Synthesis of steroidal dendrimers modified by 'click' chemistry with PAMAM dendrons as unimolecular micelles

Article abstract of DOI:10.1016/j.tetlet.2013.12.066

Novel Fréchet-PAMAM hybrid dendrimers linked by triazole units as unimolecular micelles with a hydrophobic core surrounded by a hydrophilic shell were prepared. The dendritic cores with 3 and 6 alkyne terminal groups were synthesized from 1,3,5-tribromomethyl-benzene (tBrMeB), in one case by direct coupling with 17α-ethynylestradiol (EE); in the second one the tBrMeB was reacted with bis(hydroxymethyl) phenol followed by chlorination of the hydroxyl groups and subsequent coupling to EE. With this strategy, the core can be grown by further substitutions of bis(hydroxymethyl) phenol over the halogenated terminals as Fréchet dendrimer. The hydrophilic shells used were PAMAM type dendrons of 0.5 and 1.5 generations with azide as focal point and tert-butyl ester as end groups. The unimolecular micelles were obtained by cycloaddition between an azide in the selected dendron and the alkyne terminal in the hydrophobic core to obtain a 1,4-disubstituted 1,2,3-triazole. Once the coupling was achieved, the tert-butyl ester groups were hydrolyzed in trifluoroacetic acid and the corresponding dendrimers with carboxylic acid as end groups were completely soluble in phosphate buffer solutions of pH 7.0, 7.4, and 8.0. All hybrid dendrimers were characterized by High Resolution Mass Spectrometry, ¹H and ¹³C NMR, and FTIR.

Full text of DOI:10.1016/j.tetlet.2013.12.066

Tetrahedron Letters 55 (2014) 1014–1019
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of steroidal dendrimers modified by ‘click’ chemistry with
PAMAM dendrons as unimolecular micelles
Delia Soto-Castro a, Nancy E. Magaña-Vergara a, Norberto Farfán b, Rosa Santillan a,
⇑
a Departamento de Química, Centro de Investigación y de Estudios Avanzados del IPN, México, D.F., Apdo. Postal 14-740, 07000, Mexico
b Facultad de Química, Departamento de Química Orgánica, Universidad Nacional Autónoma de México, México, D.F. 04510, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel Fréchet–PAMAM hybrid dendrimers linked by triazole units as unimolecular micelles with a
hydrophobic core surrounded by a hydrophilic shell were prepared. The dendritic cores with 3 and 6
alkyne terminal groups were synthesized from 1,3,5-tribromomethyl-benzene (tBrMeB), in one case by
Received 4 November 2013
Revised 13 December 2013
Accepted 17 December 2013
Available online 27 December 2013
direct coupling with 17a-ethynylestradiol (EE); in the second one the tBrMeB was reacted with
bis(hydroxymethyl) phenol followed by chlorination of the hydroxyl groups and subsequent coupling
to EE. With this strategy, the core can be grown by further substitutions of bis(hydroxymethyl) phenol
over the halogenated terminals as Fréchet dendrimer. The hydrophilic shells used were PAMAM type
dendrons of 0.5 and 1.5 generations with azide as focal point and tert-butyl ester as end groups. The uni-
molecular micelles were obtained by cycloaddition between an azide in the selected dendron and the
alkyne terminal in the hydrophobic core to obtain a 1,4-disubstituted 1,2,3-triazole. Once the coupling
was achieved, the tert-butyl ester groups were hydrolyzed in trifluoroacetic acid and the corresponding
dendrimers with carboxylic acid as end groups were completely soluble in phosphate buffer solutions of
pH 7.0, 7.4, and 8.0. All hybrid dendrimers were characterized by High Resolution Mass Spectrometry, 1H
and 13C NMR, and FTIR.
Keywords:
Unimolecular micelles
Ethynylestradiol
Triazole
Click chemistry
Ó 2013 Elsevier Ltd. All rights reserved.
Introduction
new proposal for hybrid dendrimers as unimolecular micelles con-
sisting of a hydrophobic interior (Fréchet type) functionalized with
Nowadays, dendrimers have been gathering much attention as
interesting functional and high-performance materials. Due to
their molecular structure, they have been considered as unimolec-
ular micelles1–5 comprising a hydrophobic core and a polar surface
since their structural parameters are comparable to those of nor-
mal or traditional micelles.6 From the architectural point of view,
they have a covalent monomer connectivity that provides better
stability toward pH, temperature, and ionic strength changes.
Additionally, the possibility to tailor the internal core provides
the opportunity to apply them in aqueous solubilization of inher-
ently water insoluble species.7–9 They are an attractive and prom-
ising alternative to carry and deliver drugs since the hydrophobic
interior can encapsulate drug molecules10–13 to solve problems
such as the low aqueous solubility, lack of permeability, and in
some cases enhance the activity of bioactive molecules.14
ethynylestradiol and hydrophilic exterior (PAMAM type). This type
of dendrimers would make good candidates for carrying bioactive
compounds in two scenarios: (i) encapsulation mediated by hydro-
phobic interactions, for example steroids17 and (ii) conjugation
where drug molecules are covalently attached at the peripheral
groups.18
Results and discussion
Taking into account the key features of unimolecular micelles,
we designed and synthesized dendritic compounds addressing
three important elements: (i) hydrophobic core that can be
progressively enlarged to favor the encapsulation events; (ii) ethy-
nylestradiol as linker because it can promote the affinity to other
biological steroid; and envisaging possible biological applications
and (iii) PAMAM type hydrophilic surface with carboxylic acids
as end groups mainly because it has been reported that dendrimers
with anionic components are less toxic and less hemolytic than
their cationic counterparts.19–22 The carboxylic end groups can
undergo an acid–base reaction with NaOH to produce the corre-
sponding carboxylates leading to the formation of anionic entities
that prevent adhesion to the walls of blood vessels by electrostatic
repulsions and to avoid acidic solutions.23
It is well known that dendrimers possess a well-defined
structure and high number of terminal groups which can undergo
further modifications in order to obtain new properties or manip-
ulate the existing ones, this fact makes them very promising raw
materials even at low generations.15,16 In this paper we present a
⇑
Corresponding author. Tel.: +52 555 747 3725.
E-mail address: rsantill@cinvestav.mx (R. Santillan).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.12.066

