Thermoresponsive dendrimers based on oligoethylene glycols: Design, synthesis and cytotoxic activity against MCF-7 breast cancer cells

Article abstract of DOI:10.1016/j.ejmech.2013.09.019

Three interesting thermoresponsive branched oligoethylene glycol dendrimers based on tetrabromohydroquinone were efficiently synthesized from tetrabromohydroquinone and three different oligoethylene glycol derivatives. By visual inspection, all these dend

Full text of DOI:10.1016/j.ejmech.2013.09.019

European Journal of Medicinal Chemistry 69 (2013) 848e854
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Thermoresponsive dendrimers based on oligoethylene glycols:
Design, synthesis and cytotoxic activity against MCF-7 breast cancer
cells
,
b
Mona A. Abdel-Rahman a , Ahmed M. Al-Abd
*
a Chemistry Department, Polymer Lab. 122, Faculty of Science, Assiut University, PB 71516, Assiut, Egypt
b Department of Pharmacology, Medical Division, National Research Center, Dokki, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Three interesting thermoresponsive branched oligoethylene glycol dendrimers based on tetra-
bromohydroquinone were efficiently synthesized from tetrabromohydroquinone and three different
oligoethylene glycol derivatives. By visual inspection, all these dendrimers are water-soluble at room
temperature. The thermoresponsive behaviors were investigated by using UV/vis turbidity measurement
at different temperatures for 0.25 wt% of aqueous solutions from D1, D2 and D3. The cytotoxicity of the
prepared dendrimers was tested against MCF-7 breast cancer cells. All tested dendrimers showed
considerable results, where D2 dendrimer gave the best result; it showed cytotoxicity against MCF-7 cell
Received 6 November 2012
Received in revised form
6 September 2013
Accepted 7 September 2013
Available online 19 September 2013
Keywords:
Cytotoxicity
Tetrabromohydroquinone
Thermoresponsive dendrimers
Synthesis
line with IC50 of 1.07
m
g/mL and resistant fraction equals 1.97%, the other two dendrimers showed a
modest cytotoxic profile.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Water-soluble dendrimers
1. Introduction
promise in tissue targeting applications, controlled drug release;
moreover, their interesting nanoscopic architecture might allow
easier passage across biological barriers by transcytosis [18].
Attention has further been paid to thermally responsive den-
drimers for wider application purposes [19e21], like having ma-
terials with sharp, fast, and fully reversible phase transitions
covering a broad temperature range [1,22,23].
The central core of these new dendrimers is hydroquinone
moiety. Quinones and their reduced form hydroquinones are
ubiquitous in nature. Several of them are obtained from natural
sources [24,25]. The sustained interest in quinone/hydroquinone is
in part due to the several biological activities, which related to their
redox potential [26e29]. These compounds provided interesting
applications as anticancer, potential antitumor and antifungal
agents [30e33]. Moreover, some hydroquinone derivatives showed
inhibition in tumor cell respiration [30,34].
In the course of a medicinal chemistry program which aimed to
the synthesis of new hydroquinone derivatives, three thermores-
ponsive branched oligoethylene glycol dendrimers based on tet-
rabromohydroquinone were synthesized and characterized by 1H
NMR, 13C NMR and mass analyses. Thermoresponsive behavior and
cytotoxicity of these dendrimers against MCF-7 breast cancer cells
were tested in the present work.
Dendrimers are highly branched macromolecules of low poly-
dispersity that provide many exciting applications in areas such as
biomaterials [1e4], molecular devices [5e8], and catalysis [9]. The
properties of dendrimers are dominated by the functional groups
on the molecular surface; however, there are examples of den-
drimers with internal functionality [10e12]. Dendritic encapsula-
tion of functional molecules allows for the isolation of the active
site, a structure which mimics that of active sites in biomaterials
[13e15]. Also, it is possible to make water-soluble dendrimers,
unlike most polymers, by functionalizing their outer shell with
charged species or other hydrophilic groups. In addition, control-
lable properties of dendrimers include toxicity, crystallinity, tecto-
dendrimer formation, and chirality [16]. The high structural and
chemical homogeneity of dendrimers structure allows bio-
distribution and pharmacokinetics to be tuned and their degrada-
tion to be controlled [17]. As a consequence, dendrimers hold
* Corresponding author. Fax: þ20 88 2342708.
