Dany Pechalrieu

Daniel Dauzonne

Paola B Arimondo

Marie Lopez
email: marie.lopez@cnrs.fr

Synthesis of novel 3-halo-3-nitroflavanones and their activities as DNA methyltransferase inhibitors in cancer cells

Keywords: Epigenetics, DNA methylation, flavanone, DNMT inhibitor, anticancer therapy

Highlights

• New 3-halo-3-nitroflavanones to be used as DNMT inhibitors in cancers were prepared using a new synthesis pathway.

• 3-Bromo-3-nitroflavanones show an increased stability compared to the chlorinated analogues.

• The new DNMT inhibitors identified show similar activity on purified DNMT and several of them exhibit an increased efficacy in cells.

Introduction

Epigenetic modifications play an essential role in the establishment and regulation of gene expression. While these changes do not modify the DNA sequence, they regulate gene expression by altering DNA methylation patterns of genes, chemical modifications of histones, expression profiles of RNAs, and nucleosome positioning [START_REF] Bestor | Epigenetic effects in eukaryotic gene expression[END_REF]. Among these modifications, DNA methylation is one of the most studied together with histone acetylation. In cancers, hypermethylation of tumour suppressor gene (TSG) promoters is associated with TSG silencing, which participates to tumour formation, maintenance and progression [START_REF] Baylin | DNA methylation and gene silencing in cancer[END_REF]. DNA methylation is catalysed by DNA methyltransferases (DNMTs), comprising two families of active DNMTs, i.e. DNMT1 and DNMT3, mainly involved in maintenance and de novo DNA methylation, respectively [START_REF] Chen | Structure and function of eukaryotic DNA methyltransferases[END_REF][START_REF] Smith | DNA methylation: roles in mammalian development[END_REF]. The methyl group is added on position 5 of cytosine, mostly at CpG sites, by using S-adenosyl-L-methionine (SAM) as a cofactor. In recent years, a growing number of researches aimed at targeting epigenetic effectors to treat cancer. To date, only two nucleoside DNMT inhibitors (DNMTi), 5-azacytidine and 5-aza-2'-deoxycytidine, received FDA approval for the treatment of leukaemia [START_REF] Christman | 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy[END_REF]. Despite their high efficiency, their mechanism of action requires incorporation into DNA leading to undesired side effects, which, together with chemical instability, limits their use [START_REF] Issa | Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in hematopoietic malignancies[END_REF]. Due to these observations, recent research efforts have been focused on the identification of non-nucleoside DNMTi. Several DNMTi families were reported but to date none show enough potency to be considered as drug candidates [START_REF] Lopez | DNA Methyltransferase Inhibitors: Development and Applications[END_REF][START_REF] Pechalrieu | DNA methyltransferase inhibitors in cancer: From pharmacology to translational studies[END_REF]. Our group previously carried out a screen and identified 3-chloro-3nitroflavanones as potent inhibitors of murine Dnmt3A/3L complex [START_REF] Ceccaldi | C5-DNA Methyltransferase Inhibitors: From Screening to Effects on Zebrafish Embryo Development[END_REF]. However, despite these promising results, these flavonoid compounds were not potent enough and were shown to be too unstable to be considered as suitable potential drug candidates, due to the possibility of undergoing a β-elimination with the loss of HCl to lead to the corresponding 3nitroflavones. This encouraged us to pursue our research on new flavonoid DNMTi, making a breakthrough in the design and synthesis to improve potency and compound stability. Here, we report the synthesis and biological evaluation of new flavonoid DNMTi. Reprogramming cells by restoring a normal DNA methylation pattern instead of killing them is the purpose of DNMTi. Therefore, stability and cell toxicity of the compounds was also measured to assure that an epigenetic effect is observed without the cytotoxicity associated with conventional chemotherapies.

Results and discussion

Chemical synthesis

Aiming at optimising the flavanone biological activity and chemical stability, and considering that 3-chloro-3-nitroflavanones are susceptible to HCl-elimination leading to corresponding inactive flavones, we modulated the substitution of position 3 of the 3-chloro-3nitroflavanones. As we previously showed, the nitro group is crucial for inhibition activity [START_REF] Ceccaldi | C5-DNA Methyltransferase Inhibitors: From Screening to Effects on Zebrafish Embryo Development[END_REF][START_REF] Menon | Modifications of DNA methylation by non-nucleoside inhibitors induce specific chromatin changes[END_REF], thus this group was maintained and we focused on the replacement of the chloride atom (Figure 1).

Figure 1. General structure of target 3-halo-3-nitroflavanones 1 -11

In addition to a potential improvement of activity, we expected these new compounds to be more stable, limiting their ability to undergo β-elimination, which leads to inactive flavones. Such modification has never been reported before. Therefore, we first adapted the synthesis pathway described for the synthesis of 3-chloro-3-nitroflavanones [START_REF] Dauzonne | Synthesis of 2-Aryl-3-nitro-4H-1-benzopyran-4-ones[END_REF][START_REF] Dauzonne | Synthesis and in vitro cytotoxicity of a series of 3-aminoflavones[END_REF] to the preparation of 3-methyl-3-nitro-(1) and 3-bromo-3-nitro-(2) substituted compounds (Scheme 1). a) KF (0.2 equiv.), xylene, reflux, 7h; b) salicylaldehyde (5 equiv.), Et 3 N (0.7 equiv.), THF, rt; c) PCC (2 equiv.), THF, rt, sonication.

