Synthesis of angelicin heteroanalogues: Preliminary photobiological and pharmacological studies

Article abstract of DOI:10.1016/S0014-827X(98)00076-7

A series of angelicin heteroanalogues, in which the furan was replaced by thiophene or a 1-substituted pyrazole moiety, was synthesised in order to obtain potential therapeutic agents with antiproliferative and/or other biological activities. In general,

Full text of DOI:10.1016/S0014-827X(98)00076-7

Il Farmaco 53 (1998) 602±610
Synthesis of angelicin heteroanalogues: preliminary photobiological and
pharmacological studies
a,
Luisa Mosti , Eleonora Lo Presti , Giulia Menozzi , Christine Marzano ,
a
a
b
*
Francarosa Baccichettib, Giuseppe Falconec, Walter Filippellic, Brunella Piuccic
a
Á
Dipartimento di Scienze Farmaceutiche, Universita degli Studi, Viale Benedetto XV 3, 16132 Genoa, Italy
b
Á
Dipartimento di Scienze Farmaceutiche, Universita degli Studi, Via Marzolo 5, 35123 Padua, Italy
c
Á Á
Istituto di Farmacologia e Tossicologia, Facolta di Medicina e Chirurgia, II Universita degli Studi, Via S. Andrea delle Dame 8, 80138 Naples, Italy
Received 30 July 1998; accepted 20 October 1998
Abstract
A series of angelicin heteroanalogues, in which the furan was replaced by thiophene or a 1-substituted pyrazole moiety, was synthesised in
order to obtain potential therapeutic agents with antiproliferative and/or other biological activities. In general, the antiproliferative activity of
the new thioangelicin, tested in different biological substrates, appeared to be higher than that of the angelicin, the natural parent compound,
but lower than that of 8-MOP, the furocoumarin ordinarily used in PUVA therapy and photopheresis. Thioangelicin 6 induced strong
inhibition of T2 bacteriophage infectivity and was able to signi®cantly repress the DNA synthesis in Ehrlich ascites cells and the clonal
growth in HeLa cells. The pyrazolocoumarins did not show any noticeable effect upon UVA irradiation in all the biological systems
considered. All the new angelicin heteroanalogues appeared to be free of the known phototoxicity of furocoumarins on the skin. The
pyrazolocoumarins have also been tested as anti-in¯ammatory, analgesic, antipyretic, local anaesthetic, anti-arrhythmic and platelet anti-
aggregating agents by standard procedures. In this class of derivatives, 10a showed good anti-in¯ammatory and antipyretic properties, while
9a and 11a showed signi®cant local anaesthetic activity. q 1998 Elsevier Science S.A. All rights reserved.
Keywords: Angelicin heteroanalogues; NSAI analogues; Photosensitizers
1. Introduction
these effects were associated with the induction of covalent
adducts in DNA, mostly to inter-strand cross-links [5,6].
Thus, several monofunctional derivatives have been
prepared and studied, in particular angelicin analogues
(iso-psoralens), in which the angular structure prevents,
for geometrical reasons, the formation of inter-strand
cross-links [7±10]. Some of these compounds have an
evident antiproliferative activity with a genotoxicity lower
than that shown by psoralens [7].
Recently, one interesting ®eld of research, aimed at
obtaining new derivatives having better features than 8-
MOP, was the insertion of a heteroatom into the furocou-
marin nucleus thus obtaining furocoumarin isosters, for
example azapsoralens [11±13] and some sulfur and sele-
nium derivatives [14]. These heteroanalogues exhibit a
strongly increased DNA photoinactivation property and
appear to be very promising for photochemotherapy [15±
17].
Psoralens, a well known group of linear furocoumarins,
are natural and synthetic compounds with interesting photo-
sensitizing activity. Some are commonly used in PUVA
photochemotherapy (psoralen plus UVA) of a variety of
skin diseases characterized by hyperproliferative conditions
[1]. Recently, psoralens have been used in extracorporeal
photochemotherapy to treat cutaneous T-cell lymphomas
and other autoimmune diseases [2]. Furthermore, psoralen
derivatives are also used as tools for biophysical studies on
nucleic acids [3] and are now recognized as effective viru-
cidal agents, especially against enveloped viruses such as
the herpes simplex virus or human immunode®ciency virus
type 1 (HIV-1) [4].
However, 8-methoxypsoralen (8-MOP), the compound
generally used in therapy, leads to short-term side effects
(erythema) and to long-term risks such as genotoxicity;
Other interesting angelicin isosters were also studied, in
particular some furoquinolinones, in which a nitrogen atom
replaces the oxygen at the pyrone ring [18]; they showed a
strong antiproliferative effect both in the dark and under light
* Corresponding author. Tel: 1 39-10-3538350; fax: 1 39-10-
3538358; e-mail: mosti@csita.unige.it
0014-827X/98/$ - see front matter q 1998 Elsevier Science S.A. All rights reserved.
PII S0014-827X(98)00076-7

L. Mosti et al. / Il Farmaco 53 (1998) 602±610
603
activation [19,20]. In addition, some 4-amino-3-chloro-
angelicin derivatives carrying a sulfur atom substituting the
oxygen on the furan ring [21] and, more recently, some
methyl derivatives of thienocoumarins have been also
synthesised [22]. The synthesis of a new sulfur analogue of
angelicin characterized by the presence of the thiopyrone in
place of the pyrone moiety has been carried out and the
evaluation of its photochemical and photobiological activity
is in progress [23].
These ®ndings prompted us to synthesize and study the
preliminary photobiological activity of some new angelicin
heteroanalogues in which the furan is replaced by thiophene
(4a,b, 5, 6) or 1-substituted pyrazole moiety (10a,b and
11a,b) and position 3 of the a-pyrone ring retains an elec-
tron-withdrawing group, as in 3-carbethoxyangelicin [24] in
order to obtain photochemotherapeutic agents with a mono-
functional pro®le and more signi®cant photoreactive prop-
erties.
