As a first step in the synthesis and the study of DNA polyintercalating drugs, dimers of acridines have been prepared. Their DNA binding properties have been studied. It has been determined that wen the chain separating the two aromatic rings is longer than a critical distance, bisintercalation is actually observed and that the DNA binding affinity becomes quite large (>108-109 M-1). It is shown also that the optical characteristics of these molecules are dependent on the sequences of DNA. The fluorescence intensity of one of these dimers when bound to DNA varies as the fourth power of its A+T content. This derivative could be used as a fluorescent probe of DNA sequence.

Thirty-three porphyrins or metalloporphyrins corresponding to the general formula [meso-[N-methyl-4(or 3 or 2)-pyridiniumyl]n(aryl)4-nporphyrin]M (M = H2, CuII, or ClFeIII), with n = 2-4, have been synthesized and characterized by UV-visible and 1H NMR spectroscopy and mass spectrometry. These porphyrins differ not only in the number (2-4) and position of their cationic charges but also in the steric requirements to reach even temporarily a completely planar geometry. In particular, they contain 0, 1, 2, 3, or 4 meso-aryl substituents not able to rotate. Interaction of these porphyrins or metalloporphyrins with calf thymus DNA has been studied and their apparent affinity binding constants have been determined by use of a competition method with ethidium bromide which was applicable not only for all the free base porphyrins but also for their copper(II) or iron(III) complexes. Whatever their mode of binding may be, their apparent affinity binding constants were relatively high (Kapp between 1.2 x 10(7) and 5 x 10(4) M-1 under our conditions), and a linear decrease of log Kapp with the number of porphyrin charges was observed. Studies of porphyrin-DNA interactions by UV and fluorescence spectroscopy, viscosimetry, and fluorescence energy transfer experiments showed that not only the tetracationic meso-tetrakis[N-methyl-4(or 3)-pyridiniumyl]porphyrins, which both involved four freely rotating meso-aryl groups, but also the corresponding tri- and dicationic porphyrins were able to intercalate into calf thymus DNA. Moreover, the cis dicationic meso-bis(N-methyl-2-pyridiniumyl)diphenylporphyrin, which involved only two freely rotating meso-aryl groups in a cis position, was also able to intercalate. The other meso-(N-methyl-2-pyridiniumyl)n(phenyl)4-nporphyrins, which involved either zero, one, or two trans freely rotating meso-aryl groups, could not intercalate into DNA. These results show that only half of the porphyrin ring is necessary for intercalation to occur.

A novel series of omega-aminoalkanoic acid derivatives of betulinic acid were synthesized and evaluated for their activity against human immunodeficiency virus (HIV). The anti-HIV-1 activity of several members of this new series was found to be in the nanomolar range in CEM 4 and MT-4 cell cultures. The optimization of the omega-aminoalkanoic acid side chain is described. The presence of an amide function within the side chain was found important for optimal activity. RPR 103611 (14g), a statine derivative, was found to be inactive against HIV-1 protease, reverse transcriptase, and integrase as well as on gp120/CD4 binding. "Time of addition" experiments suggested interaction with an early step of HIV-1 replication. As syncytium formation, but not virus-cell binding, seems to be affected, betulinic acid derivatives are assumed to interact with the postbinding virus-cell fusion process.

An ethidium homodimer and acridine ethidium heterodimer have been synthesized (Gaugain, B., Barbet, J., Oberlin, R., Roques, B. P., & Le Pecq, J. B. (1978) Biochemistry 17 (preceding paper in this issue)). The binding of these molecules to DNA has been studied. We show that these dimers intercalate only one of their chromophores in DNA. At high salt concentration (Na+ greater than 1 M) only a single type of DNA-binding site exists. Binding affinity constants can then be measured directly using the Mc Ghee& Von Hippel treatment (Mc Ghee, J. D., & Von Hippel, P. H. (1974) J. Mol. Biol. 86, 469). In these conditions the dimers cover four base pairs when bound to DNA. Binding affinities have been deduced from competition experiments in 0.2 M Na+ and are in agreement with the extrapolated values determined from direct DNA-binding measurements at high ionic strength. As expected, the intrinsic binding constant of these dimers is considerably larger than the affinity of the monomer (ethidium dimer K = 2 X 10(8) M-1; ethidium bromide K = 1.5 X 10(5) M-1 in 0.2 M Na+). The fluorescence properties of these molecules have also been studied. The efficiency of the energy transfer from the acridine to the phenanthridinium chromophore, in the acridine ethidium heterodimer when bound to DNA, depends on the square of the AT base pair content. The large increase of fluorescence on binding to DNA combined with a high affinity constant for nucleic acid fluorescent probes. In particular, such molecules can be used in competition experiments to determine the DNA binding constant of ligands of high binding affinity such as bifunctional intercalators.

