Fatty acyl amide derivatives of doxorubicin: Synthesis and in vitro anticancer activities

Article abstract of DOI:10.1016/j.ejmech.2011.02.056

Doxorubicin is extensively used in anticancer therapy. Doxorubicin is highly hydrophilic, has short half-life, and its use is associated with severe side effects at high doses. Fatty acyl amide derivatives of doxorubicin were synthesized with the expectation to improve the lipophilicity and anticancer activity of the drug. The lipophilicity was enhanced with the increase in chain length of fatty acyl moiety. Conjugation of 4′-amino group with fatty acids through an amide bond reduced the anticancer activity in leukemia, breast, ovarian, and colon cancer cell lines, suggesting that the presence of free amino group is required for anticancer activity of doxorubicin. Dodecanoyl-doxorubicin derivative was consistently the most effective among the synthesized derivatives and inhibited the proliferation of colon (HT-29) and ovarian (SK-OV-3) cancer cells by 64% and 58%, respectively, at a concentration of 1 μM after 96 h incubation.

Full text of DOI:10.1016/j.ejmech.2011.02.056

European Journal of Medicinal Chemistry 46 (2011) 2037e2042
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Fatty acyl amide derivatives of doxorubicin: Synthesis and in vitro anticancer
activities
,
Bhupender S. Chhikara a, Nicole St. Jean a, Deendayal Mandal a, Anil Kumar b, Keykavous Parang a
*
a Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, 41 Lower College Road, Kingston, RI 02881, USA
b Chemistry Group, Birla Institute of Technology and Science, Pilani 333031, Rajasthan, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Doxorubicin is extensively used in anticancer therapy. Doxorubicin is highly hydrophilic, has short half-
life, and its use is associated with severe side effects at high doses. Fatty acyl amide derivatives of
doxorubicin were synthesized with the expectation to improve the lipophilicity and anticancer activity of
the drug. The lipophilicity was enhanced with the increase in chain length of fatty acyl moiety. Conju-
gation of 40-amino group with fatty acids through an amide bond reduced the anticancer activity in
leukemia, breast, ovarian, and colon cancer cell lines, suggesting that the presence of free amino group is
required for anticancer activity of doxorubicin. Dodecanoyl-doxorubicin derivative was consistently the
most effective among the synthesized derivatives and inhibited the proliferation of colon (HT-29) and
Received 11 October 2010
Received in revised form
14 February 2011
Accepted 22 February 2011
Available online 3 March 2011
Keywords:
Doxorubicin
Prodrug
ovarian (SK-OV-3) cancer cells by 64% and 58%, respectively, at a concentration of 1
incubation.
mM after 96 h
Fatty acids
Ó 2011 Elsevier Masson SAS. All rights reserved.
Anticancer
Lipophilicity
Sustained delivery
1. Introduction
create change in its circulation in blood and improve the target
accumulation by enhanced permeation rate (EPR).
Doxorubicin is an anthracycline antibiotic produced by the
fungus Streptomyces peucetius [1]. The drug is widely used as an
anticancer agent in the treatment of leukemia, breast carcinoma,
and other solid tumors [2]. Doxorubicin (Fig.1) has high hydrophilic
nature and low bioavailability. To circumvent problems associated
with low bioavailability, higher dose is used clinically that may lead
to dose-dependent side effects, such as cumulative cardiotoxicity,
myelosuppression, nephrotoxicity and extravasation. Doxorubicin
has been called as “red devil” or “red death” because of its high
antibiotic activity and red color [3].
Development of compounds with higher therapeutic index is
a subject of major interest for the treatment of different cancers. It is
widely accepted concept that biological activity and toxicity of low
molecular weight antitumor drugs are dependent on their physico-
chemical and pharmacokinetic properties, such as biodistribution
[4e6]. The toxicityand side effects associated withan anticancerdrug
can be counteracted by altering the pharmacokinetic behavior of the
compound. The pharmacological modulations of an anticancer drug
Various approaches have been applied to improve the drug
delivery by chemical modification in a parent drug (prodrug
strategy) [7], by attaching the drug molecules to longer circulating
particles [8] like polymeric nanoparticles [9], metal nanoparticles
[10] and other nanoparticles [11], or by encapsulation in protective
sheaths like liposomes, niosomes [12,13], nanomicelle [14], den-
drimers [15] or hydrogels/organogels [16].
Doxorubicin has a short half-life in the blood stream and high
volume of distribution [17]. In case of doxorubicin, the liposomal
formulations have been successfully used in improving its thera-
peutic efficacy [18e22]. Furthermore, the conjugation of docosa-
hexanoic acid (DHA) to doxorubicin using a hydrazone linker at
position 13 formed a lipophilic prodrug, which was able to release
doxorubicin at a lower pH. DHAedoxorubicin displayed lower
cytotoxicity in vitro as compared to that of doxorubicin against
a lymphocytic leukemia cell line L1210. Following intraperitoneal
injection in L1210 leukemia ascites model, DHAedoxorubicin
showed considerably higher anticancer activity than free doxoru-
bicin in vivo [23].
In continuation of our efforts to modulate and improve the
therapeutic potential of anticancer drugs through chemical modi-
fications [24], herein we report the synthesis of fatty acyl amide
* Corresponding author. Tel.: þ1 401 874 4471; fax: þ1 401 874 5787.
E-mail address: kparang@uri.edu (K. Parang).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.056

2038
B.S. Chhikara et al. / European Journal of Medicinal Chemistry 46 (2011) 2037e2042
O
O
OH
OH
O
O
OH
O
4
OH
7
5
6
13
OH
4a
5a
6a
8
3
2
OH
O
14
OH
R-COOH
1
9
10
H
10a
HBTU,
DIPEA, DCM
12a
11a
O
O
12
1
11
H
O
O
OH
O
O
R = -(CH2)nCH3
1'
6'
5'
2
n = 0
2'
3'
OH
R
3
n = 6
4
n = 8
HN
1
4'
5
n = 10
n = 12
n = 14
n = 16
n= 18
OH
6
8
NH2
O
9
10
Fig. 1. Chemical structure of doxorubicin i.e. (8S,10S)-10-(4-amino-5-hydroxy-6-
methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-
methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione.
