Synthesis of 5-aryl-1,4-benzodiazepine derivatives attached in resorcinaren-PAMAM dendrimers and their anti-cancer activity

Article abstract of DOI:10.1016/j.bmc.2011.10.070

A series of resorcinaren-PAMAM dendrimers with benzodiazepines in the periphery were synthesized and their anticancer properties studied. The synthesized dendrimers showed potential anticancer activities, which were enhanced in the presence of a chloro-su

Full text of DOI:10.1016/j.bmc.2011.10.070

Bioorganic & Medicinal Chemistry 20 (2012) 415–421
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Bioorganic & Medicinal Chemistry
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Synthesis of 5-aryl-1,4-benzodiazepine derivatives attached
in resorcinaren-PAMAM dendrimers and their anti-cancer activity
Sandra Cortez-Maya a, Simón Hernández-Ortega a, Teresa Ramírez-Apan a, Irina V. Lijanova b,
Marcos Martínez-García a,
⇑
a Instituto de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria, Circuito Exterior, Coyoacán, C.P. 04510, México D.F., Mexico
b Instituto Politécnico Nacional, CIITEC, Cerrada Cecati S/N, Colonia Santa Catarina de Azcapotzalco, C.P. 02250, México D.F., Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of resorcinaren-PAMAM dendrimers with benzodiazepines in the periphery were synthesized
and their anticancer properties studied. The synthesized dendrimers showed potential anticancer
activities, which were enhanced in the presence of a chloro-substituent in the second ring of the 5-
aryl-1,4-benzodiazepine. The dendrimers were characterized by IR, 1H and 13C NMR, UV–vis absorption,
electrospray (ES) and/or MALDI-TOF mass spectrometries.
Received 27 August 2011
Revised 17 October 2011
Accepted 23 October 2011
Available online 31 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Anticancer activity
Benzodiazepines
Resorcinarenes
PAMAM
Dendrimers
1. Introduction
2. Synthesis
Dendrimers1 are synthetic, nanometer-sized macromolecules
that can be modified to suit a specific application with unique archi-
tecture, given that they have well-defined, compartmentalized
structure and narrow polydispersity. They have been extensively
investigated as drug delivery carriers2–7 due to the presence of nano-
compartments that can encapsulate guest molecules.8–10 Dendri-
mers are commercially available, being PAMAM dendrimers the
most extensively studied for biological applications.11–15 They have
a unique architecture based on b-alanine subunits with primary
amine groups on the surface that are available for the attachment
of several types of biological materials.16 Their aqueous solubility
and biocompatibility are well suited to carry ligands, fluorochromes,
and drugs for targeting, imaging,17 and drug delivery.18,1,19,20
Benzodiazepines represent a large and still expanding group of
synthetic heterocyclic derivatives, which have attracted much
attention in the field of drugs and pharmaceuticals in the last
30 years. The biological relevance of the benzodiazepine family
explains our interest in exploring the anticancer activities of the
dendrimers with benzodiazepines. In the present paper, we describe
the synthesis of 5-aryl-1,4-benzodiazepines derivatives attached to
resorcinaren-PAMAM dendrimers and their anticancer activity.
It has been established that the introduction of activated ester
groups on the upper rim of resorcinarenes provides an excellent pos-
sibility for further chemical modification of resorcinarenes. The es-
ter group of resorcinarenes can be ammonolysized by diamine to
obtain amide derivatives with terminal amino groups. Resorcina-
rene-core amine dendrimers reacting with esters will introduce a
benzodiazepine at the periphery of resorcinarenes. The synthetic
routes for the preparation of the first and second generations of den-
dritic resorcinaren-polyamidoamine and Schiff bases are shown in
Scheme 1.
The dendritic core resorcinarenes 1 and 2 were prepared by the
reaction of resorcinol with dodecanaldehyde or hydrocinnamalde-
hyde,21 they have eight functionalized phenolic hydroxyl groups,
which were primarily full alkylated with methyl bromoacetate in
a K2CO3/acetone system to afford active ester derivatives 3, 4. Com-
pounds 3 and 4 were used as the starting building blocks for con-
structing high-generation dendrimers. Refluxing compounds 3 and
4 with a large excess of 1,2-diaminoethane in a mixture of metha-
nol and benzene 1:1 for 24 h gave the corresponding first genera-
tion amide dendrimers 5 and 6 containing free terminal amino
groups in excellent yields (90% and 88%) (Scheme 1).
Generation 1.5 was obtained from a mixture of methyl acrylate
in MeOH cooled to 0 °C, this was added to compounds 5 or 6 in
MeOH cooled to 0 °C. The MeOH and excess of methyl acrylate,
as volatiles, were evaporated on a rotary evaporator at 34 °C, and
⇑
Corresponding author. Tel.: +52 5622 4451; fax: +52 5616 22032.
E-mail address: margar@servidor.unam.mx (M. Martínez-García).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.070

416
S. Cortez-Maya et al. / Bioorg. Med. Chem. 20 (2012) 415–421
H2N
NH2
NH
O
HN
O
O
O
O
O
O
Br
NH2
Methanol
O
H2N
O
OH
R
O
O
HO
O
K2CO3
Acetone
Benzene
R
4
4
4
R
3=(CH2)-Ph
5=(CH2)-Ph
1=(CH2)-Ph
4=(CH2)-CH3
6=(CH2)-CH3
2=(CH2)-CH3
NH2
H2N
H2N
NH2
NH
NH
O
O
NH
O
O
O
O
O
HN
O
O
O
O
O
N
N
N
N
NH2
O
O
MeOH
H2N
NH
O
Methanol
NH
O
HN
HN
Benzene
O
O
O
O
R
O
O
R
4
4
9=(CH2)-Ph
7=(CH2)-Ph
8=(CH2)-CH3
10=(CH2)-CH3
Scheme 1. Synthesis of the resorcinaren-PAMAM dendrimers first and second generation.
