Photobiological studies of new cyclopentene-psoralens

Article abstract of DOI:10.1016/S0014-827X(98)00079-2

Psoralen analogues bearing a cyclopentane ring fused to either the 4',5' double bond (compound 4) or the 3,4 double bond (compound 7) of the tricyclic furocoumarin structure were prepared. AM1 theoretical calculations performed for these compounds indicated that the electronic properties of their reactive double bonds were very similar to those of psoralen and its derivative 8-methoxypsoralen (8-MOP), though the overall molecular geometries were clearly different, particularly as regards the change in molecular curvature produced by the introduction of the cyclopentane ring. Compound 4 showed a capacity similar to that of 8-MOP to inhibit the growth of human cervix adenocarcinoma cells (HeLa) and to induce mutagenic effects, but it was definitely less phototoxic to skin than 8-MOP. Its ability to photoadd to DNA and to cross-link DNA strands was also demonstrated. Instead, compound 7 was practically devoid of biological activity and no interaction with the macromolecule could be detected. These differences in behaviour between 4 and 7 are probably due to the molecular curvature resulting from the introduction of the cyclopentane ring.

Full text of DOI:10.1016/S0014-827X(98)00079-2

Il Farmaco 53 (1998) 638–644
Photobiological studies of new cyclopentene–psoralensꢀ
Lisa Dalla Via a,*, Ornella Gia a, Giampietro Viola a, Giulio Bertoloni b,
Lourdes Santana c, Eugenio Uriarte c
a Department of Pharmaceutical Sciences, Uni6ersity of Pado6a,
and Centro di Studio sulla Chimica del Farmaco e dei Prodotti Biologicamente Atti6i del CNR (associated with the National Institute for the
Chemistry of Biological Systems), 6ia Marzolo 5, 35131 Padua, Italy
b Institute of Microbiology, Uni6ersity of Pado6a, 6ia A. Gabelli 63, 35100 Padua, Italy
c Department of Organic Chemistry, Uni6ersity of Santiago de Compostela, Santiago de Compostela, Spain
Abstract
Psoralen analogues bearing a cyclopentane ring fused to either the 4%,5% double bond (compound 4) or the 3,4 double bond
(compound 7) of the tricyclic furocoumarin structure were prepared. AM1 theoretical calculations performed for these compounds
indicated that the electronic properties of their reactive double bonds were very similar to those of psoralen and its derivative
8-methoxypsoralen (8-MOP), though the overall molecular geometries were clearly different, particularly as regards the change in
molecular curvature produced by the introduction of the cyclopentane ring. Compound 4 showed a capacity similar to that of
8-MOP to inhibit the growth of human cervix adenocarcinoma cells (HeLa) and to induce mutagenic effects, but it was definitely
less phototoxic to skin than 8-MOP. Its ability to photoadd to DNA and to cross-link DNA strands was also demonstrated.
Instead, compound 7 was practically devoid of biological activity and no interaction with the macromolecule could be detected.
These differences in behaviour between 4 and 7 are probably due to the molecular curvature resulting from the introduction of
the cyclopentane ring. © 1998 Elsevier Science S.A. All rights reserved.
Keywords: Synthesis; Cyclopentene–psoralens; Photobiological properties; Mutagenesis; Phototoxicity
1. Introduction
both strands of the macromolecule [3,4]. Although both
mono- and bifunctional photoaddition can inhibit the
synthesis of cellular DNA, the compounds which cause
bifunctional damage to the macromolecule are usually
more mutagenic and provoke skin phototoxicity [5–7].
For this reason, several research groups have been
working to develop analogue psoralen derivatives that
retain high affinity towards the macromolecule while
giving only monoaddition. This result may be reached
by deactivating one of the two photoreactive sites,
either changing electronic properties or by means of
steric hindrance [8,9]. In the present work we synthe-
sized two new analogues bearing a cyclopentane ring
fused to either the 4%,5% double bond (compound 4) or
the 3,4 double bond (compound 7) of the tricyclic
furocoumarin structure, aiming at selective prevention
of the photoaddition of the affected double bond with-
out altering the planarity of the molecule. The effects of
these modifications on the photobiological activity of 4
and 7 were also evaluated.
