the Construct Program of the Key
Discipline in Hunan Province. Prof. Qiang-Guo Li (E-mail:
liqiangguo@163.com) is the chief-director of these fund projects.
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