New tetracyclic analogues of photochemotherapeutic drugs 5-MOP and 8- MOP: Synthesis, DNA interaction, and antiproliferative activity

Article abstract of DOI:10.1021/jm9910829

The synthesis of new tetrahydrobenzo- and benzopsoralen derivatives carrying at position 5 or 8 of the furocoumarin moiety a methoxy, hydroxy, or dimethylaminopropoxy side chain is reported. The study of their photoantiproliferative activity and ability t

Full text of DOI:10.1021/jm9910829

J . Med. Chem. 1999, 42, 4405-4413
4405
New Tetr a cyclic An a logu es of P h otoch em oth er a p eu tic Dr u gs 5-MOP a n d
8-MOP : Syn th esis, DNA In ter a ction , a n d An tip r olifer a tive Activity
Lisa Dalla Via,*,§ Ornella Gia,§ Sebastiano Marciani Magno,§ Lourdes Santana,‡ Marta Teijeira,‡ and
Eugenio Uriarte‡
Department of Pharmaceutical Sciences, University of Padua, and “Centro di Studio sulla Chimica del Farmaco e dei Prodotti
Biologicamente Attivi del CNR” (associated with the National Institute for the Chemistry of Biological Systems - CNR),
via Marzolo 5, 35131 Padova, Italy, and Department of Organic Chemistry, University of Santiago de Compostela, Spain
Received May 17, 1999
The synthesis of new tetrahydrobenzo- and benzopsoralen derivatives carrying at position 5
or 8 of the furocoumarin moiety a methoxy, hydroxy, or dimethylaminopropoxy side chain is
reported. The study of their photoantiproliferative activity and ability to induce erythema on
guinea pig skin allows us to state that the derivatives carrying the dimethylaminopropoxy
side chain exhibit a very interesting photobiological pattern. Indeed, if compared with the lead
compounds 5-MOP and 8-MOP, they exert a higher cytotoxic activity devoid of significant skin
phototoxicity. Between them, the more interesting appears to be 16, a nonphototoxic compound
whose antiproliferative activity on HeLa cells is 2 orders of magnitude higher than that of the
reference drug 8-MOP. Photoreaction experiments have revealed that, like classic furocou-
marins, A-T is the preferred nucleic base pair in its photobinding. Moreover, the extent of
covalent photoaddition to DNA correlates well with the photobiological activity. For this
compound a certain effect was also observed in the dark. Evaluation of the ability to induce
DNA cleavage in the presence of human topoisomerase II has suggested that this enzyme is
probably the target accountable for this effect.
In tr od u ction
Several approaches have been followed in order to
obtain new psoralen analogues with good photoreactiv-
ity along with diminished undesirable side effects. One
of the more interesting is the inactivation of one of the
two photoreactive double bonds by means of the con-
densation of a benzenic or cyclohexenyl ring at the furan
side of the psoralen molecule to achieve the inactivation
of the 4′,5′ reactive site.12-14 Indeed the 3,4 position
represents the unique photoreactive site for benzo-
psoralens, while the tetrahydro congeners are still able
to form interstrand cross-links because the cyclohex-
enyl ring does not prevent reactivity of the furan side.
The derivatives obtainedsbenzo- and tetrahydrobenzo-
psoralenssshowed a good capacity to photoreact with
calf thymus DNA in vitro. Furthermore, the skin
photosensitizing potency, evaluated as erythema induc-
tion on guinea pig skin, appears totally absent for all
the benzo derivatives and is greatly reduced for the
tetrahydrobenzo derivatives.12-14
Natural and synthetic psoralens have been used in
PUVA therapy (psoralen plus ultraviolet-A radiation)
for at least 20 years, and nowadays numerous skin
diseases are treated with 5-methoxypsoralen (5-MOP)
or 8-methoxypsoralen (8-MOP).1-3 Their therapeutic
application is by topical or oral administration followed
by ultraviolet (320-400 nm) exposure. A more recent
development in this field has been photopheresis, an
extracorporeal photochemotherapy, employed in the
treatment of cutaneous T-cell lymphoma and auto-
immune disorders such as pemphigus vulgaris and
scleroderma.4,5 Notwithstanding the unquestionable
benefits obtained from the therapeutic use of these
photosensitizers, it has to be underlined that photo-
chemotherapy with psoralen provokes some serious side
effects, such as skin photosensitization, genotoxicity,
and the induction of cutaneous tumors.6-8
The biological effects have been mainly related to the
combination with cell DNA. The intercalative complex
formed in the dark between two base pairs of the
macromolecule gives a photoaddition, upon irradiation
with UVA light, that involves the 5,6 double bond of
the pyrimidine bases and the 4′,5′ and/or 3,4 psoralen
double bonds, forming mono- and diadducts in DNA.9-11
Although both mono- and bifunctional adducts may be
responsible for inhibition of cellular DNA synthesis, the
compounds which cause bifunctional damage to the
macromolecule are usually more phototoxic.6-8
On the basis of these results, we have synthesized a
new series of tetrahydrobenzo- and benzopsoralens,
closely related to the photochemotherapeutic drugs
5-MOP and 8-MOP. The new derivatives present a
methoxy group in the same position as the lead com-
pounds. Furthermore, the presence of a dimethyl-
aminopropoxy side chain in position 5 or 8 of the
psoralen moiety was set with a view to increasing the
low solubility of the tetracyclic derivatives in aqueous
media. The latter have been synthesized from the
hydroxy derivative analogues which were also taken
into account.
* To whom correspondence should be addressed. Phone: +39 049
In this paper we describe the synthesis of six deriva-
tives of 5-MOP (compounds 5-10) and six of 8-MOP
(compounds 11-16), tetrahydrobenzo and benzo con-
827 5712. Fax: +39 049 827 5366. E-mail: dallavia@dsfarm.unipd.it.
§ Italy.
‡ Spain.
10.1021/jm9910829 CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/25/1999

4406 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Dalla Via et al.
Sch em e 1a
Ta ble 1. Cell Growth Inhibition in the Presence of Examined
Compounds and 5-MOP and 8-MOP as Reference Drugs
cell lines IC50 (µM)
HeLa
HL-60
compd
dark
>20
UVA
dark
UVA
5
6
7
8
9
10
11
12
13
14
15
16
5-MOP
8-MOP
4.6 ( 0.5
>20
>20
>20
4.9 ( 1.5
7.3 ( 0.5
3.4 ( 0.4
4.5 ( 1.1
>20
0.73 ( 0.16
3.5 ( 0.3
>20
0.60 ( 0.12
6.4 ( 1.1
>20
0.60 ( 0.09
3.35 ( 0.35
5.4 ( 0.7
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
9.6 ( 1.4
>20
>20
3.7 ( 0.2
>20
13.8 ( 3.6
>20
>20
>20
2.8 ( 0.3
>20
9.2 ( 1.1
>20
>20
>20
1.2 ( 0.7
>20
12.4 ( 1.5
>20
>20
>20
0.08 ( 0.02
16.3 ( 0.8
10 ( 3
4.2 ( 0.4
>20
>20
psoralen derivatives were significantly better than those
of the corresponding 5-methoxypsoralen derivatives and
that the yields of the tetrahydrobenzo compounds were
likewise better than those of the corresponding benzo
compounds.
P h otobiologica l Activity. The antiproliferative ac-
tivity of the new derivatives 5-16 was evaluated by
means of an in vitro test carried out on human cervix
adenocarcinoma (HeLa) and on human promyelocytic
leukemia (HL-60) cell lines, determining the concentra-
tion of compound able to cause the death of 50% of the
initial cell concentration. The results of the experiments,
performed as indicated in the Experimental Section, are
shown in Table 1; as reference we took both 5-MOP and
8-MOP.
a
Reagents: (a) 2-chlorocyclohexanone, K2CO3, acetone; (b)
NaOH; (c) DDQ, toluene; (d) HI, AcOH, Ac2O; (e) AlCl3, CH2Cl2;
(f) 3-chloro-N,N-dimethylpropylamine, NaH, NaI, DMF.
geners; their DNA interaction and photobiological prop-
erties were also studied. The availability of the 11-
dimethylaminopropoxy derivative 16 as a tritiated
compound allowed us to examine its photocombination
ability in depth.