A series of omega-undecanoic amides of lup-20(29)-en-28-oic acid derivatives were synthesized and evaluated for activity in CEM 4 and MT-4 cell cultures against human immunodeficiency virus type 1 (HIV-1) strain IIIB/LAI. The potent HIV inhibitors which emerged, compounds 5a, 16a, and 17b, were all derivatives of betulinic acid (3beta-hydroxylup-20(29)-en-28-oic acid). No activity was found against HIV-2 strain ROD. Compound 5a showed no inhibition of HIV-1 reverse transcriptase activity with poly(C).oligo(dG) as template/primer, nor did it inhibit HIV-1 protease. Additional mechanistic studies revealed that this class of compounds interfere with HIV-1 entry in the cells at a postbinding step.

In order to obtain antitumor agents, various 7H-pyridocarbazole dimers were prepared by quaternization of the pyridinic nitrogens of the different isomeric 7H-pyridocarbazole rings with halogenoamino alicyclic or aliphatic chains. The dimers interact with DNA more markedly than with the corresponding monomers, and the bisintercalation depends upon the nature, the flexibility, and the ionization state of the linking chains. They most often bisintercalate at pH 5 where the chain is protonated and monointercalate at pH 7.4. The apparent binding constants (kap) range from 10(8) to 10(9) M-1 at pH 5 and from 5 X 10(5) to 2 X 10(7) M-1 at pH 7.4. The bisintercalating dimers covered four DNA base pairs, whereas most of the monointercalating dimers covered two bases pairs. The antitumor activity against L1210 murine leukemia is strongly dependent on the position of attachment, the nature of the linking chain, and its rigidity. Three highly active dimers were obtained in the series of 7H-pyrido[4,3-c]carbazole dimers with rigid bis(ethylpiperidinyl) chains. On the other hand, two ellipticine dimers were prepared which were found completely inactive on L1210. These results show that in the series of 7H-pyridocarbazoles the process of dimerization leads to very active antitumor compounds.

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Abstract. Evidence for an internal oscillatory Brownian motion in the DNA helix is obtained from the measurement of the decay of the fluorescence emission anisotropy of the ethidium bromide-DNA complex. The amplitude of the oscillation is found to be equal to 350 and the relaxation time equal to 28 nanoseconds.The dye ethidium bromide has been found to bind specifically to doublestranded nucleic acid helix. The interesting characteristics of this binding are:(1) The fluorescence quantum efficiency of the dye increases considerably on binding to double-stranded nucleic acids." 2 (2) Binding occurs by intercalation.2' Then ethidium bromide is rigidly bound to the double-stranded helix and the direction of the absorption and emission transition moments which are in the plane of the dye have a known direction with reference to the helix axis.2 Then, ethidium bromide is an ideal candidate as a fluorescence label for fluorescence depolarization measurements since the ethidium bromide lifetime of fluorescence is relatively large.4It was first observed5 that the polarization fluorescence coefficient for ethidium bromide bound to DNA was dependent on the viscosity of the solution. In other words, in aqueous solvent a depolarization of fluorescence was observed. Since the DNA is a large molecule, the extent of its rotation during the time of the ethidium bromide excited state can be assumed negligible. Then, two hypotheses had to be postulated, either the DNA was not rigid and was able to undergo some kind of internal motion, or ethidium bromide was not really rigidly bound as predicted by the intercalation model.6 In these conditions it was of interest to further study this problem by measuring the emission anisotropy during the fluorescence decay. This method has been already used with macromolecules bearing covalently bound fluorescent chromophores. Detailed information on the Brownian motion and the internal deformation of these macromolecules have been obtained.7'0 Methods. Let Ill(t), I1(t), be, respectively, the decay of fluorescence intensity of the horizontal and perpendicular components when fluorescence excitation is made by an infinitely short flash of natural light. The time of the flash is taken as the origin.