7 R = -(CH2)11SCH2CH3
Scheme 1. Chemical synthesis of fatty acyl-doxorubicin derivatives.
2.2. Biological activity
derivatives of doxorubicin and evaluation of their in vitro anticancer
activity in different cancer cell lines. The conjugates were synthe-
sized with the expectation that lipophilic nature of fatty acyl chains
will enhance the sustained effect and biological activity of the
parent drug.
The effect of compounds on the cell proliferation of cancer cells
was evaluated in vitro in human leukemia cell line CCRF-CEM,
breast adenocarcinoma MDA-MB-468, ovarian adenocarcinoma SK-
OV-3, and colon adenocarcinoma HT-29 cell lines up to 120 h at
1
mM.
At 24 h of incubation, the inhibition of cell proliferation by the
2. Result and discussion
derivatives was negligible when incubated with breast cancer cell
line (MDA-MB-468) while at 96 h dodecanoyl (5) and tetradecanoyl
(6) doxorubicin derivatives inhibited the cell proliferation up to 38%
and 31%, respectively, when compared to the control (Fig. 3a).
These data suggest that even though the compounds 2e10 have
higher lipophilicity than that of doxorubicin, they were probably
stable intracellularly and did not release substantial amount of
doxorubicin even after 96 h in breast cancer cell line.
Furthermore, the effect of compounds on cell proliferation of
other cell lines was evaluated to determine whether fatty acyl-
doxorubicin conjugates demonstrate any cell-specific activity. In
case of leukemia cell line (CCRF-CEM), the anti-proliferation
activity was negligible when compared to doxorubicin (1). The
tetradecanoyl derivative (6) showed only 27% growth inhibition at
96 h (Fig. 3b). Because of high lipophilicity and the prodrug nature,
these compounds were expected to have enhanced cellular uptake
and/or sustained effect. Thus, the lack of inhibitory activity is
postulated to be because of limited intracellular hydrolysis of amide
conjugates.
2.1. Chemistry
Monosubstituted fatty acyl-doxorubicin conjugates were
synthesized by reaction of doxorubicin with fatty acids (i.e.,
CH3(CH2)nCOOH (n ¼ 0e18), CH3CH2S(CH2)11COOH) (one equivalent)
in the presence of 1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) and N,N-diisopropylethylamine (DIPEA) (Scheme 1). The 40-
primary amino group of doxorubicin was selectively reacted with the
carboxylic acid moiety of fatty acid under these conditions due to its
higher nucleophilic nature compared to the hydroxyl groups. The
structures of the synthesized derivatives were confirmed by using
infrared spectroscopy, one-dimensional nuclear magnetic resonance
(NMR) spectroscopy (1H, 13C or DEPT), 2-dimensional correlative
NMR (HSQC and COSY), and high-resolution time-of-flight electro-
spray mass spectrometry. The IR spectra showed the peak at
1642e1645 cmꢀ1 confirming the presence of an amide bond in the
final products. Furthermore, C40 peak in 13C NMR was significantly
deshielded after conjugation, suggesting the involvement of 40-
amino group in amide formation. While the doxorubicin was highly
soluble in aqueous medium, the fatty acyl amide doxorubicin deriv-
atives were soluble in organic solvents. All the derivatives had dark
red brown color. The palmityl and stearyl derivatives of doxorubicin
have been reported earlier in Refs. [25,26] and were resynthesized
here for evaluation of their anticancer activity and showed similar
spectral properties to those reported in the literature.
The anti-proliferation profile of compounds 1e10 in SK-OV-3
cell line is shown in Fig. 3c. A number of fatty acyl amide doxoru-
bicin conjugates show efficient inhibition of cell proliferation at
Partition coefficients (Log P) of 1e7 were determined using
biphasic n-octanol/water shake flask system. The concentration of
the compounds was measured in both phases using UV spectros-
copy. Lipophilicity of many compounds is usually correlated with
the membrane permeability and/or biological activity in quantita-
tive structureeactivity relationship studies.
Lipophilicity of doxorubicin Log P ¼ 0.6 was consistent with the
reported value [27]. The Log P values of the doxorubicin derivatives
showed a gradual increase in the lipophilicity of the derivatives
with the increase in chain length of the attached fatty acyl groups.
For example, the partition coefficient increased in compound 2
with the introduction of acetyl group (Log P ¼ 1.18) (Fig. 2).
Fig. 2. Log P of fatty acyl-doxorubicin conjugates (2e10).

B.S. Chhikara et al. / European Journal of Medicinal Chemistry 46 (2011) 2037e2042
2039
a
b
120
120
100
80
60
40
20
0
100
80
60
24h
96h
24h
96h
40
20
0
c
d
120
100
80
60
40
20
0
120
100
80
60
24h
24h
96h
40
120h
20
0
Fig. 3. Anti-proliferative activity of 1e10 (1
mM) in (a) MDA-MB-468, (b) CCRF-CEM, (c) SK-OV-3, and (d) HT-29 cells after 24 h, 96 h, or 120 h. The results are shown as the
percentage of the control that has no compound (set at 100%). All the experiments were performed in triplicate.
96 h incubation in ovarian cancer cell line. The derivatives showed
peculiar parabolic correlation on inhibition of cell proliferation of
SK-OV-3 cells. Anti-proliferation activity enhanced with increase in
methylene numbers in the fatty acyl chain in compounds 2e4,
reached a maxima in compounds 5 and 6 and then decreased with
increase in carbon number (8e10). 12-Thiododecanoyl derivative 7
that has different side chain containing heteroatom sulfur than the
other derivatives did not follow the pattern. The maximum activity
was shown by dodecanoyl derivative 5. Dodecanoyl 5 and tetra-
decanoyl 6 derivatives inhibited the cell proliferation by 58% and
50%, respectively, when compared to the control after 96 h of
incubation.
