Synthesis and antimycobacterial activity of analogues of the bioactive natural products sampangine and cleistopholine

European Journal of Medicinal Chemistry 67 (2013) 98e110

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Original article

Synthesis and antimycobacterial activity of analogues of the bioactive natural products sampangine and cleistopholine Pieter Claes a,1, Davie Cappoen b,1, Blaise Mavinga Mbala a, Jan Jacobs a, Birgit Mertens d, Vanessa Mathys c, Luc Verschaeve d, e, Kris Huygen b, *, Norbert De Kimpe a, ** a

Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium Scientific Service Immunology, O.D. Communicable & Infectious Diseases, Scientific Institute of Public Health (Site Ukkel), Engelandstraat 642, B-21180 Ukkel, Belgium c Program Tuberculosis and Mycobacteria, O.D. Communicable & Infectious Diseases, Scientific Institute of Public Health (Site Ukkel), Engelandstraat 642, B-1180 Ukkel, Belgium d Program Toxicology, O.D. Public Health and Surveillance, Scientific Institute of Public Health (Site Elsene), J. Wytsmanstraat 14, B-1050 Brussels, Belgium e Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 April 2013 Received in revised form 5 June 2013 Accepted 7 June 2013 Available online 18 June 2013

Identification and investigation of novel classes and compounds for the treatment of tuberculosis remains of utmost importance in the fight against the disease. Despite many efforts, the weakly gram positive Mycobacterium tuberculosis keeps demanding its toll in human lives. For this reason a small library of substituted and unsubstituted aza analogues of cleistopholine and sampangine were synthesized in a short and straightforward manner and tested in vitro against M.tb. The compounds showed promising activity against the M.tb H37Rv strain and Minimal Inhibitory Concentrations (MIC) could be observed as low as 0.88 mM. Accompanied by moderate acute toxicity against C3A hepatocytes, the therapeutic index showed an acceptable range. Further tests confirmed the inhibition by up to 74% of intracellular growth of M.tb inside macrophages conferred by1-hydroxybenzo[g]isoquinoline-5,10diones. Activity of the library against other clinically relevant mycobacterial species such as Mycobacterium bovis, Mycobacterium avium and Mycobacterium ulcerans was confirmed. Furthermore the activity against a multi-drug-resistant MDR LAM-1 M.tb strain was tested and the MIC value situated around 1 mM. The lacking genotoxicity of a group of enamine substituted cleistopholine analogues indicates this group as a hit and encourages their use as a scaffold for further studies. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Sampangine Cleistopholine Pyridinium ylids Tuberculosis Antibiotics Acute toxicity

1. Introduction Despite the progress made in diagnostics, hygiene and overall welfare, tuberculosis (TB) remains a major challenge to healthcare worldwide. In 2012 Mycobacterium tuberculosis (M.tb) maintained its status as a notorious killer with 1.4 million casualties worldwide [1]. Close hostepathogen co evolution enabled M.tb to evolve in a highly resilient organism with a high tolerance for xenobiotics effective against most other bacteria. The reason for this high tolerance can partly be attributed to the unique lipid-rich

* Corresponding author. Tel.: þ32 (0)2 373 33 70; fax: þ32 (0)2 373 33 67. ** Corresponding author. Tel.: þ32 (0)9 264 59 51; fax: þ32 (0)9 264 62 43. E-mail addresses: [email protected] (K. Huygen), norbert.dekimpe@ ugent.be (N. De Kimpe). 1 Both authors contributed equally to the paper. 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.06.010

mycobacterial cell envelope. This protective barrier originated to block the effector molecules produced by the host’s cellular immune response, but also protects M.tb adequately against a broad range of antibiotics [2]. M.tb is an intracellular pathogen that mostly resides inside macrophages, evading enzymatic degradation by preventing phagosomeelysosome fusion. To be effective, antibiotics have to reach the early endosome in which the M.tb bacilli reside and replicates. Evolution has enabled M.tb to adopt a protective metabolic mechanism fit for the harsh conditions within the host. As a result, part of the mycobacterial population inside the host shifts to a metabolic quiescence. Ultimately forming persister cells against which no known drug is active [3]. Treatment of tuberculosis requires a long chemotherapy composed of multiple antibiotics. As a poverty disease, the requirements for efficient treatment become compromised in settings such as third world countries, where healthcare is less obvious.

P. Claes et al. / European Journal of Medicinal Chemistry 67 (2013) 98e110

and 2-aminomethyl-2-methyl-1,3-dioxolane 8 [12b] (Scheme 1). Subsequent acid induced ring closure with sulfuric acid in dichloromethane and spontaneous oxidation yielded cleistopholine analogue 10 in a 50% yield. Next, cleistopholine analogue 10 was reacted with 5 equivalents of N,N-dimethylformamide dimethyl acetal (DMF-DMA) in DMF at 125  C, yielding (E)-4-[2(dimethylamino)vinyl]benzo[g]isoquinoline-5,10-dione 11 in 86% yield. Then, this enamine 11 was reacted with an excess of ammonium chloride in boiling acetic acid as described in the synthesis of sampangine 3 [8]. However, a complex reaction mixture was obtained. After testing several reaction conditions, (E)-4-[2(dimethylamino)vinyl]benzo[g]isoquinoline-5,10-dione 11 was converted in excellent yield towards unsubstituted sampangine analogue 12, i.e.7H-naphtho[3,2,1-i,j]-2,6-naphthiridin-6-one, by heating with ammonium acetate in methanol. Next, the synthesis of substituted sampangine analogues 17 was envisaged starting from 3-substituted-1-methylbenzo[g]isoquinoline-5,10-diones 15. The latter were prepared by applying a microwave protocol previously developed at our research department for the synthesis of 1-hydroxydbenzo[g]isoquinoline-5,10-diones 19 [13]. This reaction involved the Michael addition of the appropriate Pyridinium ylids across the enone moiety followed by elimination of pyridine to generate the intermediate 3-acylmethylquinones which underwent cyclization with ammonia to provide tricyclic compounds 19. Thus, 2-acetyl-1,4-naphthoquinone 13 and different pyridinium salts 14 were irradiated for 6 min in a 5% (w/v) solution of ammonium acetate in methanol forming 1-methylbenzo[g]isoquinoline-5,10-diones 15 in 38e67% yield. Subsequently, these 2azaanthraquinones 15 were reacted with an excess of DMFeDMA in DMF at 125  C for 15 h to yield 3-substituted-1-[2-(dimethylamino)vinyl]benzo[g]isoquinoline-5,10-diones 16 in good to excellent yields with the exception of 3-isopropyl-1-[2-(dimethylamino) vinyl]benzo[g]isoquinoline-5,10-dione 16g which was not accessible due to the formation of complex reaction mixture. Boiling under reflux of 1-[2-(dimethylamino) vinyl]benzo[g]isoquinoline5,10-diones 16 in a 5% (w/v) solution of NH4OAc in methanol afforded 5-substituted-7H-naphtho[3,2,1-de]naphthyridine-7-ones 17 in 64e95% yield (Scheme 2). In order to obtain a clear view of the biological activity of 2azaanthraquinones 10 and 15, the known 1-methylbenzo[g]isoquinoline-5,10-dione 18 [14] and 1-hydroxy benzo[g]isoquinoline5,10-diones 19 [11] were included in the screening as well (Scheme 3).

Although combination chemotherapy can be very effective, the limitation of antibiotics and their inadequate use has led to the generation of multi-drug-resistant strains. MDR strains are at least resistant to isoniazid and rifampicin, while extensively drugresistant (XDR) strains show additional resistance to fluoroquinolones and injectable drugs [4]. We have focused our attention on a series of analogues of the natural products cleistopholine 1 and sampangine 3 which are two strongly related polycyclic aromatic alkaloids isolated from different plants belonging to the Annonaceae family with a wide range of interesting biological activities [5]. Cleistopholine 1 is a tricyclic 1-azaanthraquinone alkaloid isolated from the root bark of Cleistopholis paten [6a] (Fig. 1). Sampangine 3 is a tetratracyclic naphthyridine alkaloid isolated from the stem bark of Cananga odorata [6b]. Cleistopholine 1 showed fungitoxic activity against Candida albicans and Cryptococcus neoformans, which are opportunistic fungi in AIDS patients [5e]. In addition to exhibiting a powerful activity against Mycobacterium intracellulare with a minimum inhibitory concentration (MIC ¼ 0.78 mg/ml) which is lower than the MIC for current anti-TB drugs as rifampicin (MIC ¼ 0.5e 0.9 mg/ml) and streptomycin (MIC ¼ 2e8 mg/ml) [7], sampangine 3 is known to possess strong antifungal activity [5d]. Synthetic analogues 2, 4 and 5 showed strong activity against M. intracellulare. Ascididemin 6, a metabolite from the marine tunicate Didemnum sp. is very active against the rapidly growing Mycobacterium aurum A þ strain [7]. In the literature, a straightforward synthesis of sampangine 3 starting from cleistopholine 1 has been reported [8]. Since it is known that 2-azaanthraquinones are more bioactive than their corresponding 1-aza analogues [9a], a short and efficient synthesis of 2-aza analogues of cleistopholine 1 and sampangine 3 was performed. Even though numerous analogues have been synthesized with the nitrogen atom at the 1-position [5d,10,16], these analogues with the nitrogen atom at the 2-position have not been reported yet in the literature. 2. Results and discussion 2.1. Synthesis of the target compounds Initially, the synthesis of an unsubstituted aza analogue of sampangine 3 was envisaged, starting from cleistopholine analogue 10. This 4-methylbenzo[g]isoquinoline-5,10-dione 10 was previously obtained by an intramolecular Heck reaction but the yield was low and large amounts of palladium(II) acetate were necessary [11]. Therefore, an alternative route using a PomeranzeFritsch reaction was developed. Thus, amine 9 was synthesized by means of a reductive amination of 2-formyl-1,4-dimethoxynaphthalene7 [12a]

O

R

2.2. Biological activity assays In previous publications, we and others have shown that luminometry is a rapid and reproducible tool to test the antimycobacterial

R3

N

N R2

N

N O 1 R = Me Cleistopholin MIC = 12.5 µg/ml 2 R = Et MIC = 12.5 µg/ml

99

Z

R1

O 3 Sampangin: R1 = R2 = R3 = H, MIC = 0.78 µg/ml 4a R1 = R3, R2 = Br, MIC = 3.12 µg/ml 4b R1 = OEt, R2 = Br, R3 = Br, MIC = 25 µg/ml 4c R1 = R3, R2 = Cl, MIC = 3.12 µg/ml 4d R1 = R3, R2 = OCH3, MIC = 3.12 µg/ml 4d R1 = R2, R3 = OCH3, MIC = 1.56 µg/ml 4e R1 = R2, R3 = CH3, MIC = 0.39 µg/ml

N O 5 Z = CH MIC = 0.39 µg/ml 6 Ascididemin: Z = N MIC = 0.25 µg/ml

Fig. 1. Structure and activity of natural and synthetic alkaloids against M. intracellulare (1e5) and M. aurum (6).