Furthermore, taking into account the biological signi®-
cance of the indazole nucleus and the results published in
previous papers in which some of us reported the interesting
analgesic, anti-in¯ammatory, antipyretic and local anaes-
thetic activities of a number of derivatives related to pyra-
zole coumarins [25], we decided to select and test a set of
these compounds characterized by an indazole moiety
(10a,b, 11a) and of their dihydroprecursors (8b, 9a,b) for
the activities described. The evaluation of a potential anti-
arrhythmic and platelet anti-aggregating activity of the
above compounds was realized to complete their biological
screening.
2. Chemistry
The synthetic route for the preparation of methyl and
ethyl esters of 2-oxo-2H-thieno[2,3-h]-1-benzopyran-3-
carboxylic acid 4a,b and of 1-methyl or 1-phenyl substi-
tuted 5-oxo-5H-pyran[2,3-e]indazole-6-carboxylic acid
10a,b and 11a,b are presented in Schemes 1 and 2.
The precursors 2 [26] and 7a,b [27], obtained in the past
in two steps from the corresponding a-hydroxymethylene
derivatives with dimethylamine at room temperature, were
synthesized directly, in good yield, by re¯uxing a solution
of the suitable commercially available ketone in excess of
N,N-dimethylformamide dimethylacetal.
In the past Maitte et al. [28] studied the reaction between
malonic derivatives and some bicyclic enaminoketones in a
basic medium followed by acid hydrolysis and obtained the
corresponding a-pyrone derivatives only when a cyano-
methylene active nucleophile was used. On the contrary,
in our case, the reaction of bicyclic enaminoketones 2 and
7 with the appropriate dialkyl malonate took place without
adding a base to afford, generally in good yield, the desired
adducts 3a,b, 8a,b and 9a,b. This reaction probably ®rst
occurs between the nucleophilic methylene of the malonic
ester and the a-enamino-carbon atom, which is the most
electrophilic site of the enaminone. Subsequently, the
dimethylamine released in situ causes the reaction to
proceed towards the direct closure of the 2-pyrone ring
without isolation of intermediates.
Full aromatization of adducts 3a,b, 8a,b and 9a,b was
accomplished with 2,3-dichloro-5,6-dicyano-1,4-benzoqui-
none (DDQ) in re¯uxing anhydrous toluene (method A) [24]
to afford, generally in satisfactory yield, the desired esters
4a,b, 10a,b and 11a,b. Only in the case of 9a and 9b, even
with a prolonged reaction time (up to 5 days), was the ®nal
yield poor.
Realizing the cyclization and following aromatization in
one step (method B), the desired angelicin heteroanalogues
4, 10 and 11 were also obtained but in generally smaller
yield than that of method A.
Scheme 1.
These experimental data showed that the full aromatiza-
tion occurred easily in the case of the adducts characterized
by the benzothiophene moiety but is less satisfactory in the
case of the adducts with a N-substituted indazol structure,
probably due to the higher basicity of the second rather than
that of the ®rst nucleus.
Methyl ester 4a was routinely converted into the
corresponding
2-oxo-2H-thieno[2,3-h]-1-benzopyran-3-
carboxylic acid 5 in a high yield by re¯uxing with a
50% mixture of hydrochloric and acetic acids.
Decarboxylation of acid 5 by re¯uxing in quinoline
containing a catalytic amount of copper powder afforded
thioangelicin 6.
Scheme 2.

604
L. Mosti et al. / Il Farmaco 53 (1998) 602±610
3. Biology
tory, analgesic, antipyretic, local anaesthetic, anti-arrhyth-
mic and platelet anti-aggregating activities, using
indomethacin, lidocaine, quinidine and acetylsalicylic acid
(ASA), respectively, as reference compounds.
All the new angelicin heteroanalogues were investigated
for the following photobiological activities: skin phototoxi-
city; DNA synthesis inhibition in Ehrlich ascites tumor
cells; T2 phage inactivation; HeLa cells growth inhibition.
Both angelicin and 8-MOP were used as reference
compounds.
4. Results and discussion
The indazole esters 10a,b, 11a and dihydroprecursors 8b
and 9a,b, were submitted to a screening for: anti-in¯amma-
4.1. Photobiology
The indazole esters 10, 11 and related dihydroprecursors
8 and 9 were entirely ineffective in inhibiting T2 phage
infectivity, DNA synthesis in Ehrlich cells and clonal
growth capacity of in vitro HeLa cells (data not shown).
Fig. 1 shows the results regarding the ability of thioange-
licin derivatives of inactivating T2 phage. Unsubstituted
thioangelicin 6 (5 mM at the range 0.0±0.7 kJ £ m22
UVA) induced a strong reduction of the surviving fraction
at every UVA dose, while all the other derivatives were
essentially ineffective. Under the same experimental condi-
tions, 8-MOP and angelicin also appeared to be poorly
effective in repressing T2 phage activity.
Fig. 2 shows the results obtained from studying the activ-
ity of thioangelicin derivatives in inhibiting the DNA synth-
esis in Ehrlich cells (20 mM at the range 0±12 kJ £ m22
UVA). Unsubstituted thioangelin 6 shows the highest activ-
ity, although carboxylic acid 5 is only a little less effective;
both compounds have an antiproliferative effect higher than
that of angelicin but much lower than that of 8-MOP. The
two esters 4a and 4b appeared to be poorly effective.
Fig. 3 shows the data obtained from studying the activity
of thioangelicin derivatives (5 mM at the range 0.00±0.25
kJ £ m22 UVA) on the clonal growth capacity of HeLa cells
cultivated in vitro. Once again, unsubstituted thioangelicin 6
was the most effective compound; its activity is higher than
that of 8-MOP at a small UVA dose (0.1 kJ £ m22) but at
larger doses the reference compound becomes more ef®-
cient. Carboxylic acid 5, like angelicin, induced only a
moderate reduction in the colony-forming ability, while
the two esters 4a and 4b appeared to be totally ineffective.
