Pyrimido[5,4-c ]pyrrolo[2,1-a ]isoquinoline: a new potential DNA-interactive ring system

The acid catalyzed decomposition of the azide 9 failed to give the title compounds, which were however obtained by a Pschorr-type cyclization on reactive 1-(6-aminopyrimidin-5-yl)-pyrroles of type 13 . Derivatives of type 14 and 15 were fully characterized by NMR data. Theoretical calculations demonstrated that the new compounds possess properties suitable for DNA-intercalation


Introduction
Polycyclic heterocycles having a planar structure can be effective pharmacophore units of drugs endowed with anti-tumor activity because they can intercalate into double-stranded DNA.Phenanthridine-and acridine-derivatives of type 1 and 2 are well known compounds possessing such a property whose principal driving forces are stacking-and charge-transfer-interactions, as well as hydrogen bonding and electrostatic forces. 1 In particular, it has been demonstrated that the biological activity of acridine derivatives of type 2 (R=NHSO 2 Me) correlates with their DNA association constants, the more active compounds being those that bind more tightly to DNA.

R H +
The ethidium derivatives 1 (R=Et) and anthracycline antitumor antibiotics 3 also bind tightly to DNA and show a strong specificity for guanine (G) and cytosine (C) residues. 3Doxorubicin (3, R=OH), in particular, interacts with the 2-amino group of guanine. 4In addition, compounds of types 2 and 3 target the topoisomerase II. 5 For a long time, we have been interested in studying heterocycles annelated with either pyrrole or indole rings as potential DNA-interactive agents.In particular several azolophenanthridine derivatives of type 4 [pyrazolo (X=CR, Y=N); triazolo (X=Y=N); pyrrolo (X=Y=CR)] have been prepared [6][7][8][9][10] and some of them showed anti-proliferative activity in in vitro tests. 11Moreover, some pyrrolo[1,2-f]phenanthridines, and in particular compound 5, showed unique properties-being able not only to reduce the HIV-induced cytopathogenicity, but also to stimulate the growth of the same MT cells at lower concentrations. 12Structureactivity relationship (SAR) studies in the class of pyrrolo[1,2-f]phenanthridines have shown that the presence of amino or methoxy groups on the 6-and/or 11-positions of the polycondensed system is relevant for the appearance of the biological activity.On the basis of these results, it was interesting to explore further this type of planar heterocycles by making isosteric modifications of the phenanthridine moiety.The introduction of one or two nitrogens could lead to several classes of hitherto unknown heterocyclic systems, annelated with the pyrrole ring, such as DiAzaPhenanthrenes (DAP) or TriAza-Phenanthrenes (TAP).In this paper, we report our approach to the new ring system TAP, namely pyrimido [5,4-c]pyrrolo[2,1a]isoquinolines, of type 6.

