Novel antileukemic agents derived from tamibarotene and nitric oxide donors

Article abstract of DOI:10.1016/j.bmcl.2011.09.103

A series of novel nitric oxide-releasing tamibarotene derivatives were synthesized by coupling nitric oxide (NO) donors with tamibarotene via various spacers, and were evaluated for their antiproliferative activities against human leukemic HL-60, NB4 and

Full text of DOI:10.1016/j.bmcl.2011.09.103

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7025–7029
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel antileukemic agents derived from tamibarotene and nitric oxide donors
⇑
⇑
Haiyong Bian, Jinhong Feng, Minyong Li , Wenfang Xu
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, Ji’nan, Shandong 250012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 June 2011
Revised 23 September 2011
Accepted 27 September 2011
Available online 1 October 2011
A series of novel nitric oxide-releasing tamibarotene derivatives were synthesized by coupling nitric
oxide (NO) donors with tamibarotene via various spacers, and were evaluated for their antiproliferative
activities against human leukemic HL-60, NB4 and K562 cell lines in vitro. The test results showed that
three compounds (7g, 9a and 9e) exhibited more potent antileukemic activity than the control tamibaro-
tene. Furthermore, the preliminary structure–activity analysis revealed that amino acids serving as spac-
ers could bring about significantly improved biological activities of NO donor hybrids. These interesting
results could provide new insights into the development of NO-based antileukemic agents.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Multi-target drugs
Nitric oxide
RAR agonist
Synthesis
Biological evaluation
Recently, multi-target drugs, which are designed as single mol-
ecules to modulate multiple physiological targets simultaneously,
have increasingly attracted the concerns of medicinal chemists,1
and it represents a very promising way to enhance efficacy and
to decrease adverse effects of drugs especially in the treatment of
complex diseases such as cancers, cardiovascular diseases, and
neurodegenerative diseases.2,3 Currently, one of the most interest-
ing cases for multi-target drugs is nitric oxide (NO) donor hybrids,
which are obtained by combining NO donors with an appropriate
reference drug, and several classes of NO-donor hybrids have been
extensively studied, including NO-donor nonsteroidal anti-inflam-
matory drug hybrids (NO-NSAIDs), NO-donor cardiovascular drug
hybrids, and NO-donor antioxidant hybrids.1,4 Nitric oxide is an
important cellular messenger molecule in vivo and has been dem-
onstrated to be involved in many physiological processes such as
vascular relaxation, neurotransmission and immune responses,
and some pathological processes including rheumatoid arthropa-
thies, hypertension and neurodegenerative diseases, etc.5,6 In par-
ticular, a variety of experimental evidences have indicated that
nitric oxide, which was found to be a cytotoxic and apoptosis-
inducing agent against tumor cells under appropriate conditions
of concentration, played a crucial role in the tumoricidal activity
of the human immune system, and could prevent cancer cells from
metastasis as well as effectively overcome tumor cell resistance to
conventional therapeutics.7 Furoxans (1,2,5-oxadiazole-2-oxides),
as an important class of NO donors, have been found to possess a
variety of NO-related biological activities such as tumor cell
apoptosis-inducing activity, vasodilator capacity, antiaggregant
and antibacterial activities. Especially, some furoxans exhibited
remarkable antileukemic activities and have been expected as
promising lead compounds to develop novel antileukemic agents.8
Tamibarotene (AM80), a selective RAR
a agonist launched in
Japan, has proved to be an effective drug for relapsed or refractory
APL. Compared to all-trans retinoic acid (ATRA), tamibarotene
exhibited higher differentiation-inducing activities for APL cells
and lower drug resistance due to its low affinity to cellular retinoic
acid binding protein (CRABP). However, the inevitable toxic and
side effects, such as hypertriglyceridemia, hypercholesterolemia,
rash, bone pain, retinoic acid syndrome and a strong teratogenic ef-
fect, would appear to hinder the clinical application of tamibaro-
tene.9 Therefore, it is interesting to develop tamibarotene
derivatives with improved safety features and similar or even bet-
ter antileukemic activities.
