Structure-Antitumor Activity Relationships of Aza- and Diaza-Anthracene-2,9,10-Triones and Their Partially Saturated Derivatives

The 1,8-Diazaanthracene-2,9,10-triones, their 5,8-dihydro derivatives, and 1,8-diazaanthracene-2,7,9,10-tetraones, structurally related to the diazaquinomycin family of natural products, were synthesized in a regioselective fashion employing Diels–Alder strategies. These libraries were studied for their cytotoxicity in a variety of human cancer cell lines in order to establish structure–activity relationships. From the results obtained, we conclude that some representatives of the 1,8-diazaanthracene-2,9,10-trione framework show potent and selective cytotoxicity against solid tumors. Similar findings were made for the related 1-azaanthracene-2,9,10-trione derivatives, structurally similar to the marcanine natural products, which showed improved activity over their natural counterparts. An enantioselective protocol based on the use of a SAMP-related chiral auxiliary derived was developed for the case of chiral 5-substituted 1,8-diazaanthracene-2,9,10-triones, and showed that their cytotoxicity was not enantiospecific.

The deoxyniboquinones [13,14] and the diazaquinomycins (DAQs) are the only natural products derived from the 1,8-diazaanthracene-2,7,9,10-trione framework, although some related natural products, like nybomycin, are known [15,16].After its isolation from a Streptomyces strain, the Ōmura group found that diazaquinomycin A (DAQA) possessed antibacterial properties [17,18], which they subsequently proposed to be due to its ability to inhibit thimidylate synthase [19].Both findings made DAQA an attractive lead compound in the field of cancer chemotherapy; although the natural product itself lacked useful antitumor activity because of its poor pharmacokinetic properties [20].On the other hand, subsequent studies by Murphy that involved the screening of an actinomycete secondary metabolite library against an ovarian cancer cell line led to the identification of the diazaquinomycins E, F and G [21], and a similar study by the same group of a library of aquatic bacterial natural product fractions led to the discovery of diazoquinomycins H and J, and also a promising in vitro activity profile for some DAQs against a panel of drug-resistant Mycobacterium tuberculosis strains [22] (Figure 1, compounds 1).Interestingly, diazaquinomycin biosynthetic gene clusters have been sequenced from marine and freshwater actinomycetes [23].
Molecules 2024, 29, 489 2 of 27 and J, and also a promising in vitro activity profile for some DAQs against a panel of drugresistant Mycobacterium tuberculosis strains [22] (Figure 1, compounds 1).Interestingly, diazaquinomycin biosynthetic gene clusters have been sequenced from marine and freshwater actinomycetes [23].The structurally related 1-azaanthracenones are another class of natural compounds with interesting anticancer activity.In 1999, Soonthornchareonnon et al. described the cytotoxic activity of marcanine A, isolated from the stem bark of Goniothalaus marcanii [24], and its derivatives [25] against several tumor cell lines (Figure 1, compounds 2).Subsequently, these compounds have also been found to exhibit antimalarial activity against Plasmodium falciparum [26,27].Jacobs et al. evaluated the cytotoxic activity of a series of compounds with an azaanthracenedione structure, finding that the quinone scaffold and the lactam moiety are important for activity [28].Their mechanism of action is not clear, although some marcanine derivatives have been found to induce apoptosis via caspase-3 activation [29].
To date, there are no conclusive data about structure-activity relationships for diazoquinomycin analogues.The preparation and study of a number of semisynthetic analogues of DAQA (compounds 3 and 4) led Ōmura to the conclusion that the bis-lactam structure of the natural product was an essential requisite for thimidylate synthase inhibition and antitumor activity [30].However, when we investigated the antitumor activity of a small set of derivatives of the structure 5, we found that they exhibited good antitumor activities, particularly towards solid tumors [31], while the introduction of a fluorine atom at C-3 led to compounds with antileukemic activity [32].In order to assist the establishment of structure-activity relationships within this class of compounds, we describe here a systematic study of the in vitro antitumor properties of a large series of derivatives of structure 5, their 5,8-dihydro derivatives 6, where one of the electron-withdrawing pyridine nitrogen atoms has been replaced by an electron-releasing enamine-like moiety, 1azaanthraquinones derived from structure 7 related to the marcanines and more simplified structures such as quinolinedione derivatives 8 where the lactam scaffold that appears to be essential for cytotoxic activity is maintained (Figure 2).The structurally related 1-azaanthracenones are another class of natural compounds with interesting anticancer activity.In 1999, Soonthornchareonnon et al. described the cytotoxic activity of marcanine A, isolated from the stem bark of Goniothalaus marcanii [24], and its derivatives [25] against several tumor cell lines (Figure 1, compounds 2).Subsequently, these compounds have also been found to exhibit antimalarial activity against Plasmodium falciparum [26,27].Jacobs et al. evaluated the cytotoxic activity of a series of compounds with an azaanthracenedione structure, finding that the quinone scaffold and the lactam moiety are important for activity [28].Their mechanism of action is not clear, although some marcanine derivatives have been found to induce apoptosis via caspase-3 activation [29].
To date, there are no conclusive data about structure-activity relationships for diazoquinomycin analogues.The preparation and study of a number of semisynthetic analogues of DAQA (compounds 3 and 4) led Ōmura to the conclusion that the bis-lactam structure of the natural product was an essential requisite for thimidylate synthase inhibition and antitumor activity [30].However, when we investigated the antitumor activity of a small set of derivatives of the structure 5, we found that they exhibited good antitumor activities, particularly towards solid tumors [31], while the introduction of a fluorine atom at C-3 led to compounds with antileukemic activity [32].In order to assist the establishment of structure-activity relationships within this class of compounds, we describe here a systematic study of the in vitro antitumor properties of a large series of derivatives of structure 5, their 5,8-dihydro derivatives 6, where one of the electron-withdrawing pyridine nitrogen atoms has been replaced by an electron-releasing enamine-like moiety, 1-azaanthraquinones derived from structure 7 related to the marcanines and more simplified structures such as quinolinedione derivatives 8 where the lactam scaffold that appears to be essential for cytotoxic activity is maintained (Figure 2).