100

P. Claes et al. / European Journal of Medicinal Chemistry 67 (2013) 98e110

Scheme 1. Synthesis of 7H-naphtho[3,2,1-i,j]-2,6-naphthiridin-6-one 12.

activity of novel antituberculous compounds [15]. Screening the present library of compounds for in vitro activity against M.tb, was done with luminescent M.tb H37Rv laboratory strain (H37Rvlux). The H37Rvlux strain has multiple copies of pSMT1 plasmid carrying the necessary genes to express the ATP independent bacterial luciferase of Vibrio harveyi driven by a constitutive hsp60 promoter. Addition of the luciferase substrate n-decanal, elicits a luminescent signal of green-blue light (490 nm) which can be measured by luminometry. The influence of compounds or other xenobiotics on the bacillary growth can be measured as a reduction in the intensity of the luminescent signal. A preliminary screen of compounds 10e12, 15aeh, 16aef, h, 17aef, h, 18, 19aed, f (Table 1) was performed at concentrations of 10 mM, 1 mM and 0.1 mM. The criterion to withhold compounds as

potential hits was a threshold of 90% M.tb growth reduction at 1 mM after 6 days exposure. Among the 1-[2-(dimethylamino)vinyl] benzo[g]isoquinolone-5,10-diones 16, 7H-naphtho[3,2,1-de]naphthyridine-7-ones 17 and 1-hydroxybenzo[g]isoquinolone-5,10diones 19 several hits were detected. No hits were found within the 1-methylbenzo[g]isoquinolone-5,10-diones 15 and 18 and this group was excluded in further studies. The phenyl substitution at position 3 showed some importance for the efficacy of the compound as the activity was significantly increased for these compounds. No further increase in potency could be detected by further substitution of the phenyl group. Some reports on analogous compounds containing a 2-(dimethylamino)vinyl substituent suggested that their activity could be attributed to in vivo hydrolysis of the dimethylaminovinyl substituent to the aldehyde [16]. However, compounds 16 were found to be very resistant to acidic hydrolysis and were bench-stable for over one year. Derivatives with a 2,5dimethoxyphenyl substituent were markedly reduced in activity against the H37Rvlux M.tb lab strain. Comparing the bioisosteres 10 and 18, the inhibition of M.tb growth by either was comparable, which leads to the conclusion that the position of the nitrogen atom in the heterocyclic system had no influence on the activity. The minimal inhibitory concentration was determined using serial dilutions of the investigated compounds (Table 2). The same method described above was used and the concentration at which at least one log10 growth reduction was detected, as compared to the untreated control cultures, was accepted as the MIC. To calculate the therapeutic index, the concentration at which a growth

O

O

OH

N R O

O 19a R = C6H5 19b R = 4-MeOC6H4 19c R = 4-FC6H4 19d R = 4-ClC6H4 19g R = 4-MeC6H4

18

Scheme 2. Synthesis of 5-substituted-7H-naphtho[3,2,1-de]naphthyridine-7-ones 17.

Scheme 3.

P. Claes et al. / European Journal of Medicinal Chemistry 67 (2013) 98e110 Table 1 M.tb growth inhibition at 10 mM, 1 mM and 0.1 mM of the compounds. Compound

10 11 12 15a 15b 15c 15d 15e 15f 15g 15h 16a 16b 16c 16d 16e 16f 16h 17a 17b 17c 17d 17e 17f 17h 18 19a 19b 19c 19d 19g INHb RIFc

R

e e e C6H5 4-MeOC6H4 4-FC6H4 4-ClC6H4 2,5-(MeO)2C6H3 4-MeC6H4 i-Pr t-Bu C6H5 4-MeOC6H4 4-FC6H4 4-ClC6H4 2,5-(MeO)2C6H3 4-MeC6H4 t-Bu C6H5 4-MeOC6H4 4-FC6H4 4-ClC6H4 C6H5C3H5 4-MeC6H4 t-Bu e C6H5 4-MeOC6H4 4-FC6H4 4-ClC6H4 4-MeC6H4

101

Table 2 Inhibitory concentrations and calculated therapeutic index of the selected compounds.

M.tb growth inhibition %a 10 mM

1 mM

0.1 mM

48 53 62 98 100 100 100 86 99 54 51 100 100 100 100 87 100 73 100 100 100 99 87 100 73 58 100 100 100 100 98 100 100

11 17 25 66 70 68 68 35 63 24 18 100 100 92 91 47 100 53 95 99 100 99 75 100 41 22 88 92 91 90 35 100 100

<10 <10 11 21 32 41 42 <10 54 <10 <10 45 42 35 34 35 39 21 67 81 53 77 39 73 32 10 15 35 19 34 21 99 99

a Growth inhibition as compared to the untreated culture, measured after 6 days (mean of triplicate cultures, SD <10%). b INH, the positive control isoniazid. c RIF rifampicin.

inhibition of 50% of the bacillary strain (GI50) was obtained, was determined as well. Acute toxicity of the compounds was examined in a Neutral Red Dye Uptake assay (NRU) [17]. This test method makes use of the ability of living cells to bind and incorporate neutral red in their lysosome structures. C3A hepatocytes were selected as a cellular model since hepatotoxicity is a problem often encountered in chemotherapy and M.tb drug development. Toxicity is measured as reduced uptake of the neutral red dye by the C3A culture and the NI50 is determined as the concentration at which this reduction reaches 50%. The therapeutic Index (TI) was calculated as a function of the NI50 divided with the GI50. 1Hydroxybenzo[g]isoquinolone-5,10-diones 19 exerted the highest acute toxicity on the hepatocytes with NI50 concentrations, varying from 1.87 mM for the methoxy substituted derivative 19b to 10.83 mM for the unsubstituted derivative resulting in low TI for compounds 19aed. Higher TI values were found for the group of enamine substituted benzo[g]isoquinolone-5,10-diones 16 with a TI of 92.5 for 1-[2-(dimethylamino)vinyl]-3-(4-methoxyphenyl) benzo[g]isoquinoline-5,10-dione 16b and phenyl substituted naphthyridine compounds 17. To avoid any influence of DMSO on the cellular growth, the maximum concentration of DMSO in the cultures did not exceed 1%. For this reason the maximum concentration of the compounds tested in the neutral red uptake assay was 50 mM. For compounds 17b, 17c and 17d this was not high enough to reduce the cellular viability with 50% so the Ni50 could not be determined. Macrophages are the most important target cell for M.tb infection [18]. For most of its infectious cycle, M.tb resides within the

Compound R

MICa (mM) GI50b (mM) NI50c (mM) TI ¼ NI50/GI50d

16a 16b 16c 16d 16f 17a 17b 17c 17d 17f 19a 19b 19c 19d INHe RIFf

1.4 0.88 0.88 1.1 1.5 1.2 0.92 1.5 1.85 1.31 2.12 0.92 1.98 1.1 0.09 0.12

C6H5 4-MeOC6H4 4-FC6H4 4-ClC6H4 4-MeC6H4 C6H5 4-MeOC6H4 4-FC6H4 4-ClC6H4 4-MeC6H4 C6H5 4-MeOC6H4 4-FC6H4 4-ClC6H4

0.52 0.36 0.41 0.55 0.69 0.55 0.37 0.69 0.72 0.43 0.86 0.45 0.77 0.58 0.01 0.03

24.4 33.3 37.3 18.8 27.9 21.5 >50 >50 >50 7.14 10.83 1.87 9.82 9.07 N.D. N.D.

46.9 92.5 91.0 34.2 40.4 39.1 >135.1 >72.4 >69.4 16.6 12.6 4.2 12.8 15.6 N.D. N.D.

a The minimal inhibitory concentration at which 90% growth reduction is observed of M.tb. b The concentration at which a 50% growth reduction could be observed. c The concentration at which the viability of C3A hepatocytes is reduced by 50%. d Therapeutic Index calculated as NI50/GI50. e INH, the positive control isoniazid and. f RIF rifampicin.

early endosome of the macrophages where it replicates. Therefore it is important to investigate if the synthesized cleistopholine and sampangine analogues are able to reach their target through the membrane structure of the macrophages. In the macrophage infection assay setup, cells were first infected with M.tb H37Rvlux. After 24 h of infection, the infected J774 murine macrophages were treated with the compounds for 4 days. Treatment was followed by washing and subsequent lysis of the J774 monolayer. The luminescence emitted from the cell lysate was compared with infected macrophages which received no treatment. If the bacterial load inside the cells was diminished it was concluded that the compound was able to reach its target and kill it. As displayed in Table 3, the compounds were indeed able to reduce the bacterial load inside the macrophages and at 2 mM displayed inhibitory properties on the growth that were satisfactory compared with the results obtained with INH. 1-Hydroxybenzo[g]isoquinolone-5,10-diones 19aed, showed a higher potency than the group of 1-[2-(dimethylamino) vinyl]benzo[g]isoquinolone-5,10-diones 16aef and sampangine analogues 17a and 17b. Cell viability of the macrophages was monitored by trypan blue coloration of control groups. Next, the susceptibility of other clinically relevant mycobacterial species was tested for the three groups of compounds. Mycobacterium bovis, together with Mycobacterium africanum and Mycobacterium microti, constitute the M. tuberculosis complex [19]. Mycobacterium avium subsp. ovum is pathogenic for birds but can cause opportunistic infections in AIDS patients, whereas Mycobacterium avium subsp. paratuberculosis is the etiological agent of Johne’s disease or paratuberculosis (in ruminants) which may be linked to Crohn’s disease in humans [20]. Mycobacterium ulcerans ultimately causes Buruli ulcer, a necrotizing skin disease affecting mostly children in certain rural areas of West-Africa that may lead to irreversible disabilities when left untreated [21]. M. bovis and M.tb showed comparable susceptibility to all compounds. However, increased concentrations of compounds 16, 17 and 19 were needed to inhibit the growth of both M. avium subspecies. M. ulcerans showed the highest tolerance and concentrations close to 10 mM were needed to reduce the growth by 50% (Table 4). To extend the study, antimicrobial activity against a multi-drugresistant M.tb isolate was determined using the radiometric BACTEC 460 TB method (Fig. 2). This standardized radiometric assay

102

P. Claes et al. / European Journal of Medicinal Chemistry 67 (2013) 98e110

Table 3 The inhibition of intracellular growth of M.tb inside macrophages by compounds 16, 17 and 19. Cmpd.

R

Intracellular growth inhibition % 2 mM

16a 16b 16c 16d 16f 17a 17b 17c 17d 17f 19a 19b 19c 19d INHa

C6H5 4-MeOC6H4 4-FC6H4 4-ClC6H4 4-MeC6H4 C6H5 4-MeOC6H4 4-FC6H4 4-ClC6H4 4-MeC6H4 C6H5 4-MeOC6H4 4-FC6H4 4-ClC6H4

49.5 47.1 60.5 54.4 48.7 48.2 51.5 65.5 44.2 47.2 68.3 42.3 74.7 51.2 97.3

              

1 mM 3.4 5.2 1.3 1.9 2.6 6.9 3.7 2.0 1.8 5.5 0.7 5.4 2.3 6.7 1.6

34.6 26.3 27.3 35.6 36.2 32.6 38.5 30.2 20.3 34.6 49.0 27.6 59.1 44.1 92.8

              

0.5 mM 6.9 0.8 1.8 1.0 0.5 0.7 1.8 8.0 6.9 5.8 3.2 8.0 2.7 7.5 2.7

13.4 9.3 10.2 19.8 15.9 14.6 10.0 15.3 2.27 7.0 36.6 21.6 45.7 19.7 76.4

              

6.8 4.1 6.0 7.6 8.9 0.2 5.5 5.3 1.9 3.5 4.7 5.2 3.9 4.2 5.3

% Growth inhibition of M.tb H37Rv lab strain inside J774 macrophages mean  SD of triplicate cultures. Treatment of the infected cells started 24 h postinfection and lasted for 4 days. a INH, isoniazid was used as a positive control.

