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A water soluble derivative of 4,5'-dimethylangelicin (I) having a long chain linking an amino group to the planar furocoumarinic moiety, that is 4'-N,N-dimethylaminoethoxymethyl-4,5'-dimethylangelicin (III), has been prepared. This compound is able to form effectively the intercalated complex with DNA like the previously prepared 4'-aminomethyl-4,5'-dimethylangelicin (II), however while the compound (II) showed very poor photobinding to DNA, the new derivative (III) shows high photobinding to the macromolecule. It is proposed that these data illustrate the importance of the geometry of intercalation for the subsequent covalent photobinding to the macromolecule. Also some photophysical data of (II) and of (III) appear to confirm the critical role of the position assumed by the chromophore of the two compounds when intercalated in duplex DNA. The compound (III) displays high photobiological effects, also in terms of antiproliferative activity as shown by its capacity to inhibit DNA and RNA synthesis in Ehrlich cells, the growth of an E. coli culture and the infectivity of T2 phage. (III) on the basis of these properties seems to deserve a clinical evaluation of its potential photochemotherapeutic activity in the treatment of psoriasis.

The photobiological properties of a series of pyranocoumarin derivatives having linear (xanthyletines) and angular structure (seselins) have been studied; while the linear derivative carrying the methyl geminal group, typical of the parent natural compound, appeared to be entirely inactive in all tests performed, very probably because of the steric hindrance in the dark interaction with DNA, 4 substances lacking in this group showed a marked capacity for inhibiting DNA synthesis in Ehrlich cells. In particular, 4,6-dimethyl-8-desmethylseselin proved to be about 7 times more active than 8-MOP. Practically all compounds were capable of inducing cross-links in DNA, but this feature, marked in the linear compounds, is very much reduced in the angular ones; this property appears to be clearly related to the mutagenicity in the light and with the skin phototoxicity, which are both marked in the former and low or absent in the latter. In the dark, while all compounds are non-mutagenic in the absence of metabolic activation, in the presence of microsomial enzymes pyranocoumarins become mutagens; only the xanthyletine derivative carrying a geminal methyl group at the 8 position was not activated, suggesting that the enzymes metabolize the pyranic ring.

T2 bacteriophage sensitization has been studied using two furocoumarins capable of linking covalently to DNA to the same extent but producing different damages, psoralen and 4,5'-dimethylangelicin. Psoralen is a well-known linear furocoumarin capable of inducing in DNA both monoadducts and cross-links; 4,5'-dimethylangelicin is a new angular compound known as a pure monofunctional reagent. In the sensitization of T2 mature virions both drugs proved very active, yielding survival curves practically superimposable; on the contrary, in the experiments with the T2 vegetative form, i. e. its DNA inside the host, 4,5'-dimethylangelicin resulted much less effective, resembling the picture observed in the inactivation of the host bacteria. This result did not appear related to an enhancement of DNA repair by a Weigle effect. The different killing activity of 4,5'-dimethylangelicin can be explained supposing that this drug is capable of inducing cross-links in T2 DNA inside the virus core, in which it exists in a very folded form, but not in the same DNA after injection into the host bacteria.

Angelicins are a group of compounds that show marked photobiologic activity on various substrates; some of them have been proposed as potential agents for the photochemotherapy of skin diseases. A good correlation exists between the photosensitizing activity of these compounds and their capacity to induce monofunctional lesions to DNA; therefore, we believe the chemical nature of these photolesions, we isolated from the products of hydrolysis of the photocombinations between 5 angelicins (angelicin, 4-methyl, 5-methyl, 5'-methyl, and 5,5'-dimethylangelicin) and DNA, the corresponding new fluorescent monoadducts between the 4',5'-double bond of the furocoumarins and the 5,6-double bond of thymine.

4,5'-Dimethylpsoralen, a bifunctional furocoumarin, can be formed as an impurity in the synthesis of its angular isomer, that is 4,5'-dimethylangelicin; the latter has recently been proposed as a potential monofunctional agent for photochemotherapy. To have precise information on the possible modifications of the photochemical and photobiological properties of synthetic 4,5'-dimethylangelicin caused by the presence of its linear isomer, we have studied the interactions of the latter with DNA in both the ground and the excited state and its photobiological activity. 4,5'-Dimethylpsoralen photobinds much more effectively to DNA than its angular isomer and is capable to form effectively inter-strand cross-linkages in DNA while dimethylangelicin is unable to form these bifunctional adducts in DNA. Dimethylpsoralen shows a strong skin-phototoxicity while angelicin lacks this activity. Moreover the antiproliferative activity of the psoralen derivative in terms of DNA synthesis inhibition in Ehrlich cells and of inhibition of infectivity of T2 phages, is about four times higher than that of the angular isomer. These data stress the necessity of the absence of the isomeric linear furocoumarin in the synthetic 4,5'-dimethylangelicin because its presence can markedly modify the photobiological and phototherapeutic properties of the angelicin derivative.

Four compounds having a molecular structure analogous to that of tilorone and tilorone itself, taken as a reference compound, were examined for complex formation ability with DNA. While the association constants of the various complexes were almost the same, the r values in saturation conditions (that is the highest number of molecules bound per nucleotide of DNA) increased with the size of the planar moiety or with the length of the two basic side chains of the molecules. Concerning the structure of the complexes, it was evidenced by means of flow dichroism measurements that the non-covalent binding to DNA occurs via an intercalative mode. Moreover, it was observed that by decreasing the ionic strength, the affinity of the drugs for the macromolecule increases, indicating that in complex formation, electrostatic forces exerted between the DNA phosphate residues and the positively charged nitrogen of the side chains of the drugs are involved. It seems also possible that, in this condition, and in the presence of high concentrations of the drug, a secondary binding consisting only of electrostatic interactions outside of the helix takes place. In connection with the complexing ability with DNA, the examined compounds proved able to inhibit DNA and RNA synthesis in Ehrlich ascites tumor cells. A correlation was found between complexing ability and inhibitory activity on nucleic acid synthesis.