Psoralens are a group of natural or synthetic linear
furocoumarins with interesting photobiological proper-
ties. Some of them are used clinically in psoralen plus
ultraviolet-A radiation therapy (PUVA) for hyperpro-
liferative skin diseases [1] and for extracorporeal treat-
ment of cutaneous T-cell lymphoma (photopheresis) [2].
The biological activity of psoralens is mainly due to
their ability to photoreact with DNA. Upon irradiation
with UVA light, photoaddition of the 4%,5% double bond
of their furane ring and/or of the 3,4 double bond of
their pyrone ring with the 5,6 double bond of pyrim-
idine bases (usually thymine) occurs, leading to the
formation of mono- or di-adducts, the latter involving
ꢀ Dedicated to Professor Antonio Maccioni.
* Corresponding author. Tel.: +39-049-8275712; fax: +39-049-
8275366.
E-mail address: dallavia@dsfarm.unipd.it (L. Dalla Via)
0014-827X/98/$ - see front matter © 1998 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(98)00079-2

L. Dalla Via et al. / Il Farmaco 53 (1998) 638–644
639
2. Chemistry
discs) were recorded on a Perkin–Elmer 1640 FT spec-
1
trometer ( in cm−1). H NMR spectra were recorded
Compounds 4 and 7 were prepared from resorcinol
(1) in parallel sequences, each comprising three steps
(Scheme 1). The first step was Pechmann reaction of 1
with ethyl acetoacetate or ethyl 2-oxocyclopentanocar-
boxylate in sulfuric acid, which gave hydroxycoumarins
2 (also available commercially from Aldrich) and 5 in
44 and 70% yields, respectively. Next, the Williamson
reaction of 2 with 2-chlorocyclopentanone, and the
corresponding reaction of 5 with chloroacetone, gave
ketoethers 3 and 6 in 58 and 97% yield, respectively.
Compounds 3 and 6 were cyclized by heating in a
strongly alkaline solution, affording cyclopentene–
on a Bru¨ker WM (250 MHz) spectrometer using TMS
as the internal standard (chemical shifts in l values and
J in Hz). Elemental analyses were determined by a
Perkin–Elmer 240B microanalyser; all were within 9
0.4% of calculated values. Flash chromatography (FC)
was performed on silica gel (Merck 60, 230–4000 mesh)
and analytical thin-layer chromatography (TLC) on
precoated silica gel plates (Merck 60 F254, 0.25 mm).
3.2. 4-Methyl-7-(2%-oxocyclopentyloxy)coumarin (3)
To a solution of 7-hydroxycoumarin 2 (5 g, 28.41
mmol) in anhydrous acetone (30 ml) 2-chlorocyclopen-
tanone (6.50 g, 55.10 mmol) and potassium carbonate
(9.5 g) were added and the mixture was heated at reflux
for 20 h. The precipitate was filtered out, the acetone
was evaporated under vacuum, and the solid residue
was purified by FC using 3:2 toluene–ethyl acetate as
the eluent. Compound 3 (4.2 g, 58%): m.p. 141–143°C.
1H NMR (CDCl3): 7.50 (d, 1H, H5, J=8.72), 6.92 (m,
2H, H8+H6), 6.16 (d, 1H, H3, J=1.12), 4.69 (dd, 1H,
CH–O, J=8.90 and 7.75), 2.55 (m, 1H, HCH–CO),
2.42 (m, 2H, HCH–CO+HCH–CH–O), 2.40 (d, 3H,
CH3, J=1.12), 2.25–1.95 (m, 3H, HCH–CH–O+
CH2–CH2–CO). IR: 3073, 2975, 2944, 2883, 1752,
1712, 1608, 1557, 1269, 1206, 1157, 1139, 848. Anal.
(C15H14O4) C, H.
furocoumarins
respectively.