Scheme 2. Synthesis of the benzodiazepine derivatives.
G-1.5 was dried in vacuum obtaining compounds 7 and 8. Finally
the compounds 7 and 8 were reacted with an excess of 1,2-diami-
noethane in a mixture of methanol and benzene 1:1 for 24 h result-
ing in the corresponding second generation amide dendrimers 9
and 10 (Scheme 1).
The 5-aryl-1,4-benzodiazepines (11–14) were synthesized in
agreement with the literature procedure22 and modified in N1 with
MeOꢀ Na+ and methyl bromoacetate (Scheme 2).
The structure of compound (11) was confirmed by X-ray diffrac-
tion analysis of
a single crystal prepared by crystallization
It should be pointed out that compounds 5, 6, 9 and 10 have
good solubility in most organic solvents. In the IR spectra of amides
5 and 6 the absorption band of C@O appears at 1675 cmꢀl, while
the absorption band of C@O in the ester derivatives 3 and 4 ap-
pears at 1750 cmꢀl, which indicates that all the ethyl ester groups
were transformed into amide groups.
from chloroform. The general view of compound (11) is shown in
Figure 1.
Finally the benzodiazepine derivatives were attached to the res-
orcinaren-PAMAM dendrimers in methanol/benzene 1:1 at reflux
for 3 days, the compounds 19–34 were washed with acetone and
methanol to remove the excess of benzodiazepine (Scheme 3).

S. Cortez-Maya et al. / Bioorg. Med. Chem. 20 (2012) 415–421
417
Figure 1. Crystal structure and crystal packing of compound 11. Selected bond lengths (Å): C(1)–C(7) 1.736(2); N(1)–C(18) 1.458(3); N(1)–C(18) 1.458(3); N(1)–C(2)
1.367(3); C(5)–C(11) 1.486(3); C(5)–C(12) 1.499(3); Cl(1)–C(7) 1.736(2). Selected bond angles (°): C(11)–C(5)–C(12) 118.83(17); N(4)–C(5)–C(11) 124.53(17);
C(2)–N(1)–C(10) 123.91(16); C(2)–N(1)–C(18) 115.44(17).
Scheme 3. Synthesis of resorcinaren-PAMAM with benzodiazepine derivatives in the periphery.

418
S. Cortez-Maya et al. / Bioorg. Med. Chem. 20 (2012) 415–421
Table 1
In the case of the second generation dendrimers 17 and 18, the den-
drimer 18 was the most active.
Inhibition on the growth (%) of human tumor cell lines for (1, 2, 5, 6, 9, 10–18) at
50 M in DMSO
l
The results of the present experiments clearly indicate that com-
pounds 15–18 inhibit, in all cell lines, the proliferation of cancer
cells. The rank order of potency was: 16 > 17 > 18 > 15, at the con-
centration assayed. Dendrimers 16 and 18 emerged as the most
promising PC-3, HCT-15 and SKLU-1 inhibitors. In summary, these
results reveal that this class of resorcinaren-PAMAM with benzodi-
azepine derivatives in the periphery gives rise to an inhibitory
growth of cancerous cells, although further investigation is war-
ranted to establish the mechanism of inhibitory activity.
Cell line
U251
PC-3
K256
HCT-15
MCF-7
SKLU-1
Compound
1
2
5
6
9
10
11
12
13
14
47.57
11.80
37.00
18.05
—
56.52
11.72
18.6
5.4
60.78
17.67
35.33
11.19
—
14.73
—
34.91
31.54
—
91.27
12.0
12.9
26.0
25.2
21.46
8.54
33.21
—
14.85
6.01
30.92
16.24
—
1.78
28.96
—
—
—
38.54
12.30
12.83
22.41
22.33
100
91.98
19.29
11.8
24.5
22.2
100
19.71
15.92
27.23
30.62
13.23
12.50
19.8
27.5
20.72
33.72
3. Conclusions
In the present work it was observed that resorcinaren-PAMAM
with benzodiazepine derivatives in the periphery can be obtained
in good yields. Biological activity tests showed that the synthesized
resorcinaren-PAMAM with benzodiazepine derivatives compounds
have potential activity against cancer. Inhibition of cancer cell pro-
liferation was enhanced in the presence of a chloro-substituent in
the second ring of the 5-aryl 1,4-benzodiazepines.
Table 2
Inhibitory concentration (IC50) (
lines for compounds (15–18) in DMSO
l
M) values obtained in PC-3, HCT-15 and SKLU-1 cell
Dendrimers
PC-3
HCT-15
SKLU-1
15
16
17
18
38.54 4.60
15.40 2.30
20.86 1.60
40.84 1.60
15.20 1.40
35.43 2.50
18.85 0.75
23.62 1.90
33.52 1.90
13.83 0.70
39.71 2.30
15.69 0.24
22.64 1.00
32.44 1.00
7.13 0.20
4. Experimental section
cis-Platin
4.1. Materials and equipments
2.1. Cytotoxic activity
Solvents and reagents were purchased as reagent grade and used
without further purification. Ether and toluene were distilled from
sodium and benzophenone, acetone was distilled over calcium chlo-
ride, and methanolwas distilled over calcium oxide powder. Column
chromatography was performed on Merck Silica Gel 60 Å (70–
230 mesh). 1H and 13C NMR spectra were recorded on a Varian
Unity-300 MHz with tetramethylsilane (TMS) as an internal refer-
ence. Infrared (IR) spectra were measured on a Nicolet FT-SSX spec-
trophotometer. Elemental analysis was determined by Galbraith
Laboratories, INC Knoxville.
