Synthesis and biological evaluation of new symmetrical derivatives as cytotoxic agents and apoptosis inducers

Article abstract of DOI:10.1016/j.bmc.2005.01.008

Based on the research of less toxic anticancer therapies, we have looked for novel compounds with anticancer activity based on a proapoptotic mechanism. The described compounds are derivatives of ether, carbamate, urea, amide, or amine. Some of the prepared compounds decreased cell viability of various tumor cell lines in a time- and dose-dependent manner, and also induced DNA fragmentation, which indicated cell apoptosis. The potential antitumoral activity of the compounds was evaluated in vitro by examining their cytotoxic effects against human mama, colon, and bladder cancer cell lines (MD-MBA-231, HT-29, and T-24). Compounds showing cytotoxic activity were subjected to an apoptosis assay. In addition, some of the synthesized compounds provoked a rapid and dose-dependent increase in the level of caspase-3, an enzyme, which is considered to be one of the principal executing caspases in which all of the biochemical routes involved in the apoptosis response converge. The most promising compounds, with respect to cytotoxicity and apoptosis induction capability, were the 4-nitrophenylcarbamate derivative of 2,2′- methylenebis(4-chlorophenyl) 3c, the naphthylurea derivative 4d, and the n-propylurea derivative 4c, from 4,4′-methylenebisphenyl, all of which displayed cytotoxic activity and showed very interesting levels of apoptosis. Furthermore, good levels of apoptosis induction were achieved for 3a and 4b in the T-24 cell line. Therefore, compounds such as 7b, a pyrido[2,3-d]pyrimidine derivative, show a significant in vitro cytotoxicity, with IC₅₀ values between 3 and 8 μm in the three cell lines tested. This compound also produced a rapid and dose-dependent increase of the caspase-3 level and induced apoptosis in HT-29 cells. Other profiles have been found, such as those presented by 5c and 7c, which are cytotoxic and apoptotic but do not provoke an increase in the level of caspase-3, or those presented by 1c, 1d, and 2a, which are cytotoxic, without showing any other activity. The different types of behavior of each compound are not necessarily parallel in the three cell lines tested. A great number of these compounds of interest show no cytotoxicity in nontumoral human cells such as CRL-8799, a nontumoral line of mama. Subsequent modulation of these lead structures permits advances in the design of potent cytotoxic and proapoptotic anticancer drugs.

Full text of DOI:10.1016/j.bmc.2005.01.008

Bioorganic & Medicinal Chemistry 13 (2005) 2031–2044
Synthesis and biological evaluation of new symmetrical
derivatives as cytotoxic agents and apoptosis inducers
a
Carmen Sanmartın, Mikel Echeverrıa, Beatriz Mendıvil, Lucıa Cordeu, Elena Cubedo,
a
a
b
b
´
Jesus Garcıa-Foncillas, Marıa Font and Juan Antonio Palop
´
´
´
b
c
a,
*
´
´
´
a
´ ´
Seccion de Sıntesis, Departamento de Quımica Organica y Farmaceutica, Universidad de Navarra,
´
Irunlarrea, 1, E-31008 Pamplona, Spain
´
´
b
ꢀ
´
´
´
Laboratorio de Biotecnologıa y Genomica, Area de Terapia Celular, Clınica Universitaria de Navarra, Pamplona, Spain
c
´ ´ ´ ´
Seccion de Modelizacion Molecular, Departamento de Quımica Organica y Farmaceutica,
Universidad de Navarra, Pamplona, Spain
´
Received 19 November 2004; revised 23 December 2004; accepted 7 January 2005
Available online 29 January 2005
Abstract—Based on the research of less toxic anticancer therapies, we have looked for novel compounds with anticancer activity
based on a proapoptotic mechanism. The described compounds are derivatives of ether, carbamate, urea, amide, or amine. Some
of the prepared compounds decreased cell viability of various tumor cell lines in a time- and dose-dependent manner, and also
induced DNA fragmentation, which indicated cell apoptosis. The potential antitumoral activity of the compounds was evaluated
in vitro by examining their cytotoxic effects against human mama, colon, and bladder cancer cell lines (MD-MBA-231, HT-29,
and T-24). Compounds showing cytotoxic activity were subjected to an apoptosis assay. In addition, some of the synthesized com-
pounds provoked a rapid and dose-dependent increase in the level of caspase-3, an enzyme, which is considered to be one of the
principal executing caspases in which all of the biochemical routes involved in the apoptosis response converge. The most promising
compounds, with respect to cytotoxicity and apoptosis induction capability, were the 4-nitrophenylcarbamate derivative of 2,20-
methylenebis(4-chlorophenyl) 3c, the naphthylurea derivative 4d, and the n-propylurea derivative 4c, from 4,40-methylenebisphenyl,
all of which displayed cytotoxic activity and showed very interesting levels of apoptosis. Furthermore, good levels of apoptosis
induction were achieved for 3a and 4b in the T-24 cell line. Therefore, compounds such as 7b, a pyrido[2,3-d]pyrimidine derivative,
show a significant in vitro cytotoxicity, with IC50 values between 3 and 8 lm in the three cell lines tested. This compound also pro-
duced a rapid and dose-dependent increase of the caspase-3 level and induced apoptosis in HT-29 cells. Other profiles have been
found, such as those presented by 5c and 7c, which are cytotoxic and apoptotic but do not provoke an increase in the level of cas-
pase-3, or those presented by 1c, 1d, and 2a, which are cytotoxic, without showing any other activity. The different types of behavior
of each compound are not necessarily parallel in the three cell lines tested. A great number of these compounds of interest show no
cytotoxicity in nontumoral human cells such as CRL-8799, a nontumoral line of mama. Subsequent modulation of these lead struc-
tures permits advances in the design of potent cytotoxic and proapoptotic anticancer drugs.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction and design considerations
cytotoxic agents, which has led to the use of a group
of compounds whose mechanism is related to the alter-
ation, at different levels, of the correct functioning of
DNA, there has been a recent shift of emphasis toward
novel mechanistic targets that have arisen directly from
the in-depth study of the underlying genetic changes re-
lated to the cancerous state. Thus, for instance, it is now
accepted that tumor growth may principally be due to
reduced rates of cell death rather than to enhanced pro-
liferation.1–3 The process of programmed cell death is
now recognized as an important component of multi-
step carcinogenesis.4–10 Apoptotic pathways might be
significantly altered in cancer cells with respect to
Progressive elucidation of the molecular mechanisms in-
volved in cancer has opened up a new horizon for the
development of new antitumoral compounds. While
the traditional cancer therapies were based on the theory
that rapidly dividing cancer cells are more sensitive to
Keywords: Symmetrical ethers; Carbamates; Ureas; Amides and
amines; Cytotoxics; Apoptosis; Caspase-3.
*
Corresponding author. Tel.: +34 948 425 600; fax: +34 948 425
649; e-mail: jpalop@unav.es
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.008

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C. Sanmartın et al. / Bioorg. Med. Chem. 13 (2005) 2031–2044
2032
untransformed cells, and these differences might present
a therapeutic window that can be exploited for the
development of cancer drugs.11–13
basically responds to molecules, which contain three
entities. These molecules have a central nucleus made
up of an aromatic system or a cyclic or linear aliphatic
chain of variable length and flexibility, connected by a
variable functional group, upon which two identical lat-
eral arms, consisting of heterocycles (pyridine, quino-
line, indole, pyrido[2,3-d]pyrimidine) with different
substituents, are introduced. These arms can bond to
the central element by means of a variable functional
group (ether, amine, amide, urea, and carbamate) (Fig-
ure 1).
It is a well-known fact that the regulating mechanisms of
apoptosis are extremely complex, but it is also known
that the way in which the apoptotic processes function
differs, depending on whether the cells involved are tu-
moral or healthy. These differences are due to the fact
that the tumoral lines are more sensitive when these
mechanisms are activated, while the healthy cells possess
the ability to make repairs, which often counteracts this
apoptotic process, maintaining survival of the cell.14
The general scheme of distribution used for these struc-
tural elements initially results in the formation a trian-
gle, with one of its vertices being the central nucleus
and the other two vertices being made up of the ends
of the arms.
Among the mechanisms involved, the activation of a
group of enzymes pertaining to the cistein-protease fam-
ily, known as caspases, stands out. They are probably
the most important effect-provoking molecules related
to the induction of apoptosis, and all of them are synthe-
sized in the form of inactive precursors (proenzymes),
which are subsequently activated by self-proteolysis
(autocatalytic cleavage) or other proteases.15 It is as-
sumed that a series of adaptive molecules exist, which
interact with a specific point of the amino-terminal
prodomains of the procaspases. If two proenzymes,
which carry these adaptive structures, meet each other
within an adequate distance, they are able to aggregate
to each other. This fact is sufficient enough so that, by
means of a self-activating internal self-proteolysis pro-
cess, the two large units and the two small ones are re-
leased. These four units associate with each other,
forming a heterotetramer, the active form of the cas-
pase. At the same time, this active form of caspase can
act on other procaspases, thereby initiating what is
known as a cascade of the caspases, an amplifying sys-
tem of the apoptotic signals, fruit of the original signal.
Selection of the chemical groups of the new molecules is
based on the diverse reference literature that was con-
sulted. Structural analysis is then carried out on com-
pounds with recognized efficacy in cancer treatment
via induction of apoptosis. Among the many structural
groups identified, the following are included: (1) carbo-
aromatics and p-deficient monocyclic aromatic systems,
such as pyridine16,17 or bicyclic aromatic systems, such
as quinoline,18,19 indole,20,21 and pyrido[2,3-d]pyrim-
idine,22 as well as variable aliphatic chains at the ends
(R) of the molecule. (2) Functional groups, Y, have been
selected for the ends, maintaining a gradient in their
ability to form hydrogen bonds and establish electro-
static interactions. These groups include: ether
group,23–25 a low polarity structure of great flexibility,
with the ability to establish hydrogen bonds; amide
groups directly linked to the heterocycle or near it;26,27
carbamate group,28–30 a structure of medium polarity,
capable of forming hydrogen bonds as donor and recep-
tor; a urea group,31,32 in which the substitution of oxy-
gen for nitrogen increases the ability of interaction and
the formation of hydrogen bonds and finally, an amine
In designing new structures, a general pattern derived
from the reference literature has been adopted. This pat-
tern, while flexible in geometry and chemical structure,
R
Y
W
R
Y
W
Y
R
-(CH2)n-
Alkyl
Aryl
-O-
-NH-
Heteroaryl
Alkylaryl
N
N
N
-NH-CO-
-O-NH-CO-
-NH-CO-NH-
N
N
N
N
N
X
X
Figure 1.

