A new psoralen derivative with enlarged antiproliferative properties

Article abstract of DOI:10.1016/j.bmcl.2009.03.073

Following our results with benzopsoralens as potent photochemotherapeutic agents, we report the antiproliferative evaluation of nitrogenated isoster upon and without UVA irradiation. The evaluated pyridazinopsoralen showed a higher photochemotherapeutic a

Full text of DOI:10.1016/j.bmcl.2009.03.073

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2874–2876
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Bioorganic & Medicinal Chemistry Letters
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A new psoralen derivative with enlarged antiproliferative properties
Lisa Dalla Via a, Jose Carlos González-Gómez b, , Lázaro Guillermo Pérez-Montoto b, Lourdes Santana b,
Eugenio Uriarte b, Sebastiano Marciani Magno a, Ornella Gia a,
*
a Department of Pharmaceutical Sciences, School of Pharmacy, University of Padova, Via Marzolo 5, 35131 Padova, Italy
b Department of Organic Chemistry, Universidad de Santiago de Compostela, 15782, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Following our results with benzopsoralens as potent photochemotherapeutic agents, we report the anti-
proliferative evaluation of nitrogenated isoster upon and without UVA irradiation. The evaluated pyridaz-
inopsoralen showed a higher photochemotherapeutic activity with respect to the well-known drug, 8-
MOP, and a significant cytotoxicity, also in the dark. This result enlarges the interest in this tetracyclic
psoralen derivative skeleton in the search of new anticancer agents.
Received 28 January 2009
Revised 13 March 2009
Accepted 19 March 2009
Available online 24 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Pyridazinopsoralen
Antiproliferative activity
Photobiology
Furocoumarins are a class of compounds of high pharmaco-
logical interest due to their capacity to link covalently to DNA
upon irradiation with long-wavelength UV light (365 nm). This
photoreactivity is generally greatest for linear furocoumarins
(psoralens), among which 8-methoxypsoralen (8-MOP) is cur-
rently the most common reference drug for PUVA (Psora-
len + UVA) therapy of various skin diseases.1–4 Nevertheless,
some serious side-effects both short-term (erythema, hyperpig-
mentation) and long-term (premalignant keratoses, skin cancers)
have been detected.5–7 We have previously shown that widening
the tricyclic psoralen nucleus, by condensing a cyclohexane or
benzene to 40,50-furan-side double bond, and inserting a dimeth-
ylaminopropoxy side chain in position 5 or 8 of the psoralen
moiety with a view to increase the low aqueous solubility, very
interesting results are produced.8 In particular, all tetracyclic
derivatives showed a significant decrease of skin phototoxicity,
and the disappearance of this undesired side effect was obtained
with benzopsoralens. Moreover, the photoantiproliferative activ-
ity resulted considerably high for benzopsoralen carrying the
protonable side chain in position 8 which actually appeared
100 times more efficient in inhibiting HeLa growth, than 8-
MOP. These results stimulated further studies; in fact, tetracyclic
congeners with a cyclohexane or benzene ring condensed at 3,4-
derivative, the disappearance of skin phototoxicity was con-
firmed, but it showed a significantly lower photoantiproliferative
ability on HeLa cells with respect to the furan-side analogue.8,9
Furthermore, the comparison of the photobiological properties
of a series of tetracyclic psoralens, carrying a cyclopentane,
cyclohexane or benzene ring, evidenced that the intensity of skin
phototoxicity strongly depends on the type of the condensed
ring.10 In particular, the planar six membered aromatic nucleus
appears to be the most suitable.
According to these previous results, we designed and synthes-
ised a series of tetracyclic psoralens carrying a fourth pyridazino
ring fused at the level of the 40,50 photoreactive double bond which
should ensure the weakening of skin phototoxicity.11,12 Moreover,
on the basis of the crucial role played in the photoantiproliferative
effect, a dimethylaminopropoxy side-chain was inserted in posi-
tion 8 of the new tetracyclic chromophore.
We describe here the synthesis of a new pyridazino[4,3-h]psor-
alen and the biological activity evaluation.
Compound 3 was prepared from commercially available 8-MOP
as follows (Scheme 1). Pyridazinopsoralen 1 was obtained in an
excellent 98% yield from 8-MOP and 3,6-dichlorotetrazine, in a
Diels–Alder reaction followed by intramolecular cyclization.13 This
procedure was successfully used in a 2-g scale and the compound
crystallized with high purity from the reaction mixture.14,15
Hydrolysis of compound 1 to the corresponding pyridazinone,
followed by triflate preparation and palladium catalyzed hydrog-
pyrone-side double bond, and
a dimethylaminopropoxy side
chain in position 8, were prepared and studied.9 For the benzo
enolysis under transfer conditions, afforded compound
2 in
* Corresponding author. Tel.: +39 0498275710; fax: +39 0498275366.
E-mail address: ornellamaria.gia@unipd.it (O. Gia).
Present address: Department of Organic Chemistry, Universidad de Alicante,
Apdo. 99, 03080, Spain.
about 65% yield over three steps.13 Hydrolysis of methoxy group
of compound 2, using AlCl3/CH2Cl2 at room temperature, was
followed by alkylation under Williamson conditions, with 3-
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.073

