Lignopurines: A new family of hybrids between cyclolignans and purines. Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2012.10.026

A new family of hybrids between cyclolignans related to podophyllic aldehyde, a non-lactonic cyclolignan, and purines were prepared and evaluated against several human tumour cell lines. Both fragments, cyclolignan and purine, were linked through aliphatic and aromatic chains. The influence on the cytotoxicity of the purine substitution and the nature of the linker is analyzed. The new family was slightly less cytotoxic than the parent podophyllic aldehyde, although the selectivity is maintained or even improved and among the linkers used, the presence of an aromatic ring gave the most potent and selective derivatives within the new series tested. Cell cycle and confocal studies demonstrate that these derivatives interfere with the tubulin polymerization and arrest cells at the G₂/M phase, in the same way than the parent compounds podophyllotoxin and podophyllic aldehyde do.

Full text of DOI:10.1016/j.ejmech.2012.10.026

European Journal of Medicinal Chemistry 58 (2012) 377e389
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Lignopurines: A new family of hybrids between cyclolignans and purines.
Synthesis and biological evaluation
,
a
a
a
a
a
b
Ma Ángeles Castro a , José M . Miguel del Corral , Pablo A. García , M Victoria Rojo , Ana C. Bento ,
*
Faustino Mollinedo b, Andrés M. Francesch c, Arturo San Feliciano a
a Departamento de Química Farmacéutica, Facultad de Farmacia, CIETUS-IBSAL, Campus Miguel de Unamuno, Universidad de Salamanca, E-37007 Salamanca, Spain
b Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, CSIC-USAL, Campus Miguel de Unamuno, E-37007 Salamanca, Spain
c PharmaMar S.A. Avda. de los Reyes, P.I. La Mina Norte, E-28770 Colmenar Viejo, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A new family of hybrids between cyclolignans related to podophyllic aldehyde, a non-lactonic cyclo-
lignan, and purines were prepared and evaluated against several human tumour cell lines. Both frag-
ments, cyclolignan and purine, were linked through aliphatic and aromatic chains. The influence on the
cytotoxicity of the purine substitution and the nature of the linker is analyzed. The new family was
slightly less cytotoxic than the parent podophyllic aldehyde, although the selectivity is maintained or
even improved and among the linkers used, the presence of an aromatic ring gave the most potent and
selective derivatives within the new series tested. Cell cycle and confocal studies demonstrate that these
derivatives interfere with the tubulin polymerization and arrest cells at the G2/M phase, in the same way
than the parent compounds podophyllotoxin and podophyllic aldehyde do.
Received 25 July 2012
Received in revised form
15 October 2012
Accepted 16 October 2012
Available online 26 October 2012
Keywords:
Cyclolignans
Hybrids
Podophyllic aldehyde
Purines
Ó 2012 Elsevier Masson SAS. All rights reserved.
Cytotoxicity
1. Introduction
diversity of structures due to the large possibilities of combination.
The use of hybridization as a way to obtain new drugs is especially
Hybrid formation is a classic strategy in drug design based in
combining different bioactive fragments or molecules to get the
corresponding “hybrids” or “conjugates” [1]. It is based in the fact
that many natural products present hybrid structures and their
activities are the resultant of combining those from the individual
fragments [2,3]. In many cases, conjugates are able to improve their
precursors’ properties, to overcome resistance associated to indi-
vidual fragments or to show different activities or even different
mechanisms of action to that of their precursors [1e3]. In some
cases the presence of an endogenous metabolite, as a part of the
conjugate drug, serves to transport the other active part towards
the biological target where the metabolite is well recognized [3].
The promising biological properties observed in several cases
increased the interest of the scientific community in this strategy of
drug design with the aim of generating new activities or modu-
lating the already existing ones [2e8]. It also provides a very high
interesting in research related to chemotherapy, where natural
products play a relevant role in the development of new drugs [9].
Among those natural products, our research group has been
interested in the chemomodulation of the antineoplastic activity of
cyclolignans related to podophyllotoxin 1 (Fig. 1), the main compo-
nent of Podophyllum resin, whose pharmacological properties
(antiviral, cytotoxic, antirheumatic, cathartic, etc.) have been well
recognized for centuries [10]. In this sense, our group has been
engaged in the design and synthesis of new and better podo-
phyllotoxin derivatives. We have prepared a large number of
podophyllotoxin analogues through chemical modification of most
rings and positions of the lignan skeleton [11,12] and very recently
some of us have reported on the importance of attaching bulky
substituents at position 7
a [13]. Along these studies, we also
prepared a potent and selective derivative that lacked the lactone
ring, the podophyllic aldehyde 2 [14] (Fig. 1). The lactone ring is a
structural feature that was previously considered essential for
the antineoplastic activity of podophyllotoxin analogues. This
compound showed an important cytotoxicity and selectivity against
HT-29 human colon carcinoma and certain breast and brain cancer
cell lines [14a] and became our lead compound for further structural
modifications, including those presented here.
* Corresponding author. Departamento de Química Farmacéutica, Facultad de
Farmacia, Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Sala-
manca, Spain. Tel.: þ34 923 294528; fax: þ34 923 294515.
E-mail address: macg@usal.es (MaÁ. Castro).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.026

378
MaÁ. Castro et al. / European Journal of Medicinal Chemistry 58 (2012) 377e389
R
R1
Compound
A
B
R
9
7
6
CHO
7
9
O
8
1
OH
Podophyllotoxin1
CH3
O
10
A
B
C
D
O
9'
O
COOCH3
9'
O
4
8'
H
O
Etoposide
Etopophos
7'
O
OH
3
O
1'
O
O
O
2'
HO
OH
E
4'
P
H3CO
OCH3
OH
H3CO
12'
OCH3
10'
OCH3
11'
OR1
O
O
H
Teniposide
O
S
O
OH
Podophyllic aldehyde 2
HO
Fig. 1. Structures of cyclolignans in clinical use (A) and structure of selective podophyllic aldehyde (B).
It is worth to point out that a few semisynthetic derivatives of
g-lactone ring is not an essential feature for cytotoxicity, whereas
podophyllotoxin are in clinical use for the treatment of different
malignancies, including lung and testicular carcinoma, lymphoma
or nonlymphocytic leukaemia [15,16]. Those derivatives are eto-
poside, teniposide and etopophos (Fig. 1), which could be consid-
ered themselves as hybrids of podophyllotoxin and modified
glucose, being examples of conjugates that show a different
mechanism of action to that of their precursors. While podo-
phyllotoxin inhibits the assembly of tubulin into microtubules,
avoiding the formation of the achromatic spindle and arresting cell
division in metaphase, etoposide and analogues inhibit DNA
topoisomerase II, preventing re-ligation of the double-stranded
breaks. Another podophyllotoxin derivative is tafluposide,
currently undergoing clinical trials, which is a dual Topo I and Topo
II inhibitor [17]. Tafluposide has the hydroxyl groups in the sugar
moiety of etopophos acylated with the pentafluorophenoxyacetyl
group and it is another example of a different mechanism of action
to that of their precursors.
Additionally, podophyllotoxin and its derivatives have also been
hybridized with different cytotoxic agents in an attempt to generate
compounds, which can act simultaneously on different biological
targets such as topoisomerases and microtubules [18]. Thus,
conjugates of epipodophyllotoxin (Fig. 2) with camptothecin [19]
and paclitaxel [20] have been described by Cheng and Lee. The
first ones showed both anti Topo-I and Topo-II activities and those
hybridized with paclitaxel were more potent than the parent
compounds. Passarella and col. have hybridized podophyllotoxin
with thiocolchicine [21] and different vinca alkaloids [22].
We have also found in literature studies where different
analogues of podophyllotoxin have been conjugated with artesu-
nate [23] and lexitropsines [24]. Bisepipodophyllotoxins [25] with
interesting cytotoxicity have been described in literature as well.
However, and despite the significant role of nitrogenated bases in
many biological processes and the important biological activities
described either for pyrimidine or purine nucleosides, only a few
synthetic hybrids of cyclolignans and pyrimidines have been re-
ported [26] and we have not found any example of structures
conjugating podophyllotoxin analogues with purines. However,
there are several examples of natural conjugates between purine
and terpenoids, such as asmarines, agelasines and agelasimines
that showed a very interesting antineoplastic cytotoxicity [27]. This
fact and the potential interest of this family of compounds as
antitumour agents lead us to consider the development of “ligno-
purines” as a new type of bioactive podophyllotoxin derivatives.
In our previous work [14,28] we described that podophyllic
aldehyde 2 itself, its imino derivatives at C-9 and other aldehydes
containing longer and structurally varied ester chains or bio-
isosteric amides at C-90 can inhibit tubulin polymerization by
interacting with the microtubule network. Those compounds were
also able to induce delayed cellular apoptosis after 48 h of treat-
ment [14a] and these biological properties confirmed that the
the presence of an electrophilic function at C-7 and/or C-9 seems
important for the antineoplastic potency observed for this type of
cyclolignans.
We now describe the synthesis and biological evaluation of
lignopurines, a novel family of hybrids between podophyllic alde-
hyde derivatives and different substituted purines. Both precursors
are attached through the position C-90 of the cyclolignan and the
nitrogen N-9(N-7) of the purine using aliphatic and aromatic
linkers. The cytotoxicity evaluation and cell-cycle arrest studies for
these derivatives are presented here as well.
2. Results and discussion
2.1. Chemistry
The general structure for the new lignopurines presented in this
work is illustrated in the retrosynthetic Scheme 1 by the conjugates
(“hybrid model”) formed by the junction of a cyclolignan and
a purine through different spacers. The numbering for the cyclo-
lignan component (Fig. 1) used in this work is in accordance with
IUPAC rules for lignans [29].
The lignopurine objective could be obtained through two main
synthetic strategies, depending on whether the linker is firstly
attached to the purine system (Scheme 1, route A) or to the lignan
derivative (Scheme 1, route B). Thus, route A involves the alkylation
of the purine with the linker and a later condensation with dihy-
droxyacid 3, generating the aldehyde group in the last step by
oxidation under Swern conditions. Route B implies an initial
esterification of the lignan with the corresponding linker and a final
condensation with the purine. The cyclolignan fragment was pic-
ropodophyllic acid, 3, which is the precursor of the lead compound
podophyllic aldehyde 2. Compound 3 can be easily prepared from
podophyllotoxin 1 [14b] and the later was isolated from Podo-
phyllum resin as previously described by us [30].
The purines used for these transformations were purine itself
and 6-chloropurine, which were commercially available. To study
the effect that the presence of a nucleophile at C-6 has on the
cytotoxicity of the conjugates, the chlorine in 6-chloropurine was
replaced by a propylamine group. The substitution [31] was per-
formed by treatment of 6-chloropurine with propylamine yielding
N6-propyladenine in a 60% yield after chromatography. Since
bifunctionalized hydrocarbon chains are reported in the literature
as the most frequently used spacers [2,32] and to take advantage of
our experience in cyclolignan derivatisation as esters [28], we
decided to use dibromides as linker agents (Scheme 2). Two
different alkylene dibromides (1,3-dibromopropane and 1,6-
dibromohexane) and one phenylene analogue (a,a
0-dibromo-p-
xylene) were selected for the preparation of the hybrids in order to
study the effect that the size of the spacer would have on the
cytotoxic activity of the conjugates. All esterification reactions

