Triple helix-specific ligands.

A triple helix is formed upon binding of an oligodeoxynucleotide to the major groove of duplex DNA. A benzo[e]pyridoindole derivative (BePI) strongly stabilized this structure and showed preferential binding to a triplex rather than to a duplex. Energy transfer experiments suggest that BePI intercalates within the triple helix. Sequence-specific inhibition of transcription initiation of a specific gene by Escherichia coli RNA polymerase by a triplex-forming oligodeoxynucleotide is strongly enhanced when the triplex is stabilized by BePI. Upon irradiation with ultraviolet light, BePI induces covalent modifications of the target within the triple helix structure.

Likelihood of the new antitumoral drug 10-[gamma-diethylaminopropylamino]-6-methyl-5H-pyrido[3',4':4,5]pyrrolo [2,3-g]isoquinoline (BD-40), a pyridopyrroloisoquinoline derivative, to induce DNA strand breaks in vivo and its nonmutagenicity in yeast.

BD-40, a pyridopyrroloisoquinoline analogue of ellipticines, has dose-dependent cytostatic and cytotoxic effects on cultures of Saccharomyces cerevisiae. These inhibitory effects take place only in growing cells and are enhanced in the presence of oxygen. Among the different repair-deficient mutants examined, a mutant defective in DNA strand break repair (rad52-1) was found to be the most sensitive to such a toxic effect. A triple mutant blocked in the excision (rad2), the mutagenic (rad6), and the recombinogenic (rad52) repair pathways demonstrated the same sensitivity as the single rad52 mutant. Nuclear reversion and forward mutations as well as mitochondrial "petite" mutation were not induced by BD-40. These results indicate that: (a) the lesions induced in vivo by BD-40 are likely to be DNA strand breaks; (b) such damage is repairable in the wild type and is not of the mutagenic type; and (c) the excision pathway is not involved in such a repair of BD-40-induced lesions, and the mutagenic pathway plays a minor role. Since DNA strand breaks were not detected in vitro whether exposure of DNA to BD-40 was achieved in the presence or the absence of microsomal S-9 mix, it is suggested that an oxygen-dependent enzymatic processing, not linked to the microsomal monooxygenase complex, is required for the development of the cytotoxic activity of BD-40.

Intoplicine (RP 60475) and its derivatives, a new class of antitumor agents inhibiting both topoisomerase I and II activities.

Intoplicine (RP 60475, NSC 645008) is an antitumor derivative in the 7H-benzo[e]pyrido[4,3-b]indole series which is now being tested in clinical trials. Intoplicine strongly binds DNA (KA = 2 x 10(5) M-1) and thereby increases the length of linear DNA. These properties are consistent with DNA unwinding by intoplicine. Intoplicine was found to be a dual topoisomerase I and II inhibitor, with DNA sites of enzyme inhibition being different for these two enzymes. In this study, 22 analogues of intoplicine were evaluated for their effects on topoisomerase I- and II-mediated DNA cleavage reactions by using enzymes purified from calf thymus. Site-specific DNA cleavage mediated by topoisomerase I was observed with 7H-benzo[e]pyrido[4,3-b]indole derivatives but not with 11H-benzo[g]-pyrido[4,3-b]indole derivatives. Site-specific DNA cleavage mediated by topoisomerase II occurred with derivatives having hydroxyl groups at the 3-position on the 7H-benzo[e]pyrido[4,3-b]indole ring or at the 4-position on the 11H-benzo[g]pyrido[4,3-b]indole ring. Study of the relationships between the in vivo antitumor activity on P388 leukemia and the topoisomerase I- and/or II-mediated DNA cleavage activity revealed that the most highly active antitumor compounds possessed both topoisomerase I-and II-inhibitory properties. Compounds selectively inhibiting either topoisomerase I or II were less active. These results suggest that dual topoisomerase I and II inhibition is critical for the antitumor activity of this new series of antitumor compounds.

Biological effects and repair of damage photoinduced by a derivative of psoralen substituted at the 3,4 reaction site: photoreactivity of this compound and lethal effect in yeast.