In a time dependent study to evaluate the effect of incubation
time (more than 96 h) on cell proliferation inhibitory activity of the
derivatives, colon cancer cells HT-29 were incubated for 120 h with
the compounds. The compounds showed similar activity pattern in
HT-29 cells as seen in SK-OV-3 cells. Dodecanoyl doxorubicin
derivative 5 showed inhibited proliferative activity of the cells by
64% while tetradecanoyl derivative 6 exhibited approximately 41%
inhibition after 120 h of incubation (Fig. 3d).
Doxorubicin acts as a DNA intercalator, inhibiting the ability of
the enzyme topoisomerase II to reseal the DNA double helix strands
during the replication and thereby stops the reproduction of cells.
Several crystal structures suggest that replication inhibition is
directly due to the structure of the doxorubicin; the planar aromatic
portion of the molecule intercalates between two base pairs of the
DNA helix, while the carbohydrate portion of the drug is positioned
to intercalate with the flanking base pairs [28e30]. Thus, the
carbohydrate part of doxorubicin is involved in the binding with
bases of replicating DNA during its mechanism of action and amine
groupshould be freelyavailable for interaction. Thus, this systematic
study performed by fatty acylation of doxorubicin at 40-amino group
of the carbohydrate moiety and the anti-proliferative profile further
confirms the requirement for availability of the sugar moiety in
doxorubicin. Stable amide was only partially hydrolyzed even after
long incubation time providing anti-proliferative activity in specific
cell lines. The inhibitory activity for 5 at 24 h and 96 h suggests that
the intracellular hydrolysis of the prodrug to doxorubicin leads to
improved anti-proliferative activity after longer incubation period,
suggesting the potential benefit of this compound for sustained
release effect.
3. Conclusions
Fatty acyl amide derivatives of doxorubicin were synthesized by
coupling of 40-amino group with the fatty acids and were found to
be more lipophilic when compared to doxorubicin. In general, the
fatty acyl amide derivatives exhibited more anti-proliferative
activity in ovarian and colon cell lines when compared to leukemia
and breast cancer cells. The overall pattern for the synthesized
derivatives for anti-proliferation activity in different cell lines
showed that dodecanoyl derivative was the most effective in in vitro
studies. This compound may have potential application for slow
delivery of doxorubicin. Further optimization will be required to
generate the lead compounds with optimal anticancer activity.
Future studies on designing fatty acyl conjugates of doxorubicin
should be also directed on conjugation of other functional groups.
4. Experimental protocols
4.1. Materials and methods
Doxorubicin was purchased from EuroAsian Chemical Ltd
(Mumbai, India). HBTU, anhydrous dichloromethane (DCM), and
other chemicals and reagents were purchased from Sigma-Aldrich
Chemical Co. (Milwaukee, WI). 12-Thioethyldodecanoic acid was
synthesized from 12-bromododecanoic acid and thioethanol as
described previously in Ref. [31]. HBTU in DCM was used as

2040
B.S. Chhikara et al. / European Journal of Medicinal Chemistry 46 (2011) 2037e2042
a coupling reagent. The chemical structures of final products were
characterized by nuclear magnetic resonance spectra (1H NMR, 13
(57 mg, 92.5% yield). IR (cmꢀ1, ATR): 3346. 2923, 2848, 1716 (C]O),
1642 (C]O str, amide), 1618, 1575, 1445, 1407, 1373, 1279, 1233, 1203,
C
NMR) determined on a Varian NMR spectrometer (500 MHz). 13C
NMR spectra are fully decoupled. Chemical shifts were reported in
parts per millions (ppm) using deuterated solvent peak or tetra-
methylsilane (internal) as the internal standards. The chemical
structures of final products were confirmed by a high-resolution
Biosystems QStar Elite time-of-flight electrospray mass spectrom-
eter. Details of procedures and spectroscopic data of the respective
compounds are presented below. Final compounds were purified
1109, 1068, 1015, 844, 764; UV/vis
(500 MHz, CDCl3, ppm): 13.98 (s,1H, PhOH),13.25 (s,1H, PhOH), 8.04
l
(nm): 478, 290, 254, 234; 1H NMR
d
(d, J ¼ 7.7 Hz, 1H, ArC2H), 7.79 (t, 1H, J ¼ 7.8 Hz, ArC3H), 7.39 (d, 1H,
J ¼ 8.5 Hz, ArC4H), 7.26 (s,1H, NH), 5.80 (d,1H, J ¼ 8.2 Hz, OH), 5.50 (d,
1H, J ¼ 3.3 Hz, H-20), 5.28 (s, 1H, H-50), 4.76 (s, 2H, O]C13eCH2),
4.14e4.19 (m, 2H, H-10 and H-40), 4.08 (s, 3H, OCH3), 3.63 (s, 1H, H-60),
3.28 (d,1H, J ¼ 12.8 Hz, H-9eq), 3.02 (d,1H, J ¼ 18.8 Hz, H-9ax), 2.33 (d,
1H, J ¼ 14.5 Hz, H-30eq), 2.17 (dd, J ¼ 14.6 Hz, J ¼ 3.8 Hz, 1H, H-30ax),
2.10e2.14 (m, 2H, O]CeCH2), 1.87 (dd, 1H, J ¼ 13.5 Hz, J ¼ 5.1 Hz, H-
7ax), 1.72e1.77 (m, 1H, H-7eq), 1.56 (t, 2H, J ¼ 6.5 Hz, NHCOCH2CH2),
1.29 (d, 3H, J ¼ 6.5 Hz, 60-CH3), 1.25 (br s, 8H, 4 ꢁ CH2, methylene
envelope), 0.85 (t, 3H, J ¼ 5.9 Hz, CH3); 13C NMR (125 MHz, CDCl3,
on a Phenomenex Prodigy 10
mm ODS reversed-phase column
(2.1 cm ꢁ 25 cm) with a Hitachi HPLC system using a gradient
system of acetonitrile or methanol and water (CH3CN or CH3OH/
H2O, 0ꢀ100%, pH 7.0, 60 min). The purity of final products (>99%)
was confirmed by analytical HPLC. The analytical HPLC was per-
formed on a Hitachi analytical HPLC system using a C18 Shimadzu
d
ppm): 213.94 (C13]O), 187.10 (C12]O), 186.65 (C5]O), 172.61
(NHeC]O), 161.01 (C1), 156.01 (C6), 155.64 (C11), 135.77 (C3) 135.48
(C10a), 133.61 (C4a), 133.55 (C6a), 120.83 (C12a), 119.86 (C2), 118.41
(C4), 111.57 (C11a), 111.37 (C5a), 100.70 (C20), 76.53 (C8), 69.66 (C60),
69.63 (C50), 67.21 (C10), 65.56 (O]CeCH2OH), 56.66 (OCH3), 44.99
(NH-C40), 36.76 (NHCOCH2), 35.71 (C30), 33.95 (C9), 29.98 (C7), 31.63,
29.16, 28.98 (methylene carbons), 25.68 (NHCOCH2CH2), 22.58
(CH2CH3), 16.83 (60-CH3) 14.04 (CH3); HR-MS (ESI-TOF): calcd. for
C35H43NO12 669.2785; found, 692.1190 [M þ Na]þ.