The formation of nitrostyrenes (12 and 13) was carried out using commercially available nitroethane [START_REF] Fioravanti | Facile and highly stereoselective one-pot synthesis of either (E)-or (Z)-nitro alkenes[END_REF] or bromonitromethane, respectively. This condensation reaction leads exclusively to nitrostyrenes with the nitro group and the aromatic ring located on the opposite sides of the double bond (Z-2-bromo-2-nitrostyrene and E-2-methyl-2-nitrostyrene). For the formation of methylstyrene derivative 12, the yield was quite low (21%) as the secondary carbon of nitroethane is much less electrophile compared to chloronitromethane or bromonitromethane. The structure of compound 12 was confirmed by crystal structure determination (Figure 2, Table SI- Then, cyclisation via a Henry-type reaction with an ortho-hydroxybenzaldehyde results in the corresponding flavan-4-ol 14 and 15. In some cases cyclisation yields were relatively low due to the possible retrocyclisation as previously reported [START_REF] Dauzonne | A Convenient Synthesis of 3-Chloro-3,4-dihydro-4hydroxy-3-nitro-2-phenyl-2H-1-benzopyrans[END_REF], occurring in basic conditions in presence of traces of water, which gives back the starting materials. Finally, oxidation with PCC using ultrasonic assistance, gave the expected flavanones 1 and 2. These compounds are stable as long as stored dry or in an organic solvent (e.g. DMSO), exempt from any trace of water.

H O H X NO 2 X NO 2 O OH X + NO 2 a b c 12 X = -CH 3 , R' = -NO 2 , 21% 13 X = Br, R' = H, 32% R' R' R' 14 X = -CH 3 , R' = -NO 2 , 24% 15 X = Br, R' = H 1 X = -CH 3 , R' = -NO 2, 61% 2 X = Br, R' = H, 34% over 2 steps (racemate) (racemate) O O X NO 2 R'
Although this synthesis pathway allowed access the target molecules, yields were quite low. Therefore, we next sought for a more versatile method in order to diversify the nature of the substituents on carbon-3 with better yields. Two publications from 1968 and 1981 [START_REF] Michalska | Chemical transformations of isonitrosoflavanone. I. Synthesis of 3nitroflavone[END_REF][START_REF] Paparao | A One-Step Synthesis of 3-Nitroflavanones and their Conversion to 3-Nitroflavones[END_REF] reported the synthesis of 3-bromo-3-nitroflavanones as intermediates for the synthesis of flavone analogues, by bromination of 3-nitroflavanones. We decided to adapt this strategy for the synthesis of our target compounds (Scheme 2).

Scheme 2. Synthesis pathway of 3-halo-3-nitroflavanones.

a) aqueous NaOH solution (5%, w/w, 8 equiv.), 60 °C, 2 h; b) benzaldehyde derivative (1.0 equiv.), Na 2 CO 3 (1.5 equiv.) L-Proline (0.5 equiv.), EtOH, 40 °C, 5 h to o/n; c) NFSI, NBS or NIS (1.7 equiv.), Na 2 CO 3 (1.3 equiv.), THF, rt, 1-5 h.

First, 2'-hydroxy-2-nitroacetophenone 16 was prepared from commercially available 4hydroxy-3-nitrocoumarin by treatment with an aqueous sodium hydroxide solution. Then, cyclisation in basic conditions afforded 3-nitroflavanones 17-20. Finally, halogenation with N-bromosuccinimide (NBS) afforded expected compounds 2', 3-5 in good to high yields.

Noteworthy, contrarily to the first synthesis pathway, here both diastereoisomers can be obtained, since halogenation can occur on both sides of the molecules. For compounds 3 and 4 both diastereoisomers (3a (anti)-3b (syn) and 4a (anti)-4b (syn)) were separated by flash chromatography, then were tested separately to compare their activity and stability.

Compound 2' was formed as a mixture of syn/anti diastereoisomers. A majority of the syndiastereoisomer was obtained as confirmed by comparison of 1 H and 13 C NMR signals with those of compound 2 obtained via the first synthesis pathway (Scheme 1) and thus via the Z-2-bromo-2-nitrostyrene intermediate. Noteworthy, H-2 signal is more deshielded for the syndiastereoisomer (δ = 6.08 ppm) than for the anti-analogue (δ = 5.75 ppm) and C-2 signal shifted from δ = 82.9 ppm for the syn-isomer to δ = 86.9 ppm for the anti-analogue. For compound 5 only one diastereoisomer was formed and the relative stereochemistry syn was confirmed by crystal structure determination (Figure 3). In order to explore more halide

O NO 2 OH O OH NO 2 O H O R 2 R 1 R 1 O O NO 2 R 2 2': X = Br, R 1 = H, R 2 = H 3: X = Br, R 1 = NO 2 , R 2 = H 4: X = Br, R 1 = NO 2 , R 2 = OMe 5: X = Br, R 1 = F, R 2 = H R 1 O O NO 2 R 2 X a b c 16 17-20 6: X = F, R 1 = H, R 2 = H 7: X = F, R 1 = NO 2 , R 2 = H 8: X = F, R 1 = NO 2 , R 2 = OMe 9: X = F, R 1 = F, R 2 = H 10: X = I, R 1 = NO 2 , R 2 = H 11: X = I, R 1 = NO 2 , R 2 = OMe 2', 3-11 (racemate)
substitutions on 3-position of the flavanone, N-fluorobenzenesulfonimide (NFSI) was used instead of NBS to access fluoride-substituted compounds 6-9 [START_REF] Cui | Facile Stereoselective Synthesis of Fluorinated Flavanone Derivatives via a One-Pot Tandem Reaction[END_REF]. Crystal structure determination and NMR studies showed that compounds 6 and 9 were obtained as anti isomer, compound 7 as syn isomer. Compound 8 was obtained as the anti diastereoisomer but with an unidentified impurity that could not be separated. 3-Iodo-3-nitroflavanones 10-11 were prepared similarly using N-iodosuccinimide. In this case, only syn diastereoisomers were obtained as determined by NMR spectra. 1 H NMR aromatic signals, especially H-6' and H-3' signals, of 10 are similar to those obtained for chlorine (Flv69) and bromine (3b) analogues and significantly different from the anti-derivative 3a (spectra in supporting information).

Similarly, the aromatic region of compound 11 matches the one of Flv880 and 4b, synconformation, and is different from compound 4a, anti-conformation.

For compounds 3a, 3b, 5 and 9, the structure was confirmed by crystal structures (Figure 3) with a good resolution (Tables SI-1 to SI-4). These structure determinations provided the relative configuration of the compounds obtained as a racemic mixture.

Interestingly, crystal structures showed that the ring C always adopts a half-chair conformation, with the phenyl ring (ring B) in equatorial position.