Preparation of 1,8-Diazaanthracene-2,9,10-Trione Derivatives
Compounds 5 and 6 were prepared using hetero Diels-Alder chemistry [33][34][35][36][37].The starting materials were, on the one hand, the unsaturated dimethylhydrazones 9, obtained [38,39] from N,N-dimethylhydrazine and the corresponding aldehydes, which are commercially available with the exception of 2-methyl-2-hexenal, synthesized using a literature procedure [40] (Table 1).On the other hand, the quinones 8 acting as dienophiles were obtained from simple aromatic starting materials using literature methods based on the Knorr [41][42][43][44] and Meth-Cohn [45] quinoline syntheses, followed by an oxidative demethylation reaction (Table 2).The course of the reaction between compounds 9 and 8 depended on their substituent distribution (Scheme 1); thus, combinations of hydrazones 9 and quinones 8 (R 4 , R 5 ̸ = H) gave 5,8-dihydro-1,8-diazaanthraquinones 6 as the sole cycloadducts, while other combinations gave the fully aromatic compounds 5 [33,46] with the exception of the reaction between azadiene 9b and quinone 8h, which, together with 5p, gave a significant amount of the corresponding dihydro derivative 6p that could not be isolated in pure form.In this particular case, the reaction crude was treated with manganese dioxide to force the dehydrogenation of 6p.As a secondary product of the reaction, compound 10 was obtained from the addition of dimethylamine to the starting quinones 8, followed by spontaneous air oxidation (Table 3, method A).These side products were formed in a higher yield when dimethylhydrazone derivatives of crotonaldehyde were used; in these cases, a method previously described by our group based on the use of a silica gel support [34] allowed us to considerably reduce the formation of 10 (Table 3, method B).Alternatively, some of the reactions were performed in the presence of a chloroformyl polystyrene resin in order to trap the liberated dimethylamine [35] (Table 3, method C).The course of the reaction between compounds 9 and 8 depended on their substituent distribution (Scheme 1); thus, combinations of hydrazones 9 and quinones 8 (R4, R5 ≠ H) gave 5,8-dihydro-1,8-diazaanthraquinones 6 as the sole cycloadducts, while other combinations gave the fully aromatic compounds 5 [33,46] with the exception of the reaction between azadiene 9b and quinone 8h, which, together with 5p, gave a significant amount of the corresponding dihydro derivative 6p that could not be isolated in pure form.In this particular case, the reaction crude was treated with manganese dioxide to force the dehydrogenation of 6p.As a secondary product of the reaction, compound 10 was obtained from the addition of dimethylamine to the starting quinones 8, followed by spontaneous air oxidation (Table 3, method A).These side products were formed in a higher yield when dimethylhydrazone derivatives of crotonaldehyde were used; in these cases, a method previously described by our group based on the use of a silica gel support [34] allowed us to considerably reduce the formation of 10 (Table 3, method B).Alternatively, some of the reactions were performed in the presence of a chloroformyl polystyrene resin in order to trap the liberated dimethylamine [35] (Table 3, method C).Scheme 1. Hetero Diels-Alder reaction between azadienes 9 and quinones 8.  [33].c Data obtained from reference [34].d Data obtained from reference [36].e Data obtained from reference [46].f Data obtained from reference [35].g Data obtained from reference [44].
The reaction between azadiene 9b and quinone 8f afforded compound 5n as the main reaction product, together with a small amount of a derivative oxidized at the C 4 benzylic position (5ah) (Scheme 2).
The reaction between azadiene 9b and quinone 8f afforded compound 5n as the m reaction product, together with a small amount of a derivative oxidized at the C4 benzy position (5ah) (Scheme 2).The hetero Diels-Alder reactions performed were usually regioselective, accord to the previous experience of our group in similar reactions [33,46], and afforded only 1,8-diazanthracene-2,9,10-trione structure.The selectivity of the process towards the end can be attributed to the combination of two electronic factors: the conjugation of carbonyl at C-5 with the lactam nitrogen and the deficiency created at C8=O as a con quence of its conjugation with the C2=O group and the existence of an intramolecular drogen bond with N1-H [33].In one case, as shown in Scheme 3, the reaction of 1-(4-tol 1-H-2,5,8-quinolinetrione (8h) with azadiene 9b afforded significant amounts of the re oisomer 1,5-diaza adduct 11 (5p:11 = 2:1).In this case, the presence of an aryl group on of the quinone may alter the first effect mentioned above because of the partial transfer the nitrogen charge towards the aromatic ring; on the other hand, the presence of a su stituent on N1 may affect the coplanarity of the system, hindering the conjugative effe described above and also leading to the loss of the N1-H bond.Scheme 3. A mixture of 1,5-diaza-and 1,8-diaza-regioisomers was isolated in the reaction of the aryl quinolinetrione 8h with azadiene 9b.The hetero Diels-Alder reactions performed were usually regioselective, according to the previous experience of our group in similar reactions [33,46], and afforded only the 1,8-diazanthracene-2,9,10-trione structure.The selectivity of the process towards the C 6 end can be attributed to the combination of two electronic factors: the conjugation of the carbonyl at C-5 with the lactam nitrogen and the deficiency created at C 8 =O as a consequence of its conjugation with the C 2 =O group and the existence of an intramolecular hydrogen bond with N 1 -H [33].In one case, as shown in Scheme 3, the reaction of 1-(4tolyl)-1-H-2,5,8-quinolinetrione (8h) with azadiene 9b afforded significant amounts of the regioisomer 1,5-diaza adduct 11 (5p:11 = 2:1).In this case, the presence of an aryl group on N 1 of the quinone may alter the first effect mentioned above because of the partial transfer of the nitrogen charge towards the aromatic ring; on the other hand, the presence of a substituent on N 1 may affect the coplanarity of the system, hindering the conjugative effects described above and also leading to the loss of the N 1 -H bond.
The reaction of C 4 -substituted 1-dimethylamino-1-azadienes affords C 5 -substituted 5,8-dihydro-1,8-diazaanthracene-2,9,10-trione systems, which are chiral and contain a stereogenic center at C 5 .It was considered of interest to obtain one of the compounds of this series in enantiomerically pure or enriched form in order to evaluate the stereospecificity of the antitumor activity.To achieve asymmetric induction in the Diels-Alder reaction, it was decided to employ chiral auxiliaries attached to the azadiene structure, which were synthetized from chiral 1-aminopyrrolidine derivatives obtained from (S)-N-benzyl ethyl prolinate and (R)-N-benzyl ethyl prolinate using a route described in the literature [47].The only chiral dimethylamino-1-azadiene previously studied as a Diels-Alder diene is an analogue of compound 12a, containing an additional methyl at C-2 and derived from the Enders SAMP chiral auxiliary, which gave cycloadducts with maleic anhydride and maleimide derivatives in 76-98% enantiomeric excess (ee) [48].Therefore, the reaction of 12a with quinone 8c was employed for optimizing our process.After some unsuccessful initial attempts at room temperature, we found that the reaction between 12a and 8c in refluxing chloroform for 3.5 h gave the desired adduct 6ai although in moderate yield (40%) and enantioselectivity (40% ee).The more hindered diphenyl diene 12b also gave poor results, but its dimethyl analogue 12c afforded 6ai in 40% yield and 85% ee.Similarly, the reaction of quinone 8c with 12d, the enantiomer of 12c, under the same conditions gave 6aj in 30% yield and 83% ee.The configuration of the C 5 stereocenter in these compounds was proposed according to the endo transition state shown in Scheme 4, according to a literature proposal for this type of azadienes [48].The level of stereoselection achieved with azadienes 12c and 12d was considered enough for a comparison of the activities of enantiomeric derivatives of the structure 6.
The hetero Diels-Alder reactions performed were usually regioselective, according to the previous experience of our group in similar reactions [33,46], and afforded only the 1,8-diazanthracene-2,9,10-trione structure.The selectivity of the process towards the C6 end can be attributed to the combination of two electronic factors: the conjugation of the carbonyl at C-5 with the lactam nitrogen and the deficiency created at C8=O as a consequence of its conjugation with the C2=O group and the existence of an intramolecular hydrogen bond with N1-H [33].In one case, as shown in Scheme 3, the reaction of 1-(4-tolyl)-1-H-2,5,8-quinolinetrione (8h) with azadiene 9b afforded significant amounts of the regioisomer 1,5-diaza adduct 11 (5p:11 = 2:1).In this case, the presence of an aryl group on N1 of the quinone may alter the first effect mentioned above because of the partial transfer of the nitrogen charge towards the aromatic ring; on the other hand, the presence of a substituent on N1 may affect the coplanarity of the system, hindering the conjugative effects described above and also leading to the loss of the N1-H bond.The reaction of C4-substituted 1-dimethylamino-1-azadienes affords C5-substituted 5,8-dihydro-1,8-diazaanthracene-2,9,10-trione systems, which are chiral and contain a stereogenic center at C5.It was considered of interest to obtain one of the compounds of this series in enantiomerically pure or enriched form in order to evaluate the stereospecificity of the antitumor activity.To achieve asymmetric induction in the Diels-Alder reaction, it was decided to employ chiral auxiliaries attached to the azadiene structure, which were synthetized from chiral 1-aminopyrrolidine derivatives obtained from (S)-N-benzyl ethyl prolinate and (R)-N-benzyl ethyl prolinate using a route described in the literature [47].The only chiral dimethylamino-1-azadiene previously studied as a Diels-Alder diene is an analogue of compound 12a, containing an additional methyl at C-2 and derived from the Enders SAMP chiral auxiliary, which gave cycloadducts with maleic anhydride and maleimide derivatives in 76-98% enantiomeric excess (ee) [48].Therefore, the reaction of 12a with quinone 8c was employed for optimizing our process.After some unsuccessful initial attempts at room temperature, we found that the reaction between 12a and 8c in refluxing chloroform for 3.5 h gave the desired adduct 6ai although in moderate yield (40%) and enantioselectivity (40% ee).The more hindered diphenyl diene 12b also gave poor results, but its dimethyl analogue 12c afforded 6ai in 40% yield and 85% ee.Similarly, the reaction of quinone 8c with 12d, the enantiomer of 12c, under the same conditions gave 6aj in 30% yield and 83% ee.The configuration of the C5 stereocenter in these compounds was proposed according to the endo transition state shown in Scheme 4, according to a literature proposal for this type of azadienes [48].The level of stereoselection achieved with azadienes 12c and 12d was considered enough for a comparison of the activities of enantiomeric derivatives of the structure 6. Scheme 4. Synthesis of enantio-enriched compound 6ai and its enantiomer 6aj.The chiral 1-aminopyrrolidine derivatives were obtained according to reference [47] Scheme 4. Synthesis of enantio-enriched compound 6ai and its enantiomer 6aj.The chiral 1aminopyrrolidine derivatives were obtained according to reference [47].
In order to complete the library of compounds 5, the dehydrogenation of their dihydro derivatives 6 (Scheme 5) was examined by comparing two methods, namely treatment with manganese dioxide (method A), which afforded good to excellent yields under mild conditions and air oxidation at high temperature (method B), which was efficient but rather slow.In the case of compound 5r, air oxidation was carried out in the presence of NaOH at room temperature (method C) (Table 4).
Molecules 2024, 29,489 In order to complete the library of compounds 5, the dehydrogenation of the dro derivatives 6 (Scheme 5) was examined by comparing two methods, namely tre with manganese dioxide (method A), which afforded good to excellent yields und conditions and air oxidation at high temperature (method B), which was efficient ther slow.In the case of compound 5r, air oxidation was carried out in the pres NaOH at room temperature (method C) (Table 4).
In order to complete the library of compounds 5, the dehydrogenation of thei dro derivatives 6 (Scheme 5) was examined by comparing two methods, namely trea with manganese dioxide (method A), which afforded good to excellent yields unde conditions and air oxidation at high temperature (method B), which was efficient ther slow.In the case of compound 5r, air oxidation was carried out in the prese NaOH at room temperature (method C) (Table 4).
Scheme 6. Diels-Alder reactions of dienes 13 and quinones 8.In some cases, it was not possible to obtain the aromatic compound 7 in a single step, although the level of oxidation of the obtained cycloadducts could be controlled by modifying the reaction conditions.Thus, the reaction between quinone 8b and 2,3-dimethyl-1,3-butadiene in ethyl acetate at 100 • C led to the formation of the primary Diels-Alder adduct 14.The reaction in the same solvent under increased reaction temperature (120 • C) gave a mixture of the aromatic derivative 7g and 14, which by reaction with DDQ in benzene at reflux for 7 h finally afforded the target compound 7g as a single product in 56% overall yield (Scheme 7).
Molecules 2024, 29 In some cases, it was not possible to obtain the aromatic compound 7 in a although the level of oxidation of the obtained cycloadducts could be controll ifying the reaction conditions.Thus, the reaction between quinone 8b and 2,3 1,3-butadiene in ethyl acetate at 100 °C led to the formation of the primary D adduct 14.The reaction in the same solvent under increased reaction temperatu gave a mixture of the aromatic derivative 7g and 14, which by reaction with D zene at reflux for 7 h finally afforded the target compound 7g as a single prod overall yield (Scheme 7).In order to study the activity of some 5,8-dihydro derivatives of the 1-aza etrione framework, we resorted to modulating the reaction conditions to prev aromatization of the primary Diels-Alder adduct.Thus, compound 15 was o gether with the corresponding aromatic derivative 7d, as a minor product of t of quinone 8b with piperylene (1,3-pentadiene) in chloroform at 100 °C in a for 24 h.Additional 5,8-dihydro-1-azaanthracenetrione derivatives were obta the diene anthracene (compound 16) and cyclopentadiene.In the latter case compound 18 was obtained together with the corresponding hydroquinone mixture was transformed into 18 by a simple reflux in methanolic sodium (Scheme 8).In order to study the activity of some 5,8-dihydro derivatives of the 1-azaanthracenetrione framework, we resorted to modulating the reaction conditions to prevent the full aromatization of the primary Diels-Alder adduct.Thus, compound 15 was obtained, together with the corresponding aromatic derivative 7d, as a minor product of the reaction of quinone 8b with piperylene (1,3-pentadiene) in chloroform at 100 • C in a sealed tube for 24 h.Additional 5,8-dihydro-1-azaanthracenetrione derivatives were obtained using the diene anthracene (compound 16) and cyclopentadiene.In the latter case, the target compound 18 was obtained together with the corresponding hydroquinone 17, but this mixture was transformed into 18 by a simple reflux in methanolic sodium hydroxide (Scheme 8).Scheme 8. Synthesis of 5,8-dihydro-1-azaanthracenetrione derivatives.