Toward HeLa cells after exposure to UVA light, the
derivatives carrying the dimethylaminopropoxy side
chain (7, 10, 13, 16) exert the higher activity. Further-
more, the 11-derivatives are more reactive than the
5-congeners and the benzo more than the tetrahydro
analogues. It is worth noting the effect of compound 16
which is 100 times more efficient in inhibiting cell
growth than 8-MOP and compound 13 which is about 8
times more efficient.
The four above-mentioned hydrophilic compounds
appear to be more active on HL-60 cells as well as on
HeLa. Moreover, the methoxy derivatives, both tetrahy-
drobenzo and benzo, also exert a certain cytotoxic
activity; indeed the IC50 values are similar to those of
8-MOP.
Resu lts a n d Discu ssion
Ch em istr y. The compounds studied were obtained
as shown in Scheme 1. The preparation of 3, 5, and 8
in accordance with a general approach to the psoralen
skeleton15 has been described elsewhere;16 compounds
4, 11, and 14 were prepared analogously, as follows.
Williamson reaction of 2 with 2-chlorocyclohexanone for
24 h in refluxing acetone/K2CO3 gave the oxo ether 4
in 47% yield. Cyclization of 4 by heating in strong
alkaline solution afforded the tetrahydrobenzofuro-
coumarin 11 in 82% yield. Finally, the unsubstituted
terminal ring of 11 was aromatized by heating with
DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) in re-
fluxing toluene, which afforded the benzofurocoumarin
14 in 63.5% yield.
The 5-methoxypsoralen derivatives 5 and 8 were
transformed to 6 and 9, respectively, in almost quan-
titative yield by hydrolysis of their methoxy groups to
hydroxyl groups with hydroiodic acid in a refluxing
mixture of acetic acid and acetic anhydride. The 8-meth-
oxypsoralen derivatives 11 and 14 were hydrolyzed to
compounds 12 and 15 in 97% and 85.5% yields, respec-
tively, by treatment with aluminum trichloride in
refluxing methylene chloride.
In some cases a certain activity in the dark was also
observed, but it appears significantly less than that
exerted upon UVA irradiation.
Experiments were carried out to determine the skin
photosensitizing potency of the new tetracyclic psoralen,
tested on guinea pigs. The results reported in Table 2
indicate that all the benzo derivatives are nonphoto-
toxic. Regarding the tetrahydrobenzo compounds, only
the very active 7 and 13 show a photosensitization
capacity even if between them there is a significant
difference in behavior. Indeed, the derivative carrying
the dimethylaminopropoxy side chain in the 5 position
of the tricyclic psoralen structure is clearly weaker in
its ability to induce skin erythema with respect to the
corresponding 11-derivative. Moreover it is noteworthy
that, in both cases, the phototoxic effect is considerably
Finally, treatment of compounds 6, 9, 12, and 15 with
3-chloro-N,N-dimethylpropylamine and NaH in the
presence of NaI in refluxing dimethylformamide, so as
to replace their hydroxyl groups, afforded compounds
7, 10, 13, and 16 in 50%, 30.5%, 83%, and 55.5% yields,
respectively. Note that the yields of the 8-methoxy-

New Tetracyclic Analogues of 5-MOP and 8-MOP
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4407
Ta ble 2. Skin Phototoxicity in Guinea Pigs After Exposure to
the Tested Compounds as Indicated in Experimental Section
and 20 kJ m-2 of UVA
compd
formation of erythemaa
5
6
7
8
9
10
11
12
13
14
15
16
5-MOP
8-MOP
- - -
- - -
+ - -
- - -
- - -
- - -
- - -
- - -
+ + + (without edema)
- - -
- - -
- - -
+ + + (without edema)
+ + + (with edema)
a
+ + +, strong; + - -, mild; - - -, absent.
lower than that of the reference drugs because the new
compounds induce a detectable effect only when used
at a molar concentration about 3 times higher than that
of the 5-MOP and 8-MOP.
Non cova len t Bin d in g to DNA. The formation of a
molecular complex between the new psoralen analogues
and DNA was observed by means of flow linear dichro-
ism experiments (LD). The spectra of DNA solutions in
the presence of increasing amounts of compounds with
the dimethylaminopropoxy side chain (7, 10, 13, 16) are
reported (Figure 1).
The DNA spectra are negative at the lowest wave-
length region (260 nm), as expected for this macromol-
ecule (traces a). In the region at longer wavelengths
(320-380 nm), where only the added drug exhibits
absorption, a dichroic signal was also observed. Since
these small molecules cannot themselves become ori-
ented in the flow field, the existence of a dichroic signal
in this region allows us to state that these compounds
form a complex with DNA. Moreover the sign of this
signal is negative such as that observed for the strong
LD band around 260 nm due to the DNA base pairs. In
the case of planar aromatic molecules, all strong ab-
sorptions are πfπ* transitions, which are polarized in
the plane of the chromophore. Thus, the negative LD
sign is in qualitative agreement with an orientation of
the molecular plane preferentially parallel to the plane
of the DNA bases. This orientation is to be expected if
the drug is intercalated between two adjacent base
pairs.
Specific information on the geometry of the binding
process has been obtained by the LDr calculations and
using the equation reported in the Experimental Sec-
tion. These values, determined at the wavelength region
where the added drug absorbs, were close to those
observed for the DNA absorption band (260 nm). The
average angle R is 73° for the two tetrahydrobenzo
analogues (7 and 13). This value is consistent with a
geometry in which the molecular residues are in close
proximity to the nucleic bases in a pseudo-intercalative
conformation; this means that they are not in perfect
parallel alignment with the base pairs of DNA. On the
other hand, for the two benzo derivatives (16 and 10)
the value is 90° as for typical intercalators. This
difference in the complexation ability is probably at-
tributable to the hydrogenated ring of the tetrahy-
F igu r e 1. Linear flow dichroism spectra for compounds 7 (A),
10 (B), 13 (C), and 16 (D) at different [drug]/[DNA] ratios: a
) 0; b ) 0.02; c ) 0.04; d ) 0.08; [DNA] ) 1.89 × 10-3 M.

4408 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Dalla Via et al.
F igu r e 3. Absorption spectrum of pyrone cycloadduct ob-
tained from calf thymus DNA and compound 16, as reported
in the Experimental Section.
F igu r e 2. Fluorescence intensity of 4′,5′-cycloadducts ob-
tained by hydrolysis of DNA irradiated in the presence of
tetrahydrobenzo derivatives as a function of irradiation time.
drobenzopsoralen which is not planar with respect to
the tricyclic moiety.
P h otobin d in g to DNA. The tetrahydrobenzo- and
benzopsoralen DNA photoaddition process, consequent
to the irradiation of the intercalated furocoumarin
moiety into the double helix, was studied evaluating the
photoadduct formation. Regarding the tetrahydroben-
zopsoralens, it has already been demonstrated that the
cyclohexenyl ring does not inhibit the photoreactivity
of the furan side of the furocumarinic moiety,13 so that
the possibility of forming adducts at both reactive
positions of the furocoumarinic nucleus is substantially
maintained. The presence of 4′,5′-cycloadducts, obtained
after acid hydrolysis as described in the Experimental
Section, can be verified from their strong fluorescence,
the intensity of which increases with the amount of
compound photobound to DNA. Fluorescence was de-
tected after various increasing periods of irradiation at
365 nm, and the results are reported in Figure 2.
Intensity is greater for the derivatives carrying the
hydrophilic side chain. In particular the derivative with
the substituent in the 11 position (13) appears more
reactive than the 5-analogue (7). Among the others, the
methoxy derivatives seem to be more photoreactive than
the hydroxyl, whose fluorescence intensity is somewhat
low.