L. Dalla Via et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2874–2876
2875
Cl
Cl
N
N
N
N
a
N
N
N
N
b, c, d
+
O
O
O
O
O
O
O
O
O
OMe
OMe
OMe
Cl
8-MOP
1
2
e, f
N
N
O
O
O
NMe2
O
3
Scheme 1. Reagents and conditions: (a) CH2Cl2, 140 °C, then i-Pr2NEt; (b) 20:1 AcOH/H2O, reflux; (c) Tf2O, Py, rt; (d) HCO2H, cat. Pd(OAc)2/dppf, i-Pr2NEt, DMF; (e) AlCl3,
CH2Cl2, then H2O; (f) Cl(CH2)3NMe2, NaH, DMF.
chloro-N,N-dimethyl-propylamine hydrochloride, to afford target
compound 3 in 40% yield over two steps.16
The antiproliferative activity of compound 3 was evaluated by
means of a growth inhibition assay on HeLa, human cervix adeno-
carcinoma, cell line and expressed as IC50 values, that is, the con-
derivatives8,9 an antiproliferative effect in the dark was also ob-
served; nevertheless, for the pyridazinopsoralen 3 the difference
between photobiological and biological activity decreases. In this
connection, it is well known that in ground state, psoralens and
psoralen analogues are able to form a molecular complex with
DNA through an intercalative mode of binding. Figure 1 shows
centration (lM) of compound able to induce 50% of cell death
with respect to the control culture. Experiments were performed
both in the absence and in the presence of UVA light (365 nm,
0.793 J cmꢀ2) following previously established procedures.17 The
well-known photochemotherapeutic drug 8-MOP was taken as ref-
erence compound and the results are reported in Table 1.
Table 1
HeLa cell growth inhibition and skin phototoxicity in guinea pigs in the presence of 3
and 8-MOP as reference drug
After exposure to UVA light, compound 3 showed a notable
capacity to inhibit cell growth. Indeed, the measured IC50 value is
significantly lower than that obtained for the reference drug. More-
over, the new pyridazinopsoralen showed a cytotoxic activity even
in the dark, and this latter effect is about four times lower with re-
spect to that exerted upon UVA irradiation. For previous tetracyclic
Compd
IC50
(
l
M)
Formation of erythema
Dark
UVA
Intensitya
l
mol cmꢀ2
3
13.5 1.7
>20
2.9 0.5
10
+ꢀ
0.046
0.046
8-MOP
3
++
a
Symbols: ++, erythema with edema; +ꢀ, mild.
0.005
0.000
a
b
-0.005
-0.010
-0.015
x 5
-0.020
a
-0.025
b
-0.030
250
300
350
400
450
wavelength (nm)
Figure 1. Linear flow dichroism spectra for compound 3 at [drug]/[DNA] ratios: a = 0, b = 0.04; [DNA] = 1.8 ꢁ 10ꢀ3 M.