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C. Sanmartın et al. / Bioorg. Med. Chem. 13 (2005) 2031–2044
2033
group,33,34 which greatly increases polarity and the pos-
sibility of the formation of hydrogen bonds. (3) Diverse
antitumoral compounds described in the reference liter-
ature,35,36 which present two central aromatic systems
separated by aliphatic chains or other structural groups.
Variable, linear, and cyclic aliphatic chains as well as the
heterocycle, pyrido[2,3-d]pyrimidine in position W of
the general formula, have also been assayed. Finally, it
has been observed that many of the molecules studied
possess high symmetry.32,37–42
a
OR
HO
OH
RO
X
X
X
X
1a-d
Chain
Ref
X
R
position
1a
1b
1c
1d
4,4’
4,4’
4,4’
2,2’
H
H
-CH2-(4-C6H4Cl)
-(CH2)3-CH3
2-CH2-C5H4N
-CH2-C6H5
H
Cl
Scheme 1. Reagents and conditions: (a) R–Cl, Br/KOH/dimethylsulf-
oxide/rt.
2. Chemistry
Compounds 1a–d were prepared from 4,40-methylenebi-
sphenol or 2,20-methylenebis(4-chlorophenol) and the
corresponding alkyl halide by Williamson synthesis, as
shown in Scheme 1. The use of dimethylsulfoxide and
potassium hydroxide enhances the yield and allows mild
reaction conditions for the ether synthesis.43
trated hydrochloric acid (8:2, v:v). Pure compounds
were afforded after purification by flash chromatogra-
phy, as reported in the Experimental section.
Carbamates 3a–d were afforded by reaction of the corre-
sponding diphenol or dialcohol, with an isocyanate and
catalytic copper(I) chloride in dry dioxane at room tem-
perature45 (Scheme 3).
For the synthesis of secondary amines 2a–b, 4,40-methy-
lenebisaniline was first condensed with the aldehyde in
˚
the presence of molecular sieves (4 A) to give an imine,
which was isolated and then reduced by treatment with
sodium cyanoborohydride,44 as shown in Scheme 2.
Reduction requires acidic conditions, which were
achieved by using a mixture of methanol and concen-
Compounds 4a–d were prepared by nucleophilic substi-
tution from a primary amine46 and the corresponding
isocyanate. Reaction was carried out at room tempera-
ture in diethyl ether, and the derivatives were obtained
in good yields. The starting reagent for compounds
a
H2N
NH2
R
N
N
R
b
R
2a:
C6H5
2b: 4-C6H4NO2
R
N
H
N
H
R
2a-b
˚
Scheme 2. Reagents and conditions: (a) R–CHO/diethyl ether/molecular sieves (4 A)/rt; (b) methanol:HCl(c) (8:2, v:v)/NaCBH3/rt.
O
O
R
R
a
O
N
H
N
H
O
HO
HO
OH
X
X
X
X
3a-c
NO2
O2N
b
O
O
(CH2)5
OH
(CH2)5
O
N
H
N
O
H
3d
Chain
Ref
X
R
position
4,4’
3a
3b
3c
H
H
4-C6H4NO2
-CH2-CH2-Cl
4-C6H4NO2
4,4’
2,2’
Cl
Scheme 3. Reagents and conditions: (a) R–NCO/dioxane/CuCl/rt; (b) (4-NO2C6H4)–NCO/dioxane/CuCl/rt.

´
C. Sanmartın et al. / Bioorg. Med. Chem. 13 (2005) 2031–2044
2034
4a–c was 4,40-methylenebisaniline, whereas compound
4d was synthesized from 4,40-methylenebis-(phenylisocy-
anate), as reported in Scheme 4.
in the presence of equimolecular amounts of trieth-
ylamine.
Synthesis of the diamides, 5a–b, was carried out by reac-
tion of acyl chlorides, obtained from the corresponding
carboxylic acid by means of reflux with thionyl chloride,
with the appropriate amines and in the presence of equi-
molecular amounts of triethylamine. In the case of the
acyl chloride prepared for compound 5b, chloroform
was added as the solvent in order to provide milder con-
ditions and avoid halogenation of the quinoline ring
(Scheme 5). Compound 5c, 4-methoxy-N-(3-{[(4-
methoxy-7-nitroquinolin-2-yl)carbonyl]amine}propyl)-
5-nitroquinoline-2-carboxamide, was obtained by
reacting 1,3-propanediamine with the reaction mixture
obtained by means of the following steps: (1) nitration
of 4-methoxy-2-quinolinic acid with nitric acid, and
later, (2) reaction with thionyl chloride for acyl chloride
formation from carboxylic acid. Compound 5c and
N,N0-propane-1,3-diylbis(4-methoxy-5-nitroquinoline-
2-carboxamide) were principally formed, and the latter,
a symmetrical isomer, which did not present any cyto-
toxic activity, was then separated.
3. Biological evaluation
3.1. Cytotoxicity
The cytotoxic activity of the synthesized compounds is
determined on three human cancer cell lines: mama
(MD-MBA-231), bladder (T-24), and colon (HT-29),
using the neutral red assay. Survival percentage is deter-
mined after a 72-h period, at screening concentrations of
100 and 20 lm and using the survival percentage ob-
tained with the cell treated only with the solvent (DMSO
at 0.5%) as reference. The results are expressed as the
average of triplicate assays. IC50 values were calculated
for those compounds showing survival <45% (100 lm).
Table 1 shows the biological results obtained for the
compounds.
With regard to selectivity and as an orientative measure,
the cytotoxicity has been determined in cell cultures of
two nontumoral lines, one of mama (CRL-8799) and an-
other of liver (CRL-11233). The same experimental pro-
cedure has been used and the highest IC50 calculated in
the three tumoral lines was selected as the test concen-
tration. The data obtained using these healthy cell lines
can be found in the section titled ꢀResults and
discussionꢁ.
Compound 6a was obtained by direct reaction of piper-
azine with 1-bromo-3-phenylpropane, in refluxing chlo-
roform and in the presence of triethylamine.
The synthesis of compounds 7a–b and 7c was carried out
in three steps (Scheme 6). 2-aminonicotinic acid was
condensed with an excess of urea or formamide, afford-
ing pyrido[2,3-d]pyrimidine-2,4-diol and pyrido[2,3-
d]pyrimidine-4-ol, respectively.47 The hydroxyl groups
were replaced by chlorine after treatment with refluxing
phosphorus oxychloride and N,N-dimethylformamide
was added as the catalyst.47 In the third step, the chlo-
rines were substituted with the corresponding amines
3.2. Apoptosis
Once it has been determined, which compounds are ac-
tive in the cytotoxicity assay, they are subjected to a test,
which determines whether or not they also act as induc-
ers of apoptosis. Their ability to induce DNA fragmen-
tation in cell cultures of the lines MD-MBA-231, T-24
H
N
H
O
R
N
O
W
b
a
H2N W NH2
O=C=N W N=C=O
NH
HN
R
4a-d
Ref
4a
W
R
Method
4,4'-methylenebisphenyle -CH2-CH2Cl
4,4'-methylenebisphenyle 4-C6H4NO2
4,4'-methylenebisphenyle -(CH2)2-CH3
4,4'-methylenebisphenyle Naphth-1-yl
a
a
a
b
4b
4c
4d
Scheme 4. Reagents and conditions: (a) R–NCO/diethyl ether/rt; (b) Ar–NH2/diethyl ether/rt.
SOCl2
H2N-(CH2)n-NH2
Et3N/ CHCl3
O
O
O
O
Ar
Ar
Ar
Ar
Cl
(CH2)n
N
H
OH
N
H
5a-b
Scheme 5. 5a: Ar: pyrid-3-yl; n = 12; 5b: Ar: quinol-3-yl; n = 3.

´
C. Sanmartın et al. / Bioorg. Med. Chem. 13 (2005) 2031–2044
2035
COOH
O
O
N
NH2
H2N
OH
NH2
H
NH2
OH
N
N
N
N
N
OH
N
POCl3
POCl3
Cl
N
Cl
N
N
N
N
Cl
N
H2N-(CH2)8-NH2
CHCl3, Et3N
R
EtOH, Et3N
H2N
(CH2)8
R
HN
N
HN
N
NH
N
N
R
N
N
N
H
N
N
N
7a-b
7c
7a: R= pyrid-2-yl
7b: R= indol-3-yl
Scheme 6. 7a: R = pyrid-2-yl; 7b: R = indol-3-yl.
and HT-29, which were incubated with the compound
under study for 24 h, is also tested, using the Cell Death
Detection ELISA Plus Kit (Roche). With regard to
apoptosis induction, a result is considered positive when
the obtained level of DNA fragmentation at least dou-
bles the values obtained for the control cultures that
were treated only with the solvent. The results express
the number of times in which the culture containing
the test product surpasses the control culture in the abil-
ity to induce DNA fragmentation, having assigned a rel-
ative value of 1 at the level of apoptosis detected in said
control culture. Table 1 shows the results obtained for
the selected compounds as well as for the reference sub-
stance, camptothecin.
data for the determination of the mechanism of action.
The results of this semi-quantitative assay are expressed
using the following symbols: (ꢀ) when no increase is
detected in caspase-3 level with respect to the control,
(+) when there is an increase of approximately 50%,
and (++) when there is an increase of 100% or more
(Table 1).
4. Results and discussion
4.1. Cytotoxicity
Of all of the compounds synthesized by our group for
this research, we only make reference to those, which
have presented an acceptable level of cytotoxicity in at
least one of the tested cell lines. In general, the com-
pounds which contain the ether group did not present
notable cytotoxicity values.
3.3. Caspase-3
The compounds which show cytotoxicity and sufficient
ability to induce apoptosis are subjected to the cas-
pase-3 assay because caspase-3 is considered to be one
of the principal executing caspases in which all of the
biochemical routes involved in the apoptosis response
converge. The test used, Active-Caspase-3 FITC Mab
apoptosis kit from Pharmingen, detects the quantity of
caspase-3 dimerized in the apoptotic cells and permits
confirmation of the involvement of this enzyme in the
cell death process; this is considered to be preliminary
Compound 1b displayed selective cytotoxic activity
against the MD-MBA-231 cell line, while compounds
1a and 1d exhibited cytotoxicity only against T-24 cells.
Nevertheless, compound 1c has demonstrated cytotoxic-
ity against all cancer cell lines tested, showing more
selectivity for MD-MBA-231 (12.3 lm) and HT-29
(15.4 lm). Good cytotoxicity levels were obtained for