In the present study, a series of novel Tamibarotene-NO donor
hybrids were developed as antileukemic agents by attaching
phenyl-substituted furoxans as NO-donors to the reference drug
tamibarotene through various spacers such as ethylene glycol,
ethanolamine, ethylenediamine, and amino acids. Furoxans as
NO-donors might contribute to reduce the side effects of tamibaro-
tene, such as hypertriglyceridemia and hypercholesterolemia, by
releasing NO in vivo, as well as effectively avoid the nitrate toler-
ance from organic nitrates as classical NO donors.10 Particularly,
these hybrids were proposed to release NO and tamibarotene
through metabolism in vivo to exert synergistic effects at multiple
target sites and to bring about significantly enhanced efficacy. So it
is anticipated that more potent antileukemic agents would be
found out from these Tamibarotene-NO donor hybrids. In addition,
we also attempted to seek a better paradigm for multi-target-di-
⇑
Corresponding authors. Tel./fax: +86 531 88382264 (W.X.); tel./fax: +86 531
88382076 (M.L.).
E-mail addresses: mli@sdu.edu.cn (M. Li), wfxu@yahoo.cn (W. Xu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.103

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H. Bian et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7025–7029
rected drug design strategy by the structure–activity studies of
these pharmacodynamic hybrids. In this Letter, we report the syn-
thesis of Tamibarotene-NO donor hybrids, their NO-releasing
capacities, and the antiproliferative activities against human leuke-
mic HL-60, NB4 and K562 cell lines.
compounds 8a–e in tetrahydrofuran under the catalysis of 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDCI) and 1-hydroxybenzotrizole (HOBt). All of the target com-
pounds were purified by column chromatography on silica gel
and then characterized by IR, 1H NMR and HR-MS.13
The synthetic route of several key intermediates (compounds 3,
5) was outlined in Scheme 1. Compound 3 was prepared in 48.4%
overall yield from cinnamyl alcohol (1) by modified procedures de-
scribed in the literature.11,12 Compound 4 was synthesized in 83.4%
yield by the reaction of compound 3 with 4-(methoxycarbonyl)
phenol in DMF in the presence of anhydrous potassium carbonate
and potassium iodide at room temperature, and then compound 5
was obtained in 72.9% yield by the selective hydrolysis of com-
In order to evaluate the influence of the NO-releasing properties
on biological activities of the target compounds, the percentage of
NO released in vitro from these Tamibarotene-NO donor hybrids
was determined by Griess test (Fig. 1).14 It has been found that a
reduced thiol group from endogenous L-cysteine, glutathione or
proteins could mediate the release of NO from furoxan deriva-
tives.10,15 According to that principle, the evaluation of NO released
from furoxan derivatives in vitro is generally performed upon incu-
bation in phosphate buffered saline (PBS) solution with a large ex-
pound
4 in DMF–H2O solution on treatment with lithium
hydroxide.
cess of
L-cysteine. The resulting data showed that all of the target
The preparation of target compounds 7a–g and 9a–e was shown
in Schemes 2 and 3 respectively. Tamibarotene was reacted with
short chain alkylene glycols containing from two to five carbon
atoms, ethanolamine or ethylenediamine by the catalysis of N,N0-
carbonyldiimidazole (CDI) in dry tetrahydrofuran at 50 °C to give
compounds 6a–g in excellent yield (81.9–91.1%). Compound 3
was reacted with some N-tert-butoxycarbonyl protected amino
acids (such as N-t-Boc-Gly, N-t-Boc-L-Ala, N-t-Boc-L-Leu, N-t-
Boc-L-Val, and N-t-Boc-L-Phe) in the presence of cesium carbonate
and potassium iodide in dry DMF at room temperature, followed
by the treatment of trifluoroacetic acid in dichloromethane to af-
ford compounds 8a–e in moderate yield (50.6–72.2%). Finally, tar-
get compounds 7a–g were obtained in good yield (75.1–88.3%) by
esterification of compound 5 with compounds 6a–g in tetrahydro-
furan at room temperature under the catalysis of 1,3-dicyclohexyl-
carbodiimide (DCC) and 4-dimethylaminopyridine (DMAP), while
target compounds 9a–e were also stereoselectively synthesized
in various yield (68.3–87.5%) by amidation of tamibarotene with
compounds released significant levels of NO in
L
-cysteine solution,
and this evidence also suggested that the antileukemic effects of
the target compounds might partly come from NO released from
these Tamibarotene-NO donor hybrids.