relies on the ability of mycobacteria to produce 14CO2 from palmitic-(carboxy-14C) acid. The quantity of 14CO2 expelled by the bacteria is measured and expressed as the growth index (G.I.), a measure for the bacterial growth [22]. The MIC90 and MIC99 of a compound were deduced by comparing the G.I. of the M.tb culture exposed to compound concentration with the G.I. of an untreated control culture which is diluted 10 times (C/10) and 100 times (C/ 100), respectively. As a resistant M.tb strain, an LAM-1 clinical isolate was selected [23]. This MDR M.tb strain shows resistance against isoniazid, rifampicin, rifabutine and prothionamide. The susceptibility to compounds 16b, 17b and 19b was tested at concentrations of 5 mM, 1 mM and 0.5 mM, respectively. A growth inhibition of 90% and of 99% could be observed for all three compounds at 1 mM and 5 mM, respectively. Early signs of genotoxicity can be observed by studying the activation of DNA repair mechanisms. The regulatory SOS operon is key to the repair of early cellular DNA damage and its activation is mediated by a multiple component system. As a result of DNA interruption, binding of ssDNA fragments to the recA protein increases the enzymatic affinity for the lexA repressor protein. The decrease in lexA frees the SOS box consensus sequence and thereby facilitates the binding of the general transcription machinery to the recN promotor sequence and transcription of the SOS operon. Based

on this molecular mechanism, the VITOTOX model from Gentaur can detect activation of the SOS operon by an integrated lux operon. The model employs two recombinant Salmonella typhimurium reporter strains. The integrated lux operon of the Genox strain T104 (recN2-4) is expressed under transcriptional control of the recN promoter and in the Cytox pr1 strain it is driven by the strong constitutive pr1 promotor, the latter strain mainly functioning as an internal control. Both recombinant reporter strains lack the necessary oxidative machinery to metabolize xenobiotics. By adding S9 liver fraction to genotoxicity of the investigated substance can be distinguished from that of its metabolites [23]. Closely correlated with the AMES test (91%), with a high specificity of 94%, this model allows an early elimination of novel compound classes due to genotoxicity [24]. Emission of luminescence by the strains was recorded in real time after the addition of the compounds 16b, 17b and 19b, every five minutes during a 4-h period. Compounds were tested at 10 mM, 3 mM, 1 mM, 0.3 mM and 0.1 mM in the presence and absence of the metabolizing S9 fraction. Genotoxicity was deduced from the signal to noise ratio (S/N), being the light produced by the bacterial suspension exposed to the compounds divided by the light produced by a non-exposed bacterial suspension. S/N values for the Genox (recN2-4) higher than 1.5 were interpreted as positive and implied genotoxic properties by the compounds. For the Cytox (pr1) strain, S/N values above 1.5 are considered as direct influence of the compound on the bacterial luciferase or the luminescent signal itself and regarded a false positive. S/N values lower than 0.8 for the Cytox strain are indicative of direct cytotoxicity and exclude false negatives. As shown in Fig. 3, compounds 16b, 17b and 19b resulted in values of the Cytox control strain between 0.8 and 1.5 with and without the S9 liver extract. The luminescence emitted from the Genox strain did not exceed S/N values of 1.5 after addition of the compounds both in absence and presence of S9 extract. Hence it was concluded that there was no activation of the SOS operon neither by the compounds nor by their metabolites. Further study of the genotoxic properties was performed for compounds 16b, 17b and 19b using a COMET assay [25]. With this assay, fragmentation of the genomic DNA can be detected. Briefly, C3A cells were exposed to a compound concentration and immobilized onto agarose. Immobilized cells were lysed and the remaining nuclei were submitted to electrophoresis. Through pore exclusion of the agarose, fragmented DNA migrated further with the applied current. After staining with gel red it can be observed that the genomic DNA of an undamaged nucleus maintains its spherical organization (HEAD) whereas fragmented DNA migrates further across the gel (TAIL). The ratio DNA in the TAIL/DNA in the

Table 4 The 50% in vitro growth inhibitory concentration of the derivatives against other Mycobacteria. Compound

R

16a 16b 16c 16d 16f 17a 17b 17c 19a 19b 19c 19d

C6H5 4-MeOC6H4 4-FC6H4 4-ClC6H4 4-MeC6H4 C6H5 4-MeOC6H4 4-FC6H4 C6H5 4-MeOC6H4 C6H5F C6H5Cl

M. tuberculosis H37Rvlux

M. bovis AN5lux

M. avium ssp paratuberculosis ATCC 15769lux

M. avium ssp avium ATCC 19698lux

M. ulcerans 1615lux

0.64 0.47 0.48 0.49 0.58 0.67 0.52 0.67 0.80 0.38 0.85 0.65

3.68 3.06 3.23 3.95 4.83 3.21 2.54 3.56 4.71 3.52 5.52 4.72

4.89 2.98 3.65 3.88 3.69 4.22 3.56 3.98 5.02 3.01 4.25 3.89

8.65 7.65 7.49 7.69 8.59 10.56 8.56 9.23 12.56 7.55 8.56 9.54

50% growth inhibition (mM) 0.52 0.36 0.41 0.55 0.69 0.55 0.37 0.69 0.86 0.45 0.77 0.58

In vitro growth inhibition was measured by luminescence after 6 days exposure to the compounds. Inhibition calculated from triplicates cultures (SD values <10%).

P. Claes et al. / European Journal of Medicinal Chemistry 67 (2013) 98e110

A

B

103

C

Fig. 2. Susceptibility of an MDR LAM-1 M.tb strain for the derivatives. Growth of MDR M.tb was monitored in BACTEC 460 TB. Resistance was confirmed with isoniazid (INH). Growth was compared to bacterial suspensions diluted 10 times (C/10) and 100 times (C/100) to study the MIC.

HEAD is a measure of DNA fragmentation due to the genotoxicity of the compounds for the C3A hepatocytes. At 1 mM and 3 mM, no DNA fragmentation was detected in the C3A cells exposed to the compounds as summarized in Fig. 4. However, at 10 mM there was a significant increase in genomic DNA damage for compound 19b and compound 17b (P < 0.0001 Manne Whitney test). This was not the case for 1-[2-(dimethylamino)vinyl]-3-(4-methoxyphenyl) benzo[g]isoquinoline-5,10-dione 16b, for which no significant increase of DNA in the tail was measured. It must be pointed out, however, that for compound 19b, there was also significant toxicity within the range of 10 mM what renders the result by the COMET assay inconclusive for this compound. 3. Conclusion Recent success and breakthrough in the development M.tb therapeutics are most promising. However, history has taught us the importance of perseverance in compound research and the investigation of novel compounds to reduce tuberculosis. Here, we report on the screening of a library of sampangine and cleistopholine analogues for their ability to inhibit the in vitro growth of M.tb. Some compounds showed a high potency towards M.tb. This observation combined with low acute toxicity resulted in favorable therapeutic indices. Although initially genotoxicity was not observed by the VITOTOX, a significant increase in DNA fragmentation caused by compounds 19b and 17b was noticed by the COMET assay at concentrations of 10 mM. This was not the case for compound 16b for which no significant increase in DNA fragmentation was detected. The different outcome in both assays can be due to the choice of model organism and its organization of genomic DNA (prokaryotic versus eukaryotic). The time of exposure differs as well, 4 h in the case of the VITOTOX versus 24 h with the

COMET assay. One possibility could be that the genotoxicity caused by the compounds simply does not activate the regulatory SOS operon. All compounds tested showed stronger antimycobacterial activity against M.tb and M. bovis than against both M. avium subspecies and M. ulcerans. This could be attributed to the close phylogenetic relationship between M.tb and M. bovis but an increasing distance with both the M. avium species and finally M. ulcerans. Therefore, the unknown bacillary target could be more closely conserved between M.tb and M. bovis. Promising bacillary growth inhibition could be observed when macrophages infected with M.tb were subsequently treated with the compounds, indicating that the compounds are able to reach the early endosomes to target M.tb. Furthermore, the ability to inhibit the MDR LAM-1 confirmed the potency against MDR M.tb. Although further in vitro and in vivo studies are needed to confirm this hit compound, the findings in this paper are promising. 1-[2(Dimethylamino)vinyl]-3-(4-methoxyphenyl)benzo[g]isoquinoline-5,10-dione 16b with an observed M.tb GI50 of 0.36 mM and a NI50 of 33.3 mM against C3A cells showed an acceptable therapeutic index of 92. The lack of genotoxicity observed in our assay encourages us to promote this compound as a hit and to use it as a scaffold for further investigation. 4. Experimental part 4.1. Biological data 4.1.1. Materials 7H9 growth medium was purchased as a powder from BD Science (Franklin Lakes, NJ, USA). DMEM, glutamax, non-essential amino acids, sodium pyruvate, gentamycin, 2-mercaptoethanol,

Fig. 3. Detection of early signs of genotoxicity of compounds 16b, 17b and 19b by VITOTOX. Maximum recorded S/N in a time span of 4 h by the Genox (recN2-4) and Cytox (pr1) reporter strains. 4NQO; 4-nitroquinoline-1-oxide genotoxic positive control in samples without S9 liver fraction. Bap; benzo[a]pyrene, the positive control, only turns genotoxic after S9 metabolization.

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Fig. 4. Detection of DNA fragmentation by COMET assay. A. Genomic DNA fragmentation after 24 h exposure to different concentrations of compounds 16b, 17b and 19b. Manne Whitney was used to test the significance at P < 0.0001. B. Nuclei of C3A cells, lysed after exposure and stained with gel red.

PBS were from GIBCO Invitrogen (Carlsbad, CA, USA). OADC, mycobactin J, penicillin, fungizone and hygromycin were from Roche (Basel, CH) and Triton X-100, glycerol, Tween 80 and ndecanal were purchased from Sigma Aldrich (St. Louis, MO, USA).

(Difco). To measure the luminescence, 100 mL of 1% n-decanal in ethanol was added to the Eppendorf tube and light emission was measured over 10 s using a Turner Modulus Single Tube Luminometer from Biosystems.

4.1.2. Strains and growth conditions M. tuberculosis H37Rv (American Type Culture Collection 27294) is known to be sensitive to the five first line anti-tuberculosis drugs (streptomycin, isoniazid, rifampin, ethambutol and pyrazinamide). The LAM-1 strain is a clinical isolate from a patient diagnosed with TB. This strain has been spoligotyped for identification and characterized for antibiotic resistance by the national TB reference lab of the Institute of Public Health of Belgium. With resistance to isoniazid, rifampicin, rifabutin and prothionamide this strain was classified as a multi-drug-resistant (MDR) strain [15b]. Four other strains used for screening were M. bovis strain AN5, M. avium ssp. avium ATCC 15769, M. avium ssp. paratuberculosis ATCC19698 and M. ulcerans 1615. All strains were cultivated in 7H9 medium supplemented with 10% oleic acidealbuminedextroseecatalase (OADC) and 0.2% glycerol. For M. bovis, the glycerol was replaced with 0.05% Tween 80. The growth medium of M. avium ssp. paratuberculosis was further supplemented with mycobactin J (2 mg/ml) required for optimal in vitro growth. Both M. avium strains and the M. bovis strain were grown at 39  C, M. ulcerans was grown at 32  C and M.tb was grown at 37  C.

4.1.4. Activity against multi-drug-resistant M. tuberculosis Antimicrobial activity of the compounds was tested using the LAM-1 strain using the BACTEC 460 TB detection system [15b]. The BACTEC system relies on the unique capability of mycobacteria to metabolize palmitic acid expelling CO2. By using radioactively labeled palmitic acid, 14CO2 expelled in the gaseous phase and is measured in a Beta counter and interpreted as a measurement of bacillary growth inside the BACTEC tubes [15b]. The compounds were solubilized in DMSO at stock concentrations of 10 mM. Serial dilutions of each compound were made in 7H9 containing 10% OADC, at 40-fold the final concentrations. M.tb LAM-1 was precultured in a 4 ml BACTEC vial to a growth index (GI) of 300. Then 100 mL of this pre-culture was inoculated into a new 4 ml BACTEC vial together with 100 mL of the serial dilutions of the compounds. As a positive control for resistance, the MDR LAM-1 culture was inoculated with 0.1 mg/ml isoniazid. The GI was measured each day. To determine the MIC90, the cultures, treated with the compounds were compared with an untreated culture.