MaÁ. Castro et al. / European Journal of Medicinal Chemistry 58 (2012) 377e389
379
AcO
O
A
OH
O
C6H5
O
R1
N
H
O
O
O
O
H
OBz
HO
HC
N
OAc
N
HN
N
O
O
O
O
O
O
N
O
O
O
O
O
H3CO
OCH3
H3CO
OCH3
OH
OH
NH
B
O
O
O
O
S
S
OCH3
OCH3
O
MeO
O
NH
H
N
O
n
n
(CH2)n
H
HO
COOMe
O
O
O
O
O
O
OCH3
O
O
O
H3CS
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
n = 2 / 8
n = 1-2
Fig. 2. A) Hybrids of epipodophyllotoxin with camptothecin (left) and paclitaxel (right). B) Hybrids of podophyllotoxin with thiocolchicine (left) and vinorelbine (right).
described in this work were carried out using the minimum
volume of dry DMF. This aspect was critical because dilution leads
to re-lactonization with formation of the 80-epimeric lactone, pic-
ropodophyllin [28].
esters of 3 (Scheme 3). The reactions were performed in DMF, at
room temperature and in the presence of potassium carbonate [28].
When the purines with the shorter linker (4 and 5) were used,
a complex reaction crude was obtained where the expected prod-
ucts 10 and 11, which would lead to aldehydes 16 and 17, were not
found. In the case of the purines with longer linkers, the hybrid
dihydroxyesters 12e15 were obtained in variable yields and traces
of picropodophyllin were always detected. When mixtures of N-9
and N-7 alkyl purines 7 and 9 were used for the condensation
process, the corresponding regioisomer derivatives could be iden-
tified in the reaction products.
2.1.1. Route A: condensation of the lignans with previously alkylated
purines
As mentioned before, route A involves the reaction of dihy-
droxyacid 3 (easily obtained from podophyllotoxin 1 isolated from
Podophyllum resin), with purines that had been previously alky-
lated with different linkers.
The linkers were attached to the purines by alkylation at room
temperature using potassium carbonate as a base [33] and dime-
thylformamide as the solvent. In general, N-9 alkylated purines 4e9
were generated as the main reaction products, being the N-7
alkylated derivatives 7a and 9a, when detected, the minor reaction
product (Scheme 2). The alkylated purines were used to prepare the
Due to their lability and lactonization tendency, conjugated
dihydroxyesters 12e15 were not purified and the crude products
were directly transformed into the corresponding aldehydes 18e21
by oxidation under Swern conditions. Again, when mixtures of N-9
and N-7 alkyl purines were used, both regioisomeric aldehydes
could be identified in the reaction products. Only in the case of 19,
X
7
9
7
N
6
CHO
O
N 1
8
9'
2
O
N
COO
link
N
9
H3CO
OCH3
OCH3
Route B
Route A
Hybrid model
OH
Ar
OH
Ar
X
N
X
N
CH2OH
CH2OH
COO
N
N
N
+
N
N
+
COOH
link Br
N
H
link
Br
3
Scheme 1. Retrosynthetic scheme for the two main strategies to get the cyclolignan conjugates.

380
MaÁ. Castro et al. / European Journal of Medicinal Chemistry 58 (2012) 377e389
X
X
N
link Br
1) K2CO3
X
N
N9
link Br
N-9 Alkyl purines
N
N
N
2) Br-LINKER-Br
+
N 7
N
N
N
H
N
N
X
H
N-7 Alkyl purines
(major regioisomer) (minor regioisomer "a")
Purine
6-Chloropurine
N6-Propyladenine
Cl
X
LINK
Purine
NH-Pr
L1= -(CH2)3-
L1= -(CH2)3-
L2= -(CH2)6-
L2= -(CH2)6-
L3= -CH2-Ph-CH2-
L2= -(CH2)6-
H
Cl
H
Cl
Cl
4
5
6
7(7a) (3:1)
8
NH-Pr
9(9a) (2:1)
Scheme 2. Preparation of the alkylated purines.
X
N
OH
OH
Ar
X
N
N
N
CH2OH 1) K2CO3 / DMF
CH2OH
COO
O
O
CHO
COO
N
N
N
N
X
COOH
Swern
link
Diol
10
11
12
13
14
N
link
N
2)
Ar
N
N
10-15
link Br
16-21
X
H
Link
L1
L1
L2
L2
Aldehyde
16
H3CO
OCH3
OCH3
3
Cl
H
Cl
Cl
17
18
19(19a)
20
L3
15 NH-Pr
L2
21(21a)
Scheme 3. Condensation of the lignans with previously alkylated purines following route A.
both regioisomers (19 and 19a) were isolated and characterized by
two-dimensional NMR experiments.
protected, as its acetonide, in order to avoid re-lactonization of the
cyclolignan. These possibilities are shown in Scheme 4.
According with this planning, the linkers were attached to the
cyclolignans through ester-type bonds. Reaction of dihydroxyacid 3
with different dibromides at room temperature [28], in small
volumes of dimethylformamide and using potassium carbonate as
a base gave esters 22e24 (Scheme 5), which were the starting
points for the different possibilities of route B.
2.1.2. Route B: treatment of the lignan with the linker and final
condensation with the purine
In view of the aldehydes 16 and 17 were not obtained by
previous route A, further studies were devoted to explore an
alternative way to condensate purines and lignan derivatives. Thus,
route B implied an initial reaction of the lignan carboxylate with the
corresponding dibromide and a final condensation with the purine.
In this case, the aldehyde formation could be carried out either
before or after the condensation process. If the purine is attached
before the formation of the unsaturated aldehyde, the dihydrox-
yesters could be conjugated with the purine either as a free diol or
2.1.2.1. B1. Generation of the aldehyde prior to condensation with the
purine. The bromide esters 22e24 were oxidized under Swern
conditions to generate the corresponding aldehydes 25e27 in
good yields. All of them were purified and characterized by 1H and
13C NMR. Different reaction conditions were studied for the
X
OH
CHO
O
O
N
N
CH2OH
N
COO
N
link
COO
Br
link
X
N
Ar
Ar
O
N
Route B2
N
+
N
H
Route B1
O
X
N
CHO
N
COO
Br
N
link
+
COO
Br
link
Ar
N
H
Ar
Route B3
Scheme 4. Retrosynthetic analysis for the preparation of the cyclolignan hybrids by route B.

MaÁ. Castro et al. / European Journal of Medicinal Chemistry 58 (2012) 377e389
381
OH
OH
CH2OH
CH2OH
COOH
1) K2CO3
O
O
O
O
2,2-DMP
p-TsOH
2) Br-Link-Br
COO Link
Br
DMF
COO
Br
Ar
32
H3CO
OCH3
OCH3
Link Diol
H3CO
OCH3
OCH3
3
L1
L2
L3
22
23
24
Purine
K2CO3
K2CO3 / Purine
DMF, 100 °C
DMF,
100°C
Swern
X
N
N
O
O
N
OH
CHO
COO Link
CH2OH
COO
N
Br
COO
Ar
Ar
Link
Purine
Ar
Link Aldehyde
Diol
33
X
H
Cl
L1
L2
L3
25
26
27
34
p-TsOH
K2CO3 / Purine
H2O/acetone
DMF
X
X
N
N
N
CHO
10, 11 (Scheme 3)
Swern
N
N
CHO
+
N
N
N
COO
COO
Link
Link
Ar
Ar
16-20
28-31
X
H
Cl
Cl
Cl
Link
L1
L1
L2
L3
16, 17 (Scheme 3)
16
17
19
20
28
29
30
31
Scheme 5. Preparation of the cyclolignans hybrids following route B.
condensation process between those esters and the purines, and
various solvents (DMF, acetonitrile) and bases (K2CO3, NaH) were
explored. Reaction times were generally long (15 h to 3 d) and, as
happened before, the best results were obtained when reactions
were carried out in DMF, at room temperature and using potassium
carbonate as the base. However, partial aromatization of the cyclo-
lignan skeleton was observed and mixtures of the hybrid aldehydes
(16, 17, 19 and 20) and their derivatives with aromatized C-ring
(28, 29, 30 and 31) were always found. Both aldehydes co-eluted in
chromatography, so their separation was difficult and only small
amounts of the aromatic aldehydes 28 and 29 could be isolated.
Working in this way, when the mixture of both aldehydes 16 and 28
was kept under the same reaction conditions used for the conden-
sation, transformation of 16 into 28 was observed. In all these cases,
only N-9 alkylated derivates were found in the reaction crudes
(Scheme 5).
2.1.2.2. B2. Condensation of dihydroxyesters with purines. Due to the
issues arose from the aromatization of the aldehydes we explored
the formation of hybrids before the Swern oxidation, generating
the a,b-unsaturated aldehydes at the end of the route. Thus, dihy-
droxyesters 22 and 23 were condensed with 6-chloropurine in DMF
Cl
Cl
N
N
H
COO
NaBH4
MeOH
N
N
Pr-NH2
MgSO4
N
N
N
N
19
20
COO
N
Link
Link
Link
Link
N
Ar
Ar
35
36
L2
L3
37
38
L2
L3
Scheme 6. Preparation of hybrids containing nitrogen at C-9.

