Synthesis of non-cytotoxic poly(ester-amine) dendrimers as potential solubility enhancers for drugs: Methotrexate as a case study

Article abstract of DOI:10.3390/molecules15118082

This study describes the synthesis of two new families of dendrimers based on the esterification of N-alkylated 3-amine-1-propanol with two different cores, adipic acid (1^st and 2^nd generations) and ethylenediamine (generation 1.5), both with carboxylic acid end groups, offering a wide variety of further modifications at the periphery. According to the cytotoxic evaluation of the dendrimers and their possible degradation products within cell lines, these materials could be considered as innocuous. In preliminary studies, the synthesized dendrimers proved to be potential enhancers of solubility of highly hydrophobic drugs, like methotrexate, widely used in chemotherapy.

Full text of DOI:10.3390/molecules15118082

Molecules 2010, 15, 8082-8097; doi:10.3390/molecules15118082
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Non-Cytotoxic Poly(Ester-Amine) Dendrimers as
Potential Solubility Enhancers for Drugs: Methotrexate as a
Case Study
Delia Soto-Castro 1, Jorge A. Cruz-Morales 1, María Teresa Ramírez Apan 2 and
Patricia Guadarrama 1,*
1
Instituto de Investigaciones en Materiales, Universidad Nacional Autónoma de México, Apartado
Postal 70-360, CU, Coyoacán, Mexico, DF, 04510, Mexico;
E-Mails: sotodelia@hotmail.com (D.S.C.); sirarmando@hotmail.com (J.A.C.M.)
2
Instituto de Química, Universidad Nacional Autónoma de México, Apartado Postal 70-213, CU,
Coyoacán, Mexico, DF, 04510, Mexico; E-Mail: mtrapan@servidor.unam.mx (M.T.R.A.)
* Author to whom correspondence should be addressed; E-Mail: patriciagua@iim.unam.mx.
Received: 8 October 2010; in revised form: 20 October 2010 / Accepted: 4 November 2010 /
Published: 9 November 2010
Abstract: This study describes the synthesis of two new families of dendrimers based on
the esterification of N-alkylated 3-amine-1-propanol with two different cores, adipic acid
(1st and 2nd generations) and ethylenediamine (generation 1.5), both with carboxylic acid
end groups, offering a wide variety of further modifications at the periphery. According to
the cytotoxic evaluation of the dendrimers and their possible degradation products within
cell lines, these materials could be considered as innocuous. In preliminary studies, the
synthesized dendrimers proved to be potential enhancers of solubility of highly
hydrophobic drugs, like methotrexate, widely used in chemotherapy.
Keywords: non-cytotoxic dendrimers; solubility enhancer; methotrexate
1. Introduction
After at least three decades of study, dendrimers remain as one of the most exciting and promising
molecular architectures for a great variety of applications in important fields such as catalysis [1],
alternative sources of energy [2,3], and biomedical applications for both medical diagnostics and

Molecules 2010, 15
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therapeutic applications, among others [4–8]. In the area of drug delivery, there is a constant search of
new vehicles for many drugs with serious solubility and cytotoxicity problems that make it difficult for
them to reach their main targets. Recently, progress has been made in the new field of nanomedicine,
where nanoscale engineered materials, such as PAMAM (polyamidoamine) dendrimers, are used in
several medical applications, as well as dendritic scaffolds in biology [9,10].
In this regard, the unique architecture and functionality of dendrimers at this scale make them
exceptional carrier molecule candidates for use in medical applications. Thus, the present study
describes the synthesis of two new families of poly(ester-amine) dendrimers, designed to be
considered as flexible non-cytotoxic nanocarriers of poorly water-soluble drugs, like methotrexate
(MTX, Figure 1), widely used in chemotherapy [11].
Figure 1. Chemical structure of methotrexate (MTX).
H2N
N
N
N
N
N
O
H
NH2
N
OH
OH
O
O
To evaluate the cytotoxicity of the dendrimers and their possible degradation products, assays on
different cell lines were performed. In addition, these new materials were also tested as enhancers of
MTX solubility in water. Preliminary theoretical simulations were carried out to visualize the
hydrophobic/hydrophilic balance once MTX has interacted with the dendrimers.
2. Results and Discussion
Taking into account the key features that materials with bio-applications should have, we designed
and synthesized dendritic compounds addressing three important elements: i) high flexibility to
enforce the encapsulation events and to improve solubility; ii) biodegradability by the presence of ester
groups, since they are cleaved in the body and, at the same time, induce formation of hydrogen bonds;
and iii) non-cytotoxicity by selecting carboxylic acids as end groups (pKa ∼5), which should be
practically deprotonated at the blood’s pH (7.4), leading to the formation of anionic entities that
prevent adhesion to the walls of blood vessels by electrostatic repulsions [12]. Dendrimers with
anionic components have shown to be less toxic and less hemolytic than their cationic
counterparts [13–16]. To avoid acidic solutions, the carboxylic end groups can undergo an acid-base
reaction with NaOH to produce the corresponding carboxylates.
The synthesis of compound 3 [17] (Scheme 1), from 3-aminopropan-1-ol (1) and tert-butyl acrylate
(2) enabled us to obtain a 1st generation dendron bearing a single reactive point, a OH group, ready to
be coupled via an esterification reaction to -COOH end groups of proposed cores (see Schemes 2 and 3).
The reaction was allowed to proceed for 24 h, then the solvent and excess reactants were evaporated to
give the product, quantitatively. The structure was corroborated by common spectroscopic techniques.