Finally, skin phototoxicity of the new angelicin hetero-
analogues was evaluated on albino guinea-pig skin. As
reported in Table 1, the compounds, which also appeared
more effective in the other biological tests, are not able to
induce a detectable erythema even when tested at 30 mM £
cm22 and with 5 kJ £ m22 UVA. Under these experimental
conditions, angelicin was also ineffective. 8-MOP induced a
barely visible erythema up to 4 mM £ cm22 of applied drug
at the UVA dose of 5 kJ £ m22, while at a drug concentra-
tion of 30 mM and with the same UVA dose it induced a
very strong erythema with edema.
Fig. 1. T2 phage infectivity; virus particles were exposed to increasing
UVA doses in the presence of thioangelicin derivatives (5 mM) and the
number of the plaque forming units was determined. The symbols are:
compound 4a (O); compound 4b (P); compound 5 (X); compound 6
Fig. 2. Inhibition of DNA synthesis observed in Ehrlich cells. The tumor
cells were incubated with tritiated thymidine and exposed to increasing
UVA doses in the presence of thioangelicin derivatives (20 mM); then
the acid insoluble radioactivity was determined. The symbols are:
compound 4a (O); compound 4b (P); compound 5 (X); compound 6
(B); 8-MOP (W); angelicin (A). The bars represent the standard errors.
Summarizing the main points regarding photobiological
studies of the new angelicin heteroanalogues, we can say
that only the substitution of the furan with the thiophene
ring increases the photoreactivity and consequently the

L. Mosti et al. / Il Farmaco 53 (1998) 602±610
605
Unlike 8-MOP, the new angelicin heteroanalogues were
entirely ineffective in inducing erythema on guinea-pig
skin, thus resembling angelicin (Table 1).
4.2. Pharmacology
The indazole esters 10a,b, 11a and their dihydroprecur-
sors 8b, 9a,b gave negligible or too weak activity in the
preliminary test for analgesic and platelet anti-aggregating
activities (data not shown).
Compounds 8b, 9a and 10a showed at a dose of 50 mg/kg
p.o. an appreciable anti-in¯ammatory activity in compari-
son to that of the indomethacin (5 mg/kg p.o.) used as
reference compound (Table 2). 10a showed an ED50 at the
third and the fourth hour of 17.73 (14.61±21.52) and 17.01
(1.61±17.94) mg/kg, respectively. 10a also exhibited a
good antipyretic effect in the yeast-induced pyrexia test
(Table 3).
On the other hand, a satisfactory local anaesthetic activity
was present in nearly all pyrazolocoumarins studied (with
the exception of 8b and 10b), the most active being 9a and
11a, which showed a 0.082% (0.066±0.095) and 0.079%
(0.066±0.095) ED50 after 5 and 30 min, respectively,
expressed as % effective concentration (Table 4). Anti-
arrhytmic activity of moderate level was shown by 9b
(Table 5).
In conclusion, from the preliminary pharmacological
data, we can con®rm that the indazole nucleus could be a
good carrier of anti-in¯ammatory and antipyretic activities
even if it does not bear a classic acid functionalization. The
presence of the ester function together with a basic centre
such as the pyrazole nucleus may justify the signi®cant local
anaesthetic effect.
Fig. 3. Clonal growth capacity of HeLa cells cultivated in vitro; the cells
were exposed to increasing UVA doses in the presence of thioangelicin
derivatives (5 mM) and then their colony forming ability was determined.
The symbols are: compound 4a (O); compound 4b (P); compound 5 (X);
compound 6 (B); 8-MOP (W); angelicin (A). The bars represent the stan-
dard errors.
phototherapeutic activity, mainly when the pyrone moiety is
unsubstituted.
In fact thioangelicin 6 is the best compound of the new
angelicin isosteres.
In a simple system such as T2 phage, compound 6 quickly
inactivated the virus infectivity under UVA irradiation and
was the most effective compound in the in vitro HeLa cell
test, resulting in a very good capacity to reduce the colony-
forming ability.
The antiproliferative effect of thioangelicin 6 on the DNA
synthesis in the Ehrlich cells was lower than that of 8-MOP
but higher than that of angelicin, the natural furocoumarin
characterized by the same structure. This is probably due to
a better sensitizing activity on the macromolecule which
forms the structure of the virus particle rather than on that
of mammalian cell.
5. Experimental
5.1. Photobiological methods
5.1.1. T2 phage inactivation
T2 phage was grown in E. coli B48 cultures prepared in
brain±heart infusion (Difco Laboratories, Detroit, Michi-
gan, USA). Irradiation was performed by diluting the
phage suspension up to 109 virus particles per ml with
Hank's solution containing the tested compound (®nal
concentration 20 mM). Phage titles were determined using
the two-layer method and E. coli B48 as host bacteria,
according to Adams [29].
Table 1
Phototoxicity on guinea-pig skin
Comp.
mM £ cm22
UVA dose (kJ m22
)
Erythema intensitya
None
4a
4b
0
30
30
30
30
30
4
10
5
2
2
2
2
2
2
1
1
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
1
5
5
5
5.1.2. DNA synthesis inhibition in Ehrlich ascites tumor
cells
6
5
Angelicin
8-MOP
8-MOP
5
The capacity of inhibiting DNA synthesis in Ehrlich
ascites tumor cells was evaluated as already described
[13]. Results were calculated as percentages of radioactivity
incorporated into the DNA with respect to untreated control
cells.
5
30
5
a The symbols are: 2
detectable erythema; 1
2
2
1 1 1 , strong erythema with edema.
2 , no erythema; 1
2 2 2 , barely

606
L. Mosti et al. / Il Farmaco 53 (1998) 602±610
Table 2
Anti-in¯ammatory activity by carrageenan-induced rat paw edema testa
Comp.
Dose
(mg/kg 4p.o.)