4.1.3. Monitoring mycobacterial growth by luminometry The minimal inhibitory concentration (MIC) against mycobacteria of all synthesized compounds was evaluated by testing serial dilutions. The in vitro assay was based on a method in which luminescent mycobacteria transformed with pSMT1 luciferase reporter plasmid is used. The synthetic compounds were solubilized in DMSO (SigmaeAldrich) at stock concentrations of 10 mM. Serial dilutions of each compound were made in liquid 7H9 medium [Middlebrook 7H9 broth based (Difco)] þ 10% FCS (Gibco). Volumes of 20 mL of the serial dilutions were added in triplicate to 96-well, flat-bottomed microwell plates. The bacterial suspension was made by thawing and dissolving a frozen Mycobacteria pellet in 7H9-10% FCS. The dissolved pellet was passed through a 5.0 mM filter (Millipore) to eliminate clumps and left for 1 h to recover at 37  C, 5% CO2. Next, the bacterial suspension was diluted in 7H9-10% FCS to obtain 50,000 Relative Light Units (RLU)/ml and a volume of 180 mL of bacteria was added to each well. Bacterial replication was analyzed by luminometry after 6 days of culture. The bacterial suspension from each well was collected, and brought in a 2.5 ml Eppendorf tube. Each well was washed four times with 200 mL PBS

4.1.5. Inhibition of intracellular M.tb growth The compounds were tested on the murine J774.A1 macrophage cell line infected with M.tb H37Rvlux. The J774 macrophages were grown at 37  C, 5% CO2 in complete DMEM medium until a semiconfluent layer was formed. The macrophages were washed in fresh complete DMEM medium and seeded in a flat-bottomed 96well microtiter plate at a cell density of 40,000 cells per well. The cells were left to recover overnight and were washed three times in complete DMEM medium. M.tb H37Rvlux was grown at 37  C in 7H9 containing 10% FCS and 0.2% hygromycin to an OD580 of 0.6e1.0. The fully-grown bacterial suspension was measured and brought into complete DMEM-Pen/Fung [DMEM medium containing 0.1% penicillin and 0.8% fungizone but without gentamicin]. The synthesized compounds were solubilized in DMSO at stock concentrations of 10 mM. Serial dilutions of the peptides were made in DMEM-Pen/Fung at two times the concentration of each compound to be tested. A volume of 100 mL of the bacterial suspension in DMEM-Pen/Fung containing 4000 RLU of bacteria (multiplicity of infection of 0.1) and 100 mL of the serial compound dilutions were added to the macrophage cultures. To measure the effects of the compounds on intracellular growth of M.tb, the infected macrophages were washed three times on day 5 to remove all

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extracellular bacteria, incubated 1 h with 1% gentamicin to kill residual extracellular bacteria, lysed with 200 mL 1% Triton X-100 (Sigma) and the wells washed four times with 200 mL PBS. The lysate was transferred in a 2.5 ml tube together with the 4 PBS washings. One hundred microliter of 1% n-decanal in ethanol was added to the tube and the luminescence was measured. RLU values shown were obtained from six replicate cultures. Cell viability of the macrophage culture is observed by Trypan blue with a microscope. 4.1.6. Assessment of cytotoxicity Inhibitory effects on C3A human hepatocytes were determined for the derivatives by a neutral red uptake assay as described before. The C3A cells were grown in DMEM þ 10% FCS until a semi confluent layer of cells was obtained. The cells were trypsinized, washed and 40,000 cells were seeded per well of a 96-well plate. The cells were subsequently incubated at 37  C and 5% CO2 to recover. The following days, the compounds were solubilized in DMSO to stock concentrations of 10 mM. A serial dilution of each compound was made in DMEM þ 10% FCS. The C3A cells were washed and exposed to the derivatives by adding the serial dilutions of the compounds to the wells. The plates were left for incubation at 37  C, 5% CO2 for 24 h. After exposure, the cells were washed with 200 mL PBS and 200 mL neutral red working solution (Sigma) was added per well. Subsequently the plates were incubated for 3 h at 37  C, 5% CO2. The wells were washed with 200 mL PBS and 200 mL of an ethanol/acetic acid (50%) mixture. The plates were left on the shaker until the color became homogeneous purple and the optical density was measured at 530 nm (NR max) and 620 nM (reference wavelength) with the Paradigm detection platform. 4.1.7. Vitotox assay Observations on genotoxicity were done with Vitotox (Gentaur, Kampenhout, BE) and the included protocol was followed. In brief, TA104 RecN2-4 (genox) and TA104 (cytox) S. typhimurium bacteria were cultivated at 36  C for 16 h in poor 869 medium. The bacterial culture was diluted 250 times, incubated for 1 h at 36  C and kept on ice. The bacterial culture was divided by 10 once more. S9 was added to the designated þ S9 cultures to test the genotoxic effects of the metabolites of the compounds. The bacterial suspensions were then incubated, shaking at 36  C, and the luminescent signal was measured for 4 h with a 5 min interval. 4.1.8. Comet assay Possible DNA breakage effects of the derivatives on C3A cells were investigated by the alkaline comet assay. The C3A cells were grown in DMEM þ 10% FCS until a semi-confluent layer of cells was obtained. The cells were trypsinized, washed and seeded at 100,000 cells per well of a 24-well plate and left for recovery at 37  C, 5% CO2. The following day, the testing compounds were dissolved in DMSO as a stock concentration of 1 mM. Serial dilutions of each compound were made in DMEM þ 10% FCS to obtain the final concentrations (10 mM, 3 mM, 1 mM, 0.3 mM, 0.1 mM). The C3A cells were washed and exposed to the derivatives by adding 1 ml of the serial dilutions to each well. The plates were incubated at 37  C, 5% CO2 for 24 h. After incubation, the cells were trypsinized, washed with PBS, and 10 mL of cell suspension was dissolved in 300 mL low melting point agarose. The dissolved cell suspension was then placed onto a frosted microscope slide and left on ice for 5 min. The slide was subsequently placed in a jar containing lysing solution for 1 h. After lysis, the agarose cell suspension was subjected to electrophoresis for 20 min at 300 mA. The slides were washed with neutralization buffer for 5 min and dried in ice cold ethanol for 10 min. Staining of the DNA was done

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with gel red (SigmaeAldrich). For the quantification of the DNA migration, a fluorescence microscope was used and the percentage DNA in the comet tail of the cells nuclei core was calculated in proportion to the total DNA present in the nuclei (comet head þ tail) by appropriate imager software from Metasystems (Altlussheim, Germany). 4.2. Chemical data Column chromatography was carried out using a glass column with silica gel (Aldrich, particle size 0.035e0.070 mm, pore diameter ca. 6 nm). Solvent systems were determined via initial TLC analysis on silica gel (Merck, Kieselgel 60F254, precoated 0.25 mm). Compounds were revealed by UV light or KMnO4 oxidation. 1H NMR (300 MHz), 13C NMR (75 MHz) and 19F NMR (282 MHz) spectra were recorded with a JEOL JNM-EX 300 NMR spectrometer. Peak assignments were performed with the aid of the DEPT, 2D COSY, HSQC, HMBC techniques. The NMR samples were prepared with commercially available deuterated solvents with SiMe4 or CFCl3 as an internal standard. Low resolution mass spectra were recorded using an Agilent 1100 series VS (ESI, 4000 V) mass spectrometer via a direct inlet or via LCeMS coupling [Phenomenex luna column; 250  3 mm length, 5 mm particle size, 100 A pore size with 5 mM NH4OAc in H2O and acetonitrile as eluents]. High resolution mass spectra were recorded on a Finnigan MAT 95 XPAPIGC-Trap tandem mass spectrometer or a tandem spectrometer Agilent 6220 TOF-LC/MS. Infrared spectra were recorded with a Perkin Elmer BX FT-IR spectrometer. Melting points were recorded on a Buchi Melting point B-540 apparatus and are uncorrected. Microwave reactions were performed in a CEM DiscoverÒ microwave. Dichloromethane and DMF were dried over CaH2/benzophenone, while methanol was dried by distillation over magnesium/iodine. All reagent were used without further purification and all glassware was oven-dried prior to use. 4.2.1. Synthesis of 2-[N-(1,4-dimethoxy-2-naphthyl)aminomethyl]-2-methyl-1,3-dioxolane 9 1,4-Dimethoxy-2-formylnaphthalene 7 [12a] (1.0 g, 4.62 mmol), 2-aminomethyl-2-methyl-1,3-dioxolane 8 [12b] (0.57 g, 4.62 mmol), and magnesium(II) sulfate (1.12 g, 9.24 mmol) were dissolved in dichloromethane in a flask fitted with a calcium chloride tube and the mixture was stirred for 2 h at room temperature. After filtration and evaporation of the solvent in vacuo, the crude imine was dissolved in methanol (10 ml) and sodium borohydride (0.35 g, 9.24 mmol) was added portion-wise at 0  C. Then, the reaction mixture was stirred for 16 h at room temperature in a flask fitted with a calcium chloride tube. The reaction was quenched by careful addition of water (5 ml) and the aqueous solution was extracted with dichloromethane (3  10 ml). The combined organic extracts were washed with brine, dried (MgSO4), and evaporated in vacuo to afford 2-[N-(1,4-dimethoxy-2-naphthyl)aminomethyl]-2-methyl-1,3dioxolane 9 (1.45 g, quant.) as a viscous oil. As it had only a limited stability, it was immediately used in the next step. Quant., viscous oil. 1H NMR (CDCl3): d 1.40 (3H, s, CH3), 2.76 (2H, s, ArCH2N), 3.89 (3H, s, CH3O), 3.96 (4H, m, OCH2CH2O), 3.99 (3H, s, CH3O), 4.04 (2H, s, NHCH2), 6.86 (1H, s, H-3), 7.45 (1H, dt, J ¼ 1.7, 7.8 Hz, H-6 or H-7), 7.52 (1H, dt, J ¼ 1.7, 7.8 Hz, H-6 or H-7), 8.04 (1H, dd, J ¼ 1.7, 7.8 Hz, H-5 or H-8), 8.22 (1H, dd, J ¼ 1.7, 7.8 Hz, H-5 or H-8). 13C NMR (CDCl3): d 23.00 (CH3), 48.44 (ArCH2N), 55.54 (OCH3), 55.7, (OCH3), 62.36 (NCH2), 65.07 (2  CH2O), 103.88 (Oe CeO), 104.79 (CH), 109.54 (Cquat), 121.88 (CH), 122.38 (CH), 125.21 (CH), 126.57 (CH), 128.23 (Cquat), 128.61 (Cquat), 146.45 (Cquat), 151.26 (Cquat). IR (ATR): nmax 3332, 3069, 2936, 1595, 1459, 1368, 1266, 1213, 1162, 1121, 1091, 1055, 768 cm1. MS m/z (%): 318 ([M þ H]þ, 100).