4 and 7 in 43 and 66% yields,
3. Experimental
3.1. Chemistry
Melting points were determined on a Koffler hot-
stage microscope and are uncorrected. IR spectra (KBr
3.3. 4-Methylcyclopentenefuro[3,2-g]coumarin (4)
A mixture of coumarin 3 (1.5 g, 5.81 mmol) and 0.1
N NaOH (500 ml) was heated at reflux for 24 h. It was
then acidified with 1 N HCl, and the precipitating solid
was filtered out, washed with water and purified by FC
using 9:1 hexane–ethyl acetate as the eluent. Com-
pound 4 (600 mg, 43%): m.p. 222°C. 1H NMR
(CDCl3): 7.55 (s, 1H, H5), 7.38 (s, 1H, H8), 6.25 (d, 1H,
H3, J=1.10), 2.89, 2.77 and 2.61 (3m, 3×2H, (CH2)3),
2.49 (d, 3H, CH3, J=1.10). IR: 3067, 2930, 2861, 1701,
1639, 1568, 1445, 1126. Anal. (C15H12O3) C, H.
3.4. 7-Hydroxy-3,4-cyclopentenecoumarin (5)
A mixture of resorcinol (1, 1 g, 9.08 mmol), ethyl
2-oxocyclopentanecarboxylate (1.6 g, 10.21 mmol) in
concentrated H2SO4 (5 ml) was stirred at room temper-
ature for 8 h and then poured into cold water (50 ml).
The resulting precipitate was filtered out, washed with
water, and purified by FC with 1:1 hexane–ethyl ac-
etate as the eluent. Compound 5 (1.36 g, 70%): m.p.
1
250–252°C. H NMR (DMSO-d6): 10.42 (s, 1H, OH),
7.41 (d, 1H, J=8.40, H5), 6.76 (m, 2H, H6 and H8),
3.01 (t, 2H, allylic CH2, J=7.50), 2.70 (t, 2H, allylic
CH2, J=7.50), 2.07 (quintet, 2H, CH2–CH2–CH2, J=
Scheme 1.

640
L. Dalla Via et al. / Il Farmaco 53 (1998) 638–644
7.50). IR: 3208, 1675, 1621, 1559, 1393, 1307, 1133,
1075. Anal. (C12H10O3) C, H.
ter (model 97503-00, Cole–Parmer Instrument, IL),
was 4.24 mW/cm2.
3.5. 7-Acetonyloxy-3,4-cyclopentenecoumarin (6)
3.9.2. Assay of inhibition of human cell growth
Human cervix adenocarcinoma cells (HeLa) were
grown in nutrient mixture F12 [HAM] medium (Sigma,
N 6760), supplemented with 10% heat-inactivated fetal
calf serum (Seromed), 100 U/ml penicillin, 100 mg/ml
streptomycin and 0.25 mg/ml amphotericin B (Sigma).
The cells were cultured at 37°C in a humidified atmo-
sphere with 5% carbon dioxide in air.
To a solution of 7-hydroxycoumarin 5 (500 mg, 2.47
mmol) in anhydrous acetone (100 ml) were added
chloroacetone (0.60 ml, 7.53 mmol) and potassium
carbonate (2 g) and the mixture was heated at reflux for
15 h. The precipitate was filtered out, the acetone was
evaporated under vacuum, and the solid residue was
purified by FC using 1:1 hexane–ethyl acetate as the
A total of 1×105 cells were seeded into each well of
a 24-well microtitex plate. After incubation for 24 h,
the medium was replaced with an equal volume of
Dulbecco’s modified Eagle’s medium without phenol
red (DMEM, Sigma, D2902), and various concentra-
tions of the test compound were added.
The cells were kept in the dark for 1 h and then
irradiated with a UVA dose of 0.79 J/cm2. After irradi-
ation, the medium containing the compounds was re-
moved and the cells were incubated in complete F12
medium for 24 h. Cell viability was determined by
means of the trypan blue exclusion method. Cytotoxic-
ity was expressed as IC50, i.e. the concentration of test
compound inducing a 50% reduction in cell number
with respect to control cultures.