Next, the stability of the synthesised compound was measured and compared to that of the previously prepared 3-chloro-3-nitroflavanones Flv69 [START_REF] Ceccaldi | C5-DNA Methyltransferase Inhibitors: From Screening to Effects on Zebrafish Embryo Development[END_REF] and Flv880 [START_REF] Menon | Modifications of DNA methylation by non-nucleoside inhibitors induce specific chromatin changes[END_REF].

Chemical stability assays

The stability experiments were carried out in the assay buffer used for DNMT3A-c methylation tests and were followed by HPLC (Figure 4). Percentage of remaining compound is followed by HPLC analysis at 250 nm and the mean of at least 2 experiments is reported.

3-Fluoro-3-nitroflavanones 7-8 (Figure 4 green curves) and 6, 9 (data not shown) are totally instable in the enzymatic buffer, even at t = 0 min no remaining compound was detected by HPLC. Brominated derivative 3b showed a marginal increase in stability with half-life time of 1.7 h (Figure 4A) when compared to the chlorinated compound Flv69 presenting a half-life time of 1 h. More interestingly, for compounds bearing 2'-nitro-5'-methoxy substitution on cycle B, i.e. Flv880 and 4b (Figure 4B), the stability of brominated compound 4b is highly increased and the half-time life is more than doubled going from 3.4 h for Flv880 to 7 h for compound 4b. In addition, the stability of both diastereoisomers 3a and 3b is compared in Figure 4A (grey circles). Whereas syn compound 3b exhibits a half-life time of 1.7 h, this value drops down to 0.7 h for anti derivative 3a. This observation is of high interest as it shows that syn derivative 3b, with an antiperiplanar orientation of the C 3 -Br and C 2 -H bonds, is the most stable diastereoisomer (Figure 4). This presumes that, in the case of brominated derivatives, the instability is not due to a β-elimination as it was observed for the 3-chloro-3nitroflavanones, since such a reaction would be favoured by an antiperiplanar configuration of the C 3 -Br and C 2 -H bonds. In such a hypothesis, compound 3b should then be less stable than compound 3a. Finally, both compounds containing an iodine atom 10 and 11 are extremely more stable than their brominated and a fortiori chlorinated analogues with no degradation observed after 15 h.

Considering this stability results, only brominated and iodinated were further evaluated for their biological activities.

Biological results

This new library of 3-halo-3-nitroflavanones was first tested to evaluate the methylation inhibition activities using purified human DNMT3A-c. Second, a luciferase reexpression cellular model assay dependent on promoter DNA methylation was carried out with promising inhibitors. Finally, their cytotoxicity in different cancer cell lines was evaluated.

DNMT inhibition

The results of the inhibition of hDNMT3A-c by 3-nitroflavanones fact that inhibition activity is independent from the relative stereochemistry of carbon-2 and -3 of these 3-halo-3-nitroflavanones. Regarding 3-iodo-3-nitroflavanones 10 and 11, inhibition activity is slightly improved compared to 3-bromo-and 3-chloro-analogues with EC 50 = 2.8 ± 1.6 µM and 2.0 ± 1.6 µM for 10 and 11, respectively.

In summary, first the substitution of the chlorine atom on 3-position of ring C of the 3-chloro-3-nitroflavanones does not improve the in-vitro DNMT inhibition activity, which is maintained at micromolar levels. Second, maintenance of micromolar activity was observed for all the 3-halo-3-nitroflavanones compared to the chlorinated reference, with slightly better activity for iodinated compounds. Importantly, when both diastereoisomers were obtained, they showed the same level of in-vitro DNMT inhibition activity.

Cellular activity in a reporter gene reexpression model

Next, cell activity was evaluated in KG-1 cells, stably transfected with the firefly luciferase reporter gene under the control of a methylated cytomegalovirus (CMV) promoter. DNMTi, decrease promoter methylation inducing an increase of the luciferase expression, which is measured by luminescence [START_REF] Rilova | Design, Synthesis and Biological Evaluation of 4-Amino-N-(4-aminophenyl)benzamide Analogues of Quinoline-Based SGI-1027 as Inhibitors of DNA Methylation[END_REF]. Results are reported in induction fold (IF) as a ratio of the luminescence level in the presence of the inhibitor to DMSO-treated cells. Thus, the more active is the inhibitor, the higher the IF (Figure 5). (3b) at 5 µM compared to its chlorinated analogue Flv69 (IF = 2.0 at 5 µM). In summary, iodinated compounds 10-11, despite good in vitro inhibition activities, did not show any activity in cells. Interestingly, brominated analogues (3a-3b), with an IF 3-fold higher than parent compounds Flv69 and Flv880, are now the most promising flavonoid DNMTi.

Antiproliferative activity in cancer cell lines

In the field of epigenetic reprogramming, the Holy Grail is to identify compounds with inhibition activities on purified enzymes as well as capability of reinducing targeted gene expression in cellular models with no or weak cell cytotoxicity at the used concentrations.

This strategy, limiting cytotoxic effects and favouring cell reprogramming through tumour suppressor genes reexpression, will be a real breakthrough in the development of anticancer therapies. This reprogramming will allow epi-drug to be used in combination as anticancer treatments, while minimizing the severe side effects observed with cytotoxic anticancer treatments. We showed in this work that compounds 3a-4b, maintain in vitro inhibition activity and a higher capability to reinduce the expression of the luciferase gene in a cellular gene reporter model compared to their previously reported chlorinated analogues. We also demonstrated that, contrarily to fluorinated compounds 6-9, brominated compounds 3a-4b