A newly synthesized linear psoralen derivative, 3-carbethoxypsoralen is shown to bind to yeast nucleic acids after 365 nm light treatment. As compared to 8-methoxypsoralen, a well-known bifunctional furocoumarin, 3-carbethoxypsoralen exhibits a high photoaffinity for DNA in vivo. Both compounds bind and photoreact more efficiently in vivo than in vitro. In contrast to 8-methoxypsoralen, 3-carbethoxypsoralen does not form cross-links in yeast DNA as demonstrated by heat denaturation-reassociation studies at least in the range of doses used. Thus 3-carbethoxypsoralen reacts as a monofunctional compound. Wild-type cells of Saccharomyces cerevisiae are 6 times more resistant to 3-carbethoxypsoralen than to 8-methoxypsoralen plus 365 nm light treatment in terms of lethal effect. In comparison to angelicin, another monofunctional (but angular) furocoumarin, 3-carbethoxypsoralen is more photoreactive. When the photoaffinity for DNA of 8-methoxypsoralen and 3-carbethoxypsoralen are considered in relation to photoinduced cell killing, it is clear that monoadducts are very efficiently repaired in wild-type cells. In contrast to the additivity obtained with 8-methoxypsoralen, a synergistic interaction of the two different repair pathways blocked by the rad2 and the rad9 mutation is observed after 3-carbethoxypsoralen plus 365 nm light. Dark holding experiments show that the excision repair function which is present in wild-type and rad9-4 cells is important for dark recovery.

Triple-helix specific ligands stabilize H-DNA conformation.

Under superhelical stress, oligopurine-oligopyrimidine mirror-repeat sequences are able to adopt H-DNA conformations where a triple-helical and a single-stranded structure co-exist. We have previously shown that a benzo[e]pyridoindole derivative (BePI), an antitumor drug interacting more tightly with triplex than with duplex DNA, strongly stabilizes intermolecular triple helices formed upon binding of homopyrimidine oligonucleotides to the major groove of double-stranded DNA at oligopurine-oligopyrimidine sequences. Here we show that an intramolecular triple helix is also strongly stabilized by this ligand. In vitro elongation performed by different DNA polymerases (bacteriophage T7, Escherichia coli or Taq polymerase) could be irreversibly inhibited by the H-DNA structure in the presence of BePI. A mirror-repeat polypurine-polypyrimidine sequence inserted between the E. coli beta-lactamase gene (conferring ampicillin resistance) and its bla promoter strongly inhibited transcription of the beta-lactamase gene in vivo. In the absence of supercoiling, transition to the H-conformation did not occur, but BePI stabilized the H-DNA structure induced by supercoiling as shown by chemical probes (chloroacetaldehyde). The results presented here open a new field of investigation for antitumor agents targeted to a novel class of genetic structures able to regulate gene expression.

Characterization of a triple helix-specific ligand. BePI (3-methoxy-7H-8-methyl-11- [(3'-amino)propylamino]-benzo[e]pyrido[4,3-b]indole) intercalates into both double-helical and triple-helical DNA.

A benzo[e]pyridoindole derivative, 3-methoxy-7H-8-methyl-11-[(3'-amino)propylamino] -benzo[e]pyrido[4,3-b]indole (BePI), and its interactions with double and triple-helical DNA have been investigated by a variety of fluorescence, spectrophotometric, hydrodynamic and molecular modeling techniques. Binding to DNA stabilizes the doubly charged (+2) form of BePI, increasing the apparent pKa of the 10-NH proton by approximately 1 pH unit. Binding to DNA also quenches the fluorescence of BePI, with a greater extent of quenching upon binding triplex relative to duplex DNA. BePI preferentially binds (and stabilizes) triple-helical relative to double-helical DNA. This preferential binding is not restricted to triplexes containing solely T x A.T base triplets. In addition, BePI preferentially stabilizes the poly(dA).poly(dT) relative to the poly[d(A-T)].poly[d(A-T)] duplex. Viscosity studies demonstrate that, upon binding, BePI induces the unwinding of negative supercoils in the pBR322 plasmid, and increases the relative contour lengths of double and triple-helical polydeoxynucleotides. Fluorescence studies reveal that energy transfer occurs from polynucleotide bases to bound BePI molecules in both BePI/duplex and BePI/triplex complexes. In a BePI/triplex complex, an average of 4.8 bases appear to transfer excitation energy totally to a bound BePI molecule, while in various BePI/duplex complexes an average of only 2.5 bases appear to do so, indicating that energy transfer is more efficient in the former complex. Measurements of fluorescence quenching indicate that BePI is protected from quenching by acrylamide when bound to either double or triple-helical polynucleotides. The viscosity and fluorescence behavior of BePI are fully consistent with the conclusion that BePI intercalates into both double and triple-helical DNA. Molecular modeling studies suggest that stronger stacking interactions between intercalated BePI and adjacent bases in BePI/triplex relative to BePI/duplex complexes may account for the enhanced thermal stability of the former complex.