E-mail addresses: manoush00@yahoo.com, mona.ali1@science.au.edu.eg
(M.A. Abdel-Rahman).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.019

M.A. Abdel-Rahman, A.M. Al-Abd / European Journal of Medicinal Chemistry 69 (2013) 848e854
849
2. Experimental part
then a white crystalline product precipitated; yield 92%: m.p.: 248e
ꢀC. The solid product was used in the next step without further
9
2.1. Chemicals and drugs
purification. C6H2Br4O2: Calc. C16.92, H 0.473, Br 75.08; Found:
C16.94, H 0.500, Br 75.42.
All dendrimers are new; details of the synthesis and charac-
terization of these dendrimers are given in the experimental part.
Tetrahydrofuran (THF) was refluxed over lithium aluminum hy-
dride (LAH) and dichloromethane (DCM) was distilled from CaH2
for drying. Sulfarhodamine was purchased from SigmaeAldrich (St.
Louis, MO, USA). Other reagents, materials and solvents were pur-
chased and used as received unless otherwise stated. All reactions
were run under a nitrogen atmosphere. Silica gel 60 M (Machereye
Nagel, 0.04e0.063 mm, 230e400 mesh) was used as the stationary
phase for column chromatography. Whenever possible, reactions
were monitored by thin-layer chromatography (TLC) using TLC
silica gel coated aluminum plates 60F254 (Merck).
2.3.3. Methyl 3,4,5-tris(2-(2-(2-ethoxyethoxy)ethoxy)ethoxy)
benzoate (3)
A mixture of methyl gallate (2.76 g, 15 mmol), tosylated com-
pound 1 (19.94 g, 60 mmol), KI (1.99 g, 12 mmol), and potassium
carbonate (K2CO3) (20.73 g, 150 mmol) in dry DMF (100 mL) was
stirred at 80 ꢀC over 24 h. After removal of DMF in vacuo, the res-
idue was dissolved in DCM and washed sequentially with saturated
NaHCO3 and brine. After drying over MgSO4, purification by column
chromatography with DCM/MeOH (20:1) afforded the product as
colorless oil; yield: 83%. 1H NMR (CDCl3):
d
¼ 1.15 (t, 9H, 3CH3),
3.45e3.87 (m, 39H,18CH2 þ CH3), 4.16 (t, 6H, 3CH2), 7.28 (s, 2H, Are
H). MS (MALDIeTOF): m/z calc, 664.78; found, 687.35 [M þ Na]þ.
2.2. Instrumentation
2.3.4. 3,4,5-Tris(2-(2-(2-ethoxyethoxy)ethoxy)ethoxy) benzyl
alcohol (4)
1H and 13C NMR spectra were recorded on a Bruker AM 300 (1H:
300 MHz) and AV 500 (1H: 500 MHz) spectrometers, and chemical
shifts are reported relative to internal Me4Si. For NMR measure-
ment of compounds D1, D2 and D3, the sample weight is from 5 to
25 mg, and 0.7 mL solvent, the sample measurement takes place
overnight. High-resolution MALDI-TOF mass spectrometry (MS),
analysis was performed by the MS-service of the Laboratorium für
Organische Chemie at ETH Zürich {dithranol (DIT) and trans-2-[3-
(4-tert-butylphenyl)-2-methyl-2-propenylidenemalononitrile]
(DCTB) were used as matrices}. The samples were dried rigorously
under vacuum prior to analysis to remove strongly-adhering sol-
vent molecules. For the evaluation of the thermoresponsive
behavior of compounds D1, D2 and D3, UV/vis turbidity measure-
ments were carried out for the lower critical solution temperature
(LCST) determination, UV/vis spectrophotometer e Perkin Elmer e
Lambda 35 (at Assiut university, Egypt), equipped with a thermo-
statically regulated bath. Solutions of the dendrimers in de-ionized
LAH (0.95 g, 25.1 mmol) was added to a solution of ester com-
pound 3 (9.97 g, 15 mmol) in dry THF (50 mL) at ꢁ5 ꢀC, the mixture
was stirred for 30 min, then warmed to r.t. and stirred for another
3 h. The reaction was quenched by dropwise addition of water
(5 mL), 10% NaCl (10 mL), and water (20 mL) successively. The
resulting precipitate was filtered and THF evaporated. The residue
was dissolved in DCM and washed with brine. After drying over
MgSO4, purification by column chromatography with DCM/MeOH
(20:1) afforded the product as colorless oil; yield: 73%. 1H NMR
(CDCl3):
d
¼ 1.2 (t, 9H, 3CH3), 2.11 (s, 1H, OH), 3.29e3.69 (m, 30H,
15CH2), 3.79 (t, 2H, CH2), 3.84 (t, 4H, 2CH2), 4.13 (t, 2H, CH2), 4.17 (t,
4H, 2CH2), 4.59 (s, 2H, CH2OH), 6.77 (s, 2H, AreH). MS (MALDI-
TOF): m/z calc, 636.77; found, 659.35 [M þ Na]þ.