have a good stability in physiological conditions. Therefore, compounds 3a-4b were evaluated for their cytotoxicity in colorectal cancer HCT116 and leukaemia KG1 cell lines after 72 h of daily treatment (Figure 6). Cell cytotoxicity ranges from micromolar to high micromolar with EC 50 values from 5.3 to 18.6 µM (Figure 6). Notably, cytotoxicity is similar for KG1 and HCT116 cell lines, respectively a leukaemia, haematological cancer cell lines, and a colorectal, i.e. solid tumour cancer cell line. For all compounds, the cytotoxicity value is higher than the in vitro EC 50 value on purified enzyme and higher than their active concentration in the reexpression cellular model. More interestingly, compounds 3b and 4a, exhibited lower cytotoxicity with EC 50 = 13.0 and 18.6 µM, respectively, in parallel with the lower in vitro EC 50 on the purified DNMT3A-c 3.2 and 4.7 µM, respectively. These compounds show the highest difference between the active concentrations and EC 50 cytotoxicity values. Although this difference is moderate it means that compounds exhibit DNMT inhibition activity and luciferase reexpression capability at concentrations at which they have limited cytotoxic effects. This is the first step toward the identification of low-cytotoxic cell-reprograming epi-drugs. In conclusion, we developed a new versatile synthesis pathway to provide access to 3-halo-3nitroflavanones and prepared a library of compounds. The structure was confirmed by crystallography and several diastereoisomers could be isolated. We assessed the stability of the compounds and showed that, with half-life time up to 7 h, 3-bromo-3-nitroflavanones analogues were twice more stable than the chlorinated analogues. Regarding 3-iodo-3nitroflavanones, they reveal a very high stability since they are totally stable up to 15 h. However, they unfortunately did not show any activity in cells. Four 3-bromo-3nitroflavanones, 3a-4b, exhibited the best profile as DNMTi for cell reprogramming that is to say they are active against the purified DNMT3A-c, induced gene expression in a cellular model much more efficiently than reference compounds and showed moderate cytotoxicity at active concentrations. This last point is crucial for epi-drugs and is a breakthrough in the way to think anticancer therapies as ideally, "epi-drugs" reprogram cell profile without being cytotoxic. Among these four 3-bromo-3-nitroflavanones, compounds 3b and 4a were identified as the most potent compounds and constitute new non-nucleoside DNMTi with good activity and stability that can now be considered as reference DNMTi for the future developments of new "epi-drugs" targeting DNA methylation.

Conclusion

Experimental section

Chemistry

Material and methods

All starting materials and reagents were purchased from commercial suppliers, Sigma Aldrich, Alfa Aesar or TCI. Solution of Girard's reagent was prepared by dilution of Girard's reagent (3.9 g) in 30 ml of EtOH/AcOH/H 2 O (8/1/1). All reactions were monitored by thin layer chromatography (TLC) and TLC plates were visualized with UV light. Flash silica chromatographies were performed on a Puriflash 430 apparatus (Interchim) using 30-µm spherical silica columns. 1 H NMR at 400 MHz and 13 C NMR at 100 MHz were acquired on a Bruker Avance 400 instrument. 1 H NMR at 500 MHz and 13 C NMR at 125 MHz were acquired on a Bruker Avance II instrument equipped with 13 C cryoprobe. For 1 H and 13 C NMR acquired in CDCl 3 chemical shifts (δ) are reported in ppm relative to the solvent residual peak: proton (δ = 7.26 ppm) and carbon (δ = 77.2 ppm). 1 H NMR and 13 C NMR spectras were assigned using 2D NMR experiments, such as, 1 H-1 H gCOSY, 1 H- 13 C HSQC and 1 H- 13 C HMBC experiments. Multiplicity is indicated as follows: s (singlet); d (doublet); t (triplet); q (quartet); m (multiplet); dd (doublet of doublets); td (triplet of doublets); qd (quartet of doublets); ddd (doublet of doublets of doublets); tdd (triplet of doublets of doublets); br (broad). Coupling constants (J) are reported in Hertz (Hz). High and low resolution electrospray ionization mass spectra were acquired using electrospray as the ionization technique in positive ion and/or negative ion modes as stated. All MS analysis samples were prepared as solutions in MeOH. Compound purity was verified to be higher than 95% using reversed-phase HPLC on an X-terra C18 MS column (3.9 100 mm; Waters) with a linear gradient 0 to 95% CH 3 CN in H 2 O, eluents containing 0.01% formic acid. The Xray crystallographic structural determinations were performed at the Service de Diffraction des Rayons X, Laboratoire de Chimie de Coordination, Toulouse, France.

Numbering of flavonoid compounds will be done as below Chemical synthesis

syn-3-Chloro-2,3-dihydro-3-nitro-2-(2'-nitrophenyl)-4H-1-benzopyran-4-one (Flv69)

The Flv69 was obtained in 3 steps following a previously described procedure [START_REF] Dauzonne | Synthesis of 2-Aryl-3-nitro-4H-1-benzopyran-4-ones[END_REF][START_REF] Dauzonne | Synthesis and in vitro cytotoxicity of a series of 3-aminoflavones[END_REF]. The Flv880 was obtained in 3 steps according to a previously described procedure [START_REF] Menon | Modifications of DNA methylation by non-nucleoside inhibitors induce specific chromatin changes[END_REF]. The compound 14 (46 mg, 0.14 mmol, 1.0 equiv.) was solubilised in anhydrous THF (2 ml).

After addition of pyridinium chlorochromate (151 mg, 0.70 mmol, 5.0 equiv.), the roundbottom flask was equipped with a condenser and a drying tube and the mixture was submitted to sonication at 40 °C for 5 h. The reaction mixture was filtered, the precipitate was rinsed with CH 2 Cl 2 and the filtrate was concentrated under vacuum. The residue was purified by flash chromatography using a linear gradient (cyclohexane/EtOAc; 100/0 to 50/50) to give the title compound 1 (28 mg, 0.08 mmol, 61% yield) as a white solid.

syn-3-Bromo-2,3-dihydro-2-phenyl-3-nitro-4H-1-benzopyran-4-one (2)

The title compound 2 was synthesised following the procedure described for the synthesis of compound

anti/syn-3-Bromo-2,3-dihydro-2-phenyl-3-nitro-4H-1-benzopyran-4-one (2')

The title compound 2' was synthesised from the compound 17 (54 mg) following the general procedure 1. The title compound 2' (65 mg) was obtained as a mixture of syn/anti diastereoisomers (75/25) as a white solid with 93% yield. R f = 0.40 (EtOAc/cyclohexane 1:5).