Biological Studies
The in vitro cytotoxicity of compounds derived from the structures 5, 6, 7, and 8 was studied in the following cell lines: mouse lymphoma (P-388), human lung carcinoma (A-549), human colon carcinoma (HT-29), human breast carcinoma (MCF-7), and human melanoma (MEL-28).For comparison purposes, we also evaluated the activity of diazaquinomycin A and some related compounds derived from structure 1 in these cell lines to establish a structure-activity relationship as conclusive as possible and determine the pharmacophore group.The results are shown in Figures 3-11 and Tables S1-S6 in the Supporting Information expressed as inhibitory concentration 50 values (IC50, µM).
We first assessed the activity of simplified structures derived from the quinoline-2,5,8(1H)-trione framework (starting quinones 8), with the results shown in Figure 3 and Table S1.These compounds generally showed relatively modest activities and selectivities towards the tumors assayed, although three of the compounds (8c, 8k and 8l) had IC50 values against the P-388 leukemia cell line at the micromolar level, with some selectivity with respect to solid tumors.Nevertheless, the comparison of these activities with those shown below for the Diels-Alder and Diels-Alder adducts reveals the need for the tricyclic system in order to achieve good anticancer activity.The unsubstituted 1-azaanthracenetrione derivative 7a showed the best antitumor activity of this series, and also selectivity against the lung cancer cell line, with an IC50 in the ten nanomolar range.Interestingly, this activity was superior to that of the marcanine Scheme 8. Synthesis of 5,8-dihydro-1-azaanthracenetrione derivatives.

