A Survey of Synthetic Routes and Antitumor Activities for Benzo[g]quinoxaline-5,10-diones

Anthracycline antibiotics play an important role in cancer chemotherapy. The need to improve their therapeutic index has stimulated an ongoing search for anthracycline analogs with enhanced properties. This review aims to summarize the common synthetic approaches to benzo[g]quinoxaline-5,10-diones and their uses in heterocyclic chemistry. Because of the valuable biological activities of the 1,4-diazaanthraquinone compounds, a summary of the most promising heterocyclic quinones is provided together with their antitumor properties.


Introduction
The substructure of 1,4-anthraquinones (anthracene-9,10-diones) is an important class of bioactive non-heterocyclic quinonoid compounds ( Figure 1). They include natural anthracyclines such as Daunomycin, mainly active against acute lymphoblastic leukemia and acute myeloid leukemia, and Doxorubicin approved for the treatment of a wide variety of liquid and solid tumors [1]. The reference synthetic 1,4-anthraquinone is Mitoxantrone, with anticancer applications but also active on multiple sclerosis through its immunosuppressant properties [2]. Both drugs have adverse side effects typical of non-selective cytotoxic drugs, with inhibitor effects on rapidly dividing tissues (hair, bone marrow and mucous membranes). Moreover, 1,4-anthraquinones produce a dose-limiting specific toxicity to the heart. This has led to the development of many analogs with reduced toxicity and improved spectrum of activity [3]. Recently pixantrone, an aza-anthracenedione, was developed to reduce cardiotoxicity typically associated with anthracyclines but without compromising antineoplastic efficacy [4].
Numerous quinones are known for their anticancer activities, such as the alkylating agent Mitomycine C [5], or Streptonigrin obtained from Streptomyces flocculus, whose application is limited by its toxicity [6].
1,4-Diazaanthraquiones exhibit promising in vitro and in vivo activity against a wide spectrum of tumor cell lines [7][8][9]. They have been reported interesting antibiotic properties against a wide range of fungal and bacterial pathogens [5]. This review includes synthetic methodologies for the preparation of linear benzo[g]quinoxaline-5,10-diones derivatives and their structural analogs, as reported in the literature. Different strategies were found to give access to this class of compound, including: • Condensations of 2,3-diamino-1,4-naphthoquinone with 1,2-dicarbonyl derivatives. • Cycloaddition via generated in situ N-substituted-quinolinedione intermediates. • Diels-Alder cyclocondensation reactions.
The second part of the review summarizes the common biological activities of benzo[g]quinoxaline-5,10-diones, with particular emphasis on their antitumor activity.
As an alternative, a Gabriel reaction can also be used. Phthalimide potassium with alkyl halides allows the synthesis of primary amines. After alkylation, the product was cleaved by reaction with hydrazine (method b-Scheme 2). In this case, the reaction of compound 1 with 2 equiv. of phthalimide potassium afforded diphthalimido naphthoquinone, which was allowed to react with hydrazine hydrate to give compound 2 also named 2,2-diamino [1,4]-naphtoquinone (Scheme 2) [15,16]. Díaz et al. [17] synthesized compounds 3 as shown in Scheme 2 (method c) by melting 1 in ethanolic ammonia solution followed by acetylation. The acetylated compound 3 was suspended in an ammonia solution to obtain product 4. Hydrochloric acid gas was then added in methanol to derivative 4 (Scheme 2).
Most of the derivatives 13 can also be obtained through a nucleophilic substitution by reacting a substituted aniline with 2,3-dichloronaphthoquinone using CeCl 3 as catalyst. Addition of this latter induces the formation of a complex between the carbonyl function and Ce 3+ . Thus, selective nucleophilic substitution takes place [35], with a chlorine atom replaced by a substituted aniline (68-90% yields with R = H; 4-OMe; 4-OEt; 2-F; 4-F; 3,4-diF; 2-OMe).
This approach is an easy way to synthesize tetracyclic linear diazaanthraquinone. The scope is limited by the variety of substituents when N-aryl is synthesized. Substituted anilines with electron-donating groups are quite reactive and afford high yields of the corresponding anilinoquinones. However, using either method with anilines substituted with electron-withdrawing groups gives quite low yields. As a consequence, these methods may not be used for the preparation of nitro substituted-2-(4-arylamino)-1,4-naphthoquinones. However, they can be obtained by an alternative two-step synthesis was developed. In the first step, 2-anilino-3-chloro-1,4-naphthoquinones were prepared by the classical Michael type addition-elimination reaction [33,36]. The phenyl group was then nitrated via direct electrophilic aromatic substitution in the second step.
Azidonaphthoquinones having an electron-withdrawing substituent favor the generation of an open-shell nitrene with a strong biradical character, which undergoes hydrogen abstraction to give N-aminonaphthoquinone (Scheme 9). In contrast, azidonaphthoquinones having a strong electron-donor like O-R favor the generation of a highly reactive singlet nitrene which undergoes insertion to give a tetracyclic compound (Scheme 10).

