Pyrrolo[2,3-h]quinolinones: Synthesis and photochemotherapic activity

Article abstract of DOI:10.1016/S0960-894X(03)00529-8

A series of derivatives of the new ring system pyrrolo[2,3-h]quinoline-2-one was synthesized and evaluated as photoreagents toward cultured human tumor cells. Remarkable phototoxycity resulted for some derivatives, especially those bearing the phenyl grou

Full text of DOI:10.1016/S0960-894X(03)00529-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2809–2811
Pyrrolo[2,3-h]quinolinones: Synthesis and Photochemotherapic
Activity
Paola Barraja,a Patrizia Diana,a Antonino Lauria,a Alessandra Montalbano,a
Anna Maria Almerico,a Gaetano Dattolo,a Girolamo Cirrincione,a,* Giampietro Violab
and Francesco Dall’Acquab
a
`
Dipartimento Farmacochimico, Tossicologico e Biologico Universita degli Studi, Via Archirafi 32, 90123 Palermo, Italy
b
`
Dipartimento di Scienze Farmaceutiche Universita degli Studi, Via Marzolo 5, 35131 Padova, Italy
Received 24 December 2002; revised 21 February 2003; accepted 27 March 2003
Abstract—A series of derivatives of the new ring system pyrrolo[2,3-h]quinoline-2-one was synthesized and evaluated as photo-
reagents toward cultured human tumor cells. Remarkable phototoxycity resulted for some derivatives, especially those bearing the
phenyl group at the 7-position.
# 2003 Elsevier Ltd. All rights reserved.
Psoralens are a family of natural occurring linear furo-
coumarins, whose lead compound is 1. They find appli-
cation in the treatment of several skin diseases, such as
psoriasis, vitiligo and tumors such as T-cell lymphoma1
when used in conjunction with long-wave (320–400 nm)
ultraviolet light (UV-A). The effectiveness of this treat-
ment, called PUVA, is connected with the specific
damage these linear compounds can induce to DNA. In
the dark, psoralen molecules can form complex with
DNA via intercalation of the furocoumarin moiety
between base pairs of the nucleic acid; upon irradiation
with UVA light a [2+2] photocycloaddition with the
pyrimidine bases, particularly thymine, can occur
involving one or both DNA strands. Thus two types of
lesions can take place, monoadducts and crosslinks,
which are responsible of short and long term side effects
respectively: first ones involve a cycloaddition between
psoralen and a pyrimidine base with a 1:1 molar ratio,
and second ones a cycloaddition between one psoralen
molecule and two pyrimidine moieties belonging to
DNA complementary strands.2,3
Many approaches have been followed to obtain new
furocoumarin analogues with better DNA photobind-
ing ability and lower toxicity. One of these involves the
synthesis of heteroanalogues of 1 and 2 replacing one or
both of the two intracyclic oxygen atom. The substitu-
tion with the nitrogen atom at the furan ring leads
respectively to the linear or angular pyrrolocoumarins4,5
3, 4 whilst the same type of replacement at the pyrone
ring leads to the furoquinolinones6 5 which showed
strong antiproliferative effect both in the dark and
under light activation.
For this reason, considerable efforts have been done in
developing angular furocoumarins such as angelicin 2,
which on account of their geometry cannot crosslink
DNA allowing only monofunctional photobinding and
therefore reducing undesirable side effects especially long
term ones such as genotoxicity and risk of skin cancer.
These findings together with our interest in antitumor
polycondensed heterocycles containing the pyrrole/indole
*Corresponding author. Tel.: +39-091-616-1606; fax: +39-091-616-
9999; e-mail: gcirrinc@unipa.it
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00529-8