Biological Studies
The in vitro cytotoxicity of compounds derived from the structures 5, 6, 7, and 8 was studied in the following cell lines: mouse lymphoma (P-388), human lung carcinoma (A-549), human colon carcinoma (HT-29), human breast carcinoma (MCF-7), and human melanoma (MEL-28).For comparison purposes, we also evaluated the activity of diazaquinomycin A and some related compounds derived from structure 1 in these cell lines to establish a structure-activity relationship as conclusive as possible and determine the pharmacophore group.The results are shown in Figures 3-11 and Tables S1-S6 in the Supporting Information expressed as inhibitory concentration 50 values (IC 50 , µM).
We first assessed the activity of simplified structures derived from the quinoline-2,5,8(1H)-trione framework (starting quinones 8), with the results shown in Figure 3 and Table S1.These compounds generally showed relatively modest activities and selectivities towards the tumors assayed, although three of the compounds (8c, 8k and 8l) had IC 50 values against the P-388 leukemia cell line at the micromolar level, with some selectivity with respect to solid tumors.Nevertheless, the comparison of these activities with those shown below for the Diels-Alder and Diels-Alder adducts reveals the need for the tricyclic system in order to achieve good anticancer activity.Scheme 8. Synthesis of 5,8-dihydro-1-azaanthracenetrione derivatives.