Scheme 9.
Proposed mechanism for the synthesis of N-aminonaphthoquinones from azidonaphthoquinones bearing electron-withdrawing substituent.

Scheme 10.
Proposed ring closure reaction mechanism for the synthesis of diazaanthraquinones. from azidonaphthoquinones bearing electron-rich substituents.

Miscellaneous Reactions
The synthesis of 6,9-bis-[(aminoalkyl)amino]-substituted benzo[g]quinoxalines was described [47][48][49][50][51]. They were prepared by displacements (S N Ar) of the fluorides from 6,9-difluoro-substituted benzo[g]quinoxaline. The diacid was converted into the anhydride by refluxing in acetic anhydride or by treatment with DCC in THF. Friedel-Crafts acylation of 1,4-difluorobenzene with the anhydride in the presence of aluminium chloride led to keto acid 46. Cyclodehydratation of 46 to 47 was obtained with fuming sulfuric acid at 140 • C. Addition of N,N-dimethylethylenediamine to a pyridine solution of 47 yielded 48a. Fluoride displacements proceeded quite slowly and the mixture was stirred for several days to complete the bis-substitution. Shortening the reaction time would the mono-substituted analog to be isolated. 48b and its mono-substituted analog were prepared by treatment of N-(tert-butoxycarbonyl)ethylenediamine in DMSO (Scheme 20). The above synthetic pathway had the disadvantage to synthesize benzo[g]quinoxalinediones in which both distal side arms were the same. Krapcho et al. [48] accomplished subsequently the synthesis of regioisomeric dihalo-substituted heterocyclic quinone named 6-chloro-9-fluorobenzo[g]quinoxaline 56. This molecule 56 can be used as starting material for the synthesis of dialkylamino-substituted heterocyclic quinones by fluoride and chloride on the C-6 and C-8 positions. The fluoride will undergo an S N Ar displacement at room temperature at a rate considerably more rapid than the chloride, on being treated with amine nucleophiles. The mono-chlorosubstituted compound being treated at a higher temperature with a different amine derivative (or any other nucleophilic species) will lead to the desired compounds. The synthesis of compound 56 is detailed on Scheme 21.
Preparation of quinoxaline-5,8-dione 76a is reported in the literature [64]. Benzo[g]quinoxaline-5,10-dione derivatives bearing 7-dialkylaminomethyl were synthesized based on the Diels-Alder reaction of quinoxaline-5,8-dione 76a with isoprene [65], as outlined in Scheme 28. Starting material 76a was treated with isoprene to give the cycloaddition adduct, which was directly aromatized by aerial oxidation in 5N ethanolic KOH under reflux to give 7-methylbenzo[g]quinoxaline-5,10-dione 78. Intermediate 78 was then treated by N-bromosuccinimide (NBS) and a catalytic amount of benzoylperoxide in anhydrous 1,2-dichloroethane under reflux for 48 h with irradiation by tungsten lamp to give bromomethyl product 79 in 30% yield (Scheme 28). Starting material 76a was treated with 2,3-dimethylbutadiene to give the cycloaddition adduct, which was directly aromatized by aerial oxidation in ethanolic KOH under reflux to give 6,7-dimethylbenzo[g]quinoxaline-5,10-dione 82 [66,67]. The required intermediate 82 was treated with N-bromosuccinimide (NBS) and a catalytic amount of benzoylperoxide under irradiation to give the corresponding bisbromomethyl product in 42% yield. Target compounds 83 containing alkyl or cycloalkyl substituents were synthesized by direct substitution reaction of the bisbromomethyl compound with the corresponding alkylamine, and afforded alkyl-substituted triazacyclopenta[b]anthracene-5,10-dione derivatives in 30% to 81% yield (Scheme 29). Further reactions of compounds 82 with 18% HNO 3 in a Parr-type A-30397 titanium pressure reactor afforded the corresponding dicarboxylic acid 84. The crude product heated to reflux in SOCl 2 , and subsequent treatment of this crude with a number of alkyl-or arylamines, led to compounds 85a-j in 16% to 31% global yields (Scheme 30) [67]. Cycloadditions with cyclic dienes are expected to occur with formation of a 1:1-cycloadduct, followed by tautomerization and ready oxidation. This yields a bridged ring system which can undergo thermal elimination of ethylene to afford diazaanthraquinone [51]. Cycloadditions of 1,3-cyclohexadiene with the other heterocyclic quinones also yielded initial 1:1-cycloadducts 86, which were isolated directly from the reaction mixture. Conversion into the oxidized quinone with silver oxide and thermal elimination of ethylene afforded diazaanthraquinones in high yields (Scheme 31). The total synthesis of pyrazine analogs of 1,1-deoxydaunomycin was reported. Treatment of sodium salts generated from tetrahydrohomophthalic anhydride (Scheme 33) with derivative 76b gave cycloadducts 92 and 93, regioselectively [68]. These adducts were converted to pyrazine analogs of 1,1-deoxydaunomycin 94 and 95. Treatment of the sodium salts cited above with mercury(II) oxide and diluted sulfuric acid, followed by bromination with bromine and AIBN, gave cis-diol 92 in 27% yield. The respective configurations of 92 and 93 were determined from proton nuclear magnetic resonance (1H-NMR) spectral data [69]. Condensation of racemic 92 with tetrahydropyrane derivative followed by base hydrolysis gave α-glycosides 94 and 95 as an inseparable 1:1 mixture of two diastereomers in 48% yield (Scheme 33).