ESI-MS

General procedure 1-Synthesis of 3-halo-3-nitro-flavanones 3-11

The appropriate 3-nitroflavanone 17 to 20 (1.0 equiv.) was solubilised in anhydrous THF under Ar. The appropriate halogenating agent, N-fluorobenzenesulfonimide, Nbromosuccinimide or N-iodosuccinimide, (1.7 equiv.) and Na 2 CO 3 (1.3 equiv.) were added.

The reaction mixture was stirred at rt until disappearance of starting material, typically 2-5 h.

After addition of CH 2 Cl 2 and H 2 O, the aqueous layer was acidified to pH 6 using a 10% citric acid solution and was extracted with CH 2 Cl 2 (x3). The combined organic layers were dried over sodium sulfate, filtered and concentrated under vacuum. Purification by flash chromatography using a linear gradient (cyclohexane/EtOAc 100:0 to 85:15) afforded the final 3-halo-3-nitroflavanones [START_REF] Chen | Structure and function of eukaryotic DNA methyltransferases[END_REF][START_REF] Smith | DNA methylation: roles in mammalian development[END_REF][START_REF] Christman | 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy[END_REF][START_REF] Issa | Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in hematopoietic malignancies[END_REF][START_REF] Lopez | DNA Methyltransferase Inhibitors: Development and Applications[END_REF][START_REF] Pechalrieu | DNA methyltransferase inhibitors in cancer: From pharmacology to translational studies[END_REF][START_REF] Ceccaldi | C5-DNA Methyltransferase Inhibitors: From Screening to Effects on Zebrafish Embryo Development[END_REF][START_REF] Menon | Modifications of DNA methylation by non-nucleoside inhibitors induce specific chromatin changes[END_REF][START_REF] Dauzonne | Synthesis of 2-Aryl-3-nitro-4H-1-benzopyran-4-ones[END_REF].

anti-3-Bromo-2,3-dihydro-3-nitro-2-(2'-nitrophenyl)-4H-1-benzopyran-4-one (3a) and syn-3-Bromo-2,3-dihydro-3-nitro-2-(2'-nitrophenyl)-4H-1-benzopyran-4-one (3b)

R 1 O O NO 2 R 2 R 1 O O NO 2 R 2 X c

17-20 3-11 (racemate)

The title compounds 3a and 3b were synthesised simultaneously from the 3-nitroflavanone 18 (54 mg) following the general procedure 1. The expected 3-bromo-3-nitroflavanones 3a (41 mg) and 3b (207 mg) were separated by flash chromatography and obtained as white solids with an overall yield of 75%. R f = 0.44 (3b), 0.50 (3a) (cyclohexane/EtOAc 5:1). ESI-MS:

m/z = 415 [M+Na] + .
Compound

anti-3-Bromo-2,3-dihydro-2-(5'-methoxy-2'-nitrophenyl)-3-nitro-4H-1-benzopyran-4-

one (4a) and syn-3-Bromo-2,3-dihydro-2-(5'-methoxy-2'-nitrophenyl)-3-nitro-4H-1-

benzopyran-4-one (4b)

The title compounds 4a and 4b were synthesised simultaneously from the 3-nitroflavanone 19 (141 mg) following the general procedure 1. The expected 3-bromo-3-nitroflavanones 4a (15 mg) and 4b (98 mg) were separated by flash chromatography and obtained as white solids with a global yield of 66%. R f = 0.35 (4b), 0.44 (4a) (cyclohexane/EtOAc 5:1). ESI-MS: m/z = 445 [M+Na] + .

Compound 4a

1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.15 (d, J = 9.2 Hz, 1H, H-3'); 8.12 (dd, J = 1.6, 7.9 Hz, 1H, H-5); 7.71 (td, J = 1.9, 7.9 Hz, 1H, H-7); 7.30 (td, J = 0.8, 7.6 Hz, 1H, H-6); 7.25 (s, 1H, H-2); 7.19 (dd, J = 0.8, 8.4 Hz, 1H, H-8); 7.17 (d, J = 2.7 Hz, 1H, H-6'); 7.07 (dd, J = 2.7, 9.2 Hz, 1H, H-4'); 3.91 (s, 1H, OCH 3 ).

syn-3-Bromo-2,3-dihydro-2-(2'-fluorophenyl)-3-nitro-4H-1-benzopyran-4-one (5)

The title compound 5 was synthesised from the 3-nitroflavanone 20 (54 mg) following the general procedure 1. The expected 3-bromo-3-nitroflavanone 5 (32 mg) was obtained as a white solid with 46% yield. R f = 0.51 (cyclohexane/EtOAc 6:4).

anti-3-Fluoro-2,3-dihydro-2-phenyl-3-nitro-4H-1-benzopyran-4-one (6)

The title compound 6 was synthesised from the 3-nitroflavanone 17 (64 mg) following the general procedure 1. The expected 3-fluoro-3-nitroflavanone 6 (46 mg) was obtained as a white solid with 67% yield. R f = 0.50 (cyclohexane/EtOAc 3:2). ESI-MS: m/z = 310

[M+Na] + . 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.06 (dd, J = 1.7, 7.9 Hz, 1H, H-5); 7.73 (td, J = 1.8, 7.9 Hz, 1H, H-7); 7.49-7.46 (m, 5H, H-2', H-3', H-4', H-5', H-6'); 7.27 (td, J = 1.1, 7.6 Hz, 1H, H-6); 7.23 (dd, J = 0.7, 8.5 Hz, 1H, H-8); 5.67 (d, J = 6.0 Hz, 1H, H-2).

syn-3-Fluoro-2,3-dihydro-3-nitro-2-(2'-nitrophenyl)-4H-1-benzopyran-4-one (7)

The title compound 7 was prepared from the 3-nitroflavanone 18 (110 mg) following the

anti-3-Fluoro-2,3-dihydro-2-(5'-methoxy-2'-nitrophenyl)-3-nitro-4H-1-benzopyran-4-

one (8)

The title compound 8 was synthesised from the nitroflavanone 19 (131 mg) following the general procedure 1. The expected 3-fluoro-3-nitroflavanone 8 was obtained with 53% purity.

anti-3-Fluoro-2,3-dihydro-2-(2'-fluorophenyl)-3-nitro-4H-1-benzopyran-4-one (9)

