Synthesis and DNA binding of 6-(alkylamino)indolo[1,2-b ][2,7]naphthyridine-5,12-quinones

We describe the synthesis of eight novel putative mono– and bis–DNA intercalators from a common precursor, 6-bromoindolo[1,2-b ][2,7]naphthyridine-5,12-dione. Of these new indoloquinones, our data indicate that two are most likely DNA mono–intercalators, but weaker than ethidium bromide, and two others are DNA bis–intercalators. Our indoloquinones are inactive against mammalian topoisomerase II.


Scheme 1
This observation fortuitously provided a convenient synthesis of the C-6 alkylamino derivatives 17a-d and the bis-compounds 18a-d, which we describe herein.Furthermore, it was conceivable that compounds of type 17 (or 18) might irreversibly acylate DNA, given the tendency of N-acylindoles to undergo facile nucleophilic cleavage of the nitrogen-carbonyl bond 27,28 (i.e., 17 → 19) (Figure 3).
As noted earlier, our motivation to explore the reaction of 24 with various amines, and examine their DNA affinity, was that the aminoanthraquinones ametantrone (2) and mitoxantrone (3) show strong DNA affinity and good antineoplastic activity in clinical trials. 7In actuality, treatment of 24 with an excess of dimethylamine (25% aqueous solution) in THF at room temperature provided 5-dimethylaminoindolo[1,2b] [2,7]naphthyridine-6,11-dione (17a) cleanly in high yield.Similarly, compounds 17b and 17c were synthesized by allowing solutions of 24 in THF to react with excess N,N,N'-trimethylethylenediamine and 3diethylaminopropylamine, respectively.These reactions doubtlessly proceed by a typical nucleophilic addition-elimination mechanism. 29

Scheme 3
Our motivation for preparing the bis-indoloquinones 18a-d was to attain novel DNA bis-intercalators, and we were encouraged by the results from our previous studies with bis-acridines. 32Indeed, as we found with bis-acridines, adjustment of the length and flexibility of the diamino-linkage (tether) can dramatically alter the DNA-binding of these compounds.Therefore, we prepared bis-indoloquinones 18a-d using a method similar to the method used to synthesize compounds 17a (Scheme 4).The reactions were run in the presence of excess 24 so as to encourage the formation of the bis-indoloquinones.Nonetheless, some monoindoloquinones were still present in the reaction mixture after 24 hours of stirring at room temperature.

Scheme 5
Our results of DNA affinity studies for 17a-d and 18a-d are summarized in Table 1.The change in DNA melting, ∆T m , is the change in the thermal denaturation of DNA by a drug as a result of intercalation. 34From these results, it can be seen that compounds 17b and 17c are intercalators, although less effective when compared to a powerful intercalator such as the aminoacridine, quinacrine, which has a ∆T m value of 24.9. 34he other experiment was to determine the slope of the calf thymus viscometric titration.When a drug intercalates into DNA, the DNA helix lengthens.Thus, the line of concern here is the ratio of the increase in DNA contour length versus the drug/nucleotide ratio. 34Unfortunately, in further tests, neither of these compounds exhibited any activity against mammalian topoisomerase II, which are enzymes that cause doublestrand breaks in DNA, cause the DNA to unwind, and reseal the breaks. 35They are believed to be necessary for replication, since the helical winding of the DNA must be released to allow strand splitting in replication.Thus, it is currently believed that topoisomerase activity is crucial for useful anticancer activity. 35We find that 17a-d have no activity against mammalian topoisomerase II at a drug concentration of 100 µM.

Conclusions
We have synthesized eight novel indoloquinones, 17a-d and 18a-d for DNA binding studies.Of these, 17a and 17d do not bind to DNA as evidenced by the calf thymus DNA assay, suggesting that the pyridine ring system alone is insufficiently basic to become protonated in the presence of the polyphosphates of DNA.Moreover, as might be expected, the exocyclic amino group is rendered less basic by the C-5 carbonyl group (vinylogous amide).In contrast, our data indicate that 17b and 17c are probably mono-intercalators.These two quinones show a binding profile similar to other DNA intercalators, and they exhibit a GC preference.By comparison to ethidium bromide, these two compounds are somewhat weaker intercalators.In contrast, indoloquinones 18b and 18c are likely bis-intercalators, since the high CT for 18b and its biphasic nature suggests a double intercalator.The lesser DNA affinity of 18c is probably imparted by the less electronegative oxygen atoms in the tether.Our eight new compounds, 17a-d and 18a-d, will serve in future biological studies.
ARKAT USA, Inc

Experimental Section
General.Melting points were determined on a Büchi 510 apparatus and are uncorrected.Infrared (IR) spectra were recorded on a Perkin-Elmer 599 spectrometer and are referenced to the 1601 cm -1 band of polystyrene. 1 H NMR (300 MHz) and 13 C NMR (75 MHz) were recorded on a Varian XL-300 multinuclear Fourier transform spectrometer.Unitary resolution mass spectra (MS) were obtained on a Finnigan 4023 GC/MS system.High resolution mass spectra (HRMS) were recorded at the National Institutes of Health regional facility at the Massachusetts Institute of Technology.Ultraviolet (UV) spectra were recorded on a Hewlett Packard 8451A diode array spectrophotometer.Elemental analyses were performed by Atlantic Microlab Inc., Norcross, GA.Analytical thin-layer chromatography (TLC) was performed on precoated Silica Gel 60 F 254 plates from EM Reagents.Visualization was accomplished with 254 and 365 nm UV light, iodine vapor, ceric ammonium sulfate spray (3% in 10% sulfuric acid) or "van Urk's reagent" spray (p-dimethylaminobenzaldehyde in ethanolic sulfuric acid).Flash chromatography was performed with EM Reagents Silica Gel 60 (230-400 mesh).All reactions were performed under a static head of predried (CaSO 4 tower) nitrogen or argon in glassware that had been dried for at least 1 h at 135 °C.Benzenesulfonyl chloride and 3,4-pyridinedicarboxylic anhydride were distilled prior to use.

Table 1 .
DNA affinity results for 17a-d and 18 b,c a CT is sonicated calf thymus DNA.b Poly AT is sonicated poly(dA)•poly(dT) homopolymer.c Poly GC is sonicated poly(dG-dC) with alternating GCGCGC on both strands.