Synthesis and Electrophilic Substitution of Pyrido[2,3,4-kl]- acridines‡

Several new pyrido[2,3,4-kl]acridines were synthesized by reacting naphthoquinone, juglone or cyclohexan-1,3-dione with β,β’-diaminoketones in a biomimetic reaction. The structure of all new compounds was elucidated by NMR and MS spectroscopy. Electrophilic substitution, mainly nitration, of the various compounds was undertaken and the substitution positions determined. A series of derivatives was prepared and their cytotoxicity towards P-388 mouse lymphoma cells analysed. The most cytotoxic derivatives were found to have IC50’s of 0.05 and 0.1 ug/ml.

Here we report a biomimetic synthesis of additional new pyridoacridines and a study of their reactions with electrophiles or amines (in the case of the quinoneimines). Most of the new pyridoacridines were tested for in-vitro activity against tumor cells and some of them were found to be highly cytotoxic.

Results and Discussion
Several new pyridoacridines were synthesized in a two step reaction of β,β'-diaminoketones with quinones. Thus, addition of 2,2'-diaminobenzophenone 4a or 4b to 1,4-naphthoquinone afforded in the first step the arylaminonaphthoquinones 5a and 5b respectively, in approximately 50% yield (Scheme 1). The reaction took place in the presence of catalytic amounts of cerous chloride while air was bubbled through the solution to oxidize the intermediate hydroquinone [6][7]. In the second step, treatment of compounds 5a and 5b in methanol with NH 4 OH at room temperature for 7 days gave the corresponding compounds 6a and 6b in over 90% yield (Scheme 1).
The structures of 6a and 6b, possessing the required molecular ions (m/z 332 and 392, respectively) were confirmed by 1D and mainly COSY, HMQC and HMBC 2D-NMR spectra (See Table 1 for the HMBC correlations and the Experimental section for the proton and carbon chemical shift assignments).
Characteristic were the resonances of C-10 and C-14b of the quinoneimine system (ring C) and the down-field proton resonances of the spatially close protons H-4 and H-5 (δ H 9.09 and 9.18 ppm, respectively, for 6a and δ H 8.90 and 8.98 ppm, respectively, for 6b) [5].
Three four-spin systems were observed in the 1 H-NMR spectrum of 6a belonging to rings A, E and F. Rings A and E, bearing the spatially close H-4 and H-5 protons, were distinguished from ring F by NOE measurements. The differentiation between rings A and E was achieved from an NOE between H-1 and H-14 (about 3.7Å apart). This NOE was also the key for determining the structure of the nitration products 21, 23a and 23b, as described below. A second reaction that was performed with naphthoquinone was its reaction with TFA-kynuramine (7) [4] (Scheme 1). This reaction afforded 9Hbenzo[i]pyrido [2,3,4-kl]acridin-9-one (deaza-ascididemin, 8), earlier synthesized by Zjawiony by a four step reaction [8]. The structure of compound 8 (m/z 282) was confirmed by its NMR data (see Experimental) and comparison with the data in the literature [8].
The orientation of this addition was defined by the structure determination of compound 10, obtained in a second step by stirring compound 9 in methanol with Et 3 N. A key HMBC correlation in the structure elucidation was the one between C-14b (δ=147. 5 that assisted with the structure determination see Table 1. The regioselectivity of nucleophilic additions of amines to juglone was observed before by Thomson [9] in the reactions of aniline with the juglone derivatives 5-acetoxy or 5-methoxy-1,4-naphthoquinones. Performing the second step of the latter reaction with ammonia, rather than Et 3 N, as used for the preparation of compounds 6a and 6b, caused unexpectedly the disappearance of the C-10 carbonyl group. Moreover, acetylation of the obtained pyridoacridine (12) (Scheme 3) gave a mono-(13a) and a diacetate (13b). It is suggested that the carbonyl group of compound 10 is replaced in compound 12 by an imine and indeed, treatment of 10, obtained with the Et 3 N, with NH 3 gave compound 12. The position of the imine group was defined by a HMBC experiment of compound 13a namely from correlations between the 11-hydroxylic proton and carbons C-10a, C-11, and C-12 of ring F (see Table 1).  13 14 14a Quinoneacetimide systems such as ring C in compounds 13a and 13b are stable, e.g. the reported simple acetimides of naphthoquinones and benzoquinones [10]. In contrast, simple quinoneimines are unstable and were seldom isolated [10]. Thus, it was interesting to find that the quinoneimine moiety of compound 12 is stable as was also found to be the case of the natural pyridoacridine calliactine [11] whose structure was determined recently [12]. In both compound 12 and calliactine the hydroxyl group in the β position relative to the imine group seems to stabilize the quinoneimine by a hydrogen-bond. Another example for the latter behaviour was seen in compound 14, synthesized from juglone and panisidine (Scheme 3). Compound 14 in methanol with aqueous ammonia, yielded the quinoneimine 15, while compound 16 [13] without the β-OH group, did not form the quinoneimine under the same conditions. These results proved the necessity of a hydroxyl group β to the ketone for the imine formation and also suggest that the quinoneimine ring could be obtained, in the synthesis, before the rings closure of compound 10.