382
MaÁ. Castro et al. / European Journal of Medicinal Chemistry 58 (2012) 377e389
Table 1
The acetonide formation could be performed before or after
the reaction of the acid with 1,3-dibromopropane. The protection of
the dihydroxyacid 3 by treatment with 2,2-dimethoxypropane
and p-toluenesulfonic acid [34] followed by reaction with
1,3-dibromopropane provided acetonide 32 in only 33% yield, while
the protection of the already synthesized bromoester 22 in similar
conditions, generated the acetonide 32 in 86% yield. This acetonide
32 was condensed with purine and 6-chloropurine in the same
conditions described above to afford derivatives 33 and 34
respectively. Hybrids were then deprotected by reaction with
p-toluenesulfonic acid in water/acetone [35] solution to get the
diol-esters 10 and 11 and these compounds were finally oxidized
under Swern conditions. Thus, and despite the need of a longer and
more complex route, aldehydes 16 and 17 were synthesized and
purified in acceptable yields and completely characterized through
their NMR spectra.
Cytotoxicity of podophyllic aldehyde derivatives (GI50 ꢀ SD/
mM).
Compound
1a
2
16
17
18
19
19a
20
21
22
23
25
26
27
29
36
MB-231
HT-29
A-549
e
0.024
0.012
0.2 ꢀ 0.0082
7.7 ꢀ 0.5
0.039 ꢀ 0.0048
0.71 ꢀ 0.026
0.91 ꢀ 0.02
0.53 ꢀ 0.036
0.77 ꢀ 0.069
1.7 ꢀ 0.15
0.06 ꢀ 0.003
0.79 ꢀ 0.026
0.75 ꢀ 0.079
0.31 ꢀ 0.031
0.4 ꢀ 0.052
1.2 ꢀ 0.0083
0.029 ꢀ 0.0014
1.2 ꢀ 0.1
2.4 ꢀ 0.17
2.4 ꢀ 0.18
3.1 ꢀ 0.081
3.7 ꢀ 0.25
0.81 ꢀ 0.031
5.1 ꢀ 1.9
0.051 ꢀ 0.0046
1.2 ꢀ 0.16
0.16 ꢀ 0.025
0.35 ꢀ 0.02
0.13 ꢀ 0.031
1.8 ꢀ 0.048
0.7 ꢀ 0.17
5 ꢀ 0.26
0.14 ꢀ 0.0057
0.16 ꢀ 0.0091
0.14 ꢀ 0.02
0.74 ꢀ 0.026
0.077 ꢀ 0.0077
2.1 ꢀ 0.016
0.09 ꢀ 0.02
2.2 ꢀ 0.023
0.63 ꢀ 0.011
9.4 ꢀ 0.087
0.14 ꢀ 0.014
0.25 ꢀ 0.015
0.19 ꢀ 0.014
0.66 ꢀ 0.075
0.085 ꢀ 0.0043
2 ꢀ 0.07
0.74 ꢀ 0.074
3.5 ꢀ 0.1
0.059 ꢀ 0.0048
2.3 ꢀ 0.18
37
38
Etoposide
2.1.3. Hybrids containing nitrogen attached to position C-9 of the
cyclolignan
1.3 ꢀ 0.015
7.2 ꢀ 8.5
0.58 ꢀ 0.018
2.2 ꢀ 0.81
Once the new family of lignopurines was prepared, and with the
aim of studying the effect that substitution of oxygen by nitrogen at
C-9 position of the cyclolignan had on the cytotoxicity of the
previously obtained conjugates, we prepared new derivatives con-
taining a nitrogen atom at this position. The synthetic methodology
chosen for these transformations was the reductive amination of
the aldehydes [28], and propylamine was selected for these reac-
tions as it can be eliminated in vacuo and transformations can be
easily carried out in dichloromethane solution. Thus, aldehydes 19
and 20 were treated with propylamine and anhydrous magnesium
sulphate at room temperature during 3 d, generating the corre-
sponding imines 35 and 36, in quantitative yields, that were finally
reduced with sodium borohydride in methanol (Scheme 6). Crude
products were purified by column chromatography, leading to
amines 37 and 38 in low yields, despite the presence of triethyl-
amine in the chromatographic eluents, due to their liability and
GI50 values represent the mean ꢀ SD of three independent experiments.
a
Data for compound 1 was taken from our previous research [28]. Data in bold
correspond to the most potent compounds in the series.
and in the presence of potassium carbonate, but no reaction took
place in any case and mixtures of starting materials and pic-
ropodophyllin were found (Scheme 5).
2.1.2.3. B3. Condensation of dihydroxyesters, protected as acetonides,
with purines. The re-lactonization of the dihydroxyesters during
the condensation process and the fact that hybrids containing tri-
methylene chain as a spacer had not been generated yet by any
previous attempt, forced us to explore a new methodology for the
preparation of these compounds. Thus, we decided to generate
a protected derivative of bromoester 22 that would be the substrate
for the condensation with the corresponding purines (Scheme 5).
tendency to form the corresponding g-lactams [28].
Fig. 3. Effects of compounds 16e19, 19a, 20, 21 and 29 on cell cycle in HeLa cells. (A) Cells were incubated with 1
mM of compound 18 for 24 h and stained with propidium iodide
(PI). Their DNA content was analyzed by fluorescence flow cytometry. The positions of G0/G1 and G2/M peaks are indicated by arrows. (B) Quantitative measurements of G2/M arrest
following 24 h treatment of HeLa cells with 1
independent experiments.
m
M of the indicated compounds. Control untreated cells were run in parallel. Data shown are means ꢀ S.D. or representative of three

MaÁ. Castro et al. / European Journal of Medicinal Chemistry 58 (2012) 377e389
383
2.2. Cytotoxicity studies
maintained the antineoplastic cytotoxicity, being aldehyde 27 a few
times more potent against HT-29 and A-549 than against MB-231
cells.
The new family of hybrids 16e21 was slightly less cytotoxic than
the parent compound 2, although the selectivity is maintained or
even improved as in the case of 20, lignopurine containing an
aromatic link and being the most potent and selective derivative. It
showed lower GI50 values than the podophyllic aldehyde and also it
was several times more potent against A-459 and HT-29 cell lines
than MB-231 cells.
Regarding the purine fragment, the hybrids containing either
purine or 6-chloropurine showed similar GI50 values, so the pres-
ence or absence of the chlorine at the purine C-6 position has no
significant influence on the cytotoxicity of the resulting conjugates
(16 and 18 vs. 17 and 19). However, the substitution of the chlorine
by a propylamino group seems to decrease the potency of the
compound (21 vs. 19). In those cases where the separation of the
N-9 and N-7 regioisomers was possible, it has been observed that
compounds containing N-7 alkyl purines were less cytotoxic than
N-9 alkyl derivatives (19a vs. 19).
Most of the compounds prepared in this work were evaluated
in vitro in order to determine their cytotoxic activity using a color-
imetric cell growth inhibition assay [36]. Thus, cytotoxicity was
measured against the human tumour cell lines: MB-231 (breast
carcinoma), A-549 (lung carcinoma) and HT-29 (colon carcinoma),
and the results are shown in the Table 1, expressed as GI50 values
(drug concentration in mM causing a 50% growth inhibition). The
GI50 values found for the parent podophyllotoxin 1, the “lead
compound” 2, and the semisynthetic derivative in clinical use,
etoposide, have also been included as references.
All the compounds tested, considered as analogues of the
podophyllic aldehyde 2, were cytotoxic. Some of them resulted
several times more potent against HT-29 and A-459 carcinomas
than against MB-231 carcinoma cell lines, showing GI50 values in
the
etoposide.
Considering the different functionalities at positions C-9 and
C-90 of the lignan, the dihydroxyesters 22 and 23 showed GI50
values in the M range or below, and compared to 2, no significant
mM range or below and being, in general, more cytotoxic than
m
About the nature of the linker, it can be observed that there are
no significant differences between L1 and L2, however those
analogues with the aromatic linker L3 were the most potent and
differences between the three lines tested were observed. Among
the aldehyde-ester derivatives 25e27, it can be observed that they
Fig. 4. Effects of compounds 16e19, 19a, 20, 21 and 29 on the microtubule network of HeLa cells. Cells were incubated for 24 h in the absence (control) or in the presence of 1
the indicated compounds and the fixed and processed for immunofluorescence of microtubules (green fluorescence) and nuclei (blue fluorescence) as described in Experimental
section. Bar, 10 m. The photomicrographs shown are representative of at least three independent experiments performed.
mM of
m

