Anticancer activity of (1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-pyrimidines and -purines against the MCF-7 cell line: Preliminary cDNA microarray studies

Article abstract of DOI:10.1016/j.bmcl.2007.12.070

Completing a SAR study, a series of (RS)-1- or 3-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-pyrimidines and (RS)-6-substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7H- or 9H-purines have been prepared. Their antiproliferative activities on MCF-7 cells are here presented and discussed. (RS)-6-Chloro-9-[1-(9H-9-fluorenylmethoxycarbonyl)-1,2,3,5-tetrahydro-4, 1-benzoxazepine-3-yl]-9H-purine (28) is the most active (IC₅₀ = 0.67 ± 0.18 μM) of the series so far described. cDNA microarray technology reveals potential drug targets, which are mainly centred on apoptosis regulatory pathway genes.

Full text of DOI:10.1016/j.bmcl.2007.12.070

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1457–1460
Anticancer activity of (1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-
pyrimidines and -purines against the MCF-7 cell line:
Preliminary cDNA microarray studies
a
a
b,c
´
´
´
´
´
´
Monica Dıaz-Gavilan, Jose A. Gomez-Vidal, Fernando Rodrıguez-Serrano,
Juan A. Marchal,c,d Octavio Caba,b,c Antonia Aranega, Miguel A. Gallo,a
c,d
´
a
a,
*
´
Antonio Espinosa and Joaquın M. Campos
aDepartamento de Quı´mica Farmace´utica y Orga´nica, Facultad de Farmacia, c/Campus de Cartuja s/n, 18071 Granada, Spain
bDepartamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Paraje de Las Lagunillas s/n, 23071 Jae´n, Spain
cInstituto de Biopatologı´a y Medicina Regenerativa (IBIMER), Facultad de Medicina, Avenida de Madrid s/n, 18071 Granada, Spain
dDepartamento de Anatomı´a y Embriologı´a Humana, Facultad de Medicina, Avenida de Madrid s/n, 18071 Granada, Spain
Received 7 December 2007; revised 22 December 2007; accepted 27 December 2007
Available online 1 January 2008
Abstract—Completing a SAR study, a series of (RS)-1- or 3-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-pyrimidines and (RS)-6-
substituted-7- or 9-(1,2,3,5-tetrahydro-4,1-benzoxazepine-3-yl)-7H- or 9H-purines have been prepared. Their antiproliferative
activities on MCF-7 cells are here presented and discussed. (RS)-6-Chloro-9-[1-(9H-9-fluorenylmethoxycarbonyl)-1,2,3,5-tetrahy-
dro-4,1-benzoxazepine-3-yl]-9H-purine (28) is the most active (IC50 = 0.67 0.18 lM) of the series so far described. cDNA
microarray technology reveals potential drug targets, which are mainly centred on apoptosis regulatory pathway genes.
Ó 2008 Elsevier Ltd. All rights reserved.
Advance in the knowledge of molecular biology has
thrown light on many aspects of the cell-cycle regulation
mechanisms. This has allowed a change in the anticancer
therapy trends, from the classic cytotoxic strategies to
new non-harmful therapies which target the cell-cycle
regulation. One of them is the differentiation therapy,
searching for the survival of the cells once the cancerous
phenotype is eliminated.1 Despite these advances, the
successful treatment of cancer still remains a significant
challenge. We have previously reported the synthesis
and biological activity of a series of O,N-acetals with
antitumoural activity, which included seven-membered
benzoxaheterocyclic moieties and different nitrogen
bases. To complete a structure–activity relationship
study, we have prepared the bioisosteric compounds
bearing the heteroatoms O, S or N in the cyclic seven-
membered moiety and linked them to natural or unnat-
ural pyrimidines, or purines.2–8,10 Between them, the
structures 1–126,8 (Fig. 1) can be highlighted due to their
notable antitumoural activity (IC50 = 1.25–6.75 lM on
MCF-7 cells), that can be compared to that of drugs
currently used in therapy, such as the cytotoxic agent
5-fluorouracil (5-FU, IC50 = 4.32 0.02 lM, in vitro
studies on MCF-7 human breast cancer cells8).