Molecules 2010, 15
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Scheme 1. Synthesis of dendron 3.
O
N
O
O
O
i
H
HO
quantitative
N
O
O
1
2
OH
3
Reagents and Conditions: i) MeOH, rt, 24 h.
In a convergent fashion, dendron 3 was coupled to two different cores with terminal carboxylic
acids, one is adipic acid 4 (scheme 2) and the other is compound 11 (scheme 3) obtained from the
alkylation of ethylenediamine and their subsequent hydrolysis.
Scheme 2. Synthesis of dendrimers with an adipic acid core
O
O
O
O
O
O
O
HO
OH
i
O
O
N
4
O
94%
O
O
N
3
O
O
5
98% ii
OH
O
HO
O
N
O
HO
O
O
N
OH
O
O
O
6
46.4% iii
O
N
O
O
O
O
O
O
O
N
O
N
O
O
O
O
O
O
O
O
O
O
N
O
O
N
O
O
O
O
O
N
O
7
O
iv
96%
HO
N
O
O
HO
HO
OH
O
N
O
OH
OH
O
O
O
O
O
O
N
O
N
O
O
N
O
O
O
O
O
O
N
HO
O
OH
8
Reagents and Conditions: i) DMF freshly distilled, DCC, DMAP, 24 h; ii) and iv) TFA with 3
drops of water, 2 h; iii) DMF freshly distilled, EDC, DMAP, 24 h.

Molecules 2010, 15
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From Scheme 2, adipic acid and dendron 3 (1:6 mole ratio) were coupled in the presence of
DCC/DMAP (dicyclohexylcarbodiimide/dimethylaminopyridine) to obtain compound 5 in high yield
(96%) as an amber oil. The DMF solvent must be completely dried and freshly distilled; otherwise the
yield drops to 30–35%. This new compound was characterized by 1H NMR, where the ratio of signals
corresponding to the tert-butyl groups [36 H, 1.42 ppm (s)] was compared with those of the adipic core
(8H) distributed in two groups of signals in 1.64 and 1.76 ppm. In the 13C-NMR spectrum, only those
carbons related to ester groups in 171 and 173 ppm, corresponding to tert-butyl, and internal esters
were observed, respectively. Finally, through FAB+ a m/z value of 773, corresponding to compound 5,
was obtained.
The hydrolysis of compound 5 with trifluoroacetic acid (TFA) takes place in a selective manner to
generate compound 6 (G1 dendrimer with carboxylic acid terminals) in 98% yield. The structure was
1
corroborated by H-NMR, where the signal in 1.42 ppm, associated with the tert-butyl groups, had
disappeared.
The DCC/DMAP coupling system was used to couple dendron 3 to compound 6 in order to obtain
compound 7 (Scheme 2), like the coupling of adipic acid to dendron 3. Even though the DCC/DMAP
coupling system usually gives excellent yields (86%, in this case), a serious inconvenience arose due
to the presence of dicyclohexylurea (DCU), formed as by-product of the reaction with DCC, as the
former was strongly encapsulated by the dendrimer (compound 7), which was confirmed by 1H-NMR
(signals in 3.21 ppm, related to DCU).
Failed attempts to revert the encapsulation were made by filtration, centrifugation, and dialysis with
flexible and rigid membranes; therefore, a change in the coupling system was required. EDC (N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide) was chosen instead of DCC, since its urea is much more
polar and more easily removed than the DCU by column chromatography; although the yield dropped
to 46.4%, as shown in Scheme 2. In spite of the latter inconvenience, the strong encapsulation of
highly insoluble DCU provides preliminary evidence of the ability of these compounds to enhance the
solubility of hydrophobic compounds.
Thus, compound 7 was also obtained with the EDC/DMAP coupling system after chromatographic
1
purification with ethyl acetate/NH4OH (1% v/v) as eluent. The structure was confirmed by H-NMR,
where the ratio of signals in 1.44 and 1.65 ppm corresponds to the new relation of tert-butyl protons
and the more protected methylene protons present in the structure of the dendron. Through FAB+,
a m/z of 1,801 was obtained.
Compound 7 was hydrolyzed in TFA to obtain dendrimer 8 with 96% yield. This compound is
highly hygroscopic and completely soluble in water, a desirable characteristic in materials with
potential application as drug carriers.
On account of their chemical structure, these compounds exhibit two characteristic FT-IR bands.
One, broad and weak around 3,500-2,200 cm-1 attributed to COOH end groups and another band
around 1,650 cm-1 attributed to the carbonyl of the –COO-⋅⋅⋅+NHR3 salt. The formation of this kind of
salts can produce duplicity in the NMR signals; therefore, the addition of aqueous HCl (drops) can be
required. The hydrolyzed compound 3 shows no more duplicity of signals, hence all NMR spectra of
hydrolyzed compounds were recorded after addition of a few drops of HCl (see
Supplementary Materials).