Edema volume (ml ^ SEb) at the following times (h) after treatment (in parentheses per cent increase of volume
relative to 0 value)
0
1
2
3
4
Control
±
1.6 ^ 0.1
1.4 ^ 0.1
1.8 ^ 0.1
1.8 ^ 0.1
1.7 ^ 0.1
2.2 ^ 0.1
1.9 ^ 0.1
2.3 ^ 0.1
2.2 ^ 0.1
1.3 ^ 0.1
2.1 ^ 0.1 ( 1 31)
1.5 ^ 0.1 ( 1 7)
2.0 ^ 0.1 ( 1 11)
1.9 ^ 0.1 ( 1 6)
1.9 ^ 0.1 ( 1 12)
2.3 ^ 0.1 ( 1 5)
2.0 ^ 0.1 ( 1 5)
2.4 ^ 0.1 ( 1 4)
2.4 ^ 0.1 ( 1 9)
1.5 ^ 0.1 ( 1 15)
2.3 ^ 0.1 ( 1 44)
1.5 ^ 0.1 ( 1 7)
2.0 ^ 0.1 ( 1 11)
1.9 ^ 0.1 ( 1 6)
2.0 ^ 0.1 ( 1 18)
2.5 ^ 0.1 ( 1 13)
2.1 ^ 0.1 ( 1 10)
2.4 ^ 0.1 ( 1 4)
2.4 ^ 0.1 ( 1 9)
1.6 ^ 0.1 ( 1 23)
2.4 ^ 0.1 ( 1 50)
1.5 ^ 0.1 ( 1 7)
2.0 ^ 0.1 ( 1 11)
2.0 ^ 0.1 ( 1 11)
2.2 ^ 0.1 ( 1 29)
2.6 ^ 0.1 ( 1 18)
2.1 ^ 0.1 ( 1 10)
2.4 ^ 0.1 ( 1 4)
2.5 ^ 0.1 ( 1 14)
1.7 ^ 0.1 ( 1 31)
2.6 ^ 0.1 ( 1 63)
1.5 ^ 0.1 ( 1 7)
2.0 ^ 0.1 ( 1 11)
2.0 ^ 0.1 ( 1 11)
2.3 ^ 0.1 ( 1 35)
2.7 ^ 0.1 ( 1 23)
2.2 ^ 0.1 ( 1 16)
2.4 ^ 0.1 ( 1 4)
2.5 ^ 0.1 ( 1 14)
1.9 ^ 0.1 ( 1 46)
Indomethacin
5
8b
9a
50
50
50
12.5
25
50
50
50
9b
10a
10b
11a
a Each compound was tested on a group of ®ve albino rats (200±250 g). Compounds were given by gastric probe 30 min before carrageenan (0.1 ml of 1%
solution).
b SE was always smaller than ^ 0.10 ml and so rounded up to this value.
5.1.3. Clonal growth of HeLa cells
(outbred Dunkin±Hartley strain) as 0.1% methanol solutions
up to 5 £ 1022 mg cm22 and the area was exposed to UVA
light. The animals were observed for 48 h.
HeLa cells ((1.5±2) £ 105), grown in Nutrient Mixture F-
12 Ham medium (Sigma Chemical Co, St Louis, MO,
USA.), containing 5% foetal calf serum, were seeded in
Petri dishes in growth medium (4 ml). After 24 h, the
medium was replaced with a fresh one containing the
compound to be studied. The cells were then incubated
for 15 min at 378C in the dark and then exposed to UVA
light. Aliquots of 200 cells were seeded in the same
medium, incubated for 7 days and then the colonies were
stained and counted, discarding colonies with less than 50
cells. The ef®ciency of the clonal growth, that is the ratio
between the number of the formed colonies and the number
of the cells seeded, was then calculated. The plating ef®-
ciency was about 90%.
5.1.5. Ultraviolet light source
All irradiations were performed by Philips HPW 125 W
lamps provided with built-in Philips ®lters. The emission
spectrum was in the range 320±400 nm, with a maximum
at 365 nm (over 90% of the total). Irradiation intensity was
determined on a UVX radiometer (Ultraviolet Products Co.,
Cambridge, UK) at 5:5 £ 1023 kJ s21 m22
.
5.2. Pharmacological methods
For the most active compounds, the ED50 values were
determined by administration of three dosages (12.5, 25
and 50 mg p.o.).
The following pharmacological activities were evaluated
by standard procedures:
5.1.4. Skin phototoxicity
All new heteroangelicins were evaluated following a
standard procedure already described [30]. Compounds
were applied on the depilated skin of albino guinea-pigs
Table 3
Antipyretic activity by yeast-induced pyrexia testa
Comp.
Dose
Mean values of rectal temperature (8C ^ SE) at the following times (h) after treatment, 18 h from yeast treatment
(in parentheses difference from 0 value)b
(mg/kg p.o.)
0
1
2
3
4
Control
±
38.7 ^ 0.2
38.7 ^ 0.2
39.2 ^ 0.3
39.0 ^ 0.3
38.9 ^ 0.2
39.0 ^ 0.2
38.7 ^ 0.1
38.8 ^ 0.2
38.7 ^ 0.2 ( 1 0)
38.0 ^ 0.2 ( 2 0.7)
38.5 ^ 0.3 ( 2 0.7)
38.9 ^ 0.2 ( 2 0.1)
38.8 ^ 0.2 ( 2 0.1)
38.3 ^ 0.4 ( 2 0.7)
38.5 ^ 0.2 ( 2 0.2)
38.5 ^ 0.1 ( 2 0.3)
38.6 ^ 0.3 ( 2 0.1)
37.3 ^ 0.2 ( 2 1.4)
38.5 ^ 0.3 ( 2 0.7)
38.4 ^ 0.1 ( 2 0.6)
38.6 ^ 0.1 ( 2 0.3)
37.8 ^ 0.5 ( 2 1.2)
38.3 ^ 0.2 ( 2 0.4)
38.6 ^ 0.2 ( 2 0.2)
38.6 ^ 0.3 ( 2 0.1)
37.2 ^ 0.3 ( 2 1.5)
38.4 ^ 0.2 ( 2 0.8)
38.3 ^ 0.2 ( 2 0.7)
38.4 ^ 0.1 ( 2 0.5)
37.6 ^ 0.5 ( 2 1.4)
38.0 ^ 0.3 ( 2 0.7)
38.7 ^ 0.1 ( 2 0.1)
38.5 ^ 0.2 ( 2 0.2)
36.8 ^ 0.2 ( 2 1.9)
38.2 ^ 0.4 ( 2 1.0)
38.2 ^ 0.2 ( 2 0.8)
38.4 ^ 0.1 ( 2 0.5)
36.8 ^ 0.2 ( 2 2.2)
37.8 ^ 0.3 ( 2 0.9)
38.7 ^ 0.1 ( 2 0.1)
Indomethacin
5
8b
9a
50
50
50
50
50
50
9b
10a
10b
11a
a Each compound was tested on a group of ®ve albino rats (200±220 g).