In the case of benzo derivatives, which can only form
pyrone monoadducts because of the 4′,5′ double bond’s
delocalization in a tetracyclic aromatic system, a rough
indication of the covalent binding can be obtained by
the absorption spectrum of irradiated DNA-compound
solution after appropriate treatment, as described in the
Experimental Section. The presence of UV absorption
around 300 nm after extraction is attributable to the
benzopsoralen-pyrimidine cycloadduct.12,17 The absorp-
tion data are correlated to the extent of the photoprod-
uct. Figure 3 reports the UV absorption spectrum
obtained by irradiating the macromolecule in the pres-
ence of 16. A similar trend was also observed for 10.
The absorption values for the compounds bearing the
methoxy or hydroxy side chain are very much lower.
Cr oss-Lin k in g. The photoreactive behavior of tet-
rahydrobenzo and benzo derivatives toward the double
helix of DNA was also assessed by denaturation-
renaturation experiments. The benzopsoralen deriva-
F igu r e 4. Cross-linking of compounds 7, 13, 5-MOP, and
8-MOP to double-stranded DNA from calf thymus (nucleotide-
drug ratio ) 78) as a function of irradiation time.
tives are totally unable to cross-link the double helix of
DNA (data not shown) according to previous findings
on other benzopsoralen compounds.13 Regarding the
cyclohexenyl congeners, Figure 4 depicts the cross-
linking ability of the compounds with the dimethyl-
aminopropoxy side chain in comparison with the two
drugs 5-MOP and 8-MOP.
Compound 13 shows a higher capacity to form cross-
links than the reference drug 8-MOP, whereas for
compound 7 this ability appears to be practically absent.
The presence of a substituent in the 5 or 8 position of
the psoralen moiety plays a crucial role in the DNA
photoaddition. This fact is slightly evident if we compare
5-MOP and 8-MOP, while it is clearly evident for
compounds 7 and 13 bearing a dimethylaminopropoxy
side chain. These results are in fair agreement with the
photobiological effects and confirm previous studies on
water-soluble compounds of furocoumarins.18
Qu a n t it a t ive E va lu a t ion of P h ot ob in d in g t o
Ma cr om olecu les. The availability of compound 16 as
a tritiated sample allowed determination of the amount
covalently linked to DNA from calf thymus. Figure 5
reports the nmol of drug photobound/mg of macromol-
ecule as a function of the irradiation time: derivative
16 exerts remarkable reactivity, significantly higher
than that of 8-MOP.
It is known that in the initial period of irradiation
the photoreactions of furocoumarins toward DNA be-

New Tetracyclic Analogues of 5-MOP and 8-MOP
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4409
F igu r e 7. Photobinding of compound 16 as a function of %
A-T content in nucleic acids (irradiation time 5 min).
F igu r e 5. Photobinding of compound 16 and 8-MOP to
double-stranded DNA from calf thymus (nucleotide-drug ratio
) 80) as a function of irradiation time.
preference toward the pyrimidine base thymine as the
target of photoreaction.
Top oisom er a se II-Med ia ted Clea va ble Com p lex.
The biological effects evoked by the furocoumarin
derivatives generally require UVA light activation;
indeed the well-known drugs 5-MOP and 8-MOP are
completely ineffective in the dark. However, for some
photobiological active compounds, a certain activity in
the dark was also observed so that two different
mechanisms, one light-mediated and the other in the
dark, had to be taken into account to explain their
antiproliferative effects.20,21 Because of the interaction
with DNA, the proposed mechanism was the involve-
ment of topoisomerase I and/or II.20
DNA topoisomerases are a unique class of enzymes
that change the topological state of DNA by breaking
and re-uniting the phosphodiester backbone of the
nucleic acid. Some antitumor drugs interfere with the
activity of mammalian DNA topoisomerase II by trap-
ping a key covalent reaction intermediate, called the
“cleavable complex”. The treatment of this complex with
a strong protein denaturant such as SDS results in DNA
breakage. In particular for a benzopsoralen derivative
the capacity to inhibit the activity of mammalian
topoisomerase II in vitro in both the catenation and
decatenation assay was detected.22
Among the new tetracyclic compounds, 16 also shows
higher antiproliferative activity in the dark. Its dark
effect is remarkably lower than that exerted upon UVA
irradiation; moreover it may be responsible for some
molecular events which could contribute to the overall
antiproliferative effect. Figure 8, lanes C-E, shows the
effect on human topoisomerase II activity of compound
16 at three different concentrations, as indicated. The
presence of DNA cleavage site at the higher concentra-
tion taken into account (100 µM) is evident, while there
is no significant effect at lower concentrations, even
though at 50 µM a weak inhibition of the enzymatic
activity appears.
Figu r e 6. Photobinding of compound 16 to synthetic polydeoxy-
ribonucleotides and DNA with various A-T contents (nucleo-
tide-drug ratio ) 8) as a function of irradiation time.
have like pseudo-first-order reactions with respect to the
furocoumarins.19 The photoreactions between 16 and
DNA also show this behavior, and the calculated initial
rate constant was 1.7 × 10-1 min-1, about 6 times
higher than that of 8-MOP (3.1 × 10-2 min-1), thus
confirming the good activity of this benzo derivative.
With the aim of highlighting whether the photo-
binding process is driven by the presence of particular
base sequences along the double helix, we also studied
the photoreactivity of compound 16 toward bacterial
DNA characterized by a different base pair composition
(see Experimental Section). The results are reported in
Figure 6 in terms of a photobound drug as a function of
irradiation time. It is evident that the amount of
covalent product increases as A-T content increases. In
Figure 6 the photobinding to poly[dA-dT]-poly[dA-dT]
and poly[dG-dC]-poly[dG-dC] is also reported. The
results are in agreement with the previous ones; indeed
the amount of compound 16 covalently bound to the
polynucleotides strictly depends on the presence of A-T
pairs.
Con clu sion s
To obtain a clearer evaluation of this behavior, the
extent of covalent photobinding, at the same irradiation
time, has been plotted against the percent of A-T content
(Figure 7). The pattern obtained is quite similar to that
of classical furocoumarins, which generally exert a
A new series of 5- or 11-methoxytetrahydrobenzo- and
benzopsoralen derivatives whose chemical structure is
related to the well-known photochemotherapeutic drugs
5-MOP and 8-MOP was synthesized. With the aim of
increasing the low solubility in aqueous media, the

4410 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Dalla Via et al.
A-T content causes a linear increase in the amount of
drug covalently bound to the polynucleotide. The be-
havior shown by the new benzo derivative confirms
these previous findings and indicates a leading prefer-
ence toward A-T residues independently of the flanking
sequences.
It is well-known that the photobiological activity of
furocoumarins generally requires light activation, while
in the dark there is only negligible or no effect. Com-
pound 16, besides the high photoantiproliferative activ-
ity, also shows modest, but detectable, cytotoxicity in
the dark. Experiments performed to gain further insight
into this unusual behavior demonstrate the involvement
of the nuclear enzyme topoisomerase II. The inhibition
ability of 16 toward the catalytic activity of this enzyme
only appears at higher concentrations, and this is well-
correlated with the extent of the antiproliferative effect
in the dark.
F igu r e 8. Topoisomerase II-mediated cleavage of pBR322
DNA. Cleavage reactions were carried out as described in the
Experimental Section: lane A, pBR322 DNA control (no
enzyme); lane B, pBR322 DNA and topoisomerase II (no drug);
lanes C-E, same as lane B with 10, 50, and 100 µM 16,
respectively.
Taking into account that furocoumarins have been
successfully used in combination with UVA to manage
hyperproliferative skin diseases such as psoriasis and
mycosis fungoides, the presence of a notable antiprolif-
erative activity besides the reduction in ability to induce
skin erythema renders this new family of compounds a
very promising tool for the development of new photo-
chemotherapeutic agents.
methoxy group was replaced with a dimethylaminopro-
poxy side chain. The synthetic pathway followed to
obtain these latter compounds provides for the replace-
ment of a hydroxyl group with the dimethylaminopro-
poxy side chain so that the hydroxy derivatives were
also taken into account. Nevertheless, neither 5- nor 11-
hydroxytetrahydrobenzo- and benzopsoralens reveal any
interesting photobiological pattern.