2810
P. Barraja et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2809–2811
moiety7a,b prompted us to synthesize and study the
photobiological activity of some new angelicin hetero-
analogues in which both the oxygen atoms are replaced by
nitrogens, leading to the new ring system pyrrolo[2,3-h]-
quinoline-2-one.
irradiation at different UVA doses.10 Control experi-
ments with drugs in the presence/absence of UVA light
or drugs alone were carried out without significant
cytotoxic effects (data not shown).
It can be noted that the compounds exhibit different
values of IC50 depending on the substitution pattern,
and a remarkable dose UVA-dependence. In particular,
compounds 11 and 12 show the highest cytotoxicity also
in comparison to 8-MOP, 5-MOP and angelicin used as
references compounds. The most active compounds
require a phenyl group linked to the indole moiety,
whereas a benzyl group in which the phenyl group is not
directly linked to the indole moiety strongly reduces the
activity. On the other hand, the substitution with a
methyl group causes the disappearance of the photo-
toxic activity. In order to understand the nature of the
binding between the drugs and the biological substrate,
linear dichroism (LD) measurements on DNA–drug
complexes were performed at various molar ratios. LD
is a technique that was demonstrated to be able to
characterize complexes between ligands and nucleic
acid.11,12
Scheme 1 outlines the synthetic pathway followed for
the synthesis of the desired new ring system. According
to the Stetter and Lauterbach procedure,8 the reaction
of 1,3-dihydroresorcin 6 with chloroacetone and potas-
sium hydroxide in methanol was stirred at room tempera-
ture for 48 h to afford the 1,3-acetonylcyclohexandione 7
as a mixture of the two tautomers which was used
without any further purification. Condensation of 7
with the suitable amine in acetic acid at 60 ꢀC for 2 h
yielded the 1-substituted 1,5,6,7-tetrahydro-2-methyl-
indole-4-ones 8 (60–80%). These latter by reaction with
ethyl formate and potassium t-butoxide in benzene, under
nitrogen atmosphere gave 1-substituted 5-hydroxy-
methylene-1,5,6,7-tetrahydro-2-methyl-indole-4-ones
in good yield (60–94%).
9
The enaminoketones 10, key intermediate for the
synthesis of the new ring system pyrrolo[2,3-h]quinoli-
none, were prepared in very high yield (85-90%) from
compounds 9 and diethylamine in benzene at room
temperature for 24 h. Ring closure was accomplished by
refluxing in ethanol for 24 h the versatile precursors
obtained with cyanomethylene compounds such as phe-
nylsulphonylacetonitrile, benzoylacetonitrile and malo-
nitrile.9 Recrystallization from ethanol gave compounds
11–17 in moderate to very good yield (30–80%).
In a typical LD experiment, the long axis of the DNA
double helix is aligned along the flow lines, thus the
DNA bases, which are oriented roughly perpendicular
to the helix axis, give a negative LD signal. The same
negative LD signal is recorded for drug molecules which
are intercalated in the DNA structure and are almost
perpendicular to the helix axis. Instead, drug molecules
which are bound in the minor groove of DNA, such as
netropsin or dystamicin, generally exhibit angles of 40–
50ꢀ between the transition moment of the oriented
chromophore and the helix axis, and give a positive LD
signal.11,12
The phototoxycity of title compounds, was investigated
on a cultured cell line of human fibrosarcoma (HT-
1080). Table 1 shows the extent of cell survival expres-
sed as IC50 which is the concentration, expressed in mM,
which induce 50% of inhibition of cell growth, after
The absorbance spectra, and the LD spectra of two
derivatives (11 and 12), recorded at different [Drug]/
[DNA] ratios are shown in Figure 1. Inspection of the
LD spectra of the derivatives in the presence of salmon
testes-DNA reveals that despite a strong absorption in
the 300–500 nm region, the two compounds give no LD
bands in this region. Furthermore fluorescence titrations
Table 1. Values of IC50 (mM) obtained for the test compounds at
different UVA doses in HT-1080 human fibrosarcomaa
Compd
Cytotoxicity (IC50, mM)
2.6 (J cmÀ2 b
)
3.2 (J cmÀ2
)
6.5 (J cmÀ2
)
11
12
13
14
15
16
17
3.6Æ0.5
6.6Æ1.6
>20
>20
>20
2.3Æ0.8
4.6Æ0.9
13.1Æ1.5
>20
0.40Æ0.04
1.7Æ0.2
11.1Æ1.4
16.4Æ1.4
>20
1.8Æ0.1
1.9Æ0.02
1.5Æ0.2
0.9Æ0.3
2.5Æ0.3
>20
>20
15.1Æ0.2
9.0Æ1.1
2.1Æ0.3
1.8Æ0.4
2.6Æ0.2
14.7Æ0.9
7.8Æ0.7
8.5Æ0.6
15.7Æ1.9
8-MOP
5-MOP
Angelicin
aValues are meansÆSEM of three experiments.
Scheme 1. Synthesis of compounds 11–17: (a) AcCH2Cl, KOH,
methanol, rt; (b) RNH2, AcOH, 60 ꢀC; (c) HCOOEt, t-BuOK, ben-
zene, rt; (d) HNEt2, benzene, rt; (e) NC-CH2R1, ethanol, reflux.
bUVA dose expressed in J cmÀ2 as measured at 365 nm by a Cole
Parmer Radiometer.