Table 1. Biological profile of compounds
R
W
R
Y
Y
Ref.
W
Y
R
IC50 (lm)
Apoptosis in cell
line
Caspase-3
(d) % Surviv (e) % Surviv
(a)
(b)
(c)
(a)
(b)
(c)
1a
1b
1c
1d
2a
2b
3a
3b
3c
3d
4a
4b
4,40-Methylenebisphenyl
4,40-Methylenebisphenyl
4,40-Methylenebisphenyl
–O–
–O–
–O–
4-Chlorobenzyl
Butyl
n.a. (f) n.a.
29.9
n.a.
72.5
50.5
28.9
n.a.
44.1
81.5
23.5
n.a.
34.0
33.1
—
1.5
—
—
2. 3 ++ (T-24)
90
79
89
100
82
96
—
64
93
100
0
27.3
12.3
n.a.
7.9
n.a.
15.4
n.a.
37.3
n.a.
n.a.
n.a.
29.5
n.a.
n.a.
31.4
—
n.a.
—
Pyrid-2-ylmethyl
Benzyl
Benzyl
n.a. n.a.
—
n.a.
n.a.
2,20-Methylenebis-4-chlorophenyl –O–
—
—
—
—
4,40-Methylenebisphenyl
4,40-Methylenebisphenyl
4,40-Methylenebisphenyl
4,40-Methylenebisphenyl
–NH–
–NH–
n.a.
1.3
n.a.
—
n. a n.a.
—
53
71
73
80
95
4-Nitrophenyl
4-Nitrophenyl
2-Chloroethyl
4-Nitrophenyl
4-Nitrophenyl
0.63
—
n.a.
–O–CO–NH–
–O–CO–NH–
14.0
n.a.
4.2
—
—
8.6
+ (T-24)
++(T-24)
+ (T-24)
—
2.5
14.3
—
2,20-Methylenebis-4-chlorophenyl –O–CO–NH–
6.9
—
1.5
0
–(CH2)5–
4,40-Methylenebisphenyl
4,40-Methylenebisphenyl
–O–CO–NH–
n.a.
66.7
7.0
—
—
—
—
93
–NH–CO–NH– 2-Chloroethyl
–NH–CO–NH– 4-Nitrophenyl
1.3
n.a.
1.2
3.3
++ (T-24)
++ (T-24);
++ (HT-29)
++ (T-24)
++ (HT-29)
92
2.0
100
86
9
0
4c
4d
5a
5b
5c
6a
7a
7b
7c
4,40-Methylenebisphenyl
4,40-Methylenebisphenyl
–NH–CO–NH– Propyl
–NH–CO–NH– Naphth-2-yl
68.2
3.1
n.a.
42.9
n.a.
n.a.
n.a.
32.1
68.0
3.0
18. 6
7. 4
n.a.
n.a.
39.0
n.a.
n.a.
7.7
5.8
6.6
n.a.
1.3
—
—
2.7
25.4
—
9.0
90
0
45
–(CH2)12
–
–NH–CO–
–NH–CO–
–NH–CO–
@N–
–NH–
–NH–
–NH–
Pyrid-3-yl
Quinol-3-yl
(g)
13.0
31.0
n.a.
n.a.
n.a.
5.1
—
—
+ (MD-MBA-321) 100
+ (MD-MBA-321) 100
–(CH2)3–
–(CH2)3–
–[(CH2)2]2–
—
48
—
2.8
—
n.a.
100
42
n.d. (h)
n.d.
100
n.d.
n.d.
n.d.
3-Phenylpropyl
Pyrid-2-ylethyl
3-Indolyl
—
—
13.2
1.5
3.0
—
++ (HT-29)
+ (HT-29)
++ (HT-29)
n.a.
Pyrido[2,3-d]pyrimidine (2,4)
Pyrido[2,3-d]pyrimidine (2,4)
–(CH2)8–
—
100
56
n.a.
n.a.
n.a.
3.8
3.3
Pyrido[2,3-d]-pyrimid-4-yl
7.7
0.291
n.a.
86.0
6
n.d.
Camptothecin
0.014
0.009 2.6
2.6
n.d.
(a) cell line MD-MBA-231; (b) cell line HT-29; (c) cell line T-24; (d) survival percentage in CRL-8799 cell line; (e) survival percentage in CRL-11233 cell line; (f) n.a. = no activity observed after 48 h
incubation; (g) this compound is not symmetrical, R = 4-methoxy-5-nitroquinol-2-yl; R0 = 4-methoxy-7-nitroquinol-2-yl; (h) n.d. = no data.

´
C. Sanmartın et al. / Bioorg. Med. Chem. 13 (2005) 2031–2044
2037
both carbamates and ureas possessing an aromatic cen-
tral nucleus and a 4-nitrophenyl group at the terminal
position (3a, 3c, 4b). The best IC50 were achieved for
MD-MBA-231 cells in each case (14.0 lm for 3a,
4.2 lm for 3c, and 7.0 lm for 4b). Cytotoxic activity
decreased when the aromatic nucleus was substituted
by an aliphatic chain, so, compound 3d showed no cyto-
toxicity against any of the cell lines. The insertion of a
naphthalene ring at the terminal position (4d) led to a
compound showing good cytotoxic activity in all of
the cell lines. However, substitution of the naphthalene
ring for a heterocycle, such as quinoline or indole (not
included in this publication), resulted in a complete loss
of cytotoxicity. The insertion of the amine group re-
sulted in an increase of the cytotoxicity values. The mol-
ecules that contain the amide group were not very
active. Compounds 5a and 5b were cytotoxic against
the MD-MBA-231 cell line (13.0 and 31.0 lm, respec-
tively), while compound 5c was active against T-24
(39.0 lm). The amines synthesized (2a, 2b, 6a, 7a, 7b,
and 7c) displayed cytotoxic activity against at least
one of the cancer cell lines. Compounds 2a and 7b
showed good to moderate IC50 values in all of the cell
lines. The best cytotoxicity level was achieved for com-
pound 2b, which exhibited an IC50 value of 0.63 lm in
MD-MBA-231 cells.
Certain compounds, especially 1a, 3b, 4a, 4b, 4c, and 4d
have notably provoked an increase in the caspase-3 lev-
els in the T-24 cell line. Compounds 4b, 6a, and 7b pres-
ent the same effect on caspase-3 in the HT-29 cell line,
and compounds 3a, 3c, 5a, 5b, and 7a are also cas-
pase-3 active, but to a more moderate degree. This fact
is considered to be very interesting given the character-
istics of this enzyme; the enzyme is considered to be
one of the principal executing caspases in which all of
the biochemical routes involved in the apoptosis re-
sponse converge.
5. Conclusion
Based on a general pattern inspired by the reference lit-
erature, we have completed the synthesis and biological
evaluation of novel symmetrical compounds as potential
cytotoxic and apoptosis inducers. These compounds
were performed in a cytotoxicity in vitro assay against
three cancer cell lines: mama (MD-MBA-231), colon
(HT-29), and bladder (T-24). Some compounds showed
good anticancer activity against all of the cell lines
tested, with IC50 values in the low-medium micromolar
range. The majority of these compounds of interest
showed no significant cytotoxicity in human nontumoral
cells such as CRL-8799 or CRL-11233. Compounds
which showed cytotoxicity were evaluated in an apopto-
sis assay. Some compounds exhibited great ability in
inducing apoptosis in all of the cell lines tested. The best
apoptosis levels were achieved for compounds 4d (25.4),
3c (14.3), and 3a (8.6) in T-24 cells; for compounds 6a
(13.2) and 4d (9.0) in HT-29 cells; and for compounds
3c (6.9), 4d (6.6), and 4c (5.8) in MD-MBA-231 cells,
while for camptothecin, we obtained values between
2.6 and 3.3 for the different cell lines.
In order to study the degree of selectivity in the cyto-
toxic activity of the compounds, assays using healthy
cells have been carried out on some representative
compounds those which showed the greatest activity
in tumoral cells. The healthy cells corresponded to
CLR-8799 and CLR-11233, and the survival values
were between 90% and 100% for 1a, 1c, 2b, 3a, 3c,
4a, 4b, 4d, 5a, 5b, 5c, and 7a, in at least one of the
tested lines. In other cases, intermediate survival values
were: 1b (79–82%), 2a (53–64%), 3b (80%), 4c (86%), 6a
(42%), and 7b (56%). Only a small fraction of the
tested compounds were cytotoxic against healthy cell
lines.
Although a precise structure–activity relationship can
not be defined, it is possible to point out some general
tendencies observed for the active compounds, both
those presented in this work and others prepared by
our research team: the greatest apoptotic activity is
found in compounds with a central aromatic nucleus
(W) of two rings. With regard to the functional group
of connection Y, the best results were obtained for the
urea, carbamate, and amine groups. The most frequent
R endings in compounds with apoptotic activity are aro-
matic, fairly voluminous, either carbocyclic or hetero-
cyclic, with the case of the 4-nitrophenyl group
standing out. In general, no direct relationship has been
found between the apoptotic activity and the activation
of caspase-3, thereby suggesting the existence of different
mechanisms of action. The precise mechanism of action
of derivatives with greater apoptotic activity is currently
under study in our laboratory using microarray tech-
niques. The ability of 10 of these synthesized com-
pounds to provoke a caspase-3 accumulation has been
taken into account before choosing this technique. The
optimization of design of these compounds from a struc-
ture–activity relationship point of view and the elucida-
tion of their mechanisms of action could very well lead
to the development of a novel type of antineoplastic
drugs.
4.2. Apoptosis
Nine compounds showed notable activity (>1.50) in
inducing apoptosis in T-24 cells, while four compounds
induced significant apoptosis in MD-MBA-231 cells and
six in HT-29 cells. Compounds 3c and 4c showed very
interesting levels of apoptosis in both MD-MBA-231
and T-24 cells and compound 4d showed this behavior
in all of the cell lines. Compounds 4d and 3c exhibited
exceptional ability in inducing apoptosis against bladder
(T-24) cell line, showing apoptosis values of 25.4 and
14.3 with regard to the control cells. Furthermore, very
good levels of apoptosis induction were achieved for 3a
(8.6), 4b (3.3), and 7c (3.8) in this cell line. Another inter-
esting fact was that compounds 1a, 3b, and 5c dupli-
cated the apoptosis level. With regard to the HT-29
cell line, the compounds 6a, 4d, 7b, 4b, and 3c have
induced apoptosis, with the action of 6a being very
marked (13.2). These results are shown in Table 1 along
with values obtained for camptothecin, used as the ref-
erence substance and known for its proapoptotic
behavior.

