Synthesis and biological activity of (hydroxymethyl)- and (diethylaminomethyl)benzopsoralens

Article abstract of DOI:10.1021/jm991028s

Some benzopsoralens, carrying a hydroxymethyl or a diethylaminomethyl group at the 3, 5, 8, and 11 positions, were prepared, and their biological activity was compared with that of 4-(hydroxymethyl)benzopsoralen (BP). 5- (Hydroxymethyl)benzopsoralen (7b),

Full text of DOI:10.1021/jm991028s

2936
J . Med. Chem. 1999, 42, 2936-2945
Syn th esis a n d Biologica l Activity of (Hyd r oxym eth yl)- a n d
(Dieth yla m in om eth yl)ben zop sor a len s
Adriana Chilin,† Cristina Marzano,† Adriano Guiotto,*,‡ Paolo Manzini,‡ Francarosa Baccichetti,‡
Francesco Carlassare,‡ and Franco Bordin‡
Department of Pharmaceutical Sciences 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,
Department of Pharmaceutical Sciences, University of Padua, Via Francesco Marzolo 5, I-35131 Padova, Italy
Received February 28, 1999
Some benzopsoralens, carrying a hydroxymethyl or a diethylaminomethyl group at the 3, 5, 8,
and 11 positions, were prepared, and their biological activity was compared with that of
4-(hydroxymethyl)benzopsoralen (BP). 5-(Hydroxymethyl)benzopsoralen (7b ), 11-(hydroxy-
methyl)benzopsoralen (7c), and 11-(diethylaminomethyl)benzopsoralen (8c) induced marked
antiproliferative effects in mammalian cells by simple incubation in the dark; this activity
appeared to be related to their ability to inhibit topoisomerase II. Benzopsoralens appeared to
be more active, especially BP and 7c, upon UVA activation. Compounds carrying a methyl
group at the 4 position together with a hydroxymethyl or diethylaminomethyl at the 8 position
(7d and 8d , respectively) were also effective, although to a lower extent; instead, a substituent
at the 3 position canceled all activity. Benzopsoralens did not induce interstrand cross-links
in DNA in vitro, as seen in the induction of cytoplasmic ,petite. mutations and double-strand
breaks in yeast. This behavior is also compatible with their low mutagenic activity in E. coli
WP2 and with the absence of any phototoxicity on the skin. For these features, benzopsoralens
seem to be interesting potential drugs for PUVA photochemotherapy and photopheresis. The
activity shown in the dark is not sufficient for their possible use as antitumor drugs, but it
does offer a new model for the study of topoisomerase inhibitors.
In tr od u ction
Furocoumarins (psoralens) are well-known photosen-
sitizing drugs which have been used in photomedicine
for several years now, to cure a number of skin diseases
such as psoriasis, mycosis fungoides, lupus erythema-
F igu r e 1. Structures of 8-methoxypsoralen (8-MOP) and
tosus, etc.1 This therapeutic treatment is generally
4-(hydroxymethyl)benzopsoralen (BP).
called PUVA, from psoralen plus UVA (the wavelength
range used, 320-400 nm); its therapeutic effectiveness,
8-MOP and by several other furocoumarins, a new
particularly to treat vitiligo, has been known for cen-
aspect of their bifunctional behavior, has been re-
turies.2 In 1978, Edelson introduced extracorporeal
ported.11
photochemotherapy or photopheresis, in which lympho-
For these reasons, various monofunctional furocou-
cytes are withdrawn, submitted to furocoumarin sen-
marins have been prepared and studied.12,13 Among the
sitization, and then returned to the patient.3 This
various ways of obtaining monofunctional furocou-
therapy was approved by the FDA for the treatment of
marins, we also studied the possibility of deleting one
T-cell lymphoma, but it is also successfully used for
of the two photoreactive sites existing in the furocou-
marin molecule (3,4 and 4′,5′ double bonds) by introduc-
ing a fourth nucleus into the furocoumarin skeleton at
preventing rejection in organ transplants and to treat
various autoimmune diseases.4
The compound mostly preferred for both PUVA and
the furan ring. Following this research line, we prepared
photopheresis is 8-methoxypsoralen (8-MOP), (Figure
a series of compounds carrying a benzene or a cyclo-
1) but 5-methoxypsoralen (5-MOP) and 4,4′,8-trimeth-
hexenyl ring fused at the furan side, thus obtaining
ylpsoralen (TMP) are also used, particularly in some
benzopsoralens and tetrahydrobenzopsoralens.14 To-
PUVA treatments.5,6 All these compounds damage DNA,
gether with good photosensitizing activity, these deriva-
leading to monofunctional and bifunctional adducts
tives also induce pronounced antiproliferative effects on
(interstrand cross-links) with pyrimidine bases; the
mammalian cells in the dark,15 i.e., without UVA
bifunctional damage is generally regarded as the main
activation; this property appears to be associated with
agent responsible for the side effects observed in PUVA
an evident ability to inhibit topoisomerase II.16 The
therapy such as genotoxicity,7,8 carcinogenicity,9 and the
presence of a hydroxymethyl group at the 4 position in
formation of skin erythemas.10 Recently, the formation
the furocoumarin molecule is important for drug activity
of covalent adducts between DNA and proteins by
in both experimental conditions, upon UVA irradiation
and by incubation in the dark.15 Moreover, previous
studies carried out on the influence of a methyl group
inserted at various positions on the photosensitizing
† Department of Pharmaceutical Sciences.
‡ Centro di Studio sulla Chimica del Farmaco e dei Prodotti
Biologicamente Attivi del CNR.
10.1021/jm991028s CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/13/1999

Synthesis and Activity of Benzopsoralens
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2937
Sch em e 1
Sch em e 2
activity of both linear and angular furocoumarins17,18
suggested that the introduction of the hydroxymethyl
group variously in the benzopsoralen molecule could
also modulate the activity and features of these drugs.
We therefore studied the main biological properties
of a series of tetracyclic psoralen derivatives carrying a
hydroxymethyl group at the 3, 5, 8, or 11 position. We
also studied another series of benzopsoralens bearing
a diethylaminomethyl substituent at the same positions;
this group was introduced into the benzopsoralen mol-
ecule with the aim of improving its capacity to interact
with DNA and, as a result, of increasing its activity in
the dark.
We tested all these compounds for their activity at
the ground state, that is, by simple incubation in the
dark, and upon UVA irradiation. As reference com-
pounds we chose 8-MOP, a well-known photosensitizing
furocoumarin widely used in photobiology and in pho-
tomedicine, and 4-(hydroxymethyl)benzopsoralen, as the
most interesting tetracyclic psoralen derivative now
known.16
Bromination of 4,8-dimethylbenzopsoralen (4d ) occurs
only in the 8 position, because the 4-methyl group is
unaffected by substitution reactions.20 Intermediates
5a -d were submitted to acetylation, yielding the cor-
responding acetoxymethyl groups, which were hydro-
lyzed in alkaline conditions to the desired (hydroxy-
methyl)benzopsoralens 7a -d . Direct alkaline hydrolysis
of 5a -d to 7a -d was carried out, but the reaction time
was longer and the yield lower than indirect hydrolysis
through 6a -d , owing to the formation of many degra-
dation products. Alternatively, compounds 5a -d were
reacted with diethylamine to give the corresponding
(diethylaminomethyl)benzopsoralens 8a -d . Figure 1
shows the molecular structure of the two reference
compounds, 8-methoxypsoralen (8-MOP) and 4-(hy-
droxymethyl)benzopsoralen (BP).
Sp ectr op h otom etr ic P r op er ties. Table 1 shows the
spectrophotometric properties of the new benzopsor-
alens; because they are compounds designed for photo-
chemotherapy and photopheresis, the most important
wavelength range is 320-400 nm, and in particular 365
nm, the maximum of our UVA lamp emission. All benzo-
psoralens exhibit good absorption at this wavelength,
with values similar to those of BP and higher than those
of 8-MOP (the extinction coefficients of both compounds
are reported in Table 1 for comparison). This result may
be associated with higher photosensitizing activity.
Ch em istr y
The synthetic route consists of building the tetracyclic
nucleus already carrying the methyl group in the
appropriate position, and then transforming the methyl
function into a hydroxymethyl or diethylaminomethyl
one. According to Scheme 1, starting materials were
methyl-7-hydroxycoumarins 1a -d , which were con-
densed with 2-chlorocyclohexanone to give the corre-
sponding 7-O-(2′-oxocyclohexyloxy) ethers 2a -c or with
2-bromo-4-methylcyclohexanone to give ether 2d . Com-
pounds 2a -d were submitted to cyclization in alkaline
medium,19 yielding methyltetrahydrobenzopsoralens 3a-
d , which were aromatized in toluene solution by 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to the cor-
responding methylbenzopsoralens 4a -d .