A major target in the present investigation was the study of the electrophilic substitution reactions of pyridoacridines and dihydropyridoacridines for the preparation of derivatives for structure activity relationship studies. It was decided to start with nitration as the nitro groups are easily transformed to other functional groups. Investigating a variety of nitration conditions (HNO 3 -TFA, HNO 3 -H 2 SO 4 and NO 2 BF 4 in CH 3 CN) brought to the best conditions, namely, the use of HNO 3 -H 2 SO 4 , 1:1 vide infra.
The first substrate for nitration was dihydropyridoacridine 17a. Compounds 17a and 17b were obtained in quantitative yields by condensation of compounds 4a or 4b with 1,3-cyclohexanedione (Scheme 4). Nitration of compound 17a gave, after 1 hour, two mononitro isomers 18a and 18b and three dinitroisomers 19a, 19b and 19c after 12 hours of reaction at room temperature.
The structures of the different isomers were determined by 1 H-NMR and COSY experiments. In all the products the nitro group(s) are in ring A (or E) in positions para or ortho to the nitrogen of the attached pyridine ring (para or ortho positions). The yields of the nitration products indicate that the para position in ring A (and E) is more reactive than the ortho one under the conditions used. Nitration of quinoline with nitric acid in sulphuric acid at 0 o C was reported to yield 5-and 8-nitroquinoline in a ratio of 52% to 48% respectively [14]. The nitration experiments of compound 17a show that the para position of ring A (and E) in the dihydropyridoacridine is more reactive than position-6 of quinoline, under the same conditions [15]. Positions 4 and 5 in the pyridoacridine, which can be compared to the reactive position-5 of quinoline, are blocked by steric interference and therefore are not substituted.
The nitration of compound 6a afforded a mono-nitro product 20 in 53% yield after 12 hours at room temperature. Because of the absence of long range CH-correlations in the NMR experiments between atoms of rings A or E and F to ring C it was difficult to determine whether the nitro group is attached to ring A or E. However, the nitration position, C-3 on ring A, could be determined from a NOE between H-1 and H-14 (2%), which are ca.3.7 Å apart (see Scheme 5). It was found by 1D and 2D NMR experiments (for HMBC correlations see Table 1) that the nitration went to the para position of ring A as was found for the nitro derivatives of compound 17a which were obtained in higher yields (compounds 18b, 19b and 19c).  Nitration of compound 6b, the electron richer 2,7-dimethoxy derivative of 6a, gave a dinitro derivative 22 after 1 hour and two tetra nitro isomers 23a and 23b (Scheme 5) after 12 hours of reaction at room temperature. That the two nitro groups in 22 substituted C-1 and -8, ortho to the quinoline-nitrogen, was clear from the two AB-systems seen in the 1 H-NMR spectrum along with the aromatic four-proton system (Experimental).
The structures of 23a and 23b were also determined by 1D and 2D-NMR experiments (for HMBC correlations see Table 1). In compounds 23a and 23b only one of rings A or E was attacked by the electrophile at the para position. The structures of compounds 23a and 23b are tentatively suggested on the basis of the structure of compound 20 as depicted in Scheme 5. Because of the nitro groups at the ortho positions, it was impossible to prove by NOE that the substitution is at the para position of ring A (as in the case of compound 20). In addition to the nitration of rings A and E, ring F in 23a and 23b was also substituted due to long range activation by the methoxyl groups.
The second electrophilic substitution undertaken was bromination. Compounds 6a and 8 did not react with Br 2 in CH 2 Cl 2 , Br 2 in AcOH or NBS in CH 2 Cl 2 and it seems that severe conditions may be needed in order to brominate these compounds. The use of Lewis acids as catalyst precipitated the compound. Quinoneimine 24 [2] afforded the dibromo derivative 26 (Scheme 6) by reaction with Br 2 in AcOH; a reaction known for quinones [16]. Compound 26 like other dibromoquinones is expected to afford a variety of derivatives by cycloaddition reactions and by reactions with amines and thiols [13,[16][17].
Several of the synthesized new pyridoacridines were tested for in-vitro cytotoxicity against P-388 mouse lymphoma cells (Table 2). It was found that compounds 6a and 8 are more cytotoxic than other reported natural pyridoacridines for which IC 50 values of 0.1-0.4 ug/ml were found [1a]. The cytotoxicity of compound 6a, the electron richer dimethoxy derivative 6b and the electron poorer nitro derivatives 21 and 23b, as well as compound 12 and its acetate derivatives 13a and 13b is lower. Table 2. In-vitro cytotoxicity against P-388 mouse lymphoma cells Oxidation of compounds 17a and 17b, with cerium ammonium nitrate, afforded benzopyridoacridones 24 and 25, respectively, in high yields (Scheme 6). Amination of the latter quinoacridones (24 and 25) with several primary amines in ethanol afforded two kinds of derivatives; monoamination products (compounds 27a-32a) and symmetric diamination ones (compounds 27b-31b and 33b). The diamination products were separated easily from the monoamination products, in each reaction, by silica gel chromatography (eluting with chloroform-methanol mixtures). The diamination products are more polar than the monoamination products and the starting material. Performing the amination in acetonitrile instead of ethanol afforded mainly the monoamination products 27a and 30a with only traces of the diamination products 27b and 30b (Scheme 6). Another derivative, prepared from 24, was compound 32a (Scheme 6), which was derived from 24 by hydrazoic acid in methanol under conditions reported for synthesis of aminonaphthoquinones [18]. (c) see Table 3 and Experimental.