Recently, some molecular aspects of the biological
mechanism of these molecules have been reported.9
These results hint at a completely different mechanism
with respect to 5-FU, based on cell-cycle arrest at the
phases G0/G1 and G2/M versus the S-phase cell-cycle ar-
rest produced by 5-FU. Moreover, they induce apopto-
sis. This makes these molecules potential cell-
differentiating agents and they become excellent proto-
types for optimization studies. For compound 2, this ef-
fect is based on the decrease of the expression of cyclin
D1 and the activity of Cdk1, and also produces an incre-
ment of the activity of CDKIs such as p21 and p27. The
apoptotic activity has been found to be due to a p53
independent apoptotic DNA-fragmentation.
Keywords: Benzoxazepines; MCF-7 cell line; Microarrays; Purines;
Pyrimidines.
*
Herein we would like to report the antiproliferative
activity found for those bioisosteres containing a
Corresponding author. Tel.: +34 958243848; fax: +34 958243845;
e-mail: jmcampos@ugr.es
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.070

´ ´
M. Dıaz-Gavilan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1457–1460
1458
X
X
R2
N
N
N
X
N
O
R3
O
N
O
O
N
N
NH
O
N
R1
R3
N
R4
R4
1. X = O, R1 = OCH3, R2 = F
2. X = O, R1 = R2 = H
3. X = O, R3 = Cl, R4 = H
4. X = O, R3 = I, R4 = H
5. X = O, R3 = R4 = Cl
6. X = S, R3 = Cl, R4 = H
7. X = O, R3 = Cl, R4 = H
8. X = O, R3 = I, R4 = H
9. X = O, R3 = R4 = Cl
10. X = O, R3 = CF3, R4 = H
11. X = S, R3 = Cl, R4 = H
12. X = SO2, R3 = Cl, R4 = H
Figure 1. O,N-Acetals with antitumoural activities against MCF-7 cells.
4,1-benzoxazepine N-alkylated pyrimidine7 (13–25,
Fig. 2) or purine10 (26–38, Fig. 3).
substitution on N7-alkylated purines versus the N9-
alkylated has been previously observed.10
The structures 28, 32 and 35 were prepared from the
condensation of O,O-acetal 39 with 6-chloropurine (6-
CP), following the reported procedure10 (Scheme 1).
The rate of transformation of the starting O,O-acetal
is very similar to that found for R1 = nitrobenzenesulfo-
nyl (Ns)10 with a product yield near to 45%. The alkyl-
ation of 6-CP seems to occur to a larger extent on its N7
versus its N9 position, as the structure 35 could be
formed from the 6-chloroderivative 32. This trend was
also found when the nitrobenzenesulfonyl O,O-acetals
were condensed with 6-CP.10 The more facile chlorine
The N7- or N9-binding of the purine ring in 28 and 32 was
established from a comparison study of the H NMR
1
spectra of N9- and N7-isomers11,12: (i) H3b and H2b ap-
pear more unshielded in N7-isomers than in N9. Both
hydrogen atoms are more deshielded in 32. (ii) H2a ap-
pears at slightly lower fields in N9- than in N7-isomers.
In 28/32, the difference in chemical shift for this hydrogen
is 0.16 ppm and is more unshielded in 28. (iii) In every
case, H800 is more deshielded than H200. Both hydrogen
atoms appear at lower fields in N7- than in N9-isomers.