In ter a ction w ith DNA in Vitr o
In h ibition of Top oisom er a se II. The ability of
benzopsoralens to inhibit topoisomerase II was studied
using the relaxation test of supercoiled DNA of PM2
phage, followed by electrophoretic separation on agarose
gel. The data are reported in Figure 2. After incubation
in the presence of topoisomerase II, the supercoiled form
completely disappeared, but after incubation in the
According to Scheme 2, compounds 4a -d were re-
acted with N-bromosuccinimide, yielding bromination
of the methyl substituents, giving bromomethyl groups.

2938 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Chilin et al.
Ta ble 1. Spectrophotometric Properties of the New
Benzopsoralen Derivatives
wavelength
(nm)
extinction coefficient
compd
(M-1‚cm-1
)
7a
365
333
269
250
1570
15040
27690
31230
7b
7c
7d
365
339
287
270
252
365
337
287
270
250
365
337
270
250
365
365
336
287
271
250
365
338
288
270
250
365
336
287
271
250
365
337
270
252
365
4200
13050
20000
27350
26850
2970
9990
F igu r e 2. Inhibition of topoisomerase II activity studied by
the DNA relaxation assay. Supercoiled PM2 DNA (0.125 µg)
was incubated with topoisomerase II from D. melanogaster
embryos in the presence of different concentrations of com-
pound 7b or 7c. Agarose gel electrophoresis of the samples is
shown. Lane 1: PM2 DNA alone, which migrates as two bands.
Lane 2: PM2 DNA incubated in the presence of topoisomerase
II, so showing only the relaxed form (the slower band). Lanes
3, 4: PM2 DNA incubated in the presence of topoisomerase II
and compound 7b (10 and 1 µM, respectively). Lanes 5, 6: PM2
DNA incubated in the presence of topoisomerase II and
compound 7c (10 and 1 µM, respectively). Lane 7: PM2 DNA
incubated in the presence of topoisomerase II and BP, used
as a reference (10 µM). The arrow indicates gel mobility.
18010
24160
24210
3400
15940
31030
29760
3050
BP
8a
3080
15620
23550
29080
31010
3890
15080
30470
32140
31050
3040
12010
23210
30880
29570
3580
15000
29660
32070
990
8b
8c
8d
8-MOP
presence of compounds 7b and 7c, the band correspond-
ing to the supercoiled form of PM2 DNA was comparable
to that of the control sample. The same migration
pattern was obtained with BP (10 µM), used in the same
experimental conditions as a reference. This derivative
had already been assayed for the same activity, but by
a slightly different assay.16 All the benzopsoralen de-
rivatives were assayed in biological studies, but some-
times, to simplify data display, only the most significant
ones are reported.
F or m a tion of In ter str a n d Cr oss-Lin k s in DNA
by UVA Ir r a d ia tion . The induction of interstrand
cross-links by photosensitization in PM2-linearized
DNA in vitro was tested. The results obtained with
compound 7c and with BP and 8-MOP, used as refer-
ences, are reported in Figure 3. The DNA treated with
compound 7c produced only the band for single-stranded
DNA, identical to that of the reference nontreated DNA,
thus suggesting its complete inability to induce inter-
strand cross-links. As expected, BP gave very similar
results, whereas a significant amount of DNA irradiated
in the presence of 8-MOP appeared to be cross-linked,
and its amount increased with the UVA dose.
F igu r e 3. Interstrand cross-links formed in vitro in PM2-
linearized DNA by sensitization with benzopsoralen deriva-
tives (2 drug molecules per base pair) detected by electro-
phoresis. 8-MOP and BP were also tested in the same
experimental conditions. Lane 1: double-stranded DNA. Lane
2: denatured single-stranded DNA. Lanes 3, 4: DNA irradi-
ated in the presence of 8-MOP and exposed to 3 and 5 kJ ‚m-2
,
respectively. Lanes 5, 6: DNA irradiated in the presence of
compound 7c and exposed to 3 and 5 kJ ‚m-2, respectively.
Lanes 7, 8: DNA irradiated in the presence of BP and exposed
to 3 and 5 kJ ‚m-2, respectively. The arrow indicates the
direction of the electrophoretic migration.
experimental conditions: by incubation at the ground
state keeping cells in the dark or by UVA irradiation.
In both cases, inhibition of DNA synthesis and of clonal
growth was evaluated.
At th e Gr ou n d Sta te. Table 2 summarizes the
results obtained studying the antiproliferative activity
of benzopsoralens in the dark. The most active com-
pound appears to be BP, but compound 7c also induced
an evident antiproliferative effect; compounds 7b and
Exp er im en ts w ith Ma m m a lia n Cells
The antiproliferative activity of the new benzopsor-
alens on mammalian cells was assayed by two different

Synthesis and Activity of Benzopsoralens
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2939
Ta ble 2. Antiproliferative Activity of the New Benzopsoralens
in the Dark
IC50-dark ( SDa
compd
DNA synthesis
clonogenic assay
8-MOP
BP
7a
7b
7c
7d
8a
8b
8c
>30
>30
0.94 ( 0.23
>30
3.8 ( 1.2
>30
10.9 ( 0.31
3.0 ( 0.18
>30
>30
>30
10.7 ( 2
>30
25 ( 3.9
6.8 ( 1.9
>30
>30
>30
10.7 ( 2.1
>30
8d
a
The IC50-dark is the drug concentration (µM) necessary to
induce a 50% inhibition; the data were computed by probit analysis
and are reported together with the standard deviation.
F igu r e 4. Mutagenicity in E. coli TM9 after sensitization with
benzopsoralens. Bacteria were exposed to increasing UVA light
doses in the presence of benzopsoralens (20 µM). The surviving
fraction and the number of mutants/million survivors were
determined. Results are reported plotting revertants against
the surviving fraction obtained in the same treatment: ∆, 7a ;
O, 7b; 0, 7c; b, BP; 9, 8-MOP. The data related to the other
compounds are not shown for clarity and because they are
practically superimposable to that reported.
Ta ble 3. Antiproliferative Activity of the New Benzopsoralens
by UVA Irradiation
IC50-UVA ( SDa
compd
DNA synthesis
clonogenic assay
8-MOP
BP
7a
7b
7c
7d
8a
8b
8c
3.59 ( 0.12
0.67 ( 0.27
16.8 ( 0.37
8.3 ( 0.23
0.97 ( 0.12
0.48 ( 0.13
>30
1.23 ( 0.21
0.11 ( 0.03
1.06 ( 0.18
0.23 ( 0.02
0.19 ( 0.02
0.16 ( 0.025
>30
>30
>30
9.3 ( 0.3
2.22 ( 0.17
0.65 ( 0.19
1.83 ( 0.23
8d
a
The IC50-UVA is the dose of UVA light (kJ ‚m-2) which in the
presence of a fixed drug concentration (20 µM) induces a 50%
inhibition; the data were computed by probit analysis and are
reported together with the standard deviation.
8c showed moderate activity, while 7a , 7d , 8a , 8b, and
8d were practically inactive.
By UVA Ir r a d ia tion . Table 3 shows the data ob-
tained by UVA irradiation; these experiments were
carried out in the presence of a fixed compound concen-
tration (20 µM) and increasing UVA doses. Therefore,
in this case, an IC50 is the UVA dose which induces 50%
inhibition when delivered in the presence of the com-
pound studied. In addition, care was taken to avoid a
dark effect, as described in the Experimental Section.
Control experiments with UVA light alone were also
carried out, without any detectable effects (data not
shown). In these experimental conditions the new
benzopsoralens showed pronounced antiproliferative
activity; only two compounds appeared to be inactive,
8a and 8b. Again, BP appeared to be the most effective
derivative, but compounds 7c and 7d showed compa-
rable activity; even 7a , 8c, and 8d induced evident
antiproliferative effects.
F igu r e 5. Formation of cytoplasmic ,petite. mutants in S.
cerevisiae D7 after sensitization with benzopsoralens. Yeast
cells were irradiated with increasing UVA light doses in the
presence of benzopsoralens (20 µM). The number of mutated
colonies was plotted against the surviving fraction: O, 7b; 0,
7c; b, BP; 9, 8-MOP.
alens (data relating to other derivatives are omitted for
clarity) were capable of forming very similar levels of
revertants. Instead, 8-MOP, tested in the same experi-
mental conditions, appeared to be much more effective,
forming a higher number of mutants: this result may
be explained by recalling its ability to induce bifunc-
tional adducts in DNA.22
Mu ta gen ic Activity
The genotoxic activity of benzopsoralens was detected
in E. coli TM9, a strain very sensitive to the formation
of C4-cycloadducts of furocoumarins.21 These experi-
ments were also carried out at a fixed concentration of
test compound (20 µM) and increasing the UVA dose.