1
eluent. Compound 6 (620 mg, 97%): m.p. 139°C. H
NMR (CDCl3): 7.38 (d, 1H, H5, J=8.60), 6.88 (dd,
1H, H6, J=8.60 and 2.40), 6.81 (d, 1H, H8, J=2.40),
4.63 (s, 2H, CH2–O), 3.05 (t, 2H, allylic CH2, J=7.50),
2.91 (t, 2H, allylic CH2, J=7.50), 2.31 (s, 3H, CH3),
2.21 (quintet, 2H, CH2–CH2–CH2, J=7.50). IR: 3065,
2910, 1719, 1611, 1420, 1396, 1073. Anal. (C15H14O4) C,
H.
3.6. 3,4-Cyclopenten-4%-methylfuro[3,2-g]coumarin (7)
A mixture of 7-acetonyloxycoumarin 6 (420 mg, 1.63
mmol) and 0.1 N NaOH (200 ml) was heated at reflux
for 20 h. The mixture was acidified with 1 N HCl, and
the precipitating solid was filtered out, washed with
water and purified by FC using 9:1 hexane–ethyl ac-
etate as the eluent. Compound 7 (260 mg, 66%): m.p.
3.9.3. Nucleic acid
Salmon testes DNA was purchased from Sigma
Chemical (D 1626, St. Louis, MO); its hypochromicity
was over 35%. The DNA concentration was determined
using extinction coefficient 6600 M−1/cm at 260 nm.
1
237°C. H NMR (CDCl3): 7.51 (s, 1H, H5), 7.46 (d,
1H, HC–O, J=1.30), 7.45 (s, 1H, H8), 3.17 (t, 2H,
CH2, J=7.70), 2.96 (t, 2H, CH2, J=7.70), 2.29 (s, 3H,
CH3, J=1.30), 2.27 (quintet, 2H, CH2–CH2–CH2, J=
7.70). IR: 3070, 2921, 1718, 1637, 1128, 1067, 1052.
Anal. (C15H12O3) C, H.
3.9.4. Flow linear dichroism
Flow linear dichroism was performed on a Jasco
J500A spectropolarimeter equipped with a Jasco inter-
face linked to an IBM PC.
3.7. Theoretical calculations
The test compound and nucleic acid were mixed in
ETN buffer (10 mM Tris, 1 mM EDTA, 10 mM NaCl,
pH 7.0) in order to reach a [compound]/[DNA] ratio of
0.04. The irradiated solutions (21.2 J/cm2) were also
submitted to exhaustive extraction with chloroform to
remove the free compound. A DNA solution treated in
the same way was used as reference.
Theoretical optimization of molecular geometries was
calculated by the AM1 semi-empirical quantum me-
chanical method [10], using the AMPAC program [11]
run on an SGI work station.
3.8. Spectrophotometric measurements
Samples were oriented by a device designed by Wada
and Kosawa [12] at a shear gradient of 800 rpm; each
spectrum was accumulated four times. Reduced linear
dichroism (LDr) was determined by dividing the linear
dichroism values (LD) by the relative isotropic ab-
sorbance (LDr=LD/A). Orientation angle h was deter-
UV absorption spectra were recorded at 25°C on a
Perkin–Elmer UV–Vis spectrophotometer (model
Lambda 12).
3.9. Photobiological studies
mined using the following equation [13]:
1
3.9.1. Irradiation procedure
2
1
ꢀ
3
(LDr)L
3(LDr)DNA
n
Irradiation was performed by means of Philips HPW
125 lamps, equipped with Philips filters, so that they
emitted over 90% of their radiation at 365 nm. Irradia-
tion intensity, determined by a Cole–Parmer radiome-
hL=arc cos
−
(1)
where (LDr)L is the reduced linear dichroism for the
ligand, calculated at the chromophore absorption band;

L. Dalla Via et al. / Il Farmaco 53 (1998) 638–644
641
Table 1
(LDr)DNA is that of DNA, measured at 260 nm, and hL
Spectroscopic properties of test compounds and 8-MOP in UV region
defines the relative drug–DNA orientation.