Molecules 2010, 15
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A second family of dendrimers was synthesized starting from the alkylation of ethylenediamine
with tert-butyl acrylate (scheme 3) to obtain compound 10 as a white solid in quantitative yield. The
structure of this compound was verified by 1H-NMR with signals at 1.44 ppm (tert-butyl groups) and a
singlet at 2.53 ppm corresponding to the symmetric ethylenediamine core.
As before, the carboxylic acid end groups were obtained after the hydrolysis of compound 10 with
TFA to obtain compound 11 with 99% yield as a poorly water-soluble solid. Compound 11 is less
flexible and has less polar groups in comparison with compound 6, which exhibits the same number of
terminal groups.
1
The coupling of compound 11 and dendron 3 allowed us to obtain compound 12. Its H-NMR
reveals a singlet at 1.43 ppm corresponding to tert-butyl groups and a m/z of 1601 was found by
FAB+. Compound 12 was hydrolyzed to obtain dendrimer 13 (G1.5) in 97% yield as a highly
hygroscopic foamy solid. The FT-IR bands at 3,600 to 2,200 cm-1 and the disappearance of the singlet
at 1.43 ppm in the NMR confirmed the hydrolysis.
Scheme 3. Synthesis of dendrimers with ethylenediamine core.
O
OH
O
O
O
H2N
NH2
O
O
O
O
OH
9
i
ii
99%
O
Quantitative
O
HO
O
O
10
O
O
HO
11
2
HO
O
O
O
O
O
O
O
O
HO
HO
OH
O
O
O
O
O
OH
OH
O
O
O
O
O
O
11
3
O
iii
45%
O
O
O
O
N
iv
97%
O
N
O
N
N
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
O
12
13
OH
Reagents and Conditions: i) MeOH, rt, 48 h; ii) and iv) TFA with 3 drops of water, 2 h; iii) DMF
freshly distilled, EDC, DMAP, 24 h.
3. Biological Section
Since non-cytotoxicity is a necessary feature for any biomaterial, the harmlessness of dendrimers 8
and 13 and their possible degradation products (hydrolyzed compound 3, and compounds 6 and 11)
was evaluated by assays on human lymphocytic cells (MT2) [18]. The results of the assays (performed
in triplicate), as well as the standard error, are shown in Table 1, where a sample of polyglycidol

Molecules 2010, 15
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(synthesized in our lab, see Experimental) was included as reference parameter of innocuity [20].
Although the main interest for the biological study was the verification of non-cytotoxycity of the new
materials in normal cell lines, additional assays on human cancer cell lines were carried out for
forthcoming studies.
Table 1. Activity (%) of human cancer cell lines and human lymphocytic MT2 cells. a
Compound
MT2
U251
PC-3
K-563 HCT-15 MCF7 SKLU-1
+3.4±2.0 10.6±1.2 +6.6±5.0
6.5±1.2 7.8±1.4 24.0±1.2 17.9±1.3
3 (hydrolyzed)
+3.2±1.5 +13.7±2.9 +3.7±0.8 +12.0±1.3 +6.5±1.0 +11.1±6.0 +5.1±0.5
4
+3.0±1.5
+6.1±1.0
1.6±1.0 +5.2±0.2 15.3±5.8 +3.5±3.3 +21.4±5.4 +2.3±0.8
9.8±0.4 12.6±3.6 7.7±0.0 +5.9±2.0 +10.2±2.2 2.4±0.4
6
8
6.7±2.2 19.7±5.0 +1.7±1.0 11.8±2.4 9.9±3.3 +9.8±4.2 +5.4±1.3
11
1.3±0.6 11.2±0.5 +9.6±2.1
+5.8±0.6
1.3±0.3 +4.4±4.0 +6.6±1.2 2.1±0.1
5.3±0.2 5.5±1.5
13
Polyglycidolb
1.6±0.1 +5.4±1.8 6.4±0.08 4.8±1.5
a: Cellular activity measured in presence of different compounds at 50 µM standard error. MT2:
±
Human lymphocytes; Human cancer cell lines: U251 (human glyoblastoma); PC-3 (human
prostatic adenocarcinoma); K562 (human chronic myelogenous leukemia cells); HCT-15 (human
colorectal adenocarcinoma); MCF-7 (human mammary adenocarcinoma); SKLU-1 (human lung
adenocarcinoma); : MW = 4591; polydispersity = 1.8.
b
In Table 1 the + sign denotes cell growth compared with the control, which means that the
compounds are non cytotoxic; and the values without + sign correspond to cell growth inhibition (%).
From these results it can be observed that these new dendrimers, as well as their possible metabolites,
exhibit cytotoxycity towards some cancer cell lines; however, they are harmless towards normal cells.
The cytotoxycity observed for compound 11 and dendrimer 13 in normal cells is considered negligible.
Even though polyglycidol (reference compound) showed cytotoxycity in some cancer cell lines, it was
also non cytotoxic towards normal cells (lymphocytes).
The performance of dendrimers 8 and 13 eventually might propitiate a synergistic effect [21] when
the drug is included, since these materials already exhibit certain level of cytotoxycity toward some
cancer cell lines. Concerning the possible metabolites 3 (hydrolyzed) and 11 (hydrosoluble molecules
eliminable from the body), their differential effect in human glyoblastoma (U251) and human
mammary adenocarcinoma (MCF-7) might be related to the selectivity of these cells, in terms of
lipophilicity [22].
4. Dendrimers as Enhancers of Solubility: Preliminary Studies with MTX
MTX is an anticancer drug extensively used against breast and cervical cancer, although it is
essentially insoluble in water at neutral pH [23]; therefore, it is an ideal candidate to be solubilized by
dendrimers, ideally forming complexes. In order to avoid possible repulsive interactions owing to the
dissociation of acid groups present in both, dendrimers and MTX, the dendrimer-drug complexes were
formed in absence of water, initially grinding in a mortar the corresponding dendrimer with NaOH
(equimolar amounts) to form the salt, followed by the addition of MTX to form the complex, as a
paste, which was dissolved in deionized water up to 5 mL in a volumetric flask (see Experimental