Figure 4 shows the results obtained with 7a , 7b, 7c,
BP, and 8-MOP, plotting the number of revertants per
million survivors against the surviving fraction; thus,
genotoxic activity is estimated at the same level of
antiproliferative effect. Practically all the benzopsor-
Exp er im en ts w ith Yea st
In d u ction of Cytop la sm ic ,P etite. Mu ta tion s.
Figure 5 shows the induction of cytoplasmic ,petite.
mutants in a diploid wild-type (D7) strain of Saccharo-
myces cerevisiae as a function of survival after exposure
to increasing doses of UVA light in the presence (20 µM)
of compounds 7b, and 7c; 8-MOP and BP were used as
references. Unlike 8-MOP, the mutagenic activity of
benzopsoralens on mitochondrial DNA is very high; at

2940 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Chilin et al.
Ta ble 4. Phototoxicity on Guinea Pig Skin
UVA dose
intensity of
erythemaa
compd
µM‚cm-2
(kJ ‚m-2
)
BP
7a
7b
7c
7d
8a
8b
8c
8d
30
30
30
30
30
30
30
30
30
2
20
20
20
20
20
20
20
20
20
5
- - - -
- - - -
- - - -
- - - -
- - - -
- - - -
- - - -
- - - -
- - - -
- - - -
+ - - -
+ + + +
8-MOP
4
30
5
5
a
Symbols: - - - -, no erythema; + - - -, barely visible
erythema; + + - -, light erythema; + + + -, medium erythema;
+ + + +, strong erythema with edema.
lesions. Even failing quantitative DSB determinations,
it is clear that benzopsoralens are capable of inducing
high DNA fragmentation. This observation, together
with their monofunctional behavior, indicates that the
number of monoadducts forming by benzopsoralens in
DNA is very high.
F igu r e 6. Induction of DNA double-strand breaks during
posttreatment incubation of diploid yeast cells. Lane 1: un-
treated control. Lane 2: untreated control + 3 h of incubation.
Lanes 3-5: cells treated with compound 7c (1 µM) and UVA
(3 kJ ‚m-2) + no, + 15 min, and + 3 h of incubation,
respectively. Lanes 6-8: cells treated with BP (1 µM) and
UVA (3 kJ ‚m-2) + no, + 15 min, and + 3 h of incubation,
respectively.
Sk in P h ototoxicity
Skin phototoxicity was detected on albino guinea pig
skin (Table 4); despite the severe experimental condi-
tions used (compound concentration 30 µM‚cm-2; UVA
light dose 20 kJ ‚m-2) no erythemas were observed with
any of the compounds tested, except 8-MOP, used as a
positive reference. These results are consistent with the
hypothesis of an association between the induction of
bifunctional damage and the formation of skin erythe-
mas.
50% of survival, BP, 7b, 7c, and 8-MOP produced
respectively about 62%, 45%, 40%, and 5% of cytoplas-
mic ,petite. mutations. These results fit the mono-
functional activity of the new benzopsoralens. In fact,
as is known, damage to mitochondrial DNA is not
repaired; therefore, while interstrand cross-links form-
ing in mitochondrial DNA are nearly always lethal,
monoadducts can bypass and therefore induce mutants.
Conversely, both lesions forming in chromosomal DNA
are repaired, and thus the strong genotoxic activity of
interstrand cross-links can be scored.23
In d u ction of DNA Dou ble-Str a n d Br ea k s. Using
pulsed-field gel electrophoresis (PFGE), the induction
of double-strand breaks in DNA (DSB) in yeast strain
D7 was determined. Figure 6 shows the data obtained
by UVA irradiation in the presence of BP or 7c. After
treatment, but without any incubation after treatment
(lanes 3, 6), the 12 bands corresponding to the 16 yeast
chromosomes are clearly visible and their intensities are
comparable with those obtained with untreated controls
(lane 1). Conversely, when allowing for repair of poten-
tial lethal damage by holding treated cells in complete
growth medium, a significant increase in DSB was
observed. In fact, after 15 min of incubation postsensi-
tization with compound 7c or BP (lanes 4, 7), the first
7 chromosomal bands are scarcely visible; moreover,
after 3 h of incubation, all bands totally disappeared
(lanes 5, 8). These data suggest that neither compound
7c nor BP directly induced DSB, which are formed by
the enzymatic repair of the lesions induced in yeast
DNA by the two benzopsoralen derivatives. This result
is consistent with the data already obtained:24 it is
suggested that bifunctional damage (interstrand cross-
links) induces much more DSB in DNA than the mono-
functional one and that this fact is related to the dif-
ferent pathways of DNA repair of these two kinds of
Con clu sion s
The presence of a hydroxymethyl or diethylaminom-
ethyl group at various positions in the benzopsoralen
skeleton confers good antiproliferative activity on the
molecule. However, the results are somewhat different
according to the specific position involved and the
experimental conditions considered, i.e., with or without
UVA activation.
In fact, upon incubation in the dark, derivatives
carrying the hydroxymethyl group at the 4, 5, or 11
position (BP, 7b, 7c) showed more pronounced antipro-
liferative activity, although to different extents; among
the diethylaminomethyl series, only the 11-substituted
derivative (8c) is effective. This activity appeared to be
related to the capacity of such derivatives to inhibit
topoisomerase II, as demonstrated in an in vitro assay.
In general, upon UVA irradiation, benzopsoralens
induce higher antiproliferative effects than in the dark.
The same derivatives regarding dark activity (BP, 7b,
7c, 8c) appear to be good photosensitizing drugs;
however, compounds 7d and 8d also showed marked
activity. Among the 3-substituted benzopsoralens, only
compound 7a induced a significant antiproliferative
effect, especially in the clonogenic assay.
Summarizing, in general we can say that, in both
experimental conditions, in the dark and upon UVA
irradiation, the hydroxymethyl group is a slightly better
substituent than the diethylaminomethyl one. In the
dark, the 4 position is certainly the best choice for the

Synthesis and Activity of Benzopsoralens
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2941
hydroxymethyl-substituted compounds, followed by po-
sitions 11 and 5; in the same experimental conditions,
only the 11-diethylaminomethyl derivative was active.
psoralens upon UVA irradiation do not form DSBs;
because DSBs can be formed by DNA repair,26 their
formation by incubating yeast cells after photosensiti-
zation with benzopsoralens was also studied. In these
experimental conditions, the formation of DSBs, which
are very probably formed by an enzymatic process of
DNA repair, was observed. This result is consistent with
the data already obtained by Averbeck et al.;25 consider-
ing the reduced DSB frequency by monofunctional
damage,25 together with the high DNA fragmentation
observed, we can conclude that benzopsoralens can
induce a very high amount of monoadducts into DNA.
Last, we studied benzopsoralen phototoxicity in guinea
pig skin, as the ability of bifunctional furocoumarins
such as 8-methoxypsoralen to induce severe erythemas
on the skin is a well-known and serious side effect of
PUVA therapy.11 Benzopsoralens appeared to be en-
tirely unable to cause skin phototoxicity, even when
tested in harsh experimental conditions.
Considering all these results, we can say that ben-
zopsoralen derivatives are interesting and active po-
tential drugs for PUVA therapy and photopheresis,
showing good activity together with reduced side effects
such as genotoxicity and skin phototoxicity in compari-
son with 8-methoxypsoralen, the drug presently used
in this field. The activity shown by benzopsoralens in
the dark is also interesting, although it is not sufficient
to suggest their use as antitumor drugs. However,
benzopsoralens can be good models for the study of new
drugs having antitopoisomerase activity.
Upon UVA activation, again BP was the most effec-
tive derivative (even more than 8-MOP), but compound
7c (having the hydroxymethyl group at the 11 position)
showed similar activity, immediately followed by 7b.
Instead, compound 7a , substituted at the 3 position, was
poorly active. This last result is not surprising, consid-
ering the already known data on the photosensitizing
activity of methylpsoralens17 and methylangelicins.18
The insertion of the substituent at the 3 position
strongly reduces the sensitizing potency of the molecule,
attributed to steric hindrance generated by the methyl
group introduced near the photoreactive 3,4 double
bond. It is probably the same for the hydroxymethyl
group. Instead, the 5 and 11 positions in benzopsoralens
correspond to the 5 and 8 positions in psoralens; the
insertion of a methyl or hydroxymethyl group yields
active photosensitizers.
With diethylaminomethyl-substituted derivatives, posi-
tive results were obtained only with compounds 8c and
8d . On the basis of these results, we can say that the
kind of substituent and the position in which it is
inserted are both very important for the best activity.