2.3.5. 3,4,5-Tris(2-(2-(2-ethoxyethoxy)ethoxy)ethoxy) benzyl
chloride (5)
water (with concentration 0.25 wt%) were filtered with a 0.45
filter before adding into a cuvette (path length 1 cm), which was
m
m
4-Dimethylaminopyridine (DMAP) (0.734 g, 6 mmol) was added
to a solution of alcohol compound 4 (3.184 g, 5 mmol) in dry DCM
(30 mL) at ꢁ5 ꢀC, the mixture was stirred for 10 min, then SOCl2
(1.180 g, 25 mmol) in dry DCM (10 mL) was added to the mixture,
stirred for 30 min, then warmed to r.t. and stirred for another 1 h.
The reaction was quenched by dropwise addition of 10% NaCl
(10 mL), and water (20 mL) successively. After phase separation the
water phase was extracted three times with DCM. The combined
organic phases were dried over MgSO4 and the solvent was evap-
orated under reduced pressure, purification by column chroma-
tography with DCM/MeOH (20:1) afforded the product as yellow
placed in tꢁh1e spectrophotometer and heated or cooled at a rate of
0.2 ꢀC min
.
2.3. Monomers syntheses
2.3.1. Monotosyloxy triethylene glycol monoethylether (1)
Triethylene glycol monoethylether (20 g, 112 mmol) was dis-
solved in 20 mL THF, NaOH solution (6.73 g NaOH, 168 mmol in
15 mL H2O). The mixture was cooled down to 0 ꢀC by ice/water
bath. p-Tosyl chloride (TsCl) (25.67 g, 135 mmol) in THF (150 mL)
was added dropwise to the mixture and the reaction was started
simultaneously. The solution was stirred for another 1 h, then the
solvent was evaporated. The residue was dissolved in DCM and
washed with brine (if there is a lot of salt we can wash first the
organic layer with water). After that drying over MgSO4, the crude
product was further purified by column chromatography with
hexane/ethylacetate (2:1) as eluant, afforded the product as a
oil; yield: 85%. 1H NMR (CDCl3):
d
¼ 1.19 (t, 9H, 3CH3), 3.44e3.75 (m,
30H, 15CH2), 3.79 (t, 2H, CH2), 3.85 (t, 4H, 2CH2), 4.04 (t, 2H, CH2),
4.17 (t, 4H, 2CH2), 4.50 (s, 2H, CH2Cl), 6.58 (s, 2H, AreH). MS
(MALDI-TOF): m/z calc, 655.21; found, 677.32 [M þ Na]þ.
2.3.6. 2-(2-(2-Hydroxyethoxy)ethoxy)ethyl 4-
methylbenzenesulfonate (6)
Triethylene glycol (16.81 g, 112 mmol) was dissolved in 20 mL
THF, NaOH solution (6.73 g, 168 mmol in 15 mL H2O). The mixture
was cooled down to 0 ꢀC by ice/water bath, then TsCl (25.67 g,
135 mmol) in THF (150) was added dropwise to the mixture and the
reaction was started simultaneously. The solution was stirred for
another 1 h and the solvent was evaporated. The residue was dis-
solved in DCM and washed with brine (if there is a lot of salt we can
wash first the organic layer with water). After that drying over
MgSO4, the crude product was further purified by column chro-
matography with hexane/ethylacetate (2:1) as eluant, afforded the
colorless oil; yield: 93%. 1H NMR (CDCl3):
d
¼ 1.18 (t, 3H, CH3), 2.46
(s, 3H, CH3), 3.52e3.73 (m, 14H, 7CH2), 7.34 (d, 2H, AreH), 7.80 (d,
2H, AreH). MS (MALDI-TOF): m/z calc, 332.41; found, 355.11
[M þ Na]þ.