The title compound 9 was synthesised from the 3-nitroflavanone 20 (54 mg) following the general procedure 1. The expected 3-fluoro-3-nitroflavanone 9 (44 mg) was obtained as a white solid with 77% yield. R f = 0.52 (cyclohexane/EtOAc 6:4). ESI-MS: m/z = 328

[M+Na] + . 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.08 (dd, J = 1.7, 7.9 Hz, 1H, H-5); 7.72 (td, J = 1.7, 7.8 Hz, 1H, H-7); 7.53-7.46 (m, 2H, H-4', H-6'); 7.29-7.24 (m, 2H, H-6, H-5'); 7.22-7.17 (m, 2H, H-8, H-3'); 6.08 (d, J = 4.0 Hz, 1H, H-2).

syn-3-Iodo-2,3-dihydro-3-nitro-2-(2'-nitrophenyl)-4H-1-benzopyran-4-one (10)

The title compound 10 was synthesised from the 3-nitroflavanone 18 (105 mg) following the general procedure 1. The expected 3-iodo-3-nitroflavanone 10 (83 mg) was obtained as a white solid with 56% yield. R f = 0.52 (cyclohexane/EtOAc 6:4). 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.12 (br d, J = 7.9 Hz, 1H, H-6'); 8.07 (dd, J = 1.6, 7.9 Hz, 1H, H-5); 7.91 (dd, J = 1.0, 8.1 Hz, 1H, H-3'); 7.75 (br t, J = 7.9 Hz, 1H, H-5'); 7.71-7.64 (m, 2H, H-7, H-4'); 7.28 (br t, J = 7.3 Hz, 1H, H-6); 7.13 (d, J = 8.3 Hz, 1H, H-8); 6.14 (s, 1H, H-2).

syn-3-Iodo-2,3-dihydro-2-(5'-methoxy-2'-nitrophenyl)-3-nitro-4H-1-benzopyran-4-one (11)

The title compound 11 was synthesised from the 3-nitroflavanone 19 (72 mg) following the general procedure 1. The expected 3-iodo-3-nitroflavanone 11 (24 mg) was obtained as a white solid with 24% yield. R f = 0.57 (cyclohexane/EtOAc 1:2). ESI-MS: m/z = 493 The 2-nitrobenzaldehyde (1.1 g, 6.95 mmol, 1.0 equiv.) was solubilised in toluene (10 ml) in a round-bottom flask equipped with a Dean-Stark apparatus. Dimethylamine hydrochloride (5.4 g, 66.3 mmol, 9.5 equiv.), potassium fluoride (1.1 g, 18.0 mmol, 2.6 equiv.) and nitroethane (1.5 ml, 29.9 mmol, 1.5 equiv.) were added and the mixture was refluxed for 10 h. The reaction mixture was diluted in CH 2 Cl 2 and washed with water (x2). The aqueous layers were backextracted with CH 2 Cl 2 (x2). The combined organic layers were dried over sodium sulfate and concentrated under vacuum. The residue was purified by flash chromatography using a linear gradient (cyclohexane/EtOAc; 100/0 to 50/50) to give compound 12 (303.8 mg, 1.46 mmol, 21% yield) as slightly yellow oil. R f = 0.25 (cyclohexane/EtOAc 5:2). The benzaldehyde (1 ml, 9.89 mmol, 1.0 equiv.) was diluted in toluene (25 ml) containing 4Å activated molecular sieves. Bromonitromethane (0.7 ml, 10.4 mmol, 1.04 equiv.) and piperidine (0.06 ml, 0.61 mmol, 0.06 equiv.) were added and the mixture was refluxed for 6 h.

The reaction mixture was filtered and filtrate was concentrated under vacuum. The residue was purified by flash chromatography using a linear gradient (cyclohexane/EtOAc 100:0 to 50:50) to give compound 13 (714 mg, 3.13 mmol, 32% yield) as a bright orange oil, which tends to crystallize. R f = 0.54 (cyclohexane/EtOAc 3:1). The compound 12 (206 mg, 1.0 mmol, 1.0 equiv.) was put under argon in a round-bottom flask dried under the flame and was solubilised in anhydrous THF (2 ml). After addition of the salicylaldehyde (0.6 ml, 5.6 mmol, 5.6 equiv.) and the Et 3 N (0.1 ml, 0.72 mmol, 0.7 equiv.), reaction was stirred overnight at room temperature under Ar. After neutralization with acetic acid (0.15 ml), Girard's reagent solution was added and the reaction mixture was stirred for 2 h at rt before extraction with CH 2 Cl 2 . The organic layer was washed with H 2 O and aqueous layer was then backextracted with CH 2 Cl 2 (x2). The combined organic layers were dried with sodium sulfate, filtered and concentrated under vacuum. The crude mixture was purified by flash chromatography using a linear gradient (cyclohexane/EtOAc; 100/0 to 50/50) to give compound 14 (80 mg, 0.24 mmol, 24% yield) as slightly yellow oil. 1H, H-7); 7.13 (td, J = 1.6, 7.6 Hz, 1H, H-6); 6.93 (dd, J = 0.9, 8.2 Hz, 1H, H-8); 6.32 (s, 1H, H-2) 6.02 (s, 1H, H-4); 2.89 (br s, 1H, OH); 1.62 (s, 3H, CH 3 ).

3-Bromo-3,4-dihydro-4-hydroxy-3-nitro-2-phenyl-2H-1-benzopyran (15)

The title compound 15 was synthesised following the procedure described for the synthesis of compound 14 starting from compound 13 (49 mg, 0.22 mmol, 1.0 equiv.), using salicylaldehyde (0.12 ml, 1.13 mmol, 5.1 equiv.) and Et 3 N (15 µl, 0.11 mmol, 0.5 equiv.).

After purification by flash chromatography using a linear gradient (cyclohexane/EtOAc 100:0 to 80:20), the title compound 15 was obtained as a mixture of both diastereoisomers used as such for the synthesis of compound 2 (Method 1). R f = 0.29 (EtOAc/cyclohexane 1:5).