Compound 32 presents more unshielded signals for H800
and H200 than 28. (iv) The most shielded of the two ben-
zylic hydrogen atoms H5 is more shielded in N7- than in
N9-isomers. This hydrogen is also more shielded in 32
R1
N
R1
N
O
N
O
N
O
NH
NH
1
O
than in 28. To summarise, the H NMR spectrum of 28
O
O
is more similar to that of the remainder of the N9-deriva-
tives, while the 1H NMR spectrum of 32 is more similar to
those of the N7-isomers. All new compounds gave satis-
R2
R2
13 R1 = H, R2 = F
20 R1 = H, R2 = F
1
21 R1 = SO2-C6H4-pNO2, R2 = F
22 R1 = SO2-C6H4-oNO2, R2 = F
23 R1 = SO2-C6H4-oNH2, R2 = F
24 R1 = SO2-C6H4-pNO2, R2 = H
25 R1 = SO2-C6H4-oNO2, R2 = H
factory H NMR, 13C NMR, HR LSIMS and combus-
14 R1 = SO2-C6H4-pNO2, R2 = F
15 R1 = SO2-C6H4-pNH2, R2 = F
16 R1 = SO2-C6H4-oNO2, R2 = F
17 R1 = SO2-C6H4-oNH2, R2 = F
18 R1 = SO2-C6H4-pNO2, R2 = H
19 R1 = SO2-C6H4-oNO2, R2 = H
tion analyses.
Tables 1 and 2 show the antiproliferative activity (IC50
values) on MCF-7 human breast cancer cells found for
the pyrimidine and purine derivatives 13–38.13
NO2
SO2-C6H4-(o,p)NO2
The most potent molecules are the purine derivatives.
Compounds 28, 30, 32 and 37 present IC50 values below
1 lM. We selected 28 and 37 to identify the molecular
key targets of its anticancer activity. Completion of
the human genome sequence and the advent of DNA
microarrays using cDNAs enhanced the detection and
identification of hundreds of differentially expressed
genes in response to anticancer drugs. In this way we ob-
tained gene-expression patterns of treated human breast
cancer cells in comparison with parental MCF-7 cells.
For this purpose, we analysed the expression of about
22,000 different human genes using the Agilent 60-mer
oligo microarray platform and the Human 1A Oligo
Microarray Kit (V2) (Agilent Technologies, CA, USA).
SO2
Figure 2. New benzoxazepine O,N-acetals containing pyrimidine rings.
R1
N
R1
N
N
N
N
N
O
O
R3
N
N
R3
N
N
26 R1 = SO2-C6H4-pNO2, R3 = Cl
27 R1 = SO2-C6H4-oNO2, R3 = Cl
28 R1 = Fmoc, R3 = Cl
29 R1 = SO2-C6H4-pNO2, R3 = Cl
30 R1 = SO2-C6H4-oNO2, R3 = Cl
31 R1 = SO2-C6H4-oNH2, R3 = Cl
32 R1 = Fmoc, R3 = Cl
33 R1 = SO2-C6H4-pNO2, R3 = -OH
34 R1 = SO2-C6H4-oNO2, R3 = OH
35 R1 = Fmoc, R3 = OH
36 R1 = H, R3 = SPh
37 R1 = SO2-C6H4-pNO2, R3 = SPh
The up-regulated and the down-regulated genes include
genes that encode for different metabolic pathways, cel-
lular development process, signal molecules, response to
stress, regulation of the cell cycle and apoptosis, etc.
Analysis of the mRNAs, which are deregulated (up-reg-
ulated or down-regulated) at least 2-fold in treated cells,
38 R1 = SO2-C6H4-oNO2, R3 = SPh
Figure 3. New benzoxazepine O,N-acetals containing purine rings.

´ ´
M. Dıaz-Gavilan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1457–1460
1459
O
O
O
O
O
O
O
O
OMe
SnCl4,
HMDS, TCS
MeCN
Cl
N
N
N
N
N
N
N
N
N
N
+
+
+
N
OMe
R3
O
O
O
N
H
R3
N
N
N
N
OH
R3
N
N
39
6-CP
28 R3 = Cl (12%)
32 R3 = Cl (18%)
35 R3 = OH (10%)
40 (6%)
Scheme 1. Condensation between the O,O-acetal 39 and 6-chloropurine (6-CP). Reagents and conditions: 39 (1.0 equiv), 6-CP (1.2 equiv), HMDS
(1.2 equiv), TMSCl (1.2 equiv), SnCl4 (1.5 equiv), anhydrous MeCN, 50 °C, 48 h.