Regarding the methoxy derivatives, in most cases
they exert an antiproliferative activity comparable with
that of the tricyclic reference drug. However, unlike
5-MOP and 8-MOP, they do not induce skin erythema
in guinea pigs. Keeping in mind that skin phototoxicity
is a short-term side effect which has constituted a severe
limit to the development of photochemotherapy with
psoralens, the lack of cutaneous photosensitivity ap-
pears to be a noteworthy feature.
The new compounds bearing the dimethylaminopro-
poxy side chain have shown remarkable antiprolifera-
tive activity upon UVA irradiation, clearly higher than
that induced by reference drugs. Regarding skin photo-
sensitizing potency, only 13 induces erythema, although
to a substantially lower extent than the two reference
compounds 5-MOP and 8-MOP. The events occurring
in the photoreaction between the new tetracyclic com-
pound and DNA are similar to those of furocoumarins.
Indeed they form a preliminary complex with the
macromolecule undergoing intercalation. After irradia-
tion, the intercalated ligand photobinds covalently to
DNA giving rise to monoadducts and also diadducts in
the case of tetrahydrobenzo derivatives. Photobiological
activity is well-correlated with photoaddition to the
macromolecule.
The most antiproliferative activity is exerted by
compound 16, the benzo derivative carrying the di-
methylaminopropoxy side chain in the 11 position. The
availability of labeled 16 allowed us to examine its
photobiological pattern in depth. The ability to photoadd
with mammalian DNA is higher than that for 8-MOP,
thus confirming that the macromolecule may be con-
sidered a crucial target for photobiological events. The
investigation of photoaddition toward bacterial nucleic
acids and synthetic polydeoxyribonucleotides highlights
the fact that, like the majority of furocoumarins, A-T is
by far the preferred base pair in its photobinding.
Generally, for the classical furocoumarins increasing
Exp er im en ta l Section
Melting points are uncorrected and were determined in a
Reichert Kofler thermopan or capillary tubes in a Bu¨chi 510
apparatus. IR spectra were recorded in a Perkin-Elmer 1640FT
spectrometer (KBr disks, υ in cm-1). H NMR (300 MHz) and
1
13C NMR (75.4 MHz) spectra were recorded in a Bruker AMX
spectrometer, using TMS as internal standard (chemical shifts
in δ values, J in Hz). Mass spectrometry was carried out on a
Kratos MS-50 or Varian MAT-711 spectrometer. Elemental
analyses were performed by a Perkin-Elmer 240B microana-
lyzer and were within (0.4% of calculated values in all cases.
Flash chromatography (FC) was performed on silica gel (Merck
60, 230-400 mesh); analytical TLC was performed on pre-
coated silica gel plates (Merck 60 F254, 0.25 mm).
8-Me t h oxy-4-m e t h yl-7-(2-oxocycloh e xa n yloxy)cou -
m a r in (4). A mixture of 7-hydroxy-8-methoxycoumarin (5 g,
24.2 mmol), 2-chlorocyclohexanone (6.4 g, 36.4 mmol), and
K2CO3 (5 g, 36.2 mmol) in dry acetone (100 mL) was refluxed
for 48 h. The mixture was cooled, the precipitate collected, and
the solvent evaporated under reduced pressure. The crude
product was purified by FC with 8:2 hexane/ethyl acetate as
1
eluent, which gave pure 2 (3.40 g, 47%): mp 142-144 °C. H
NMR (CDCl3): 7.28 (d, 1H, H5, J ) 8.80), 6.73 (d, 1H, H6, J
) 8.80), 6.13 (d, 1H, H3, J ) 1.15), 4.82 (dd, 1H, H1′, J )
10.10 and 5.50), 3.96 (s, 3H, OCH3), 2.58 (m, 1H, 1H3′), 2.40
(m, 2H, 1H3′ and 1H6′), 2.35 (d, 3H, CH3, J ) 1.15), 2.10-
1.78 (m, 5H, H4′, H5′ and 1H6′). 13C NMR (CDCl3): 207.0 (C-
2′), 160.6 (C-2), 153.5 (C-4), 152.7 (C-8a), 148.0 (C-7), 137.1
(C-8), 119.2 (C-5), 115.5 (C-4a), 112.6 (C-6), 111.9 (C-3), 81.8
(C-1′), 61.5 (OCH3), 40.6 (C-3′), 34.5 (C-6′), 27.6 (C-4′), 23.1
(C-5′), 18.8 (CH3). IR: 2937, 1717, 1602, 1292, 1104. Anal.
(C17H18O4) C, H.
6,7,8,9-Tetr a h yd r o-11-m eth oxy-4-m eth ylben zofu r o[3,2-
g]cou m a r in (11). Oxo ether 4 (2.4 g, 7.9 mmol) was refluxed
in 1 M NaOH (500 mL) for 5 h. The mixture was cooled and
acidified with 1 M HCl, and the precipitate formed was
collected, washed with water, and purified by FC with 8:2
hexane/ethyl acetate as eluent, to give 11 (1.85 g, 82%): mp
1
196-197 °C. H NMR (CDCl3): 7.23 (s, 1H, H5), 6.24 (d, 1H,
H3, J ) 1.05), 4.25 (s, 3H, OCH3), 2.79 (m, 2H, H9), 2.63 (m,
2H, H6), 2.48 (d, 3H, CH3, J ) 1.05), 2.00-1.87 (m, 4H, H7
and H8). 13C NMR (CDCl3): 161.2 (C-2), 156.5 (C-4), 153.6 (C-
10a), 147.3 (C-11a), 143.0 (C-9a), 132.8 (C-11), 127.7 (C-5a),

New Tetracyclic Analogues of 5-MOP and 8-MOP
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4411
117.3 (C-4a), 113.5 (C-3), 113.4 (C-5b), 107.1 (C-5), 61.8 (OCH3),
23.9, 23.1, 22.8, 20.7, 19.9 (C-9, C-6, C-8, C-7, CH3). IR: 2949,
2841, 1716, 1582, 1441, 1063, 865. MS: m/z 285 (M+, 18), 284
(M+, 100), 256 (M+ - CO, 24), 228 (256 - C2H4, 15), 128 (10).
Anal. (C17H16O4) C, H.
849. MS: m/z 271 (M+, 18), 270 (M+, 100), 242 (M+ - CO, 39),
214 (242 - C2H4, 31), 185 (214 - CHO, 3), 115 (14). Anal.
(C16H14O4) C, H.
11-Hyd r oxy-4-m eth ylben zofu r o[3,2-g]cou m a r in (15).
This compound was prepared from 14 (100 mg) in an analogous
manner to 12. The crude product was purified by FC with 8:2
hexane/EtOAc as eluent which gave pure 15 (80 mg, 85.5%):
11-Meth oxy-4-m eth ylben zofu r o[3,2-g]cou m a r in (14). A
solution of tetrahydrobenzopsoralen 11 (5 g, 17.6 mmol) and
DDQ (8 g, 35.2 mmol) in toluene (500 mL) was heated at reflux
for 6 h. The mixture was cooled, the precipitate collected, and
the solvent evaporated under reduced pressure. The residue
was purified by FC with 8:2 hexane/EtOAc as eluent, which
gave benzopsoralen 14 (4.5 g, 91%): mp 191-193 °C. 1H NMR
(CDCl3): 7.96 (dd, 1H, H6, J ) 7.65 and 1.15), 7.82 (s, 1H,
H5), 7.62 (dd, 1H, H9, J ) 8.25 and 0.80), 7.51 (m, 1H, H8, J
) 8.25 and 1.15), 7.40 (m, 1H, H7, J ) 7.65 and 0.80), 6.31 (d,
1H, H3, J ) 1.20), 4.33 (s, 3H, OCH3), 2.57 (d, 3H, CH3, J )
1.20). 13C NMR (CDCl3): 160.3 (C-2), 156.9 (C-4), 152.8 (C-
9a), 148.9 (C-10a), 145.1 (C-11a), 132.9 (C-11), 127.9 (C-8),
123.6 (C-5a), 123.3 (C-7), 122.4 (C-5b), 120.7 (C-6), 117.2 (C-
4a), 113.5 (C-9), 112.0 (C-3), 109.3 (C-5), 61.6 (OCH3), 19.4
(CH3). IR: 3418, 1718, 1684, 1654, 1636, 1584, 1508, 1457,
1397, 1188, 1021. MS: m/z 281 (M+, 9), 280 (M+, 100), 252
(M+ - CO, 18), 237 (252 - CH3, 29), 209 (237 - C2H4, 13),
181 (209 - CO, 24), 152 (181 - CHO, 29). Anal. (C17H12O4) C,
H.