Design of a triple-helix-specific cleaving reagent.

BACKGROUND: Double-helical DNA can be recognized sequence specifically by oligonucleotides that bind in the major groove, forming a local triple helix. Triplex-forming oligonucleotides are new tools in molecular and cellular biology and their development as gene-targeting drugs is under intensive study. Intramolecular triple-helical structures (H-DNA) are expected to play an important role in the control of gene expression. There are currently no good probes available for investigating triple-helical structures. We previously reported that a pentacyclic benzoquinoquinoxaline derivative (BQQ) can strongly stabilize triple helices. RESULTS: We have designed and synthesized the first triple-helix-specific DNA cleaving reagent by covalently attaching BQQ to ethylenediaminetetraacetic acid (EDTA). The intercalative binding of BQQ should position EDTA in the minor groove of the triple helix. In the presence of Fe(2+) and a reducing agent, the BQQ-EDTA conjugate can selectively cleave an 80 base pair (bp) DNA fragment at the site where an oligonucleotide binds to form a local triple helix. The selectivity of the BQQ-EDTA conjugate for a triplex structure was sufficiently high to induce oligonucleotide-directed DNA cleavage at a single site on a 2718 bp plasmid DNA. CONCLUSIONS: This new class of structure-directed DNA cleaving reagents could be useful for cleaving DNA at specific sequences in the presence of a site-specific, triple-helix-forming oligonucleotide and also for investigating triple-helical structures, such as H-DNA, which could play an important role in the control of gene expression in vivo.

Ligand-induced formation of hoogsteen-paired parallel DNA.

INTRODUCTION: Based on molecular modeling studies, a model has been proposed for intercalation of triple-helix-specific ligands (benzopyridoindole (BPI) derivatives) into triple helices, in which the intercalating compounds interact mainly with the Hoogsteen-paired strands of the triple helix. We set out to test this model experimentally using DNA duplexes capable of forming parallel Hoogsteen base-paired structures. RESULTS: We have investigated the possible formation of a parallel DNA structure involving Hoogsteen hydrogen bonds by thermal denaturation, FTIR spectroscopy and gel-shift experiments. We show that BPI derivatives bind to Hoogsteen base-paired duplexes and stabilize them. The compounds induce a reorganization from a non-perfectly matched antiparallel Watson- Crick duplex into a perfectly matched parallel Hoogsteen-paired duplex. CONCLUSIONS: These results suggest that preferential intercalation of BPI derivatives in triple helices is due to their ability to interact specifically with the Hoogsteen-paired bases. The results are consistent with a model proposed on the basis of molecular modeling studies using energy minimization, and they open a new field of investigations regarding the biological relevance of Hoogsteen base-pairing.

Spectroscopic study of the dar interaction and of the photoreaction between a new monofunctional psoralen: 3-carbethoxy psoralen, and DNA.

3-carbethoxypsoralen (3-CPs) is a new linear psoralen derivative. Its dark interaction and photoreaction with DNA has been studied and compared with that of a well known bifunctional psoralen : 8-methoxypsoralen (8-MOP). 3-CPs is able to form in the dark a non covalent complex with native DNA. After irradiation of this complex with UV-A light (365 nm) 3-CPs is able to link covalently to DNA. Heat denaturation and renaturation patterns of treated DNA clearly show that, in contrast to 8-MOP, 3-CPs does not form DNA interstrand cross links. Fluorescence studies show that the photobinding of 3-CPs gives rise to the formation of monoadducts involving the 4',5' double bond of this molecule.