Premier 3
m
m column (150 cm ꢁ 4.6 mm) and a gradient system,
and a flow rate of 1 mL/min with detection at 490 nm.
4.2. Chemistry
4.2.1. General synthesis
Fatty acyl amide derivatives of doxorubicin (2e10) were synthe-
sized by coupling reaction of doxorubicin and the fatty acids in pres-
ence of HBTU. All derivatives were synthesized using a similar
procedure. As a representative example the synthesis of tetradecanoyl
amide derivative is described here. Doxorubicin (50 mg, 0.09 mmol)
was suspendedin dry DCM(10 mL). Myristicacid (20.9mg, 0.09 mmol)
and HBTU (115 mg, 0.30 mmol) in dry DCM (20 mL) were added slowly
tothe reaction mixture. Next, DIPEA (100 mg, 0.77 mmol) was added to
the reaction mixture at room temperature. The mixture was stirred for
3 h under nitrogen atmosphere. After completion of the reaction (TLC
product Rf ¼ 0.8, doxorubicin Rf ¼ 0.1 in DCM/Methanol (8:2 v/v)),
water (50 mL) was added to the mixture and the crude product was
extracted with DCM (3 ꢁ 40 mL). After removal of DCM under reduced
pressure, the crude product was purified by column chromatography
over silica gel using DCM/methanol (0e20%) as the eluents to afford
product (63 mg, 90.8%). The product was further purified on a reverse
phase HPLC using C18 column and gradient methanol/water as mobile
phase as described above.
4.2.1.3. (8S,10S)-10-(4-(N-decylamido)-5-hydroxy-6-methyl-tetrahy-
dro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-
methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione (4). Red brown solid
(60 mg, 93.6% yield). IR (cmꢀ1, ATR): 3346, 2923, 2848, 1719 (C]O),
1644 (C]O str, amide), 1614, 1575, 1445, 1407, 1280, 1205, 1017, 980;
UV/vis
ppm): 13.92 (s, 1H, PhOH), 13.18 (s, 1H, PhOH), 7.99 (d, J ¼ 7.7 Hz, 1H,
l
(nm): 494, 477, 288, 250, 229; 1H NMR (500 MHz, CDCl3,
d
ArC2H), 7.74 (t, 1H, J ¼ 8.2 Hz, ArC3H), 7.35 (d, 1H, J ¼ 8.5 Hz, ArC4H),
7.24 (s, 1H, NH), 5.85 (d, 1H, J ¼ 4.2 Hz, OH), 5.46 (d, 1H, J ¼ 3.7 Hz, H-
20), 5.22 (s, 1H, H-50), 4.73 (s, 2H, O]C13eCH2), 4.12e4.16 (m, 2H, H-10
and H-40), 4.04 (s, 3H, OCH3), 3.61 (s, 1H, H-60), 3.23 (d, 1H, J ¼ 18.9 Hz,
H-9eq), 2.96 (d, 1H, J ¼ 18.9 Hz, H-9ax), 2.30 (d, 1H, J ¼ 14.4 Hz, H-
30eq), 2.15 (d, J ¼ 3.9 Hz, 1H, H-30ax), 2.07e2.12 (m, 2H, O]CeCH2),
1.87 (dd, 1H, J ¼ 13.5 Hz, J ¼ 5.0 Hz, H-7ax), 1.70e1.76 (m, 1H, H-7eq),
1.52 (t, 2H, J ¼ 7.0 Hz, NHCOCH2CH2), 1.25 (d, 3H, J ¼ 6.5 Hz, 60-CH3),
1.22 (br s,12H, 6 ꢁ CH2, methylene envelope), 0.80e0.86 (m, 3H, CH3);
13C NMR (125 MHz, CDCl3,
d ppm): 213.54 (C13]O), 186.65 (C12]O),
4.2.1.1. (8S,10S)-10-(4-(N-acetylamido)-5-hydroxy-6-methyl-tetra-
hydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-
methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione (2). Red brown
solid (50 mg, 92.8% yield). IR (cmꢀ1, ATR): 2969, 1718 (C]O), 1642
(C]O str, amide),1611,1573,1404,1373,1279,1231,1200,1110,1072,
186.21 (C5]O), 172.32 (NHeC]O), 160.61 (C1), 155.80 (C6), 155.23
(C11), 135.35 (C3), 135.08 (C10a), 133.23 (C4a), 133.17 (C6a), 120.44
(C12a), 119.45 (C2), 118.02 (C4), 111.15 (C11a), 110.96 (C5a), 100.29
(C20), 76.14 (C8), 69.24 (C60), 69.22 (C50), 66.84 (C10), 65.16 (O]
CeCH2OH), 56.26 (OCH3), 44.67 (NH-C40), 36.35 (NHCOCH2), 35.32
(C30), 33.55 (C9), 29.32 (C7), 31.43, 29.01, 28.97, 28.92, 28.85, 28.82,
(methylene carbons), 25.30 (NHCOCH2CH2), 22.24 (CH2CH3), 16.43
(60-CH3) 13.69 (CH3); HR-MS (ESI-TOF): calcd. for C37H47NO12
697.3098; found, 720.1878 [M þ Na]þ.