Molecules 2010, 15
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section). Water soluble complexes were obtained after sonication, centrifugation, and filtration of the
precipitated MTX. The complexes remained stable at neutral pH during several days of storage
in darkness.
Due to the very poor solubility of MTX in deionized water, our attempts to construct a calibration
curve by UV-Vis at different concentrations failed, hence concentration values of MTX in the presence
of dendrimers were unobtainable; however, from the measured absorbance values of dendrimer-MTX
complex solutions in water in an appropriate dilution, and considering the Lambert-Beer law, it was
possible to establish a ratio of concentrations to clearly show that both dendrimers, 8 and 13,
significantly improve MTX solubility in water. To determine such a ratio of concentrations, it was
necessary first to measure at least one absorbance value corresponding to MTX in water, which was
achieved by means of the formation of a saturated solution of MTX in deionized water, and making the
required dilutions to apply the Lambert-Beer law.
After a dilution of a saturated solution (0.3 mL up to 5 mL) of MTX, an absorbance (A302) of 0.457
at 302 nm was obtained. Applying Equations 1 and 2 and by algebraic treatment yields the
concentration of MTX in water as a function of the constant (k):
7.61
CM0 TX
=
Aλ = k ⋅C
(1)
(2)
k
C0 ⋅V 0 = C1 ⋅V1
In a similar way, and making the appropriate dilutions (0.2 mL up to 5 mL), the measured
absorbance values in water at 302 nm for the complexes formed between dendrimer 8 and 13 with
MTX were 1.261 and 0.827 respectively.
Based on equations 1 and 2, and the maximum concentartion of MTX in water, the ratio of
concentrations (the concentration of MTX in the presence of dendrimer (CM0 TX −8 ) by the concentration
of MTX in water alone (CM0 TX )), can be expressed as follows:
CM0 TX −8
CM0 TX
CM0 TX −13
CM0 TX
= 4.14
= 2.71
Thus, the presence of dendrimer 8 increases 4.14-times the concentration of MTX in water, while
dendrimer 13 increases it 2.71-times. Figure 2 shows the UV-VIS spectra of MTX alone in water and
dendrimer-MTX complexes. From Figure 2, a slight shift and best definition of maximum of MTX at
372 nm can be observed in the presence of dendrimers, presumably due to dendrimer-MTX
interactions via π-π* transitions.
With the experimental information described before it is possible to estimate a stoichiometry of
interaction between dendrimers and MTX. Considering that the same initial concentration of each
dendrimer (CD=1.7 mM) was used to form the complexes dendrimer-MTX, and comparing their
performance to interact with MTX, we assumed a 1:1 stoichiometry for the dendrimer 8-MTX
complex since the largest increment of MTX concentration in water is achieved with dendrimer 8.
Under this hypothesis, a 5:3 stoichiometry for dendrimer 13-MTX complex was estimated, taking
into account the obtained concentration ratios by algebraic manipulation (see Experimental).

Molecules 2010, 15
Figure 2. UV-VIS spectra of dendrimer8-MTX and dendrimer13-MTX complexes and
8089
MTX in deionized water. The relative concentrations with regard to MTX in water are 2.71
to 13-MTX complex and 4.14 to 8-MTX complex.
3,0
2,5
dendrimer8-MTX
2,0
dendrimer13-MTX
MTX
1,5
1,0
0,5
0,0
150
200
250
300
350
400
450
500
550
λ(nm)
Considering the estimated stoichiometries, possible conformations of the dendrimer-MTX
complexes were explored by conformational search calculations in aqueous medium (continuum
dielectric constant = 81). The Monte Carlo multiple minimum method (MCMM) was used [24,25],
where random changes were made in torsion angles during the search.
Thus, considering, on one hand, carboxylate end groups (scenario at pH ∼7), and, on the other hand,
the experimentally observed complexation stoichiometry, ∼1:1 and 5:3 complexes between dendrimer
8 and MTX and dendrimer 13 and MTX were constructed, respectively. From Figure 3a it can be
suggested that the interaction dendrimer-drug is viable. Due to the flexible adipic core, the dendrimer
exhibits a relaxed structure that surrounds the MTX and interacts with it via hydrogen bonds. In order
to obtain additional information about the ability of dendrimers for isolating the hydrophobic MTX
from polar environments (aqueous medium), surfaces that display hydrophobic and hydrophilic
regions (in orange and blue respectively) were generated by an active-site mapping procedure that
follows the Goodford algorithm [26].
Figure 3. (a) dendrimer 8-MTX; (b) Hydrophilic/hydrophobic balance.
A
B

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In case of complex 8-MTX (Figure 3b), the drug is visibly shielded by the dendrimer, with the
anionic end groups exposed to the aqueous medium, forming a hydrophilic bag with the drug inside.
In case of the less flexible dendrimer 13, the proposed complexation stoichiometry of 5:3 was
minimized to obtain the conformation shown in Figure 4a, where the group of dendrimeric molecules
surrounds the MTX, and several hydrogen bonds are formed.
According to the hydrophobic/hydrophilic balance, a homogeneous protection of MTX in an
isolated bag formed by the dendrimeric cluster is observed (figure 4b). Thus, even when the
experimental observation is the most reliable evidence of the interaction between the dendrimers and
MTX, these preliminary calculations offer an illustrative rationalization of the experimental results.
Figure 4. (a) dendrimer 13-MTX (5:3); (b) Hydrophilic/hydrophobic balance.
5. Conclusions
The synthesis of two new families of ester-amine dendrimers constructed by esterification between
3-amine-1-propanol, previously N-alkylated, and two different cores was carried out. All dendrimers
are monodisperse, highly soluble in water and both dendrimers and their metabolites, are innocuous to
human cells. Studies on solubilization of MTX place these materials as potential solubility enhancers
of hydrophobic molecules. According to the dendrimer: MTX ratio, estimated by UV-VIS spectra, the
stoichiometry of the complexes depends on the molecular flexibility, directly related to the flexibility
of the cores. Finally, the preliminary calculations to explore the conformations of the complexes
dendrimer-MTX showed an effective isolation of MTX, where the hydrophilic surface generated by
the dendrimers can be related with the increase of solubility observed for the MTX in aqueous media.
6. Experimental
6.1. General
Unless stated otherwise, chemicals were obtained from commercial sources and used without
further purification. DMF was dried over CaH2 (5% w/v) overnight, filtered and freshly distilled at
reduced pressure prior to its use. Uncorrected melting points were determined on an electrothermal
9100 Fisher and NMR spectra were recorded on a Bruker Avance 400 instrument. FT-IR spectra were
measured on a Nicolet 6700 spectrometer. MS (FAB) mass spectra were measured with a