The antiproliferative activities of all target compounds against
human leukemic HL-60, NB4 and K562 cell lines were respectively
evaluated by MTT cell proliferation assay and the results are sum-
marized in Table 1.16 The activity data indicated that all the Tam-
ibarotene-NO donor hybrids exhibited higher antiproliferative
activity against HL-60 and NB4 cell lines (acute myeloid leukemia)
than against K562 cell lines (chronic myeloid leukemia). Three of
the target compounds (7g, 9a and 9e) displayed significantly stron-
ger antiproliferative effects against all of the three human leuke-
mic cell lines than the positive control tamibarotene. In
particular, compound 9a exhibited the best antiproliferative activ-
ities against HL-60, NB4 and K562 cells with the IC50 of 0.16
0.19 M and 7.12 Mm, respectively, which were 56-, 25- and 6-fold
higher than that of tamibarotene. Another compound (9d) exhib-
lM,
l
OH
Cl
a
b
OH
N
2
N
d
N
3
N
O
O
O
O
1
O
O
O
O
c
O
OH
N
N
N
N
5
O
O
O
O
4
Scheme 1. Reagents and conditions: (a) NaNO2, HOAc, 62.4%; (b) SOCl2, Py, CH2Cl2, 77.6%; (c) 4-(methoxycarbonyl) phenol, K2CO3, KI, DMF, 83.4%; (d) LiOH, DMF, H2O, 72.9%.
O
O
ROH/NH2
OH
H
N
H
N
a
6a ROH/NH2=O(CH2)2OH 6b ROH/NH2=O(CH2)3OH
6c ROH/NH2=O(CH2)4OH 6d ROH/NH2=O(CH2)5OH
6e ROH/NH2=O(CH2)6OH 6f ROH/NH2=NH(CH2)2OH
6g ROH/NH2=NH(CH2)2NH2
O
O
AM80
O
O
O
7a R1=O(CH2)2O 7b R1=O(CH2)3O
b
R1
H
7c R1=O(CH2)4O 7d R1=O(CH2)5O
7e R1=O(CH2)6O 7f R1=O(CH2)2NH
7g R1=NH(CH2)2NH
N
N
N
O
O
O
Scheme 2. Reagents and conditions: (a) HO(CH2)nOH (n = 2–6), H2N(CH2)2OH and H2N(CH2)2NH2, CDI, THF, 50 °C, 81.9–91.1%; (b) compound 5, DCC, DMAP, THF, 75.1–88.3%.

H. Bian et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7025–7029
7027
R2
Cl
O
8a R2=H 8b R2=Me
NH2
a
b
8c R2=i-Pr 8d R2=i-Bu
8e R2=Bn
O
N
N
N
N
O
O
O
O
O
3
R2
O
O
9a R2=H
9b R2=Me
N
H
c
H
9c R2=i-Pr 9d R2=i-Bu
9e R2=Bn
O
N
N
N
O
O
Scheme 3. Reagents and conditions: (a) N-Boc amino acid, Cs2CO3, KI, DMF; (b) TFA, CH2Cl2, 50.6–72.2%; (c) tamibarotene, EDCI, HOBt, THF, 68.3–87.5%.
By comparing compounds 7f–g and 7a–e, it was found that the
types of chemical bonds associated with spacers make a strong im-
pact on the biological activity of these Tamibarotene-NO donor hy-
brids, and the amide bonds, formed between spacers and
tamibarotene moieties, might give rise to a significant increase in
antileukemic activities of the target compounds. That might par-
tially due to the fact that the amide groups, as excellent hydrogen
bond donors and acceptors, generally exhibit much stronger inter-
action with biomacromolecules than ester groups, which can only
act as hydrogen bond acceptors. Particularly, by comparing com-
pounds 9a–e and 7a–e, we concluded that those target compounds
with amino acids as spacers usually exhibited more potent antipro-
liferative activity against human leukemic cell lines than the other
compounds. This occurrence might be partially due to the short
and chiral structure of amino acid spacers, which can help facilitate
stereoselective enzyme-catalyzed metabolic reactions for these fi-
nal derivatives to produce NO and tamibarotene to exert synergis-
tic antileukemic effects. However, the precise mechanisms
underlying the differential antileukemic activity of these com-
pounds still remain unclear. Furthermore, the statistical signifi-
cance test was performed to compare compounds 9a–e group
with 7a–e group on the average percentage of NO released
in vitro using a dual-tailed t-test by the SPSS software. The results
showed that there were significant differences in the average per-
centage of NO released between two groups of compounds (P
<0.01), which was in accordance with the results of MTT cell pro-
liferation assay (Fig. 2). Altogether, it was suggested that amino
acids acting as spacers might bring about markedly enhanced bio-
logical activities for NO-donor hybrids.