6,7,8,9-Tetr a h yd r o-5-h yd r oxy-4-m eth ylben zofu r o[3,2-
g]cou m a r in (6). A mixture of 5 (150 mg, 0.528 mmol), acetic
acid (2 mL), acetic anhydride (2 mL), and HI (4 mL) was
refluxed for 2 h. The solvent was then eliminated under
vacuum and the residue purified by FC using 8:2 hexane/
EtOAc as eluent to yield 6 (140 mg, 98%): mp 270-272 °C.
1H NMR (MeOD): 6.53 (s, 1H, H11), 6.06 (d, 1H, H3, J ) 0.95),
2.85 (m, 2H, H9), 2.75 (m, 2H, H6), 2.70 (d, 3H, CH3, J ) 0.95),
1.96 (m, 2H, H8), 1.86 (m, 2H, H7). 13C NMR (MeOD): 164.5
(C-2), 157.2, 155.6, 154.8, 154.4, 153.0 (C-10a, C-11a, C-5, C-4,
C-9a), 116.8 (C-5a), 114.3 (C-5b), 110.6 (C-3), 102.4 (C-4a), 97.9
(C-11), 24.6, 24.3, 24.2, 23.3, 22.5 (C-9, C-6, C-8, C-7, CH3).
IR: 3496, 3125, 2944, 1676, 1594, 1419, 1206, 843. MS: m/z
271 (M+, 18), 270 (M+, 100), 242 (M+ - CO, 51), 214 (242 -
C2H4, 39), 185 (214 - CHO, 4), 115 (12). Anal. (C16H14O4) C,
H.
5-Hyd r oxy-4-m eth ylben zofu r o[3,2-g]cou m a r in (9). This
compound was prepared from 8 (150 mg) in an analogous
manner to 6. The crude product was purified by FC with 8:2
hexane/EtOAc as eluent which gave pure 9 (140 mg, 98%): mp
260 °C. 1H NMR (DMSO-d6): 10.75 (bs, 1H, OH), 8.26 (dd,
1H, H6, J ) 7.40 and 1.35), 7.67 (dd, 1H, H9, J ) 8.10 and
1.05), 7.47 (m, H8, J ) 8.10, 7.40 and 1.35), 7.41 (m, 1H, H7,
J ) 7.40 and 1.05), 7.33 (s, 1H, H11), 6.17 (d, 1H, H3, J )
1.15), 2.73 (d, 3H, CH3, J ) 1.15). 13C NMR (DMSO-d6): 159.9
(C-2), 157.8 (C-10a), 155.6, 155.3, 155.0 (C-9a, C-11a, C-5),
151.6 (C-4), 127.2 (C-8), 123.8, 122.9 (C-7, C-6), 122.6 (C-5b),
112.3, 112.0, 111.5 (C-5a, C-3, C-9), 108.1 (C-4a), 93.5 (C-11),
24.3 (CH3). IR: 3378, 1684, 1639, 1601, 1573, 1438, 747. MS:
m/z 267 (M+, 11), 266 (M+, 100), 238 (M+ - CO, 71), 209 (238
- CHO, 6), 181 (209 - CO, 28), 152 (181 - CHO, 15), 119 (5).
Anal. (C16H10O4) C, H.
1
mp 263-265 °C. H NMR (DMSO-d6): 8.29 (s, 1H, OH), 8.20
(d, 1H, H6, J ) 7.30), 8.06 (s, 1H, H5), 7.73 (d, 1H, H9, J )
8.15), 7.54 (m, 1H, H8, J ) 8.15, 7.30 and 1.30), 7.44 (t, 1H,
H7, J ) 7.30), 6.38 (s, 1H, H3), 2.55 (s, 3H, CH3). 13C NMR
(DMSO-d6): 158.0 (C-2), 154.5 (C-4), 152.5 (C-9a), 144.4 (C-
10a), 140.2 (C-11a), 128.5 (C-11), 126.2 (C-8), 121.9 (C-5a),
121.8 (C-7), 119.8 (C-5b), 119.4 (C-6), 115.3 (C-4a), 110.9 (C-
9), 110.1 (C-3), 105.6 (C-5), 17.2 (CH3). IR: 3277, 1700, 1653,
1597, 1056. MS: m/z 267 (M+, 18), 266 (M+, 100), 238 (M+
CO, 38), 210 (238 - C2H4, 5), 181 (210 - CHO, 15), 152 (181
- CHO, 16). Anal. (C16H10O4) C, H.
-
6,7,8,9-Tetr a h yd r o-5-d im eth yla m in op r op oxy-4-m eth -
ylben zofu r o[3,2-g]cou m a r in (7). A mixture of hydroxytet-
rahydrobenzofurocoumarin 6 (110 mg, 0.407 mmol), 3-chloro-
N,N-dimethylpropylamine hydrochloride (77 mg, 0.488 mmol),
60% NaH (48 mg, 1.22 mmol), NaI (73 mg, 0.488 mmol), and
dimethylformamide (12 mL) was heated 2 h at 100 °C. It was
then concentrated under reduced pressure and the residue
purified by FC with CHCl3 as eluent to give pure 7‚HCl (80
mg, 50%): mp 238-240 °C. 1H NMR (MeOD): 6.70 (s, 1H,
H11), 6.10 (d, 1H, H3, J ) 1.15), 4.26 (t, 2H, H1′, J ) 5.90),
3.24 (m, 2H, H3′), 2.84 (s, 6H, H4′), 2.83 (m, 4H, H6 and H9),
2.66 (d, 3H, CH3, J ) 1.15), 2.28 (m, 2H, H2′), 2.00-1.87 (m,
4H, H7 and H8). 13C NMR (MeOD): 163.9 (C-2), 157.2, 155.4,
155.0, 154.4, 152.3 (C-10a, C-11a, C-5, C-4, C-9a), 117.1, 114.1,
111.9 (C-5a, C-5b, C-3), 103.5 (C-4a), 95.7 (C-11), 67.4 (C′), 57.1
(C-3′), 44.6 (2C, C-4′), 26.7, 24.6, 24.3, 24.1, 23.6, 22.4 (C-2,
C-9, C-6, C-8, C-7, CH3). IR: 3452, 2926, 1718, 1604, 1386,
1106. MS: m/z 356 (M+, 2), 355 (M+, 8), 269 (M+ - (CH3)2N-
(CH2)3, 5), 86 ((CH3)2N(CH2)3+, 71), 58 ((CH3)2NCH2+, 100).
Anal. (C21H26Cl NO4) C, H, Cl, N.
5-Dim et h yla m in op r op oxy-4-m et h ylb en zofu r o[3,2-g]-
cou m a r in (10). This compound was prepared from 9 (100 mg)
in an analogous manner to 7. The crude product was purified
by FC with CHCl3 as eluent and treated with ion-exchange
resin IRA-4000 to give pure 10 (40 mg, 30.5%): mp 103-105
1
°C. H NMR (CDCl3): 8.02 (d, 1H, H6, J ) 7.50), 7.57 (d, 1H,
H9, J ) 8.10), 7.49 (m, 1H, H8), 7.40 (m, 1H, H7), 7.31 (s, 1H,
H11), 6.16 (s, 1H, H3), 4.16 (t, 2H, H1′, J ) 6.65), 2.72 (s, 3H,
CH3), 2.67 (m, 2H, H3′), 2.38 (s, 6H, H4′), 2.25 (m, 2H, H2′).