Because the formation of interstrand cross-links (ISC)
in DNA is postulated to be mainly responsible for PUVA
toxicity, we tested the ability of the new benzopsoralens
to induce this lesion by an in vitro assay, using linear-
ized DNA of PM2 plasmid and electrophoresis; all
benzopsoralens appeared to be entirely incapable of
inducing ISC. This result is consistent with the hypoth-
esis of a complete block of the photoreactive site at the
furan side by fusing the fourth benzene nucleus. To
confirm the monofunctional behavior of benzopsoralens
and to check their genotoxicity, additional experiments
on bacteria were carried out. E. coli TM9 was chosen, a
strain reverted by base substitution mutagens. Because
it is defective in DNA repair (uvrA) and contains a
plasmid (R46) coding error-prone DNA repair, it is very
sensitive to the formation of furocoumarin C4-cycload-
ducts. In such a system, benzopsoralens showed very
similar, low mutagenic activity, while 8-methoxypsor-
alen, the drug currently used in human therapy (used
here as a reference), appeared to be a very strong
mutagen. This result may be explained by considering
the ability of 8-methoxypsoralen to induce ISC, unlike
benzopsoralens, which cannot form this lesion.
Benzopsoralens were also studied in yeast, scoring the
cytoplasmic ,petite. mutations, as consequences of
damage to mitochondrial DNA. It is well-known that
these mutations are better induced by monofunctional
damage.24 In fact, with bifunctional compounds (e.g.,
8-methoxypsoralen), the main effects which are scored
are the levels of chromosomal DNA.25 Therefore, the
high activity of benzopsoralens in inducing ,petite.
mutations consists of the induction of a monofunctional
damage in DNA.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Gallen-
kamp MFB-595-010M melting point apparatus and are uncor-
rected. Analytical TLC was performed on precoated 60 F254
silica gel plates (0.25 mm; Merck) developing with an EtOAc/
cyclohexane mixture (1:1) unless overwise indicated. Prepara-
tive column chromatography was performed using silica gel
60 (0.063-0.100 mm; Merck), eluting with CHCl3. 1H NMR
spectra were recorded on a Varian Gemini-200 spectrometer
with TMS as internal standard. UV spectra were taken in
absolute ethanol using a Kontron UVIKON-930 UV-visible
spectrophotometer. Elemental analyses were obtained on all
intermediates and are within (0.4% of theoretical values.
Starting 7-hydroxycoumarins 1b27 and 1c28 were prepared
according to literature methods.
3-Meth yl-7-h yd r oxycou m a r in (1a ). A mixture of 2,4-
dihydroxybenzaldehyde (25.0 g, 181.0 mmol), propionic anhy-
dride (50.8 g, 50.0 mL, 389.9 mmol), sodium propionate (25.0
g), and piperidine (0.5 mL) was refluxed until aldehyde
disappeared (1 h, TLC). H2SO4 (25 mL) was cautiously added
to the reaction mixture, and the solution was kept at room
temperature for 0.5 h. The mixture was poured into cold water
(2 L), and the precipitate was collected, washed with water,
and crystallized from MeOH to give 1a (12.7 g, 40%): mp 232
°C; 1H NMR (DMSO-d6) δ 10.40 (br s, 1H, -OH), 7.77 (q, 1H,
J ) 1.2 Hz, 4-H), 7.75 (d, 1H, J ) 8.4 Hz, 5-H), 6.78 (dd, 1H,
J ) 8.4, 2.3 Hz, 6-H), 6.71 (d, 1H, J ) 2.3 Hz, 8-H), 2.05 (d,
3H, J ) 1.2 Hz, 3-Me). Anal. (C10H8O3) C,H.
Meth yl-7-(2′-oxocycloh exyloxy)cou m a r in s 2a -d . Gen -
er a l P r oced u r e. A mixture of 1 (70.0 mmol), 2-chlorocyclo-
hexanone (105.0 mmol), and anhydrous K2CO3 (40.0 g) in
acetone (800 mL) was refluxed until 1 disappeared (72 h, TLC).
After cooling, the solid was filtered off and washed with fresh
acetone. The solvent was evaporated from the combined filtrate
and washings, and the residue was crystallized from MeOH
to give 2.
Using the technique of pulse field electrophoresis, the
formation of double-strand breaks (DSB) was checked
in yeast chromosomal DNA. DSBs are known to be
severe lesions with very dangerous consequences, like
the formation of chromosomal aberrations.25 We ascer-
tained that photochemical reactions caused by benzo-
3-Meth yl-7-(2′-oxocycloh exyloxy)cou m a r in (2a ): yield
1
75%; mp 184 °C; H NMR (CDCl3) δ 7.43 (q, 1H, J ) 1.3 Hz,

2942 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Chilin et al.
4-H), 7.29 (d, 1H, J ) 8.5 Hz, 5-H), 6.80 (dd, 1H, J ) 8.5, 2.4
Hz, 6-H), 6.69 (d, 1H, J ) 2.4 Hz, 8-H), 4.72 (dd, 1H, J ) 9.9,
5.5 Hz, 1′-H), 2.66-2.34 (m, 4H, 3′-H and 6′-H), 2.16 (d, 3H, J
) 1.3 Hz, 3-Me), 2.13-1.74 (m, 4H, 4′-H and 5′-H). Anal.
(C16H16O4) C,H.
7.5, 1.5, 0.7 Hz, 9-H), 7.95 (s, 1H, 5-H), 7.95 (qd, 1H, J ) 1.4,
0.6 Hz, 4-H), 7.59 (ddd, 1H, J ) 8.1, 1.4, 0.7 Hz, 6-H), 7.51 (d,
1H, J ) 0.6 Hz, 11-H), 7.50 (ddd, 1H, J ) 8.1, 7.1, 1.5 Hz,
7-H), 7.39 (ddd, 1H, J ) 7.5, 7.1, 1.4 Hz, 8-H), 2.26 (d, 3H, J
) 1.4 Hz, 3-Me). Anal. (C16H10O3) C,H.
5-Meth yl-7-(2′-oxocycloh exyloxy)cou m a r in (2b): yield
71%; mp 178 °C; H NMR (CDCl3) δ 7.80 (d, 1H, J ) 9.9 Hz,
5-Meth yl-2H-ben zofu r o[3,2-g]-1-ben zopyr an -2-on e (4b):
yield 87%; mp 241 °C; 1H NMR (CDCl3) δ 8.04 (d, 1H, J ) 9.9
Hz, 4-H), 8.00 (ddd, 1H, J ) 7.2, 1.4, 0.7 Hz, 9-H), 7.54 (ddd,
1H, J ) 8.0, 1.5, 0.7 Hz, 6-H), 7.47 (ddd, 1H, J ) 8.0, 6.9, 1.4
Hz, 7-H), 7.37 (ddd, 1H, J ) 7.2, 6.9, 1.5 Hz, 8-H), 7.27 (s, 1H,
11-H), 6.36 (d, 1H, J ) 9.9 Hz, 3-H), 2.87 (s, 3H, 5-Me). Anal.
(C16H10O3) C,H.
1
4-H), 6.68 (d, 1H, J ) 2.2 Hz, 8-H), 6.51 (d, 1H, J ) 2.2 Hz,
6-H), 6.24 (d, 1H, J ) 9.9 Hz, 3-H), 4.73 (dd, 1H, J ) 9.5, 5.5
Hz, 1′-H), 2.65-2.35 (m, 4H, 3′-H and 6′-H), 2.45 (s, 3H, 5-Me),
2.09-1.73 (m, 4H, 4′-H and 5′-H). Anal. (C16H16O4) C,H.
8-Meth yl-7-(2′-oxocycloh exyloxy)cou m a r in (2c): yield
1
72%; mp 171 °C; H NMR (DMSO-d6) δ 7.97 (d, 1H, J ) 9.4
11-Meth yl-2H-ben zofu r o[3,2-g]-1-ben zopyr an -2-on e (4c):
1
Hz, 4-H), 7.47 (d, 1H, J ) 8.8 Hz, 5-H), 6.89 (d, 1H, J ) 8.8
Hz, 6-H), 6.29 (d, 1H, J ) 9.4 Hz, 3-H), 5.23 (dd, 1H, J ) 9.7,
5.7 Hz, 1′-H), 2.66-2.29 (m, 4H, 3′-H and 6′-H), 2.23 (s, 3H,
8-Me), 2.07-1.55 (m, 4H, 4′-H and 5′-H). Anal. (C16H16O4) C,H.