1.2
1.1
1
Percentage of NO release
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
7a 7b 7c 7d 7e 7f 7g 9a 9b 9c 9d 9e
Compounds
2
Figure 1. NO releasing properties of compounds 2, 7a–g and 9a–e in vitro (all
values are mean SD; n = 3).
Table 1
Antiproliferative activity of compounds 7a–g, 9a–e and Tamibarotene against human
leukemic cell lines in vitro
a
Compound
IC50
NB4
(lM)
HL-60
K562
7a
7b
7c
7d
7e
7f
7g
9a
9b
9c
9d
9e
394.84 29.15
184.70 26.37
650.89 59.95
281.74 25.82
486.18 62.13
12.31 2.39
1.14 0.28
0.16 0.06
6.97 1.14
5.85 1.61
0.74 0.17
118.92 15.61
139.72 13.19
951.95 87.55
239.68 38.24
>1000
10.41 3.65
1.93 0.51
0.19 0.09
5.10 0.95
4.39 1.91
0.97 0.25
0.75 0.13
4.81 2.03
>1000
>1000
>1000
>1000
>1000
>1000
Biological activities of drug molecules are mainly dependent on
their physicochemical properties including electronic, steric, and
hydrophobic profiles. To help design more potent NO donor hy-
13.32 1.91
7.12 0.96
60.40 8.25
191.80 28.26
64.04 12.32
22.59 3.67
42.37 5.62
0.50 0.19
8.94 3.16
Tamibaroteneb
** * *
*
*
*
6.0
5.0
4.0
3.0
2.0
1.0
a
All values are mean SD (n = 3).
Tamibarotene was used as a positive control drug in MTT assay.
b
ited remarkably stronger antiproliferative effects against HL-60
and NB4 cells than tamibarotene, while exhibited similar biological
activity against K562 cells with tamibarotene. Moreover, two com-
pounds (9b–c) exhibited similar antiproliferative activity against
HL-60 and NB4 cells with tamibarotene as well. In contrast, five
of the target compounds (7a–e) showed a significant decrease in
antiproliferative activity against HL-60, NB4 and K562 cell lines.
Obviously, the different biological activities of the tested com-
pounds should be attributed to the different spacers between
NO-donor and tamibarotene moieties.
HL-60
NB4
K562
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
% NO release
Figure 2. The correlations between the percentages of NO released in vitro from the
target compounds and the antiproliferative activities against three human leukemic
cell lines. (⁄)The response IC50 values of these compounds are more than 1000
lM.

7028
H. Bian et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7025–7029
** *
* * **
*
* * * * **
A
B
C
******
*
6.0
5.0
4.0
3.0
2.0
1.0
6.0
5.0
4.0
3.0
2.0
1.0
6.0
5.0
4.0
3.0
2.0
1.0
HL-60
NB4
HL-60
NB4
HL-60
NB4
K562
K562
K562
6.0 7.0 8.0 9.0 10.0 11.0 12.0
logP
12.7 13.2 13.7 14.2 14.7 15.2
pKa
160.0 170.0 180.0 190.0 200.0 210.0
MR (cm3
/mol)
Figure 3. The correlations between the physicochemical properties of the target compounds and the antiproliferative activities against three human leukemic cell lines.
(⁄)The response IC50 values of these compounds are more than 1000 M.
l
2. Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini,
M.; Melchiorre, C. J. Med. Chem. 2008, 51, 347.
brids as antileukemic agents, the correlations between antileuke-
mic activity of these target compounds and their physicochemical
parameters including logP, molar refraction (MR) and pKa (calcu-
lated by ACD/Labs software), were well examined (Fig. 3). The re-
sults showed that there was no simple linear relationship
between the biological activity and the corresponding physico-
chemical parameter values. However, in the overall trend, a signif-
icant decrease in antileukemic activity of these final compounds
was depicted with the increase of logP or MR values, while a
remarkable increase in the antileukemic activity was showed with
the increase of pKa values. These results suggested that small-
sized, hydrophilic and alkalinous spacers might bring about en-
hanced biological activity for NO donor hybrids. Since amino acids
are common small-sized and hydrophilic molecules and also can
react with amines or carboxylic acids to give weak alkalinous
amide groups, it could be expected that amino acids serving as
spacers in NO donor hybrids might afford more potent antileuke-
mic agents. This finding has also been confirmed by the present
research.