2.3.2. Tetrabromohydroquinone (2)
Tetrabromo-p-benzoquinone (4.23 g, 10 mmol) was dissolved in
20 mL glacial acetic acid, stannous chloride (13 g, 6 mmol in 15 mL
HCl). The mixture was refluxed for 3 h, while the yellow color
turned to off white (or colorless). The reaction mixture was cooled,
product as yellow oil; yield: 89%. 1H NMR (CDCl3):
d
¼ 2.44 (s, 3H,

850
M.A. Abdel-Rahman, A.M. Al-Abd / European Journal of Medicinal Chemistry 69 (2013) 848e854
CH3), 3.51e3.70 (m, 12H, 6CH2), 7.34 (d, 2H, AreH), 7.80 (d, 2H, Are
H). MS (MALDI-TOF): m/z calc, 304.1; found, 327.21 [M þ Na]þ.
(CDCl3):
d
¼ 1.20 (t, 9H, 3CH2), 2.18 (s, 3H, CH3), 3.49e3.70 (m,
38H, 16CH2), 3.74 (t, 4H, 2CH2), 3.84 (t, 4H, 2CH2), 4.08e4.19 (m,
8H, 4CH2), 4.45 (s, 2H, CH2), 6.59 (s, 2H, AreH), 7.34 (d, 2H, Are
H), 7.80 (d, 2H, AreH). MS (MALDI-TOF): m/z calc, 923.11; found,
945.44 [M þ Na]þ.
2.3.7. 2-(2-(2-(Tetrahydro-2H-pyran-2-yloxy)ethoxy)ethoxy)ethyl-
4-methyl-benzenesulfonate (7)
3,4-Dihydro-2H-pyran (DHP) (13.04 g, 155 mmol) and pyr-
idinium toluenesulfonate (PPTS) (3.89 g, 15.5 mmol) were added to
a solution of compound 6 (7.854 g, 25.83 mmol) in dry DCM at
ꢁ5 ꢀC, and the mixture was stirred for another 6 h at room tem-
perature. After successive washing with aqueous NaHCO3 solution
and brine, the organic phase was dried over MgSO4. The crude
product was further purified by column chromatography with
hexane/ethyl acetate (2:1) as eluant, afforded the product as
2.4. Dendrimers syntheses
2.4.1. 1,2,4,5-Tetrabromo-3,6-bis(2-(2-(2-ethoxyethoxy)ethoxy)
ethoxy)benzene (D1)
A mixture of tetrabromohydroquinone (1.70 g, 4.20 mmol),
tosylated compound
1 (4.18 g, 12.60 mmol), KI (0.418 g,
2.52 mmol), and K2CO3 (4.35 g, 31.6 mmol) in dry DMF (40 mL)
was stirred at 80 ꢀC over 24 h. After removal of DMF in vacuo, the
residue was dissolved in DCM and washed sequentially with
saturated NaHCO3 and brine. After drying over MgSO4, purification
by column chromatography with hexane/ethylacetate (3:1) affor-
colorless oil; yield: 89%. 1H NMR (CDCl3):
d
¼ 1.48 (m, 2H, CH2), 1.55
(m, 2H, CH2),1.68e1.71 (m,1H, CH2),1.79e1.80 (m,1H, CH2), 2.44 (s,
3H, CH3), 3.51e3.80 (m, 12H, 6CH2), 4.14 (t, 2H, CH2), 4.60 (s, H, CH),
7.39 (d, 2H, AreH), 7.80 (d, 2H, AreH). MS (MALDI-TOF): m/z calc,
388.16; found, 411.14 [M þ Na]þ.
ded the product as pink oil. 1H NMR (CDCl3):
d
¼ 1.19 (t, 6H, 2CH3),
3.50e3.78 (m, 20H, 10CH2), 3.92 (t, 4H, 2CH2), 4.15 (t, 4H, 2CH2).
13C NMR (CDCl3):
2.3.8. 2-(2-(2-(2-(3,4,5-Tris(2-(2-(2-ethoxyethoxy)ethoxy)ethoxy)
benzyloxy) ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran (8)
Compound 7 (1.60 g, 4.16 mmol) in dry THF (30 mL) was added
d
¼ 15.58, 67.04, 70.24, 70.33, 71.08, 71.16, 71.28,
72.84, 121.78, 152.23. MS (MALDI-TOF): m/z calc, 746.12; found,
768.69 [M þ Na]þ.