´
C. Sanmartın et al. / Bioorg. Med. Chem. 13 (2005) 2031–2044
2038
6. Experimental
for Column Chromatography and Alugramꢁ SIL G/
UV254 (Layer: 0.2 mm) (Macherey-Nagel GmbH & Co.
KG. Postfach 101352. D-52313 Duren, Germany) was
¨
6.1. Synthesis
used for Thin layer chromatography. Chemicals were
purchased from E. Merck (Darmstadt, Germany), Schar-
Melting points were determined with
a Mettler
´
lau (F.E.R.O.S.A., Barcelona, Spain), Panreac Quımica
S.A. (Montcada i Reixac, Barcelona, Spain), Sigma–Al-
FP82+FP80 apparatus (Greifense, Switzerland) and have
not been corrected. The 1H NMR spectra were recorded
on either a Bruker AC-200E (Rheinstetten, Germany) or
a Bruker 400 UltrashieldTM (Rheinstetten, Germany),
using TMS as the internal standard (Table 2). The IR
spectra were performed on a Thermo Nicolet FT-IR
Nexus in KBr pellets (Table 2). Elemental microanalyses
were carried out on vacuum-dried samples (Table 2) using
an Elemental Analyzer (LECO, CHN-900 Elemental
Analyzer). Silica gel 60 (0.040–0.063 mm) 1.09385.2500
(Merck KGaA, 64271 Darmstadt, Germany) was used
´
drich Quımica, S.A., (Alcobendas, Madrid, Spain), Acros
Organics (Janssen Pharmaceuticalaan 3a, 2440 Geel, Bel-
gie¨) and Lancaster (Bischheim-Strasbourg, France).
6.2. General procedure for compounds 1a–d
Powdered KOH (20 mmol) was added to DMSO
(15 mL). After stirring for 10 min, the appropriate
diphenol, 4,40-methylenebisphenol for compounds 1a–c,
Table 2. Spectroscopic data (IR and 1H NMR) and elemental analysis for synthesized compounds
Ref.
IR (cmꢀ1
)
1H NMR (d ppm, J in Hz)
Elemental analysis for
(%) calcd/found
1a
3030, 1245, 1014, 810
a3.78 (s, 2H, C6H4–CH2–C6H4); 5.05 (s, 4H, 2(CH2–O)); 6.88–6.92 (d,
C27H22Cl2O2: C, 72.19/
72.42; H, 4.94/4.85
0
0
0
J2–38, 4H, H3, H3 , H5, H5 C6H4); 7.08–7.12 (d, J2–38, 4H, H2, H2 , H6,
0
H6 C6H4); 7.44 (br s, 8H, 2C6H4–Cl)
1b
1c
3030, 2934–2872, 1244,
812
b0.90–0.98 (t, 6H, 2CH3); 1.43–1.50 (m, 4H, 2(CH3–CH2)); 1.69–1.76 (m,
4H, 2(CH3–CH2–CH2)); 3.82 (s, 2H, C6H4–CH2–C6H4); 3.87–3.93 (t, 4H,
C21H28O2: C, 80.77/
80.54; H, 8.97/9.21
0
2(O–CH2)); 6.76–6.81 (d, J2–3 8, 4H, H3, H3 , H5, H5 C6H4); 7.03–7.07 (d,
0
0
J2–3 8, 4H, H2, H2 , H6, H6 C6H4)
0
3035, 2961–2881, 1243
b3.85 (s, 2H, C6H4–CH2–C6H4); 5.17 (s, 4H, 2(CH2–O)); 6.87–6.91 (d,
C25H22N2O2: C, 78.54/
78.21; H, 5.75/5.47; N,
7.32/ 7.03
0
J2–3 = 8, 4H, H3, H3 , H5, H5 C6H4); 7.06–7.10 (d, J2–3 = 8, 4H, H2, H2 ,
0
0
0
H6, H6 C6H4); 7.18–7.22 (d, 2H, 2H3C6H4N); 7.49–7.53 (d, J3–4 = 8, 2H,
2H5C6H4N); 7.66–7.70 (m, 2H, 2H4C6H4N); 8.60 (d, J3–4 = 5, 2H,
2H6C6H4N)
1d
2a
2b
3031, 1246, 1006
3378, 1617, 811
a3.89 (s, 2H, C6H3–CH2–C6H3); 5.08 (s, 4H, 2(CH2–O)); 7.04–7.08 (d,
C27H22Cl2O2: C, 72.19/
72.10; H, 4.90/4.79
0
J3–4 = 9, 2H, H3, H3 C6H3); 7.20 (s, 2H, H6, H6 C6H3); 7.21–7.25 (d,
0
0
J3–4 = 9, 2H, H4, H4 C6H3); 7.33 (br s, 10H, 2C6H5)
a3.75 (s, 2H, C6 H4–CH2–C6H4); 3.93 (br s, 2H, 2NH); 4.29 (s, 4H,
C27H26N2: C, 85.68/
85.69; H, 6.92/7.08; N,
7.40/7.39
0
2(CH2–N)); 6.53–6.57 (d, J2–3 = 8, 4H, H2, H2 , H6, H6 C6H4); 6.95–6.99
0
0
(d, J2–3 = 8, 4H, H3, H3 , H5, H5 C6H4); 7.29–7.35 (br s, 10H, 2C6 H5)
0
3404, 1612, 1514, 1323
c3.76 (s, 2H, C6H4–CH2–C6H4); 4.24 (br s, 2H, 2NH); 4.46 (s, 4H, 2(CH2–
C27H24N4O4: C, 69.22/
68.99; H, 5.16/5.25; N,
11.96/11.77
0
N)); 6.51–6.53 (d, J2–3 = 8, 4H, H2, H2 , H6, H6 C6H4–N); 6.97–6.99 (d,
0
0
0
J2–3 = 8, 4H, H3, H3 , H5, H5 C6H4–N); 7.53–7.55 (d, J2–3 = 9, 4H, 2H2,
2H6C6H4–NO2); 8.18–8.22 (d, J2–3 = 9, 4H, 2H3, 2H5C6H4–NO2)
0
0
3a
3b
3314, 1724, 1502, 1340
3320, 2979–2879, 1715
e3.98 (s, 2H, CH2); 7.10–7.14 (d, J2–3 = 8, 4H, H2, H2 , H6, H6 C6H4–O);
C27H20N4O8:C, 61.38/
61.58; H, 3.80/3.71; N,
10.60/10.36
0
0
7.25–7.29 (d, J2–3 = 8, 4H, H3, H3 , H5, H5 C6H4–O); 7.77–7.82 (d,
J2–3 = 9, 4H, 2H2, 2H6 C6H4–NO2); 8.18–8.22 (d, J2–3 = 9, 4H, 2H3,
2H5 C6H4–NO2); 9.84 (s, 2H, 2NH)
e3.48–3.51 (m, 4H, 2(CH2–CH2–Cl)); 3.64–3.67 (t, 4H, 2(CH2–Cl)); 3.94
0
(s, 2H, C6H4–CH2–C6H4); 6.99–7.03 (d, J2–3 = 8, 4H, H2, H2 , H6, H6
C19H20 Cl2N2O4: C,
55.47/55.22; H, 4.87/
4.79; N, 6.81/6.66
0
0
C6H4); 7.18–7.22 (d, J2–3 = 8, 4H, H3, H3 , H5, H5 C6H4); 11.13 (br s, 2H,
0
2NH)
3c
3d
3318, 1758, 1510, 1330,
1010
e3.97 (s, 2H, CH2); 7.21–7.24 (br s, 6H, 2C6H3); 7.66–7.70 (d, J2–3 = 9, 4H,
2H2, 2H6 C6H4–NO2); 8.09–8.14 (d, J2–3 = 9, 4H, 2H3, 2H5 C6H4–NO2);
9.90 (s, 2H, 2NH)
C27H18 Cl2N4O8: C,
54.24/53.85; H, 3.01/
3.20; N, 9.38/9.42
3381, 2951–2879, 1734,
1512, 1333
e1.40–1.43 (m, 2H, –CH2–CH2–CH2–O); 1.64–1.66 (m, 4H, 2(CH2–CH2–
0
O)); 4.06–4.09 (t, 4H, 2(O–CH2)); 7.68–7.80 (d, J2–3 = 8, 4H, H3, H3 , H5,
C19H20N4O8: C, 52.78/
52.40; H, 4.63/4.42; N,
12.96/12.94
0
0
0
H5 C6H4); 8.07–8.09 (d, J2–3 = 8, 4H, H2, H2 , H6, H6 C6H4); 9.25 (s, 2H,
2NH)
4a
3331, 2931–2881, 1636
a3.38–3.44 (m, 4H, 2(CH2–CH2–Cl)); 3.61–3.67 (t, 4H, 2(CH2–Cl)); 3.87
(s, 2H, C6H4–CH2–C6H4); 6.34–6.40 (t, 2H, 2(NH–CH2)); 7.02–7.06 (d,
C19H22N4O2: C, 55.65/
55.69; H, 5.37/5.36; N,
13.67/13.60
0
J2–3 = 8, 4H, H2, H2 , H6, H6 C6H4); 7.26–7.30 (d, J2–3 = 8, 4H, H3, H3 ,
0
0
0
H5, H5 C6H4); 8.58 (s, 2H, 2(NH–C6H4))