Biological Studies
The in vitro cytotoxicity of compounds derived from the structures 5, 6, 7, an studied in the following cell lines: mouse lymphoma (P-388), human lung carcino 549), human colon carcinoma (HT-29), human breast carcinoma (MCF-7), and hum anoma (MEL-28).For comparison purposes, we also evaluated the activity of diaz mycin A and some related compounds derived from structure 1 in these cell line tablish a structure-activity relationship as conclusive as possible and determine th macophore group.The results are shown in We first assessed the activity of simplified structures derived from the qu 2,5,8(1H)-trione framework (starting quinones 8), with the results shown in Figur Table S1.These compounds generally showed relatively modest activities and sele towards the tumors assayed, although three of the compounds (8c, 8k and 8l) h values against the P-388 leukemia cell line at the micromolar level, with some sel with respect to solid tumors.Nevertheless, the comparison of these activities wit shown below for the Diels-Alder and Diels-Alder adducts reveals the need for th clic system in order to achieve good anticancer activity.The unsubstituted 1-azaanthracenetrione derivative 7a showed the best an activity of this series, and also selectivity against the lung cancer cell line, with an the ten nanomolar range.Interestingly, this activity was superior to that of the ma B, C, and G natural products [24,25].The presence of substituents at C7 and C8 considerably decreases their activity, as shown by the data for compounds 7d-7g.It is also relevant to note that the primary Diels-Alder adduct showed a higher activity than its aromatic counterpart 7g (Figure 4 and Table S2).The dihydro derivatives of the 1-azaanthracenetrione framework (compounds 15-18) substituted at C5 and C8 also maintained the anticancer activity, although with low selectivity (Figure 4 and Table S3).S2 and S3 (Supporting Information).
Regarding the activity of derivatives of structure 5 (Figures 5-7 and Table S4), our observations lead to the following conclusions: (a) Aromatic substituents at N-1 lower both activity and selectivity against solid tumors, as shown by the comparison of the data for compounds 5d and 5p.(b) Aromatic substituents at C-3 do not improve the activity on solid tumor cell lines with respect to alkyl groups when directly attached to the anthraquinone ring (5q vs. 5l and 5w vs. 5r) (c) On the other hand, when an aromatic ring is attached to C3 via a methylidene spacer, not only is considerable antitumor activity observed, but also a notable selectivity towards certain solid tumors.Thus, compound 5m is ten times more active in lung carcinoma and melanoma than in colon carcinoma and ca.50 times more active than on the P-388 lymphoma cells.This difference also exists but is not so marked in the derivatives, which only present alkyl substituents at C3 such as 5l.(d) The presence of simultaneous substituents at C3, C4, and C5, leading to a steric interaction between the C10=O groups with R4 and R4, increases the activity against P-388 compared to the same compounds when they only have substituents at C4 and C5 or C3 and C4 (5u vs. 5t).(e) An increase in the length of the R5 chain seems to be accompanied by a slight decrease in activity in lung cancer cells (compounds 5g, 5j, and 5k).Aromatic substituents, especially when bearing an electron-releasing group, also lead to decreased activity in these cells (compounds 5h and 5i).(f) The presence of alkyl substituents at R6 is generally favorable for activity on the human colorectal adenocarcinoma cell line, as shown by the comparison of the data for 5c and 5d-f.(g) Electron-withdrawing groups decrease the activity when they are in the C3 and C4 positions with respect to their alkyl counterparts, as shown by the comparison between the compounds (5y, 5z, and 5aa vs. 5ad and 5ah vs. 5d).S2 and S3 (Supporting Information).
The unsubstituted 1-azaanthracenetrione derivative 7a showed the best antitumor activity of this series, and also selectivity against the lung cancer cell line, with an IC 50 in the ten nanomolar range.Interestingly, this activity was superior to that of the marcanine B, C, and G natural products [24,25].The presence of substituents at C 7 and C 8 considerably decreases their activity, as shown by the data for compounds 7d-7g.It is also relevant to note that the primary Diels-Alder adduct showed a higher activity than its aromatic counterpart 7g (Figure 4 and Table S2).The dihydro derivatives of the 1-azaanthracenetrione framework (compounds 15-18) substituted at C 5 and C 8 also maintained the anticancer activity, although with low selectivity (Figure 4 and Table S3).S4 (Supporting Information).
The 1,5-diazaanthracenetrione framework seems to provide less active compounds than its 1,8-diaza isomer when substituted at C-7, as shown by the comparison of the activity data of compounds 5p and 12 (Figures 6 and 8 and Table S4).S4 (Supporting Information).
Molecules 2024, 29, 489 The 1,5-diazaanthracenetrione framework seems to provide less active com than its 1,8-diaza isomer when substituted at C-7, as shown by the comparison of tivity data of compounds 5p and 12 (Figures 6 and 8 and Table S4).S5), the following sions were obtained: (a) Generally speaking, the aromatic compounds 5 and their 5,8-dihydro counte show similar activities, but some exceptions to this rule were observed, as s    S5 (Supporting Information).
Diazaquinomycins have been synthesized using double Knorr reactions, a strategy developed by Kelly [50] and Sun [51], or hetero Diels-Alder chemistry followed by Noxidation and rearrangement, as described by us [44].Diazaquinomycin A (1a) thus prepared exhibited a lower activity and selectivity than their corresponding derivative with a monolactam system (5u) and its 5,8-dihydro derivative (6u).Furthermore, additional structures derived from the diazaquinomycin framework (compounds 1b-e) showed a higher activity than the natural product but still lacked selectivity in their cytotoxicity (Figure 11 and Table S6).S5 (Supporting Information).
Diazaquinomycins have been synthesized using double Knorr reactions, a strategy developed by Kelly [50] and Sun [51], or hetero Diels-Alder chemistry followed by Noxidation and rearrangement, as described by us [44].Diazaquinomycin A (1a) thus prepared exhibited a lower activity and selectivity than their corresponding derivative with a monolactam system (5u) and its 5,8-dihydro derivative (6u).Furthermore, additional structures derived from the diazaquinomycin framework (compounds 1b-e) showed a higher activity than the natural product but still lacked selectivity in their cytotoxicity (Figure 11 and Table S6).Due to the importance of the multidrug resistance phenomenon (MD therapy, we also examined the effect of the representative compound 6h on Regarding the activity of derivatives of structure 5 (Figures 5-7 and Table S4), our observations lead to the following conclusions: (a) Aromatic substituents at N-1 lower both activity and selectivity against solid tumors, as shown by the comparison of the data for compounds 5d and 5p.(b) Aromatic substituents at C-3 do not improve the activity on solid tumor cell lines with respect to alkyl groups when directly attached to the anthraquinone ring (5q vs. 5l and 5w vs. 5r) (c) On the other hand, when an aromatic ring is attached to C 3 via a methylidene spacer, not only is considerable antitumor activity observed, but also a notable selectivity towards certain solid tumors.Thus, compound 5m is ten times more active in lung carcinoma and melanoma than in colon carcinoma and ca.50 times more active than on the P-388 lymphoma cells.This difference also exists but is not so marked in the derivatives, which only present alkyl substituents at C 3 such as 5l.(d) The presence of simultaneous substituents at C 3 , C 4 , and C 5 , leading to a steric interaction between the C 10 =O groups with R 4 and R 4 , increases the activity against P-388 compared to the same compounds when they only have substituents at C 4 and C 5 or C 3 and C 4 (5u vs. 5t).(e) An increase in the length of the R 5 chain seems to be accompanied by a slight decrease in activity in lung cancer cells (compounds 5g, 5j, and 5k).Aromatic substituents, especially when bearing an electron-releasing group, also lead to decreased activity in these cells (compounds 5h and 5i).(f) The presence of alkyl substituents at R 6 is generally favorable for activity on the human colorectal adenocarcinoma cell line, as shown by the comparison of the data for 5c and 5d-f.(g) Electron-withdrawing groups decrease the activity when they are in the C 3 and C 4 positions with respect to their alkyl counterparts, as shown by the comparison between the compounds (5y, 5z, and 5aa vs. 5ad and 5ah vs. 5d).
The 1,5-diazaanthracenetrione framework seems to provide less active compounds than its 1,8-diaza isomer when substituted at C-7, as shown by the comparison of the activity data of compounds 5p and 12 (Figures 6 and 8 and Table S4).
For 5,8-dihydro derivatives 6 (Figures 9 and 10 and Table S5), the following conclusions were obtained: (a) Generally speaking, the aromatic compounds 5 and their 5,8-dihydro counterparts 6 show similar activities, but some exceptions to this rule were observed, as summarized below.(b) The presence of aromatic derivatives at C-5 increases the activity and selectivity against colon cancer cells in the dihydro derivatives such as 6h and 6i compared to their aromatized counterparts 5h and 5i.For N-aryl substitution, dihydro derivatives seem less selective for solid tumors (5v vs. 6v).(c) On the other hand, for the case of alkyl substitution the selectivity trend is the opposite one, with the aromatic systems 5 showing a higher selectivity for solid tumors than the corresponding compounds 6 (5s vs. 6s, 5t vs. 6t, 5u vs. 6u).Even though a comparison cannot be established in these cases, the lack of selectivity found in compounds 6ae, 6af, and 6ag supports this conclusion.(d) A comparison of the activities of the enantio-enriched compounds 6ai and 6aj leads to the conclusion that the configuration of the stereogenic center at C-5 does not greatly influence the anti-tumor activity.
Diazaquinomycins have been synthesized using double Knorr reactions, a strategy developed by Kelly [50] and Sun [51], or hetero Diels-Alder chemistry followed by Noxidation and rearrangement, as described by us [44].Diazaquinomycin A (1a) thus prepared exhibited a lower activity and selectivity than their corresponding derivative with a monolactam system (5u) and its 5,8-dihydro derivative (6u).Furthermore, additional structures derived from the diazaquinomycin framework (compounds 1b-e) showed a higher activity than the natural product but still lacked selectivity in their cytotoxicity (Figure 11 and Table S6).
Due to the importance of the multidrug resistance phenomenon (MDR) in cancer therapy, we also examined the effect of the representative compound 6h on two cell lines showing multidrug resistance due to the overexpression of glycoprotein Pgp-170, namely the HTC-15 colon and the UO-21 kidney cancer lines.As shown in Figure 12, compound 6h showed good activity in both cases.S6 (Supporting Information).
Due to the importance of the multidrug resistance phenomenon (MDR) in cancer therapy, we also examined the effect of the representative compound 6h on two cell lines showing multidrug resistance due to the overexpression of glycoprotein Pgp-170, namely the HTC-15 colon and the UO-21 kidney cancer lines.As shown in Figure 12, compound 6h showed good activity in both cases.Regarding the cytotoxicity mechanism of the compounds described here, compound 6h gave negative results in the following potential anticancer targets: thymidylate synthase (the putative target of diazaquinomycin A [18,19]), dihydrofolate reductase, topoisomerases I and II, ADN polymerases and ARN polymerases.It also failed to block DNA, RNA, or protein synthesis.Moreover, no clear-cut connection can be established between the half-wave redox potential of the quinone moieties in our compounds [25] and their activities.Additional studies will be needed to gain insight into the mechanism of action of our compounds, and in this connection, studies on the ability of our compounds to block the cell cycle at a particular stage will perhaps be informative.Due to the facts that the formation of DNA-damaging anion-radicals often contributes to the anticancer activity of quinones [52,53] and that the deoxynyboquinones have been shown to generate reactive oxygen species via their activation by quinone reductase NQO1 [14], this possibility will also be explored.