Compound
u (nm)
m (M−1/cm)a
3.9.5. Cross-linking e6aluation
4
292
338
365
9500
5480
2800
Solutions of DNA and the tested cyclopentene–furo-
coumarin in ETN buffer ([DNA]/[compound] ratio=
75) were irradiated at 365 nm for various times. After
irradiation, the DNA was denatured thermally (100°C
for 10 min) and quickly cooled in ice. The renaturation
capacity of DNA, due to cross-link formation, was
investigated recording absorbance at 260 nm. Data are
expressed in terms of log % of non-renaturated fraction
of irradiated psoralen–DNA complex relative to that of
irradiated DNA, as suggested by Blais et al. [9].
7
299
328
365
11 200
8800
850
8-MOP
300
365
12 300
970
a Calculated in ethanol solution.
3.9.6. Phototoxicity e6aluation
perimental conditions were the same as those of the cell
survival assay.
Skin phototoxicity was tested on depilated albino
guinea pigs (Dunkin–Hartley) as described by Carlas-
sare et al. [14]. An ethanol solution of each new com-
pound was applied topically on the skin up to 50
mg/cm2. For 8-MOP the concentration used was 10
mg/cm2. The animals were kept in the dark for 45 min
and then the treated skin was irradiated with 20 kJ/m2
of UVA. The degree of erythema was estimated 72 h
after irradiation.
4. Results and discussion
4.1. Spectroscopic properties
Table 1 shows the major UV-absorption peaks of
cyclopentene–furocoumarins 4 and 7 in comparison
with those of the tricyclic compound 8-MOP. Both 4
and 7 show umax at around 300 nm, like 8-MOP, but
with a shoulder at around 325–340 nm. Note that at
365 nm (the irradiation wavelength used in the photobi-
ological experiments; vide infra), the molar extinction
coefficient of compound 7 is very similar to that of
8-MOP, while compound 4 shows higher absorption.
3.9.7. Growth conditions and cell sur6i6al assay
Salmonella typhimurium strain TA100 was grown in
brain heart infusion (Difco) and cultivated at 37°C in
aerobic conditions.
Cells from broth cultures in the logarithmic growth
phase were harvested by centrifugation at 2000×g for
15 min, washed twice with 10 mM phosphate-buffered
saline (PBS), pH 7.2, containing 0.15 M NaCl and 2.7
mM KCl, and then diluted in the same buffer in order
to obtain a final A650=0.700, corresponding to 8×107
cells per ml. Small measured volumes of concentrated
solutions in DMSO of the tested compounds were
added to the cell suspensions to reach a final concentra-
tion of 2×10−6 M. The cells were incubated in the
dark for 1 h and then irradiated with a UVA dose of
0.79 J/cm2.
Control experiments in the presence and absence of
an equal volume of DMSO alone were also carried out.
Unirradiated and irradiated cell samples were ten-
fold serially diluted in the above-mentioned buffer and
each dilution was plated in duplicate on brain heart
agar (Difco). After incubation of the plates at 37°C for
24 h, the number of colony-forming units per ml (c.f.u./
ml) was counted.
4.2. Theoretical calculations
Theoretical calculations performed for compounds 4
and 7 by the semi-empirical method AM1 (see Section
3) indicated that both these tetracycles were practically
planar. The molecular curvatures of the compounds, as
measured by the angle between the line joining the
centres of the three rings of psoralen and that joining
the centres of the cyclopentane and furane or pyrone
rings were 32° for compound 4 and 58° for compound
7 (Fig. 1). The energies of the frontier molecular or-
bitals of compounds 4 and 7 were very similar (HO-
MOs= −8.73 and −8.74 eV, LUMOs= −0.94 and
−1.02 eV, respectively), and closely agreed with those
of 8-MOP (−9.01 and −1.02 eV). In theory, these
compounds should thus show similar reactivity to 8-
MOP in 2+2 cycloaddition reactions. Moreover, the
HOMO and LUMO orbital coefficients of 4, 7, 8-MOP
and psoralen are similar (Fig. 1), suggesting that these
compounds should also show similar regioselectivity in
their 2+2 cycloaddition reactions.
3.9.8. Mutagenesis
Compounds were tested for mutagenic activity on
Salmonella typhimurium strain TA100 according to the
plate incorporation test of Maron and Ames [15]. Ex-

642
L. Dalla Via et al. / Il Farmaco 53 (1998) 638–644
Table 2
Biological activity on HeLa cells and skin phototoxicity evaluation
(8-MOP as reference drug)
Compound
IC50 (mM)
Erythema intensitya
4
7
7.091.9
2594
+ − −
− − −
8-MOP
1093
+ + + (with edema)
a Symbols: + + +=strong; + − −=mild; − − −=absent.