4-Met h yl-7-(2′-oxo-5′-m et h ylcycloh exyloxy)cou m a r in
(2d ). This compound was prepared according to the general
procedure, using 2-bromo-4-methylcyclohexanone. This re-
agent was obtained reacting pyrrolidone hydrotribromide (57.0
g, 115.0 mmol) with 4-methylcyclohexanone (12.9 g, 14.1 mL,
115.0 mmol) in THF (400 mL) at room temperature for 6 h,
filtering off the solid and removing the solvent from the filtrate
under reduced pressure: the residue so obtained was used
without further purification. Compound 2d : yield 87%; mp
207 °C; 1H NMR (CDCl3) δ 7.48 (d, 1H, J ) 8.8 Hz, 5-H), 6.84
(dd, 1H, J ) 8.8, 2.5 Hz, 6-H), 6.65 (d, 1H, J ) 2.5 Hz, 8-H),
6.13 (q, 1H, J ) 1.2 Hz, 3-H), 4.83 (dd, 1H, J ) 12.2, 6.2 Hz,
1′-H), 2.52 (m, 2H, 3′-H), 2.43 (m, 1H, 6′-H), 2.38 (d, 3H, J )
1.2 Hz, 4-Me), 2.16-2.04 (m, 1H, 4′-H), 1.78-1.39 (m, 3H, 6′-H
and 5′-H), 1.12 (d, 3H, J ) 6.3 Hz, 5′-Me). Anal. (C17H18O4)
C,H.
yield 76%; mp 224 °C; H NMR (CDCl3) δ 7.94 (ddd, 1H, J )
7.5, 1.5, 0.8 Hz, 9-H), 7.85 (s, 1H, 5-H), 7.84 (d, 1H, J ) 9.5
Hz, 4-H), 7.61 (ddd, 1H, J ) 8.1, 1.3, 0.8 Hz, 6-H), 7.50 (ddd,
1H, J ) 8.1, 7.2, 1.4 Hz, 7-H), 7.38 (ddd, 1H, J ) 7.5, 7.2, 1.3
Hz, 8-H), 6.40 (d, 1H, J ) 9.5 Hz, 3-H), 2.67 (s, 3H, 11-Me).
Anal. (C16H10O3) C,H.
4,8-Dim e t h yl-2H -b e n zofu r o[3,2-g]-1-b e n zop yr a n -2-
1
on e (4d ): yield 88%; mp 237 °C; H NMR (CDCl3) δ 7.97 (s,
1H, 5-H), 7.79 (d, 1H, J ) 7.9 Hz, 6-H), 7.40 (s, 1H, 11-H),
7.34 (dq, 1H, J ) 0.7, 0.7 Hz, 9-H), 7.19 (dd, 1H, J ) 7.9, 0.7
Hz, 7-H), 6.25 (q, 1H, J ) 1.2 Hz, 3-H), 2.53 (d, 3H, J ) 0.7
Hz, 8-Me), 2.52 (d, 3H, J ) 1.2 Hz, 4-Me). Anal. (C17H12O3)
C,H.
(Br om om et h yl)-2H-b en zofu r o[3,2-g]-1-b en zop yr a n -2-
on es 5a -d . Gen er a l P r oced u r e. A mixture of 4 (25 mmol)
and N-bromosuccinimide (37.5 mmol) in anhydrous benzene
(500 mL) was refluxed until starting product had disappeared
(10-24 h, TLC: CHCl3/MeOH, 95:5). After cooling, the solvent
was evaporated under reduced pressure. The residue was
purified by column chromatography and crystallized from
EtOAc to give 5.
Meth yl-6,7,8,9-tetr a h yd r o-2H-ben zofu r o[3,2-g]-1-ben -
zop yr a n -2-on es 3a -d . Gen er a l P r oced u r e.19 To an etha-
nolic solution (200 mL) of 2 (50.0 mmol) was added a 5%
ethanolic potassium hydroxide solution (600 mL), and the
mixture was refluxed in the dark for 1 h. The solution was
cooled, diluted with water (1 L), and acidified with diluted HCl.
The precipitate obtained was collected and crystallized from
MeOH, to give 3.
3-Meth yl-6,7,8,9-tetr a h yd r o-2H-ben zofu r o[3,2-g]-1-ben -
zop yr a n -2-on e (3a ): yield 85%; mp 195 °C; 1H NMR (CDCl3)
δ 7.58 (q, 1H, J ) 1.2 Hz, 4-H), 7.36 (s, 1H, 5-H), 7.34 (s, 1H,
11-H), 2.77-2.70 (m, 2H, 9-H), 2.66-2.59 (m, 2H, 6-H), 2.21
(d, 3H, J ) 1.2 Hz, 3-Me), 1.98-1.83 (m, 4H, 7-H and 8-H).
Anal. (C16H14O3) C,H.
5-Meth yl-6,7,8,9-tetr a h yd r o-2H-ben zofu r o[3,2-g]-1-ben -
zop yr a n -2-on e (3b): yield 57%; mp 201 °C; 1H NMR (CDCl3)
δ 7.97 (d, 1H, J ) 9.9 Hz, 4-H), 7.16 (s, 1H, 11-H), 6.30 (d, 1H,
J ) 9.9 Hz, 3-H), 2.89-2.83 (m, 2H, 9-H), 2.74-2.69 (m, 2H,
6-H), 2.67 (s, 3H, 5-Me), 1.95-1.83 (m, 4H, 7-H and 8-H). Anal.
(C16H14O3) C,H.
11-Met h yl-6,7,8,9-t et r a h yd r o-2H -b en zofu r o[3,2-g]-1-
ben zop yr a n -2-on e (3c): yield 68%; mp 182 °C; 1H NMR
(CDCl3) δ 7.77 (d, 1H, J ) 9.5 Hz, 4-H), 7.28 (s, 1H, 5-H), 7.33
(d, 1H, J ) 9.5 Hz, 3-H), 2.80-2.73 (m, 2H, 9-H), 2.66-2.59
(m, 2H, 6-H), 2.57 (s, 3H, 11-Me), 1.99-1.85 (m, 4H, 7-H and
8-H). Anal. (C16H14O3) C,H.
4,8-Dim eth yl-6,7,8,9-tetr a h yd r o-2H-ben zofu r o[3,2-g]-1-
ben zop yr a n -2-on e (3d ): yield 82%; mp 192 °C; 1H NMR
(CDCl3) δ 7.54 (s, 1H, 5-H), 7.36 (s, 1H, 11-H), 6.24 (q, 1H, J
) 1.2 Hz, 3-H), 2.91-1.93 (m, 6H, 6-H, 7-H, 8-H and 9-H),
2.50 (d, 1H, J ) 1.2 Hz, 4-Me), 1.58-1.46 (m, 1H, 8-H), 1.16
(d, 1H, J ) 6.6 Hz, 8-Me). Anal. (C17H16O3) C,H.
Meth yl-2H-ben zofu r o[3,2-g]-1-ben zop yr a n -2-on es 4a -
d . Gen er a l P r oced u r e. A mixture of 3 (30 mmol) and 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (75 mmol) in anhydrous
toluene (800 mL) was refluxed for 24 h. After cooling, the solid
was filtered off and the solvent evaporated under reduced
pressure. The residue was purified by column chromatography
and crystallized from MeOH to give 4.
3-(Br om om et h yl)-2H-b en zofu r o[3,2-g]-1-b en zop yr a n -
2-on e (5a ): yield 81%; mp 262 °C; 1H NMR (CDCl3) δ 8.04 (s,
1H, 5-H), 8.02 (dt, 1H, J ) 1.3, 0.6 Hz, 4-H), 7.97 (ddd, 1H, J
) 7.6, 1.5, 0.7 Hz, 9-H), 7.61 (ddd, 1H, J ) 8.2, 1.4, 0.7 Hz,
6-H), 7.54 (d, 1H, J ) 0.6 Hz, 11-H), 7.53 (ddd, 1H, J ) 8.2,
7.1, 1.5 Hz, 7-H), 7.41 (ddd, 1H, J ) 7.6, 7.1, 1.4 Hz, 8-H),
4.49 (d, 2H, J ) 1.3 Hz, 3-CH2Br). Anal. (C16H9BrO3) C,H,Br.
5-(Br om om et h yl)-2H-b en zofu r o[3,2-g]-1-b en zop yr a n -
2-on e (5b): yield 76%; mp 253 °C; 1H NMR (CDCl3) δ 8.16
(dd, 1H, J ) 9.9, 0.7 Hz, 4-H), 8.12 (ddd, 1H, J ) 7.5, 1.6, 0.8
Hz, 9-H), 7.65 (ddd, 1H, J ) 8.0, 1.7, 0.8 Hz, 6-H), 7.58 (ddd,
1H, J ) 8.0, 6.9, 1.6 Hz, 7-H), 7.52 (d, 1H, J ) 0.7 Hz, 11-H),
7.48 (ddd, 1H, J ) 7.5, 6.9, 1.7 Hz, 8-H), 6.56 (d, 1H, J ) 9.9
Hz, 3-H), 5.15 (s, 2H, 5-CH2Br). Anal. (C16H9BrO3) C,H,Br.