b Mean value of ®ve determinations.

L. Mosti et al. / Il Farmaco 53 (1998) 602±610
607
Table 4
In®ltration anesthesia by pinch-tail testa
Table 5
Activity against ventricular ®brillation caused by aconitine in albino rats
(anti-arrhythmic activity)a
Comp.
Doseb
Activityc
5 min
Comp.
Dose
Appearance
time
Death time
30 min
(mg/
(s ^ SE)
kg p.o.)
(s ^ SE) of
extrasystoles
Lidocaine
8b
9a
0.1
0.1
80
20
60
40
40
20
10
30
60
100
40
60
40
40
40
10
30
60
b
0.1
Control (aconitine HCl)
±
187 ^ 18.3
376 ^ 24.8d 1086 ^ 32.4d
652 ^ 22.3
9b
10a
0.1
Quinidine
8b
9a
25
50
50
50
50
50
50
0.1
0.1
192 ^ 18.4
244 ^ 20.5c
344 ^ 25.9d
210 ^ 20.4
254 ^ 22.3c
184 ^ 21.8
676 ^ 28.2
746 ^ 34.3c
856 ^ 41.2d
723 ^ 31.4
718 ^ 19.4c
672 ^ 28.4
10b
11a
0.025
0.05
0.1
9b
10a
10b
11a
a Ten mice (20±25 g)/group.
b Percent of glycofurol solution (0.2 ml).
c Percentage of animals showing anesthesia, 5 and 30 min after in®ltration
of test compounds into the tail root. Dieffenbach tweezers were applied for
10 s.
a Five animals (200±250 g)/group.
b 15 mg/kg i.v./min.
d Statistically signi®cant value calculated in comparison with the test
performed with aconitine only (P , 0:01).
c Statistically signi®cant value calculated in comparison with the test
performed with aconitine only (P , 0:05).
1. anti-in¯ammatory activity, evaluated by carrageenan-
induced paw edema in rats (Table 2);
2. analgesic activity evaluated by acetic acid writhing test
in mice;
3. antipyretic activity (Table 3);
the inhibitory test of platelet aggregation induced by
collagen.
5.3. Chemistry
4. local anaesthetic activity, evaluated as in®ltration anaes-
thesia in rats (Table 4);
Melting points were determined with a Fisher-Johns
apparatus and are uncorrected. UV spectra were measured
in 95% ethanol on a Perkin Elmer Lambda 3 spectrophot-
ometer. IR spectra were registered on a Perkin Elmer 398
5. anti-arrhythmic activity, evaluated as protection index
against ecgraphic effects from aconitine in rats (Table 5);
6. platelet anti-aggregating activity, evaluated in vitro by
Table 6
Physical and spectral data of compounds 3a,b, 8a,b and 9a,b
Comp.
R
R0
Yield (%)
48
M.p. (8C)a
Formula
IR (cm21
(CHCl3)
)
1H NMR (d, ppm) (CDCl3)
3a
CH3
190±192
C13H10O4S
1752, 1698
1755, 1690
3.03 (m, 4H, CH2-5 1 CH2-6), 3.96 (s, 3H,
CH3O), 7.24 (d, J ˆ 5, 1H, H-9), 7.41 (d,
J ˆ 5, 1H, H-8), 8.26 (s, 1H, H-4)
1.36 (t, J ˆ 7, 3H, CH3), 3.02 (m, 4H, CH2-5 1
CH2-6), 4.38 (q, J ˆ 7, 2H, CH2), 7.25 (d,
J ˆ 5, 1H, H-9), 7.42 (d, J ˆ 5, 1H, H-8), 8.22
(s, 1H, H-4)
3b
C2H5
40
130±132
C14H12O4S
8a
8b
CH3
CH3
CH3
43
54
250±252 (dec)
194±195 (dec)
C13H12N2O4
C14H14N2O4
1745, 1695
1758, 1690
2.93 (s, 4H, CH2-8 1 CH2-9), 3.84 (s, 3H,
CH3N), 3.88 (s, 3H, CH3O), 7.81 (s, 1H, H-7),
8.19 (s, 1H, H-3)
C2H5
1.37 (t, J ˆ 7, 3H, CH3±CH2), 2.98 (s, 4H, CH2-
8 1 CH2-9), 3.87 (s, 3H, CH3N), 4.37 (q,
J ˆ 7, 2H, CH3±CH2), 7.84 (s, 1H, H-7), 8.20
(s, 1H, H-3)
9a
9b
CH3
C6H5
C6H5
56
60
238±239 (dec)
178±179 (dec)
C18H14N2O4
C19H16N2O4
1753,1700
1757, 1692
3.05 (m, 4H, CH2-8 1 CH2-9), 3.89 (s, 3H,
CH3O), 7.56 (s, 5H, C6H5), 8.07 (s, 1H, H-7),
8.27 (s, 1H, H-3)
C2H5
1.37 (t, J ˆ 7, 3H, CH3±CH2), 3.06 (m, 4H,
CH2-8 1 CH2-9), 4.37 (q, J ˆ 7, 2H, CH3±
CH2), 7.56 (s, 5H, C6H5), 8.06 (s, 1H, H-7), 8.27
(s, 1H, H-3)
a From ethyl acetate.