Molecules 2010, 15
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JEOL AX-505 spectrometer and by ESI with a Bruker Daltonic DataAnalysis 3.2. Molecules 8, 13,
MTX, and the complexes were equilibrated by conformational search in aqueous media with a
continuum dielectric constant of 81 (Force Field OPLS2001) [27]), using the Monte Carlo statistical
method [28] included in the Macromodel software. The algorithm of multiple minima (MCMM)
[24,25] without limits on the number of variable torsions allowed in the search was used. Hydrophobic
and hydrophilic surfaces were calculated using Maestro [29] with a box of 6 Å.
6.1.1. General Procedure for the Synthesis of tert-Butyl Ester Terminated Dendrimer
To an ice-cold suspension of carboxylic acid compound, a mixture of dendron (compound 3) and
DMAP in dry DMF (in 12 mL), a solution of EDC (1-ethyl-3-[(3-dimethylamino)propyl]-
carbodiimide) or DCC (dicyclohexilcarbodiimide) in DMF were added dropwise. The reaction mixture
was stirred at 0°C for 1 h and then at room temperature, monitored by thin layer chromatography
(TLC) until no more changes were visible (for these products 24 h were enough). The solvent was
removed under reduced pressure to give an amber oil, which was purified by column chromatography
on silica gel using ethyl acetate/ammonium hydroxide (1% v/v) as eluent.
(tert-Butyl 3-[(2-tert-butoxycarbonyl-ethyl)-(3-hydroxy-propyl)-amino]-propionate) (3). In a round-
bottom flask purged with nitrogen and maintained in darkness, 3-aminopropan-1-ol (1, 5 g,
66.57 mmol) was diluted in methanol (30 mL) and then tert-butyl acrylate (2, 22.183 g, 173.08 mmol)
was added. The mixture was stirred at room temperature for 24 h, the excess of tert-butyl acrylate and
solvent were removed at vacuum to give a colorless oil in quantitative yield. FT-IR (cm-1): 3,413
(OH), 2,976, 2,939 (C-H aliph.), 1,723 (C=O); 1H-NMR (CDCl3) δ: 1.39 (s, 18H, -C(CH3)3), 1.64 (q,
2H, -CCH2C-), 2.35 (t, 4H, -CCH2COO-), 2.57 (t, 2H, -C(CH2)N-), 2.69 (t, 4H, -NCH2C-) ppm; 13C-
NMR δ: (CDCl3, 77), 27.9 (-C(CH3)3), 28.2 (-C(CH2)2C-), 33.1 (-CCH2COO-), 49.2 (-NCH2C-), 52.7
(-C(CH2)2N-), 62.8 (HOCH2C-), 80.4(-COOC(CH3)3) and 171.6(-CCOOC-) ppm.
(4-Cascade:adipic acid [2-1,8]:(1-azabutylidyne):tert-butylpropyl ester) (5). To an ice-cold
suspension of adipic acid (4, 1g, 6.84 mmol) compound 3 (13.6 g, 41.05 mmol) and DMAP (0.167 g,
1.37 mmol) dissolved in dry DMF (12 mL) and DCC (3.38 g, 16.41 mmol) in DMF (5 mL) were
added in portions. When the reaction was finished the mixture was purified, as described above to give
1
5 (4.97 g, 94%) as an amber oil. FT-IR (cm-1): 2,973, 2,933 (C-H aliph.), 1,722 (C=O); H-NMR
(acetone d6) δ: 1.42 (s, 36H, -C(CH3)3), 1.64 (m, 4H, -C(CH2)2C-), 1.76 (q, 4H, -CCH2C-),
2.34 (t, 12H, -CCH2COO-), 2.50 (t, 4H, -CCH2N-), 2.70 (t, 8H, -NCH2C-), 4.08 (t, 4H, -COOCH2C-)
ppm; 13C-NMR δ: (acetone d6), 25 (-C(CH2)2C-), 27 (-CCH2COO-), 28 (-C(CH3)3),
34 (-(CH2)3CH2COO-), 34 (-CCH2COO-), 49.8 (-NCH2C-), 50 (-CCH2N-), 63 (-COOCH2C-),
79 (-COOC(CH3)3), 171 (-COO(CH3)3) and 173 (-CCOOC-) ppm; MS m/z (FAB+) 773.
(8-cascade:adipic acid [2-1,8]:(1-azabutylidyne):(6-oxo-5-oxa-1-azaoctylidyne):tert-butylpropyl
ester) (7). To an ice-cold suspension of 6 (2.4 g, 4.39 mmol) compound 3 (11.65 g, 35.16 mmol) and
DMAP (0.726 g, 5.86 mmol) in dry DMF (12 mL), and EDC (8.18 g, 52.69 mmol) in DMF (5 mL)
were added in portions. When the reaction was finished, the mixture was purified as described above
to give 7 (3.67 g, 46.4%) as an amber oil. FT-IR (cm-1): 2,973, 2,933 (C-H aliph.), 1,722 (C=O);