From the series of synthesized compounds, resorcinarenes 1
and 2, as well as the resorcinaren-dendrimers of first and second
generation 5, 6, 9, 10, were tested for anticancer activity and were
screened in vitro against five human cancer cell lines. U251 (hu-
man glyoblastoma), PC-3 (human prostatic adenocarcinoma),
K562 (human chronic myelogenous leukemia cells), HCT-15 (hu-
man colorectal adenocarcinoma), MCF-7 (human mammary ade-
nocarcinoma) and SKLU-1 (human lung adenocarcinoma). Cell
lines were supplied by the National Cancer Institute (USA). The hu-
man tumor cytotoxicity was determined using the protein-binding
dye sulforhodamine B (SRB) in microculture assay to measure cell
growth, as described in the protocols established by the NCI
(Monks et al., 1991).23 The cell lines were cultured in RPMI-1640
4.2. General procedure for preparation of dendrimers,
generation 0.5
medium supplemented with 10% fetal bovine serum, 2 mM
mine, 10,000 units/ml penicillin G sodium, 10,000 g/ml strepto-
mycin sulfate and 25 g/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 exceeds 95% as determined with trypan blue.
The initially obtained cytotoxic screening data (Table 1) shows that
they have significant anticancer activities, especially compounds 6,
9, 11 and 12, and that their activity depends on the nature of the
human cancer cell line.
L-gluta-
A mixture of the methyl bromoacetate (1 mmol), potassium car-
bonate (3 mmol) in dry acetone (50 mL) was heated to reflux and
stirred vigorously under an N2 atmosphere for 20 min. The com-
pounds 1 or 2 (0.2 mmol) dissolved in dry acetone (40 mL) were
added drop wise and the reaction was continued for 7 days. The
mixture was cooled and the precipitate filtered. The filtrate was
evaporated to dryness under pressure. The residue dissolved in
diethyl ether was washed with an aqueous solution of 5% Na2CO3
(three times). The organic layer was dried and evaporated to obtain
the esters 3 and 4.
l
l
The inhibitory concentration (IC50) values were only determined
for those lines (PC-3, HCT-15 and SKLU-1), for four resorcinaren-PA-
MAM with benzodizepine derivatives samples, and were compared
to the results obtained with the cis-platin cytotoxic agent (Table 2).
For the three selected cell lines, the first generation dendrimer 6
and the second generation dendrimer 9 showed good activity and
in the case of the benzodiazepine derivatives 11, 12 showed promis-
ing activity as well. The first generation dendrimer with the benzodi-
azepine attached (16) was found to be the most potent among the
benzodiazepines studied, exhibiting the lowest inhibitoryconcentra-
4.2.1. Compound 3
Yield: 93%. Brown solid. mp: >300 °C. 1H NMR (300 MHz, CDCl3)
dH: 2.20 (br, 8H, CH2), 2.66 (br, 8H, CH2), 3.75 (s, 24H, O–CH3), 4.28
(s, 16H, CH2–O), 4.72 (t, 4H, CH, J = 7.0 Hz), 6.23 (s, 4H, Ar), 6,76 (s,
4H, Ar), 7.06–7.15 (m, 20H, Ar); 13C NMR (75 MHz, CDCl3) dC: 34.3
(CH2), 35.58 (CH2), 36.7 (CH), 51.9 (O–CH3), 66.9 (CH2-O), 100.5
(Ar), 125.4 (Ar), 126.2 (Aripso), 128.0 (Ar), 128.1 (Ar), 128.3 (Ar),
142.5 (Aripso), 154.2 (Ar-O), 169.7 (C@O). MS (MALDI-TOF) m/z:
1480 (M+). Anal. Calcd for C84H88O24: C, 68.10; H, 5.99. Found: C,
68.10; H, 5.97.
tions (15.40 2.3, 18.85 0.75 and 15.69 0.24 lM, Table 2). The
comparison of the activity of chloro-derivative (dendrimer 16) with
that of the corresponding hydrogen-derivative (dendrimer 15) indi-
cates that the presence of chloro in the second ringof the benzodiaze-
pines leads to better results in inhibition of cancer cell proliferation.
4.2.2. Compound 4
Yield: 95%. Brown solid. mp: >300 °C. 1H NMR: (300 MHz,
CDCl3) dH: 0.86 (t,12H, CH3, J = 6.8 Hz), 1.23 (s, 56H, CH2), 1.29 (s,

S. Cortez-Maya et al. / Bioorg. Med. Chem. 20 (2012) 415–421
419
16H, CH2), 1.83 (m, 8H, CH2), 3.75 (s, 24H, O–CH3), 4.27 (s, 16H,
CH2–O), 4.25 (t, 4H, CH, J = 7.5 Hz), 6.21 (s, 4H, Ar), 6.61 (s, 4H,
Ar); 13C NMR (75 MHz, CDCl3) dC: 14.0 (CH3), 22.6 (CH2), 28.0
(CH2), 29.3 (CH2), 29.6 (CH2), 29.7 (CH2), 29.8 (CH2), 29.9 (CH2),
31.9 (CH2), 34.5 (CH), 51.8 (CH3-O), 67.1 (CH2-O), 100.8 (Ar),
126.5 (Ar), 128.5 (Aripso), 154.4 (Ar-O), 169.7 (C@O). MS
(MALDI-TOF) m/z: 1681 (M+). Anal. Calcd for C96H144O24: C,
68.54; H, 8.63. Found: C, 68.52; H, 8.60.