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4.2.2. Synthesis of 4-methylbenzo[g]isoquinoline-5,10-dione 10 To a solution of 2-[N-(1,4-dimethoxy-2-naphthyl)-aminomethyl]-2-methyl-1,3-dioxolane 9 (0.32 mmol, 100 mg) in dichloromethane (2 ml) was added 4 ml of concentrated sulfuric acid. The mixture was stirred for 2 days at room temperature. After completion, the reaction mixture was cooled to 0  C, quenched with ice, neutralized with an aqueous solution of 2 M NaOH and extracted with dichloromethane (3  10 ml). The combined organic layers were washed with brine, dried over MgSO4 and evaporated in vacuo to give a brown solid. Preparative thin layer chromatography with ethyl acetate/petroleum ether (1/4) as eluent gave 4methylbenzo[g]isoquinoline-5,10-dione 10 (35 mg, 50% yield). Yield 50%, yellow crystals, m.p. 130.5e131.9  C. 1H NMR (CDCl3): d 2.84 (3H, s, CH3), 7.82e7.86 (2H, m, H-7 and H-8), 8.25e8.30 (2H, m, H-6 and H-9), 8.89 (1H, s, H-3), 9.45 (1H, s, H-1). 13C NMR (CDCl3): d 19.4 (CH), 126.6 (Cquat), 126.8 (CH), 127.3 (CH), 132.5 (Cquat), 133.4 (Cquat), 134.1 (Cquat), 134.5 (CH), 134.6 (CH), 135.5 (Cquat), 147.2 (CH), 157.5 (CH), 183.1 (C]O), 184.8 (C]O). IR (ATR): nmax 1678, 1638, 1617 cm1. MS m/z (%): 224 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C14H10NO2]þ: 224.0712, found 224.0707. 4.2.3. Synthesis of (E)-4-[2-(dimethylamino)vinyl]benzo[g] isoquinoline-5,10-dione 11 To a solution of 4-methylbenzo[g]isoquinoline-5,10-dione 10 (103 mg, 4.61 mmol) in DMF (3 ml), 5 equivalents of DMFeDMA were added under a nitrogen atmosphere and the reaction mixture was heated for 2.5 h in an oil bath at 125  C. After completion, the reaction mixture was cooled down to room temperature and poured in 30 ml of CH2Cl2. The organic layer was washed with 5  20 ml of brine. The organic layer was dried over MgSO4 and the solvent was evaporated in vacuo to afford (E)-4-[2-(dimethylamino) vinyl]benzo[g]isoquinoline-5,10-dione 11 (110 mg, 86% yield) in high purity. Yield 86%, dark blue solid, m.p. 201.0e201.7  C. 1H NMR (CDCl3): d 3.05 (6H, s, N(CH3)2, 6.96 (1H, d, J ¼ 14.3 Hz, H-10 ), 7.26 (1H, d, J ¼ 14.3 Hz, H-2), 7.78 (2H, m, H-7 and H-8), 8.25 (2H, m, H-6 and H9), 9.02 (1H, s, H-3), 9.13 (1H, s, H-1). 13C NMR (CDCl3): d 41.02 (N(CH3)2), 92.54 (CH), 126.52 (CH), 126.83 (Cquat), 127.22 (CH), 132.60 (Cquat), 133.68 (CH), 134.41 (CH), 135.12 (Cquat), 135.38 (2  Cquat), 146.45 (CH), 152.72 (CH), 184.17 (C]O), 185.13 (C]O). IR (ATR): nmax 2920, 1662, 1641, 1690, 1407, 1390, 1373, 1318, 1275, 1246 cm1. MS m/z (%): 279 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C17H15N2O2]þ: 279.1134, found 279.1135. 4.2.4. Synthesis of 7H-naphtho[3,2,1-i,j]-2,6-naphthiridin-6-one 12 (E)-4-[2-(Dimethylamino)vinyl]benzo[g]isoquinoline-5,10-dione 11 (133 mg, 0.48 mmol) was mixed with 10 ml of a 5%(w/v) solution of ammonium acetate in dry methanol under a nitrogen atmosphere and refluxed for 3 h. Then the solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (15 ml) and washed with a saturated sodium bicarbonate solution (25 ml) and brine (25 ml). The organic layer was dried over magnesium sulfate and concentrated in vacuo. The resulting residue was purified by column chromatography (petroleum ether/ethyl acetate) to afford 7H-naphtho[3,2,1-i,j]-2,6-naphthiridin-6-one 12 (80 mg, 91% yield). Yield 91%, yellow solid, m.p. 201.4  C. 1H NMR (CDCl3): d 7.68 (1H, td, J ¼ 7.7 and 1.1 Hz, H-8), 7.82 (2H, s and td, J ¼ 7.7 and 1.1 Hz, H-5 and H-9), 8,25 (2H, m, H-6 and H-9), 8.39 (1H, dd, J ¼ 7.7 and 1.1 Hz, H-7), 8.78 (1H, dd, J ¼ 7.7 and 1.1 Hz, H-10), 8.91 (1H, d, J ¼ 6.1 Hz, H-2), 9.56 (2H, br, d, J ¼ 6.1 Hz, H-1 and H-3). 13C NMR (CDCl3): 119.02 (CHAr), 121.22 (Cquat), 124.58 (CHAr), 125.24 (CHAr), 127.70 (CHAr), 129.06 (Cquat), 131.24 (CHAr), 132.35 (Cquat), 134.55 (CHAr), 135.86 (Cquat), 145.90 (CHAr), 146.43 (CHAr), 149.27 (Cquat), 157.09 (CHAr), 183.29 (C]O). IR (ATR): nmax 1666, 1655, 1591,

1289 cm1. MS m/z (%): 233 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C15H9N2O]þ: 233.0715, found 233.0711. 4.2.5. Synthesis of 3-substituted-1-methylbenzo[g]isoquinoline5,10-diones 15 2-Acetyl-1,4-naphthoquinone 13 (0.35 g, 1.75 mmol) and pyridinium salts 14aeh [26] (1.83 mmol) were added to a previously prepared 5 wt% solution of ammonium acetate in dry methanol (6 ml). The sealed reaction vessel was heated for 6 min at 90  C in a CEM DiscoverÒ microwave apparatus (ramp time 5 min, pmax 10.0 psi) [11]. Subsequently, the reaction mixture was cooled in ice water and filtered. The solid was washed with 20 ml of cold methanol and redissolved in chloroform. This solution was filtered over CeliteÒ and evaporated in vacuo to yield 3-aryl-1-methylbenz [g]isoquinoline-5,10-diones 15aef. 3-Alkyl-1-methyl benzo[g]isoquinoline-5,10-diones 15g and 15h did not precipitate upon cooling and were extracted with ethyl acetate (10 ml) and brine (2  10 ml) and subsequently purified by means of column chromatography. 4.2.5.1. 1-Methyl-3-phenylbenzo[g]isoquinoline-5,10-dione 15a. 97%, yellow solid, m.p. 190.3e191.7  C. 1H NMR (CDCl3): d 3.17 (3H, s, CH3), 7.47e7.56 (3H, m, H-30 , H-40 and H-50 ), 7.80 (1H, dt, J ¼ 1.7 and 7.7 Hz, H-7 or H-8), 7.86 (1H, dt, J ¼ 1.7 and 7.7 Hz, H-7 or H-8), 8.21e8.24 (2H, m, H-20 and H-60 ), 8.30 (1H, dd, J ¼ 1.7 and 7.7 Hz, H-6 or H-9), 8.33 (1H, dd, J ¼ 1.7 and 7.7 Hz, H-6 or H-9), 8.45 (1H, s, H-4). 13C NMR (CDCl3): d 26.93 (CH3), 114.34 (CH-4), 123.15 (Cquat), 126.98 (CHAr), 127.51 (CHAr), 127.65 (2  CHAr), 129.09 (2  CHAr), 130.66 (CHAr), 132.57 (Cquat), 133.90 (CHAr), 134.55 (Cquat), 135.10 (CHAr), 137.76 (Cquat), 141.12 (Cquat), 160.74 (Cquat), 162.19 (Cquat), 183.36 (C]O), 183.78 (C]O). IR (ATR): nmax 1677, 1664, 1573, 1375, 1329, 1278, 1243, 1158, 880, 735, 711, 684 cm1. MS m/z (%): 300 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C20H14NO2]þ: 300.1025, found 300.1027. 4.2.5.2. 3-(4-Methoxyphenyl)-1-methylbenzo[g]isoquinoline-5,10dione 15b. 47%, yellow solid, m.p. 192.8e193.5  C. 1H NMR (CDCl3): d 3.13 (3H, s, CH3), 3.88 (3H, s, OCH3), 7.02 (2H, d, J ¼ 8.8 Hz, H-30 and H-50 ), 7.77 (1H, dt, J ¼ 1.7, 7.6 Hz, H-7 or H-8), 7.83 (1H, dt, J ¼ 1.7, 7.6 Hz, H-7 or H-8), 8.21 (2H, d, J ¼ 8.8 Hz, H-20 and H-50 ), 8.26 (1H, dd, J ¼ 1.7, 7.6 Hz, H-6 or H-9), 8.29 (1H, dd, J ¼ 1.7, 7.6 Hz, H-6 or H-9), 8.36 (1H, s, H-4). 13C NMR (CDCl3): d 26.94 (CH3), 55.54 (OCH3), 113.39 (CH-4), 114.46 (2  CHAr), 126.92 (CHAr) 127.48 (CHAr), 129.24 (2  CHAr), 130.36 (Cquat), 131.57 (Cquat), 132.61 (Cquat), 133.74 (CHAr), 134.66 (Cquat), 135.03 (CHAr), 141.02 (Cquat), 160.30 (Cquat), 161.91 (Cquat), 162.14 (Cquat), 183.53 (C]O), 183.65 (C]O). IR (ATR): nmax 1672, 1663, 1573, 1514, 1375, 1375, 1333, 1278, 1261, 1175, 1030, 842, 713 cm1. MS m/z (%): 330 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C21H16NO3]þ: 330.1130, found 330.1134. 4.2.5.3. 3-(4-Fluorophenyl)-1-methylbenzo[g]isoquinoline-5,10dione 15c. Yield: 51%, yellow solid, m.p. 203.0e204.6  C. 1H NMR (CDCl3): d 3.13 (3H, s, CH3), 7.20 (2H, dd, JHH ¼ 8.7 Hz, JHF ¼ 8.8 Hz, H-30 and H-50 ), 7.78 (1H, dt, J ¼ 1.7, 7.5 Hz, H-7 or H-8), 7.84 (1H, dt, J ¼ 1.7, 7.5 Hz, H-7 or H-8), 8.20 (2H, dd, JHH ¼ 8.7 Hz, JHF ¼ 5.2 Hz, H20 and H-50 ), 8.26 (1H, dd, J ¼ 1.7, 7.5 Hz, H-6 or H-9), 8.28 (1H, dd, J ¼ 1.7, 7.5 Hz, H-6 or H-9), 8.36 (1H, s, H-4). 13C NMR (CDCl3): d 26.86 (CH3), 113.86 (CH-4), 116.10 (2  CHAr, JCF ¼ 21.8 Hz), 123.02 (Cquat), 126.97 (CHAr), 127.51 (CHAr), 129.66 (2  CHAr, JCF ¼ 8.7 Hz), 132.48 (Cquat), 133.88 (CHAr), 134.48 (Cquat), 135.21 (CHAr), 141.14 (Cquat), 159.46 (Cquat), 162.21 (Cquat), 164.53 (Cquat, JCF ¼ 254.6 Hz), 183.23 (C]O), 183.63 (C]O). 19F NMR (CDCl3): d 110.59 (1F, s, Cquat-F). IR (ATR): nmax 1671, 1662, 1506, 1414, 1370, 1373, 1332, 1275, 1192, 1155, 1017, 843, 748, 710 cm1. MS m/z (%): 318 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C20H13FNO2]þ: 318.0930, found 318.0925.