Photochemotherapy using pyridopsoralens.

Aiming to decrease the acute side effects and genotoxic hazards of PUVA, pyrido (3,4-C) psoralen (PP) and 7-methyl pyrido (3,4-C) psoralen (MPP) were synthesized and studied. Their UVA maximum absorption lies at 325 and 330 nm, respectively. Their photostability is comparable to that of 8-MOP. They complex to DNA in the dark, and, in the presence of UVA, produce only monoadditions to DNA, as shown by fluorescence and DNA denaturation-renaturation studies. In diploid eukaryotic yeast they are more effective than 8-MOP for the induction of lethal effects and mitochondrial damage. Their mutagenic activity per unit dose of UVA is in the same range as that of 8-MOP. However, per viable cell they are clearly less mutagenic than 8-MOP. This difference is also observed for recombinogenic activity. No oxygen effect is observed. In mammalian cells the following ranges of effectiveness are found: inhibition of DNA synthesis in human fibroblasts: MPP greater than PP greater than 8-MOP; mutagenic activity in V79 Chinese hamster cells: MPP greater than PP greater than 8-MOP; cell transforming ability in C3H embryonic mouse cells: MPP greater than 8-MOP greater than PP as a function of UVA dose, and: 8-MOP greater than MPP greater than PP as a function of survival; induction of sister chromatic exchanges (SCE) per unit dose: MPP greater than PP greater than 8-MOP in the linear part of the induction curve, and : 8-MOP greater than PP greater than MPP at the maximum level of SCE obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Aza analogues of lucanthone: synthesis and antitumor and bactericidal properties.

Three types of aza analogues of lucanthone were synthesized for evaluation as antitumor drugs. None of the compounds was found to have significant cytotoxic effects either on Friend tumor cells or on L1210 leukemia cells. However, one of the target compounds, 5,10-dihydro-10-oxo-1-[[3-(diethylamino)propyl]amino]-3-methylpyrido [4,3-b]quinoline, was shown to have noticeable antibiotic properties.

Synthesis and evaluation of new 6-amino-substituted benzo[c]phenanthridine derivatives.

Different 7,8,9,10-tetrahydrobenzo[c]phenanthridin-6(5H)-ones (10a-e) were prepared by using a one-pot procedure which includes the preparation of various 6- and 7-alkoxy-1-naphthylisocyanates from 1-naphthylamines and triphosgene, followed by addition of 1-N-morpholino-1-cyclohexenes, and cyclization of the resulting amides upon heating in the presence of hydrogen chloride. Subsequent aromatization, chlorination, and substitution with (dimethylamino)alkylamines, followed by a demethylation or a selective desisopropylation, allowed us to synthesize the derivatives 6a-i and 7a-h bearing a [(dimethylamino)alkyl]amino side chain at their 6-position. These compounds, as the other analogs 5a-b, were devised to further study the structure-activity relationships in the benzo[c]phenanthridine family of antitumor alkaloids led by fagaronine (1a) and nitidine (1b). Topoisomerases I and II cleavable complex assay and evaluation of the cytotoxicity and antitumor properties were performed. In vitro cytotoxicity (L1210 and Calc 18) shows a relationship between the cytotoxicity of these compounds and their topoisomerase poisoning properties. However, all these compounds were devoid of significant antitumor effect on the P388 murine leukemia system.

(+/-)-2-Amino-3,4-dihydro-7-[2,3-dihydroxy-4-(hydroxymethyl)-1- cyclopentyl]-7H-pyrrolo[2,3-d]pyrimidin-4-ones: new carbocyclic analogues of 7-deazaguanosine with antiviral activity.

5-Allyl-2-amino-4,6-dihydroxypyrimidine (3) was chlorinated and ozonized to yield (2-amino-4,6-dichloro-pyrimidin-5-yl)acetaldehyde (5). Acetalization of 5 with ethanol afforded a new pyrimidine intermediate 6 which can lead to 2-amino-3,4-dihydro-7-alkyl-7H-pyrrolo[2,3-d]pyrimidin-4-ones and therefore to carbocyclic analogues of 7-deazaguanosine. The 7-substituent was a cyclopentyl analogue of the arabinofuranosyl moiety in 10a, lyxofuranosyl moiety in 10b, and ribofuranosyl moiety in 10c. Compounds 10a and 10b exhibited selective inhibitory activities against the multiplication of HSV1 and HSV2 in cell culture. Repeated administration of compound 10a at 10mg/kg ip to mice infected with HSV2 increased the number of survivors and lengthened significantly the mean survival time.