Linear dichroism (LD) measurements of a solution of
the new cyclopentene–psoralens 4 and 7 in the presence
of DNA show the typical strong LD negative band
around 260 nm derived from the DNA base pairs, and
a negative LD signal between 300 and 380 nm where
DNA has no absorption (data not shown). This is the
first evidence that these small molecules are complexed
to DNA, because they cannot themselves become ori-
ented in the flow field. In the case of psoralens, all
strong absorption are p“p* transitions, which are
polarized in the molecular plane. Assuming that also
for cyclopentene–psoralens p“p* transitions are po-
larized in the molecular plane, the observed negative
signals (at wavelengths different from those of DNA)
indicate that the complexed 4 and 7 molecules assume a
position parallel to the plane of the DNA bases. This
orientation would be expected if the ligand was interca-
lated between two base pairs.
Fig. 1. HOMO (left) and LUMO (right) distributions of studied
compounds.
In previous studies by Tjerned et al. [16] and Vigny et
al. [17], the flow LD technique was used to investigate
the properties of covalent adducts derived from the
photobinding of 8-MOP and other psoralen derivatives
to DNA. Non-covalent DNA-psoralen complexes were
irradiated by UVA light and, after irradiation, the
non-covalently bound compound was removed. Flow
LD measurements allowed calculation of the angle
between the transition moment of the furan-side photo-
product and the axis of the DNA helix. For 8-MOP
and its isomer 5-MOP, the above authors found values
of 76–79° and 72–76°, respectively, in agreement with
a geometry in which the psoralen residues are in a
pseudo-intercalative conformation, taking into account
the fact that, for well-known intercalators, the value of
this angle is 90°.
For indications on covalent photobinding, we also
used the same LD technique. Fig. 2 shows the ab-
sorbance (top) and linear dichroic (bottom) spectra of a
DNA solution irradiated in the presence of compound
4. The negative linear dichroism signal in the furan-side
photoadduct region (320–360 nm) indicates that photo-
binding occurs. Eq. (1) (see Section 3) estimates the
value of orientation angle h, which ranges between 72
and 76°, according to the values reported in the above-
mentioned studies [16,17] and shows that the photo-
product has a pseudo-intercalative mode of binding.
This is consistent with the assumption that the photo-
The charge densities at the atoms of the reactive
double bonds of compounds 4 and 7 were very slightly
different from those at the corresponding atoms of the
pyrone and furane rings of psoralen and 8-MOP, due to
the inductive effect of the alkyl substituents. Thus, the
introduction of cyclopentane rings into psoralens, as in
4 and 7, is unlikely to change the course of reactions in
which electronic factors play a determining role.
4.3. Antiproliferati6e acti6ity
The new cyclopentene–furocoumarins were evalu-
ated for cytotoxic potency in vitro by the inhibition
growth assay on a cervix adenocarcinoma cell line
(HeLa) under UVA irradiation. The results are shown
in Table 2. Data are reported as IC50 values and
8-MOP was taken as reference drug. Compound 4
showed significant activity, whereas compound 7 was
practically devoid of biological activity.
4.4. Interaction with DNA
It is well-known that psoralen derivatives intercalate
inside the double helix of DNA when complexed with
the macromolecule. Under UVA irradiation, these in-
tercalated compounds undergo covalent photoconjuga-
tion to the pyrimidine bases of DNA.

L. Dalla Via et al. / Il Farmaco 53 (1998) 638–644
643
Fig. 3. Evaluation of cross-linking capacity of compound 4 and
8-MOP as reference drug.
4.5. Skin photosensitizing potency
Fig. 2. Absorption (top) and linear dichroism (bottom) spectra of
aqueous DNA solution irradiated in presence of compound 4.