1013; UV/vis
l
(nm): 496, 479, 289, 249, 229; 1H NMR (500 MHz,
ppm):7.65 (s,1H,ArC2H), 7.64 (s,1H,ArC3H), 7.38(s,
CDCl3 þ CD3OD,
d
1H, ArC4H), 5.48 (d,1H, J ¼ 5.6 Hz, H-20), 5.35 (s,1H, H-50), 4.78 (s, 2H,
O]C13eCH2), 4.16e4.25 (m, 2H, H-10 and H-C40), 4.09 (s, 3H, OCH3),
3.60 (s, 1H, H-60), 3.32 (s,1H, H-9eq), 2.95 (d,1H, J ¼ 14.5 Hz, H-9ax),
2.22e2.35 (m, 1H, H-30eq), 1.85e2.18 (m, 5H, H-30ax, H-7ax, O]
CeCH3),1.69e1.73 (m,1H, H-7eq),1.26 (d, 3H, J ¼ 4.8 Hz, 60-CH3); 13C
4.2.1.4. (8S,10S)-10-(4-(N-dodecanoylamido)-5-hydroxy-6-methyl-
tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydrox-
yacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione
(5). Red brown solid (61 mg, 91.4% yield). IR (cmꢀ1, ATR): 3346,
2923, 2848, 1719 (C]O), 1644 (C]O str, amide), 1614, 1575, 1445,
NMR (125 MHz, CDCl3 þ CD3OD,
d ppm): 214.20 (C13]O), 187.67
(C12]O),187.21 (C5]O),171.32 (NHeC]O),161.45 (C1),156.42 (C6),
155.72 (C11), 136.37 (C3), 135.84 (C10a), 134.45 (C4a), 134.19 (C6a),
121.18 (C12a), 120.20 (C2), 119.07 (C4), 111.96 (C11a), 111.70 (C5a),
101.13 (C20), 76.69 (C8), 69.88 (C60), 69.06 (C50), 67.78 (C10), 65.51
(O]CeCH2OH), 56.94 (OCH3), 45.83 (NH-C40), 36.34 (C30), 33.94
(C9), 29.94 (C7), 22.87 (O]CeCH3),16.99 (60-CH3); HR-MS (ESI-TOF):
calcd. for C29H31NO12 585.1846; found, 624.0463 [M þ K]þ.
1407,1280,1205,1017, 980; UV/vis
NMR (500 MHz, CDCl3, ppm): 13.94 (s, 1H, PhOH), 13.19 (s, 1H,
l
(nm):494, 477, 289, 249, 229; 1H
d
PhOH), 8.01 (d, J ¼ 7.6 Hz, 1H, ArC2H), 7.77 (t, 1H, J ¼ 8.3 Hz, ArC3H),
7.37 (d,1H, J ¼ 8.4 Hz, ArC4H), 7.26 (s,1H, NH), 5.86 (d,1H, J ¼ 8.0 Hz,
OH), 5.44e5.50 (m, 1H, H-20), 5.23 (s, 1H, H-50), 4.75 (s, 2H, O]
C
13eCH2), 4.14e4.16 (m, 2H, H-10 and H-40), 4.06 (s, 3H, OCH3), 3.62
4.2.1.2. (8S,10S)-10-(4-(N-octoylamido)-5-hydroxy-6-methyl-tetra-
hydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-
methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione (3). Red brown solid
(s, 1H, H-60), 3.23 (d, 1H, J ¼ 18.9 Hz, H-9eq), 2.96 (d, 1H, J ¼ 18.7 Hz,
H-9ax), 2.32 (d, 1H, J ¼ 14.0 Hz, H-30eq), 2.17 (d, J ¼ 3.1 Hz, 1H, H-
30ax), 2.09e2.12 (m, 2H, O]CeCH2), 1.84 (dd, 1H, J ¼ 13.3 Hz,

B.S. Chhikara et al. / European Journal of Medicinal Chemistry 46 (2011) 2037e2042
2041
J ¼ 5.1 Hz, H-7ax), 1.72e1.78 (m, 1H, H-7eq), 1.54 (d, 2H, J ¼ 6.5 Hz,
NHCOCH2CH2),1.25 (d, 3H, J ¼ 6.5 Hz, 60-CH3),1.25 (br s,16H, 8 ꢁ CH2,
methylene envelope), 0.84e0.87 (m, 3H, CH3); 13C NMR (125 MHz,
(NHCOCH2), 35.33 (C30), 33.07 (C9), 29.32 (C7), 31.53, 31.27, 29.57,
29.27, 29.23, 29.12, 29.04, 28.98, 28.91, 28.83, 28.54, 27.63 (meth-
ylene carbons), 25.52 (NHCOCH2CH2), 22.90 (CH2CH3), 22.31
(SCH2), 18.02 (60-CH3) 13.74 (CH3); HR-MS (ESI-TOF): calcd. for
C41H55NO12S 785.3445; found, 824.1986 [M þ K]þ.
CDCl3,
d ppm): 213.90 (C13]O), 186.99 (C12]O), 186.54 (C5]O),
172.61 (NHeC]O),160.98 (C1),156.17 (C6),155.59 (C11),135.72 (C3)
135.44 (C10a),133.59 (C4a),133.58 (C6a),120.80 (C12a),119.82 (C2),
118.40 (C4),111.52 (C11a),111.33 (C5a),100.69 (C20), 76.51 (C8), 69.64
(C60), 69.60 (C50), 67.22 (C10), 65.53 (O]CeCH2OH), 56.64 (OCH3),
45.01 (NH-C40), 36.74 (NHCOCH2), 35.68 (C30), 33.91 (C9), 29.95 (C7),
31.87, 29.68, 29.59, 29.55, 29.44, 29.32, 29.29, 29.22 (methylene
carbons), 25.67 (NHCOCH2CH2), 22.64 (CH2CH3), 16.81 (60-CH3),
14.08 (CH3); HR-MS (ESI-TOF): calcd. for C39H51NO12 725.3411;
found, 748.2137 [M þ Na]þ and 764.1691 [M þ K]þ.