Results and Discussion
The synthetic approach to TAP could involve either the acid-catalyzed decomposition of (3azidoaryl-or heteroaryl-)-pyrroles, or the Pschorr-type cyclization of (2-aminoaryl/heteroaryl)pyrroles, according to the procedures already employed successfully for the synthesis of the pyrrolo[1,2-f]phenanthridines.These methods can also allow the functionalization of the phenanthrene rings with electron-donating groups suitable for the interaction with DNA. 2 For this purpose we first utilized the method involving the acid catalyzed decomposition of (7) was prepared from a suitable 1,4-diketone and 5aminouracil according to the procedure recently reported by us. 13Reduction of the nitro group with hydrogen and Pd on charcoal afforded the amino derivative 8 in good yield.This last was diazotized with sodium nitrite in hydrochloric acid, then treated with sodium azide to give the corresponding 2-(3-azidophenyl)-pyrrole 9 (Scheme 1).The azide, dissolved in trifluoroacetic acid, was decomposed with an equivalent of trifluoromethane-sulfonic acid (TFMSA), from 0°C to room temperature.From the complex reaction mixture it was possible to isolate only the hydroxyphenyl derivative 12, and no traces of a polycyclic product of type 6 could be isolated.In this acid catalyzed decomposition of the azide, the intermediate cation of type 10 is involved.Such an intermediate, being a πcarbocation, needs a sufficiently high electron density on the attacking substrates under the reaction conditions.We have already demonstrated that a phenyl group is not sufficient to bring about the cyclization reaction, whereas increasing yields of cyclized product were obtained by introducing an activated aryl or an electron rich pyrrole moiety. 14Therefore we thought that the presence of the two hydroxyl groups could activate the pyrimidine moiety sufficiently to bring about the cyclization reaction.However the extensive protonation of the substrate reduced the amount of the reactive intermediate 10.Therefore compound 12, the only material isolable from the reaction mixture, was formed by competing intermolecular nucleophilic reactions with triflate, leading to the intermediate 11, or by reacting directly with water, as observed in our previous reports. 9,10A different approach to the title compounds of type 6, involving a Pschorr-type cyclization of 1-(6-aminopyrimidin-5-yl)-pyrroles of type 13, was then undertaken.The amino derivatives 13a,b were prepared in good yields from 1-(3-methoxyphenacyl)-1,4-pentanediones and 4,5diamino-6-hydroxypyrimidine. 13he compounds 13a,b were then diazotized in sulfuric acid with a large excess of sodium nitrite, followed by treatment with hypophosphorous acid (Scheme 2).From the complex reaction mixture, after column chromatography, it was possible to isolate two isomeric compounds identified as the 11-and 9-methoxy-substituted derivatives of types 14 and 15 respectively.
When amine 13a was the starting material, because of the presence of a large excess of nitrite, nitrosation of the pyrrole β-position during the diazotization reaction, followed by oxidation, was observed.Such a behavior is not unusual with pyrrole substrates. 15Since both the β-carbons were prone to substitution, the position of the nitro group, ortho to the phenyl group, was assigned by analogy with literature reports, taking into account the driving and/or activating effects of the substituents already present in the pyrrole moiety, 16 and on the basis of theoretical consideration of the 13 C-NMR carbon shifts (see below).
Diazotization of the amine 13a afforded the cyclized products, 14c and 15c, only when the reaction was carried out in the presence of methanol, whereas in the case of amine 13b the formation of the isomers 14d and 15d can be modulated by slight variation of the experimental conditions.When methanol is added to the reaction mixture to increase the poor solubility of the starting material, the diazotization reaction is faster and the less hindered isomer (11-methoxy, 14d) is formed.In the absence of methanol the diazotization is slow and has to be carried out at 30°C; under these conditions the 9-methoxy isomer 15d is formed preferentially.This product, although it is more constrained, once formed can be stabilized by intramolecular hydrogen bonding in the tautomeric form 15′, which was also the only form present in solution, as evidenced by NMR spectral data.
The structures of derivatives 14c,d and 15c,d were assigned on the basis of the spectral data, in particular 1 H-and 13 C-NMR.From the proton NMR data it is possible to identify the two isomers 14 and 15, deriving from cyclization on the ortho-and parapositions of the phenyl ring, by considering the different coupling patterns of the aromatic protons of the isoquinoline moiety.The position of the nitro group in derivatives of type c was determined on the basis of the chemical shift of the proton at C-2, which was found as a singlet at 6.87 and 6.51 respectively, and of the proton on C-12, shifted 0.3-0.5 ppm downfield by the nitro group, compared to the other derivatives of type d.
To assign all the resonances in the 13 C-NMR we evaluated the correlation between experimental and calculated chemical shifts.In particular, the structures of all the isomers 14 and 15 were fully optimized, 17 in vacuo and in CDCl 3 , by SCF calculation with the semi-empirical PM3 method, which gave a prediction of the 13 C-NMR chemical shifts by the neural-net technique. 18Table 1 reports the experimental and calculated values for the new TAP derivatives.Generally, we found a good linear correlation between the two sets of values, with r 2 = 0.91-0.95 in the case of derivatives c and d respectively.For the 9-methoxy-substituted isomers it was possible to establish the predominance of tautomer 15′, in which C-5 was deshielded by up to 27 ppm compared with the chemical shift of the corresponding carbon atom in the 11-methoxy derivatives.
Table 1. 13 C-NMR data (δ C , ppm) for pyrimido [5,4-c] To investigate the potential ability of derivatives of the new ring system, pyrimido[5,4c]pyrrolo[2,1-a]isoquinoline, to interact with DNA, we utilized the structures optimized in vacuo to calculate the values of some molecular descriptors 17 [accessible surface area (ASA), ionization potential, LUMO and HOMO orbital energies].Table 2 reports our findings on derivatives of type 14 and 15, together with those of amsacrine [AMSA, 2, (R=2-methoxy-4methanesulfonamido-)], and doxorubicin 3 (DOXO), chosen as reference compounds.All the new derivatives have LUMO and HOMO energies in the range calculated for the known intercalators, and also all the other parameters are of the same order of magnitude as the active compounds.Therefore, it can be assumed that the new ring system can constitute a probable pharmacophore for potential DNA-interactive compounds.
In conclusion, in this paper we report the synthesis of the new ring system, pyrimido [5,4c]pyrrolo [2,1-a]isoquinoline, through a Pschorr-type cyclization of suitable 1-(6aminopyrimidin-5-yl)-pyrroles.The derivatives of this polycyclic heterocycle, TAP, that can be considered an isostere of the known intercalators belonging to the phenanthridine class, have shown to be potential DNA-interactive compounds in preliminary molecular modeling studies.

Experimental Section
General Procedures.All melting points were taken on a Buchi-Tottoli capillary apparatus and are uncorrected; IR spectra were determined in bromoform with a Jasco FT/IR 5300 spectrophotometer; 1 H-and 13 C-NMR spectra were measured at 200-and 50.3 MHz respectively in (CD 3 ) 2 SO solution, unless otherwise specified, using a Bruker AC-E Series 200 MHz spectrometer (TMS as internal reference).Column chromatography was performed with Merck silica gel 230-400 Mesh ASTM or with Biotage FLASH40i chromatography module (prepacked cartridge system).For all new compounds, analyses were within ±0.4% of theoretical values.