General Information
All reagents and solvents were of commercial quality, purchased from Sigma-Aldrich (Madrid, Spain) and were used as received.Reactions were monitored by thin layer chromatography on aluminium plates coated with silica gel and fluorescent indicator.Separation by flash chromatography were performed on SDS 60 ACC, 230-400 mesh or Scharlau Ge 048 silica gel.Melting points were measured with Reichert 723 hot stage microscope (Vienna, Austria), or in open capillary tubes using a Büchi immersion instrument, and are uncorrected.Infrared spectra were recorded on a Perkin-Elmer Paragon 1000 Regarding the cytotoxicity mechanism of the compounds described here, compound 6h gave negative results in the following potential anticancer targets: thymidylate synthase (the putative target of diazaquinomycin A [18,19]), dihydrofolate reductase, topoisomerases I and II, ADN polymerases and ARN polymerases.It also failed to block DNA, RNA, or protein synthesis.Moreover, no clear-cut connection can be established between the halfwave redox potential of the quinone moieties in our compounds [25] and their activities.Additional studies will be needed to gain insight into the mechanism of action of our compounds, and in this connection, studies on the ability of our compounds to block the cell cycle at a particular stage will perhaps be informative.Due to the facts that the formation of DNA-damaging anion-radicals often contributes to the anticancer activity of quinones [52,53] and that the deoxynyboquinones have been shown to generate reactive oxygen species via their activation by quinone reductase NQO1 [14], this possibility will also be explored.