608
L. Mosti et al. / Il Farmaco 53 (1998) 602±610
Table 7
Physical and spectral data of compounds 4a,b, 5, 6, 10a,b and 11a,b
Comp.
R
R0
Yield
(%)
M.p. (8C) Formula
UV, lmax (nm) (log e)
IR (cm21
)
1H NMR (d, ppm)
4a
CH3
65 (A),
65 (B)
184±185a C13H8O4S
235 (4.30), 257 (4.26), 1760, 1735d 4.01 (s, 3H, CH3O), 7.30±7.80 (m, 4H,
275 (4.30), 324 (4.17),
H-5 1 H-6 1 H-8 1 H-9), 8.74 (s,
1H, H-4)f
366 (4.02)
4b
C2H5
80 (A),
44 (B)
130±132a C14H10O4S
(dec)
236 (4.31), 257 (4.27), 1760, 1735d 1.43 (t, J ˆ 7, 3H, CH3), 4.47 (q,
276 (4.32), 324 (4.18),
366 (4.03)
J ˆ 7, 2H, CH2), 7.30±8.00 (m, 4H,
H-5 1 H-6 1 H-8 1 H-9), 8.69 (s,
1H, H-4)f
5
6
93
70
253±255b C12H6O4S
(dec)
237 (4.21), 258 (4.20), 3100±2500, 7.40±8.20 (m, 4H, H-5 1 H-6 1 H-
276 (4.14), 320 (4.00), 1745, 1686e 8 1 H-9), 8.94 (s, 1H, H-4), 13.20 (br
363 (3.86)
s, 1H, CO2H; disappears with D2O)g
149±150c C11H6O2S
(dec)
236 (4.26), 258 (4.38), 1723, 1600d 6.47 (d, J ˆ 10, 1H, H-4), 7.34 (d,
268 (4.23), 308 (3.95),
342 (3.88)
J ˆ 2, 1H, H-9), 7.40±8.00 (m, 3H, H-
5 1 H-6 1 H-8), 7.87 (d, J ˆ 10,
1H, H-3)f
10a
10b
CH3 CH3 40 (A),
28 (B)
245±246b C13H10N2O4
(dec)
227 (4.29), 269 (4.26), 1762, 1730d 3.99 (s, 3H, CH3O), 4.17 (s, 3H,
278 (4.35), 323 (3.75),
368 (4.13)
CH3N), 7.20±7.80 (m, 2H, H-8 1 H-
9), 8.44 (s, 1H, H-7), 8.78 (s, 1H, H-
3)f
C2H5 CH3 42 (A),
13 (B)
174±175a C14H12N2O4
(dec)
226 (4.37), 268 (4.35), 1760, 1735d 1.42 (t, J ˆ 7, 3H, CH3±CH2), 4.17 (s,
277 (4.44), 323 (3.85),
367 (4.22)
3H, CH3N), 4.46 (q, J ˆ 7, 2H, CH3±
CH2), 7.20±7.70 (m, 2H, H-8 1 H-9),
8.39 (s, 1H, H-7), 8.72 (s, 1H, H-3)f
11a
11b
CH3 C6H5 22 (A),
13 (B)
227±228a C18H12N2O4
(dec)
242 (4.14), 277 (4.42), 1763, 1738d 4.00 (s, 3H, CH3), 7.40±7.90 (m, 7H,
368 (4.18)
C6H5 1 H-8 1 H-9), 8.62 (s, 1H, H-
7), 8.76 (s, 1H, H-3)f
C2H5 C6H5 32 (A),
58 (B)
160±161a C19H14N2O4
240 (4.22), 277 (4.42), 1765, 1738d 1.43 (t, J ˆ 7, 3H, CH3±CH2), 4.54 (q,
368 (4.18)
J ˆ 7, 2H, CH3±CH2), 7.45±7.85 (m,
7H, C6H5 1 H-8 1 H-9), 8.59 (s,
1H, H-7), 8.71 (s, 1H, H-3)f
a From ethyl acetate.
b From ethanol 958C.
c From diethyl ether.
d CHCl3.
e KBr.
f CDCl3.
g DMSO-d6. (A) Method A; (B) method B.
1
spectrophotometer. H NMR spectra were recorded on a
Hitachi Perkin Elmer R-600 (60 MHz) or a Varian Gemini
200 (200 MHz) spectrometer; chemical shifts are reported
as d (ppm) relative to TMS as internal standard; J in Hz.
Elemental analyses were performed at the Microanalytical
Laboratory of the Dipartimento di Scienze Farmaceutiche,
University of Genova, using a Carlo Erba Elemental Analy-
zer Model 1106. Analytical results for C, H and N were
within ^ 0.3% of the theoretical values.
5.3.2. General procedure for the preparation of esters of
5,6-dihydro-2-oxo-2H-thieno[2,3-h]-1-benzopyran-3-
carboxylic acid 3a,b and of 1-methyl or 1-phenyl substituted
8,9-dihydro-5-oxo-5H-pyran[2,3-e]indazol-6-carboxylic
acid 8a,b, 9a,b
A mixture of 5-dimethylaminomethylene-6,7-dihydro-
benzo[b]thiophen-4(5H)-one 2 or 5-dimethylaminomethy-
lene-1,5,6,7-tetrahydro-(1-methyl) (1-phenyl)-4H-indazol-
4-ones 7a,b (10 mmol) in the appropriate dialkylmalonate
(175 mmol) was re¯uxed for 6 h (only 1 h in the case of 7a
in dimethylmalonate). Then the malonic ester excess was
evaporated under reduced pressure, diethyl ether was added
to the resulting residue, cooled, and ®ltered to give a raw
solid which was recrystallized from ethyl acetate. Yields,
melting points, IR and 1H NMR spectral data are reported in
Table 6.