(CH2–N), 52.5 (CH2–C@O), 68.9 (CH2–O), 96.4 (Ar), 124.4 (Ar),
127.1 (Aripso), 154.7 (Ar-O), 169.2 (C@O), 173.7 (C@O). MS (MAL-
DI-TOF) m/z: 3730 (M+). Anal. Calcd for C184H336N48O32: C, 59.20;
H, 9.07; N, 18.01. Found: C, 59.23; H 9.10; N, 18.01.
4.4. General procedure for preparation of dendrimers,
generation 1.5
A mixture of each of the amides 5 and 6 (0.5 mmol) and methyl
acrylate (15 mL) in methanol (15 mL) was stirred in an atmosphere
of nitrogen at 45–50 °C for 5 d. Methanol and excessive methyl
acrylate were removed in vacuo. The residue was crystallized from
alcohol/ether to obtain the ester compounds 7 and 8.
4.3. General procedure for preparation of dendrimers,
Generation 1.0 and 2.0
A
mixture of ester-containing derivatives 3, 4, 7 and 8
(1.0 mmol) and 1,2-diaminoethane (15.0 mL) in ethanol (15 mL)
and toluene (15 mL) were refluxed for 24 h in an atmosphere of
nitrogen. The organic solvent and the excess 1,2-diaminoethane
were removed in vacuo. The residue was washed several times
with alcohol to obtain the amide products 5, 6, 9 and 10.
4.4.1. Compound 7
Yield: 78%. Brown solid. mp: >300 °C: 1H NMR: (300 MHz,
CDCl3) dH: 2.04 (s, 8H, CH2), 2.39 (br, 16H, CH2–N), 2.57 (s, 8H,
CH2), 2.73 (s,16H,CH2–NH), 3.10 (br, 32H, CH2–C@O), 3.49 (br,
32H, N–CH2), 3.58 (s, 48H, –OCH3), 4.42 (br, 16H, CH2–O), 4.46 (t,
4H, CH, J = 7.0 Hz), 5.12 (br, 8H, NH), 6.54 (s, 4H, Ar), 6.67 (s, 4H,
Ar), 6.9–7.22 (m, 20H, Ar); 13C NMR (75 MHz, CDCl3) dC: 32.1
(CH2), 32.5 (CH2), 32.8 (CH2–C@O), 34.2 (CH), 37.1 (CH2–NH),
49.2 (N–CH2–), 51.5 (O–CH3), 52.7 (CH2–N), 68.4 (CH2–O), 101.2
(Ar), 125.0 (Ar), 125.2 (Aripso), 128.3 (3-Ar), 141.8 (Aripso), 154.2
(Ar-O),167.9 (C@O, 1), 172.9 (C@O, 2). MS (MALDI-TOF) m/z:
3081 (M+). Anal. Calcd for C156H216N16O48: C, 60.77; H, 7.06; N,
7.27. Found: C, 60.78; H, 7.05; N, 7.25.
4.3.1. Compound 5
Yield: 90%. Brown solid. mp: >300 °C. 1H NMR (300 MHz, CDCl3)
dH: 1.96 (br, 16H, CH2), 2.89 (s,16H,CH2–NH2), 3.39 br, 16H, CH2–
NH), 4.37 (br, 16H, CH2–O), 4.47 (br, 16H, NH2), 4.68 (t, 4H, CH,
J = 7.0 Hz), 6.44 (s, 4H, Ar), 6,51 (s, 4H, Ar), 7.06–7.22 (m, 20H,
Ar); 13C NMR (75 MHz, CDCl3) dC: 30.9 (CH2), 34.5 (CH2), 37.2
(CH), 40.5 (CH2–NH2), 41.2 (CH2–NH), 68.5 (CH2–O), 101.2 (Ar),
125.7 (Ar), 127.2 (Aripso), 128.4 (Ar), 128.6 (Ar), 141.6 (Aripso),
147.9 (Ar-O), 162.3 (C@O). MS (MALDI-TOF) m/z: 1704 (M+). Anal.
Calcd for C92H120N16O16: C 64.77, H 7.09; N, 13.14. Found: C, 64.74;
H, 7.11; N, 13.12.
4.4.2. Compound 8
Yield: 75%. Brown solid. mp: >300 °C: 1H NMR: (300 MHz,
CDCl3) dH: 0.83 (t, 12 H, CH3, J = 7.2 Hz), 1.20 (s, 16H, CH2), 1.25
(s, 40H, CH2), 1.34 (s, 24H, CH2), 2.40 (br, 32H, CH2–C@O), 2.60
(br, 16H, CH2–N), 2.75 (br, 32H, N–CH2), 3.50 (br, 16H, CH2–NH),
3.62 (s, 48H, –OCH3), 4.5 (br, 16H, CH2–O), 4.69 (br, 4H, CH), 6.97
(s, 4H, Ar), 7.18 (s, 4H, Ar); 13C NMR (75 MHz, CDCl3) dC: 14.1
(CH3), 22.6 (CH2), 28.6 (CH2), 29.4 (CH2), 29.7 (CH2), 29.9 (CH2),
30.3 (CH2), 31.9 (CH2–C@O), 32.6 (CH2), 36.7 (CH2-NH), 37.2
(CH), 49.3 (N–CH2), 51.54 (–OCH3), 52.7 (CH2–N), 67.9 (CH2–O),
98.1 (Ar), 125.8 (Ar), 128.3 (Aripso), 155.4 (Ar-O), 169.7 (C@O, 1),
173.2 (C@O, 2). MS (MALDI-TOF) m/z: 3281 (M+). Anal. Calcd for
C168 H272N16 O48: C, 61.44; H, 8.35; N, 6.82. Found: C, 61.41; H,
8.37; N, 6.84.