Experiments were carried out to determine the po-
tency of the new cyclopentene–furocoumarins in induc-
ing skin erythema in guinea pigs. The results are shown
in Table 2. Compound 7 does not show any skin
phototoxicity, also confirming in this case its biological
inactivity. Interestingly, the effect of compound 4 dif-
fers from that of the reference drug. In fact, only at
concentrations five times higher than that of 8-MOP
does the new cyclopentene–psoralen 4 cause a barely
detectable erythematogenic effect.
binding of compound 4 to DNA may induce distortion
of the macromolecule structure, as hypothesized previ-
ously for 8-MOP [16,17].
With regard to compound 7, only a very weak
dichroic signal was detected, indicating that it practi-
cally does not photoreact with DNA.
4.6. Mutagenesis test
Further information regarding the photoreactivity of
compound 4 was obtained by investigating its ability to
form interstrand cross-links in the macromolecule. A
DNA solution was irradiated in the presence of test
cyclopentene–furocoumarin and thermally denatured.
After rapid cooling, the absorbance at 260 nm was
recorded for all samples. The renaturation capacity of
DNA, due to cross-link formation, was expressed as the
log of the percentual fraction of denaturated DNA at
various irradiation times (Fig. 3). It may be observed
that 4 forms cross-links practically to the same extent
as the reference drug 8-MOP. For compound 7, no
cross-link capacity was detected. From these results, it
may be concluded that, for compound 4, the steric
hindrance of the fourth ring and the molecular curva-
ture are not sufficient to hamper the photoreactivity of
the 4%,5% and 3,4 double bonds. Otherwise, the introduc-
tion of the cyclopentane ring at the pyrone side (com-
pound 7) would yield lower photoreactivity towards the
pyrimidine bases, probably due to more difficult over-
lapping between the reactive sites.
It is known that psoralens, under UVA irradiation,
may produce genotoxic effects, and the important role
played by cross-links in the production of such muta-
tions has been suggested [18,19]. In this context, it
appeared interesting to analyse the behaviour of com-
pound 4.
Salmonella typhimurium strain TA100 was used to
test mutagenic activity. Cells were treated as reported in
Section 3, and surviving and revertant numbers were
scored. The results are shown in Table 3. The be-
haviour of compound 4 is consistent with that of the
reference drug, as the number of revertants per survivor
is comparable.
5. Conclusions
With the aim of modulating the photobiological ac-
tivity of the tricyclic pharmacophoric nucleus, two cy-
clopentene–psoralen derivatives were synthesized.

644
L. Dalla Via et al. / Il Farmaco 53 (1998) 638–644
Table 3
Reversion frequencies induced in Salmonella typhimurium strain TA100 (8-MOP as reference drug)
Revertants per plate (dark)
Revertants per plate (UVA)
Revertants per survivor (%) (UVA)
Control
105(29)a
97(24)
111(25)
101(25)
111(19)
107(34)
36(9)
0.0013
0.0017
0.0406
0.0304
Solvent (DMSO)
Compound 4
8-MOP
39(10)
a Standard deviations in brackets.
[5] D. Averbeck, Recent advances in psoralen phototoxicity mecha-
nism, Photochem. Photobiol. 50 (1989) 859–882.
[6] D. Averbeck, E. Moustacchi, Decreased photo-induced muta-
genicity of mono-functional to bi-functional furocoumarins in
yeast, Photochem. Photobiol. 31 (1980) 475–478.
[7] M.A. Pathak, Mechanism in psoralen photosensitization reac-
tions: photobiologic, toxicologic and pharmacologic aspects of
psoralens, in: Monograph No. 66, M.A. Pathak, J.K. Dunnick,
(Eds.), U.S. Department of Health and Human Services, Public
Health Service, National Institute of Health, National Cancer
Institute, Bethesda, 1984, pp. 41–46.
[8] O. Gia, S. Mobilio, M. Palumbo, M.A. Pathak, Benzo- and
tetrahydrobenzo-psoralen congeners: DNA binding and photo-
biological properties, Photochem. Photobiol. 57 (3) (1993) 497–
503.
[9] J. Blais, P. Vigny, J. Moron, E. Bisagni, Spectroscopic properties
and photoreactivity with DNA of new monofunctional pyrido-
psoralens, Photochem. Photobiol. 39 (1984) 145–156.