´
C. Sanmartın et al. / Bioorg. Med. Chem. 13 (2005) 2031–2044
2039
Table 2 (continued)
Ref.
IR (cmꢀ1
)
1H NMR (d ppm, J in Hz)
Elemental analysis for
(%) calcd/found
0
0
4b
3366, 2931–2883, 1664,
1501, 1343
a3.84 (s, 2H, CH2); 7.13–7.17 (d, J2–3 = 8, 4H, H2, H2 , H6, H6 CH2–
C27H22N6O6: C, 61.61/
61.22; H, 4.18/4.29; N,
15.97/16.36
0
0
C6H4); 7.37–7.41 (d, J2–3 = 8, 4H, H3, H3 , H5, H5 CH2–C6H4); 7.65–7.69
(d, 4H, J2–3 = 9, 2H2, 2H6NO2–C6H4); 8.16–8.20 (d, J2–3 = 9, 4H, 2H3,
2H5 NO2–C6H4); 8.85 (s, 2H, 2(NH–C6H4–CH2)); 9.39 (s, 2H, 2(NH–
C6H4–NO2))
4c
4d
3321, 2932–2871, 1636
3281, 2931–2881, 1638
a 0.91–0.97 (t, 6H, 2CH3); 1.45–1.51 (m, 4H, 2(CH3–CH2)); 3.45–3.52 (m,
4H, 2(CH2–CH2–CH3)); 3.85 (s, 2H, C6H4–CH2–C6H4); 6.28–6.32 (t, 2H,
2(NH–CH2)); 7.14–7.18 (d, J2–3 = 8, 4H, 2H3, 2H5 C6H4); 7.35–7.39 (d,
J2–3 = 8, 4H, 2H2, 2H6 C6H4); 8.43 (s, 2H, 2(NH–C6H4))
C21H28N4O2: C, 68.45/
68.22; H, 7.66/7.43; N,
15.20/15.21
a3.70 (s, 2H, CH2); 6.99–7.03 (d, J2–3 = 8, 4H, H3, H3 , H5, H5 C6H4);
C35H28N4O2: C, 78.36/
78.08; H, 5.22/5.43; N,
10.45/10.67
0
0
0
0
7.26–7.31 (d, J2–3 = 8, 4H, H2, H2 , H6, H6 C6H4); 7.35–7.50 (m, 8H, 2H3,
2H5, 2H6, 2H7, C10H7); 7.76–7.80 (d, J7ꢀ8 = 7, 2H, 2H8 C10H7); 7.86–7.90
(d, J3–4 = 7, 2H, 2H4 C10H7); 7.95–7.99 (d, J2–3 = 6, 2H, 2H2 C10H7); 8.59
(s, 2H, 2NH); 8.86 (s, 2H, 2NH)
5a
5b
3321, 3081, 2921, 2850,
1630
c1.25 (br s, 16H, CH2); 1.50 (m, 4H, CH2); 3.26 (m, 4H, CH2); 7.49 (dd,
C24H34N4O2: C, 69.75/
69.81; H, 8.35/8.30; N,
13.55/13.53
0
0
J5–4 = 8, J5ꢀ6 = 5, 2H, H5, H5 ); 8.15 (d, J4ꢀ5 = 8, 2H, H4, H4 ); 8.62 (t,
J = 5, 2H, NH, NH0); 8.68 (d, J6ꢀ5 = 5, 2H, H6, H6 ); 8.96 (s, 2H, H2, H2 )
a1.91 (q, J = 7, 2H, CH2); 3.45 (m, 4H, CH2); 7.68 (dd, J6ꢀ5 = 8, J6ꢀ7 = 8,
2H, H6, H6 ); 7.86 (dd, J7–6 = 8, J7ꢀ8 = 8, 2H, H7, H7 ); 8.07 (d, J5ꢀ6 = 8,
0
2H, H5, H5 ); 8.07 (d, J8–7 = 8, 2H, H8, H8 ); 8.81 (s, 2H, H4, H4 ); 8.89 (t,
0
0
3295, 3068, 3000–2900,
1634
C23H20N4O2: C, 71.89/
71.55; H, 5.20/5.21; N,
14.57/14.57
0
0
0
0
0
0
J = 5, 2H, NH, NH ); 9.28 (s, 2H, H2, H2 )
d
0
2.05 (q, J = 7, 2H, CH2); 3.68 (m, 4H, CH2); 4.19 (s, 3H, CH3 ); 4.23 (s,
5c
3547, 3413, 3070, 3000–
2850, 1683
C25H22N6O8: C, 56.20/
55.98; H, 4.12/4.22; N,
15.72/15.89
0
3H, CH3); 7.62 (dd, J7–6 = 8, J7ꢀ8 = 8, 1H, H7); 7.84 (s, 1H, H3 ); 7.90 (s,
0
1H, H3); 8.08 (d, J5 –6 = 9, 1H, H5 ); 8.09 (d, J8–7 = 8, 1H, H8); 8.43 (d,
0
0
a
0
0
0
J6ꢀ7 = 8, 1H, H6); 8.43 (d, J6 –5 = 9, 1H, H6 ); 8.52 (t, J=6, 1H, NH );
b
8.61 (t, J=6, 1H, NH ); 9.14 (s, 1H, H8 )
0
6a
7a
3429, 3100–3000, 3000–
2900, 2306
c1.91 (m, 4H, CH2); 2.64 (t, J = 8 Hz, 4H, CH2-phenyl); 3.12 (m, 4H,
CH2–CH2–CH2-piperaz.); 3.41 (br s, 4H, CH piperaz.); 3.74 (br s, 4H, CH
piperazi.); 7.19–7.33 (m, 10H, CH phenyl); 12.03 (br s, 2H, hydrochloride)
C22H30N2 Æ 2HCl: C,
66.87/66.50; H, 8.09/
8.21; N, 7.09/6.98
3254, 3100–3000, 3000–
2900
c3.15 (br s, 4H, CH2–pyridine); 3.96 (br s, 4H, CH2–N); 7.11 (br s, 1H, H6
C21H21N7: C, 67.94/
67.58; H, 5.65/5.43; N,
26.40/26.49
0
pyridopyrimidine); 7.19 (dd, J5–4 = 8, J5ꢀ6 = 5, 2H, H5, H5 pyridine); 7.23
0
(d, J3–4 = 8, 2H, H3, H3 pyridine); 7.56 (br s, 1H, H5 pyridopyrimidine);
0
7.64 (dd, J4ꢀ3 = 8, J4ꢀ5 = 8, 2H, H4, H4 pyridine); 8.54 (br s, 1H, H7
0
pyridopyrimidine); 8.57 (d, J6ꢀ5 = 5, 2H, H6, H6 pyridine); 7.00–7.92–
8.29–8.71 (dd–br s–br s–br s, 2H, NH, NH0)
a
7b
3415, 3300, 3100–3000,
3000–2900
c3.01 (t, 2H, CH2a–indole); 3.10 (t, 2H, CH2c–indole); 3.74 (m, 2H, CH2
–
C27H25N7 Æ HCl: C,
67.03/67.27; H, 5.17/
5.09; N, 20.25/19.90
N); 3.85 (m, 2H, CH2c–N); 6.86 (dd, J5–4 = 8, J5ꢀ6 = 8, 1H, H5 indole);
0
6.97 (dd, J5 –4 = 8, J5 –6 = 8, 1H, H5 indole); 7.03 (br s, 1H, H6
0
0
0
0
0
pyridopyrimidine); 7.03 (br s, 2H, H6, H6 indole); 7.14 (br s, 1H, H2
0
indole); 7.19 (br s, 1H, H2 indole); 7.33 (d, J5ꢀ6 = 8, 1H, H5
0
pyridopyrimidine); 7.35 (d, J7–6 = 8, 2H, H7, H7 indole); 7.57 (d, J4ꢀ5 = 8,
0
2H, H4, H4 indole); 8.63 (d, J7–6 = 5, 1H, H7 pyridopyrimidine); 10.80 (br
s, 2H, NH, NH0 indole); 7.96–8.40–8.52–9.55 (br s, 3H, NH, NH0, HCl)
7c
3293, 3100–3000, 2929,
2850
c1.30 (br s, 8H, CH2); 1.61 (m, 4H, CH2); 3.51 (m, 4H, CH2); 7.51 (dd,
C21H21N7: C, 65.69/
65.67; H, 6.46/6.35; N,
27.84/27.61
0
0
J6ꢀ5 = 8, J6ꢀ7 = 4, 2H, H6, H6 ); 8.57 (s, 2H, H2, H2 ); 8.57 (br s, 2H, NH,
NH0); 8.67 (d, J5ꢀ6 = 8, 2H, H5, H5 ); 8.95 (d, J7–6 = 4, 2H, H7, H7 )
0
0
a DMSO-d6; 200 MHz.
b CDCl3; 200 MHz.
c DMSO-d6; 400 MHz.
d CDCl3; 400 MHz.
e (CD3)2CO; 200 MHz.
and 2,20-methylenebis(4-chlorophenol) for compound
1d (2.50 mmol), was added, followed by the addition
of alkyl halide (10 mmol). The mixture was stirred for
24 h and then poured into water (100 mL). Extraction
was carried out with dichloromethane (3 · 75 mL).
The combined extracts were washed with water
(3 · 75 mL) and dried over anhydrous sodium sulfate.
The solvent was removed and the residue was recrystal-
lized from ethanol and dichloromethane, unless other-
wise noted.