D. Soto-Castro et al. / Tetrahedron Letters 55 (2014) 1014–1019
1015
H2N
OH
With the aim to evaluate different sizes of hydrophobic zones,
two steroidal cores were synthesized as hydrophobic part, taking
in mind that with a larger core the encapsulating process will be
8
O
O
more efficient. For the first core, 17a-Ethynylestradiol (2) was
O
O
directly coupled by ether linkage to 1,3,5-tribromomethyl-benzene
(1) to give core 3 as a pale yellow solid in high yield (91%). As the
second core, a Fréchet type dendrimer was prepared by reaction of
compound 1 with bis(hydroxymethyl) phenol (4) to give 1,3,5-
9
10
MeOH
MeOH
O
O
O
O
tri(3,5-bis((hydroxymethyl)phenoxy)benzene
followed by chlorination using thionyl chloride to achieve the cou-
pling of 17 -Ethynylestradiol and obtain 7 as a pale yellow solid
(dendrimer
5),
a
O
N
OH
O
N
OH
(Scheme 1). The cores 3 and 7 were completely insoluble in water,
ethanol or methanol and very soluble in chloroform or DMSO.
Along with the synthesis of the cores 3 and 7 and with the aim
to synthesize the hydrophilic part, dendrons of half-generation
were obtained by the Michael addition between 3-aminopropan-
1-ol (8) and methyl acrylate (9), to give 11, as well as aminopropa-
nol and tert-butyl acrylate (10) to obtain 12 under the conditions
previously described.24 The reason to use these acrylates was
firstly, to have the possibility to obtain higher generations by
successive reactions with ethylenediamine and then with acrylate.
Secondly, the tert-butyl ester terminal obtained opens the possibil-
ity to hydrolyze them and obtain carboxylic acids as non-cytotoxic
peripheral groups.19–22 Once dendrons 11 and 12 were synthe-
sized, the focal point –OH was transformed into the iodide (com-
pounds 13 and 14) with I2 in the presence of imidazole and
triphenylphosphine25 and subsequently into the corresponding
azide derivative (15, 16) with NaN3 in DMF at 70 °C (Scheme 2).26
The structures were corroborated by 1H and 13C NMR as well as
high resolution mass spectroscopy.
O
O
12
11
Ph3P, Imidazole, I2
CH2Cl2, 1h, rt
O
O
N
O
O
O
I
O
N
I
O
O
14
13
NaN3
DMF, 20h, 70°C
O
O
O
O
N
O
N
N3
O
N3
O
O
16
15
Scheme 2. Synthesis of dendrons with the azide group as focal point.
The subsequent cycloaddition reaction between the alkyne
terminal on ethynylestradiol of hydrophobic cores 3 and 7 and
the azide group of dendron 16 led to novel Fréchet–PAMAM den-
drimers linked by steroids (Scheme 3). In order to obtain only a
1,4-disubstituted 1,2,3-triazole we used an adapted protocol estab-
lished by Shao et al.,27 where CuSO4.5H2O, sodium ascorbate, and
PhCO2H were dissolved in a mixture of t-BuOH/H2O, for core 3,
and EtOH/H2O for core 7, until the color associated with the copper
reduction was observed. Then the alkyne (3 or 7) and the azide (16)
derivatives were added and the reaction was allowed to proceed
for 2 days to give the desired tri- and hexatriazoles in yields of
80% after a simple purification procedure which involves extrac-
tion with CH2Cl2/NaHCO3 solution, evaporation of the solvents un-
der vacuum and washings with hexane to remove the unreacted
azide. The structure of these new dendrimers, 17 and 18, was
unambiguously established by 1H and 13C NMR, and HRMS.
Formation of dendrimer 17 was confirmed by comparison with
the 1H NMR spectra of 3. As seen in Figure 1, the singlet at
HO
HO
2
Br
Br
HO
R
R
K2CO3 /FTBA
Acetonitrile
O
R =
OH
OH
Br
3
1
R
HO
K2CO3 /FTBA
Acetone
4
R
Cl
HO
HO
R
Cl
HO
R
O
O
O
Cl
HO
SO Cl /Py
HO
2
O
O
O
R
Cl
OH
CH Cl (anh)
2
2
K2CO3 /FTBA
Acetonitrile
O
O
O
6
R
5
7
R
Cl
OH
Cl
HO
Scheme 1. Synthesis of hydrophobic cores with 17a-Ethynylestradiol as end groups.