4.3.2. Compound 6
Yield: 88%. Brown solid. mp: >300 °C. 1H NMR (200 MHz, CDCl3)
dH: 0.86 (t, 12H, CH3, J = 6.2 Hz), 1.21 (br, 72H, CH2), 1.95 (br, 8H,
CH2), 2.9 (br, 16H, CH2–NH2), 3.4 (s, 16H, CH2–NH), 4.58 (s, 16H,
CH2–O), 4.71 (t, 4H, CH, J = 7.0 Hz), 5.89 (br, 4H, NH2), 6.49 (s,
4H, Ar), 7.25 (s, 4H, Ar); 13C NMR: (75 MHz, CDCl3) dC: 14.0
(CH3), 22.6 (CH2), 29.3 (CH2), 29.7 (CH2), 29.8 (CH2), 31.9 (CH2),
34.5 (CH), 37.3 (CH2–NH2), 41.2 (CH2–NH), 68.1 (CH2–O), 99.0
(Ar), 126.8 (Ar), 129.5 (Aripso), 154.4 (Ar-O), 168.6 (C@O). MS (MAL-
DI-TOF) m/z: 1905 (M+). Anal. Calcd for C104H176N16O16: C, 65.51;
H, 9.30; N, 11.75. Found: C, 65.49; H, 9.32; N, 11.72.
4.3.3. Compound 9
Yield: 89%. Brown solid. mp: >300 °C: 1H NMR (300 MHz, CDCl3)
dH: 2.34 (br, 8H, CH2), 2.59 (br, 16H, CH2–N), 2.74 (br, 8H, CH2),
2.97 (s, 32H, CH2–NH2), 3.01 (s, 32H, CH2–NH), 3.10 (br, 32H, N–
CH2), 3.23 (br, 32H, CH2–C@O), 3.46 (br, 16H, CH2–NH), 4.44 (br,
16H, CH2–O), 4.80 (t, 4H, CH, J = 7.0 Hz), 6.43 (s, 4H, Ar), 6.50 (s,
4H, Ar), 7.06–7.22 (m, 20H, Ar); 13C NMR (75 MHz, CDCl3) dC:
32.2 (N–CH2), 33.9 (CH2), 34.4 (CH2), 37.2 (CH), 40.9 (CH2–NH),
41.7 (CH2–NH2), 43.6 (CH2–NH), 45.6 (CH2–N), 52.2 (N–CH2),
67.2 (CH2–O), 101.2 (Ar), 125.9 (Ar), 126.7(Aripso), 127.4 (Ar),
128.4 (Ar), 141.8 (Aripso), 154.9 (Ar-O), 169.3 (C@O), 173.6 (C@O).
4.5. General procedure for preparation of benzodiazepine
derivatives
To a solution of 1,4-benzodiazepines 11–14 (7.9 mmol) in
50 mL of anhydrous methanol, NaMeOH (9.8 mmol) was added,
the mixture was stirred for 30 min. Afterwards, was cooled at
10 °C and methyl bromoacetate (9.8 mmol) was added, the
reaction was stirred for 4 h. The solvent was evaporated under
vacuum and the product was chromatographed in a mixture of
hexanes/ethyl acetate 6:4.
MS (MALDI-TOF) m/z: 3530 (M+). Anal. Calcd for C172H280N48O32
:
4.5.1. Methyl-2-(7-chloro-2-oxo-5-phenyl-2,3-dihydro-1
H-benzo[e][1,4]diazepin-1-yl) acetate 11
C, 58.48; H, 7.99; N, 19.03. Found: C, 58.44; H, 7.95; N, 19.92.
Yield: 58%. White solid. mp: 134–135 °C. 1H NMR: (300 MHz,
CDCl3) dH: 3.71 (s, 3H, O–CH3), 3.87 (d, 1H, CH2, J = 10.8 Hz), 4.55
(s, 2H, CH2), 4.86 (d, 1H, CH2, J = 10.8 Hz), 7.29 (d, 1H, Ar-9,
J = 2.4 Hz), 7.43 (s, 1H, Ar-6), 7.44–7.46 (m, 2H, Ar-60, 20), 7.48–
7.50 (m, 1H, Ar-40), 7.51–7.53 (m, 2H, Ar-50, 30), 7.64 (d, 1H, Ar-8,
J = 7.9 Hz); 13C NMR (75 MHz, CDCl3) dC: 49.3 (CH2), 52.6 (O–
CH3), 56.0 (CH2), 123.0 (Ar-9), 128.5 (Ar-50, 30), 129.1 (Ar-7),
129.7 (Ar-60, 20), 130.1 (Ar-6), 130.5 (Ar-5a), 131.0 (Ar-40), 131.9
(Ar-8), 137.7 (Ar-10), 141.3 (Ar-9a), 168.9 (C@N), 169.0 (C@O),
4.3.4. Compound 10
Yield: 89%. Brown solid. mp: >300 °C: 1H NMR: (300 MHz,
CDCl3) dH: 0.87 (s, 12H, CH3), 1.23 (s, 80H, CH2), 2.36 (br, 16H,
CH2–N), 2.77 (br, 32H, CH2–NH2), 3.02 (br, 32H, CH2-NH), 3.27
(br, 32H, CH2–C@O), 3.36 (br, 16H, CH2–NH), 4.57 (br, 16H,
CH2–O), 4.71 (t, 4H, CH, J = 7.0 Hz), 5.96 (br, 32H, NH2), 6.37 (s,
4H, Ar), 7.25 (s, 4H, Ar); 13C NMR (75 MHz, CDCl3) dC: 14.1 (CH3),
22.7 (CH2), 29.5 (CH2), 29.8 (CH2), 29.9 (CH2), 31.2 (CH2), 34.0
(CH), 37.6 (CH2–NH), 41.1(CH2–NH2), 41.9 (CH2–NH), 50.4

420
S. Cortez-Maya et al. / Bioorg. Med. Chem. 20 (2012) 415–421
169.7 (C@O). MS (IE) m/z: 342 (M+). Anal. Calcd for C18H15ClN2O3:
C, 63.07; H, 4.41; N, 8.17. Found: C, 63.06; H, 4.40; N, 8.15.