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C. Sanmartın et al. / Bioorg. Med. Chem. 13 (2005) 2031–2044
2040
6.3. 1,10-Methylenebis[4-(4-chloro)benzyloxyphenyl] (1a)
(3 · 40 mL) and water (3 · 40 mL), and then dried over
anhydrous sodium sulfate. The solvent was removed
and the residue was recrystallized from a suitable solvent.
From 4-chlorobenzyl chloride. Yield 55%. Mp 139–
140 ꢂC.
6.11. Methylenebis[(4,1-phenylene)-N-(4-nitrophenyl)car-
bamate] (3a)
6.4. 1,10-Methylenebis(4-butoxyphenyl) (1b)
From 4-nitrophenyl isocyanate. A pure compound was
afforded after washing with diethyl ether (3 · 15 mL)
and absolute ethanol (3 · 15 mL). Yield 34%. Mp 232–
233 ꢂC.
From butyl bromide. Yield 33%. Mp 35–36 ꢂC (metha-
nol/dichloromethane).
6.5. 2,20-[Methylenebis(1,4-phenyloxymethylene)]dipyr-
idine (1c)
6.12. Methylenebis[(4,1-phenylene)-N-(2-chloroethyl)car-
bamate] (3b)
From 2-chloromethyl pyridine hydrochloride. Purified
by flash-chromatography using silica gel as the solid
support, and eluted with hexane:ethyl acetate (8:2 v:v)
mixture. Yield 18%. Mp 78–79 ꢂC.
From 2-chloroethyl isocyanate. Yield 27%. Mp 175–
176 ꢂC (ethanol).
6.13. Methylenebis[(4-chloro-2,1-phenylene)-N-(4-nitro-
phenyl)carbamate] (3c)
6.6. 1,10-Methylenebis(2-benzyloxy-5-chlorophenyl) (1d)
From benzyl chloride. Yield 33%. Mp 179–180 ꢂC.
6.7. General procedure for compounds 2a–b
From 4-nitro phenylisocyanate. A pure compound was
afforded after washing with diethyl ether (3 · 15 mL)
and absolute ethanol (3 · 15 mL). Yield 25%. Mp 131–
132 ꢂC.
˚
Molecular sieves (4 A; 2.0 mmol) and aldehyde
(15 mmol) were added to a solution of 4,40-methylenebi-
saniline (5.0 mmol) in diethyl ether (30 mL). The mix-
ture was stirred for 24 h. The solid precipitate was
filtered, and then dissolved in chloroform (30 mL). The
solution was filtered from the molecular sieves, which
were then washed with chloroform. The solvent was re-
moved from the filtrate and washings, and the residue
was washed with diethyl ether (3 · 15 mL) and dichlo-
romethane (3 · 15 mL), affording the intermediate imine
(yield: 60–75%). NaBH3CN (1.2 mmol) was added to a
solution of the imine (2.5 mmol) in methanol:HCl(c)
(8:2, v:v). The reaction mixture was stirred for 24 h.
The methanol was removed and the residue was taken
up in water (30 mL) and extracted with diethyl ether
(3 · 30 mL). The combined extracts were dried over
anhydrous sodium sulfate, and the solvent was removed.
The residue was purified by flash chromatography using
silica gel as the solid support and then eluted with
dichloromethane/methanol (95:5, v:v) mixture.
6.14. Pentane-1,5-diylbis[N-(4-nitrophenyl)carbamate]
(3d)
From 4-nitrophenylisocyanate. Yield 37%. Mp 200–
201 ꢂC (methanol/dichloromethane).
6.15. General procedure for compounds 4a–c
A solution of the appropriate isocyanate (5.0 mmol) in
diethyl ether (10 mL) was added dropwise to a solution
of 4,40-methylenebisaniline in diethyl ether (25 mL). The
reaction mixture was stirred for 30 min. The solid pre-
cipitate was filtered, washed with diethyl ether
(3 · 15 mL) and boiling ethanol (3 · 15 mL) and then
dried, unless otherwise noted.
6.16. Methylenebis[N-(4,1-phenylene)-N0-(2-chloro-
ethyl)urea] (4a)
From 2-chloro ethylisocyanate. Yield 82%. Mp 228–
229 ꢂC.
6.8. 4,40-Methylenebis(N-benzylaniline) (2a)
6.17. Methylenebis[N-(4,1-phenylene)-N0-(4-nitro-
phenyl)urea] (4b)
From benzaldehyde. Yield 18%. Mp 112–113 ꢂC.
6.9. 4,40-Methylenebis[N-(4-nitrobenzyl)aniline](2b)
From 4-nitrobenzaldehyde. Yield 22%. Mp 152–153 ꢂC.
6.10. General procedure for compounds 3a–d
From
4-nitro
phenylisocyanate.
Yield
21%.
Mp > 300 ꢂC.
6.18. Methylenebis[N-(4,1-phenylene)-N0-(n-propyl)urea]
(4c)
The corresponding isocyanate (5.0 mmol) was added to a
heterogeneous mixture of the appropriate phenol, 4,40-
methylenebisphenol for compounds 3a–b and 2,20-meth-
ylenebis(4-chlorophenol) for compound 3c, or alcohol,
1,5-pentanediol for compound 3d, (2.5 mmol), copper(I)
chloride (0.50 g), and dry dioxane (20 mL) under stir-
ring. After stirring for 5 h, the reaction mixture was di-
luted with diethyl ether (40 mL), washed with brine
From propyl isocyanate. Yield 85%. Mp 218–219 ꢂC.
6.19. Methylenebis[N-(4,1-phenylene)-N0-(naphth-1-
yl)urea] (4d)
4,40-Methylenebis(phenylisocyanate) (2.0 mmol) was
suspended in diethyl ether (30 mL) and a solution of

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C. Sanmartın et al. / Bioorg. Med. Chem. 13 (2005) 2031–2044
2041
1-amino naphthalene (4.2 mmol) in diethyl ether
(15 mL) was added dropwise. The reaction mixture
was stirred for 5 h. The solid precipitate was filtered,
washed with methanol (3 · 10 mL), and boiling acetone
(3 · 10 mL) and then dried, unless otherwise noted.
Yield 46%. Mp > 300 ꢂC.
amine (0.18 mL, 2.2 mmol) and triethylamine
(0.61 mL, 4.43 mmol) in dry chloroform. The mixture
was then heated under reflux for 4 h. The solvent
was removed. The resulting residue was suspended in
water (100 mL), filtered, washed with water
(4 · 25 mL), and purified by flash chromatography,
using silica gel as the solid support and with n-hex-
ane/ethyl acetate (1:1, v:v) as the eluent. Compounds
5c (yield 26%. Mp 224–225 ꢂC) and N,N0-propane-
1,3-diylbis(4-methoxy-5-nitroquinoline-2-carboxamide)
(yield 19%. Mp 273–274 ꢂC) were obtained.
6.20. N,N0-Dodecane-1,12-diyldinicotinamide (5a)
A solution of nicotinic acid (4.00 g, 32.5 mmol) in thio-
nyl chloride (15–20 mL) was stirred under reflux for 2 h.
The solvent was removed and the residue was suspended
in dry chloroform (30 mL). Triethylamine was then
added (4.52 mL, 32.5 mmol). This solution was added
dropwise at room temperature for 1 h to a mixture of
the 1,12-dodecanediamine (3.25 g, 16.2 mmol) and tri-
ethylamine (4.52 mL, 32.5 mmol) in dry chloroform.
After said addition, the mixture was heated under reflux
for 5 h. The solvent was removed and the resulting res-
idue was suspended in water (100 mL), filtered, washed
with water, and recrystallized. Yield 30%. Mp 152–
153 ꢂC (ethanol).
6.23. 1,4-Bis(3-phenylpropyl)piperazine dihydrochloride
(6a)
A solution of piperazine (0.65 g, 7.5 mmol) and triethyl-
amine (2.10 g, 15.1 mmol) in chloroform (50 mL) was
stirred under reflux. A solution of 3-phenylpropane bro-
mide (2.29 mL, 15.1 mmol) in chloroform (20 mL) was
added dropwise for 2 h. The mixture was stirred under
reflux for 48 h. The solvent was removed, and the result-
ing residue was dissolved in water (50 mL) and extracted
with dichloromethane (3 · 50 mL). The combined ex-
tracts were washed with water (2 · 25 mL) and dried
over anhydrous sodium sulfate. The solvent was re-
moved and HCl 1.5 N (15 mL) was added. The mixture
was extracted with dichloromethane (3 · 30 mL). The
solution was stirred vigorously after each addition,
and the precipitate that appeared was filtered. The com-
bined solids were recrystallized. Yield 39%. Mp 226–
227 ꢂC (ethanol).
6.21. N,N0-Propane-1,3-diyldiquinoline-3-carboxamide
(5b)
A solution of 3-quinolinic acid (2.00 g, 11.5 mmol), and
thionyl chloride (15–20 mL) in dry chloroform (50 mL)
was stirred under reflux for 2 h. The solvent was re-
moved and the residue was suspended in dry chloroform
(30 mL). Triethylamine was then added (1.61 mL,
11.6 mmol). This solution was added dropwise to a mix-
ture of 1,3-propanediamine (0.48 mL, 5.8 mmol) and tri-
ethylamine (1.61 mL, 11.6 mmol) in dry chloroform at
room temperature for 1 h. The mixture was then heated
under reflux for 4 h and the solvent was removed. The
residue was suspended in water (100 mL) and filtered
off. The resulting solid was washed with boiling water
(4 · 25 mL) and recrystallized from n-hexane/ethanol.
Yield 66%. Mp 200–201 ꢂC.
6.24. General procedure for compounds 7a and 7b
(a) Preparation of pyrido[2,3-d]pyrimidin-2,4-diol: a
mixture of 2-aminonicotinic acid (5.00 g, 36.2 mmol)
and urea (9.00 g, 150 mmol) was pulverized and heated
to 210 ꢂC (15 min at this temperature). After cooling
to room temperature, NaOH 2 N (50 mL) was added.
The solution was heated until complete dissolution,
and the mixture was added to solid CO2. The solid
was filtered, washed with cool water (2 · 10 mL), and
suspended in boiling acetic acid. The solution was fil-
tered and washed with diethyl ether. Pyrido[2,3-d]pyrim-
idin-2,4-diol was obtained. Yield 62.3%. IR: 3406, 3176,
6.22. 4-Methoxy-N-(3-{[(4-methoxy-7-nitroquinolin-2-
yl)carbonyl]amino}propyl)-5-nitro quinoline-2-carboxam-
ide (5c)
A solution of 4-methoxy-2-quinolinic acid (2.50 g,
11.7 mmol) in H2SO4 (c) (15 mL) and HNO3 (c)
(5 mL) was stirred and heated to 60 ꢂC for 4 h. The
mixture was cooled to 0 ꢂC. Next, while under stirring,
NaOH 20 N (30 mL) was added until pH 6–7. The
solution was filtered and the solid was dissolved in
boiling methanol. After cooling to room temperature,
the solution was filtered, and the methanol of the fil-
trate was removed under low pressure. The residue ob-
tained was the nitrated 4-methoxy-2-quinolinic acid
(1.10 g, 4.43 mmol). A solution of nitrated 4-meth-
oxy-2-quinolinic acid (1.10 g, 4.43 mmol) and thionyl
chloride (10 mL) in dry chloroform (50 mL) was stir-
red under reflux for 2 h. The solvent was removed
and the residue was suspended in dry chloroform
(30 mL). Triethylamine was then added (0.61 mL,
4.43 mmol). This solution was added dropwise at room
temperature for 1 h to the mixture of 1,3-propanedi-
3100–3000, 2850, 2754, 1720, 1671 cmꢀ1 1H NMR
.
(400 MHz, DMSO-d6, d): 6.76 (dd, J6–5 = 8 Hz,
J6–7 = 5 Hz, 1H, H6); 7.99 (d, J5–6 = 8 Hz, 1H, H5);
8.35 (d, J7–6 = 5 Hz, 1H, H7); 11.8 (br s, 2H, OH). Anal.
Calcd for C7H5N3O2 (%): C, 51.55; H, 3.06; N, 25.75.
Found (%): C, 51.55; H, 3.07; N, 25.72. (b) Preparation
of 2,4-dichloropyrido[2,3-d]pyrimidine: a solution of
pyrido[2,3-d]pyrimidin-2,4-diol (0.60 g, 3.7 mmol) and
N,N-dimethylformamide (two drops) in phosphorus
oxychloride (40 mL) was stirred under reflux for 24 h.
The solvent was removed and ice (50 g) was added to
the resulting residue. The solution was extracted with
chloroform (5 · 50 mL). The combined extracts were
washed with water (2 · 50 mL) and dried over anhy-
drous sodium sulfate. The solvent was then removed
and 2,4-dichloropyrido[2,3-d]pyrimidine (0.74 g) was
obtained and used immediately without further
purification.