1016
D. Soto-Castro et al. / Tetrahedron Letters 55 (2014) 1014–1019
O
Core 7
Core 3
O
O
N
N3
O
16
CuSO4.5H2O, NaAsc, PhCO2H
EtOH/H2O, 2 days, 40°C
CuSO4.5H2O, NaAsc, PhCO2H
tBuOH/H2O, 2 days, rt
O
O
O
O
O
O
O
N
O
N
N
N
N
N
N
O
N
O
O
HO
OH
O
N
N
N
O
O
N
HO
O
N
O
O
O
O
N
N
N
OH
N
O
O
OH
N
N
O
N
O
O
O
O
O
O
O
O
O
HO
N N
O
O
O
N
OH
N
N
N
N
O
O
O
OH
N
O
HO
N
O
N
N
N
N
N
O
17
18
O
N
N
O
O
O
O
O
O
O
Scheme 3. Synthesis of hybrid Fréchet–PAMAM dendrimers linked via triazole units.
OH
12 18
ore
3
C
11
1
19
2
9
10
13
14
17
20
3
=
R
O
16
R
8
15
5
4
2
7
6
1´
O
N
O
3´
OH
2´
12
1
8
N
11
N
1
R
R
19
9
10
13
22
25
17
N
3
26
O
en r mer
D
d i
17
0.5
0.4
0.3
0.2
0.1
0.0
27
=
R
O
16
14
21
23
20
8
24
15
5
4
28
O
7
6
1H NMR
3
ore
C
20
7.5
7.0
6.5
6.0
5.5
5.0
f1 (ppm)
4.5
4.0
3.5
3.0
2.5
0.15
1
en r mer 17
H NMR D
d i
20
0.10
0.05
0.00
21
7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2
f1 (ppm)
Figure 1. 1H NMR spectra of core 3 and dendrimer 17.

D. Soto-Castro et al. / Tetrahedron Letters 55 (2014) 1014–1019
1017
OH
12 18
ore
3
C
11
1
19
2
9
10
13
14
17
20
3
=
R
O
16
R
8
15
5
4
2
7
6
1´
O
N
O
3´
OH
2´
en r mer
17
d i
12 18
13
D
N
11
N
1
R
R
19
9
10
22
25
17
N
3
26
O
27
=
R
O
16
14
21
23
20
8
24
15
5
4
28
O
7
6
13
ore
C
NMR
3
C
0.15
0.10
0.05
0.00
20
17
19
170 165 160 155 150 145 140 135 130 125 120 115 110 105 100
f1 (ppm)
95
90
85
80
75
70
65
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
13
en r mer 17
NMR D
C
d i
27
26
17
20
19
170 165 160 155 150 145 140 135 130 125 120 115 110 105 100
f1 (ppm)
95
90
85
80
75
70
65
Figure 2. 13C NMR spectra of core 3 and dendrimer 17.
Dendrimer 17
Dendrimer 18
TFA
2h rt
TFA
2h rt
HO
O
N
OH
N
OH
O
O
HO
O
HO
N
HO
O
N
N
N
N
O
N
HO
OH
N
N
N
N
O
O
HO
O
OH
O
O
HO
N
OH
N
N
OH
N
N
O
OH
N
N
O
O
O
HO
O
N
O
O
O
O
HO
N N
O
O
HO
N
OH
O
N
N
N
N
O
O
HO
OH
N
OH
N
HO
N
19
N
N
N
OH
O
N
O
OH
N
20
N
O
O
HO
O
HO
OH
Scheme 4. Hydrolisis of tert-butyl ester dendrimers to obtain unimolecular micelles with carboxylic acids as end groups.