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4.2.5.4. 3-(4-Chlorophenyl)-1-methylbenzo[g]isoquinoline-5,10dione 15d. Yield: 67%, yellow solid, m.p. 223.8e225.4  C. 1H NMR (CDCl3): d 3.16 (3H, s, CH3), 7.51 (2H, d, J ¼ 8.5 Hz, H-30 and H-50 ), 7.81 (1H, dt, J ¼ 1.7, 7.7 Hz, H-7 or H-8), 7.87 (1H, dt, J ¼ 1.7, 7.7 Hz, H-7 or H-8), 8.19 (2H, d, J ¼ 8.5 Hz, H-20 ), 8.29 (1H, dd, J ¼ 1.7, 7.7 Hz, H-6 or H-9), 8.32 (1H, dd, J ¼ 1.7, 7.7 Hz, H-6 or H-9), 8.42 (1H, s, H4). 13C NMR (CDCl3): d 26.88 (CH3), 114.09 (CH-4), 123.22 (Cquat), 127.02 (CHAr), 127.57 (CHAr), 128.92 (2  CHAr), 129.31 (2  CHAr), 132.52 (Cquat), 133.97 (CHAr), 134.54 (Cquat), 135.19 (CHAr), 136.15 (Cquat), 136.97 (Cquat), 141.25 (Cquat), 159.39 (Cquat), 162.30 (Cquat), 183.25 (C]O), 183.68 (C]O). IR (ATR): nmax 1670, 1661, 1570, 1408, 1370, 1329, 1279, 1092, 845, 706 cm1. MS m/z (%): 334 ([M þ H]þ, 100). No satisfactory HRMS could be obtained, a mass spectrometric phenomenon which is typical for certain quinones. 4.2.5.5. 3-(2,5-Dimethoxyphenyl)-1-methylbenzo[g]isoquinoline5,10-dione 15e. Yield: 52%, orange solid, m.p. 213.1  C. 1H NMR (CDCl3): d 3.17 (3H, s, CH3), 3.87 (3H, s, OCH3), 3.90 (3H, s, OCH3), 6.98 (1H, dd, J ¼ 1.1, 8.3 Hz, CH-30 ), 7.02 (1H, dd, J ¼ 1.1, 8.3 Hz, CH40 ), 7.61 (1H, dd, J ¼ 2.2, 1.1 Hz, CH-60 ), 7.80 (1H, dt, J ¼ 1.7, 7.5 Hz, CH-7 or CH-8), 7.86 (1H, dt, J ¼ 1.7, 7.5 Hz, CH-7 or CH-8), 8.29 (1H, dd, J ¼ 1.7, 7.5 Hz, CH-6 or CH-9), 8.32 (1H, dd, J ¼ 1.7, 7.5 Hz, CH-6 or CH-9), 8.67 (1H, s, CH-4). 13C NMR (CDCl3): d 26.82 (CH3), 55.97 (OCH3), 56.36 (OCH3), 113.05 (CH-4), 116.00 (CHAr), 117.25 (CHAr), 119.59 (CHAr) 122.78 (Cquat), 126.90 (CHAr), 127.45 (CHAr), 128.02 (Cquat), 132.72 (Cquat), 133.85 (CHAr), 134.58 (Cquat), 134.96 (CHAr), 140.20 (Cquat), 152.34 (Cquat), 153.99 (Cquat), 159.61 (Cquat), 161.55 (Cquat), 183.61 (C]O), 183.99 (C]O). IR (ATR): nmax 1677, 1660, 1571, 1282, 1261 cm1. MS m/z (%): 360 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C22H18NO4]þ: 360.1236, found 360.1220. 4.2.5.6. 1-Methyl-3-(4-methylphenyl)benzo[g]isoquinoline-5,10dione 15f. Yield: 93%, yellow solid, m.p. 203.4e204.7  C. 1H NMR (CDCl3): d 2.44 (3H, s, CH3), 3.16 (3H, s, CH3), 7.35 (2H, d, J ¼ 8.3 Hz, CH-30 and CH-50 ), 7.81 (1H, dt, J ¼ 1.3, 7.8 Hz, CH-7 or CH-8), 7.87 (1H, dt, J ¼ 1.3, 7.8 Hz, CH-7 or CH-8), 8.15 (2H, d, J ¼ 8.3 Hz, CH-20 and CH-60 ), 8.30 (1H, dd, J ¼ 1.3, 7.8 Hz, CH-6 or CH-9), 8.33 (1H, dd, J ¼ 1.3, 7.8 Hz, CH-6 or CH-9), 8.45 (1H, s, CH-4). 13C NMR (CDCl3): d 21.55 (CH3), 26.93 (CH3), 113.96 (CH-4), 122.90 (Cquat), 126.96 (CHAr), 127.51 (CHAr), 127.57 (2  CHAr), 129.24 (2  CHAr), 129.24 (Cquat), 132.63 (Cquat), 133.82 (CHAr), 134.64 (Cquat), 135.06 (CHAr), 135.06 (Cquat), 141.09 (Cquat), 160.77 (Cquat), 162.16 (Cquat), 183.49 (C]O), 183.76 (C]O). IR (ATR): nmax 1671, 1662, 1570, 1511, 1374, 1332, 1274, 1261, 1155, 1018, 882, 844, 708 cm1. MS m/z (%): 314 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C21H16NO2]þ: 314.1181, found 314.1180. 4.2.5.7. 1-Methyl-3-isopropylbenzo[g]isoquinoline-5,10-dione 15g. Yield: 40%, yellow solid, m.p. 137.1  C. 1H NMR (CDCl3): d 1.38 (6H, d, J ¼ 6.8 Hz, CH(CH3)2), 3.09 (3H, s, CH3), 3.21 (1H, septet, J ¼ 6.8 Hz),7.79 (1H, dt, J ¼ 1.7, 7.6 Hz, CH-7 or CH-8), 7.85 (1H, dt, J ¼ 1.7, 7.6 Hz, CH-7 or CH-8), 7.90 (1H, s, CH-4), 8.27 (1H, dd, J ¼ 1.7, 7.6 Hz, CH-6 or CH-9), 8.30 (1H, dd, J ¼ 1.7, 7.6 Hz, CH-6 or CH-9). 13C NMR (CDCl3): d 22.25 (CH(CH3)2), 26.56 (CH3), 37.10 (CH(CH3)2), 114.57 (CH-4), 122.60 (Cquat), 126.72 (CHAr), 127.32 (CHAr), 132.41 (Cquat), 133.67 (CHAr), 134.71 (Cquat), 134.84 (CHAr), 140.57 (Cquat), 161.44 (Cquat), 172.60 (Cquat), 183.35 (C]O), 183.67 (C]O). IR (ATR): nmax 2928, 1676, 1666, 1573, 1336, 1279, 715 cm1. MS m/z (%): 266 ([M  H], 100). HRMS (ESþ) calcd. for [C17H16NO2]þ: 266.1181, found 266.1175. 4.2.5.8. 1-Methyl-3-tert-butylbenzo[g]isoquinoline-5,10-dione 15h. Yield: 38%, orange solid, m.p. 90.7  C. 1H NMR (CDCl3): d 1.44 (9H, s, C(CH3)3), 3.08 (3H, s, CH3), 7.77 (1H, dt, J ¼ 1.7, 7.4 Hz, CH-7 or CH8), 7.83 (1H, dt, J ¼ 1.7, 7.4 Hz, CH-7 or CH-8), 8.03 (1H, s, CH-4), 8.25

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(1H, dd, J ¼ 1.7, 7.4 Hz, CH-6 or CH-9), 8.28 (1H, dd, J ¼ 1.7, 7.4 Hz, CH-6 or CH-9). 13C NMR (CDCl3): d 26.77 (CH3), 29.90 (C(CH3)3), 38.55 (C(CH3)3), 113.29 (CH-4), 122.23 (Cquat), 126.86 (CHAr), 127.39 (CHAr), 132.64 (Cquat), 133.76 (CHAr), 134.46 (Cquat), 134.90 (CHAr), 140.54 (Cquat), 160.95 (Cquat), 174.69 (Cquat), 183.82 (C]O), 184.04 (C]O). IR (ATR): nmax 2958, 2925, 1676, 1575, 1276, 711 cm1. MS m/ z (%): 280 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C18H18NO2]þ: 280.1338, found 280.1340. 4.2.6. Synthesis of 3-substituted-1-[2-(dimethylamino)vinyl]benzo [g] isoquinoline-5,10-diones 16 To a solution of 3-substituted-1-methylbenzo[g]isoquinoline5,10-diones 15 (250 mg) in dry DMF (5 ml), 10 equivalents of DMFe DMA were added under a nitrogen atmosphere and the reaction mixture was heated for 15 h in an oil bath at 125  C. Next, the reaction mixture was poured in 30 ml of water, and extracted with 3  10 ml of CH2Cl2. The organic phase was washed three times with brine, dried over MgSO4, evaporated and concentrated under high vacuum to remove residual traces of DMF, thus affording3substitited-1-[2-(dimethylamino)vinyl]benzo[g]isoquinoline-5,10diones 16 in high purity. Due to coalescence of the 2-(dimethylamino) vinyl system, the dimethylamino group is not visible in 13C NMR at 25  C. In order to view this peak, 13C spectra have to be recorded at 50  C (CDCl3). 4.2.6.1. 1-[2-(Dimethylamino)vinyl]-3-phenylbenzo[g]isoquinoline5,10-dione 16a. Yield: 52%, deep blue solid, m.p. 215.3e216.9  C. 1H NMR (CDCl3): d 3.12 (6H, s, N(CH3)2), 7.24 (1H, d, J ¼ 12.4 Hz, CH] CHeN(CH3)2), 7.46e7.55 (3H, m, H-30 , H-40 and H-50 ), 7.72 (1H, dt, J ¼ 1.4, 7.6 Hz, H-7 or H-8), 7.81 (1H, dt, J ¼ 1.4, 7.6 Hz, H-7 or H-8), 8.03 (1H, s, H-4), 8.19 (2H, dd, J ¼ 1.9, 8.0 Hz, H-20 and H-50 ), 8.23 (1H, dd, J ¼ 1.4, 7.6 Hz, H-6 or H-9), 8.31 (1H, dd, J ¼ 1.4, 7.6 Hz, H-6 or H-9), 8.41 (1H, d, J ¼ 12.4 Hz, CH]CHeN(CH3)2). 13C NMR (CDCl3): d 41.14 (N(CH3)2), 95.43 (]CH), 109.97 (CH-4), 115.31 (Cquat), 126.52 (CHAr), 127.25 (CHAr), 127.51 (2  CHAr), 128.78 (2  CHAr), 130.14 (CHAr), 132.46 (Cquat), 132.83 (CHAr), 134.72 (CHAr), 135.48 (Cquat), 138.84 (Cquat), 141.96 (Cquat), 151.12 (]CH), 160.20 (Cquat), 183.55 (C]O), 184.40 (C]O). IR (ATR): nmax 3062, 2908, 1667, 1602, 1557, 1531, 1492, 1425, 1360, 1304, 1240, 1090, 1061, 964, 907, 859 cm1. MS m/z (%): 355 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C23H18N2O2]þ: 355.1447, found 355.1449. 4.2.6.2. 1-[2-(Dimethylamino)vinyl]-3-(4-methoxyphenyl)benzo[g] isoquinoline-5,10-dione 16b. Yield: 70%, deep purple solid, m.p. 233.3e235.9  C. 1H NMR (CDCl3): d 3.12 (6H, s, CH3), 3.89 (3H, s, OCH3), 7.03 (2H, d, J ¼ 8.8 Hz, H-30 and H-50 ), 7.25 (1H, d, J ¼ 12.7 Hz, CH]CHN(CH3)2), 7.71 (1H, dt, J ¼ 1.1, 7.7 Hz, H-7 or H-8), 7.80 (1H, dt, J ¼ 1.1, 7.7 Hz, H-7 or H-8), 8.06 (1H, s, H-4), 8.18 (2H, d, J ¼ 8.8 Hz, H-20 and H-50 ), 8.23 (1H, dd, J ¼ 1.1, 7.7 Hz, H-6 or H-9), 8.30 (1H, dd, J ¼ 1.1, 7.7 Hz, H-6 or H-9), 8.40 (1H, d, J ¼ 12.7 Hz, CH]CHN(CH3)2). 13C NMR (CDCl3): d 41.17 (N(CH3)2), 55.51 (OCH3), 95.50 (]CH), 109.36 (CH-4), 114.14 (2  CHAr), 114.80 (Cquat), 126.48 (CHAr) 127.22 (CHAr), 129.05 (2  CHAr), 131.39 (Cquat), 132.45 (Cquat), 132.74 (CHAr), 134.69 (CHAr), 135.54 (Cquat), 141.77 (Cquat), 150.95 (] CH), 159.65 (Cquat), 160.07 (Cquat), 161.50 (Cquat), 183.35 (C]O), 184.52 (C]O). IR (ATR): nmax 2923, 1669, 1636, 1599, 1527, 1428, 1361, 1302, 1236, 1174, 1103, 1015, 834, 709 cm1. MS m/z (%): 385 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C24H21N2O3]þ: 385.1552, found 385.1550. 4.2.6.3. 3-(4-Fluorophenyl)-1-[2-(dimethylamino)vinyl]benzo[g]isoquinoline-5,10-dione 16c. Yield: quant., deep blue solid, m.p. 224.5  C. 1H NMR (CDCl3): d 3.11 (6H, s, N(CH3)2), 7.18 (2H, dd, JHF ¼ 8.5, JHH ¼ 8.8 Hz, H-30 and H-50 ), 7.21 (1H, d, J ¼ 12.7 Hz, CH] CHN(CH3)2), 7.70 (1H, t, J ¼ 7.7 Hz, H-7 or H-8), 7.80 (1H, t, J ¼ 7.7 Hz,