11-(Br om om eth yl)-2H-ben zofu r o[3,2-g]-1-ben zop yr a n -
2-on e (5c): yield 72%; mp 267 °C; 1H NMR (CDCl3) δ 7.96 (s,
1H, 5-H), 7.94 (dd, 1H, J ) 7.5, 1.4 Hz, 9-H), 7.83 (d, 1H, J )
9.6 Hz, 4-H), 7.66 (dd, 1H, J ) 8.1, 1.2 Hz, 6-H), 7.53 (ddd,
1H, J ) 8.1, 7.3, 1.4 Hz, 7-H), 7.41 (ddd, 1H, J ) 7.5, 7.3, 1.2
Hz, 8-H), 6.43 (d, 1H, J ) 9.6 Hz, 3-H), 5.04 (s, 2H, 11-CH2-
Br). Anal. (C16H9BrO3) C,H,Br.
8-(Br om om eth yl)-4-m eth yl-2H-ben zofu r o[3,2-g]-1-ben -
zop yr a n -2-on e (5d ): yield 57%; mp 245 °C; 1H NMR (CDCl3)
δ 8.11 (s, 1H, 5-H), 7.94 (dd, 1H, J ) 8.0, 0.6 Hz, 6-H), 7.63
(dd, 1H, J ) 1.5, 0.6 Hz, 9-H), 7.51 (s, 1H, 11-H), 7.43 (dd,
1H, J ) 8.0, 1.5 Hz, 7-H), 6.31 (q, 1H, J ) 1.3 Hz, 3-H), 4.67
(s, 2H, 8-CH2Br), 2.58 (d, 3H, J ) 1.3 Hz, 4-Me). Anal. (C17H11
BrO3) C,H,Br.
-
(Acetoxym eth yl)-2H-ben zofu r o[3,2-g]-1-ben zop yr a n -2-
on es 6a -d . Gen er a l P r oced u r e. A mixture of 5 (10 mmol)
and anhydrous AcONa (1.0 g) in acetic anhydride (50 mL) was
refluxed for 1 h. The mixture was cautiously diluted with water
(50 mL) and poured into cold water (250 mL). The precipitate
obtained was filtered, washed with water, and crystallized
from MeOH to give 6.
3-(Acetoxym eth yl)-2H-ben zofu r o[3,2-g]-1-ben zop yr a n -
2-on e (6a ): yield 91%; mp 230 °C; 1H NMR (CDCl3) δ 8.05 (t,
1H, J ) 1.0 Hz, 4-H), 7.96 (ddd, 1H, J ) 7.7, 1.5, 0.7 Hz, 9-H),
7.93 (s, 1H, 5-H), 7.61 (ddd, 1H, J ) 8.2, 1.4, 0.7 Hz, 6-H),
7.53 (s, 1H, 11-H), 7.52 (ddd, 1H, J ) 8.2, 7.1, 1.5 Hz, 7-H),
3-Meth yl-2H-ben zofu r o[3,2-g]-1-ben zopyr an -2-on e (4a):
1
yield 56%; mp 267 °C; H NMR (CDCl3) δ 7.96 (ddd, 1H, J )

Synthesis and Activity of Benzopsoralens
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2943
7.41 (ddd, 1H, J ) 7.7, 7.1, 1.4 Hz, 8-H), 5.11 (d, 2H, J ) 1.0
Hz, 3-CH2OAc), 2.18 (s, 3H, Ac). Anal. (C18H12O5) C,H.
5-(Acetoxym eth yl)-2H-ben zofu r o[3,2-g]-1-ben zop yr a n -
2-on e (6b): yield 98%; mp 248 °C; 1H NMR (CDCl3) δ 8.31 (d,
1H, J ) 9.9 Hz, 4-H), 8.13 (ddd, 1H, J ) 7.5, 1.3, 0.8 Hz, 9-H),
7.62 (ddd, 1H, J ) 8.1, 1.7, 0.8 Hz, 6-H), 7.55 (ddd, 1H, J )
8.1, 7.0, 1.3 Hz, 7-H), 7.53 (s, 1H, 11-H), 7.44 (ddd, 1H, J )
7.6, 7.0, 1.6 Hz, 8-H), 6.50 (d, 1H, J ) 9.9 Hz, 3-H), 5.82 (s,
2H, 5-CH2OAc), 2.10 (s, 3H, Ac). Anal. (C18H12O5) C,H.
11-(Acetoxym eth yl)-2H-ben zofu r o[3,2-g]-1-ben zopyr an -
2-on e (6c): yield 85%; mp 204 °C; 1H NMR (CDCl3) δ 8.04 (s,
1H, 5-H), 7.96 (dd, 1H, J ) 7.5, 1.4 Hz, 9-H), 7.87 (d, 1H, J )
9.6 Hz, 4-H), 7.64 (dd, 1H, J ) 8.1, 1.3 Hz, 6-H), 7.53 (ddd,
1H, J ) 8.1, 7.2, 1.4 Hz, 7-H), 7.41 (ddd, 1H, J ) 7.5, 7.2, 1.3
Hz, 8-H), 6.44 (d, 1H, J ) 9.6 Hz, 3-H), 5.70 (s, 2H, 11-CH2-
OAc), 2.13 (s, 3H, Ac). Anal. (C18H12O5) C,H.
8-(Acetoxym eth yl)-4-m eth yl-2H-ben zofu r o[3,2-g]-1-ben -
zop yr a n -2-on e (6d ): yield 95%; mp 220 °C; 1H NMR (CDCl3)
δ 8.10 (s, 1H, 5-H), 7.94 (dd, 1H, J ) 7.9, 0.6 Hz, 6-H), 7.59
(dd, 1H, J ) 1.4, 0.6 Hz, 9-H), 7.48 (s, 1H, 11-H), 7.39 (dd,
1H, J ) 7.9, 1.4 Hz, 7-H), 6.29 (q, 1H, J ) 1.3 Hz, 3-H), 5.27
(s, 2H, 8-CH2OAc), 2.56 (d, 3H, J ) 1.3 Hz, 4-Me), 2.15 (s, 3H,
Ac). Anal. (C19H14O5) C,H.
(Hyd r oxym eth yl)-2H-ben zofu r o[3,2-g]-1-ben zop yr a n -
2-on es 7a -d . Gen er a l P r oced u r e. To a methanolic solution
(100 mL) of 6 (9.0 mmol) was added a 5% methanolic
potassium hydroxide solution (100 mL), and the mixture was
refluxed in the dark for 1 h. The solution was cooled, diluted
with cold water (500 mL), and acidified with diluted HCl. The
precipitate obtained was collected and crystallized from EtOAc
to give 7.
3-(Hydr oxym eth yl)-2H-ben zofu r o[3,2-g]-1-ben zopyr an -
2-on e (7a ): yield 59%; mp 272 °C; 1H NMR (DMSO-d6) δ 8.55
(s, 1H, 5-H), 8.17 (ddd, 1H, J ) 7.5, 1.6, 0.7 Hz, 9-H), 8.12 (t,
1H, J ) 1.5 Hz, 4-H), 7.85 (s, 1H, 11-H), 7.76 (ddd, 1H, J )
8.1, 1.2, 0.7 Hz, 6-H), 7.59 (ddd, 1H, J ) 8.1, 7.4, 1.6 Hz, 7-H),
7.48 (ddd, 1H, J ) 7.5, 7.4, 1.2 Hz, 8-H), 5.51 (t, 1H, J ) 5.5
Hz, -OH), 4.42 (dd, 2H, J ) 5.5, 1.5 Hz, 3-CH2OH). Anal.
(C16H10O4) C,H.
5-(Hydr oxym eth yl)-2H-ben zofu r o[3,2-g]-1-ben zopyr an -
2-on e (7b): yield 70%; mp 252 °C; 1H NMR (DMSO-d6) δ 8.55
(dd, 1H, J ) 10.0, 0.7 Hz, 4-H), 8.32 (ddd, 1H, J ) 7.7, 1.5, 0.7
Hz, 9-H), 7.70 (ddd, 1H, J ) 8.1, 1.3, 0.7 Hz, 6-H), 7.59 (ddd,
1H, J ) 8.1, 7.2, 1.5 Hz, 7-H), 7.57 (d, 1H, J ) 0.7 Hz, 11-H),
7.47 (ddd, 1H, J ) 7.7, 7.2, 1.3 Hz, 8-H), 6.44 (d, 1H, J ) 10.0
Hz, 3-H), 5.42 (s, 2H, 5-CH2OH). Anal. (C16H10O4) C,H.