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C. Sanmartın et al. / Bioorg. Med. Chem. 13 (2005) 2031–2044
2042
6.25. N,N0-Bis(2-pyridin-2-ylethyl)pyrido[2,3-d]pyrim-
idine-2,4-diamine (7a)
A solution of 4-chloropyrido[2,3-d]pyrimidine (0.84 g,
5.1 mmol), triethylamine (0.70 mL, 5.1 mmol) and 1,8-
octanediamine (0.37 g, 2.5 mmol) in chloroform
(50 mL) was stirred under reflux for 48 h. The solvent
was removed under low pressure. The resulting residue
was suspended in water (50 mL), filtered, washed with
water (3 · 20 mL), and recrystallized. Yield 20%. Mp
195–196 ꢂC (ethyl acetate/ethanol).
A
solution of 2,4-dichloropyrido[2,3-d]pyrimidine
(0.74 g, 3.7 mmol), triethylamine (1.0 mL, 7.4 mmol)
and 2-(2-aminoethyl)pyridine (0.95 g, 7.4 mmol) in etha-
nol (50 mL) was stirred under reflux for 24 h. The sol-
vent was removed, and the resulting residue was
dissolved in HCl 1 N (25 mL) and washed with chloro-
form (3 · 25 mL). NaOH 10% was added until pH 9–
10, and the solution was extracted with chloroform
(4 · 25 mL). The combined extracts were washed with
water (2 · 25 mL) and dried over anhydrous sodium sul-
fate. The solvent was removed and ethyl acetate/isopro-
panol (100 mL, 1:1, v:v) was added to the resultant oil.
The residue was then filtered and recrystallized. Yield
15%. Mp 156–157 ꢂC (ethyl acetate/2-propanol).
6.28. Evaluation of cytotoxic potential against human
cancer cell lines
An assay using neutral red48 staining was employed.
Cells were cultured in McCoy medium for HT-29 and
T-24 cell lines and Leibovitz medium for MD-MBA-
231 cell line supplemented with 10% heat-inactivated
FCS and 1% penicillin/streptomicin. For this assay, cells
were obtained from 80–90% confluent T-75 flasks by
detaching the cells with PBS/EDTA. Cells (100 lm) were
seeded at a density of 20 · 103/well in 96-well plates, but
only the 60 inner wells were used in order to avoid pos-
sible border effects. Before adding the compounds, cells
were allowed to attach to the bottom of the wells for
12 h. Compounds were diluted in complete medium.
The plating density permitted several rounds of cell pro-
liferation before confluent monolayers were formed.
After 3 days of incubation, 0.05 mL of neutral red solu-
tion (0.05 mg/mL diluted in saline) was added to the
cells in the existing growth medium (0.2 mL) for 1 h
30 min at 37 ꢂC. The plates were flicked and 0.1 mL of
0.05 M sodium phosphate (monobasic) in 50% ethanol
was added. The plates were vortexed in a plate shaker,
incubated for 10 min at room temperature and then read
using a plate reader at 540 nm absorbance. Data was
calculated as a percentage of total absorbance found
for cells in nondrug-treated wells.
6.26. N,N0-Bis[2-(1H-indol-3-yl)ethyl]pyrido[2,3-d]pyrim-
idine-2,4-diamine (7b)
A
solution of 2,4-dichloropyrido[2,3-d]pyrimidine
(0.74 g, 3.7 mmol) and 3-(2-aminoethyl)indole (2.38 g,
14.8 mmol) in ethanol (50 mL) was stirred under reflux
for 24 h. The solvent was removed, and water (25 mL)
and chloroform (25 mL) were added to the resulting res-
idue. The mixture was stirred and filtered. The solid was
dissolved in boiling isopropanol and the solvent was
removed. The resultant residue was purified by flash
chromatography, using silica gel as the solid support
and with dichloromethane/methanol (95:5, v:v) as the
eluent. Once the solvent was removed, the residue was
recrystallized. Yield 10%. Mp 162–163 ꢂC (n-hexane/
methanol).
6.27. Procedure for compound N,N0-dipyrido[2,3-d]pyr-
imidin-4-yloctane-1,8-diamine (7c)
6.29. Evaluation of potential selectivity
(a) Preparation of pyrido[2,3-d]pyrimidin-4-ol: a mix-
ture of 2-aminonicotinic acid (8.00 g, 57.9 mmol) and
formamide (16.0 g, 355 mmol) was pulverized and
heated to 170 ꢂC (2 h at this temperature). After cooling
to room temperature, water (50 mL) was added. The
solution was then filtered and washed with water
(2 · 10 mL). The solid was recrystallized from water
and pyrido[2,3-d]pyrimidin-4-ol was obtained. Yield
55%. IR: 3422, 3100–3000, 3000–2900. 1H NMR
Cytotoxic activity was determined by performing the
aforementioned neutral red assay. However, this time,
the cell lines used were CRL-8799 from mama epithe-
lium and CLR-11233 from liver. The test concentration
used was the highest IC50 determined for each com-
pound in the three cultures in tumor cell lines.
6.30. Evaluation of apoptosis induction in human cancer
cell lines
(400 MHz, DMSO-d6, d): 7.56 (dd, J6–5 = 8 Hz, J6–7
=
5 Hz, 1H, H6); 8.32 (s, 1H, H2); 8.51 (d, J5–6 = 8 Hz,
1H, H5); 8.95 (d, J7–6 = 5 Hz, 2H, H7); 12.55 (br s, 1H,
OH). Anal. Calcd for C7H5N3O (%): C, 57.16; H,
3.40; N, 28.56. Found (%): C, 56.88; H, 3.42; N, 28.89.
(b) Preparation of 4-chloropyrido[2,3-d]pyrimidine: a
Apoptosis was quantified using a detection kit called the
Cell Death Detection ELISAPlus (Roche Biochemicals).
This cellular test detects nucleosomes in cytoplasm prior
to disintegration of the plasma membrane, a well-known
hallmark of apoptosis.49,50 The assay is based on a
quantitative sandwich-enzyme-immunoassay principle:
monoclonal mouse antibodies directed against DNA
and histones (H1, H2A, H2B, H3, and H4) specifically
detect mono- and oligonucleosomes. Apoptosis was
measured with the aid of this kit, following the instruc-
tions provided by the manufacturer, and using the IC50
values determined in the previous cytotoxicity assay for
each one of the cell lines as the test concentrations. The
apoptosis measurements were taken after 48 h of incu-
solution
of
pyrido[2,3-d]pyrimidin-4-ol
(0.75 g,
5.1 mmol) in phosphorus oxychloride (40 mL) was stir-
red under reflux for 1 h. The solvent was removed under
low pressure, and ice (50 g) was added to the resulting
residue. The solution was extracted with chloroform
(10 · 40 mL). The combined extracts were washed with
water (2 · 80 mL) and dried over anhydrous sodium
sulfate. The solvent was removed, and 4-chloropyrido[2,3-
d]pyrimidine (0.84 g) was obtained and used immedi-
ately without further purification.