384
MaÁ. Castro et al. / European Journal of Medicinal Chemistry 58 (2012) 377e389
selective derivatives (20, 27 and 36). This is in accordance with our
previous work [28] in which we also found that an aromatic ring
in the alkyl fragment of the ester function increased the
cytotoxicity.
themselves, as the purine moiety, could lead to better anticancer
agents.
4. Experimental section
Transformation of the lignan aldehyde group at C-9 position into
nitrogen derivatives led to a decrease of the cytotoxicity values
against A-549 and HT-29 cell lines (37 and 38 vs. 19 and 20),
although the analogue 38, which has the aromatic linker, kept the
GI50 in the same range than the hybrids with linear linkers. The only
derivative with an imine group at this position, 36, maintained the
cytotoxicity of its precursor aldehyde in the same level as described
previously [28].
4.1. Chemistry
NMR spectra were recorded on a Bruker AC 200 at 200 MHz for
1H and 50.3 MHz for 13C in deuterochloroform with TMS as internal
standard. Chemical shift (d) values are expressed in ppm followed
by multiplicity and coupling constants (J) in Hz. The complete NMR
signals are given for the first compound of each series described
here, and only characteristic signals are indicated for the remainder.
Optical rotations were recorded on a PerkineElmer 241 polarimeter
in chloroform solution and UV spectra on a Hitachi 100-60 spec-
trophotometer in ethanol solution. IR spectra were obtained on
a Nicolet Impact 410 spectrophotometer and wavenumbers are
given in cmꢁ1. HRMS were run in a VG-TS-250 spectrometer
working at 70 eV. Elemental analyses (C, H, N) were obtained with
a LECO CHNS-932 and were within ꢀ0.4% of the theoretical values.
Solvents and reagents were purified by standard procedures as
necessary and the chlorinated solvents, including deuterochloro-
form, were filtered through sodium bicarbonate prior its use, in
order to eliminate acid traces. Dimethylformamide was dried over
molecular sieves and treated with K2CO3 for the same reason.
2.3. Cell cycle analysis
To gain further insight into the mode of action of these
compounds, we analyzed the effects of compounds 16e19, 19a, 20,
21 and 29 on cell cycle by flow cytometry in human cervical
cancer HeLa cells, which were also sensitive to the podophyllic
aldehyde derivatives at the low micromolar range (data not
shown). Fig. 3 shows that these compounds (1
mM) induced an
almost total G2/M arrest after 24 h incubation. Fig. 3A shows
a representative histogram of the cell cycle profile shown after
HeLa cell incubation with compound 18. Similar cell cycle profiles
were obtained after incubation with the other podophyllotoxin
derivatives as shown in Fig. 3B, where the percentages of cells
showing G2/M arrest upon incubation with the indicated podo-
phyllotoxin derivatives were determined.
After this initial cell cycle arrest, the onset of apoptosis was
observed following 72 h incubation (data not shown). Because
these compounds are podophyllotoxin derivatives, it could be
feasible that the G2/M cell cycle arrest might be due to the
disruption of the microtubule network [37]. This notion was sup-
ported by analyzing the effect of the above compounds on the
microtubule network by confocal microscopy. We found that
compounds 16e19, 19a, 20, 21 and 29 totally disrupted microtu-
bules in human cancer HeLa cells when compared to untreated cells
(Fig. 4). Thus, the strong G2/M arrest following treatment with the
above podophyllotoxin derivatives seems to be mediated by
microtubule disruption, which eventually leads to apoptosis
through a series of mechanisms that are not yet completely
understood [37].
4.1.1. General method for the synthesis of alkylated purines 4e9
A solution of purine and K2CO3 in dry DMF was stirred at room
temperature for 15 min. The alkylating agent was then added and the
mixture stirred at room temperature for 1 h. The crude product was
diluted with EtOAc and filtered, and the solution was washed with
sat aq NaCl, dried over Na2SO4 and filtered. The solvent was removed
under vacuum to give the alkylated purines 9-(3-bromopropyl)
purine 4, 9-(3-bromopropyl)-6-chloropurine 5, 9-(6-bromohexyl)
purine 6, 9-(6-bromohexyl)-6-chloropurine 7, 7-(6-bromohexyl)-6-
chloropurine 7a, 9-(a
0-bromo-p-xylenyl)-6-chloropurine 8, 9-(6-
bromohexyl)-N6-propyladenine 9. Detailed experimental proce-
dure is described in Supplementary data.
4.1.2. General method for the preparation of hybrid aldehydes 18e21
using route A
A mixture of picropodophyllic acid 3 and K2CO3 was dissolved in
dry DMF and stirred at room temperature for 30 min. Then the
alkylated purine was added and the mixture stirred at room
temperature for 1e16 h. The crude product was diluted with EtOAc
and filtered, and the organic phases were washed with sat aq NaCl,
dried over Na2SO4 and filtered. The solvent was removed under
vacuum to give the corresponding dihydroxyesters (12e15) that
were transformed, without further purification, into the corre-
sponding aldehydes 18e21 by oxidation under Swern conditions.
Swern oxidation: To a precooled (ꢁ55 ꢂC) and stirred solution of
oxalyl chloride (3 equiv) in dry CH2Cl2 (5 mL) was added dropwise
a solution of DMSO (6 equiv) in dry CH2Cl2 (2 mL). After 5 min
at ꢁ55 ꢂC, a solution of the corresponding dihydroxyester (1 equiv)
in dry CH2Cl2 was slowly added. The mixture was stirred at the
same temperature for 30 min and then triethylamine (10 equiv)
was added dropwise. The mixture was warmed to 0 ꢂC over 1 h,
quenched with water and extracted with CH2Cl2. The organic phase
was washed with 2 N HCl and sat aq solutions of NaHCO3 and NaCl
and the solvent was evaporated off. Column chromatography of the
reaction products gave the corresponding aldehydes.
3. Conclusions
A new family of hybrids, named lignopurines, have been
prepared by the junction of the non-lactonic podophyllic aldehyde
and purines. Both fragments were linked through aliphatic and
aromatic chains and evaluated against several tumour cell lines.
The cyclolignan derivatives reported here retain the antineoplastic
cytotoxicity of their chemical precursors, podophyllotoxin 1 and
podophyllic aldehyde 2. The most potent compounds have a doubly
benzylic aromatic fragment as the linker, and within them, 20 and
36 actually correspond to desired lignopurine hybrids, while
derivative 27 is an intermediate in their synthesis bearing
a brominated fragment, which could contribute to its cytotoxicity
by an additional alkylating mechanism. The lignopurines synthe-
sized also retain the antimitotic mode of action of the parent
compounds, based on the inhibition of tubulin polymerization, as it
is demonstrated for compounds 16e19 and 29 (Figs. 3 and 4). These
data suggest that the approach described here to generate novel
podophyllotoxin derivatives keeps the features of the parent drug,
acting on microtubules and cell cycle arrest in tumour cells. Thus
incorporating new structural fragments that could be bioactive by
4.1.2.1. 6-(Purin-9-yl)hexyl 9-deoxy-9-oxo-a-apopicropodophyllate
18. From 3 (200 mg, 0.463 mmol), K2CO3 (191 mg, 1.39 mmol) and
purine 6 (130 mg, 0.64 mmol) in dry DMF (2.0 mL) using the general