Molecules 2010, 15
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1H-NMR (MeOD) δ: 1.44 (s, 72H, -C(CH3)3), 1.65 (m, 4H, -C(CH2)2C-), 1.76 (q, 12H, -CCH2C-),
2.33 (t, 28H, -CCH2COO-), 2.50 (t, 12H-CCH2N-), 2.70 (t, 24H, -NCH2C-), 4.08 (t, 12H,
-COOCH2C-). 13C (δ (ppm)): (MeOD): 24.3 (-C(CH2)2C-), 25.4 (-CCH2C-), 26.6 (-CCH2C-),
27.5 (-C(CH3)3), 32.2 (-CCH2COO-), 33.4 (-(CH2)3CH2COO-), 33.7 (-CCH2COO-), 49.6 (-NCH2C-,
CCH2N-), 62.2 (-COOCH2C-), 79.3 (-COOC(CH3)3), 171.3 (-COO(CH3)3) 172.4 (-CCOOC-) ppm;
MS: m/z (FAB+) 1,801 and 1,803 by ESI.
(4-Cascade:ethylenediamine[4-N,N,N´,N´]: tert-butyl propyl ester) (10). In a round-bottom flask
purged with nitrogen and maintained in darkness, ethylenediamine (2 g, 33.27 mmol) was diluted in
methanol (30 mL), and then tert-butyl acrylate (22.183 g, 173.08 mmol) was added. The mixture was
stirred at room temperature for 24 h, the excess of tert-butyl acrylate and solvent were removed at
vacuum to give a white solid in quantitative yield (19 g). p.f. 42 ºC. FT-IR (cm-1): 2,940, 2,818 (C-H
1
aliph.); 1,715 (C=O); H-NMR (MeOD) δ: 1.47 (s, 36H, -C(CH3)3); 2.36 (t, 8H, -CCH2COO-);
13
2.53 (s, 4H, -N(CH2)2N-); 2.73 (t, 8H, -NCH2C-) ppm; C-NMR δ: (MeOD); 27 (-C(CH3)3); 33
(-CCH2COO-); 55 (-N(CH2)2N-); 80 (-COOC(CH3)3); 172 (-COO(CH3)3) ppm.
(4-Cascade:ethylenediamine[4-N,N,N´,N´]:(6-oxo-5-oxa-1-azaoctylidyne):tert-butylpropyl ester) (12).
To an ice-cold suspension of 11 (1.5 g, 4.3 mmol), compound 3 (11.51 g, 34.72 mmol) and DMAP
(0.726 g, 5.68 mmol) in dry DMF (12 mL) and EDC (8.02 g, 51.66 mmol) in DMF (5 mL) were added
in portions. When the reaction was finished, the mixture was purified as described above, to give 12
(3.1 g, 45%) as an amber oil. FT-IR (cm-1): 2,975, 2,818 (C-H aliph.) 1,723 (C=O); 1H-NMR (MeOD)
δ: 1.43 (s, 72H, -C(CH3)3); 1.74 (q, 8H, -CCH2C-); 2.36 (t, 16H, -CCH2COO-); 2.48 (t,
8H, -CCH2COO-); 2.52 (t, 8H, -NCH2C-); 2.56 (s, 4H, -N(CH2)2N-); 2.72 (t, 16H, -NCH2C-); 2.80 (t,
8H, -CCH2N-); 4.10 (t, 8H, -COOCH2C-) ppm; 13C-NMR (MeOD) δ: 25 (-CCH2C-); 27 (-C(CH3)3);
33 (-CCH2COO-); 35 (-CCH2COO-); 48 (-NCH2C-); 49 (- NCH2C-); 50 (-CCH2N-); 55 (-N(CH2)2N-);
62 (-COOCH2C-); 80 (-COOC(CH3)3); 172 (-COO(CH3)3); 173 (-CCOOC-)ppm; MS m/z (FAB+)
1,601 and 1,603 by ESI.
6.1.2. General Procedure for the Hydrolysis of tert-Butyl Esters
tert-Butyl ester-terminated dendrimer (1 g) was dissolved in trifluoroacetic acid (TFA, 2 mL) with
3 drops of water. The hydrolysis was monitored by TLC. The TFA was evaporated under reduced
pressure to give a viscous brown light oil, which was dissolved in methanol (10 mL) and evaporated
under reduced pressure; this step was repeated from 3 to 5 times until no more TFA was present. As an
alternative, the viscous oil can be triturated several times in hexane-dichloromethane (hex-DCM,
50/50) until no more TFA is present. Finally, the product was dried under high vacuum.
(4-Cascade:adipic acid [2-1,8]:(1-azabutylidyne):propionic acid) (6). Compound 5 (2 g, 2.58 mmol)
was dissolved in TFA (4 mL). Once the general procedure was applied, the product was dried under
high vacuum to give 6 (1.4 g, 98%) as a white, foamy and very hygroscopic product. FT-IR (cm-1):
1
2,220 to 3,600 (COOH broad and weak), 2,953 (C-H aliph), 1,738 and 1,662 (C=O); H-NMR
(MeOH+HCl) δ: 1.66 (m, 4H, -C(CH2)2C-), 2.20 (q, 4H, -CCH2C-), 2.42 (t, 4H, -(CH2)3CH2COO -),
2.88 (t, 8H, -CCH2COO-), 3.48 (t, 8H, -NCH2C-), 3.74 (t, 4H-CCH2N-), 4.21 (t, 4H, -COOCH2C-)