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Umansky, V. Blood 1999, 93, 2342.
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Balligand, J. L. Pharmacol. Ther. 2005, 108, 225.
11. Gasco, A. M.; Fruttero, R.; Sorba, G.; Gasco, A. Liebigs Ann. Chem. 1991, 1991,
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González, M.; Onetto, S.; Seoane, G.; Suescun, L.; Mariezcurrena, R. Pharmazie
1998, 53, 758.
13. All the target compounds 7a–g and 9a–e provided acceptable IR, 1H NMR and
HR-MS spectra that exhibit no discernible impurities. The HPLC analysis data of
representative compounds 7b, 7g and 9a were reported in Supplementary
data. Compound 7b: mp 103–105 °C; IR (KBr, cmꢀ1): 3331.41, 2959.45,
2926.47, 2860.44, 1717.15, 1654.17, 1606.24, 1580.25, 1507.49, 1457.15; 1H
NMR (300 MHz, DMSO-d6) d: 1.225 (s, 6H, 2ꢁCH3), 1.234 (s, 6H, 2ꢁCH3), 1.632
(s, 4H, 2ꢁCH2), 2.156–2.240 (quint, J = 6.3 Hz, 2H, C–CH2), 4.400–4.491 (m, 4H,
2ꢁO–CH2), 5.296 (s, 2H, O–CH2), 7.067–7.097 (m, 2H, 2ꢁPh-H), 7.260–7.289 (d,
J = 8.7 Hz, 1H, Ph-H), 7.545–7.643 (m, 4H, 4ꢁPh-H), 7.675–7.681 (d, J = 1.8 Hz,
1H, Ph-H), 7.777–7.808 (m, 2H, 2ꢁPh-H), 7.905–7.935 (m, 2H, 2ꢁPh-H), 8.027–
8.100 (m, 4H, 4ꢁPh-H), 10.269 (s, 1H, NH); HR-MS (ESI-TOF) (m/z): 704.2949
[M+H]+ (calcd for [C41H41N3O8+H]+: 704.2967). Compound 7g: mp 193–194 °C;
IR(KBr, cmꢀ1): 3340.87, 2959.40, 2929.70, 2858.63, 1644.17, 1608.76, 1579.53,
1501.89, 1457.2; 1H NMR (300 MHz, DMSO-d6) d: 1.238 (s, 6H, 2ꢁCH3), 1.252
(s, 6H, 2ꢁCH3), 1.648 (s, 4H, 2ꢁCH2), 3.455 (br s, 4H, 2ꢁN–CH2), 5.287 (s, 2H,
O–CH2), 7.054–7.084 (m, 2H, 2ꢁPh-H), 7.278–7.307 (d, J = 8.7 Hz, 1H, Ph-H),
7.574–7.650 (m, 4H, 4ꢁPh-H), 7.680–7.688 (m, 1H, Ph-H), 7.806–7.857 (m, 4H,
4ꢁPh-H), 7.956–7.985 (d, J = 8.7 Hz, 2H, 2ꢁPh-H), 8.020–8.049 (d, J = 8.7 Hz,
2H, 2ꢁPh-H), 8.544 (br s, 1H, NH), 8.767 (br s, 1H, NH), 10.197 (s, 1H, NH); HR-
MS (ESI-TOF) (m/z): 688.3107 [M+H]+ (calcd for [C40H41N5O6+H]+: 688.3130).