Antitumor Activities
Tricyclic diazaquinones exhibit strong anticancer activity [48]. One of the cytostatic action mechanisms of coplanar polycyclic compounds is their intercalation with human DNA. This caused enzymatic blockade and reading errors during the replication process [74]. Compounds having three to four coplanar rings, appear to give the optimal intercalation. More annelated heterocyclic quinones were reported to increase antitumor activity [75]. The electrochemical properties of quinone compounds are obviously very important for their bioreduction to semiquinone and/or hydroquinone. The replacement of two carbons at the phenyl ring of the 1,4-naphthoquinone core by two nitrogen atoms increased the oxidant nature of the molecules in accordance with both redox potential and substrate efficiencies [76].
2,3-Diethyl-5,10-pyrazino[2,3-g]quinoxalinedione 39d, prepared from 6,7-dichloroqui-noxaline-5,8-dione 37 in 59% overall yield in three steps [39] exhibited potent cytotoxic activity against human gastric adenocarcinoma cells (IC 50 = 1.30 and 7.61 µM, respectively). Both compounds bearing bulky side chains are supposed to interact with DNA and form a stable complex (Figure 2). Many heterocyclic quinones act as topoisomerase inhibitors via DNA-intercalation. Topoisomerases are DNA-modifying enzymes essential to the control of DNA topology. They are involved in all cellular processes (replication, transcription, chromatin condensation and recombination) in which the topology of the DNA molecule must be changed without changing its chemical structure. Some pyridophenazinediones [77] showed potent activity against human stomach cancer cells (Figure 3). The possible mechanism of this action is suggested to be a DNA topo I and topo II inhibition. The best representatives are compounds 101 (IC 50 = 0.06 µM on SK-OV-3), prepared from 6,7-dichloroquinoline-5,8-dione in 38% overall yield in two steps, and 102 (IC 50 = 0.06 µM on XF-498), prepared from 6,7-dichloroquinoline-5,8-dione in 23% overall yield in two steps. Giorgi-Renault prepared benzoquinoxalinediones and examined their antitumor activities [18]. 2,3-Bis(bromomethyl)-5,10-benzo[g]quinoxalinedione 6c, prepared from 2,2-diamino [1,4]-naphtoquinone 2 in 75% yield in one step, exhibited cytotoxicity and was highly active, especially against sarcoma ( Figure 4). The tumor suppressor p53 is a central mediator of apoptosis from chemically induced stress. Doxorubicin causes activation of p53 in both diploid and tetraploid cells due to a lack of polyploid cell line-specific selectivity. Recently, 2,3-diphenyl-1,4-diazaanthraquinone (DPBQ) 62 was proven to be a selective lead compound for the treatment of high-ploidy breast cancer, which activates p53 and triggers apoptosis of tumor cells ( Figure 5) [78]. This latter compound appears to