General Information
All reagents and solvents were of commercial quality, purchased from Sigma-Aldrich (Madrid, Spain) and were used as received.Reactions were monitored by thin layer chromatography on aluminium plates coated with silica gel and fluorescent indicator.Separation by flash chromatography were performed on SDS 60 ACC, 230-400 mesh or Scharlau Ge 048 silica gel.Melting points were measured with Reichert 723 hot stage microscope (Vienna, Austria), or in open capillary tubes using a Büchi immersion instrument, and are uncorrected.Infrared spectra were recorded on a Perkin-Elmer Paragon 1000 spectrophotometer (Tres Cantos, Spain), with solid compounds compressed into KBr pellets and liquid compounds placed between two NaCl disks.NMR spectra data were obtained using Bruker spectrometers (Rivas-Vaciamadrid, Spain) maintained by the CAI de Resonancia Magnética, UCM, operating at 250 and 300 MHz for 1 H NMR and, 63 and 75 MHz for 13 C NMR; chemical shifts (δ) are given in parts per million and coupling constants (J) in Hertz.Elemental analyses were determined by the CAI de Microanálisis, Universidad Complutense, using a Leco CHNS-932 combustion microanalyzer (Tres Cantos, Spain).The enantiomeric excess of compounds 6ai and 6aj was assessed by 1 H-NMR in the presence of 1.5 equivalents of (+) tris(3-heptafluoropropylhydroxymethylene) europium camphorate.

Preparation of Enantiomerically Pure 1-Azadienes-General Procedure
To a solution of crotonaldehyde in dry ethyl ether (5 mL) is added a catalytic amount of acetic acid (0.02 mL) and 1.00-3.10mmol of suitable chiral auxiliary.The resulting solution is heated at reflux in a bath at 45 • C in the absence of moisture for 30 min.After checking by TLC that the reaction is complete, it is allowed to cool at room temperature and washed with saturated aqueous sodium bicarbonate solution (1 mL × 2).The organic phase is dried with anhydrous sodium sulfate and evaporated, obtaining the desirable products.

8-General Procedure
Method A: To a solution of quinones 8 (0.05 to 0.55 mmol) in chloroform or dry THF (5-20 mL) was added the suitable azadiene 9 (1.05 to 2 equiv.).The solution was stirred at room temperature (5 min-1 h), and the solvent was evaporated.The residue was chromatographed on silica gel eluting with a gradient from neat dichloromethane or ethyl ether to neat ethyl acetate to yield compounds 5 or 6 in the yields shown in Table 2.
Method B: To silica gel-supported suitable quinone 8 (0.06-0.55 mmol), the suitable dimethylhydrazone 9 was rapidly added (2 eq).The column was then eluted with a gradient from dichloromethane or ethyl ether to ethyl acetate, affording compounds 5 or 6 in the yields shown in Table 2.
Method C: A suspension of carboxy polystyrene resin, prepared from 2% cross-linked Merrifield resin (2-2.5 meq Cl/g, 200-400 mesh) using a literature method [55] (5.0 g) in dry toluene (20 mL) was stirred at room temperature under an argon atmosphere for 30 min.Thionyl chloride (20 mL) was added, and the suspension was refluxed for 24 h with vigorous stirring and then cooled.The chloroformyl polystyrene resin thus obtained was filtered and washed with toluene (2 × 25 mL) and ethyl ether (2 × 25 mL).If stored at −18 • C under argon, it could be used for 2-3 months without a significant loss of activity.
To a stirred suspension of the suitable 2,5,8(1H)-quinolinetrione 8 (0.25 mmol) and the chloroformyl polystyrene resin (1.25 g, 5 eq) in chloroform (30 mL) was added the suitable dimethylhydrazone 9 (0.60 mmol).The suspension was stirred at room temperature for 5 min and filtered.The filtrate was evaporated and then residue was chromatographed on silica gel, affording compounds 5 or 6 in the yields shown in Table 2.
Characterization data for new compounds follow.Exchangeable spectral assignments are marked with asterisks.  13   13  After carrying out the Diels Alder reaction on 19 mg (0.07 mmol) of 8h and 16 mg (0.13 mmol) of 9b according to method B, the reaction crude was dissolved in 10 mL of dichloromethane and 62 mg (0.72 mmol) of 85% activated manganese oxide was added.The solution was stirred at room temperature for 24 h and filtered through a layer of celite which was washed twice with 30 mL chloroform.The combined organic phases were dried over anhydrous sodium sulphate, evaporated and purified by silica gel column chromatography eluting with a gradient from dichloromethane to ethyl acetate, yielding 12 mg (52%) of 5p, 6 mg (26%) of 11 and 2 mg (9%) of the corresponding quinone 10.