1-(2-hydroxyphenyl)-2-nitroethanone (16)

The 4-hydroxy-3-nitrocoumarine (3.12 g, 15.1 mmol, 1 equiv.) was solubilised in NaOH solution (5%, w/v in H 2 O, 100 ml). The reaction mixture was stirred for 2 h at 60 °C and turned from yellow to bright dark orange. The mixture was cooled down in ice and acidified by slow addition of acetic acid (30 ml). The precipitate was filtered to give the expected compound 16 (2.35 g 13.0 mmol, 86% yield) as an off-white solid.

General procedure 2 -Synthesis of 3-nitroflavanones 17-20

The compound 16 (1.0 equiv.) was solubilised in absolute ethanol. The appropriate benzaldehyde (1.0 equiv.), D-proline (0.5 equiv.) and Na 2 CO 3 (1.5 equiv.) were added to the mixture and the reaction was stirred overnight at rt. After dilution in CH 2 Cl 2 , compound was extracted from organic layer with H 2 O. Aqueous layer was acidified to pH 2-3 using HCl 1M and extracted with CH 2 Cl 2 (x3). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under vacuum. The crude mixture was purified by flash chromatography using a linear gradient (CH 2 Cl 2 /MeOH 100:0 to 70:30) to give expected 3nitroflavanones 17-20.

trans-2,3-Dihydro-2-phenyl-3-nitro-4H-1-benzopyran-4-one (17)

The title compound 17 was synthesised from the compound 16 (299 mg, 1.65 mmol) and benzaldehyde (0.17 ml, 1.68 mmol), following the general procedure 2. The title compound 17 (301 mg, 1.12 mmol) was obtained after flash chromatography as a white solid with 68%

O NO 2 OH H O R 2 R 1 R 1 O O NO 2 R 2 16

Stability measurement

The compound stability was measured by HPLC analysis using an X-terra column (100

Biology

DNMT3A-c expression and purification

The human catalytic DNMT3A (DNMT3A-c) was expressed in Escherichia coli Rosetta-gami™ 2(DE3) pLysS competent cells (Novagen) carrying the plasmid pET28a-DNMT3A(624-912) containing a 6-His tag used by Gros et al. [START_REF] Gros | Development of a universal radioactive DNA methyltransferase inhibition test for highthroughput screening and mechanistic studies[END_REF]. Briefly, after bacterial transformation and preculture, cells were grown in lysogeny broth (Lennox) supplemented with 25 µg/mL of kanamycin and 20 µg/mL of chloramphenicol at 37 °C to OD 600 nm ≈ 0.5.

Then, culture was cooled down to 20 °C and induced overnight with 1 mM isopropyl β-D-1thiogalactopyranoside or cooled down to 34 °C and induced during 4 hours. Protein purification was carried out at 4 °C based on Gowher et al protocol [START_REF] Gowher | Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpG [correction of non-CpA] sites[END_REF]. Polyhistidine-tagged proteins were purified by affinity chromatography, on Ni-NTA agarose resin (Qiagen).

Coomassie-stained SDS-PAGE gel was used to evaluate the purity more than 90% and the DNMT3A-c concentration was determined by Bradford assay or by using NanoDrop 2000 spectrophotometer (Thermo Scientific) with an estimated extinction coefficient.

DNMT inhibition assay

Compound activities were determined with a fluorescence-based assay [START_REF] Ceccaldi | C5-DNA Methyltransferase Inhibitors: From Screening to Effects on Zebrafish Embryo Development[END_REF]. In brief, a doublestrand DNA, comprising a unique CpG site overlaying an endonuclease restriction site for methylation-sensitive enzyme was used. This oligonucleotide comprises a 6-FAM at one end and biotin on the other end allowing immobilization into a 384-well plate (Perkin Elmer) precoated with avidin. Compounds to be evaluated and SAM as methyl donor were added followed by DNMT3A-c to start the methylation reaction, which was prolonged 1 h at 37 °C.

After several washing, with PBS tween (0.05 %) containing NaCl (0.5 M) and PBS tween (0.05 %). restriction step was performed with HpyCH4IV (New England, BioLabs) to hand on only the specific fluorescence signal. Fluorescence was quantified on an EnVision 2103 Multilabel Reader (Perkin Elmer). Inhibition percentages are defined as 100x(100-(X meth -X restri )/(X DNA -X restri ), where X meth , X restri and X DNA are respectively the fluorescence signals of the compound methylation, restriction and DNA controls. These percentages for a concentration range were then used to calculate the half maximal effective concentration (EC 50 ) (average of at least 2 experiments) for the compound of interest by using the nonlinear regression with sigmoidal dose-response (variable slope) tool of GraphPad Prism software.

Luciferase expression measurement in cmv-Luc KG1 cell model [START_REF] Menon | Modifications of DNA methylation by non-nucleoside inhibitors induce specific chromatin changes[END_REF][START_REF] Rilova | Design, Synthesis and Biological Evaluation of 4-Amino-N-(4-aminophenyl)benzamide Analogues of Quinoline-Based SGI-1027 as Inhibitors of DNA Methylation[END_REF] KG-1 cells, stably transfected with the firefly luciferase (luc+ from pGL3 by Promega) reporter gene under the control of the cytomegalovirus (CMV) promoter (from pEGFP-N1 by Clontech Laboratories Inc.) partially methylated (50 %), was seeded at 20 000 cells per well in a 96-well plate. After 24 h incubation in the presence of the test compound or solvent (DMSO), the induction of the promoter was measured by quantification of luciferase using the One-Glo assay (Promega) according to the manufacturer′s protocol. The luminescence was measured on an EnVision multilabel plate reader (PerkinElmer), and the data are expressed as the fold induction as compared with the DMSO control. The mean of three experiments and the standard error is reported.

Antiproliferative activity

For antiproliferative activity, cells were seeded (2x10 4 cells/ml for KG-1 in RPMI-1640 medium supplemented with 15 % foetal calf serum (Lonza, France) and 1.5x10 4 cells/ml for HCT116 in MEM medium containing HEPES, L-glutamine and supplemented with 10% foetal calf serum (Lonza), at 37 °C under 5 % CO 2 ) at day 0 in 96-well plates. Cells were treated with tested compound solutions at a dose range from 3.2x10 -9 M to 1x10 -5 M.