5.3.1. General procedure for the preparation of N,N-
dimethylaminomethyleneketones (2) [25] and 7a,b [26]
This procedure was realized directly by re¯uxing of the
suitable ketone (20 mmol) in N,N-dimethylformamide
dimethylacetal (3.57 g, 30 mmol) for different times (2, 6
h, 51%; 7a, 24 h, 40%; 7b, 24 h, 55%).

L. Mosti et al. / Il Farmaco 53 (1998) 602±610
609
5.3.3. General procedure for the preparation of esters of
References
2-oxo-2H-thieno[2,3-h]-1-benzopyran-3-carboxylic acid
4a,b and of 1-methyl or 1-phenyl substituted 5-oxo-5H-
pyran[2,3-e]indazol-6-carboxylic acid 10a,b, 11a,b
[1] J.A. Parrish, R.S. Stern, M.A. Pathak, T.B. Fitzpatrick, Photoche-
motherapy of skin disease, in: J.D. Regan, J.A. Parrish (Eds.), The
Science of Photomedicine, Plenum, New York, 1982, p. 595.
[2] F.P. Gasparro, Extracorporeal photochemotherapy, Clinical aspects
and the molecular basis for ef®cacy, R.G. Landes, Austin, TX, 1994.
[3] G.D. Cimino, H.B. Gamper, S.T. Isaacs, J.E. Hearst, Psoralens as
photoreactive probes of nucleic acid structure and function: organic
chemistry, photochemistry and biochemistry, Annu. Rev. Biochem.
54 (1985) 1151±1193.
Method A. A warm solution of DDQ (2.27 g, 10 mmol) in
anhydrous toluene (50 ml) was added dropwise to a warm
and stirred solution of dihydroprecursors 3a,b, 8a,b and
9a,b (10 mmol) in the same solvent (100±120 ml). After
the addition was completed, the reaction mixture was
re¯uxed (24 h for 3a,b and 8a,b; up to 5 days for 9a,b)
and then ®ltered. The ®ltrate was washed twice with 1 M
sodium hydroxide and once with water, dried (MgSO4) and
evaporated under reduced pressure. The solid residue was
recrystallized from a suitable solvent. Yields, melting
[4] J. North, H. Neyndorff, J.P. Levy, Photosensitizers as virucidal
agents, J. Photochem. Photobiol. B: Biol. 17 (1993) 99±108.
[5] F. Dall'Acqua, S. Marciani Magno, F. Zambon, G. Rodighiero,
Kinetic analysis of the photoreaction (365 nm) between psoralen
and DNA, Photochem. Photobiol. 29 (1979) 489±495.
1
points, UV, IR and H NMR spectral data are reported in
Table 7.
[6] R.S. Stern, R. Lange, Members of the photochemotherapy follow-up
study. Non-melanoma skin cancer occurring in patients treated with
PUVA ®ve to ten years after ®rst treatment, J. Invest. Dermatol. 91
(1988) 120±124.
Method B. A warm solution of DDQ (2.27 g, 10 mmol) in
anhydrous toluene (50 ml) was added dropwise to a warm
and stirred solution of 2 or 7a,b (10 mmol) and the appro-
priate dialkylmalonate (175 mmol) in the same solvent (100
ml). After the addition was completed, the reaction mixture
was re¯uxed for 24 h and then ®ltered. The ®ltrate was
washed twice with 1 M sodium hydroxide and once with
water, dried (MgSO4) and evaporated under reduced pres-
sure. The solid raw residue was recrystallized from a suita-
ble solvent as in Method A.
[7] A. Guiotto, P. Rodighiero, P. Manzini, G. Pastorini, F. Bordin, F.
Baccichetti, F. Carlassare, D. Vedaldi, F. Dall'Acqua, M. Tamaro,
G. Recchia, M. Cristofolini, 6-Methylangelicins: a new series of
potential photochemoterapeutic agents for the treatment of psoriasis,
J. Med. Chem. 27 (1984) 959±967 and references cited therein.
[8] D. Vedaldi, F. Dall'Acqua, F. Baccichetti, F. Carlassare, F. Bordin, P.
Rodighiero, P. Manzini, A. Guiotto, Methylangelicins: structure
activity studies on the role of methyl groups present in 3, 4 and 40,
50 photoreactive sites, Farmaco 46 (1991) 1381±1406.
[9] L. Mosti, P. Schenone, G. Menozzi, G. Romussi, F. Baccichetti, F.
Carlassare, D. Vedaldi, F. Bordin, Synthesis and photobiological
activity of some 4-amino-3-chloroangelicin derivatives, Eur. J.
Med. Chem. 18 (1983) 113±120.
5.3.4. 2-Oxo-2H-thieno[2,3-h]-1-benzopyran-3-carboxylic
acid 5
[10] L. Mosti, G. Menozzi, P. Schenone, F. Carlassare, F. Baccichetti, F.
Bordin, Synthesis and photobiological activity of new N,N-disubsti-
tuted 4-amino-3-chloroangelicins, Farmaco 43 (1988) 775±792.
[11] D. Vedaldi, F. Dall'Acqua, S. Caf®eri, F. Baccichetti, F. Carlassare, F.
Bordin, A. Chilin, A. Guiotto, 4,40,50-trimethyl-8-azapsoralen, a new
photoactive and non skin-phototoxic bifunctional bioisoster of psor-
alen, Photochem. Photobiol. 53 (1991) 143±148.
A solution of ester 4a (2.60 g, 10 mmol) in a 50% hydro-
chloric and acetic acid mixture (100 ml) was re¯uxed for 24
h. After cooling, the solid residue was ®ltered, washed with
water and recrystallized from 95% ethanol. Yields, melting
1
points, UV, IR and H NMR spectral data are reported in
Table 7.
[12] B. Pani, M. Tamaro, F. Baccichetti, F. Carlassare, M. Simonato, F.
Bordin, Aspects of genotoxic activity of some 8-azapsoralen deriva-
tives, Med. Biol. Environ. 20 (1992) 117±124.