2876
L. Dalla Via et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2874–2876
the spectra of DNA alone (line a) and in the presence of 3 (line b) at
[drug]/[DNA] = 0.04, obtained from flow dichroism experiments
performed as previously reported.17 The occurrence of a negative
dichroic signal at wavelengths at which the pyridazinopsoralen
chromophore absorbs (300–380 nm), also confirmed for derivative
3 the ability to intercalate between base pairs of the macromole-
cule and this property could account for the occurrence of the
UVA-independent cytotoxicity.
Skin phototoxicity was evaluated for compound 3 on depilated
skin in guinea pigs in comparison with 8-MOP (Table 1).17 Interest-
ingly, the pyridazinopsoralen derivative is significantly less photo-
toxic than the reference drug and this property acquires more
significance thanks to the evidenced difference in photocytotoxic-
ity between the two compounds.
Acknowledgments
Thanks the Spanish Ministry of Sanidad y Consumo (PI061457)
and Xunta de Galicia (PXIB20304PR) for partial financial support.
References and notes
1. Santana, L.; Uriarte, E.; Roleira, F.; Milhazes, N.; Borges, F. Curr. Med. Chem.
2004, 11, 3239.
2. Pathak, M. A.; Fitzpatrick, T. B. J. Photochem. Photobiol. B 1992, 14, 3.
3. Averbeck, D. Photochem. Photobiol. 1989, 50, 859.
4. Lowes, M. A.; Bowcock, A. M.; Krueger, J. G. Nature 2007, 445, 866.
5. Stern, R. S. J. Am. Acad. Dermatol. 2001, 44, 755.
6. Morison, W. L. Photodermatol. Photoimmunol. Photomed. 2004, 20, 315.
7. Stern, R. S. N. Engl. J. Med. 2007, 357, 682.
8. Dalla Via, L.; Gia, O.; Marciani Magno, S.; Santana, L.; Teijeira, M.; Uriarte, E. J.
Med. Chem. 1999, 42, 4405.
In conclusion, the new pyridazinopsoralen derivative 3 ap-
pears to be characterized by some peculiar biological properties.
Indeed, 3 is endowed with an interesting photobiological profile
deriving from both a significant photoantiproliferative activity
and a weak skin phototoxicity. Moreover, a significant cytotoxic
effect in the dark occurs. This capacity was also exhibited by the
benzo analogue,8 nevertheless the difference in the cytotoxicity
in the presence of UVA light and in the dark is less for 3. This
behavior can be attributed to an efficient DNA binding process
mediated by the pyridazine ring. This latter, thanks to the ten-
dency to protonation of its nitrogens,18,19 with respect to ben-
zene ring, can make the intercalative capacity between DNA
base pairs more suitable for the antiproliferative activity in the
dark.
A further contribution in DNA interaction ability can derive
from the insertion of the dimethylaminopropoxy side-chain. In-
deed, a cationic side-chain linked to a planar structure, can gener-
ate additional electrostatic interactions with the polyanionic DNA
target, hence favouring the binding process.20 Both the pyridazine
ring and the protonable side-chain can account for the significant
shift of the psoralen derivative 3 toward a dark antiproliferative
activity and actually indicate the possibility to modulate the
photo-dependent and -independent cellular effect by just modify-
ing suitably the intercalative arrangement of the tetracyclic psora-
len skeleton.
9. Dalla Via, L.; Uriarte, E.; Quezada, E.; Dolmella, A.; Ferlin, M. G.; Gia, O. J. Med.
Chem. 2003, 46, 3800.
10. Dalla Via, L.; Uriarte, E.; Santana, L.; Marciani Magno, S.; Gia, O. Arkivoc 2004, V,
131.
11. González-Gómez, J. C.; Santana, L.; Uriarte, E. Synthesis 2002, 43.
12. González-Gómez, J. C.; Santana, L.; Uriarte, E. Tetrahedron 2003, 59, 8171.
13. González-Gómez, J. C.; Uriarte, E. Synlett 2003, 2225.
14. For leading references in Diels–Alder reaction of s-tetrazines see: (a) Lee, L.;
Snyder, J. K.. In Advances in Cycloadditions; Harmata, M., Ed.; JAI: Stamford, CT,
2001; Vol. 6, pp 119–171; (b) Boger, D. L. J. Heterocycl. Chem. 1998, 35, 1001; (c)
Boger, D. L. J. Heterocycl. Chem. 1996, 33, 1519; (d) Boger, D. L.; Weinreb, S. M. In
Hetero Diels–Alder Methodology in Organic Synthesis; Wasserman, H. H., Ed.;
Academic: New York, 1987; Vol. 47, (e) Boger, D. L. Chem. Rev. 1986, 86, 781.
15. (a) For the preparation of 3,6-dichlorotetrazine, see: Schirmer, U.; Wuerzer, B.;
Meyer, N.; Neugebauer, F. A.; Fisher, H. Ger. Patent 35-08214-A1, 1986; Chem.
Abstr. 1987, 106, 45718; (b) In our hands 3,6-dichlorotetrazine was obtained in
68% yield (1.02 g) from 3,6-bisthiomethyltetrazine (1.74 g).
16. 6-(N,N-Dimethylaminopropyloxy)pyridazino[4,3-h]psoralen (3) 1H NMR
(CDCl3): 9.31 (d, J = 5.0, 1H), 8.04 (d, J = 5.0, 1H), 7.86 (s, 1H), 7.83 (d, J = 9.6,
1H), 6.50 (d, J = 9.6), 4.70 (t, J = 6.3, 2H), 2.67 (m, 2H), 2.32 (s, 6H), 2.12 (m, 2H).
13C NMR (CDCl3): 166.2, 158.9, 148.3, 147.6 (CH), 143.0 (CH), 132.7, 120.8, 119.2
(CH), 118.1, 117.3, 116.1 (CH), 115.6 (CH), 72.8 (CþH2), 55.9 (CH2), 45.5 (CH3), 28.2
(CH2). HRMS (EI) found 339.1229, C18H17ClN3O4 requires 339.1219.
17. Dalla Via, L.; Mammi, S.; Uriarte, E.; Santana, L.; Lampronti, I.; Gambari, R.; Gia,
O. J. Med. Chem. 2006, 49, 4317.
18. Adams, A. Med. Chem. Rev. 2004, 1, 405.
19. Neidle, S. Nucleic Acid Structure and Recognition; Oxford University Press:
Oxford, 2002. 100.
20. Pons, M.; Campayo, L.; Martinez-Balbas, M. A.; Azorin, F.; Navarro, P.; Giralt, E.
J. Med. Chem. 1991, 34, 82.