4.2.1.7. (8S,10S)-10-(4-(N-hexadecanoylamido)-5-hydroxy-6-
methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-
hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione
(8). Red brown solid (66 mg, 91.8% yield). IR (cmꢀ1, ATR): 3346.
2923, 2848, 1716 (C]O), 1642 (C]O str, amide), 1618, 1575, 1445,
1407, 1373, 1279, 1233, 1203, 1109, 1068, 1015, 844, 764; UV/vis
l
(nm): 478, 290, 254, 234; 1H NMR (500 MHz, CDCl3,
d
ppm): 13.98
(s,1H, PhOH),13.28 (s,1H, PhOH), 8.06 (d, J ¼ 7.8 Hz,1H, ArC2H), 7.80
(t, 1H, J ¼ 8.1 Hz, ArC3H), 7.40 (d, 1H, J ¼ 8.6 Hz, ArC4H), 7.26 (s, 1H,
NH), 5.76 (d,1H, J ¼ 8.5 Hz, OH), 5.50 (d, 1H, J ¼ 3.0 Hz, H-20), 5.30 (s,
1H, H-50), 4.76 (s, 2H, O]C13eCH2), 4.19e4.13 (m, 2H, H-10 and H-
40), 4.09 (s, 3H, OCH3), 3.64 (s, 1H, H-60), 3.30 (d, 1H, J ¼ 19.2 Hz, H-
9eq), 3.05 (d,1H, J ¼ 20.1 Hz, H-9ax), 2.34 (d,1H, J ¼ 13.4 Hz, H-30eq),
2.18 (dd, J ¼ 14.8 Hz, J ¼ 3.7 Hz, 1H, H-30ax), 2.10e2.13 (m, 2H, O]
CeCH2),1.87 (dd,1H, J ¼ 13.7 Hz, J ¼ 4.5 Hz, H-7ax),1.71e1.77 (m,1H,
H-7eq), 1.54e1.62 (m, 2H, NHCOCH2CH2), 1.29 (d, 3H, J ¼ 5.3 Hz, 60-
CH3), 1.23e1.25 (br s, 24H, 12 ꢁ CH2, methylene envelope),
4.2.1.5. (8S,10S)-10-(4-(N-tetradecanoylamido)-5-hydroxy-6-
methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-
hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione
(6). Red brown solid (63 mg, 90.8% yield). IR (cmꢀ1, ATR): 3346,
2923, 2848, 1719 (C]O), 1644 (C]O str, amide), 1614, 1575, 1445,
1407, 1280, 1205, 1017, 980; UV/vis
l (nm): 494, 477, 289, 249, 229;
1H NMR (500 MHz, CDCl3,
d
ppm): 13.92 (s, 1H, PhOH), 13.18 (s, 1H,
PhOH), 7.99 (d, J ¼ 7.6 Hz, 1H, ArC2H), 7.73 (t, 1H, J ¼ 8.3 Hz, ArC3H),
7.34 (d,1H, J ¼ 8.4 Hz, ArC4H), 7.24 (s,1H, NH), 5.85 (d,1H, J ¼ 8.4 Hz,
OH), 5.46 (d, 1H, J ¼ 3.7 Hz, H-20), 5.22 (s, 1H, H-50), 4.73 (s, 2H, O]
0.86e0.89 m, 3H, CH3); 13C NMR (125 MHz, CDCl3,
d ppm): 213.94
C
13eCH2), 4.12e4.16 (m, 2H, H-10 and H-40), 4.04 (s, 3H, OCH3), 3.61
(C13]O), 187.10 (C12]O), 186.65 (C5]O), 172.61 (NHeC]O), 161.01
(C1), 156.01 (C6), 155.64 (C11), 135.77 (C3) 135.48 (C10a), 133.61
(C4a), 133.55 (C6a), 120.83 (C12a), 119.46 (C2), 118.00 (C4), 111.16
(C11a),110.97 (C5a),104.60 (C20), 76.37 (C8), 69.26 (C60), 69.24 (C50),
66.83 (C10), 65.18 (O]CeCH2OH), 56.66 (OCH3), 44.60 (NH-C40),
36.38 (NHCOCH2), 33.56 (C30), 33.09 (C9), 29.58 (C7), 33.07, 33.04,
31.54, 31.53, 29.32, 29.28, 29.26, 29.24, 29.08, 29.04, 28.98, 28.96,
28.85 (methylene carbons), 25.31 (NHCOCH2CH2), 22.90 (CH2CH3),
16.44 (60-CH3) 13.74 (CH3); HR-MS (ESI-TOF): calcd. for C43H59NO12
781.4037; found, 804.2106 [M þ Na]þ.
(s, 1H, H-60), 3.22 (d, 1H, J ¼ 18.9 Hz, H-9eq), 2.95 (d, 1H, J ¼ 18.7 Hz,
H-9ax), 2.30 (d, 1H, J ¼ 14.4 Hz, H-30eq), 2.15 (d, J ¼ 3.1 Hz, 1H, H-
30ax), 2.06e2.13 (m, 2H, O]CeCH2), 1.82 (dd, 1H, J ¼ 13.3 Hz,
J ¼ 5.1 Hz, H-7ax), 1.68e1.77 (m, 1H, H-7eq), 1.49e1.55 (m, 2H,
NHCOCH2CH2), 1.25 (d, 3H, J ¼ 6.5 Hz, 60-CH3), 1.23 (br s, 20H,
10 ꢁ CH2, methylene envelope), 0.80e0.88 (m, 3H, Hz, CH3); 13C
NMR (125 MHz, CDCl3,
d ppm): 213.56 (C13]O), 186.68 (C12]O),
186.22 (C5]O), 172.20 (NHeC]O), 160.61 (C1), 155.81 (C6), 155.25
(C11), 135.37 (C3) 135.07 (C10a), 133.25 (C4a), 133.17 (C6a), 120.46
(C12a), 119.46 (C2), 118.00 (C4), 111.16 (C11a), 110.97 (C5a), 100.29
(C20), 76.17 (C8), 69.26 (C60), 69.24 (C50), 66.83 (C10), 65.18 (O]
CeCH2OH), 56.26 (OCH3), 44.60 (NH-C40), 36.40 (NHCOCH2), 35.34
(C30), 33.57 (C9), 29.61 (C7), 31.53, 29.29, 29.25, 29.29, 29.44, 29.08,
28.85, 28.87 (methylene carbons), 25.33 (NHCOCH2CH2), 22.31
(CH2CH3), 16.44 (60-CH3), 13.74 (CH3); HR-MS (ESI-TOF): calcd. for
4.2.1.8. (8S,10S)-10-(4-(N-octadecanoylamido)-5-hydroxy-6-methyl-
tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydrox-
yacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione (9). Red
brown solid (65 mg, 87.3% yield). IR (cmꢀ1, ATR): 3346. 2923, 2848,
1716 (C]O),1642(C]O str, amide), 1618, 1575, 1445, 1407, 1373, 1279,
C41H55NO12 753.3724; found, 776.2407 [M þ Na]þ.