(CH2), 29.5 (CH2), 29.8 (CH2), 29.9 (CH2), 30.2 (CH2), 32.1 (CH2),
34.7 (CH), 37.2 (CH2–NH), 40.1 (CH2–N), 52.9 (CH2 Bz), 56.6 (CH2
Bz), 67.5 (CH2-O), 123.8 (Ar, Ar-9 Bz), 126.3 (Ar-ipso, Ar-5a Bz),
128.4 (Ar-6 Bz), 128.6 (Ar-30,50 Bz), 128.7 (Ar-20,60 Bz), 128.9 (Ar-
7 Bz), 130.6 (Ar-8 Bz), 131.3 (Ar-40 Bz), 138.4 (Ar-10), 141.6 (Ar-
O), 142.5 (Ar-9a Bz), 169.6 (C@N, C@0 Bz), 169.8(C@O Bz), 169.9
(C@O). MS (MALDI-TOF) m/z: 6903.17 (M+Na). Anal. Calcd for
4.5.2. Methyl-2-(7-chloro-5-(2-chlorophenyl)-2-oxo-2,
3-dihydro-1H-benzo[e][1,4] diazepin-1-yl)acetate 12
Yield: 64%. White solid. mp: 113–114 °C. 1H NMR: (300 MHz,
CDCl3) dH: 3.72 (s, 3H, O–CH3), 4.22 (d, 2H, CH2, J = 11.5 Hz), 4.6
(s, 2H, CH2), 7.12 (d,1H, Ar-9, J = 8.7 Hz), 7.37 (d, 1H, Ar-6,
J = 2.4 Hz), 7.42–7.45 (m, 1H, Ar-30), 7.47–7.49 (m, 1H, Ar-40),
7.52–7.53 (m, 1H, Ar-50), 7.55–7.58 (m, 1H, Ar-8), 7.63–7.65 (m,
1H, Ar-60); 13C NMR (75 MHz, CDCl3) dC: 50.1 (CH2), 52.5 (–
OCH3), 56.8 (CH2), 122.8 (Ar-9), 127.0 (Ar-50), 129.0 (Ar-5a),
129.2 (Ar-7), 129.3 (Ar-6), 130.2 (Ar-30), 131.0 (Ar-40, Ar-60),
131.9 (Ar-8), 133.2 (Ar-20), 136.6 (Ar-10), 138.3 (Ar-9a), 169.4
(C@N), 171.3 (C@O), 173.2 (C@O). MS (IE) m/z: 376 (M+). Anal.
Calcd for C18H14Cl2N2O3: C, 57.31; H, 3.74; N, 7.43. Found: C,
57.30; H, 3.73; N, 7.44.
C376H352Cl16N48O48: C, 65.66; H, 5.16; N.9.77. Found: C, 60.68; H,
5.17; N, 9.76.
4.6.1.2. Dendrimer 16. Yield: 84%. White solid. mp: >300 °C. 1H
NMR: (300 MHz, CDCl3) dH: 0.88 (t, 12H, CH3, J = 6.2 Hz), 1.25 (br,
72H, CH2), 1.95 (br, 8H, CH2), 3.02 (br, 16H, CH2–NH), 3.54 (s,
16H, CH2–N), 3.86 (d, 16H, CH2 Bz, J = 10.9 Hz), 4.35 (s, 16H,
CH2–O), 4.63 (s, 32H, CH2 Bz), 4.80 (d, 16H, CH2 Bz, J = 10.9 Hz),
6.25 (s, 4H, Ar), 6.84 (s, 4H, Ar), 7.14 (d, 16H, Ar-9 Bz, J = 8.7 Hz),
7.37 (d, 16H, Ar-6 Bz, J = 2.4 Hz), 7.45–7.49 (m, 16H, Ar-30 Bz),
7.52–7.56 (m,16H, Ar-40 Bz), 7.57–7.59 (m, 16H, Ar-50 Bz), 7.63–
7.66 (m, 16H, Ar-8 Bz), 7.70–7.72 (m, 16H, Ar-60 Bz); 13C NMR
(75 MHz, CDCl3) dC: 14.1 (CH3), 22.7 (CH2), 29.4 (CH2), 29.7 (CH2),
29.8 (CH2), 31.9 (CH2), 34.5 (CH), 36.1 (CH2–NH), 40.9 (CH2–N),
52.3 (CH2 Bz), 57.1 (CH2 Bz), 67.4 (CH2–O), 98.2 (Ar), 122.8 (Ar-
9), 125.5 (Ar), 126.6 (Ar-5a), 127.0 (Ar-50), 127.8 (Aripso), 129.2
(Ar-7), 129.4 (Ar-6), 130.1 (Ar-30), 131.0 (Ar-40, Ar-60), 131.4 (Ar-
8), 133.2 (Ar-20), 137.4 (Ar-10), 138.3 (Ar-9a), 156.4 (Ar-O), 167.2
(C@N, C@O Bz), 168.3 (C@O), 169.3 (C@O). MS (MALDI-TOF) m/z:
7411.55 (M+). Anal. Calcd for C376H336Cl32N48O48: C, 60.79; H,
4.56; N, 9.05. Found: C, 60.78; H, 4.55; N, 9.05.