dropwise to
a mixture of alcohol compound 4 (8.743 g,
13.73 mmol), KI (1.975 g, 11.90 mmol), 15-crown-5 (0.916 g,
4.16 mmol), and NaH (0.998 g, 41.6 mmol) in dry THF (60 mL). The
mixture was stirred for 24 h at r.t. before addition of MeOH to
quench the excess NaH. After evaporation of solvent in vacuo, the
residue was dissolved in DCM and successively washed with
saturated NaHCO3 and brine. After drying over MgSO4, purification
by column chromatography with DCM/MeOH (15:1) afforded the
2.4.2. 1,2,4,5-Tetrabromo-3,6-bis(3,4,5-tris(2-(2-(2-ethoxyethoxy)
ethoxy)ethoxy) benzyloxy)benzene (D2)
A mixture of tetrabromohydroquinone (0.49 g, 1.15 mmol),
compound 6 (1.00 g, 1.50 mmol), KI (0.047 g, 0.287 mmol) and
Cs2CO3 (0.93 g, 2.87 mmol) in dry DMF (15 mL) was stirred at
80 ꢀC for 12 h. After removal of DMF in vacuo, the residue was
dissolved in DCM and washed sequentially with saturated NaHCO3
and brine. After drying over MgSO4, purification by column
chromatography with DCM/MeOH (10:1) afforded the product as
product as yellow oil; yield: 94%. 1H NMR (CDCl3):
d
¼ 1.17e1.20 (m,
9H, CH3), 1.51e1.56 (m, 4H, 2CH2), 1.69e1.72 (m, 1H, CH2), 1.80e1.81
(m, 1H, CH2), 3.46e3.70 (m, 42H, 21CH2), 3.76 (t, 2H, CH2), 3.80e
3.85 (m, 6H, 3CH2), 4.10 (t, 2H, CH2), 4.14e4.16 (m, 4H, CH2), 4.43 (s,
2H, CH2), 4.59 (t, 1H, CH), 6.59 (s, 2H, AreH). MS (MALDI-TOF): m/z
calc, 853.04; found, 875.49 [M þ Na]þ.
yellowish brown oil. 1H NMR (CDCl3):
3.46e3.70 (m, 60H, 30CH2), 3.76 (t, 4H, 2CH2), 3.84 (t, 8H, 4CH2),
d
¼ 1.15e1.17 (m, 18H, 6CH3),
4.12 (t, 4H, 2CH2), 4.16 (t, 8H, 4CH2), 4.87 (s, 4H, 2CH2), 6.80 (s, 4H,
AreH). 13C NMR (CDCl3):
d
¼ 15.54, 66.96, 69.32, 69.97, 70.09,
70.18, 71.03, 71.05, 71.20, 72.70, 108.58, 122.14, 131.40, 139.16,
151.88, 153.08. MS (MALDI-TOF): m/z calc, 1663.20; found, 1685.17
[M þ Na]þ.
2.3.9. 2-(2-(2-(3,4,5-Tris(2-(2-(2-ethoxyethoxy)ethoxy)ethoxy)
benzyloxy)ethoxy) ethoxy) ethanol (9)
p-Toluenesulfonic acid (PTSA) (0.215 g, 1.25 mmol) was added to
a solution of compound 8 (7.10 g, 8.32 mmol) in MeOH (50 mL), and
the mixture was stirred for 2 h at room temperature. Then MeOH
was evaporated under vacuum at room temperature. The residue
was dissolved in DCM and successively washed with aqueous
NaHCO3 solution and brine. After drying over MgSO4, purification
by column chromatography with DCM/MeOH (10:1) afforded the
2.4.3. 1,2,4,5-Tetrabromo-3,6-bis(2-(2-(2-(3,4,5-tris(2-(2-(2-
ethoxyethoxy)ethoxy) ethoxy) benzyloxy)ethoxy)ethoxy)ethoxy)
benzene (D3)
A mixture of tetrabromohydroquinone (0.85 g, 2.10 mmol),
tosylated compound 12 (5.81 g, 6.30 mmol), KI (0.209 g, 1.26 mmol)
and K2CO3 (2.17 g, 15.8 mmol) in dry DMF (20 mL) was stirred at
80 ꢀC over 24 h. After removal of DMF in vacuo, the residue was
dissolved in DCM and washed sequentially with saturated NaHCO3
and brine. After drying over MgSO4, purification by column chro-
matography with hexane/ethylacetate (4:1) afforded the product as
product as colorless oil; yield: 90%. 1H NMR (CDCl3):
d
¼ 1.16e1.20
(m, 9H, 3CH3), 2.59 (s, 1H, OH), 3.49e3.70 (m, 42H, 21CH2), 3.74 (t,
2H, CH2), 3.84 (t, 4H, 2CH2), 4.10e4.15 (m, 6H, 3CH2), 4.43 (s, 2H,
CH2), 6.59 (s, 2H,þAreH). MS (MALDI-TOF): m/z calc, 768.93; found,
791.44 [M þ Na]
.