MaÁ. Castro et al. / European Journal of Medicinal Chemistry 58 (2012) 377e389
385
method during 16 h. The crude hybrid dihydroxyester 12 (185 mg,
0.29 mmol) was transformed to the corresponding aldehyde under
Swern conditions and the reaction product was chromatographed
on silica gel, eluting with 10% EtOH/CH2Cl2 to give compound 18
1.33 mmol) and purine 8 (150 mg, 0.445 mmol) in dry DMF (2.8 mL)
using the general method during 1.5 h to give dihydroxyester 14
(270 mg, 0.392 mmol, 88%) that was transformed to the corre-
sponding aldehyde under Swern conditions. The reaction product
was chromatographed on silica gel, eluting with 60% EtOAc/CH2Cl2
(62 mg, 35%). 1H NMR (CDCl3):
d 1.25 (m, 4H, COOe(CH2)2e(CH2)2e
(CH2)2eN9), 1.50 (m, 2H, COOeCH2eCH2e(CH2)4eN9), 1.85 (m, 2H,
COOe(CH2)4eCH2eCH2eN9), 3.70 (m, 1H, H80), 3.73 (s, 6H, H100,
H120), 3.78 (s, 3H, H110), 3.95 (m, 2H, COOeCH2e(CH2)5eN9), 4.27
(t, 2H, J ¼ 6.8 Hz, COOe(CH2)5eCH2eN9), 4.57 (d,1H, J ¼ 4.7 Hz, H70),
5.97 (d,1H, J ¼ 1.1 Hz, H10a), 5.99 (d,1H, J ¼ 1.1 Hz, H10b), 6.22 (s, 2H,
H20, H60), 6.62 (s,1H, H3), 6.87 (s,1H, H6), 7.33 (s,1H, H7), 8.18 (s,1H,
H8-purine), 8.99 (s, 1H, H2-purine), 9.15 (s, 1H, H6-purine),
to give compound 20 (164 mg, 62%). 1H NMR (CDCl3):
d 4.94 (d, 1H,
J ¼ 12.8 Hz, COOeCH2eC6H4eCH2eN9), 5.10 (d, 1H, J ¼ 12.8 Hz,
COOeCH2eC6H4eCH2eN9), 5.41 (s, 2H, COOeCH2eC6H4eCH2eN9),
7.08, 7.19 (2d, 4H, J ¼ 8.2 Hz, COOeCH2eC6H4eCH2eN9), 7.32 (s, 1H,
H7), 8.14 (s, 1H, H8-purine), 8.75 (s, 1H, H8-purine), 9.56 (s, 1H, H9);
13C NMR (CDCl3):
d 47. 5 (COOeCH2eC6H4eCH2eN9), 66.1 (COOe
CH2eC6H4eCH2eN9), 128.0, 128.3 (4CH, COOeCH2eC6H4eCH2e
N9), 131.5 (C5-purine), 134.5 (1C, COOeCH2eC6H4eCH2eN9),
136.6 (1C, COOeCH2eC6H4eCH2eN9), 145.3 (C8-purine), 145.9
9.59 (s, 1H, H9); 13C NMR (CDCl3):
d 25.1 (COOe(CH2)3eCH2e
(CH2)2eN9), 26.1 (COOe(CH2)2eCH2e(CH2)3eN9), 28.2 (COOe
CH2eCH2e(CH2)4eN9), 29.7 (COOe(CH2)4eCH2eCH2eN9), 43.9
(COOe(CH2)5eCH2eN9), 45.0 (C80), 46.6 (C70), 56.1 (2CH3, C100,
C120), 60.8 (C110), 65.0 (COOeCH2e(CH2)5eN9), 101.9 (C10), 104.9
(2CH, C20, C60), 108.9 (C6), 110.0 (C3), 125.1 (C1), 133.4 (C8), 133.9
(C2), 137.0 (C70), 137.1 (C10), 145.7 (C7), 146.0 (C8-purine), 147.4 (C5),
147.7 (C6-purine), 150.5 (C4-purine), 151.9 (C2-purine), 153.3 (2C,
(C7), 151.1 (C6-purine), 151.9 (C4-purine), 152.2 (C2-purine), 171.8
20
(C90), 191.2 (C9). IR (film, cmꢁ1): 1731 (COOR), 1668 (CHO). [
a]
(l
): ꢁ151ꢂ (c 1%). UV lmax (log
3
): 208 (4.2), 259 (4.3), 360 (4.5).
Anal. (C35H29N4O8Cl) C, H, N. HRMS: calcd for C35H29N4O8Cl þ H:
669.1747 u; found: 669.1749 m/z.
C30, C50), 172.0 (C90), 191.3 (C9). IR (film, cmꢁ1): 1726 (COOR), 1669
4.1.2.4. 6-(6-Propylaminopurin-9(7)-yl)hexyl 9-deoxy-9-oxo-a-apo-
20
(CHO). [
a]
(l
): ꢁ104ꢂ (c 0.5%). Anal. (C33H34N4O8) C, H, N. HRMS:
picropodophyllate 21(21a). From 3 (85 mg, 0.19 mmol), K2CO3
(40 mg, 1.5 mmol) and purines 9(9a) (100 mg, 1.50 mmol) in dry
DMF (1.8 mL) using the general method during 13 h. The crude
hybrid dihydroxyester 15 (120 mg), was transformed to the corre-
sponding aldehyde under Swern conditions and the reaction
product was chromatographed on silica gel, eluting with 50%
acetone/CH2Cl2 to give a mixture of compounds 21 and 21a (24 mg,
calcd for C33H34N4O8 þ H: 615.2449 u; found: 615.2466 m/z.
4.1.2.2. 6-(6-Chloropurin-9-yl)hexyl 9-deoxy-9-oxo-a-apopicropo-
dophyllate 19 and 6-(6-chloropurin-7-yl)hexyl 9-deoxy-9-oxo-
a-
apopicropodophyllate 19a. From 3 (205 mg, 0.475 mmol), K2CO3
(197 mg, 1.42 mmol) and a mixture of purines 7/7a (150 mg,
0.475 mmol) in dry DMF (2.3 mL) using the general method during
15 h to give a mixture of products 13/13a (279 mg, 88%). The crude
hybrid dihydroxyesters 13 (180 mg, 0.269 mmol) was transformed
to the corresponding aldehyde under Swern conditions and the
reaction product was chromatographed on silica gel, eluting with
10% acetone/CH2Cl2 to give compound 19 (48 mg, 28%) and with
30% acetone/CH2Cl2 to give compound 19a (13 mg, 8%).
20%) which could not be separated. 1H NMR (CDCl3):
d 1.00 (t, 3H,
J ¼ 7.5 Hz, NHeCH2eCH2eCH3), 1.20 (m, 4H, COOe(CH2)2e(CH2)2e
(CH2)2eN9/7), 1.40 (m, 2H, NHeCH2eCH2eCH3), 1.75 (m, 4H, COOe
CH2eCH2e(CH2)2eCH2eCH2eN9/7), 3.36 (t, 2H, J ¼ 6.5 Hz, NHe
CH2eCH2eCH3, 21), 3.49 (t, 2H, J ¼ 6.5 Hz, NHeCH2eCH2eCH3,
21a), 3.96 (m, 2H, COOeCH2e(CH2)5eN9/7), 4.13 (m, 2H, COOe
(CH2)5eCH2eN9/7), 7.32 (s, 1H, H7), 7.71 (s, 1H, H8-purine, 21a),
7.74 (s, 1H, H8-purine, 21), 8.37 (s, 1H, H2-purine), 9.58 (1H, H9);
4.1.2.2.1. Compound 19. 1H NMR (CDCl3):
d 1.25 (m, 4H, COOe
(CH2)2e(CH2)2e(CH2)2eN9), 1.50 (m, 2H, COOeCH2eCH2e(CH2)4e
N9), 1.85 (m, 2H, COOe(CH2)4eCH2eCH2eN9), 4.01 (m, 2H, COOe
CH2e(CH2)5eN9), 4.28 (t, 2H, J ¼ 7.2 Hz, COOe(CH2)5eCH2eN9),
7.33 (s, 1H, H7), 8.21 (s, 1H, H8-purine), 8.75 (s, 1H, H2-purine), 9.59
13C NMR (CDCl3):
d 11.5 (NHeCH2eCH2eCH3), 23.1 (NHeCH2e
CH2eCH3), 25.9, 27.6, 32.5, 33.6 (4CH2, COOeCH2e(CH2)4eCH2e
N7, 21b), 25.2 (COOe(CH2)3eCH2e(CH2)2eN9), 26.1 (COOe
(CH2)2eCH2e(CH2)3eN9),
28.3
(COOeCH2eCH2e(CH2)4eN9),
(s, 1H, H9); 13C NMR (CDCl3):
d
25.1 (COOe(CH2)3eCH2e(CH2)2eN9),
30.1 (COOe(CH2)4eCH2eCH2eN9), 42.7 (COOeCH2e(CH2)5eN9/7),
43.7 (NHeCH2eCH2eCH3), 65.1 (COOeCH2e(CH2)5eN9/7), 119.7
(C6-purine), 139.5 (C8-purine, 21), 139.6 (C8-purine, 21a), 145.5
(C7), 149.1 (C5-purine), 153.2 (C2-purine), 154.9 (C4-purine), 171.8
(C90), 191.3 (C9). Anal. (C36H41N5O8) C, H, N. HRMS: calcd for
26.0 (COOe(CH2)2eCH2e(CH2)3eN9), 28.2 (COOeCH2eCH2e
(CH2)4eN9), 29.7 (COOe(CH2)4eCH2eCH2eN9), 44.3 (COOe(CH2)5e
CH2eN9), 64.9 (COOeCH2e(CH2)5eN9), 131.6 (C5-purine), 145.5
(C8-purine), 145.7 (C7), 151.0 (C6-purine), 151.9 (2C, C2-purine,
C4-purine),172.0 (C90),191.3 (C9). IR (film, cmꢁ1): 1732 (COOR),1669
C36H41N5O8 þ H: 672.3028 u; found: 672.3030 m/z.
20
(CHO). [
a]
(l
): ꢁ103ꢂ (c 1%). UV lmax (log
3
): 206 (4.1), 258 (4.2), 358
(4.4). Anal. (C33H33N4O8Cl) C, H, N. HRMS: calcd for C33H33N4O8ClNa:
4.1.3. General method for the preparation of hybrid aldehydes 16
and 17 using route B1
671.1879 u; found: 671.1890 m/z.
4.1.2.2.2. Compound 19a. 1H NMR (CDCl3):
d
1.25 (m, 4H, COOe
A mixture of picropodophyllic acid 3 and K2CO3 was dissolved in
dry DMF and stirred at room temperature for 30 min. Then the
alkylating agent was added and the mixture stirred at room
temperature for 1e2 h. The crude product was diluted with EtOAc
and filtered, and the organics were washed with sat aq NaCl, dried
over Na2SO4, filtered and concentrated under vacuum. The ob-
tained dihydroxyesters 22e24 were oxidized to the corresponding
aldehydes 25e27 under Swern conditions as described above. After
purification by column chromatography, each aldehyde was added
to a solution of the corresponding purine and K2CO3 in dry DMF and
the mixture stirred at room temperature for 3e6 d. The crude
product was diluted with EtOAc and filtered, and the organics were
washed with sat aq NaCl, dried over Na2SO4 and filtered. The
solvent was removed under vacuum to give a mixture of the hybrid
aldehydes (16, 17, 19, 20) and their derivatives aromatized at the
C-ring (28, 29, 30, 31).
(CH2)2e(CH2)2e(CH2)2eN7), 1.50 (m, 2H, COOeCH2eCH2e(CH2)4e
N7), 1.85 (m, 2H, COOe(CH2)4eCH2eCH2eN7), 4.01 (m, 2H, COOe
CH2e(CH2)5eN7), 4.45 (t, 2H, J ¼ 7.5 Hz, COOe(CH2)5eCH2eN7),
7.34 (s, 1H, H7), 8.32 (s, 1H, H8-purine), 8.88 (s, 1H, H2-purine),
9.59 (s, 1H, H9); 13C NMR (CDCl3):
d 25.1, 25.8 (2CH2, COOe(CH2)2e
(CH2)2e(CH2)2eN9), 28.2 (COOeCH2eCH2e(CH2)4eN9), 31.5 (COOe
(CH2)4eCH2eCH2eN9), 47.3 (COOe(CH2)5eCH2eN9), 64.8 (COOe
CH2e(CH2)5eN9), 122.4 (C5-purine), 143.0 (C6-purine), 145.7 (C7),
149.3 (C8-purine), 152.4 (C2-purine), 162.0 (C4-purine), 172.0 (C90),
20
191.3 (C9). IR (film, cmꢁ1): 1731 (COOR), 1669 (CHO). [
a]
(l
): ꢁ88ꢂ
(c 1%). HRMS: calcd for C33H33N4O8ClNa: 671.1879 u; found:
671.1891 m/z.
4.1.2.3.
cropodophyllate 20. From 3 (192 mg, 0.445 mmol), K2CO3 (184 mg,
a a-apopi-
0-(6-Chloropurin-9-yl)-p-xylenyl 9-deoxy-9-oxo-