Table 1. Antiproliferative activities against the MCF-7 cells for N1-
(13–19) and N3- (20–25) pyrimidines
member of the Rho family of the GTPase key down-
stream target in Ras signalling.16
a
Compound
IC50 (lM)
Compound
IC50 (lM)
Among the pyrimidine derivatives 13–25, those contain-
ing 5-fluorouracil (R2 = F) show better activities than
those derived from uracil (R2 = H). Bonding of the
pyrimidine ring through N1 or N3 affects the activity
though only slightly, when R1 = H, 13 versus 20.
13
14
15
16
17
18
19
>100
20
21
22
23
24
25
72.40 11.3
19.81 0.08
22.63 0.11
43.70 0.09
44.28 4.65
50.90 3.87
19.33 1.04
14.37 0.69
19.70 0.15
54.82 1.04
39.78 2.60
45.17 0.48
The substitution on C6 of the purine ring is essential for
the activity. Bulky, lipophilic groups afford the best val-
ues of IC50 [R3 = Cl (26–32), -SPh (37–38)] while puri-
none compounds [R3 = OH (33–35)] are comparable to
the pyrimidine analogues in terms of activity. Bonding
of the purine ring through N7 or N9 affects the activity
to a lesser extent, and the positive or negative character
of this effect depends on the nature of R1 (see 26/29
R1 = pNs, 27/30 R1 = oNs, 28/32 R1 = Fmoc).
a The data are means SEM of three independent determinations.
Table 2. Antiproliferative activities against the MCF-7 cells for N9-
(26–28) and N7-purines (29–38)
a
Compound
IC50 (lM)
Compound
IC50 (lM)
26
27
28
29
30
31
32
2.73 0.17
2.10 0.69
0.67 0.18
1.22 0.12
0.92 0.01
9.14 1.24
0.84 0.09
33
34
35
36
37
38
>100
19.66 5.27
53.57 13.1
48.92 9.89
0.86 0.12
2.59 0.57
Both, pyrimidine and purine derivatives, are more po-
tent when R1 is not hydrogen. The lipophilic character
of R1 increases the activity, and no limit of volume
has been observed for the studied groups. The elec-
tron-withdrawing character of R1 could help to increase
the activity.4 Carbonyl derivatives are more potent than
the sulfonyl ones.
a The data are means SEM of three independent determinations.
revealed the following results: 26 genes up-regulated and
59 genes down-regulated in 28-MCF-7 treated cells; and
26 genes up-regulated and 17 genes down-regulated in
37-treated human breast cancer cells. Each compound
revealed a somewhat unique expression pattern together
with the up-regulation of significant genes involved in
different cellular functions and a significant down-regu-
lation of genes for 28. One of the more important results
in the current study was the ability of 28 to modulate the
expression of genes involved in apoptosis or its delay of
mitosis. This effect can be explained by the accumulation
of cells in the G2/M checkpoint of cell cycle, particularly
GP132, the receptor for an unknown ligand, which acti-
vates a G2a protein. This is transcriptionally up-regu-
lated by stress-inducing and cell-damaging agents and
is involved in caspase-mediated apoptosis.14 Similarly,
the ERN1 gene that belongs to the Ser/Thr protein ki-
nase family is a potent unfolded-protein response tran-
scriptional activator and acts by triggering growth
arrest and apoptosis.15 However, 37 induced the
down-regulation of a gene involved in the metastatic
progression of cancer such as RAC1, a Ras-like protein
When R1 = benzensulfonamido, it can be observed for
pyrimidine and purine derivatives, that the ortho-substi-
tution on R1 is preferred to para, in terms of potency,
and the nitro group renders better results than the ami-
no one. As an exception, compound 37 (R1 = pNs,
R3 = SPh) is more potent than 38 (R1 = oNs, R3 = SPh).
New related acyclic acetals 41–51 (Fig. 4) were obtained
as minor products in the condensation reaction between
the O,O-acetals and pyrimidine7 or purine10 bases. Their
antiproliferative activities have also been studied on
MCF-7 human breast cancer cells and the IC50 values
obtained are shown in Table 3.
Acyclic purine O,N-acetals (49–51) show higher potency
than the pyrimidine acyclic derivatives (41–48). The N7-
alkylated purine 50 presented an excellent value of IC50.