Three derivatives of angelicin (1) [4'-methyl-, 4,4'-dimethyl-, and 4',5-dimethylangelicin (2a-c)] have been prepared with the aim of obtaining new agents for the photochemotherapy of psoriasis. These compounds form a complex in the dark with DNA that shows an affinity for the macromolecule higher than that of the parent angelicin (1). A correlation between their octanol/water partition coefficients and the association constants of the complexes has been observed. Compounds 2a-c photobind to DNA to a much higher extent than 1 and also more effectively than 8-methoxypsoralen (8-MOP), taken as reference compound. When activated with UV-A, the three compounds strongly inactivate T2 phage and inhibit epidermal DNA synthesis in mice. Moreover, they show a mutagenic activity markedly lower than that of 8-methoxypsoralen on Escherichia coli wild-type strain. Due to its lack of skin phototoxicity, its low mutagenic activity, and its antiproliferative activity, 2c was chosen for clinical evaluation. It proved to be effective in clearing psoriasis in two patients.

Furocoumarin photosensitization induces lambda-prophage from lysogenic Escherichia coli cells; this effect is clearly due to the photoreaction that furocoumarins give with DNA, and it appears connected with the formation of monoadducts rather than of diadducts (cross-links).

7-Methyl- (1 a) and 4,7-dimethylallopsoralen (1 b), prepared about ten years ago and till now considered non-photosensitizing agents, have been re-investigated for studying their photochemical and photobiological properties. They form a molecular complex with DNA in the ground state, undergoing intercalation between two base pairs of the macromolecule. By successive irradiation with U.V.-A they covalently photobind to DNA inducing only the formation of mono-adducts. They are not able to induce erythema on guinea pig skin, but show evident antiproliferative activity on various biological substrates. Compound (1 b) shows both photochemical and photobiological properties at a degree higher than that of compound (1 a).

In addition to the bifunctional adducts (cross-linkages), that furocoumarins on radiation at 365 nm form in DNA, monofunctional adducts also proved able to inhibit the nucleic acid synthesis in Ehrlich ascites tumor cells.

With the aim of obtaining new bifunctional intercalators for DNA, a new series of bis-pyridoquinolines were synthesized by joining two tricyclic planar moieties through a flexible chain. For preparing the corresponding potential monofunctional intercalators also a series of mono-pyridoquinolines were prepared by chemical synthesis by joining the same chromophore with a flexible side-chain. The melting profiles of the complexes between two bis-pyridoquinolines and DNA showed two different thermal transitions supporting the bis-intercalation of the ligand with the macromolecule. On the other hand, two mono-pyridoquinolines showed only one transition in the melting profile indicating a behaviour consistent with a mono-intercalation. The antiproliferative activity was tested in terms of DNA synthesis inhibition in Ehrlich ascite tumor cells; the examined bis-intercalating agent showed to be much more active than the tested mono-intercalating compound.

Some aspects of the interactions between DNA and 8-methoxypsoralen (8-MOP) in its ground state (complex formation) or in its excited state (photobinding) have been investigated. 8-MOP shows a low affinity towards DNA in the complex formation; this fact minimizes the possible biological consequences deriving from this interaction, when it occurs in vivo. In covalent photobinding to DNA, 8-MOP forms mainly monofunctional adducts, and to a lesser extent bifunctional adducts, showing a behavior similar to that of other linearly condensed furocoumarins (psoralens); the ratio between mono- and bifunctional adducts was found to be 9:1. The covalent photobinding to DNA does not occur at random along the macromolecule, but preferentially at the level of specific receptor sites. The regions having an alternate sequence of A-T seem to be the best receptor sites for the formation of monoadducts while the regions containing an alternate sequence of A-T and C-G appeared to be the preferential sites for the cross-linkage formation.

With the aim of obtaining new agents for the photochemotherapy of psoriasis, we have prepared monofunctional reagents for DNA by starting from 4,5'-dimethylangelicin (2), an angular furocoumarin, and introducing in a 4'-(hydroxymethyl) (3), 4'-(methoxymethyl) (4), or 4'-(aminomethyl) group (5), in way analogous to what other authors have done previously on trioxsalen, a DNA bifunctional reagent. These new compounds form complexes with DNA in the ground state and by successive irradiation (UV-A) undergo monofunctional photoaddition to the macromolecule. Photobinding to DNA was highest for 3 and gradually lower for 4 and 5, respectively. These compounds do not form interstrand photocross-linkages in DNA and do not show any skin phototoxicity. Fluorimetric studies show that their 4',5' double bond is involved in the photoaddition to DNA. Their photobiological activity evaluated on Ehrlich ascites tumor cells and on T2 phages was strictly connected with their photobinding to DNA. The effect of the introduction of hydroxymethyl and methoxymethyl groups in angular 2 is somewhat similar to that previously described for trioxsalen: the introduction of an aminomethyl group in 2 markedly increases the affinity in the dark for DNA but under UV-A irradiation strongly inhibits photobinding to the macromolecule. By contrast, in the analogous derivative of trioxsalen both the affinity for DNA in the dark and the photobinding to DNA increased.