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MaÁ. Castro et al. / European Journal of Medicinal Chemistry 58 (2012) 377e389
4.1.3.1. 3-Bromopropyl picropodophyllate 22. From
3
(250 mg,
reaction product (20 mg) was redissolved in DMF (2.0 mL) and
reacted with K2CO3 (10 mg, 0.07 mmol) for 40 h at room temper-
ature. The crude was processed according to the above general
methodology to give compound 28 (8 mg, 40%). 1H NMR (CDCl3):
0.58 mmol), K2CO3 (240 mg, 1.74 mmol) and 1,3-dibromopropane
(0.177 mL, 1.74 mmol) in dry DMF (1.8 mL) using the general
method B1 during 1 h to give compound 22 (263 mg, 82%) [26].
d
2.07 (m, 2H, COOeCH2eCH2eCH2eBr), 3.68 (s, 3H, H110), 3.69
4.1.3.2. 6-Bromohexyl picropodophyllate 23. From
3
(200 mg,
(s, 6H, H100, H120), 4.03 (t, 2H, J ¼ 5.8 Hz, COOeCH2eCH2eCH2eBr),
4.20 (t, 2H, J ¼ 6.5 Hz, COOeCH2eCH2eCH2eBr), 6.03 (s, 2H, H10),
6.45 (s, 2H, H20, H60), 6.74 (s, 1H, H3), 7.36 (s, 1H, H6), 8.24 (s, 1H,
H7), 8.36 (s, 1H, H8-purine), 8.78 (s, 1H, H2-purine), 8.82 (s, 1H, H6-
purine), 9.88 (s, 1H, H9); IR (film, cmꢁ1): 1728 (COOR), 1689 (CHO).
HRMS: calcd for C30H26N4O8Na: 593.1643 u; found: 593.1641 m/z.
0.463 mmol), K2CO3 (192 mg, 1.39 mmol) and 1,6-dibromohexane
(0.214 mL, 1.39 mmol) in dry DMF (1.5 mL) using the above
method B1 during 1 h to give compound 23 (175 mg, 63%). 1H NMR
(CDCl3):
d 1.10e1.60 (m, 6H, COOe(CH2)2e(CH2)3eCH2eBr), 1.80
(m, 2H, COOeCH2eCH2e(CH2)4eBr), 2.47 (m, 1H, H8), 3.35 (m, 1H,
H80), 3.37 (t, 2H, J ¼ 6.5 Hz, COOe(CH2)5eCH2eBr), 3.67 (m, 1H, H9),
3.76 (s, 6H, H100, H120), 3.81 (s, 1H, H110), 4.00 (t, 2H, J ¼ 6.2 Hz,
COOeCH2e(CH2)5eBr), 4.27 (d, 1H, J ¼ 7.9 Hz, H70), 4.83 (d, 1H,
J ¼ 5.4 Hz, H7), 5.90 (s, 2H, H10), 6.35 (s, 1H, H3), 6.85 (s, 1H, H6);
4.1.3.7. 3-(6-Chloropurin-9-yl)propyl 9-deoxy-9-oxodidehyd-
ropodophyllate 29. From 25 (275 mg, 0.516 mmol), K2CO3 (214 mg,
1.55 mmol) and 6-chloropurine (80 mg, 0.52 mmol) in dry DMF
(3.8 mL) using the general method B1 during 3 d. The reaction
product was a mixture of compounds 17 and 29 on a 1:1 ratio, from
which compound 29 (25 mg, 8%) was purified by column chroma-
tography on silica gel, eluting with 70% EtOAc/CH2Cl2. 1H NMR
13C NMR (CDCl3):
d 27.4, 27.7, 28.4 (3CH2, COOe(CH2)2e(CH2)3e
CH2eBr), 32.5 (COOeCH2eCH2e(CH2)4eBr), 33.8 (COOe(CH2)5e
CH2eBr), 43.5 (C8), 45.7 (C70), 47.3 (C80), 56.2 (2CH3, C100, C120),
60.9 (C110), 62.7 (C9), 64.8 (COOeCH2e(CH2)5eBr), 69.8 (C7), 101.2
(C10), 106.3 (2CH, C20, C60), 108.4 (6), 109.3 (C3), 130.2 (C1), 130.7
(C2), 136.7 (C40), 140.0 (C10), 146.8 (C4), 147.7 (C5), 153.1 (2C, C30,
(CDCl3):
d
2.07 (m, 2H, COOeCH2eCH2eCH2eBr), 3.68 (s, 6H, H100,
H120), 3.69 (s, 3H, H110), 4.03 (t, 2H, J ¼ 6.5 Hz, COOeCH2eCH2eCH2e
Br), 4.26 (t, 2H, J ¼ 6.5 Hz, COOeCH2eCH2eCH2eBr), 6.12 (s, 2H,
H10), 6.54 (s, 2H, H20, H60), 6.96 (s, 1H, H3), 7.32 (s, 1H, H6), 8.18 (s,
1H, H7), 8.28 (s, 1H, H8-purine), 8.68 (s, 1H, H2-purine),10.06 (s, 1H,
C50),174.4 (C90). IR (film, cmꢁ1): 3407,1125,1037 (OH),1725 (COOR).
20
[
a]
(
l
): ꢁ56ꢂ (c 0.93%). HRMS: calcd for C28H35O9BrNa:
617.1357 u; found: 617.1335 m/z.
H9); 13C NMR (CDCl3):
d 28.8 (COOeCH2eCH2eCH2eBr), 41.5 (COOe
4.1.3.3. 3-Bromopropyl 9-deoxy-9-oxo-
From 22 (630 mg, 1.14 mmol) under Swern conditions to yield 25
(520 mg, 86%) [28].
a
-apopicropodophyllate 25.
CH2eCH2eCH2eBr), 56.3 (2CH3, C100, C120), 61.1 (C110), 61.7 (COOe
CH2eCH2eCH2eBr), 102.2 (C10), 103.8 (C6), 105.2 (C3), 107.5 (3CH,
C7, C20, C60), 127.3 (C1), 129.0 (C70), 130.1 (C80), 133.1 (C8), 131.8 (C5-
purine), 134.8 (C2), 137.8 (2C, C10, C40), 146.1 (2C, C5, C8-purine),
149.2 (C4), 150.9 (C6-purine), 151.3 (C4-purine), 151.8 (C2-purine),
153.1 (2C, C30, C50), 168.6 (C90), 190.2 (C9). IR (film, cmꢁ1): 1728
(COOR), 1690 (CHO). Anal. (C30H25N4O8Cl) C, H, N. HRMS: calcd for
4.1.3.4. 6-Bromohexyl 9-deoxy-9-oxo-a-apopicropodophyllate 26.
From 23 (210 mg, 0.353 mmol). The crude product from the Swern
reaction was chromatographed on silica gel, eluting with 5% EtOAc/
CH2Cl2 to give compound 26 (100 mg, 49%).1H NMR (CDCl3):
d
1.15e
C30H25N4O8ClNa: 627.1253 u; found: 627.1222 m/z.
1.60 (m, 6H, COOe(CH2)2e(CH2)3eCH2eBr),1.80 (m, 2H, COOeCH2e
CH2e(CH2)4eBr), 3.38 (t, 2H, J ¼ 6.5 Hz, COOe(CH2)5eCH2eBr), 4.0
(m. 2H, COOeCH2e(CH2)5eBr), 7.34 (s, 1H, H7), 9.60 (1H, H9); 13C
4.1.3.8. 6-(6-Chloropurin-9-yl)hexyl 9-deoxy-9-oxo-
a
-apopicropo-
dophyllate 19 and 6-(6-chloropurin-9-yl)hexyl 9-deoxy-9-
oxodidehydropodophyllate 30. From 26 (70 mg, 0.131 mmol),
K2CO3 (54 mg, 0.393 mmol) and 6-chloropurine (41 mg, 0.26 mmol)
in dry DMF (2.3 mL) using the general method B1 during 3 d. The
reaction product was a mixture of compounds 19 and 30 in a 2:1
ratio that were inseparable.
NMR (CDCl3):
d 24.9, 27.7, 28.3 (3CH2, COOe(CH2)2e(CH2)3eCH2e
Br), 32.6 (COOeCH2eCH2e(CH2)4eBr), 33.8 (COOe(CH2)5eCH2e
Br), 65.2 (COOeCH2e(CH2)5eBr), 145.5 (C7), 171.8 (C90), 191.3 (C9).
20
IR (film, cmꢁ1): 1727 (COOR), 1671 (CHO). [
a]
(l
): ꢁ95ꢂ (c 0.93%).
HRMS: calcd for C28H31O8BrNa: 597.1094 u; found: 597.1109 m/z.
4.1.3.5.
27. From 3 (150 mg, 0.347 mmol), K2CO3 (96 mg, 0.69 mmol) and
0-dibromo-p-xylene (183 mg, 0.694 mmol) in dry DMF (1.3 mL)
a
0-Bromo-p-xylenyl 9-deoxy-9-oxo-
a
-apopicropodophyllate
4.1.3.9.
cropodophyllate 20 and
a
0-(6-Chloropurin-9-yl)-p-xylenyl 9-deoxy-9-oxo-
0-(6-chloropurin-9-yl)-p-xylenyl 9-deoxy-9-
a-apopi-
a
a
,a
oxodidehydropodophyllate 31. From 27 (78 mg, 0.131 mmol), K2CO3
(54 mg, 0.393 mmol) and 6-chloropurine (41 mg, 0.26 mmol) in dry
DMF (2.3 mL) using the general method B1 during 3 d. The reaction
product was a complex mixture containing compounds 20 and 31
in a 1:1 ratio that were inseparable.
using the general method B1 during 2 h. The crude ester 24
(300 mg), was transformed to the corresponding aldehyde under
Swern conditions and the reaction product was chromatographed
on silica gel, eluting with 40% EtOAc/CH2Cl2 to give compound 27
(112 mg, 40%). 1H NMR (CDCl3):
d 4.45 (s, 2H, COOeCH2eC6H4e
CH2eBr), 5.04 ((AB system, 2H, J = 12.8 Hz, COOeCH2eC6H4e
CH2eBr), 7.10, 7.30 (AB system, 4H, J ¼ 8.2 Hz, COOeCH2eC6H4e
CH2eBr), 7.35 (s, 1H, H7), 9.60 (s, 1H, H9); 13C NMR (CDCl3):
4.1.4. Preparation of hybrid aldehydes 16 and 17 using route B3
A solution of acetonide 32 (obtained from 22) in dry DMF was
added to a solution of the corresponding purine and K2CO3 in dry
DMF and the mixture stirred at 100 ꢂC for 24 h. Water was added to
the reaction mixture and the crude was extracted with EtOAc. The
organics were washed with sat aq NaCl, dried over Na2SO4 and
filtered. The solvent was removed under vacuum and the crude was
purified by column chromatography to give the corresponding
protected conjugates (33 and 34). The hybrids were dissolved in
a water/acetone (2:8) solution (10 mL) and deprotected by reaction
with p-toluenesulfonic acid at room temperature during 40e48 h.
After this time, the acetone was concentrated under vacuum and
the crude mixture was diluted with sat aq NaHCO3 and extracted
with EtOAc. The organics were washed with sat aq NaCl, dried over
Na2SO4 and filtered. The obtained hybrid dihydroxyesters (10, 11)
d
33.1 (COOeCH2eC6H4eCH2eBr), 65.5 (COOeCH2eC6H4eCH2e
N9), 128.0, 128.7, 129.2 (4CH, COOeCH2eC6H4eCH2eN9), 133.8
(1C, COOeCH2eC6H4eCH2eN9), 136.0 (1C, COOeCH2eC6H4eCH2e
N9), 145.8 (C7), 171.7 (C90), 191.3 (C9). IR (film, cmꢁ1): 1732
20
(COOR), 1668 (CHO). [
C
a
]
(l
): ꢁ132ꢂ (c 1%). HRMS: calcd for
30H27O8BrNa: 617.0781 u; found: 617.0804 m/z.
4.1.3.6. 3-(Purin-9-yl)propyl 9-deoxy-9-oxodidehydropodophyllate
28. From 25 (80 mg, 0.15 mmol), K2CO3 (21 mg, 0.15 mmol) and
purine (25 mg, 0.21 mmol) in dry DMF (2.0 mL) using the general
method B1 during 6 d. The reaction product was a mixture con-
taining compounds 16 and 28 on a 1:1 ratio. A fraction of this