In contrast to the cyclic analogues, the presence of an
oNO2 or pNO2 group does not modify the activity of
the N9-isomers (49 and 51).

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M. Dıaz-Gavilan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1457–1460
1460
2. Saniger, E.; Campos, J.; Entrena, A.; Marchal, J. A.;
R1 OMe
R1 OMe
´
Su´arez, I.; Aranega, A.; Choquecillo, D.; Niclos, J.; Gallo,
M. A.; Espinosa, A. Tetrahedron 2003, 59, 5457.
´
N
N
R2
O
N
N
N
O
N
H
3. Saniger, E.; Campos, J.; Entrena, A.; Marchal, J. A.;
N
R3
OH
OH
´
Boulaiz, H.; Aranega, A.; Gallo, M. A.; Espinosa, A.
Tetrahedron 2003, 59, 8017.
N
41 R1 = H, R2 = F
42 R1 = CO-C6H5, R2 = F
49 R1 = SO2-C6H4-
50 R1 = SO2-C6H4-
51 R1 = SO2-C6H4-
p
o
o
NO2, R3 = Cl
NO2, R3 = Cl (
NO2, R3 = Cl
´
4. Dıaz-Gavilan, M.; Rodrıguez-Serrano, F.; Gomez-Vidal,
J. A.; Marchal, J. A.; Aranega, A.; Gallo, M. A.;
´
´
´
N7)
43 R1 = SO2-C6H4-
44 R1 = SO2-C6H4-
45 R1 = SO2-C6H4-
46 R1 = SO2-C6H4-
47 R1 = SO2-C6H4-
48 R1 = SO2-C6H4-
p
o
o
p
p
o
NO2, R2 = F
NO2, R2 = F
NH2, R2 = F
NO2, R2 = H
NO2, R2 = H (
NO2, R2 = H
´
Espinosa, A.; Campos, J. M. Tetrahedron 2004, 60,
11547.
´
5. Nu´n˜ez, M. C.; Entrena, A.; Rodrıguez-Serrano, F.;
N
3)
´
Marchal, J. A.; Aranega, A.; Gallo, M. A.; Espinosa,
A.; Campos, J. M. Tetrahedron 2005, 61, 10363.
Figure 4. New acyclic O,N-acetals containing pyrimidine and purine
moieties.
´
´
´
6. Nu´n˜ez, M. C.; Pavani, M. G.; Dıaz-Gavilan, M.; Rodrı-
guez-Serrano, F.; Gomez-Vidal, J. A.; Marchal, J. A.;
´
´
Aranega, A.; Gallo, M. A.; Espinosa, A.; Campos, J. M.
Tetrahedron 2006, 62, 11724.
Table 3. Antiproliferative activities against the MCF-7 cell line for
acyclic N1- and N3-pyrimidines (40–47) and N9- and N7-purines (48–
50)
´
´
´
7. Dıaz-Gavilan, M.; Gomez-Vidal, J. A.; Entrena, A.;
Gallo, M. A.; Espinosa, A.; Campos, J. M. J. Org. Chem.
2006, 71, 1043.
a
Compound
IC50 (lM)
Compound
IC50 (lM)
´
8. Nu´n˜ez, M. C.; Rodrıguez-Serrano, F.; Marchal, J. A.;
Caba, O.; Aranega, A.; Gallo, M. A.; Espinosa, A.;
41
42
43
44
45
46
35.97 0.40
16.14 0.77
55.22 12.1
90.99 6.06
>100
47
48
49
50
51
55.22 12.1
64.81 0.05
18.70 0.08
3.25 0.23
11.30 1.27
´
Campos, J. M. Tetrahedron 2007, 63, 183.
9. Marchal, J. A.; Nu´n˜ez, M. C.; Su´arez, I.; Dıaz-Gavilan,
´
M.; Gomez-Vidal, J. A.; Boulaiz, H.; Rodrıguez-Serrano,
´
´
F.; Gallo, M. A.; Espinosa, A.; Aranega, A.; Campos, J.
´
´
M. Breast Cancer Res. Treat. 2007, 105, 237.
45.76 2.45
a The data are means SEM of three independent determinations.