yellowish brown oil. 1H NMR (CDCl3):
3.70 (m, 76H, 38CH2), 3.75 (t, 8H, 4CH2), 3.79 (t, 8H, 4CH2), 3.90 (t,
d
¼ 1.17 (t, 18H, 6CH3), 3.44e
2.3.10. 2-(2-(2-(3,4,5-Tris(2-(2-(2-ethoxyethoxy)ethoxy)ethoxy)
benzyloxy)ethoxy) ethoxy)ethyl-4-methylbenzenesulfonate (10)
Compound 9 (8.60 g, 11.2 mmol) was dissolved in 10 mL THF,
NaOH solution (0.673 g, 16.8 mmol in 15 mL H2O). The mixture
was cooled down to 0 ꢀC by ice/water bath. Then TsCl (2.56 g,
13.5 mmol) in THF 50 mL was added dropwise to the mixture and
the reaction was started simultaneously. The solution was stirred
for another 1 h, then the solvent was evaporated. The residue
was dissolved in DCM and washed with brine (if there is a lot of
salt we can wash first the organic layer with water). After that
drying over MgSO4, the crude product was further purified by
column chromatography with hexane/ethylacetate (2:1) as
eluant, afforded the product as colorless oil; yield: 83%. 1H NMR
4H, 2CH2), 4.09e4.14 (m, 12H, 6CH2), 4.43 (s, 4H, 2CH2), 6.80 (s, 4H,
AreH). 13C NMR (CDCl3):
d
¼ 15.55, 66.98, 69.19, 69.76, 70.19, 70.87,
71.00, 71.04, 71.06, 71.11, 71.18, 71.25, 72.66, 73.61, 107.58, 121.77,
134.14, 138.16, 152.22, 152.99. MS (MALDI-TOF): m/z calc, 1927.52;
found, 1949.21 [M þ Na]þ.
2.5. Cell culture
Human breast cancer cell line, MCF-7, was obtained from the
Vaccera (Giza, Egypt). Cells were maintained in RPMI-1640 sup-
plemented with 100 mg/mL streptomycin, 100 units/mL penicillin
and 10% heat-inactivated fetal bovine serum in a humidified, 5% (v/
v) CO2 atmosphere at 37 ꢀC.

M.A. Abdel-Rahman, A.M. Al-Abd / European Journal of Medicinal Chemistry 69 (2013) 848e854
851
SO2Cl
O
S
O
NaOH/H2O, THF
0oC, 2 h
O
+
HO
O
O
O
O
O
O
O
1
Br
OH
Br
KI, K2CO3 / DMF
80oC, 24 h
Br
Br
Br
Br
O
O
1
+
O
O
O
O
O
Br
Br
OH
2
D1
Scheme 1. Synthetic sequence of dendrimer D1.
O
O
O
O
O
O
OH
O
O
HO
OH
O
O
KI, K2CO3 / DMF
80oC, 24 h
+
O
O
O
S
O
O
O
O
O
O
O
O
O
3
1
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
LAH / THF
(-5oC/0.5 h), (rt./3 h)
SOCl2, DMAP / DCM
rt./1 h
O
O
O
O
OH
Cl
5
4
O
O
O
O
O
O
O
OH
O
O
O
O
Br
Br
Br
Br
Br
Br
O
O
O
O
5
KI, Cs2CO3 / DMF
80oC, 12 h
+
O
O
O
O
O
O
Br
Br
O
OH
O
O
2
O
O
D2
Scheme 2. Synthetic sequence of dendrimer D2.
2.6. Cytotoxicity assays
2.6.1. Data analysis
The dose response curve of compounds was analyzed using Emax
The cytotoxicity of prepared dendrimers was tested against
MCF-7 cells by SRB assay as previously described [35]. Exponen-
tially growing cells were collected using 0.25% TrypsineEDTA and
plated in 96-well plates at 1000e2000 cells/well. Cells were
exposed to test compounds for 72 h and subsequently fixed with
TCA (10%) for 1 h at 4 ꢀC. After several washings, cells were exposed
to 0.4% SRB solution for 10 min in dark place and subsequently
washed with 1% glacial acetic acid. After drying overnight, TriseHCl
was used to dissolve the SRB-stained cells and color intensity was
measured at 540 nm.
model (Eq. (1)).