1018
D. Soto-Castro et al. / Tetrahedron Letters 55 (2014) 1014–1019
O
O
O
O
N
H2N
NH2
O
O
1)
2)
O
O
O
MeOH, 5 days, rt
NH
O
N
N3
O
N
O
MeOH, 2 days, rt
N3
15
HN
O
O
21
N
O
O
Core 3
CuSO4.5H2O, NaAsc, PhCO2H
tBuOH/H2O, 2 days, rt
O
O
O
O
O
O
O
N
O
N
O
O
O
NH
O
H
N
NH
O
N
N
O
N
O
O
O
N
N
O
N
OH
HN
N
N
HO
N
N
O
O
O
O
O
22
OH
N
N
N
N
O
HN
O
O
O
HN
O
N
O
N
O
O
O
O
Scheme 5. Synthesis of hybrid dendrimer with dendron of G 1.5.
2.61 ppm corresponding to the alkyne proton is absent in 17 and a
new signal at 7.63 ppm assigned to H-20 from the triazole ring was
evident, along with the signals assigned to the dendron which are
overlapped with those of the steroid. Additionally in 13C NMR,
(Fig. 2) the signals corresponding to C-17, C-19, and C-20 are shifted
due to the new chemical environment.
Finally for compound 17, HRMS provides the m/z values at
2073.2785, 1037.1602, and 691.7754 attributed to [M+H]+,
[M+2H]+2, and [M+3H]+3, respectively, being the latter the most
abundant peak due to the high symmetry of the molecule. A similar
procedure was used to characterize dendrimer 18.
Hydrolysis of 17 and 18 was carried out in trifluoroacetic acid
(TFA) to obtain dendrimers 19 and 20, respectively, in yields
>90% bearing carboxylic acids as terminal groups (see Scheme 4).
The structure of these compounds was corroborated by NMR. The
1H NMR spectra (Supporting information) show a shift of the peak
of the triazole ring proton from 7.62 to 7.83 ppm, and the disap-
pearance of the tert-butyl at 1.46 ppm which indicates that the
hydrolysis of the ester groups was successfully achieved. Addition-
ally, a broadening in all signals could be attributed to the formation
of a salt between the tertiary amine and the carboxylic acid in the
dendrimer, promoting very short relaxation times.28 In the 13C
NMR spectra the quaternary carbon and methylenes of the tert-bu-
tyl group are absent.
The new dendrimers 19 and 20 show complete solubility in
DMSO and in phosphate buffer solutions of pH 7.0, 7.4, and 8.0.
This characteristic is favorable taking in consideration that the
pH of blood is 7.4 and in this environment these compounds could
find application as drug carriers by forming inclusion compounds.
Additionally, in order to obtain a unimolecular micelle, the solubil-
ity in water can be improved by the acid–base reaction between
carboxylic acids and NaOH to obtain a Fréchet–PAMAM type mi-
celle. The hydrophobic core and hydrophilic terminals place these
new materials as potential drug carriers for inclusion tentatively
with high affinity toward steroids with biological importance.
In order to evidence the possibility to grow the hydrophilic
shell, a dendron of 1.5 generation was synthesized. Thus com-
pound 15 was reacted with ethylenediamine, thereafter with
tert-butyl acrylate to give 21, as amber oil, see Scheme 5. The cyclo-
addition between the core 3 and dendron 21 provided the corre-
sponding tri-triazole as
a green light solid in 68% yield,
Scheme 5. The structure of 22 was corroborated by NMR and HRMS
and demonstrates the feasibility of this approach for the synthesis
of unimolecular micelles where growth in both zones is possible.

D. Soto-Castro et al. / Tetrahedron Letters 55 (2014) 1014–1019
6. C.N. Moorefield, G.R. Newkome, C. R. Chimie 6 (2003).
1019
In summary we present an efficient methodology to synthe-
sized unimolecular micelles that are designed to tailor both, the
hydrophobic core and the hydrophilic shell in order to reach the
desired necessities. The evaluation of encapsulating properties is
currently under investigation.
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Acknowledgments
The authors thank CONACyT and PAPIIT IN 214513 for financial
support, Ma. T. Cortez for NMR spectra and Cuéllar Rivera Geiser
for HRMS. A postdoctoral fellowship to D. Soto from CONACyT
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Supplementary data
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