Molecules 2010, 15
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ppm; 13C-NMR δ: (MeOD): 23.0 (-C(CH2)2C-), 28.0 (-CCH2C-), 28.4 (-CCH2COO-),
33.0 (-(CH2)3CH2COO -), 48.5 (-NCH2C-), 49.5 (-CCH2N-), 61 (-COOCH2C-), 172 (-CCOOC-) and
171 (-CCOOH) ppm; MS m/z (ESI) 550.9.
(8-Cascade:adipic acid [2-1,8]:(1-azabutylidyne):(6-oxo-5-oxa-1-azaoctylidyne):propionic acid) (8).
Compound 7 (1.5 g, 0.83 mmol) was dissolved in TFA (3 mL). Once the general procedure was
applied, the product was dried under high vacuum to give 8 (1.08 g, 96%) as a white, foamy, very
hygroscopic product. FT-IR (cm-1): 2,220 to 3,600 (COOH broad and weak), 2,943 (C-H aliph), 1,720
and 1,662 (C=O); 1H-NMR (MeOD) δ: 1.65 (q, 4H, -C(CH2)2C-), 1.96 (q, 12H, -CCH2C-), 2.25 (t, 4H,
-OCCH2(CH2)2CH2CO-), 2.86-2.96 (m, 24H, -NCH2CH2COO-), 3.28 (m, 4H,-CCH2N-), 3.41-3.47
(2t, 24H, -NCH2C-), 3.64 (t, 8, -CCH2Nf-), 4.20 (t, 12H, -COOCH2C-)ppm; 13C-NMR δ: 25.2
(-C(CH2)2C-), 26.7 (-OCCH2(CH2)2CH2CO-), 29.2, 34.4(-CCH2COO-), 50.8 (-NCH2C-,-CCH2N-),
60.6 (-COOCH2C-), 172.5, 175.7 (-CCOOC-) and 172.5 (-COOH) ppm; MS m/z (ESI) 1354.
(4-Cascade:ethylenediamine[4-N,N,N´,N´]: propionic acid) (11). Compound 10 (1 g, 1.89 mmol) was
dissolved in TFA (2 mL). Once the general procedure was applied, the product obtained was triturated
in dichlorometane-hexane (50/50) several times until compound 11 (0.66 g, 99%), a white solid, was
precipitated. FT-IR (cm-1): 2,256 to 3,120 (COOH broad and weak); 2,992-3,011 (C-H aliph), 1,709,
1,651 (C=O); 1H-NMR (D2O) δ: 2.85 (t, 8H, -CCH2COOH), 3.46 (t, 8H, -NCH2C-),
3.72 (s, 4H, -N(CH2)2N-) ppm; 13C-NMR (D2O) δ: 28.3(-CCH2COO-), 47.8(-N(CH2)2N-),
50.3 (-NCH2C-) and 174.0 (-CCOOH) ppm.
(4-Cascade:ethylenediamine[4-N,N,N´,N´]:(6-oxo-5-oxa-1-azaoctylidyne):propionic
acid)
(13).
Compound 12 (1 g, 0.62 mmol) was dissolved in TFA (2 mL). Once the general procedure was
applied, the product was dried under high vacuum to give compound 13 (0.70 g, 97%) as a slightly
brown, foamy, very hygroscopic product. FT-IR (cm-1): 2,220 to 3,600 (COOH broad and weak),
1
2,981 (C-H aliph), 1,724, 1,660 (C=O); H-NMR (MeOD + HCl) δ: 2.92 (q, 8H, -CCH2C-), 2.88 (t,
16H, -CCH2COO-), 2.69 (t, 8H, -CCH2COO-), 3.28 (s, 4H, -N(CH2)2N-), 3.43 (t, 8H, -NCH2C-), 3.64
(t, 16H, -NCH2C-), 3.84 (t, 8H, -CCH2N-), 4.21 (t, 8H, -COOCH2C-) ppm; 13C-NMR δ:
(MeOD+HCl): 26.9 (-CCH2C-), 29.6 (-CCH2COO-), 50.7 (-CCH2N-, (-NCH2C- ), 54.2 (-N(CH2)2N-),
60.5 (-COOCH2C-), 171.9 (-CCOOC-) and 172.5 (-COOH) ppm; MS m/z (ESI) 1154.
6.1.3. Polyglycidol (Reference Compound for Biological Assays).
In a round bottom flask under N2 atmosphere, a solution of potassium methoxide (CH3OK) was
prepared with potassium (0.8 g, 11.3 mmol) in previously distilled methanol (15 mL), and the solution
was heated at 60 °C during 10 min. Afterwards, a solution of glycidol monomer (9.5 mL, 0.1427 mol)
in methanol (5 mL) was added dropwise during 3 h. The mixture was heated at 85 °C for 24 h. Drops
of 10% HCl were added to the cold solution until a pH = 7 was reached; next, the solution was filtered
and evaporated under vacuum to give a yellow pale oil product. Mw = 4591, PDI = 1.8, FT-IR (cm-1)
1
3,261 (OH), 2,877 (CH2,CH), 1,059 and 1,127 (C-O); H-NMR (D2O) δ: 3.08-3.68 (m, CH, CH2),
13
2.8–2.85 (m, CH,CH2 oxirane ring); C-NMR (D2O) δ: 58.7-62.9 (CH2OH), 79.4-78.1 (C-HOR),
71.7–73.7(CH2OR) and 71.0-68.8 (CHOH) ppm.