13C NMR (MeOD): 162.5 (C-2), 159.8 (C-10a), 157.9 (C-9a),
156.0 (C-11a), 155.6 (C-5), 153.7 (C-4), 129.3 (C-8), 125.5 (C-
7), 123.9 (C-6), 123.0 (C-5b), 116.9 (C-5a), 115.1 (C-3), 113.0
(C-9), 111.8 (C-4a), 98.3 (C-11), 79.9 (C-1′), 56.9 (C-3′), 45.2
(C-4′), 31.0 (C-4′), 28.4 (C-2′), 24.0 (CH3). IR: 3452, 2923, 1725,
1602, 1383, 747. MS: m/z 352 (M+, 3), 351 (M+, 9), 279 (M+
-
(CH3)2N(CH2)2, 4), 266 (M+ - (CH3)2N(CH2)3, 30), 128 (20), 58
(100). Anal. (C21H21O4N) C, H, N.
6,7,8,9-Tetr a h yd r o-11-d im eth yla m in op r op oxy-4-m eth -
ylben zofu r o[3,2-g]cou m a r in (13). This compound was pre-
pared from 12 (100 mg) in an analogous manner to 7. The
crude product was purified by FC with 9:1 CHCl3/MeOH as
eluent to give pure 13‚HCl (120 mg, 83%): mp 198-201 °C.
1H NMR (CDCl3): 7.17 (s, 1H, H5), 6.12 (d, 1H, H3, J ) 1.00),
4.50 (t, 2H, H1′, J ) 5.50), 3.52 (t, 2H, H3′, J ) 7.50), 2.87 (s,
6H, H4′), 2.71 (t, 2H, H9, J ) 5.70), 2.55 (t, 2H, H6, J ) 5.70),
2.40 (d, 3H, CH3, J ) 1.00), 2.33 (m, 2H, H2′), 1.81 (m, 4H,
H7 and H8). 13C NMR (CDCl3): 161.1 (C-2), 156.8 (C-4), 154.1
(C-10a), 147.4 (C-11a), 143.0 (C-9a), 130.8 (C-11), 127.8 (C-
5a), 117.1 (C-4a), 113.5 (C-3), 113.1 (C-5b), 108.1 (C-5), 71.6
(C-1′), 56.6 (C-3′), 44.2 (C-4′), 30.0 (C-4′), 26.1 (C-2′), 23.9 (C-
9), 23.0 (C-6), 22.7 (C-8), 20.7 (C-7), 19.9 (CH3). IR: 3445, 2923,
2852, 2693, 1716, 1585, 1456, 1352, 1070. MS: m/z 356 (M+,
6,7,8,9-Tetr a h yd r o-11-h yd r oxy-4-m eth ylben zofu r o[3,2-
g]cou m a r in (12). A mixture of AlCl3 (141 mg, 0.105 mmol)
and anhydrous CH2Cl2 (1.5 mL) was stirred 2 h at room
temperature. The compound 11 (100 mg, 0.352 mmol) in
anhydrous CH2Cl2 (1 mL) was added and the mixture stirred
other 2 h. The reaction was then acidified with HCl and
extracted with CH2Cl2 (100 mL × 3). The extract was dried
(Na2SO4) and the solvent evaporated under reduced pressure
to leave a residue which was purified by FC with 8:2 hexane/
EtOAc as eluent to give pure 12 (260 mg, 97%): mp 285-286
1
°C. H NMR (CDCl3): 7.14 (s, 1H, H5), 6.24 (d, 1H, H3, J )
1.15), 2.79 (m, 2H, H9), 2.63 (m, 2H, H6), 2.51 (d, 3H, CH3, J
) 1.15), 1.98-1.85 (m, 4H, H7 and H8). 13C NMR (CDCl3):
160.8 (C-2), 157.0 (C-4), 154.5 (C-10a), 143.7 (C-11a), 138.8
(C-9a), 129.2 (C-11), 127.7 (C-5a), 116.6 (C-4a), 113.4 (C-5b),
113.0 (C-3), 104.8 (C-5), 23.9, 23.1, 22.8, 20.8, 19.8 (C-9, C-6,
C-8, C-7, CH3). IR: 3346, 2949, 1707, 1593, 1457, 1421, 1051,
1), 355 (M+, 5), 296 (M+ - (CH3)2NHCH2, 1), 270 (M+
-
(CH3)2N(CH2)3, 2), 128 (6), 115 (5), 86 ((CH3)2NH(CH2)3+, 8),
58 ((CH3)2NCH2+, 100). Anal. (C21H26ClNO4) C, H, Cl, N.

4412 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Dalla Via et al.
11-Dim eth yla m in op r op oxy-4-m eth ylben zofu r o[3,2-g]-
cou m a r in (16). This compound was prepared from 15 (100
mg) in an analogous manner to 7. The crude product was
purified by FC with 9:1 CHCl3/MeOH as eluent to give pure
16‚HCl (80 mg, 55.5%): mp 227-229 °C. 1H NMR (MeOD):
8.16 (s, 1H, H5), 8.13 (m, 1H, H6), 7.65 (m, 1H, H9), 7.58 (m,
1H, H8), 7.46 (m, 1H, H7), 6.36 (d, 1H, H3, J ) 1.10), 4.64 (t,
2H, H1′, J ) 5.55), 3.61 (t, 2H, H3′, J ) 7.35), 3.04 (s, 6H,
H4′), 2.60 (d, 3H, CH3, J ) 1.10), 2.34 (m, 2H, H2′). 13C NMR
(MeOD): 162.7 (C-2), 158.8 (C-4), 156.6 (C-9a), 150.7 (C-10a),
146.8 (C-11a), 132.6 (C-11), 129.8 (C-8), 125.5 (C-7), 124.9 (C-
5a), 124.5 (C-5b), 122.8 (C-6), 119.0 (C-4a), 114.1 (C-9), 113.2
(C-3), 112.7 (C-5), 73.3 (C-1′), 57.9 (C-3′), 44.5 (C-4′), 26.9 (C-
2′), 19.9 (CH3). IR: 3153, 2923, 1702, 1654, 1636, 1583, 1508,
1458, 1437, 1403, 1340, 1258, 1195, 1155, 1075, 1031, 753.
MS: m/z 352 (M+, 3), 351 (M+, 9), 266 (M+ - (CH3)2N(CH2)3,
25), 237 (3), 181 (5), 128 (17), 58 (100). Anal. (C21H22ClNO4)
C, H, Cl, N.
P h otobiologica l Meth od s. Cell Cu ltu r es. HL-60 and
HeLa cells were grown in RPMI 1640 (Sigma Chemical Co.)
supplemented with 15% heat-inactivated fetal calf serum
(Seromed) and Nutrient Mixture F-12 (HAM) (Sigma Chemical
Co.) supplemented with 10% heat-inactivated fetal calf serum
(Seromed), respectively; 100 U/mL penicillin, 100 µg/mL
streptomycin, and 0.25 µg/mL amphotericin B (Sigma Chemi-
cal Co.) were added to both media. The cells were cultured at
37 °C in a moist atmosphere of 5% carbon dioxide in air.
Linear dichroism is defined as:
LD(λ) ) A||(λ) - A
(λ)
where A and A correspond to the absorbances of the sample
||
when polarized light is oriented parallel or perpendicular to
the flow direction, respectively. The orientation is produced
by a device designed by Wada and Kozawa24 at a shear
gradient of 500-700 rpm.
The reduced linear dichroism is defined as:
LDr ) LD(λ)/Aiso(λ)
where Aiso(λ) is the absorbance of the sample in the absence of
flow. This quantity may be related to an orientation factor (S)
and the angle between the active transition moment in the
chromophore and the DNA helix axis, R:25,26
LDr ) 3/2(3 cos2R - 1)S
Assuming a value of R ) 90° for the DNA base pair
chromophore with respect to a local helix axis, it is possible to
evaluate RL for a given ligand:
1/2
RL ) arccos[1/3 - (LDr)L/3(LDr)DNA
]
where (LDr)L is the reduced linear dichroism for the ligand,
(LDr)DNA is the reduced LD for DNA, and RL defines the
ligand-DNA relative orientation. For the intercalated system,
(LDr)L ≈ (LDr)DNA and RL = 90°.