!
m
½Dꢃ
Kdm þ ½Dꢃ
% Cell viability ¼ ð100 ꢁ RÞ ꢂ 1 ꢁ
þ R
(1)
m
where [R] is the residual unaffected fraction (the resistance frac-
tion), [D] is the drug concentration used, [Kd] is the drug concen-
tration that produces a 50% reduction of the maximum inhibition
rate and [m] is a Hill-type coefficient. IC50 was defined as the drug

852
M.A. Abdel-Rahman, A.M. Al-Abd / European Journal of Medicinal Chemistry 69 (2013) 848e854
SO Cl
O
S
O
O
S
O
DHP, PPTS, DCM
-5 to 25 °C, 6 h
O
NaOH/H O, THF
0 C, 2 h
O
HO
O
OH
O
+
O
OH
O
O
O
O
O
O
7
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
7
O
O
O
O
O
O
O
O
TSCl, NaOH/H O
THF, 0 °C, 2 h
KI, 15-crown-5, NaH
THF, r.t., 24 h
+
PTSA, MeOH
50 °C, 2 h
O
O
OH
O
4
8
O
9
10
O
O
O
O
O
O
O
S O
OH
O
O
O
O
O
O
O
O
OH
O
Br
Br
Br
Br
Br
Br
Br
O
O
O
KI, K CO / DMF
80 C, 24 h
O
O
O
10
O
O
+
O
O
O
O
O
O
O
O
O
O
Br
OH
O
O
O
O
O
D3
2
O
O
Scheme 3. Synthetic sequence of dendrimer D3.
concentration required to reduce fluorescence to 50% of that of the
control (i.e., Kd ¼ IC50 when R ¼ 0 and Emax ¼ 100 ꢁ R) [36]. Data are
presented as mean ꢄ SD.
compound 9 [38]. Compound 10 was reacted with tetrabromohy-
droquinone giving dendrimer D3 as delineate in Scheme 3.
Dendrimers D1 and D2 are considered as first-generation den-
drimers, but with different numbers of the branched oligoethylene
glycols, while dendrimer D3 can be considered as second-
generation dendrimer produced from dendrimer D1.
3. Results and discussion
All new compounds and dendrimers gave fully assignable 1H
and 13C NMR spectra (see Supporting Information) and showed the
molecular ion peaks in the matrix-assisted laser desorption/ioni-
zation time-of-flight (MALDI-TOF) mass spectra. All dendrimers
gave a single spot in TLC.
3.1. Synthesis of dendrimers and macromonomers
Three new thermoresponsive dendrimers were prepared. These
dendrimers consist of branched oligoethylene glycol dendrimers
based on tetrabromohydroquinone. Tosylated triethylene glycol
monoethylether (Et-TEG-Ts) was reacted with tetrabromohy-
droquinone to give dendrimer D1 as delineate in Scheme 1. Methyl
gallate was reacted with Et-TEG-Ts according to an established
procedure [37] to give compound 3. The reduction of compound 3
to give compound 4 with a focal alcohol group was attempted with
LAH, which was obtained with excellent yield (89%). The latter was
reacted with SOCl2 in the presence of 4-dimethylaminopyridine
(DMAP) [1], to afford the corresponding benzyl chloride 5. Com-
pound 5 was reacted with tetrabromohydroquinone giving den-
drimer D2 (see Scheme 2). The hydroxyl group of monotosylated
triethylene glycol (TEG-Ts) was THP protected to give compound 7.
Compound 4 was reacted with compound 7 according to a known
procedure using NaH as a base in the presence of KI/15-crown-5
[23] to give compound 8, which deprotected successfully producing
compound 9. Compound 10 was produced by tosylation of
3.2. Thermoresponsive behavior
At room temperature, all dendrimers are water-soluble. How-
ever, when they are heated the clear aqueous solutions turn turbid.
The thermoresponsive behavior of these dendrimers was investi-
gated by turbidity measurements using UV/vis spectroscopy [1].