[13] F. Baccichetti, F. Bordin, M. Simonato, L. Toniolo, C. Marzano, P.
Rodighiero, A. Chilin, F. Carlassare, Photobiological activity of
certain new methylazapsoralens, Farmaco 47 (1992) 1529±1541.
[14] A.E. Jakobs, L.E. Christiaens, M.J. Renson, Synthesis of monosulfur
and monoselenium analogues of psoralen, Tetrahedron 50 (1994)
9315±9320.
5.3.5. 2H-Thieno[2,3-h]-1-benzopyran-2-one 6
A solution of acid 5 (1.23 g, 10 mmol) in quinoline (20
ml) containing copper powder (0.16 g) was re¯uxed for 3 h.
The hot mixture was ®ltered, the liquid was cooled and
chloroform (20±30 ml) was added. The solution was
extracted twice with 6 M hydrochloric acid, washed once
with water, dried (MgSO4) and evaporated under reduced
pressure. The solid residue was recrystallized from diethyl
[15] A.E. Jakobs, J. Piette, Photobiological activity of sulfur and selenium
analogues of psoralen, J. Photochem. Photobiol. B: Biol. 22 (1994) 9±
15.
[16] D. Vedaldi, S. Caf®eri, S. Frank, F. Dall'Acqua, A.E. Jakobs, J. Piette,
Sulfur and selenium analogues of psoralen as novel potential photo-
chemotherapeutic agents, Farmaco 50 (1995) 527±536.
1
ether. Yields, melting points, UV, IR and H NMR spectral
data are reported in Table 7.
[17] D. Vedaldi, G. Piazza, S. Moro, S. Caf®eri, G. Miolo, G.G. Aloisi, F.
Elisei, F. Dall'Acqua, 1-Thiopsoralen, a new photobiologically active
heteropsoralen. Photophysical, photochemical and computer aided
studies, Farmaco 52 (1997) 645±652.
[18] P. Rodighiero, A. Guiotto, A. Chilin, F. Bordin, F. Baccichetti, F.
Carlassare, D. Vedaldi, S. Caf®eri, A. Pozzan, F. Dall'Acqua, Angular
furoquinolinones, psoralen analogs: novel antiproliferative agents for
skin disease. Synthesis, biological activity, mechanism of action and
computer-aided studies, J. Med. Chem. 39 (1996) 1293±1302.
[19] F. Bordin, C. Marzano, F. Carlassare, P. Rodighiero, A. Guiotto, S.
Caf®eri, F. Baccichetti, Photobiological properties of a new tetra-
Acknowledgements
Financial support from C.N.R., from Ministero dell'Uni-
Á
versita e della Ricerca Scienti®ca e Tecnologica (MURST)
and from Universita di Genova is gratefully acknowledged.
Á

610
L. Mosti et al. / Il Farmaco 53 (1998) 602±610
methylfuroquinolinone, J. Photochem. Photobiol. B: Biol. 34 (1996)
159±168.
[25] L. Mosti, L. Sansebastiano, P. Fossa, P. Schenone, F. Mattioli, 1-
Phenyl-1H-indazole derivatives with analgesic and anti-in¯ammatory
activities, Farmaco 47 (1992) 357±365 and references cited therein.
[26] L. Mosti, P. Schenone, G. Menozzi, G. Romussi, Reaction of sulfene
with heterocyclic N,N-disubstituted a-aminomethyleneketones. X.
Synthesis of thieno[2,3-h]-1,2-benzoxathiin derivatives, J. Hetero-
cycl. Chem. 19 (1982) 1057±1059.
[20] C. Marzano, E. Severin, B. Pani, A. Guiotto, F. Bordin, DNA damage
and cytotoxicity induced in mammalian cells by a tetramethylfuro-
quinolinone derivative, Environ. Mol. Mutagen. 29 (1997) 256±264.
[21] L. Mosti, P. Schenone, G. Menozzi, G. Romussi, F. Baccichetti, F.
Carlassare, F. Bordin, Synthesis and photobiological activity of N,N-
disubstituted 4-amino-3-chloro-2H-thieno[2,3-h]-1-benzopyran-2-
ones 7-thioisosters of 4-amino-3-chloroangelicins, Farmaco (1984)
81±94.
[27] L. Mosti, G. Menozzi, P. Schenone, Reaction of dichloroketene and
sulfene with N,N-disubstituted a-aminomethyleneketones. Synthesis
of pyrano[2,3-e]indazole and 1,2-oxathiino[6,5-e]indazole deriva-
tives, J. Heterocycl. Chem. 21 (1984) 361±367.
[22] A. Guiotto, A. Chilin, P. Manzini, F. Dall'Acqua, F. Bordin, P.
Rodighiero, Synthesis and antiproliferative activity of furocoumarin
isosters, Farmaco 50 (1995) 479±488.
[28] M.C. Bellassoued-Fargeau, B. Graffe, M.C. Sacquet, P. Maitte, Addi-
Â
tion de derives maloniques sur des b-enaminones cycliques, J.
Â
Â
[23] A.E. Jakobs, L.E. Christiaens, A convenient synthesis of thiopyr-
ano[2,3-e]benzofuran: a new sulfur analogue of angelicin, J. Org.
Chem. 61 (1996) 4842±4844.
Heterocycl. Chem. 22 (1984) 713±717.
[29] M.H. Adams, Bacteriophages, Interscience, New York, 1959.
[30] F. Bordin, F. Carlassare, F. Baccichetti, A. Guiotto, P. Rodighiero, D.
Vedaldi, F. Dall'Acqua, 4,50-Dimethylangelicin: a new DNA-photo-
binding monofunctional agent, Photochem. Photobiol. 29 (1979)
1063±1070.
[24] M. Iester, P. Fossa, G. Menozzi, L. Mosti, F. Baccichetti, C. Marzano,
M. Simonato, Synthesis and photobiological properties of 3-acylan-
gelicins, 3-alkoxycarbonylangelicins and related derivatives, Farm-
aco 50 (1995) 669±678.