´
´
´
10. Dıaz-Gavilan, M.; Choquesillo-Lazarte, D.; Gonzalez-
Perez, J. M.; Gallo, M. A.; Espinosa, A.; Campos, J. M.
´
To summarise, the in vitro anticancer activity on MCF-
7 cells of new 4,1-benzoxazepines containing pyrimidine
or purine rings has been studied. The change to 4,1-ben-
zoxazepine rings has afforded an enhancement of the
antiproliferative activity on MCF-7 cells in relation to
the previous 4,1-benzoxathiepin8 and 1,4-benzodioxe-
pin,2,6,17 with an improvement on the values of IC50
from 1.25 lM (for 5) to 0.67 lM (for 28). Moreover,
the studies by microarray technology showed that the
main molecular targets of some of these compounds
(28 and 37) are pro-apoptotic genes with protein kinase
activity such as GP132, ERN1 or RAC1, which prevent
the metastatic progression. More detailed results of
these key targets will be published in future papers.
Tetrahedron 2007, 63, 5274.
11. For details about the spectroscopic assignment of the N9-
and N7-isomers in alkylated purines, see Ref. 10.
12. There are characteristic differences for each type of isomer
in 13C NMR: (i) The most representative features are the
chemical shifts (d) of the quaternary carbons of the purine
ring: C400 is approximately 10 ppm more deshielded in N7-
isomers than in the N9 ones (dC4N7 = 161 vs
dC4N9 = 151 ppm for our compounds), C500 appears
approximately 9 ppm more deshielded in N9-isomers than
in
the
corresponding
N7
(dC5N9 = 130
vs
dC5N7 = 121 ppm). The differences in chemical shifts of
C400 and C500 are 40 ppm in the N7-isomers and 20 ppm in
the N9 ones. C400 is more deshielded than C200 in N7-
isomers, while the opposite occurs to N9-isomers. C600 is
approximately 7 ppm more deshielded in N9 than in N7-
isomers (dC6N9 = 149 ppm between C200 and C800) versus
dC6N7 = 142 ppm (higher than C200 and C800). (ii) In
agreement with previous reports, C200 and C800 were found
to resonate at lower fields in N7 than in N9-isomers
(0.26 ppm for C200 and 2.44 ppm for C800). The value of
dC2–dC8 is approximately 4 ppm for N7-isomers and
approximately 6 ppm for isomers N9. To summarise,
those carbon atoms of the purine ring that are orientated
towards the benzoxazepine fragment appear more shielded
than those situated towards the outer part of the molecule.
Acknowledgments
We thank the Instituto de Salud Carlos III [Fondo de
´
Investigacion Sanitaria (FIS) Project No. PI070227]
and the Consejerıa de Innovacion, Ciencia y Empresa
´
´
´
of the Junta de Andalucıa (Excellence Research Project
No. 00636) for financial supports.
´
This agrees with previous bibliographical data: Hockova,
D.; Budesinsky, M.; Marek, R.; Marek, J.; Holy, A. Eur.
J. Org. Chem. 1999, 2675; Garner, P.; Ramakanth, S.
J. Org. Chem. 1988, 53, 1294.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.12.070.
13. For the biological methods see Ref. 4.
14. Lin, P.; Ye, R. D. J. Biol. Chem. 2003, 278, 14379.
15. Yoneda, T.; Imaizumi, K.; Oono, K.; Yui, D.; Gomi, F.;
Katayama, T.; Tohyama, M. J. Biol. Chem. 2001, 276,
13935.
References and notes
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awardhane, S. F. Breast Cancer Res. 2005, 7, R965.
´
17. Campos, J. M.; Espinosa, A.; Gallo, M. A.; Gomez-Vidal,
J. A.; Nu´n˜ez, M. C.; Aranega, A.; Marchal, J. A.;
´
1. Gomez-Vidal, J. A.; Campos, J.; Marchal, J. A.; Boulaiz,
H.; Gallo, M. A.; Carrillo, E.; Espinosa, A.; Aranega, A.
´
´
´
Rodrıguez-Serrano, F. Spanish Patent, P200601538, 2006.
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