4.5.3. Methyl-2-(7-chloro-5-(2-fluorophenyl)-2-oxo-2,
3-dihydro-1H-benzo[e][1,4] diazepin-1-yl)acetate 13
Yield: 70%. Brown solid. mp: 114–115 °C. 1H NMR: (300 MHz,
CDCl3) dH: 3.76 (s, 3H, O-CH3), 3.89 (dd, 1H, CH2, J = 11.8 Hz),
4.53 (s, 2H, CH2), 4.92 (d, 1H, CH2, J = 11.8 Hz), 7.05 (d, 1H, Ar-30,
J = 0.9 Hz), 7.18 (d, 1H, Ar-9, J = 2.4 Hz), 7.25 (d, 1H, Ar-50,
J = 4.6 Hz), 7.43 (s, 1H, Ar-6), 7.45–7.48 (m, 1H, Ar-40), 7.67 (d,
1H, Ar-8, J = 1.8 Hz), 7.70 (d, 1H, Ar-60, J = 1.8 Hz); 13C NMR
(75 MHz, CDCl3) dC: 49.7 (CH2), 52.6 (O-CH3), 56.5 (CH2), 116.4
(Ar-30), 122.8 (Ar-9), 124.5 (Ar-50), 126.6 (Ar-10), 126.7 (Ar-5a),
130.4 (Ar-6), 131.3 (Ar-60), 131.4 (Ar-7), 131.6 (Ar-8),132.4 (Ar-
40), 140.2 (Ar-9a), 162.15 (Ar-20), 166.0 (C@N), 168.7 (C@O),
169.1 (C@O). MS (IE) m/z: 360 (M+). Anal. Calcd for C18H14ClFN2O3:
C, 59.93; H, 3.91; N, 7.76. Found: C, 59.91; H, 3.92; N, 7.75.
4.6.2. Second generation
4.6.2.1. Dendrimer 17. Yield: 89%. Brown solid. mp: >300 °C. 1H
NMR: (300 MHz, CDCl3) dH: 2.38 (br, 8H, CH2), 2.61 (br, 8H, CH2),
2.8 (br, 16H, CH2–N), 3.05 (s, 32H, CH2–NH), 3.12 (s, 32H, CH2–
NH), 3.20 (br, 32H, CH2–C@O), 3.41 (br, 32H, CH2–NH), 3.9 (d, 32H,
CH2, J = 10.8 Hz), 4.54 (br, 16H, CH2–O), 4.60 (d, 64H, CH2-Bz,
J = 4.8 Hz), 4.77 (d, 32H, CH2, J = 10.8 Hz), 4.80 (t, 4H, CH,
J = 7.0 Hz), 6.43 (s, 4H, Ar), 6.50 (s, 4H, Ar), 7.10–7.20 (m, 20H, Ar),
7.27 (d, 1H, Ar-9 Bz, J = 2.4 Hz), 7.33 (s, 2H, Ar-60,20 Bz), 7.36 (s, 1H,
Ar-50,30 Bz), 7.45–7.50 (m, 2H, Ar-40 Bz), 7.54 (d, 1H, Ar-6 Bz,
J = 2.4 Hz), 7.60 (d, 1H, Ar-8 Bz, J = 6.9 Hz); 13C NMR (75 MHz, CDCl3)
dC: 29.8 (CH2), 29.9 (CH2), 34.5 (CH), 37.8 (CH2–NH), 40.1 (CH2–N),
52.8 (CH2-Bz, CH2–N), 56.4 (CH2–C@O, CH2-Bz), 67.2 (CH2–O),
101.2 (Ar), 123.2 (Ar-9 Bz), 123.5 (Ar), 126.1 (Ar-5a, Bz), 128.5 (Ar),
128.8 (Ar), 129.8 (Ar-50,30-Bz), 130.4 (Ar-60,20,40-Bz), 130.8 (Ar-7-
Bz), 131.2 (Aripso), 131.3 (Ar-6 Bz), 132.4 (Ar-8 Bz), 138.3 (Ar-10 Bz),
141.4 (Aripso), 142.3 (Ar-9a, Bz), 157.9 (Ar-O), 169.5 (C@O, Bz,
C@N),169.7 (C@O, 2, Bz), 170.1 (C@O, 1). MS (MALDI-TOF) m/z:
13,442.95 (M+). Anal. Calcd for C716H632Cl32N112NaO96: C, 63.71;
H, 4.72; N, 11.62. Found: C, 63.68; H, 4.69; N, 11.59.
4.5.4. Methyl-2-(7-nitro-2-oxo-5-phenyl-2,3-dihydro-1
H-benzo[e][1,4]diazepin-1-yl) acetate 14
Yield: 70%. Brown solid. mp: 120–121 °C. 1H NMR: (300 MHz,
CDCl3) dH: 3.75 (s, 3H. O–CH3), 4.22 (d, 1H, CH2, J = 9.4 Hz), 5.28 (s,
2H, CH2), 6.57 (d, 1H, CH2, J = 9.4 Hz), 7.14 (d, 1H, Ar-9, J = 2.2 Hz),
7.48–7.50 (m, 2H, Ar-30, 50), 7.51–7.53 (m, 1H, Ar-40), 7.53–7.56
(m, 2H, Ar-20, 60), 8.10 (s, 1H, Ar-6), 8.14 (d, 1H, Ar-8, J = 2.6 Hz);
13C NMR (75 MHz, CDCl3) dC: 49.7 (CH2), 52.6 (O-CH3), 56.5 (CH2),
116.4 (Ar-30), 122.8 (Ar-9), 124.5 (Ar-50), 126.6 (Ar-10), 126.7 (Ar-
5a), 130.4 (Ar-6), 131.3 (Ar-60), 131.4 (Ar-7), 131.6 (Ar-8), 132.4
(Ar-40), 140.2 (Ar-9a), 162.15 (Ar-20), 166.0 (C@N), 168.7 (C@O),
169.1 (C@O). MS (IE) m/z: 353 (M+). Anal. Calcd for C18H15N3O5: C,
61.19; H, 4.28; N, 11.89. Found: C, 61.17; H, 4.27; N, 11.87.