Scheme 6
27a -32a (Table 3) 27b -31b, 33b ( The structures of the diamination products were determined by their 1 H-and 13 C-NMR spectra which indicated symmetric structures (see Experimental). The substitution site in the monoamination product 27a was determined by a HMBC experiment which showed correlations between proton H-11 and carbons C-9a and C-12a and correlations between the substituent NH-proton and carbons C-12a and C-11 (Scheme 7). As seen in Table 3, the symmetric diamination products are more cytotoxic than the monoamination ones and most of the diamination products are more toxic than their parent compounds 24 and 25 (Table 2). Most active are the symmetric derivatives obtained with isobutylamine and methylamine (compounds 27b-29b) while the more lypophilic derivative obtained with dodecylamine (compound 31a) (as well as 31b) and the more hydrophilic derivative obtained with serinol (compound 33b) are less active.

General
Commercially available reagents were purchased from standard chemical suppliers and were used without further purification. 2(N)-(4-methoxyaniline)-1,4-naphthoquinone (16) was synthesized by a literature method [13]. IR spectra (KBr disks) were recorded on a Nicolet 205 FT-IR spectrophotometer. MS and HRMS spectra were recorded on a Fisons Autospec Q instrument. 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker ARX-500 or a Bruker AMX-360 spectrometer. NOE experiments and 2D NMR spectra (COSY, HMQC and HMBC) were recorded on a Bruker ARX-500 instrument using standard pulse sequences. TLC was performed on Merck precoated Kieselgel 60 F 254 plates. Column chromatography was performed using Silica gel 60 H (Merck) unless otherwise stated. The silica was washed with methanol, before use, in a Soxhlet apparatus. In all cases silica gel chromatography was performed with vacuum.

General procedure for the reaction between arylamines and 1,4-naphthoquinones
The procedure of Pratt [7] was adopted. The corresponding amine (1 equiv.) was dissolved in ethanol, CeCl 3 . 7H 2 O (0.05 equiv.) was added followed by the naphthoquinone (1.5 equiv.). The resulting red solution was refluxed for 9 h. During this time air saturated with ethanol (prepared by passage of air through hot ethanol to avoid evaporation of the ethanol) was bubbled through the reaction mixture. After cooling, the ethanol was evaporated and the residue purified by chromatography on silica gel (eluting with chloroform/methanol) to afford the desired amination product.