.
1233, 1203, 1109, 1068, 1015, 844, 764; UV/vis
l (nm): 478, 290, 254,
234; 1H NMR (500 MHz, CDCl3,
d
ppm): 13.93 (s, 1H, PhOH), 13.18 (s,
4.2.1.6. (8S,10S)-10-(4-(N-(12-thioethyldodecanoylamido))-5-
hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-
8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-
dione (7). Red brown solid (66 mg, 91.4% yield). IR (cmꢀ1, ATR):
3346, 2923, 2848, 1716 (C]O), 1642 (C]O str, amide), 1618, 1575,
1445, 1407, 1373, 1279, 1233, 1203, 1109, 1068, 1015, 844, 764; UV/
1H, PhOH), 8.00 (d, J ¼ 7.7 Hz,1H, ArC2H), 7.76 (t,1H, J ¼ 8.1 Hz, ArC3H),
7.37 (d, 1H, J ¼ 8.5 Hz, ArC4H), 7.26 (s,1H, NH), 5.81 (s,1H, OH), 5.48 (br
s,1H, H-20), 5.26 (s,1H, H-50), 4.76 (s, 2H, O]C13eCH2), 4.14e4.19 (m,
2H, H-10 and H-40), 4.06 (s, 3H, OCH3), 3.67 (s, 1H, H-60), 3.24 (d, 1H,
J ¼ 18.6 Hz, H-9eq), 2.94e3.02 (m,1H, H-9ax), 2.32 (d,1H, J ¼ 13.8 Hz,
H-30eq), 2.17 (d,1H, J ¼ 3.8 Hz, H-30ax), 2.10e2.13 (m, 2H, O]CeCH2),
1.87 (dd, 1H, J ¼ 14.2 Hz, J ¼ 6.2 Hz, H-7ax),1.73e1.79 (m, 1H, H-7eq),
1.55 (t, 2H, J ¼ 6.5 Hz, NHCOCH2CH2), 1.28 (d, 3H, J ¼ 6.6 Hz, 60-CH3),
1.25 (br s, 28H, 14 ꢁ CH2, methylene envelope), 0.86e0.89 (m, 3H,
vis
l d ppm):
(nm): 478, 290, 254, 234; 1H NMR (500 MHz, CDCl3,
13.94 (s, 1H, PhOH), 13.20 (s, 1H, PhOH), 8.00 (d, J ¼ 7.6 Hz, 1H,
ArC2H), 779 (t, 1H, J ¼ 7.8 Hz, ArC3H), 7.36 (d, 1H, J ¼ 8.1 Hz, ArC4H),
7.24 (s, 1H, NH), 5.84 (d, 1H, J ¼ 7.4 Hz, OH), 5.50 (s, 1H, H-20), 5.23
(s, 1H, H-50), 4.74 (s, 2H, O]C13eCH2), 4.12e4.15 (m, 2H, H-10 and
H-40), 4.05 (s, 3H, OCH3), 3.60 (s, 1H, H-60), 3.24 (d, 1H, J ¼ 18.1 Hz,
H-9eq), 2.97 (d, 1H, J ¼ 17.7 Hz, H-9ax), 2.47e2.48 (m, 4H, SCH2),
2.30 (d, 1H, J ¼ 14.7 Hz, H-30eq), 2.14e2.19 (m, 1H, H-30a), 2.07e2.12
(m, 2H, O]CeCH2), 1.87 (s, 1H, H-7ax), 1.69e1.75 (m, 1H, H-7eq),
1.56 (br s, 4H, NHCOCH2CH2, SCH2CH2), 1.29 (s merged, 3H, 60-CH3),
1.23 (br s, 14H, 7 ꢁ CH2, methylene envelope), 0.85 (t, 3H, J ¼ 5.9 Hz,
CH3); 13C NMR (125 MHz, CDCl3,
d ppm): 213.94 (C13]O), 186.99
(C12]O),186.55 (C5]O),172.88 (NHeC]O),160.97 (C1),156.21 (C6),
155.58 (C11), 135.74 (C3), 135.43 (C10a), 133.64 (C4a), 133.61 (C6a),
120.78 (C12a), 119.83 (C2), 118.41 (C4), 111.50 (C11a), 111.37 (C5a),
100.64 (C20), 76.49 (C8), 69.60 (C60), 69.56 (C50), 67.25 (C10), 65.55
(O]CeCH2OH), 56.63 (OCH3), 45.12 (NH-C40), 36.75 (NHCOCH2),
35.72 (C30), 33.46 (C9), 29.93 (C7), 31.92, 29.74, 29.70, 29.65, 29.64,
29.63, 29.60, 29.59, 29.48, 29.46, 29.43, 29.36, 29.26, 29.24, 29.06
(methylene carbons), 25.71 (NHCOCH2CH2), 22.69 (CH2CH3), 16.82
(60-CH3), 14.12 (CH3); HR-MS (ESI-TOF): calcd. for C45H63NO12
809.4350; found, 832.2217 [M þ Na]þ.