MaÁ. Castro et al. / European Journal of Medicinal Chemistry 58 (2012) 377e389
387
were transformed into the corresponding aldehydes 16 and 17
without further purification by oxidation under Swern conditions.
(75 mg, 0.58 mmol) and 6-chloropurine (98 mg, 0.81 mmol) in dry
DMF (5.0 mL) using the general method B3 during 24 h. The reac-
tion product, containing acetonide 34, (340 mg) was treated with
p-toluenesulfonic acid (10 mg, 0.05 mmol) in a water/acetone (2:8)
solution (10 mL) during 48 h and the obtained hybrid dihydrox-
yester 11 (225 mg, 0.359 mmol) was transformed to the corre-
sponding aldehyde under Swern conditions. The reaction product
was chromatographed on silica gel, eluting with EtOAc to give
4.1.4.1. 7,9-Acetonide of 3-bromopropyl picropodophyllate 32.
A catalytic amount (8 mg) of p-toluenesulfonic acid was added to
a solution of 22 (240 mg, 0.43 mmol) in 2,2-dimethoxypropane
(10 mL, 82 mmol). The mixture was stirred at room temperature
for 6.5 h, K2CO3 (500 mg, 3.6 mmol) was then added and the
reaction mixture stirred for 1 h. After addition of water and evap-
oration of the 2,2-dimethoxypropane, the crude was extracted with
EtOAc. The organics were washed with a sat aq solution of NaCl,
dried over Na2SO4 and filtered. The solvent was removed under
vacuum to give 220 mg (86%) of compound 32. 1H NMR (CDCl3):
compound 17 (105 mg, 54%). 1H NMR (CDCl3):
d 2.16 (m, 2H, COOe
CH2eCH2eCH2eN9), 4.07 (m, 2H, COOeCH2eCH2eCH2eN9), 4.23
(m, 2H, COOeCH2eCH2eCH2eN9), 7.39 (s, 1H, H7), 8.16 (s, 1H, H8-
purine), 8.71 (s, 1H, H2-purine), 9.64 (s, 1H, H9); 13C NMR (CDCl3):
d
28.5 (COOeCH2eCH2eCH2eN9), 41.2 (COOeCH2eCH2eCH2eN9),
d
1.49, 1.58 (2s, 6H, H12, H13), 2.12 (m, 2H, COOeCH2eCH2eCH2e
61.3 (COOeCH2eCH2eCH2eN9), 131.8 (C5-purine), 133.1 (C8),
Br), 2.27 (m, 1H, H8), 2.80 (dd, 1H, J = 4.8 and 2.2 Hz, H80), 3.30
(m, 2H, COOeCH2eCH2eCH2eBr), 3.77 (s, 6H, H100, H120), 3.82
(s, 3H, H110), 4.09 (d, 1H, J ¼ 11.3 Hz, H9a), 4.14 (d, 1H, J ¼ 11.3 Hz,
H9b), 4.30 (m, 2H, COOeCH2eCH2eCH2eBr), 4.37 (d, 1H, J ¼ 2.2 Hz,
H70), 4.90 (d, 1H, J ¼ 11.2 Hz, H7), 5.90 (s, 2H, H10), 6.26 (s, 2H, H20,
145.9 (C8-purine), 146.2 (C7), 150.8 (C6-purine), 151.1 (C4-purine),
151.9 (C2-purine), 172.0 (C90), 191.5 (C9). IR (film, cmꢁ1): 1729
20
(COOR), 1667 (CHO). [
C
a
]
(l
): ꢁ102ꢂ (c 0.91%). HRMS: calcd for
30H27N4O8Cl þ H: 607.1590 u; found: 607.1644 m/z.
H60), 6.38 (s, 1H, H3), 6.99 (s, 1H, H6); 13C NMR (CDCl3):
d
19.6, 29.9
4.1.5. General methods for the synthesis of imines 35 and 36
A mixture of aldehyde (1 equiv), anhydrous MgSO4 (4 equiv) and
propylamine (2 equiv) was dissolved in CH2Cl2 (3 mL) and stirred at
room temperature during 3 d. The reaction mixture was diluted
with CH2Cl2, filtered and concentrated under vacuum to give the
imine in quantitative yield. Imines were obtained pure enough to
carry on complete characterization without being necessary any
purification step.
(2CH3, C12, C13), 29.2 (COOeCH2eCH2eCH2eBr), 31.2 (COOeCH2e
CH2eCH2eBr), 34.9 (C8), 46.8 (C70), 48.0 (C80), 56.3 (2CH3, C100,
C120), 60.9 (C110), 62.6, 63.1 (2 CH2, C9, COOeCH2eCH2eCH2eBr),
68.0 (C7), 99.8 (C11), 101.0 (C10), 104.8 (C6), 106.1 (2CH, C20, C60),
109.5 (C3), 128.7 (C1), 130.9 (C2), 136.9 (C40), 140.5 (C10), 146.9 (C5),
147.1 (C4), 153.2 (2C, C30, C50), 172.8 (C90). IR (film, cmꢁ1): 1729
(COOR). HRMS: calcd for
C28H33O9BrNa: 615.1200 u; found:
615.1221 m/z.
4.1.5.1. 6-(6-Chloropurin-9-yl)hexyl 9-deoxy-9-propylimino-a-apo-
4.1.4.2. 7,9-Acetonide of 3-(purin-9-yl)propyl picropodophyllate 33.
From acetonide 32 (220 mg, 0.370 mmol), K2CO3 (51 mg,
0.37 mmol) and purine (62 mg, 0.52 mmol) in dry DMF (5.0 mL)
using the general method B3 during 24 h. The reaction product was
chromatographed silica gel, eluting with 10% EtOH/CH2Cl2 to give
picropodophyllate 35. From aldehyde 19 (160 mg, 0.247 mmol) and
propylamine (0.041 mL, 0.49 mmol) following the above procedure
during 3 d to give compound 35 (170 mg, 99%). 1H NMR (CDCl3):
d
0.75 (t, 3H, J ¼ 7.5 Hz, NeCH2eCH2eCH3), 1.05e1.90 (m, 8H, COOe
CH2e(CH2)4eCH2eN9), 1.46 (m, 2H, NeCH2eCH2eCH3), 3.35
(m, 2H, NeCH2eCH2eCH3), 3.88 (m, 2H, COOeCH2e(CH2)4eCH2e
N9), 4.20 (t, 2H, J ¼ 7.2 Hz, COOeCH2e(CH2)4eCH2eN9), 6.74
(s, 1H, H7), 7.89 (s, 1H, H9), 8.15 (s, 1H, H8-purine), 8.68 (s, 1H, H2-
compound 33 (140 mg, 60%). 1H NMR (CDCl3):
d 1.19, 1.24 (2s, 2H,
H12, H13), 2.25 (m, 3H, H8, COOeCH2eCH2eCH2eN9), 4.07 (m, 1H,
H9), 4.11 (m, 2H, COOeCH2eCH2eCH2eN9), 4.21 (t, 2H, J ¼ 6.9 Hz,
COOeCH2eCH2eCH2eN9), 4.91 (d, 1H, J ¼ 11.0 Hz, H7), 7.88 (s, 1H,
H8-purine), 8.94 (s, 1H, H2-purine), 9.12 (s, 1H, H6-purine); 13C
purine); 13C NMR (CDCl3):
d 11.7 (NeCH2eCH2eCH3), 24.1 (NeCH2e
CH2eCH3), 25.1 (COOe(CH2)3eCH2e(CH2)2eN9), 26.1 (COOe
(CH2)2eCH2e(CH2)3eN9), 28.3 (COOeCH2eCH2e(CH2)4eN9), 29.8
(COOe(CH2)4eCH2eCH2eN9), 44.4 (COOe(CH2)5eCH2eN9), 63.0
(NeCH2eCH2eCH3), 64.4 (COOeCH2e(CH2)5eN9), 131.7 (2C, C8,
C5-purine), 135.2 (C7), 145.3 (C8-purine), 151.0 (C6-purine), 151.9
(2C, C2-purine, C4-purine), 161.1 (C9), 172.9 (C90). IR (film, cmꢁ1):
1724 (COOR), HRMS: calcd for C36H40N5O7Cl þ H: 690.2689 u;
found: 690.2667 m/z.
NMR (CDCl3):
d 19.6, 29.9 (2CH3, C12, C13), 29.1 (COOeCH2eCH2e
CH2eN9), 35.0 (C8), 40.6 (COOeCH2eCH2eCH2eN9), 61.3 (COOe
CH2eCH2eCH2eN9), 63.0 (C9), 67.9 (C7), 99.8 (C11), 134.1
(C5-purine),145.1 (C8-purine),148.8 (C6-purine),151.4 (C4-purine),
152.7 (C2-purine), 174.6 (C90). IR (film, cmꢁ1): 1729 (COOR), HRMS:
calcd for C33H36N4O9Na: 655.2374 u; found: 655.2377 m/z.
4.1.4.3. 3-(Purin-9-yl)propyl 9-deoxy-9-oxo-a-apopicropodophyllate
16. From 33 (80 mg, 0.13 mmol) and p-toluenesulfonic acid (10 mg,
0.05 mmol) in a water/acetone (2:8) solution (10 mL) using the
general deprotection described in method B3 during 40 h, to yield
the hybrid ester 10 (55 mg, 74%) that was transformed to the cor-
responding aldehyde 16 under Swern conditions (40 mg, 75%). 1H
4.1.5.2. a a-
0-(6-Chloropurin-9-yl)-p-xylenyl 9-deoxy-9-propylimino-
apopicropodophyllate 36. Fromaldehyde20(120mg, 0.18mmol)and
propylamine (0.029 mL, 0.36 mmol) following the general procedure
during 3 d to give compound 36 (125 mg, 94%). 1H NMR (CDCl3):
d
0.75(t, 3H, J ¼ 7.5 Hz, NeCH2eCH2eCH3),1.47 (m, 2H, NeCH2eCH2e
NMR (CDCl3):
d
2.18 (m, 2H, COOeCH2eCH2eCH2eN9), 4.00 (t, 2H,
CH3), 3.35 (m, 2H, NeCH2eCH2eCH3), 4.93 (d, 1H, J ¼ 12.9 Hz, COOe
CH2eC6H4eCH2eN9), 5.07 (d,1H, J ¼ 12.9 Hz, COOeCH2eC6H4eCH2e
N9), 5.41 (s, 2H, COOeCH2eC6H4eCH2eN9), 6.78 (s,1H, H7), 7.07, 7.18
(AB system, 4H, J ¼ 7.9 Hz, COOeCH2eC6H4eCH2eN9), 7.93 (s, 1H,
H9), 8.13 (s,1H, H8-purine), 8.76 (s,1H, H8-purine); 13C NMR (CDCl3):
J ¼ 6.2 Hz, COOeCH2eCH2eCH2eN9), 4.25 (t, 2H, J ¼ 6.6 Hz, COOe
CH2eCH2eCH2eN9), 7.38 (s,1H, H7), 8.09 (s,1H, H8-purine), 8.94 (s,
1H, H2-purine), 9.11 (s, 1H, H6-purine), 9.64 (s, 1H, H9); 13C NMR
(CDCl3):
d 28.5 (COOeCH2eCH2eCH2eN9), 40.5 (COOeCH2eCH2e
CH2eN9), 61.4 (COOeCH2eCH2eCH2eN9), 133.1 (C8), 134.2 (C5-
d 11.7 (NeCH2eCH2eCH3), 24.1 (NeCH2eCH2eCH3), 47. 5 (COOe
purine), 146.0 (C7), 146.2 (C8-purine), 148.5 (C6-purine), 151.4
CH2eC6H4eCH2eN9), 62.9 (NeCH2eCH2eCH3), 65.9 (COOeCH2e
C6H4eCH2eN9), 127.8, 128.3 (4CH, COOeCH2eC6H4eCH2eN9), 131.7
(2C, C8, C5-purine),134.3 (1C, COOeCH2eC6H4eCH2eN9),135.3 (C7),
136.8 (1C, COOeCH2eC6H4eCH2eN9), 145.2 (C8-purine), 151.0 (C6-
purine), 151.8 (C4-purine), 152.1 (C2-purine), 161.0 (C9), 172.6 (C90).
IR (film, cmꢁ1): 1728 (COOR). HRMS: calcd for C38H36N5O7Cl þ H:
710.2376 u; found: 710.2372 m/z.
(C4-purine), 152.5 (C2-purine), 171.9 (C90), 191.5 (C9). IR (film,
20
cmꢁ1): 1729 (COOR), 1667 (CHO). [
a]
(
l
): ꢁ134ꢂ (c 0.83%). HRMS:
calcd for C30H28N4O8 þ H: 573.1980 u; found: 573.1979 m/z.
4.1.4.4. 3-(6-Chloropurin-9-yl)propyl 9-deoxy-9-oxo-a-apopicropo-
dophyllate 17. From acetonide 32 (345 mg, 0.582 mmol), K2CO3