The transmittance during heating and cooling processes of
aqueous solutions for dendrimers D1, D2 and D3 is plotted in figures
(see Supporting Information). The lower critical solution tempera-
ture (LCST) for dendrimer D1 is at 28.2 ꢀC, LCST for dendrimer D2 is
at 28.4 ꢀC, while dendrimer D3 shows LCSTat 36 ꢀC. The conclusions
from these data are: (a) The transmittance is near 100% at temper-
atures below LCST but near zero % at temperatures above LCST. (b)
All investigated dendrimers show sharp transitions in both heating

M.A. Abdel-Rahman, A.M. Al-Abd / European Journal of Medicinal Chemistry 69 (2013) 848e854
853
and cooling directions (the difference between the two directions is
less than 0.6 ꢀC). (c) LCSTs are in range of 28e36 ꢀC.
The effect of the concentration (from 0.1% to 1%) of D1, D2 and
D3 on their LCSTs was also studied (see Supporting Information).
By increasing the dendrimer concentration (from 0.1% to 1%)
there is slightly decrease in its LCST. The decrease in LCST is less
than 2ꢀ. These results show that the dendrimer concentration has
insignificant influence on its LCST. LCST of dendrimer D3 is
higher than that for dendrimers D1 and D2, indicating that LCST
depends on the degree of generation for the dendrimer. These
results indicate that the second-generation of ethoxy terminated
dendrimer is more hydrophilic than the first-generation den-
drimers, these results are in accordance to the previously pub-
lished data [1].
3.3. Cytotoxicity assessment
SRB-U assay was used to assess the cytotoxicity of the tested
dendrimers against MCF-7 breast cancer cell line. The cytotoxicity
parameters, IC50 and R-fraction were calculated using Emax model as
described in the experimental part. All tested dendrimers showed
considerable cytotoxicity against MCF-7 cell line with IC50 ranging
from 1.07 to 25.4
dendrimers were found to be ranging from 1.97 to 11.22%. D2
g/mL)
mg/mL. Resistant fractions of MCF-7 cells to tested
dendrimer showed the most potent cytotoxicity (IC50 ¼ 1.1
m
with gradual drop in viability with increasing concentration (see
Fig. 1).
It was proposed that dendrimers shape and charge play a part in
forming dendrimer-lipid vesicles by removing individual lipid
molecules from the membrane [7]. The mechanism of drug delivery
to the inside of the cell is favored by the dendrimer lipid bilayer
interaction. In dendrimers having a charged group, the interaction
between these groups with the cell membrane disrupts the cell
membrane. The ratio of lipid head groups (L) in contact with the
dendrimer surface to the number of dendrimer peripheral end
groups (P) seems to be a determining factor in whole formation
[7,39].
The obtained results showed that in relation to the cytotoxicity
of the dendrimers, dendrimer D2 has more cytotoxic effect than
dendrimer D1, and dendrimer D3 is the least one. A possible
explanation for these results is according to the thermoresponsive
behavior of these dendrimers. Dendrimer D3 is more hydrophilic
than dendrimers D1 and D2, its cytotoxic effect on the cancer cells
will be the least one, because by increasing the hydrophilicity of
the compound the ability of penetration of the cell membrane will
decrease. Another explanation for these results is according to the
number of the terminal branches. The number of the terminal
branches of dendrimer D2 is more than that of dendrimer D1. By
increasing the number of the terminal branches the cytotoxic ef-
fect on the cancer cells will increase. By increasing the numbers of
water molecules which surround the dendrimers molecules
through hydrogen bondings, the size of the dendrimer will in-
crease and the ability of penetration the cell membrane will
decrease.
4. Conclusion
In this research a novel thermoresponsive branched oligo-
ethylene glycol dendrimers based on tetrabromohydroquinone
were prepared. These dendrimers are water-soluble at room tem-
perature. The cytotoxicity of these dendrimers was tested against
MCF-7 breast cancer cells showing considerable cytotoxic effect.
Acknowledgments
The authors cordially thank Prof. A. D. Schlüter, for helping in
making some analysis through his group in ETH Zürich. Prof. A.
Zhang is cordially thanked for his kind help in general laboratory
matters.
Fig. 1. Doseeresponse curve of testing dendrimers against MCF-7 breast cancer solid
tumor cell line. Cells were exposed to the tested dendrimers for 72 h. Cell viability was
determined using SRB-U assay and data are expressed as mean ꢄ SD (n ¼ 3).

854
M.A. Abdel-Rahman, A.M. Al-Abd / European Journal of Medicinal Chemistry 69 (2013) 848e854
ture and biocompatibility in vitro, and preliminary studies on the bio-
distribution of 125I-labelled polyamidoamine dendrimers in vivo, J. Control.
Release 65 (2000) 133e148.
Appendix A. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.ejmech.
2013.09.019.
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