be limited to high-ploidy cell types with intact p53. It does not inhibit topoisomerase or bind DNA. Mechanistic analysis demonstrates that DPBQ 54, obtained from the National Cancer Institute (NCI) elicits a hypoxia gene signature and its effect is replicated, in part, by enhancing oxidative stress. A number of 6,7-modified 5,8-quinoxalinedione derivatives containing nitrogen, sulfur and oxygen exhibited cytotoxic effects on human lung, gastric and colon adenocarcinoma cells when compared with cis-platin and adriamycin, commonly used anticancer drugs [79].
The cytotoxicity of 6,7-modified-5,8-quinoxalinedione derivatives and heterocyclic quinoxaline derivatives containing nitrogen (compounds were obtained from the NCI Open Compound Repository, Drug Synthesis and Chemistry Branch, NCI) was evaluated in vitro using an MTT assay on human lung adenocarcinoma cells (PC 14), human gastric adenocarcinoma cells (MKN 45), and human colon adenocarcinoma cells (colon 205). Pyrido [1,2-a]imidazo [4,5-g]quinoxaline-6,11-dione 44 was markedly cytotoxic against MKN 45 compared with adriamycin and cis-platin used as reference drugs. The IC 50 value of compound 44 was 0.073 µM while those of adriamycin and cis-platin were 0.12 µM and 2.67 µM, respectively. In this study, the relationship between structure, redox cycling, and cytotoxicity in the MCF-7 and HL-60 cell lines was investigated ( Figure 6) [80]. To evaluate their reactivity for bioreductive activation, the levels of free radicals under aerobic conditions were quantified. Direct ESR evidence of formation was provided. The data suggest that good levels of free radicals are generated in the HL-60 (e.g., the HL-60 myeloid leukemia) and MCF-7 cells (e.g., the MCF-7 breast carcinoma) by compound B. Group B compounds showed good redox cycling in both cell lines (HL-60 and MCF-7 cells). They were cytotoxic in the MCF-7 cell at concentrations down to 10 µM (Figure 7).

Conclusions
Despite the effort made to design benzo[g]quinoxalinedione compounds, there are few efficient synthetic approaches, especially green chemistry approaches. 1,4-diazaanthraquinones stand out because of their wide range of biological activities, including anti-cancer activity. However, exploration of these activities has been limited. These drugs offer a larger repertoire of activities in cancer cells than is currently exploited. The heterocyclic quinones that appeared to be very promising in vitro proved ineffective in studies. One reason for this failure is insufficient understanding of the mechanisms of action of these compounds, in part due to the numerous modes of action of these families of derivatives. The versatility of heterocyclic quinones containing a quinoxaline core and their potential to be selectively toxic to tumor cells hold great promise for anticancer therapy.