Ir r a d ia tion P r oced u r e. Irradiations were performed by
means of Philips HPW 125 lamps equipped with a Philips filter
emitting over 90% at 365 nm. Irradiation intensity was
checked on a UV-X radiometer (Ultraviolet Products Inc.,
Cambridge, U.K.) for each experimental procedure.
A solution of calf thymus DNA (1.5 × 10-3 M) in ETN buffer
(containing 10 mM TRIS, 10 mM NaCl, and 10 mM EDTA,
pH ) 7) was used. Spectra were recorded at 25 °C at different
[DNA]/[drug] ratios.
In h ibition Gr ow th Assa ys. HeLa cells (105) were seeded
into each well of a 24-well cell culture plate. After incubation
for 24 h, the medium was replaced with an equal volume of
Dulbecco’s modified Eagle medium (DMEM; Sigma Chemical
Co.) without phenol red, and various concentrations of the test
agent were added. One hour later the cells were irradiated
with a UVA dose of 0.793 J cm-2. After irradiation, the medium
containing the compounds was removed, and the cells were
incubated in complete F-12 medium for 24 h. For the experi-
ments carried out in the dark, the replacement of the incuba-
tion medium was omitted and the cells were incubated in
complete F-12 medium in the presence of the test compound
for 24 h.
F lu or im etr ic Deter m in a tion s. Fluorimetric determina-
tions were essentially made following previous indications.27
Volumes of concentrated solutions of the examined compound
were added to calf thymus DNA in ETN solution to achieve a
DNA/compound ratio of about 80. Aliquots of these solutions
were introduced into calibrated glass tubes, immersed in a
thermostatically controlled bath, and then irradiated for
various periods of time. After irradiation the DNA was
precipitated with NaCl and cool ethanol, washed with ethanol
80%, and collected by centrifugation. The pellet was then
dissolved in a measured volume of buffer in order to reach an
exact DNA concentration. The final solution was made 0.5 N
with HCl, heated at 100 °C for 1 h, and neutralized; the
fluorescence intensity was determined by means of a Perkin-
Elmer LS50B luminescence spectrometer at λex ) 320 nm and
λem ) 420 nm.
HL-60 cells (105) were seeded into each well of a 24-well
cell culture plate. After incubation for 24 h, various concentra-
tions of the test agents were added in complete medium. The
cells were kept in the dark for 1 h, irradiated with a UVA dose
of 0.793 J cm-2, and then incubated for a further 24 h.
Sp ect r op h ot om et r ic Det er m in a t ion s. Spectrophoto-
metric determinations were performed as described in ref 17
with some modifications. Briefly, volumes of concentrated
solutions of the examined compound were added to calf thymus
DNA in ETN solution to achieve a DNA/compound ratio of
about 20. Aliquots of these solutions were introduced into
calibrated glass tubes, immersed in a thermostatically con-
trolled bath, and then irradiated for 90 min. After irradiation
the DNA was precipitated with NaCl and cool ethanol, washed
with ethanol 80%, and collected by centrifugation. The pellet
was then dissolved in a measured volume of buffer in order to
reach an exact DNA concentration. The final solution was
made 0.5 N with HCl, heated at 100 °C for 1 h, neutralized,
and extracted exhaustively with CHCl3 for the hydroxy and
methoxy derivatives and CHCl3/methanol (2:1) for the di-
methylaminopropoxy derivatives. After this procedure the
organic layers were collected, dried under high vacuum, and
dissolved in ethanol. UV spectra were recorded on a Perkin-
Elmer model Lambda 5 spectrophotometer.
A trypan blue assay was performed to determine cell
viability. Cytotoxicity data were expressed as IC50 values, i.e.,
the concentrations of the test agent inducing 50% reduction
in cell numbers compared with control cultures.
Sk in P h ototoxicity. Skin phototoxicity was tested on
depilated albino guinea pigs (outbred Dunkin-Hartley strain),
as previously reported.15 An ethanol solution of each new
compound was applied topically to the skin up to 50 µg cm-2
.
.
For 5-MOP and 8-MOP the concentration used was 10 µg cm-2
The animals were then kept in the dark for 45 min, and the
treated skin was irradiated with 20 kJ m-2 of UVA; erythema
was scored after 48 h.
Nu cleic Acid s. Calf thymus DNA was purchased from
Sigma Chemical Co. Its hypochromicity, determined according
to Marmur and Doty,23 was over 35%. DNA from Micrococcus
lysodeikticus (Cat. D-8259), Escherichia coli (Cat. D-2001), and
Clostridium perfringens (Cat. D-1760), poly[dA-dT]‚poly[dA-
dT] (Cat. P-0883), and poly[dG-dC]‚poly[dG-dC] (Cat. P-9389)
also came from Sigma.
Eva lu a tion of In ter str a n d Cr oss-Lin k s in Vitr o. After
irradiation in the presence of the examined compound, the
DNA solutions ([DNA]/[drug] ) 78) were thermally denatured
(95 °C for 15 min) and quickly cooled in ice. One milliliter of
each of these solutions was chromatographed on a column of
Lin ea r F low Dich r oism . LD measurements were per-
formed on a J asco J 500A circular dichroism spectropolarimeter
converted for LD and equipped with an IBM PC and a J asco
J interface.

New Tetracyclic Analogues of 5-MOP and 8-MOP
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4413
(5) Edelson, R. L. Photopheresis: present and future aspects. J .
Photochem. Photobiol., B: Biol. 1991, 10, 165-174.
(6) Pathak, M. A. Mechanism of psoralen photosensitization reac-
tions: photobiologic, toxicologic and pharmacologic aspects of
psoralens. In Monograph No. 66; Pathak, M. A., Dunnick, J . K.,
Eds.; U.S. National Institute of Health, National Cancer Insti-
tute: Bethesda, MD, 1984; p 41.
(7) Averbeck, D. Recent advances in psoralen phototoxicity mech-
anism. Photochem. Photobiol. 1989, 50, 859-882.
(8) Gunther, E. J .; Yeasky, T. M.; Gasparro, F. P.; Glazer, P.
Mutagenesis by 8-methoxypsoralen and 5-methylangelicin pho-
toadducts in mouse fibroblasts: mutations at cross-linkable sites
induced by monoadducts as well as cross-links. Cancer Res. 1995,
55, 1283-1288.
hydroxylapatite (Bio-Gel HTP, 130-0420, Biorad) developing
with a linear gradient of 0.005-0.5 M phosphate buffer, pH
) 7; the flow rate was 15 drops/min. Fractions of 2 mL were
collected, and the absorbance at 260 nm was recorded.
Ra d ioch em ica l Deter m in a tion s. Radioactivity measure-
ments were made by means of a Packard model TRI-CARB
4000 liquid scintillation spectrometer. The efficiency of the
apparatus for counting tritium, calculated by using an internal
standard, was within 35-40%.
Compound 16 was tritium-labeled by Amersham Interna-
tional (Buckinghamshire, U.K.) and purified on thin-layer
chromatography plates (silica gel, Merck, 2 mm). Plates were
developed with CHCl3/methanol, 8:2 (v/v); purified product
showed a specific activity of 514 Ci/mol.
(9) Cantor, C. R. Photocross-linking studies of nucleic acid structure.
Ann. N. Y. Acad. Sci. 1980, 346, 379-385.