11-(Hydr oxym eth yl)-2H-ben zofu r o[3,2-g]-1-ben zopyr an -
2-on e (7c): yield 34%; mp 253 °C; 1H NMR (DMSO-d6) δ 8.51
(s, 1H, 5-H), 8.33 (br s, 1H, -OH), 8.25 (d, 1H, J ) 9.6 Hz,
4-H), 8.20 (dd, 1H, J ) 7.5, 1.5 Hz, 9-H), 7.82 (dd, 1H, J )
8.1, 1.2 Hz, 6-H), 7.60 (ddd, 1H, J ) 8.1, 7.4, 1.5 Hz, 7-H),
7.49 (ddd, 1H, J ) 7.5, 7.4, 1.2 Hz, 8-H), 6.54 (d, 1H, J ) 9.6
Hz, 3-H), 4.92 (s, 2H, 11-CH2OH). Anal. (C16H10O4) C,H.
8-(H yd r oxym et h yl)-4-m et h yl-2H -b en zofu r o[3,2-g]-1-
ben zop yr a n -2-on e (7d ): yield 91%; mp 279 °C; 1H NMR
(DMSO-d6) δ 8.56 (s, 1H, 5-H), 8.17 (d, 1H, J ) 7.9 Hz, 6-H),
7.78 (s, 1H, 11-H), 7.66 (d, 1H, J ) 1.0 Hz, 9-H), 7.42 (dd, 1H,
J ) 7.9, 1.0 Hz, 7-H), 6.42 (q, 1H, J ) 1.2 Hz, 3-H), 5.44 (t,
1H, J ) 5.8 Hz, -OH), 4.69 (d, 2H, J ) 5.8 Hz, 8-CH2OH),
2.58 (d, 3H, J ) 1.2 Hz, 4-Me). Anal. (C17H12O4) C,H.
(Dieth yla m in om eth yl)-2H-ben zofu r o[3,2-g]-1-ben zop y-
r a n -2-on es 8a -d . Gen er a l P r oced u r e. A mixture of 5 (10
mmol) and diethylamine (100 mmol) in anhydrous toluene (200
mL) was refluxed for 10 h (TLC: AcOEt/cyclohexane, 80:20).
After cooling, the solvent was evaporated under reduced
pressure. The residue was purified by column chromatography
and crystallized from MeOH to give 8.
-N-CH2-CH3), 1.12 (t, 6H, J ) 7.1 Hz, -N-CH2-CH3). Anal.
(C20H19NO3) C,H,N.
5-(Diet h yla m in om et h yl)-2H -b en zofu r o[3,2-g]-1-b en -
zop yr a n -2-on e (8b): yield 54%; mp 162 °C; 1H NMR (CDCl3)
δ 8.59 (d, 1H, J ) 9.9 Hz, 4-H), 8.23 (dd, 1H, J ) 7.6, 1.3 Hz,
9-H), 7.60 (dd, 1H, J ) 8.1, 1.3 Hz, 6-H), 7.51 (ddd, 1H, J )
8.1, 7.2, 1.3 Hz, 7-H), 7.45 (s, 1H, 11-H), 7.39 (ddd, 1H, J )
7.6, 7.2, 1.3 Hz, 8-H), 6.40 (d, 1H, J ) 9.9 Hz, 3-H), 4.30 (s,
2H, 5-CH2-N-CH2-CH3), 2.64 (q, 4H, J ) 7.1 Hz, -N-CH2-
CH3), 1.06 (t, 6H, J ) 7.1 Hz, -N-CH2-CH3). Anal. (C20H19
NO3) C,H,N.
-
11-(Diet h yla m in om et h yl)-2H -b en zofu r o[3,2-g]-1-b en -
zop yr a n -2-on e (8c): yield 45%; mp 148 °C; 1H NMR (CDCl3)
δ 7.98 (s, 1H, 5-H), 7.96 (dd, 1H, J ) 7.5, 1.4 Hz, 9-H), 7.87 (d,
1H, J ) 9.6 Hz, 4-H), 7.63 (dd, 1H, J ) 8.1, 1.2 Hz, 6-H), 7.52
(ddd, 1H, J ) 8.1, 7.3, 1.4 Hz, 7-H), 7.40 (ddd, 1H, J ) 7.5,
7.3, 1.2 Hz, 8-H), 6.43 (d, 1H, J ) 9.6 Hz, 3-H), 4.27 (s, 2H,
11-CH2-N-CH2-CH3), 2.79 (q, 4H, J ) 7.3 Hz, -N-CH2-
CH3), 1.26 (t, 6H, J ) 7.3 Hz, -N-CH2-CH3). Anal. (C20H19
NO3) C,H,N.
-
8-(Diet h yla m in om et h yl)-4-m et h yl-2H -b en zofu r o[3,2-
g]-1-ben zop yr a n -2-on e (8d ): yield 82%; mp 164 °C; 1H NMR
(CDCl3) δ 8.05 (s, 1H, 5-H), 7.86 (dd, 1H, J ) 7.9, 0.6 Hz, 6-H),
7.58 (dd, 1H, J ) 1.4, 0.6 Hz, 9-H), 7.44 (s, 1H, 11-H), 7.37
(dd, 1H, J ) 7.9, 1.4 Hz, 7-H), 6.26 (q, 1H, J ) 1.3 Hz, 3-H),
3.72 (s, 2H, 8-CH2-N-CH2-CH3), 2.58 (q, 4H, J ) 7.1 Hz,
-N-CH2-CH3), 2.54 (d, 3H, J ) 1.3 Hz, 4-Me), 1.08 (t, 6H, J
) 7.1 Hz, -N-CH2-CH3). Anal. (C21H21NO3) C,H,N.
Biologica l Section . Compounds 7a -d and 8a -d were
dissolved in dimethyl sulfoxide (DMSO; 4.5 mM) and the
solutions kept at -20 °C in the dark. J ust before the experi-
ment, a calculated amount of compound solution was added
in the dark to PBS or to the growth medium containing cells,
to a final DMSO concentration which never exceeded 0.5%.
UVA Ir r a d ia tion . Cell suspensions containing the test
compound were incubated at room temperature for 15 min in
the dark, put into Petri dishes (5 cm in diameter, 3 mL), and
exposed to UVA light. UVA exposure were performed with a
Philips HPW 125 lamp, provided with a built-in Philips filter.
Emission was in the 320-400 nm range, with a maximum,
over 90% of the total, at 365 nm; irradiation intensity,
determined by a radiometer (model 97503, Cole-Parmer In-
strument Co., Niles, IL), was 0.9 × 10-6 W‚m-2
.
In ter a ction w ith DNA in Vitr o: In h ibition of Top o-
isom er a se II. The inhibition of topoisomerase II activity was
studied using a purified enzyme from Drosophila melanogaster
embryos (USB, Amersham Italia S.r.l.). PM2 DNA (0.125 µg;
Boehringer Mannheim GmbH, Germany) was incubated for
15 min at 30 °C in the presence of 2 units of topoisomerase II
(1 unit is defined as the activity capable of relaxing 0.3 µg of
supercoiled DNA) in the reaction buffer containing 10 mM
Tris-HCl (pH 7.9), 50 mM NaCl, 50 mM KCl, 5 mM MgCl2,
0.1 mM EDTA, 15 µg‚mL-1 BSA, 1 mM ATP. Aliquots of
compound solution (2 µL) were added to reach the following
final concentrations: 1, 10, 20, 40, 160 µM. A suitable amount
of reaction buffer was then added to every sample to reach
the final volume of 20 µL. The reaction was blocked by adding
7 mM EDTA (3 µL) containing 0.77% SDS. Bromophenol blue
(0.2 µL) containing 15% glycerol was added to the samples,
which were then analyzed by agarose gel (0.7%) containing
TAE (40 mM Tris-sodium acetate, pH 8.2; 1 mM EDTA) at 50
V for 90 min. The gel was stained for 1 h in aqueous ethidium
bromide (0.5 µg‚mL-1) and then photographed by a Polaroid
camera placed over a UV TM36 transilluminator (UVP Inc.,
San Gabriel, CA).