5-General Procedures
Method A: To a solution of the suitable dihydro derivative 6 (0.01-0.20 mmol) in dichloromethane (1-10 mL) was added activated 85% MnO 2 (5 eq-10 eq), the solution was stirred (10 min-24 h) at r.t. and then it was filtered through celite pad and washed twice with 30 mL of dichloromethane.The solvent was evaporated and, when necessary, chromatographed on silica gel, eluting with ethyl acetate, to afford compounds 6.
Method B: A solution of the suitable dihydro derivate 6 (0.06 to 0.30 mmol) in xylene (50-60 mL) was heated to reflux while a stream of air was bubbled through the solution.Xylene was added periodically to avoid complete evaporation of the solution.After the time indicated in each case, the solution was evaporated, and the crude was chromatographed on silica gel eluting with ethyl acetate to afford compounds 6.
Method C: A solution of NaOH 10% (2 mL) was added to the suitable dihydro derivative 6 (0.10 mmol).The suspension was stirred for 30 min at r.t., then it was diluted with 10 mL of water and extracted with ethyl acetate (3 × 20 mL).The resulting organic layer was evaporated and purified by chromatography on silica gel, eluting with ethyl acetate.The solid thus obtained was washed with ethyl ether to afford compounds 6.A solution of quinone 8b (370 mg, 1.90 mmol) and cyclopentadiene (0.32 mL, 3.90 mmol) in chloroform (130 mL) was heating at 110 • C for 18 h in a sealed tube.The solution was cooled and evaporated under vacuum.The residue was chromatographed on silica gel eluting with a gradient from dichloromethane to dichloromethane/ethyl acetate (1:1), to yield 40 mg of compound 17 (23%) and 118 mg of compound 18 (71%).
To determine and compare the antitumor activity of these compounds, simple screening procedures were performed using an adapted form of the method described by Bergerom et al. [56,57] The tumor cells used were P-388 (BDA/2 mouse lymphoid neoplasm suspension culture), A-549 (human lung carcinoma monolayer culture), HT-29 (human colon carcinoma monolayer culture), MEL-29 (human melanoma monolayer culture).P-388 cells were seeded in 16 mm wells at 1 × 10 4 cells per well in aliquots to 1 mL of 5FCS MEM containing the indicated drug concentration.Separately, a batch of drug-free cultures was seeded as a growth control to ensure that the cells remained in log phase of growth.All determinations were performed in triplicate.After three days of incubation at 37 • C, 10% CO 2 in an atmosphere of 98% humidity, the approximate IC 50 was determined by comparing growth in the drug wells and growth in the control wells.
A-549, HT-29 and MEL-28 cells were seeded in 16 mm wells at 2 × 10 4 cells per well in 1 mL aliquots of MEM 10FCS containing the indicated drug concentration.Separately, a drug-free culture batch was seeded as a growth control to ensure that the cells remained in log phase of growth.All determinations were performed in duplicate.After three days of incubation at 37 • C, 10% CO 2 in an atmosphere of 98% humidity, the wells were stained with 0.1% Crystal Violet.The IC 50 value was determined by comparing the growth in the drug wells and the growth in the control wells.

Conclusions
The synthesis of libraries of derivatives of the 1,8-diazaanthracene-2,9,10-trione, including their 5,8-dihydro derivatives, 1,8-diazaanthracene-2,7,9,10-tetraone and 1-azaanthracene-2,9,10-trione frameworks, structurally related to the diazaquinomycin and marcanine families of natural products, was achieved using Diels-Alder strategies.Many of them were found to have potent and selective cytotoxicity against some solid tumors.In particular, 1,8-diazaanthracene-2,9,10-triones and their 5,8-dihydro derivatives were particularly active against a human lung cancer cell line.The use of a 1-azadiene comprising a SAMPrelated chiral auxiliary allowed the enantioselective synthesis of two representatives of chiral 5-substituted 1,8-diazaanthracene-2,9,10-triones, and their study showed that their cytotoxicity was not enantiospecific.The following targets and processes were discarded as explanations of the mechanism of action of our compounds: thymidylate synthase, dihydrofolate reductase, topoisomerases I and II, ADN polymerases, ARN polymerases, DNA, RNA and protein synthesis.Further mechanistic work will be needed to characterize the mechanism of action of these interesting compounds.

Figure 3 .
Figure 3. Cytotoxicity of quinolinetriones 8. Numerical data can be found in Table S1 (Supporting Information).

Figure 3 .
Figure 3. Cytotoxicity of quinolinetriones 8. Numerical data can be found in Table S1 (Su Information).

Figure 3 .
Figure 3. Cytotoxicity of quinolinetriones 8. Numerical data can be found in Table S1 (Supporting Information).

Figure 11 .
Figure 11.Cytotoxicity of diazaaquinomycins 1. Numerical data can be found in Table S6 (Supporting Information).

Table 3 .
Scope and yields of the hetero Diels-Alder reactions.

Table 3 .
Scope and yields of the hetero Diels-Alder reactions.
b Data obtained from reference

Table 4 .
Scope and yields obtained in the dehydrogenation of compounds 6.

Table 5 .
Structures and yields of compounds 7.

Table 4 .
Scope and yields obtained in the dehydrogenation of compounds 6.

Table 4 .
Scope and yields obtained in the dehydrogenation of compounds 6.

Table 5 .
Structures and yields of compounds 7.