P h otobin d in g to DNA in Vitr o. Small measured volumes
of concentrated ethanol solutions of the labeled compound were
added to aqueous 0.05% calf thymus DNA (containing 10 mM
TRIS, 10 mM NaCl, and 10 mM EDTA, pH ) 7) to achieve a
DNA/compound molar ratio of about 80. Aliquots of these
solutions were introduced into calibrated glass tubes, im-
mersed in a thermostatically controlled bath, and then irradi-
ated for various periods of time. After irradiation, the DNA
was precipitated with NaCl and ethanol and washed with
ethanol 80%, and the pellets were dissolved in the initial
volume of buffer before radiochemical measurements were
performed.
In the experiments on bacterial DNA and synthetic poly-
nucleotides, buffer solutions of nucleic acids (1.5 × 10-4 M)
were added to the labeled furocoumarins (1.85 × 10-5 M).
Irradiations were performed as above. After irradiation, the
solutions were extracted with chloroform in order to remove
the nonphotobound drug and other low-molecular-weight
products. Afterward, the aqueous solutions were used for
radiochemical measurements.
Top oisom er a se II-Med ia ted DNA Clea va ge. The topo-
isomerase II-mediated DNA cleavage assay was performed as
reported by Tewey et al.28 with some modifications. Briefly,
pBR322 plasmid DNA (Sigma) was linearized with EcoRI
(Sigma) and then end-labeled at the 3′ termini with the Klenow
fragment of DNA polymerase I (Sigma) in the presence of
[R-32P]dATP (Amersham). Unincorporated triphosphates were
removed by ethanol precipitation in the presence of 2 M
ammonium acetate. The linear plasmid was further digested
with HindIII restriction endonuclease (Sigma) to remove a 31-
base pair small fragment.
(10) Cimino, G. D.; Gamper, H. B.; Isaacs, S. T.; Hearst, J . E.
Psoralens as photoactive probes of nucleic acid structure and
function: organic chemistry, photochemistry and biochemistry.
Annu. Rev. Biochem. 1985, 54, 1151-1193.
(11) Dall’Acqua, F.; Caffieri, S. Recent and selected aspects of
furocoumarin photochemistry and photobiology. Photochem.
Photobiol. 1988, 10, 1-46.
(12) Palumbo, M.; Rodighiero, P.; Gia, O.; Guiotto, A.; Marciani
Magno, S. Benzofurocoumarins: new monofunctional DNA-
photobinding agents. Photochem. Photobiol. 1986, 44, 1-4.
(13) Gia, O.; Mobilio, S.; Palumbo, M.; Pathak, M. A. Benzo- and
tetrahydrobenzo-psoralen congeners: DNA binding and photo-
biological properties. Photochem. Photobiol. 1993, 57, 497-503.
(14) Gia, O.; Conconi, M. T.; Uriarte, E. Benzo- and tetrahydrobenzo-
furocoumarins: new potential photochemotherapeutic drugs.
Farmaco 1997, 5, 289-293.
(15) Gia, O.; Anselmo, A.; Conconi, M. T.; Antonello, C.; Uriarte, E.;
Caffieri, S. 4′-Methyl derivatives of 5-MOP and 5-MOA: syn-
thesis, photoreactivity, and photobiological activity. J . Med.
Chem. 1996, 39, 4489-4496.
(16) Teran, C.; Miranda, R.; Santana, L.; Teijeira, M.; Uriarte, E.
Synthesis of linear and angular benzofurocoumarins. Synthesis
1997, 12, 1384-1386.
(17) Gia, O.; Mobilio, S.; Chilin, A.; Rodighiero, P. Pyrrolocoumarin
derivatives: DNA binding properties. J . Photochem. Photobiol.,
B: Biol. 1988, 2, 435-442.
(18) Marciani Magno, S.; Antonello, C.; Baccichetti, F.; Gia, O.;
Rodighiero, G. Photoreactive water-soluble derivatives of psor-
alen. Farmaco 1981, 1, 13-22.
(19) Rodighiero, G.; Musajo, L.; Dall’Acqua, F.; Marciani, S.; Caporale,
G.; Ciavatta, L. Mechanism of skin photosensitization by furo-
coumarins. Photoreactivity of various furocoumarins with native
DNA and with ribosomal RNA. Biochim. Biophys. Acta 1970,
217, 40-49.
The cleavage reaction was performed in a reaction mixture
containing 40 mM TRIS-HCl (pH ) 7.6), 100 mM KCl, 10 mM
MgCl2, 0.5 mM dithiothreitol, 0.5 mM EDTA, 30 µg/mL bovine
serum albumin, 1.6 mM ATP, 50 ng of the uniquely end-
labeled pBR322 DNA, 20 ng of human type II topoisomerase
(TopoGEN, Inc.), and test compound as indicated. Reactions
were incubated at 37 °C for 30 min and then terminated by
the addition of 0.5% SDS and 75 µg/mL proteinase K (Sigma).
DNA samples were analyzed on a 1% agarose gel in TBE buffer
(89 mM Tris-borate (pH ) 8.3) and 2 mM EDTA). Gels were
dried and autoradiographed for 1 or 2 days.
(20) Bordin, F.; Carlassare, F.; Conconi, M. T.; Capozzi, A.; Majone,
F.; Guiotto, A.; Baccichetti, F. Biological properties of some
benzopsoralen derivatives. Photochem. Photobiol. 1992, 55, 221-
229.
(21) Rodighiero, P.; et al. Angular furoquinolinones, psoralen ana-
logues: novel antiproliferative agents for skin diseases. Syn-
thesis, biological activity, mechanism of action, and computer-
aided studies. J . Med. Chem. 1996, 39, 1293-1302.
(22) Pani, B.; Barbisin, M.; Russo, E.; Tamaro, M.; Bacchichetti, F.;
Carlassare, F.; Marzano, C.; Rodighiero, P.; Bordin, F. DNA
damage and topoisomerase II inhibition induced by a benzo-
psoralen derivative. Mutat. Res. 1994, 311, 277-285.
(23) Marmur, J .; Doty, P. Determination of the base composition of
the deoxyribonucleic acid from its thermal denaturation tem-
perature. J . Mol. Biol. 1962, 5, 109.
(24) Wada, A.; Kozawa, S. Instrument for the studies of differential
flow dichroism of polymer solutions. J . Polym. Sci., Part A 1964,
2, 853-864.
(25) Norde´n, B. Applications of linear dichroism spectroscopy. Appl.
Spectrosc. Rev. 1978, 14, 157-248.
(26) Norde´n, B.; Kubista, M.; Kurucsev, T. Linear dichroism spec-
troscopy of nucleic acid. Q. Rev. Biophys. 1992, 25, 51-170.
(27) Dall’Acqua, F.; Caffieri, S.; Rodighiero, G. Fluorimetric deter-
mination of 4′,5′-cycloadducts in the DNA-psoralen photoreac-
tion. Photochem. Photobiol. 1978, 27, 77-79.
(28) Tewey, K. M.; Rowe, T. C.; Yang, L.; Halligan, B. D.; Liu, L. F.
Adriamycin-induced DNA damage mediated by mammalian
DNA topoisomerase II. Science 1984, 226, 466-468.
Ack n ow led gm en t. The authors are grateful to Mr.
Mariano Schiavon for his skillful technical assistance.
Refer en ces
(1) Song, P. S.; Tapley, K. J ., J r. Photochemistry and photobiology
of psoralen. Photochem. Photobiol. 1979, 29, 1177-1197.
(2) Pathak, M. A.; Fitzpatrick, T. B. The evolution of photochemo-
therapy with psoralens and UVA (PUVA): 2000 BC to 1992 AD.
J . Photochem. Photobiol. B 1992, 14, 3-22.
(3) Bethea, D.; Fullmer, B.; Syed, S.; Seltzer, G.; Tiano, J .; Rischko,
C.; Gillespie, L.; Brown, D.; Gasparro, F. P. Psoralen photo-
biology and photochemotherapy: 50 years of science and medi-
cine. J . Dermatol. Sci. 1999, 19, 78-88.
(4) Edelson, R.; Berger, C.; Gasparro, F. P.; et al. Treatment of
cutaneous T-cell lymphoma by extracorporeal photochemo-
therapy. Preliminary results. N. Engl. J . Med. 1987, 100, 187-
192.
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