[10] M.J.S. Dewar, E.G. Zoebisch, E.F. Healy, J.J.P. Stewart, AM1:
a new general purpose quantum mechanical molecular model, J.
Am. Chem. Soc. 107 (1985) 3902–3909.
[11] BIOSYM Technologies, 10065 Barnes Canyon Road, San Diego,
CA 92121.
[12] A. Wada, S. Kosawa, Instrument for the studies of differential
flow dichroism of polymer solutions, J. Polym. Sci. 2 (1964)
853–864.
[13] B. Norden, M. Kubista, T. Kurucsev, Linear dichroism spec-
troscopy of nucleic acids, Q. Rev. Biophys. 25 (1992) 51–170.
[14] F. Carlassare, F. Baccichetti, A. Guiotto, P. Rodighiero, O. Gia,
A. Capozzi, G. Pastorini, F. Bordin, Synthesis and photobiolog-
ical properties of acetylpsoralen derivatives, J. Photochem. Pho-
tobiol. B: Biol. 5 (1990) 25–39.
[15] D.M. Maron, B.N. Ames, Revised methods for the Salmonella
mutagenicity test, Mutat. Res. 113 (1983) 173–215.
[16] F. Tjerneld, B. Norden, B. Ljunggren, Interaction between DNA
and 8-methoxypsoralen studied by linear dichroism, Photochem.
Photobiol. 29 (1979) 1115–1118.
[17] P. Vigny, J. Blais, V. Ibanez, Flow linear dichroism study of the
orientation of 4%-5%-psoralen–DNA photoadducts, Photochem.
Photobiol. 45 (1987) 601–607.
[18] E. Sage, Molecular basis of UV and psoralen mutagenesis, J.
Photochem. Photobiol. B: Biol. 20 (1993) 211–217.
[19] M. Collet, J. Piette, Analysis of mutations induced by 4%-hydroxy-
methyl-4,5%,8-trimethylpsoralen and UVA in Escherichia coli lac
Z gene and its regulatory region, J. Photochem. Photobiol. B:
Biol. 20 (1993) 145–152.
When the cyclopentane ring is fused to the 4%,5%
double bond, a derivative (4) which exerts antiprolifera-
tive activity comparable to that of the reference drug
8-MOP, is obtained. Compound 4 is also able to photo-
bind with DNA forming cross-links, and damage to the
macromolecule is responsible for the antiproliferative
activity. Due to this peculiar mechanism of action, a
mutagenic effect is also observed. Otherwise, the capac-
ity to cause skin erythema is greatly reduced. It is to be
noted that this side-effect represents a severe restriction
for photochemotherapy.
When the cyclopentane ring is fused to the 3,4 double
bond, a compound devoid of biological activity is ob-
tained (7). Furthermore, in our experimental condi-
tions, it did not show any interaction ability towards
DNA.
The different behaviour between 4 and 7 may be
explained by theoretical calculations which demonstrate
that the introduction of the cyclopentane ring leads to
different molecular curvatures.
Interestingly, it should be stressed that appropriate
structural modifications can greatly reduce skin photo-
toxicity without altering either molecular events or in
vitro photobiological activity.
References
[1] J.A. Parrish, R.S. Stern, M.A. Pathak, T.B. Fitzpatrick, Pho-
tochemotherapy of skin diseases, in: J.D. Reagan, J.A. Parrish
(Eds.), The Science of Photomedicine, Plenum, New York, 1982,
pp. 595–624.
[2] R. Edelson, C. Berger, F.P. Gasparro et al., Treatment of
cutaneous T-cell lymphoma by extracorporeal photochemother-
apy. Preliminary results, N. Engl. J. Med. 316 (1987) 297–303.
[3] C.R. Cantor, Photo-cross-linking studies of nucleic acid struc-
.
ture, Ann. N.Y. Acad. Sci. 346 (1980) 379–385.
[4] F. Dall’Acqua, S. Caffieri, Recent and selected aspects of furo-
coumarin photochemistry and photobiology, Photomed. Photo-
biol. 10 (1988) 1–46.