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H-7 or H-8), 7.94 (1H, s, H-4), 8.16 (2H, dd, JHF ¼ 5.5, JHH ¼ 8.8 Hz, H-20 and H-60 ), 8.21 (1H, d, J ¼ 7.7 Hz, H-6 or H-9), 8.29 (1H, d, J ¼ 7.7 Hz, H6 or H-9), 8.35 (1H, d, J ¼ 12.7 Hz, CH]CHN(CH3)2). 13C NMR (CDCl3): d 40.24 (N(CH3)2), 95.31 (]CH), 109.48 (CH-4), 115.16 (Cquat), 115.73 (2  CHAr, JCF ¼ 21.9 Hz), 126.86 (2  CHAr, JCF ¼ 56.5 Hz), 129.39 (CHAr), 129.50 (CHAr), 132.35 (Cquat), 132.83 (CHAr), 134.73 (CHAr), 134.92 (Cquat, JCF ¼ 2.3 Hz),135.41 (Cquat),141.94 (Cquat),151.03 (]CH), 158.92 (Cquat), 160.08 (Cquat), 164.26 (Cquat, JCF ¼ 250.4 Hz), 183.38 (C]O), 184.26 (C]O). 19F NMR (CDCl3): d 111.55 (1F, s, Cquat-F). IR (ATR): nmax 2925, 1668, 1599, 1537, 1506, 1361 cm1. MS m/z (%): 373 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C24H21N2O3]þ: 373.1352, found 373.1353. 4.2.6.4. 3-(4-Chlorophenyl)-1-[2-(dimethylamino)vinyl]benzo[g]isoquinoline-5,10-dione 16d. Yield: 77%, dark blue solid, m.p. 248.9e 251.0  C. 1H NMR (CDCl3): d 3.12 (6H, s, N(CH3)2), 7.22 (1H, d, J ¼ 12.7 Hz, CH]CHeN(CH3)2), 7.48 (2H, td, J ¼ 2.2, 8.3 Hz, H-30 and H-50 ), 7.73 (1H, dt, J ¼ 1.1, 7.7 Hz, H-7 or H-8), 7.82 (1H, dt, J ¼ 1.1, 7.7 Hz, H-7 or H-8), 7.98 (1H, s, H-4), 8.13 (2H, td, J ¼ 2.2, 8.3 Hz, H-20 and H-50 ), 8.23 (1H, dd, J ¼ 1.1, 7.7 Hz, H-6 or H-9), 8.30 (1H, dd, J ¼ 1.1, 7.7 Hz, H-6 or H-9), 8.38 (1H, d, J ¼ 12.7 Hz, CH]CHe N(CH3)2). 13C NMR (CDCl3): d 41.20 (N(CH3)2), 95.31 (]CH), 109.59 (CH-4), 115.45 (Cquat), 126.55 (CHAr), 127.28 (CHAr), 128.78 (2  CHAr), 128.99 (2  CHAr), 132.38 (Cquat), 132.90 (CHAr), 134.80 (CHAr), 135.44 (Cquat), 136.34 (Cquat), 137.25 (Cquat), 142.06 (Cquat), 151.12 (]CH), 158.89 (Cquat), 160.19 (Cquat), 183.47 (C]O), 184.26 (C]O). IR (ATR): nmax 2908, 1667, 1602, 1557, 1531, 1492, 1425, 1360, 1304, 1240, 1090, 1061, 964, 907, 859 cm1. MS m/z (%): 389 ([M þ H]þ, 100). No satisfactory HRMS analysis could be obtained. 4.2.6.5. 1-[2-(Dimethylamino)vinyl]-3-(2,5-dimethoxyphenyl)benzo [g]isoquinoline-5,10-dione 16e. Yield: 87%, dark blue solid, m.p. 193.3  C. 1H NMR (CDCl3): d 3.01 (6H, s, N(CH3)2), 3.81 (3H, s, OCH3), 3.84 (3H, s, OCH3), 6.90 (2H, d, J ¼ 1.7 Hz, CH-30 and CH-40 ), 7.13 (1H, d, J ¼ 12.7 Hz, CH]CHN(CH3)2), 7.84 (1H, t, J ¼ 1.7 Hz, CH-60 ), 7.64 (1H, dt, J ¼ 1.1, 7.7 Hz, H-7 or H-8), 7.73 (1H, dt, J ¼ 1.1, 7.7 Hz, H-7 or H-8), 8.11 (1H, s, H-4), 8.15 (1H, d, J ¼ 7.7 Hz, H-6 or H-9), 8.22 (1H, d, J ¼ 7.7 Hz, H-6 or H-9), 8.24 (1H, d, J ¼ 12.7 Hz, CH]CHN(CH3)2). 13 C NMR (CDCl3): d 41.07 (N(CH3)2), 55.85 (OCH3), 56.33 (OCH3), 95.31 (]CH), 112.90 (CH-4), 112.90 (CHAr), 114.84 (Cquat), 115.15 (CHAr), 116.09 (CHAr), 116.41 (CHAr), 126.32 (CHAr), 127.13 (CHAr), 129.24 (Cquat), 132.49 (Cquat), 132.69 (CHAr), 134.49 (CHAr), 135.41 (Cquat), 140.7s0 (Cquat), 150.77 (]CH), 152.46 (Cquat), 153.68 (Cquat), 159.19 (Cquat), 159.73 (Cquat), 183.49 (C]O), 184.45 (C]O). IR (ATR): nmax 1669, 1604, 1554, 1215, 1100, 715 cm1. MS m/z (%): 415 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C25H23N2O4]þ: 415.1658, found 415.1663. 4.2.6.6. 1-[2-(Dimethylamino)vinyl]-3-(4-methylphenyl)benzo[g]isoquinoline-5,10-dione 16f. Yield: 97%, deep blue solid, m.p. 228.6e 229.5  C. 1H NMR (CDCl3): d 2.43 (3H, s, CH3), 3.11 (6H, s, N(CH3)2), 7.22 (1H, d, J ¼ 12.7 Hz, CH]CHN(CH3)2), 7.31 (2H, d, J ¼ 8.3 Hz, H30 and H-50 ), 7.71 (1H, dt, J ¼ 1.1, 7.5 Hz, H-7 or H-8), 7.80 (1H, dt, J ¼ 1.1, 7.5 Hz, H-7 or H-8), 8.00 (1H, s, H-4), 8.08 (2H, d, J ¼ 8.3 Hz, H-20 ), 8.21 (1H, dd, J ¼ 1.1, 7.5 Hz, H-6 or H-9), 8.29 (1H, dd, J ¼ 1.1, 7.5 Hz, H-6 or H-9), 8.39 (1H, d, J ¼ 12.7 Hz, CH]CHN(CH3)2). 13C NMR (CDCl3): d 21.54 (CH3), 41.11 (N(CH3)2), 95.46 (]CH), 109.77 (CH-4), 115.09 (Cquat), 126.49 (CHAr), 127.24 (CHAr), 127.43 (2  CHAr), 127.43 (Cquat), 129.53 (2  CHAr), 132.46 (Cquat), 132.78 (CHAr), 134.69 (CHAr), 135.52 (Cquat), 136.05 (Cquat), 140.43 (Cquat), 141.84 (Cquat), 151.06 (]CH), 160.10 (Cquat), 183.46 (C]O), 183.76 (C]O). IR (ATR): nmax 2924, 1667, 1606, 1560, 1535, 1507, 1361, 1302, 1269, 1242, 1102, 806, 844, 709 cm1. MS m/z (%): 391 ([M þ Na]þ, 100). HRMS (ESþ) calcd. for [C24H21N2O2]þ: 369.1603, found 369.1603.