4.6. Dendrimers with benzodiazepines in the periphery
To a solution of dendrimers 5, 6, 9 and 10 (0.5 mmol) in meth-
anol (15 mL), the benzodiazepine ester 11–14 (0.5 mmol) in meth-
anol (15 mL) were added, the mixture was stirred in an
atmosphere of nitrogen at 45–50 °C for 5 d. The methanol was re-
moved in vacuo. The residue was washed several times with meth-
anol, after with CH2Cl2 to obtain desired products 15–18.
4.6.2.2. Dendrimer 18. Yield: 87%. White solid. mp: >300 °C. 1H
NMR: (300 MHz, CDCl3) dH: 2.38 (br, 8H, CH2), 2.61 (br, 8H, CH2),
2.25 (br, 16H, CH2–N), 2.77 (br, 32H, CH2–C@O), 2.92 (br, 32H, N–
CH2), 3.29 (br, 48H, CH2–NH), 3.27 (br, 32H, CH2–N), 3.77 (d, 32H,
CH2 Bz, J = 10.8 Hz), 4.49 (br, 16H, CH2–O), 4.44 (d, 32H, CH2, Bz
J = 3.3 Hz), 4.72 (d, 64 H, CH2 Bz, J = 10.8 Hz), 6.43 (s, 4H, Ar), 6.50
(s, 4H, Ar), 7.10–7.20 (m, 20H, Ar), 7.29–7.41 (m, 16H, Ar-20, Ar-60,
Bz), 7.18 (d, 32H, Ar-9, Bz, J = 3.3 Hz), 7.48 (d, 32H, Ar-6, Bz,
J = 1.8 Hz), 7.51 (s, 32H, Ar-8, Bz); 13C NMR (75 MHz, CDCl3) dC:
14.3 (CH3), 22.8 (CH2), 29.5 (CH2), 29.8 (CH2), 29.9 (CH2), 30.2
(CH2), 32.1 (CH2), 34.0 (CH), 49.0 (CH2–C@O), 49.3 (CH2–NH), 49.5
(CH), 49.8 (CH2–NH), 50.2 (CH2–N), 50.5 (CH2–N), 52.7 (N–CH2,
CH2-Bz), 56.4 (CH2-Bz), 56.7 (CH2–O), 96.4 (Ar), 122.6 (Aripso),
123.0 (Ar, Ar-9 Bz), 128.6 (Ar-30,50 Bz), 129.6 (Ar-6 Bz) 130.2(Ar-
20,60 Bz), 130.4(Ar-7 Bz), 131.0 (Ar-8 Bz), 131.9 (Ar-40 Bz), 138.1
(Ar-10 Bz), 142.8 (Ar-9a Bz), 169.2 (C@N, C@0 Bz), 169.4 (C@O, 3,
4.6.1. First generation
4.6.1.1. Dendrimer 15. Yield: 84%. White solid. mp: >300 °C. 1H
NMR: (300 MHz, CDCl3) dH: 0.82 (s, 12H, CH3), 1.03 (s, 40H, CH2),
1.25 (s, 40H, CH2), 3.21 (br, 16H, CH2–NH), 3.38 (br, 16H, CH2–N),
3.70 (s, 16H, CH2–O), 3.9 (d, 16H, CH2, Bz, J = 10.8 Hz), 4.6 (d,
32H, CH2, Bz, J = 4.8 Hz), 4.8 (d, 16H, CH2, Bz, J = 10.8 Hz), 7.09
(br, 4H, Ar), 7.14 (br, 4H, Ar), 7.26 (d, 16H, Ar-9 Bz, J = 2.4 Hz),
7.32–7.35 (m, Ar-20,60 Bz), 7.41–7.45 (m, 16H, Ar-30,50 Bz), 7.49–
7.51 (m, 16H, Ar-40 Bz), 7.53–7.56 (m, 16H, Ar-6 Bz), 7.58–7.60
(m, 16H, Ar-8 Bz); 13C NMR (75 MHz, CDCl3) dC: 14.1 (CH3), 22.8

S. Cortez-Maya et al. / Bioorg. Med. Chem. 20 (2012) 415–421
421
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716H600Cl64N112NaO96: C, 58.90; H, 4.14; N, 10.74. Found: C,
58.93; H, 4.18; N, 10.76.
C
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4.7. Crystal structure determination
A suitable crystal of compound 11 (obtained by crystallization
from CHCl3 at room temperature) was rolled in epoxy resin and
mounted on a glass fiber. Bruker Apex AXS CCD area detector
X-ray diffractometer was the instrument used for determination.
The data were first reduced and corrected for absorption using
psi-scans, and then solved using the program SHELL-XS. All non-
hydrogen atoms were refined using the anisotropic thermal
parameters and the hydrogen atoms were refined at calculated
positions applying thermal parameters constrained to the carbon
atom on which they were attached. CCDC 848697 contains the sup-
plementary crystallographic data for this paper. These data can be
obtained free of charge via www.ccdc.cam.ac.uk/conts/retriev-
ing.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ,
UK; fax: +44 1223 336033; (e-mail: deposit@ccdc.cam.ac.uk)
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This work was supported by DGAPA-UNAM (IN202010-3) grant.
We would also like to thank O. H. Rios, L. Velasco, S. E. Huerta, M.
M. R. Patiño, M. A. Peña Gonzalez for technical assistance.
References and notes
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