Molecules 2010, 15
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6.2. Cell Lines Culture and Culture Medium
The dendrimers were screened in vitro (in triplicate) against human cancer cell lines: HCT-15
(human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human
chronic myelogenous leukemia), U251 (human glyoblastoma), PC-3 (human prostatic
adenocarcinoma), SKLU-1 (human lung adenocarcinoma), cell lines were supplied by the National
Cancer Institute (USA). Besides human lymphocytes MT2 cell lines, human tumor cytotoxicity was
also determined by using the protein-binding dye sulforhodamine B (SRB) in microculture assays to
measure cell growth, as described in the protocols established by the NCI [30]. The cell lines were
cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine,
10,000 units/mL penicillin G sodium, 10,000 μg/mL streptomycin sulfate, 25 μg/mL amphotericin B
(Gibco), and 1% non-essential amino acids (Gibco). They were maintained at 37 °C in a humidified
atmosphere with 5% CO2. The viability of the cells used in the experiments exceeded 95%, as
determined with trypan blue.
6.3. Cytotoxicity Assay
Cytotoxicity after treatment of the cancer cell lines and normal cells with the test compounds was
determined using the protein-binding dye sulforhodamine B (SRB) in microculture assays to measure
cell growth, as described in Monks et al., 1991). The cells were removed from the tissue culture flasks
by treatment with trypsin, and diluted with fresh media. From these cell suspensions, 100 μL,
containing 5,000—10,000 cells per well, were pipetted into 96-well microtiter plates (Costar) and the
material was incubated at 37 °C for 24 h in a 5% CO2 atmosphere. Subsequently, a solution of the
dendrimers obtained by diluting the stocks (100 μL) was added to each well. The cultures were
exposed for 48 h to the compound at a concentration of 50 µM. After the incubation period, cells were
fixed to the plastic substratum by the addition of 50 μL of cold 50% aqueous trichloroacetic acid. The
plates were incubated at 4 °C for 1 h, washed with tap H2O, and air-dried. The trichloroacetic-acid-
fixed cells were stained by the addition of 0.4% SRB. Free SRB solution was then removed by
washing with 1% aqueous acetic-acid. The plates were then air-dried, and the bound dye was
solubilized by adding 10 mM unbuffered Tris base (100 μL). The plates were placed on a shaker for
5 min, and the absorption was determined at 515 nm using an ELISA plates reader
(Bio-Tex Instruments).
6.4. Formation of Dendrimer-MTX Complexes
A general procedure for complex formation involved, firstly, the salt formation of the
corresponding dendrimer with NaOH (equimolar amounts), grinding in an agate mortar without
solvent, followed by the addition of five equivalents of MTX to obtain a dendrimer-salt/MTX paste.
This paste was dissolved in deionized water until reaching a 5 mL volume in a volumetric flask to give
a water-soluble complex. The complex solution was sonicated for 20 min. and allowed to equilibrate in
darkness overnight. Afterwards, the solution was centrifuged in a Cole Parmer 17250 centrifuge for
10 min. at 3400 rpm. The supernatant was filtered through an ANOTOP 25 syringe filter with a
400 nm pore size.

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To determine the solubility of MTX in water, 5 mg of MTX was placed in a 5 mL volumetric flask,
the solution was stirred for 30 min., sonicated for 20 min. and allowed to equilibrate in darkness
overnight. Then, the solution was centrifuged for 10 min. at 3400 rpm. The supernatant was filtered
through an ANOTOP 25 syringe filter with a 400 nm pore size. A dilution of 0.3 mL up to 5 mL was
made to obtain a reading using the Lambert-Beer law. All data were recorded in triplicate in a UV-VIS
spectrometer. The absorbance of MTX in deionized water was 0.457.
6.4.1. Dendrimer 8-MTX Complex
Compound 8 (11.7 mg, 0.866 × 10-5 mol) was ground with NaOH (2.7 mg, 6.93 × 10-5 mol) in a
mortar, then MTX (25.5 mg, 5.61 × 10-5 mol) was added and ground for 20 min. This paste was
dissolved in deionized water in a volumetric flask up to 5 mL. The solution was sonicated, centrifuged,
and filtered according to the general procedure. In order to get a UV-VIS spectrum, a dilution of
0.2 mL up to 5 mL was made and the absorbances at 302 was recorded in triplicate. The absorbance
was 1.261.
6.4.2. Dendrimer 13-MTX Complex
Compound 13 (14 mg, 0.866 × 10-5 mol) was ground with NaOH (2.7 mg, 6.93 × 10-5 mol) in an
agate mortar; immediately MTX (25.5 mg, 5.61 × 10-5 mol) was added and ground for 20 min. This
paste was dissolved in deionized water in a volumetric flask up to 5 mL. The solution was sonicated,
centrifuged, and filtered according to the general procedure. In order to get a UV-VIS spectrum, a
dilution of 0.2 mL up to 5 mL was made and the absorbances at 302 was recorded in triplicate. The
absorbance was 0.827.
6.4.3. Calculation of Complex Stoichiometry
CMTX −8
CM0 TX
CMTX −8
CD
CM0 TX
CD
Starting from
= 4.14; isolating CMTX-8 and dividing by CD, we have:
= 4.14⋅
.
CMTX −8
1.7
If
=1 and CD = 1.7 mM, C0 will be: CM0 TX
=
= 0.407mM . With this result and evoking
MTX
CD
4.14
the concentration ratio for dendrimer 13, we have:
CMTX −13
CM0 TX
= 2.71, CMTX −13 = 2.71×0.407mM =1.1mM
Thus, an estimated stoichiometry of 5:3 for dendrimer13-MTX complex can be obtained, multiplying
and dividing by 3 to get a integer numbers, the follow expression:
CMTX −13 1.1mM
3
=
≈
CD
1.7mM
5
Supplementary Materials
Supplementary materials can be seen at http://www.mdpi.com/1420-3049/15/11/8082/s1.

Molecules 2010, 15
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Acknowledgments
This research was carried out with the support of Grants 81924 from CONACyT, IN101109 from
PAPIIT and 4316 from ICyTDF. The authors kindly thank Gerardo Cedillo Valverde and Carmen
Márquez for their technical assistance.
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Sample Availability: Samples of the compounds are available from the authors.
© 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).