4.2.6.7. 1-[2-(Dimethylamino)vinyl]-3-tert-butylbenzo[g]isoquinoline-5,10-dione 16h. Yield: 88%, dark blue solid, m.p. 189.5  C. 1H NMR (CDCl3): d 1.41 (9H, s, C(CH3)3), 3.08 (6H, s, N(CH3)2), 7.16 (1H, d, J ¼ 12.4 Hz, CH]CHN(CH3)2), 7.62 (1H, s, H-4), 7.66 (1H, dt, J ¼ 1.3, 7.6 Hz, H-7 or H-8), 7.76 (1H, dt, J ¼ 1.3, 7.6 Hz, H-7 or H8),8.17 (1H, dd, J ¼ 1.3, 7.6 Hz, H-6 or H-9), 8.26 (1H, dd, J ¼ 1.3, 7.6 Hz, H-6 or H-9), 8.29 (1H, d, J ¼ 12.4 Hz, CH]CHN(CH3)2). 13C NMR (CDCl3): d 29.81 (C(CH3)3), 29.81 (C(CH3)3), 41.16 (N(CH3)2), 95.53 (]CH), 109.30 (CH-4), 114.23 (Cquat), 126.31 (CHAr), 127.06 (CHAr), 132.41 (Cquat), 132.61 (CHAr), 134.45 (CHAr), 135.27 (Cquat), 141.19 (Cquat), 150.84 (]CH), 160.00 (Cquat), 173.46 (Cquat), 183.35 (C]O), 184.52 (C]O). IR (ATR): nmax 1660, 1608, 1566, 1307, 1245, 1096, 715 cm1. MS m/z (%): 335 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C21H23N2O2]þ: 335.1760, found 335.1763. 4.2.7. Synthesis of 5-substituted-7H-naphtho[3,2,1-de] naphthyridine-7-ones 17 3-substituted-1-[2-(dimethylamino)vinyl]-benzo[g]isoquinoline-5,10-diones 16 (0.45 mmol) were mixed with 10 ml of a 5% (w/ v) solution of ammonium acetate in dry methanol and refluxed for 3 h. After completion of the reaction, the solvent was evaporated and the residue was redissolved in dichloromethane (15 ml). Then, the reaction mixture was washed with saturated sodium bicarbonate solution (25 ml) and brine (25 ml). The organic layer was dried over magnesium sulfate and concentrated in vacuo. The resulting residue was purified by column chromatography using dichloromethane as eluent to afford 5-substituted-7H-naphtho [3,2,1-de]naphthyridine-7-ones 17. Due to their high insolubility, no 13 C NMR could be recorded for compounds 17c and 17d. Recording was attempted with 10,000 scans and a relaxation delay of 3 s in CDCl3, acetone-d6, DMSO-d6, CS2 and CF3CO2D. 4.2.7.1. 5-Phenyl-7H-naphtho[3,2,1-de]naphthyridine-7-one 17a. Yield: 89%, yellow solid, m.p. 222.0e223.3  C. 1H NMR (CDCl3): d 7.54e7.62 (3H, m, H-30 , H-40 and H-50 ), 7.68 (1H, dt, J ¼ 1.1, 7.4 Hz, H-9 or H-10), 7.87 (1H, dt, J ¼ 1.1, 7.4 Hz, H-9 or H-10), 8.02 (1H, d, J ¼ 5.9 Hz, H-3), 8.32 (2H, dd, J ¼ 7.7 and 1.7 Hz, H-20 and H-60 ), 8.41 (1H, d, J ¼ 7.7 Hz, H-11), 8.81 (1H, s, H-6), 8.87 (1H, d, J ¼ 7.7 Hz, H-8), 8.95 (1H, d, 5.9 Hz, H-2). 13C NMR (CDCl3): d 116.72 (Cquat), 118.66 (CH), 122.31 (CH), 125.50 (CH), 128.06 (Cquat), 128.06 (2  CH), 129.27 (2  CH), 130.84 (CH), 131.01 (CH), 131.93 (Cquat), 135.19 (CH), 135.38 (Cquat), 136.48 (Cquat), 138.19 (Cquat), 148.06 (CH), 149.91 (Cquat), 151.00 (Cquat), 162.80 (Cquat), 183.55 (C]O). IR (ATR): nmax 1670, 1592, 1448, 1380, 1367, 1340, 1243, 1233, 1027, 756, 697 cm1. MS: m/z (%) 309 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C21H13N2O]þ: 309.1028, found 309.1031. 4.2.7.2. 5-(3-Methoxyphenyl)-7H-naphtho[3,2,1-de]naphthyridine7-one 17b. Yield: 73%, yellow solid, m.p. 214.9  C. 1H NMR (CDCl3): d 3.92 (3H, s, OCH3), 7.08 (2H, d, J ¼ 8.5 Hz, H-30 and H-50 ), 7.66 (1H, dt, J ¼ 1.1, 7.6 Hz, H-9 or H-10), 7.82 (1H, dt, J ¼ 1.1, 7.6 Hz, H-9 or H10), 7.94 (1H, d, J ¼ 7.6 Hz, H-8 or H-11), 8.28 (2H, d, J ¼ 8.5 Hz, H-20 and H-60 ), 8.38 (1H, d, J ¼ 6.9 Hz, H-3), 8.72 (1H, s, CH-6), 8.83 (1H, d, J ¼ 7.6 Hz, H-8 or H-11), 8.89 (1H, d, J ¼ 6.9 Hz, H-2). 13C NMR (CDCl3): d 55.56 (OCH3) 114.57 (2  CHAr), 116.37 (Cquat), 118.03 (CHAr), 122.11 (CHAr), 125.41 (CHAr), 127.92 (CHAr), 129.56 (2  CHAr), 130.57 (Cquat), 130.66 (CHAr), 131.84 (Cquat), 135.04 (CHAr), 136.45 (Cquat), 147.91 (CHAr), 149.58 (Cquat), 150.92 (Cquat), 161.99 (Cquat), 162.14 (Cquat), 183.52 (C]O). IR (ATR): nmax 1668, 1590, 1381, 1255, 1174, 708 cm1. MS: m/z (%) 339 ([M þ H]þ, 100). HRMS (ESþ) calcd. for [C22H15N2O2]þ: 339.1134, found 339.1133. 4.2.7.3. 5-(4-Fluorophenyl)-7H-naphtho[3,2,1-de]naphthyridine-7one 17c. Yield: 64%, yellow solid, m.p. 250.0  C. 1H NMR (DMSOd6): d 7.50 (2H, dd, JHH ¼ 9.0, JHF ¼ 9.1, H-30 and H-50 ), 7.83 (1H, dt,

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J ¼ 1.1, 7.7 Hz, H-9 or H-10), 8.01 (1H, dt, J ¼ 1.1, 7.7 Hz, H-9 or H-10), 8.12 (1H, d, J ¼ 7.2 Hz, H-3), 8.34 (1H, dd, J ¼ 1.1, 7.7 Hz, H-8 or H-11), 8.49e8.56 (2H, dd, JHH ¼ 9.0, JHF ¼ 5.5, H-20 and H-60 ), 8.80 (1H, s, H6), 8.83 (1H, dd, J ¼ 1.1, 7.7 Hz, H-8 or H-11), 9.03 (1H, d, J ¼ 7.2 Hz, H-2). Due to the high insolubility of this compound, no 13C NMR could be recorded. 19F NMR (CDCl3): d 109.95 (1F, s, Cquat-F). IR (ATR): nmax 1667, 1591, 1340, 1226, 759 cm1. MS: m/z (%) 327 ([M þ H]þ, 100). No satisfactory HRMS could be obtained.

Acknowledgments

4.2.7.4. 5-(4-Chlorophenyl)-7H-naphtho[3,2,1-de]naphthyridine-7one 17d. Yield: 64%, yellow solid, m.p. 279.2e280.9  C. 1H NMR (CDCl3): d 7.57 (2H, d, J ¼ 8.5 Hz, H-30 and H-50 ), 7.69 (1H, t, J ¼ 7.5 Hz, H-9 or H-10), 7.88 (1H, t, J ¼ 7.5 Hz, H-9 or H-10), 8.05 (1H, d, J ¼ 5.5 Hz, H-3), 8.28 (2H, d, J ¼ 8.5 Hz, H-20 and H-60 ), 8.41 (1H, d, J ¼ 7.5 Hz, H-8 or H-11), 8.77 (1H, s, H-6), 8.87 (1H, d, J ¼ 7.5 Hz, H-8 or H-11), 8.96 (1H, d, 5.5 Hz, H-2). Due to the high insolubility of this compound, no 13C NMR could be recorded. IR (ATR): nmax 1668, 1590, 1446, 1380, 1367, 1338, 1280, 1231, 1091, 1012, 836, 758, 704 cm1. MS: m/z (%) 343 ([M þ H]þ, 100). HRMS (ESI) calcd. for [C21H11ClN2O]þ: 343.0560, found 343.0626.

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2013.06.010.

4.2.7.5. 5-(2,5-Dimethoxyphenyl)-7H-naphtho[3,2,1-de]naphthyridine-7-one 17e. Yield: 95%, yellow solid, m.p. 225.4  C. 1H NMR (CDCl3): d 3.87 (3H, s, OCH3), 3.89 (3H, s, OCH3), 7.00 (1H, d, J ¼ 8.1 Hz, H-30 ), 7.03 (1H, dd, J ¼ 2.8, 8.1 Hz, H-40 ), 7.53 (1H, d, J ¼ 2.8 Hz, H-60 ), 7.64 (1H, dt, J ¼ 1.1, 7.7 Hz, H-9 or H-10), 7.83 (1H, dt, J ¼ 1.1, 7.7 Hz, H9 or H-10), 7.99 (1H, d, J ¼ 5.5 Hz, H-3), 8.36 (1H, dd, J ¼ 1.1, 7.7 Hz, H-8 or H-11), 8.85 (1H, dd, J ¼ 1.1, 7.7 Hz, H-8 or H-11), 8.85 (1H, s, H-6), 8.90 (1H, d, J ¼ 5.5 Hz, H-3). 13C NMR (CDCl3): d 56.00 (OCH3), 56.35 (OCH3), 113.02 (CHAr), 116.12 (CHAr), 116.55 (Cquat), 117.67 (CHAr), 122.23 (CHAr), 123.73 (CHAr), 125.39 (CHAr), 127.88 (CHAr), 128.58 (Cquat), 130.72 (CHAr), 132.02 (Cquat), 133.82 (Cquat), 134.98 (CHAr), 136.43 (Cquat), 147.62 (CHAr), 149.74 (Cquat), 150.77 (Cquat), 152.06 (Cquat), 154.12 (Cquat), 162.65 (Cquat), 183.67 (C]O). IR (ATR): nmax 1664, 1593, 1499, 1416, 1226, 1035 cm1. MS: m/z (%) 369 ([M þ H]þ, 100). HRMS (ESI) calcd. for [C23H17N2O3]þ: 369.1239, found 369.1244. 4.2.7.6. 5-(3-Methylphenyl)-7H-naphtho[3,2,1-de]naphthyridine-7one 17f. Yield: 91%, yellow solid, m.p. 229.3e230.4  C. 1H NMR (CDCl3): d 2.56 (3H, s, CH3), 7.39 (2H, d, J ¼ 8.0 Hz, H-30 and H-50 ), 7.66 (1H, t, J ¼ 7.7 Hz, H-9 or H-10), 7.82 (1H, t, J ¼ 7.7 Hz, H-9 or H10), 7.99 (1H, d, J ¼ 5.8 Hz, H-3), 8.21 (2H, d, J ¼ 8.0 Hz, H-20 and H60 ), 8.40 (1H, d, J ¼ 7.7 Hz, H-8 or H-11), 8.76 (1H, s, H-6), 8.85 (1H, d, J ¼ 7.7 Hz, H-8 or H-11), 8.92 (1H, d, 5.8 Hz, H-2). 13C NMR (CDCl3): d 21.58 (CH3) 116.59 (Cquat), 118.41 (CH), 122.23 (CH), 125.44 (CH), 127.92 (2  CH), 129.97 (2  CH), 130.73 (CH), 131.90 (Cquat), 135.09 (CH), 135.16 (CH), 135.31 (Cquat), 136.46 (Cquat), 141.48 (Cquat), 147.94 (CH), 149.73 (Cquat), 150.93 (Cquat), 162.58 (Cquat), 183.52 (C]O). IR (ATR): nmax 1665, 1591, 1442, 1377, 1365, 1339, 1285, 1234, 1073, 817, 760, 706 cm1. MS: m/z (%) 323 ([M þ H]þ, 100). HRMS (ESI) calcd. for [C22H14N2O]þ: 323.1106, found 323.1176. 4.2.7.7. 5-tert-Butyl-7H-naphtho[3,2,1-de]naphthyridine-7-one 17h. Yield: 72%, yellow solid, m.p. 168.0  C. 1H NMR (CDCl3): d 1.54 (9H, s, C(CH3)3), 7.61 (1H, dt, J ¼ 1.1, 7.6 Hz, H-9 or H-10), 7.81 (1H, dt, J ¼ 1.1, 7.6 Hz, H-9 or H-10), 7.91 (1H, d, J ¼ 5.8 Hz, H-3), 8.35 (1H, dd, J ¼ 1.1, 7.6 Hz, H-8 or H-11), 8.43 (1H, s, CH-6), 8.81 (1H, dd, J ¼ 1.1, 7.6 Hz, H-8 or H-11), 8.88 (1H, d, J ¼ 5.6 Hz, H-3). 13C NMR (CDCl3): d 30.00 (C(CH3)3), 39.42 (C(CH3)3), 116.11 (Cquat), 118.15 (CHAr), 122.18 (CHAr), 125.25 (CHAr), 127.79 (CHAr), 128.58 (Cquat), 130.54 (CHAr), 131.82 (Cquat), 133.82 (Cquat), 134.52(Cquat), 134.86 (CHAr), 136.29 (Cquat), 147.48 (CHAr), 149.38 (Cquat), 149.99 (Cquat), 175.84 (Cquat), 183.58 (C]O). IR (ATR): nmax 1729, 1661, 1592, 1270, 1227, 705 cm1. MS: m/z (%) 289 ([M þ H]þ, 100). HRMS (ESI) calcd. for [C18H17N2O]þ: 289.1341, found 289.1339.

This work was partially supported by the Research Foundation Flanders (FWO-Vlaanderen) (Grant G.0020.10N) and by funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement FP7-223681. Appendix A. Supplementary data

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