In ter a ction w ith DNA in Vitr o: Detection of In ter -
str a n d Cr oss-Lin k s. Supercoiled circular DNA of PM2 bac-
teriophage (4 µg; Boehringer Mannheim GmbH, Germany) was
linearized by incubating at 37 °C for 2 h with PstI restriction
enzyme (18 U‚µL-1; Amersham International Inc., U.K.) in 100
µL of aqueous solution containing 10 mM NaCl, 50 mM Tris-
HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 10 µg BSA. The
solution was filtered through a Microcon 100 (Amicon, Beverly,
MA), centrifuging at 500g for 5 min. The membrane was
3-(Diet h yla m in om et h yl)-2H -b en zofu r o[3,2-g]-1-b en -
zop yr a n -2-on e (8a ): yield 73%; mp 124 °C; 1H NMR (CDCl3)
δ 8.09 (t, 1H, J ) 1.4 Hz, 4-H), 8.05 (s, 1H, 5-H), 7.60 (ddd,
1H, J ) 7.5, 1.5, 0.8 Hz, 9-H), 7.52 (ddd, 1H, J ) 8.1, 1.4, 0.8
Hz, 6-H), 7.52 (s, 1H, 11-H), 7.50 (ddd, 1H, J ) 8.1, 7.2, 1.5
Hz, 7-H), 7.39 (ddd, 1H, J ) 7.5, 7.2, 1.4 Hz, 8-H), 3.58 (d, 2H,
J ) 1.4 Hz, 3-CH2-N-CH2-CH3), 2.67 (q, 4H, J ) 7.1 Hz,

2944 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Chilin et al.
washed by adding 100 µL of TE (40 mM Tris-HCl, pH 7.5; 1
mM EDTA), and the solution was stirred and centrifuged at
500g for a further 5 min. The solution over the membrane was
then recovered by inverting the filter and centrifuging at 1000g
for 3 min. The remaining solution was diluted by adding TE
to obtain a DNA concentration of 80 ng‚µL-1 and then stirred
at 4 °C until use.
the ratio between the numbers of formed colonies and of cells
seeded, was then calculated. Plating efficiency was about 80%.
Every experiment was carried out at least three times.
Mu ta gen ic Activity. The strain used was E. coli TM9
(WP2, uvrA, R46) carrying a nonsense mutation in the trpE
gene, which is reverted by UV light and base pair substitution
mutagens21 and therefore by formation of C4-cycloadducts of
furocoumarins.7 Bacteria were grown overnight in a minimal
Davis-Mingioli salt glucose medium supplemented with tryp-
tophan (20 mg‚L-1). E. coli cells were washed and then
suspended in phosphate-buffered saline (pH 7.0) containing
PM2-linearized DNA (2 µL for each sample) was added to a
solution of the test compound (2 µL; 4× in DMSO) at the
appropriate concentration, so that the final molar ratio drug/
base pairs was 2; TE (4 µL) was then added to each sample
(final volume 8 µL). Two samples were prepared for each
compound: both were incubated at room temperature in the
dark for 15 min and then exposed to UVA light. As a control,
PM2-linearized DNA (2 µL) was added to DMSO (2 µL) and
TE (4 µL) and incubated as described above, but not irradiated.
Then 2-µL aliquots of a solution containing 1.5 M sodium
acetate and 100 mM EDTA were added to the samples, which
were then stirred and centrifuged at 10000g for 1 min. DNA
was precipitated with three volumes (30 µL) of 95% ethanol,
and the samples were kept at -80 °C for at least 1 h; DNA
was recovered by centrifugation at 2000g for 20 min at 4 °C.
Precipitated DNA was then suspended in TAE (7 µL) contain-
ing 0.04% bromophenol blue, 10% glycerol, and 30% DMSO,
denatured by heating at 90 °C for 2 min, and quickly cooled
in ice. Samples were analyzed by agarose gel (0.7%) electro-
the test compound (2 × 10-5 M) at a density of 108 cells‚mL-1
.
Bacteria were irradiated with UVA, as described for DNA
solutions. For the mutagenesis test, 0.1-mL aliquots of the
irradiated suspensions were added to 2 mL of molten 0.6%
top agar and poured onto plates containing 20 mL of SEM agar
(MMA fortified with 0.1 mg‚mL-1 Difco nutrient broth). To
determine the surviving fraction, the irradiated cells (0.1 mL)
were diluted with phosphate buffer, added to 2 mL of molten
0.6% agar, and plated on Davis-Mingioli minimal medium
supplemented with tryptophan. Plates were incubated for 48
h at 37 °C in the dark, and the colonies were then counted.
The mutation frequency was expressed as mutants per 106
survivors, computed by dividing the number of revertants
observed per plate by the number of surviving bacteria at the
same treatment and subtracting from the result the number
of revertant colonies per million survivors observed in controls.
In this test, all manipulations were done under red light.
Exp er im en ts w ith Yea st: In d u ction of Cytop la sm ic
,P etite. Mu ta tion s. Experiments with yeast were per-
formed using diploid strain D7 (S. cerevisiae), a kind gift of
Prof. F. K. Zimmermann (Technische Hochschule, Darmstadt,
Germany). Yeast cells were grown in complete growth medium
containing 1% yeast extract (Difco Laboratories, Detroit, MI),
2% bactopeptone (Difco), 2% glucose, and distilled water. Cells
(107‚mL-1) were incubated in the dark for 15 min in the
presence of 20 µM of the test compound and exposed to various
doses of UVA light. Suitable dilutions of samples were plated
on complete yeast-extract-peptone-glucose medium solidified
by 2.6% bactoagar (Difco). Cytoplasmic ,petite. mutants, i.e.,
respiratory-deficient mutants, were detected using the tetra-
zolium overlay technique.23
Exp er im en ts w ith Yea st: Detection of Dou ble-Str a n d
Br ea k s. Yeast chromosomes and the appearance of DNA
DSBs were analyzed using a contour-clamped homogeneous-
field electrophoresis apparatus (CHEF MAPPER, BIO-RAD,
Richmond, VA). 1% agarose gels were made up from ultrapure
DNA grade molecular biology certified agarose (BIO-RAD,
Richmond, VA) and 0.5× TBE running buffer (5.4 g of Tris-
Base, 2.75 g of boric acid, 2 mL of 0.5 EDTA, pH 8, 1 L of
distilled water). Gels loaded with yeast chromosomal DNA
were run for 22 h at 12 °C, using the CHEF apparatus at 160
mV, 170 mA, exponentially increasing pulse times. Gels were
stained with ethidium bromide and photographed with a
Polaroid camera, as previously described.
phoresis in TAE containing ethidium bromide (0.5 µg‚mL-1
)
at 50 V for 3 h, to obtain good separation of denatured and
nondenatured (cross-linked) DNA. The gel was photographed
by a Polaroid camera, as previously described.
DNA Syn th esis in Eh r lich Cells. DNA synthesis was
assayed in Ehrlich cells (Lettre` strain) as already described.15
J ust after UVA irradiation (cell density: 2 × 107 cells‚mL-1
in PBS), the samples (106 cells in 0.5 mL of PBS) were
incubated for 30 min at 37 °C in the presence of 40 kBq‚mL-1
[3H]thymidine (4.77 TBq‚mM-1; Amersham International Inc.,
U.K.). The acid-insoluble fraction was precipitated by 5% ice-
cold trichloroacetic acid and collected on Whatman GF/C filters
(2.5 cm in diameter). After several washings with cold 1%
trichloroacetic acid, the filters were dried and counted. In the
experiments carried out in the dark, the irradiation step was
omitted; the cells were treated with the compound at room
temperature, incubated for 30 min at 37 °C in the dark in the
presence of the radioactive precursor, and then processed as
above. The results were calculated as the percentage of
radioactivity incorporated into the DNA of untreated control
cells (ca. 3-6 kBq). Filtrations were carried out using a Sample
Manifold apparatus (Millipore Corp., Bedford, U.K.). Filters
were counted by a toluene-based scintillation fluid (5 g PPO,
0.25 g dimethyl-POPOP, toluene up to 1 L of solution). All
determinations were carried out on a Packard 4430 spectrom-
eter. Every experiment was carried out at least three times.
Clon a l Gr ow th of HeLa Cells. HeLa cells were grown in
nutrient mixture F-12 Ham medium (Sigma Chemical Co., St.
Louis, MO) containing 5% fetal calf serum (increased to 10%
in clonal growth) and supplemented with antibiotics. Cell
growth was accomplished at 37 °C in a 5% carbon dioxide
atmosphere.
Sk in P h ototoxicity. Skin phototoxicity was tested on
depilated albino guinea pigs (outbred Dunkin-Hartley strain),
as described;29 compounds were applied topically as 4.5 mM
methanol solutions.
HeLa cells (1.5-2 × 105) were seeded in Petri dishes in
growth medium (4 mL); after 24 h, the medium was replaced
with a fresh one containing the test compound. For UVA
irradiation, the cells were incubated in the dark for 15 min,
to allow uptake of the compound. Samples were then exposed
to UVA; in these experimental conditions, no significant
absorption of UVA light by the medium was observed. For the
experiments carried out in the dark, in which active cell
metabolism is required, cells were incubated for 3 h at 37 °C
in a 5% carbon dioxide atmosphere in growth medium supple-
mented with fetal calf serum and containing the test com-
pound. In all cases, 0.5% DMSO was added to untreated
controls. After treatment, aliquots of 200 cells were seeded in
the same medium and incubated for 7 days, after which the
colonies were stained and counted, colonies with less than 50
cells being discarded. The efficiency of clonal growth, that is,
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