1-Amino-substituted 4-methyl-5H-pyrido[3',4':4,5]pyrrolo[3,2-c]pyridines: a new class of antineoplastic agents.

In an attempt to find new anticancer agents, a series of pyrido[3',4':4,5]pyrrolo[3,2-c]pyridines were synthesized and evaluated in the standard NCI screening. Among these new compounds, which are structurally related to 9-azaellipticines but differ by deletion of a cycle, those that have a 4-methyl group and a NHCH2CH2CH2NR2 side chain at the 1-position show significant cytotoxicity on L1210 cultured cells and antitumor properties in the in vivo P388 leukemia system. The in vivo antineoplastic activity of the most potent compounds were confirmed on the L1210 leukemia model.

1-Amino-substituted 4-methyl-5H-pyrido[4,3-b]indoles (gamma-carbolines) as tricyclic analogues of ellipticines: a new class of antineoplastic agents.

A series of 1-amino-substituted 4-methyl-5H-pyrido[4,3-b]indoles that are structurally related to ellipticines by deletion of a ring have been synthesized in order to evaluate their DNA affinity, their in vitro cytotoxicity on L1210 cultured cells, and their in vivo antitumor activity. Among 24 derivatives that have been prepared and studied for the structure-activity relationship in this new class of antineoplastic agents, those that have a NH(CH2)3N(R)2 side chain (R = CH3 or C2H5) at their 1-position, a 4-methyl group, and an 8-OH substituent, either with a 5-NH or with a 5-NCH3 group, show the most potent cytotoxicities on L1210 cultured cells and in vivo antitumor properties in P388 and L1210 leukemia systems. In vivo antineoplastic activity of the most potent products was confirmed in P388 and L1210 leukemia systems. In vivo antineoplastic activity of the most potent products was confirmed on other mouse experimental tumors from the standard NCI screening:B16 melanoma and C38 adenocarcinoma.

A new series of ellipticine derivatives (1-(alkylamino)-9-methoxyellipticine). Synthesis, DNA binding, and biological properties.

A new series of ellipticine derivatives, 1-(alkylamino)-5,11-dimethyl-9-methoxy-6H-pyrido[4,3-b]carbazoles, were synthesized as potential DNA intercalating antitumor drugs. The structure of these compounds were confirmed by 1H NMR spectroscopy and mass spectrometry. These compounds are able to bind to DNA with an affinity of about 10(6) M-1, and their intercalating characteristics (lengthening and unwinding of DNA) depend upon the length of the chain in position 1. The cytotoxicities of these compounds on L1210 and NIH-3T3 cells are quite similar, and fluorescence techniques showed that the compounds are localized mainly in the cytoplasmic granules of the cells. One of these compounds appears to show a very high antitumor activity (equivalent to the more active known ellipticine analogues: 10-[[gamma-(diethylamino)propyl]amino]-6-methyl-5H- pyrido[3',4':4,5]pyrollo[2,3-g]isoquinoleine (BD40), 1-[[gamma-(diethylamino)propyl]amino]-9-methoxyellipticine (BD84) and 2-[beta-(diethylamino)ethyl]-9-hydroxyellipticinium chloride (DEAE).

Antitumor amino-substituted pyrido[3',4':4,5]pyrrolo[2,3-g]isoquinolines and pyrido[4,3-b]carbazole derivatives: synthesis and evaluation of compounds resulting from new side chain and heterocycle modifications.