P. Barraja et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2809–2811
2811
3. Dall’Acqua, F.; Viola G. In Photobiology for the 21th cen-
tury; Coohil T. P., Valenzeno D. P. Eds. Valdenmar: Overland
Park, KS, 2001; p 325.
4. Quanten, E.; Adriaens, P.; De Schryver, F. C.; Roelandts,
R.; Degreef, H. Photochem. Photobiol. 1986, 43, 485.
5. Rodighiero, P.; Chilin, A.; Pastorini, G.; Guiotto, A. J.
Heterocyclic Chem. 1987, 24, 1041.
6. Rodighiero, P.; Guiotto, A.; Chilin, A.; Bordin, F.; Bacci-
chetti, F.; Carlassare, F.; Vedaldi, D.; Caffieri, S.; Pozzan, A.;
Dall’Acqua, F. J. Med. Chem. 1996, 39, 1293.
7. (a) Barraja, P.; Diana, P.; Lauria, A.; Passannanti, A.;
Almerico, A. M.; Minnei, C.; Longu, S.; Congiu, D.;
Musiu, C.; La Colla, P. Bioorg. Med. Chem. 1999, 7, 1591.
(b) Cirrincione, G.; Almerico, A. M.; Barraja, P.; Diana, P.;
Lauria, A.; Passannanti, A.; Pani, A.; Pinna, E.; Scintu, F.;
Musiu, C.; Pisano, L.; La Colla, P. J. Med. Chem. 1999, 42,
2561.
8. Stetter, H.; Lauterbach, R. Liebigs. Ann. Chem. 1962, 652,
40.
Figure 1. Absorbance (A), and linear dichroism (LD) spectra of mix-
tures of salmon testes DNA and compounds 11 (A1, B1) and 12 (A2,
B2) at different [Drug]/[DNA] ratio (a=0.00, b=0.02, c=0.04).
9. All final products were characterized by IR, 1H and 13C
NMRand elemental analysis. A representative example, com-
ꢀ
À1
pound 11: 80% mp>300 C; IR2921 (NH), 1635 (CO) cm
;
1H NMR(DMSO- d6): 2.02 (3H, s, CH3), 2.56 (2H, t, J=7.6
Hz, CH2), 2.80 (2H, t, J=7.6 Hz, CH2), 6.71 (1H, s, H-9), 7.38
(2H, d, J=6.8, Ar), 7.50–7.69 (6H, m, Ar), 7.96 (2H, d, J=6.8
Hz, Ar), 8.10 (1H, s, H-4), 12.24 (1H, s, NH); 13C NMR
(DMSO-d6): 12.5 (q), 20.8 (t), 25.4 (t), 103.3 (d), 107.0 (s),
111.8 (s), 118.1 (s), 127.2 (2Âd), 127.5 (2Âd), 128.4 (3Âd),
129.4 (2Âd), 130.8 (s), 132.5 (d), 136.2 (s), 137.7 (s), 141.5(s),
142.0 (d), 148.6 (s), 158.2 (s). Anal. calcd for C24H20N2SO3: C,
69.21; H, 4.84; N, 6.73. Found: C, 69.11; H, 4.69; N, 6.53.
10. Antitumor assay. Exponentially growing HT-1080 human
fibrosarcoma cells were resuspended at a density of 5Â104
cells/mL in a complete medium (DMEM containing 10% fetal
bovine serum, 100 UI/mL penicillin G and 100 mg/mL strep-
tomycin) and seeded in a 96 well culture plates which were
allowed to adhere for 18 h to culture plates before addition of
the drugs. After the medium was removed, 100 mL of the drug
solution, dissolved in DMSO and diluted with Hank’s
balanced salt solution (HBSS pH=7.2), was added to each
well. The plate was then incubated for 30 min in an atmos-
phere of 5% CO2 at 37 ꢀC, the control plate was placed in the
dark and then irradiated with two HPW 125 Philips lamp,
principally emitting at 365 nm. After irradiation, the solution
was replaced by the medium and the plates were incubated for
72 h. Cell viability was assayed by the MTT [(3-(4,5-dime-
thylthiazol-2-yl)-2,5diphenyl tetrazolium bromide)] method.13
Cell growth at each drug concentration was expressed as per-
centage of untreated controls and the concentration resulting
in 50% (IC50) growth inhibition was determined by linear
regression analysis.
carried out with the same derivatives (data not shown)
showed a poor fluorescence quenching confirming that
the title compounds are loosely bound to DNA. This
fact strongly suggests that the new derivatives do not
interact efficaciously with the macromolecule. Con-
sidering these results, the new pyrroloquinolinones,
appear as novel and potentially useful agents in photo-
chemotherapy in so far as they show a remarkable photo-
antiproliferative activity which does not appear related to
the classical mechanism (i.e., formation of mono- and
bifunctional adducts to DNA) exerted by psoralens.
On the basis of the biological evaluation, experiments
aimed at defining the targets at cellular level and the
mechanism of phototoxycity are in progress.
Acknowledgements
This work was financially supported by Ministero
dell’Istruzione dell’Universita e della Ricerca.
References and Notes
1. Pathak, M. A.; Parrish, J. A.; Fitzpatrik, T. B. Farmaco
1981, 36, 479.
2. Musajo, L.; Rodighiero, G. In Photophysiology; Giese, A.
C., Ed.; Accademic: New York, 1972; Vol. 7, p 115.
11. Norde
1992, 25, 51.
12. Norden, B.; Kurucsev, T. J. Mol. Recognit. 1994, 7, 141.
13. Mosmann, T. J. Immunol. Meth. 1983, 65, 55.
´
n, B.; Kubista, M.; Kurucsev, T. Q. Rev. Biophys.
´