CH3); 13C NMR (125 MHz, CDCl3,
d ppm): 213.94 (C13]O), 187.10
(C12]O), 186.65 (C5]O), 171.91 (NHeC]O), 161.01 (C1), 156.01
(C6), 155.64 (C11), 135.77 (C3) 135.48 (C10a), 133.61 (C4a), 133.55
(C6a), 120.83 (C12a), 119.86 (C2), 118.41 (C4), 111.57 (C11a), 111.37
(C5a), 100.70 (C20), 76.37 (C8), 69.37 (C60), 69.21 (C50), 66.83 (C10),
65.17 (O]CeCH2OH), 56.27 (OCH3), 43.37 (NH-C40), 36.36
4.2.1.9. (8S,10S)-10-(4-(N-Icosanoylamido)-5-hydroxy-6-methyl-tet-
rahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-

2042
B.S. Chhikara et al. / European Journal of Medicinal Chemistry 46 (2011) 2037e2042
1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione (10). Red brown
solid (68 mg, 88.2% yield). IR (cmꢀ1, ATR): 3346, 2923, 2848, 1716
(C]O), 1642 (C]O str, amide), 1618, 1575, 1445, 1407, 1373, 1279,
Cells were incubated with doxorubicin and its derivatives (1 mM) in
4% DMSO and tested in triplicate. Incubation was carried out at
37 ꢂC in an incubator supplied with 5% CO2 for 24e96 h. At the end
1233, 1203, 1109, 1068, 1015, 844, 764; UV/vis
l
(nm): 478, 290, 254,
of the sample exposure period (24e96 h), 20 mL CellTiter 96
234; 1H NMR (500 MHz, CDCl3,
d
ppm): 13.98 (s,1H, PhOH),13.24 (s,
aqueous solution was added. The plate was returned to the incu-
bator for 1 h in a humidified atmosphere at 37 ꢂC. The absorbance of
the formazan product was measured at 490 nm using microplate
reader. The percentage of cell survival was calculated as OD value of
cells treated with test compound e OD value of culture medium/
(OD value of control cells ꢀ OD value of culture medium) ꢁ 100%.
1H, PhOH), 8.04 (d, J ¼ 5.9 Hz, 1H, ArC2H), 7.79 (t, 1H, J ¼ 6.2 Hz,
ArC3H), 7.39 (d, 1H, J ¼ 6.7 Hz, ArC4H), 7.27 (s, 1H, NH), 5.82 (d, 1H,
J ¼ 6.4 Hz, OH), 5.50 (br s, 1H, H-20), 5.28 (s, 1H, H-50), 4.76 (s, 2H,
O]C13eCH2), 4.17e4.18 (m, 2H, H-10 and H-40), 4.07 (s, 3H, OCH3),
3.63 (s, 1H, H-60), 3.28 (d, 1H, J ¼ 18.5 Hz, H-9eq), 3.01 (d, 1H,
J ¼ 19.5 Hz, H-9ax), 2.33 (d, 1H, J ¼ 14.9 Hz, H-30eq), 2.17 (m, 1H, H-
30ax), 2.10e2.13 (m, 2H, O]CeCH2), 1.84e1.87 (m, 1H, H-7ax),
1.72e1.77 (m, 1H, H-7eq), 1.54 (t, 2H, J ¼ 6.0 Hz, NHCOCH2CH2), 1.29
(d, 3H, J ¼ 6.2 Hz, 60-CH3), 1.25 (br s, 32H, 16 ꢁ CH2, methylene
envelope), 0.89 (t, 3H, J ¼ 5.1 Hz, CH3); 13C NMR (125 MHz, CDCl3,
Acknowledgments
We acknowledge the financial support from the American
Cancer Society Grant # RSG-07-290-01-CDD and National Science
Foundation, Grant # CHE 0748555. We also acknowledge National
Center for Research Resources, NIH, and Grant Number 1 P20
RR16457 for sponsoring the core facility.
d
ppm): 213.94 (C]O), 187.10 (C12]O), 186.65 (C5]O), 172.61
(NHeC]O), 160.61 (C1), 155.80 (C6), 155.26 (C11), 135.37 (C3)
135.11 (C10a), 133.22 (C4a), 133.14 (C6a), 120.83 (C12a), 119.47 (C2),
118.00 (C4), 111.19 (C11a), 111.00 (C5a), 100.30 (C20), 76.14 (C8),
69.28 (C60), 69.24 (C50), 66.81 (C10), 65.17 (O]CeCH2OH), 56.33
(OCH3), 44.58 (NH-C40), 36.38 (NHCOCH2), 35.32 (C30), 33.59 (C9),
29.59 (C7), 33.07, 31.53, 29.30, 98.08, 29.03, 28.97, 28.85 (methy-
lene carbons), 25.30 (NHCOCH2CH2), 22.90 (CH2CH3), 16.44 (60-
CH3) 13.74 (CH3); HR-MS (ESI-TOF): calcd. for C47H67NO12 837.4663;
found 860.2712 [M þ Na]þ.
Appendix. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.02.056.
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Concentration of sample in water
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and represented as Log P where P is partition coefficient.
4.3. Cell culture
Human leukemia cell line CCRF-CEM (ATCC no. CCL-119), breast
adenocarcinoma MDA-MB-468 (ATCC no. HTB-132), ovarian
adenocarcinoma SK-OV-3 (ATCC no. HTB-77), and colon adeno-
carcinoma HT-29 (ATCC no. HTB-38) were obtained from American
Type Culture Collection. Cells were grown on 75 cm2 cell culture
flasks with RPMI-16 medium for leukemia and EMEM medium for
other cell lines and supplemented with 10% Fetal bovine serum
(FBS), and 1% penicillinestreptomycin solution (10,000 units of
penicillin and 10 mg of streptomycin in 0.9% NaCl) in a humidified
atmosphere of 5% CO2, 95% air at 37 ꢂC.
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4.4. Cell proliferation assay
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Cell proliferation assay of doxorubicin derivatives synthesized
was evaluated in MDA-MB-468, CCRF-CEM, SK-OV-3, and HT-29
cells, and was compared with that of doxorubicin. Cell proliferation
assay was carried out using CellTiter 96 aqueous one solution cell
proliferation assay kit (Promega, USA). As a representative example,
CCRF-CEM cells were suspended at 5 ꢁ 105/mL and 100
mL of the
cell suspension was placed in each well of the 96 well culture plate.