Compound 9a: mp 183–184 °C; IR(KBr, cmꢀ1): 3283.25, 2957.70, 2922.81,
2858.11, 1764.25, 1642.12, 1611.50, 1580.67, 1499.43, 1457.91; 1H NMR
(300 MHz, DMSO-d6) d: 1.239 (s, 6H, 2ꢁCH3), 1.253 (s, 6H, 2ꢁCH3), 1.649 (s,
4H, 2ꢁCH2), 4.074–4.093 (d, J = 5.7 Hz, 2H, N–CH2), 5.220 (s, 2H, O–CH2),
7.054–7.084 (m, 2H, 2ꢁPhH), 7.279–7.308 (d, J = 8.7 Hz, 1H, PhH), 7.558–7.642
(m, 4H, 4ꢁPhH), 7.657–7.686 (m, 1H, PhH), 7.778–7.810 (m, 2H, 2ꢁPhH),
7.954–8.059 (q, J = 8.4 Hz, 4H, 4ꢁPhH), 9.155–9.192 (t, J = 5.7 Hz, 1H, NH),
10.210 (s, 1H, NH); HR-MS (ESI-TOF) (m/z): 583.2511 [M+H]+ (calcd for
[C33H34N4O6+H]+: 583.2551).
In conclusion, a series of novel Tamibarotene-NO donor hybrids
were developed in the current study. Three target compounds (7g,
9a and 9e) exhibited more potent antiproliferative activity against
three human leukemic cell lines than the control tamibarotene.
Particularly, compound 9a was the most potent antileukemic agent
with a low micromolar IC50 value in vitro. Furthermore, the preli-
minary SAR analysis of these derivatives revealed that amino acids
serving as spacers might give rise to a remarkable enhance in the
biological activity of NO donor hybrids. These interesting results
provide a new insight for the development of NO-based multi-tar-
get antileukemic agents. In addition, a further pharmacological
evaluation for these derivatives is in progress and will be reported
subsequently.
Acknowledgments
We are grateful to the National Nature Science Foundation of
China (Grant No. 90713041) and National Science and Technology
Major Project of the Ministry of Science and Technology of China
(Grant No. 2009ZX09103-118) for financial support for this
research.
14. In vitro assays for NO released: A solution of the test compound in DMSO
(5.0 mM) was added to 100 mM phosphate buffer solution (pH 7.4) containing
Supplementary data
of 3.4 mM L-cysteine, and the terminal concentration of target compounds was
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.103.
0.4 mM. The mixture was incubated at 37 °C for 3 h, and then 1.5 mL of the
reaction mixture was treated with 0.5 mL of freshly prepared Griess reagent.
After 10 min at room temperature, the absorbance was measured at 540 nm by
a Simadzu UV-2550 UV–VIS scanning spectrophotometer. Potassium nitrite
References and notes
standard solutions (1–25 lM) were used to prepare the calibration curve under
the same experimental conditions. The percentage of NO released (n = 3),
which is relative to a theoretical maximum release of 1 mol NO per mol of test
compound, was calculated according to the calibration curve.
1. Alberto, G.; Donatella, B.; Konstantin, C.; Clara, C.; Antonella, D. S.; Roberta, F.;
Loretta, L.; Barbara, R.; Paolo, T. Pure Appl. Chem. 2008, 80, 1693.

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7029
15. (a) Medana, C.; Ermondi, G.; Fruttero, R.; Di Stilo, A.; Ferretti, C.; Gasco, A. J.
Med. Chem. 1994, 37, 4412; (b) Civelli, M.; Giossi, M.; Caruso, P.; Razzetti, R.;
Bergamaschi, M.; Bongrani, S.; Gasco, A. Br. J. Pharmacol. 1996, 118, 923.
16. MTT cell proliferation assay: HL-60, NB4 and K562 cells were cultured in RPMI-
1640 medium with 10% fetal bovine serum at 37 °C in a humidified incubator
to triplicate wells at various concentrations (final concentration at 0.01, 0.1, 1,
10, 100 lM) and then incubated for 48 h. Following this, 20 lL of MTT solution
(5 mg mLꢀ1 within complete medium) was added to each well and incubated
for additional 4 h. After centrifugal separation, the medium was gently
removed from the wells and the formazan crystals were dissolved in 100 lL
of DMSO. The optical density was measured at 570 nm using an enzyme-linked
immunosorbent assay (ELISA) reader.
with atmosphere of 5% CO2, then cells were planted at 10,000 cells per 100
l
L
per well in 96-well cell culture plates and incubated for 4 h at 37 °C, 5% CO2 in
a humidified incubator, after that, test compounds within medium were added