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C. Sanmartın et al. / Bioorg. Med. Chem. 13 (2005) 2031–2044
2043
21. Carson, D. A.; Leoni, L. M.; Cottam, H. B.
WO 2002012188 A2, 2002; Chem. Abstr. 2002, 136,
161347.
bation. As previously indicated, a relative value of 1 was
attributed to the apoptosis detected in the control cul-
tures in which the test compound is not present.
22. Klutchko, S. R.; Hamby, J. M.; Boschelli, D. H.; Wu, Z.;
Alan, J.; Amar, A. M.; Harti, B. G.; Shen, C.; Klohs, W.
D.; Steinkampf, R. W.; Driscoll, D. L.; Nelson, J. M.;
Elliot, W. L.; Roberts, B. J.; Stoner, C. L.; Vincent, P. W.;
Dykes, D. J.; Panek, R. L.; Lu, G. H.; Major, T. C.;
Dahring, T. K.; Hallak, H.; Bradford, L. A.; Showalter,
H. D. H.; Doherty, A. M. J. Med. Chem. 1998, 41(17),
3276–3292.
6.31. Evaluation of caspase-3 activation
Detection was carried out by means of flow cytometry,
using the Active-Caspase-3 FITC Mab apoptosis kit from
Pharmingen, which evaluates the number of cells that
are contained in the dimerized and caspase-3-activated
form. It has been determined that the range of measure-
ments considered to be effective for this enzyme is be-
tween 14 and 48 h. Therefore, measurements were
taken at 14, 24, and 48 h, and the values obtained were
compared with the control cells that express this enzyme
when they are incubated without the test compound.
The test concentrations correspond to the IC50 values
determined in the cytotoxicity assay.
23. 228501, Drug Data Rep. 1996, 18, 136.
24. Ayal-Hershkovitz, M.; Miron, D.; Koller, A.; Llan, N.;
Levy, O. WO 2002060375 A2, 2002; Chem. Abstr. 2002,
137, 135116.
25. Amils, D. V.; Bills, G. F.; Diez, M. T.; Dombrowski, A.
W.; Hensens, O. D.; Huang, L.; Huber, H. E.; Jenkins, R.
G.; Lee, S. H.; Patrick, D. R. GB 2327085 A1, 1999;
Chem. Abstr. 2000, 130, 337070.
26. Hedlung, G.; Jansson, K.; Joensson, S.; Bjoerk, A.
WO 2001030758 A1, 2001; Chem. Abstr. 2001, 134,
326412.
27. Cho, E-H.; Chung, S-G.; Lee, S-H.; Kwon, H-S.; Lee,
J-E.; Kang, D-W.; Joo, J-H. WO 9800402 A1, 1998;
Chem. Abstr. 1999, 128, 114962.
28. Camden, J. B. WO 2001089499 A2, 2001; Chem. Abstr.
2002, 136, 605.
Acknowledgements
The authors wish to express their gratitude to the Uni-
´
versity of Navarra Research Plan (Plan de Investigacion
de la Universidad de Navarra, PIUNA) for its financial
support to the project as well as thank the Department
of Education and Culture of the Navarra Government.
The authors also wish to express their gratitude to the
Ministry of Science and Technology (Spain), FPU Pro-
gram, for the grant awarded.
29. Pirisi, M. A.; Murineddu, G.; Mussinu, J. M.; Pinna, G.
A. Il Farmaco 2002, 57, 331–335.
30. Rana, T. M.; Hwang, S.; Tamilarasu, N. WO 2001025188,
2001; Chem. Abstr. 2001, 134, 311431.
31. Kim, J. Y.; Yoon, B. H.; Hwang, S. K.; Oh, C. M.; Park,
M. S.; Song, K. O.; Oh, S. S. WO 2001095856, 2001;
Chem. Abstr. 2002, 136, 53769.
32. Santora, V.; Askew, B.; Ghose, A.; Hague, A.; Kim, T. S.;
Laber, E.; Li, A.; Lian, B.; Liu, G.; Norman, M. H.;
Smith, L.; Tasker, A.; Tegley, C.; Yang, K.
WO 2002014311 A2, 2002; Chem. Abstr. 2002, 136,
200177.
References and notes
1. McDonnell, T. J.; Meyn, R. E.; Robertson, L. E. Cancer
Biol. 1995, 6, 53–60.
2. Kaufmann, S. H.; Earnshaw, W. C. Exp. Cell Res. 2000,
256, 42–49.
3. Bunz, F. Curr. Opin. Pharmacol. 2001, 1, 337–341.
4. Thompson, C. B. Science 1995, 267, 1456–1462.
5. Golstein, P. Science 1997, 275, 1081–1082.
6. Green, D. R. Cell 1998, 94, 695–698.
33. Gundogus-Ozcanli, N.; Saylir, C.; Criss, W. E. Biochem.
Pharmacol. 1999, 58, 251–254.
34. Faaland, C. A.; Thomas, T. J.; Balabhadrapathruni, S.;
Langer, T.; Mian, S. S.; Shirahata, A.; Gallo, M. A.;
Thomas, T. Biochem. Cell. Biol. 2000, 78, 415–426.
35. Cordi, A.; Desos, P.; Morris, A.D.; Atassi, G. EP 754693
A2, 1997; Chem. Abstr. 1998, 126, 157635.
7. Hengartner, M. O. Nature 2000, 407, 770–776.
8. Reed, J. C. Trends Mol. Med. 2001, 7(7), 314–319.
9. Cappello, P.; Novelli, F.; Forni, G.; Giovarelly, M.
J. Immunother. 2002, 25(1), 1–15.
10. Green, D. R.; Evan, G. I. Cancer Cell 2002, 1, 19–30.
11. Debatin, K. Toxicol. Lett. 2000, 112, 41–48.
12. Nicholson, D. W. Nature 2000, 407, 810–816.
13. Huang, P.; Oliff, A. Trends Cell Biol. 2001, 11(8), 343–348.
14. Evan, G.; Vousden, K. H. Nature 2001, 411, 342–348.
15. Steller, H. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5421–
5422.
36. Tuse, D.; Chen, X.; Laderoute, K. R.; Hiebert, C. K.;
Olsen, C. M. WO 98442328 A1, 1998; Chem. Abstr. 1999,
129, 254986.
´
37. Campos, J. M.; Nu´n˜ez, M. C.; Rodrıguez, V.; Gallo, M.
A.; Espinosa, A. Bioorg. Med. Chem. Lett. 2002, 10, 767–
770.
´
´
´
38. Conejo-Garcıa, A.; Campos, J. M.; Sanchez Martın, R.
M.; Gallo, M. A.; Espinosa, A. J. Med. Chem. 2003,
46(17), 3754–3757.
39. Jia, G.; Lown, J. W. Bioorg. Med. Chem. 2000, 8(7), 1607–
1617.
´
´
16. Campos, J. M.; Nu´n˜ez, M. C.; Sanchez, R. M.; Gomez-
40. Burns, M. R.; LaTurner, S.; Ziemer, J.; McVean, M.;
Devens, B.; Carlson, C. L.; Graminski, S. M.; Vanderwerf,
R. S.; Carreon, W.; Carreon, J. Bioorg. Med. Chem. Lett.
2002, 12, 1263–1267.
´
´
´
Vidal, J. A.; Rodrıguez-Gonzalez, A.; Ban˜ez, M.; Gallo,
M. A.; Lacal, J. C.; Espinosa, A. Bioorg. Med. Chem.
2002, 10, 2215–2231.
17. MX-76629. Drug Data Rep. 2002, 24, 382.
18. Okazaki, T.; Fukazawa, N. JP 2000072749 A2, 2000;
Chem. Abstr. 2000, 132, 189659.
19. Montgomery, G. J.; McKeown, P.; McGown, A. T.;
Robins, D. J. Anti-Cancer Drug Des. 2000, 15, 171–181.
20. Wardman, P.; Folkes, L. K.; Dachs, G. U.; Rossiter, S.;
Greco, O. WO 2002002110 A1 2002; Chem. Abstr. 2002,
136, 102235.
41. Fukuda, Y.; Oomori, Y.; Ko, H.; Terajima, A. JP
08151379 A2, 1996; Chem. Abstr. 1996, 125, 195630.
42. 272464–272466. Drug Data Rep. 1999, 21, 274.
43. Johnstone, R. A. W.; Rose, M. E. Tetrahedron 1979, 35,
2169–2173.
44. Borch, R. J. Am. Chem. Soc. 1971, 93, 2897–2904.
45. Davies, A. G.; Puddephatt, R. J. J. Chem. Soc. (C) 1968,
1749.

´
C. Sanmartın et al. / Bioorg. Med. Chem. 13 (2005) 2031–2044
2044
´
Fernandez-Alvarez, E. Eur. J. Med. Chem. 1989, 24,
´
´
´
209–216.
46. Font, M.; Sanmartın, C.; Alonso, M. L.; Gracıa, L.; Losa,
M. J.; Marquiegui, B.; Merino, I.; Nadal, E.; Ruiz, I.;
Monge, A.; Bengoechea, M. T.; Cabodevilla, F.; Elena, S.;
48. Lowik, C. W.; Alblas, M. J.; Van de Ruit, M.; Papapo-
ulos, S. E.; Van der Pluijm, G. Anal. Biochem. 1993, 213,
426–433.
´
Martınez-Irujo, J. J.; Odriozola, L.; Pen˜uelas, I.; Santiago,
E.; Homa, F.; Wathen, W. Drug Des. Discovery 2000, 16,
295–315.
47. Monge, A.; Martınez-Merino, V.; Sanmartın, C.; Fernan-
49. Duke, R. C.; Ojcms, D. M.; Young, J. D. Sci. Am. 1996,
275(6), 80–87.
50. Peitsch, M. C. Trends Cell Biol. 1994, 4, 37–41.
´
dez, F. J.; Ochoa, M. C.; Bellver, C.; Artigas, P.;
´
´