388
MaÁ. Castro et al. / European Journal of Medicinal Chemistry 58 (2012) 377e389
4.1.6. General methods for the synthesis of amines 37 and 38
The solution of the imine, obtained as described above, in MeOH
(20 mL) was treated with excess of NaBH4 at 0 ꢂC for 1 h. Water was
added to the reaction mixture and MeOH was partially removed in
vacuum to give an aqueous solution that was adjusted with sat
Na2CO3 to pH > 7 and then extracted with EtOAc. The organic phase
was washed with a sat aq solution of NaCl, dried over Na2SO4 and
filtered and the solvent removed under vacuum. Purification
by column chromatography under the presence of triethylamine
(1% v/v) yielded the corresponding amine.
for 60 min at 4 ꢂC. Plates were washed with deionised water and
dried; 100 L of SBR solution (0.4% wt/vol in 1% acetic acid) was
m
added to each microtitre well and incubated for 10 min at room
temperature. Unbound SRB was removed by washing with 1%
acetic acid. Plates were air-dried and bound stain was solubilized
with Tris buffer. Optical densities were read on an automated
spectrophotometer plate reader at single wavelength of 490 nm.
Data analyses were generated automatically by the LIMS imple-
mentation. Using control OD values (C), test OD values (T) and time
zero OD values (To), the drug concentration that caused a 50%
growth inhibition (GI50 value) was calculated from the equation:
100ꢃ[(T ꢁ To)/(C ꢁ To)] ¼ 50. Each value represents the mean from
triplicate determinations.
4.1.6.1. 6-(6-Chloropurin-9-yl)hexyl 9-deoxy-9-propylamino-a-apo-
picropodophyllate 37. From imine 35 (130 mg, 0.19 mmol) and
NaBH4 (130 mg, 3.44 mmol) following the general procedure during
1 h. The reaction product was chromatographed on silica gel, eluting
with 10% EtOAc/CH2Cl2 to give compound 37 (16 mg, 9%). 1H NMR
4.3. Cell cycle analysis
(CDCl3):
d
0.78 (t, 3H, J ¼ 7.5 Hz, NHeCH2eCH2eCH3), 1.10e1.60
For cell cycle analysis, we used the human cervical cancer HeLa
cell line grown in DMEM supplemented with 10% (v/v) heat-
(m, 6H, COOeCH2e(CH2)3e(CH2)2eN9), 1.22 (m, 2H, NHeCH2e
CH2eCH3), 1.85 (m, 2H, COOeCH2e(CH2)3eCH2eCH2eN9), 2.27
(m, 2H, NHeCH2eCH2eCH3), 3.27 (d, 1H, J ¼ 14.0 Hz, H9a), 3.41 (d,
1H, J ¼ 14.0 Hz, H9b), 3.99 (m, 2H, COOeCH2e(CH2)4eCH2eN9), 4.24
(t, 2H, J ¼ 7.2 Hz, COOeCH2e(CH2)4eCH2eN9), 6.42 (s, 1H, H7), 8.18
inactivated foetal calf serum, 2 mM
L-glutamine, 100 units/mL
penicillin, and 100
m
g/mL streptomycin at 37 ꢂC in a humidified
atmosphere of 5% CO2 and 95% air. Cells were periodically tested for
Mycoplasma infection and found to be negative. Untreated and
drug-treated cells (3e5 ꢃ 105) were centrifuged and fixed over-
night in 70% ethanol at 4 ꢂC. Then, cells were washed three times
(s, 1H, H8-purine), 8.74 (s, 1H, H2-purine); 13C NMR (CDCl3):
d 11.6
(NHeCH2eCH2eCH3), 22.8 (NHeCH2eCH2eCH3), 25.2 (COOe
(CH2)3eCH2e(CH2)2eN9), 26.2 (COOe(CH2)2eCH2e(CH2)3eN9),
28.4 (COOeCH2eCH2e(CH2)4eN9), 29.8 (COOe(CH2)4eCH2eCH2e
N9), 44.3 (COOe(CH2)5eCH2eN9), 50.4 (NHeCH2eCH2eCH3),
53.5 (C9), 64.6 (COOeCH2e(CH2)5eN9), 126.0 (C7), 131.5
(C5-purine), 145.5 (C8-purine), 150.9 (C6-purine), 151.9 (2C, C2-
purine, C4-purine), 172.9 (C90). IR (film, cmꢁ1): 3366 (NH), 1724
(COOR), HRMS: calcd for C36H42N5O7Cl þ H: 692.2845 u; found:
692.2822 m/z.
with PBS, incubated for 1 h with 1 mg/mL RNAse A and 20 mg/mL
propidium iodide at room temperature, and analyzed with a Becton
Dickinson FACSCalibur flow cytometer (San Jose, CA) as described
previously [38,39] to determine the percentage of cells in each cell
cycle stage.
4.4. Confocal microscopy
HeLa cells were grown on poly-L-lysine coated coverslips, and
4.1.6.2.
-apopicropodophyllate 38. From imine 36 (120 mg, 0.17 mmol)
a
0-(6-Chloropurin-9-yl)-p-xylenyl 9-deoxy-9-propylamino-
after drug treatment coverslips were washed three times with
HPEM (25 mM HEPES, 60 mM PIPES, 10 mM EGTA, 3 mM MgCl2,
pH6.6), fixed with 4% paraformaldehyde in HPEM buffer for 15 min,
and permeabilized with 0.5% Triton X-100 as previously described
[40]. Coverslips were incubated with a specific Ab-1 anti-alpha-
tubulin mouse monoclonal antibody (diluted 1:150 in PBS) (Cal-
biochem) for 1 h, washed 4 times with PBS, and then incubated
with CY2-conjugated anti-mouse IgG (Jackson ImmunoResearch)
for 1 h at 4 ꢂC. After washing 4 times with PBS, cell nuclei were
stained with DAPI (Sigma) for 5e10 min, washed with PBS, and
then samples were analyzed by confocal microscopy using
a
and NaBH4 (120 mg, 3.17 mmol) following the general procedure
during 1 h. The reaction product was chromatographed on silica
gel, eluting with 80% EtOAc/CH2Cl2 to give compound 38 (9 mg, 7%).
1H NMR (CDCl3):
d
0.77 (t, 3H, J ¼ 7.2 Hz, NHeCH2eCH2eCH3), 1.30
(m, 2H, NHeCH2eCH2eCH3), 2.30 (m, 2H, NHeCH2eCH2eCH3),
3.26 (d, 1H, J ¼ 14.4 Hz, H9a), 3.41 (d, 1H, J ¼ 14.4 Hz, H9b), 4.96
(d, 1H, J ¼ 12.6 Hz, COOeCH2eC6H4eCH2eN9), 5.09 (d, 1H,
J ¼ 12.6 Hz, COOeCH2eC6H4eCH2eN9), 5.44 (s, 2H, COOeCH2e
C6H4eCH2eN9), 6.43 (s, 1H, H7), 7.08, 7.20 (AB system, 4H,
J ¼ 7.9 Hz, COOeCH2eC6H4eCH2eN9), 8.16 (s, 1H, H8-purine), 8.78
a
ZeissLSM310 laser scan confocal microscope (Oberkochen,
(s, 1H, H8-purine); 13C NMR (CDCl3):
d 11.5 (NHeCH2eCH2eCH3),
Germany). A drop of SlowFade Light Antifading reagent (Molecular
Probes) was added to preserve fluorescence. Negative controls,
lacking the primary antibody or using an irrelevant antibody,
showed no staining.
23.1 (NHeCH2eCH2eCH3), 47. 5 (COOeCH2eC6H4eCH2eN9), 50.0
(NHeCH2eCH2eCH3), 53.5 (C9), 65.9 (COOeCH2eC6H4eCH2eN9),
127.4 (C7), 127.9, 128.5 (4CH, COOeCH2eC6H4eCH2eN9), 130.9
(C8), 145.2 (C8-purine), 152.3 (C4-purine), 152.5 (C2-purine), 172.3
(C90). IR (film, cmꢁ1): 3395 (NH), 1728 (COOR). HRMS: calcd for
Acknowledgements
C
38H38N5O7Cl þ H: 712.2532 u; found: 712.2501 m/z.
Financial support came from Spanish Ministerio de Ciencia e
Innovación (CTQ2008-02899, SAF2011-30518), Red Temática de
Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III
(RD06/0020/1037) and Junta de Castilla y León (CSI052A11-2,
GR15-Experimental Therapeutics and Translational Oncology
Program, SA028A10-2 and fellowship to M.V.R.) cofunded by the
Fondo Europeo de Desarrollo Regional of the European Union.
4.2. Cell growth inhibition assays
A colourimetric assay using sulforhodamine
B (SRB) was
adapted for a quantitative measurement of cell growth and
viability, following a previously described method [36]. Cells were
seeded in 96-well microtitre plates, at 5 ꢃ 103 cells per well in
aliquots of 195 mL of RPMI medium, and they were allowed to attach
to the plate surface by growing in a drug-free medium for 18 h.
Afterwards, samples were added in aliquots of 5 L (dissolved in
m
Appendix A. Supplementary data
DMSO/H2O, 3:7). After 72 h exposure, the antitumour effect was
measured by the SRB methodology: cells were fixed by adding
50 mL of cold 50% (wt/vol) trichloroacetic acid (TCA) and incubating
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.10.

MaÁ. Castro et al. / European Journal of Medicinal Chemistry 58 (2012) 377e389
389
026. These data included complete 1H and 13C data assignment and
MOL files.
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Antitumour agents 210. Synthesis and evaluation of taxoid-epipodophyllo
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