New modifications of 10-[[3-(diethylamino)propyl]amino]-6-methyl-5H-pyrido[3',4':4,5]pyrrolo[2,3-g]i sisoquinoline (1b) and 1-[[3-(diethylamino)propyl]amino]-9-methoxy-5,11-dimethyl-6H-pyrido[4,3-b]carba zole (4b), which display important antitumor properties, were performed either on the side chain or on the intercalating heterocycle. Side chains were introduced by direct substitution of the corresponding chloro derivatives and 6-N-methyl-9-hydroxypyrido[4,3-b]carbazoles analogues were prepared via 9-O-benzoyl-1-chloroellipticines. Evaluation of all new compounds shows no significant increase of in vitro cytotoxicity and percent ILS on the L1210 leukemia system by comparison with the model compounds 1b and 4b.

Synthesis and antitumor activity of 1-[[(dialkylamino)alkyl]amino]-4-methyl-5H-pyrido[4,3-b]benzo[e]- and -benzo[g])indoles. A new class of antineoplastic agents.

The thermal Fischer indolization of hydrazones resulting from 4-hydrazino-5-methyl-1H-pyridin-2-one and various beta- and alpha-tetralones led to 4-methyl-6,7-dihydro-2H,5H-pyrido[4,3- b]benzo[e]indol-1-ones and 4-methyl-11-dihydro-2H,5H-pyrido[4,3- b]benzo[g]indol-1-ones, respectively. After aromatization, these compounds were transformed by phosphorus oxychloride, giving 1-chloro-4-methyl-5H-pyrido[4,3- b]benzo[e]- and -benzo[g]indoles which were substituted by [(dialkylamino)alkyl]amines. The resulting 1-[[(dialkylamino)alkyl]amino]-4-methyl-5H-pyrido- [4,3-b]benzo[e]- and -benzo[g]indoles, as well as hydroxy derivatives obtained by demethylation of methoxylated compounds with hydrobromic acid, were tested for antitumor activity in vitro (leukemic and solid tumor cells) and in vivo on various experimental tumor models using the standard NCI protocols. 1-[[3-(Dialkylamino)propyl]-amino]-4-methyl-9-hydroxy-5H-pyrido[4,3- b]benzo[e]indoles appeared as a promising new class of antineoplastic agents.

Synthesis of new (+-)-3,5-dihydroxypentyl nucleoside analogues from 1-amino-5-(benzyloxy)pentan-3-ol and their antiviral evaluation.

The synthesis and antiviral evaluation of a series of (+-)-3,5- dihydroxypentyl nucleoside analogues related to acyclic nucleoside antiviral agents are reported. All purine and pyrimidine nucleoside analogues described in this paper have been obtained from 1-amino-5-(benzyloxy)pentan-3-ol. A synthesis of this amine is reported from 1-(benzyloxy)but-3-ene after epoxidation and regiospecific diethylaluminum chloride catalyzed opening of the epoxide by trimethylsilyl cyanide. The compounds were tested in vitro in infected MRC5 and CEM cells. None of the compounds exhibited antiviral activity against HSV-1, HCMV, and HIV-1 with the exception of the guanine derivative 7, which inhibited the cytopathic effect of HSV-1 by 50% at 12.5 micrograms/mL.

Synthesis and antiviral activity of 4-benzyl pyridinone derivatives as potent and selective non-nucleoside human immunodeficiency virus type 1 reverse transcriptase inhibitors.

Several 4-benzyl analogues of 5-ethyl-6-methyl-4-(phenylthio)pyridin-2(1H)-ones were synthesized and evaluated for their anti-HIV-l activities. Key transformations include metalation at the 4-C-position of 5-ethyl-2-methoxy-6-methyl-3-pivaloylaminopyridine (5) and its coupling with benzyl bromide or benzaldehyde derivatives. Biological studies revealed that some of the new 4-benzylpyridinones show potent HIV-1 specific reverse transcriptase inhibitory properties. Compounds 14, 19, and 27, which inhibit the replication of HIV-1 in CEM-SS cells, with IC(50) values ranging from 0.2 to 6 nM are the most active compounds in this series. Biochemical studies showed that compound 27 strongly inhibited the activity of a recombinant HIV-1 RT. Moreover, the infectivity of isolated HIV-1 particles was severely decreased after exposure to compound 27. Although cross resistance is frequently observed between non-nucleoside reverse transcriptase inhibitors, compound 27 was capable of inhibiting a virus resistant to nevirapine with an IC(50) of 40 nM.