Treatment was repeated on days 2 and 3, and, on day 4, the antiproliferative activity of compounds was measured using the ATP quantification method "ATPlite One Step Luminescence Assay System" (PerkinElmer). Raw data were analysed with Prism 4.03 to generate EC 50 values corresponding to the compound concentration required to cause a 50% decrease cell viability as compared with untreated controls. The values presented are the mean of at least two independent experiments run in duplicates.

Scheme 1 .

 1 Scheme 1. Adapted pathway for the synthesis of 3-nitroflavanones 1 and 2.

  5).

Figure 2 .

 2 Figure 2. X-Ray crystal structure of compound 12.

Figure 3 .

 3 Figure 3. X-Ray crystal structures of compounds 3a, 3b, 5, 9.

FFigure 4 .

 4 Figure 4. Stability of 3-halo-3-nitroflavanones in enzymatic buffer over 15 h.

Figure 5 .

 5 Figure 5. Luciferase reexpression level in KG-1 CMV-Luc reexpression model treated with 3-halo-3-nitroflavanones at 10, 5, 3.2, and 1 µM. Induction Fold (IF) values were determined in at least three independent experiments.

Figure 6 .

 6 Figure 6. Cytotoxicity evaluation for compounds 3a, 3b, 4a and 4b. Cells were treated daily for 72 h. Values were determined as a mean of two independent experiments.

  KG1! EC 50 = 5.3 ± 1.0 μM ! HCT116! EC 50 = 12.4 ± 1.5 μM ! KG1! EC 50 = 18.6 ± 4.0 μM ! HCT116! n.d.! KG1! EC 50 = 13.0 ± 1.9 μM ! HCT116! EC 50 = 10.7 ± 1.9 μM ! KG1! EC 50 = 12.9 ± 2.5 μM ! HCT116! EC 50 = 8.1 ± 3.5 μM ! 3a (HCT116) 3a (KG-1) 3b (HCT116) 3b (KG-1) 4a (KG-1) 4b (HCT116) 4b (KG-1)

1 HNMR (CDCl 3 ,

 13 500 MHz): δ (ppm) = 8.11 (dd, J = 1.4, 7.8 Hz, 1H, H-3'); 7.87 (ddd, J = 0.4, 1.7, 8.0 Hz, 1H, H-5); 7.93 (dd, J = 1.3, 8.1 Hz, 1H, H-6'); 7.76 (td, J = 1.4, 7.6 Hz, 1H, H-4'); 7.71 (tdd, J = 1.2, 1.7, 7.8 Hz, 1H, H-7); 7.66 (td, J = 1.5, 7.7 Hz, 1H, H-5'); 7.29 (tdd, J = 0.6, 1.0, 7.6 Hz, 1H, H-6); 7.26 (s, 1H, H-2); 7.17 (qd, J = 0.4, 1.0, 8.4 Hz, 1H, H-8). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 177.9 (C=O); 160.1 (C-9); 149.4 (C-2'); 138.5 (C-7); 132.9 (C-4'); 131.7 (C-5'); 130.6 (C-3'); 129.6 (C-5); 125.4 (C-6'); 124.3 (C-6); 124.3 (C-1'); 118.5 (C-8); 116.9 (C-10); 100.9 (C-3); 76.1 (C-2).syn-3-Chloro-2,3-dihydro-2-(5'-methoxy-2'-nitrophenyl)-3-nitro-4H-1-benzopyran-4-

13 C

 13 NMR (CDCl 3 , 125 MHz): δ (ppm) = 177.9 (C=O); 162.7 (C-5'); 160.1 (C-9); 142.0 (C-2'); 138.3 (C-7); 129.7 (C-5); 128.2 (C-3'); 126.9 (C-1'); 124.3 (C-6); 118.5 (C-8); 117.0 (C-10); 116.6 (C-6'); 115.3 (C-4'); 101.0 (C-3); 76.0 (C-2); 56.3 (OCH 3 ).

  R f = 0.21 (cyclohexane/EtOAc 3:1). ESI-MS: m/z = 351 [M+Na] + . 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.02 (dd, J = 1.7, 8.0 Hz, 1H, H-5); 7.87 (dd, J = 1.3, 8.2 Hz, 1H, H-3'); 7.74 (dd, J = 1.4, 7.9 Hz, 1H, H-6'); 7.68 (td, J = 1.3, 7.5 Hz, 1H, H-5'); 7.64 (m, 1H, H-7); 7.60 (td, J = 1.4, 7.8 Hz, 1H, H-4'); 7.22 (td, J = 0.9, 7.6 Hz, 1H, H-6); 7.11 (dd, J = 0.6, 8.5 Hz, 1H, H-8); 7.05 (s, 1H, H-2); 1.78 (s, 3H, CH 3 ). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 184.9 (C=O); 160.1 (C-9); 149.8 (C-2'); 137.8 (C-7); 132.6 (C-5'); 131.0 (C-4'); 129.4 (C-6'); 128.9 (C-5); 126.2 (C-1'); 125.5 (C-3'); 123.8 (C-6); 118.4 (C-10); 118.3 (C-8); 93.4 (C-3); 77.0 (C-2); 15.3 (CH 3 ). HRMS: Calcd for C 16 H 12 N 2 O 6 Na 351.0588, Found 351.0581.

3a 1 H 1 H

 11 NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.11 (dd, J = 1.7, 8.0 Hz, 1H, H-5); 8.06 (dd, J = 1.2, 8.2 Hz, 1H, H-3'); 7.71 (td, J = 1.8, 8.0 Hz, 1H, H-7); 7.67 (td, J = 1.9, 7.6, Hz, 1H, H-4'); 7.76-7.72 (m, 2H, H-5', H-6'); 7.30 (td, J = 1.0, 7.5 Hz, 1H, H-6); 7.18 (dd, J = 0.6, 8.6 Hz, 1H, H-8); 7.06 (s, 1H, H-2). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 177.1 (C=O); 160.6 (C-9); 149.0 (C-2'); 138.0 (C-7); 133.7 (C-5'); 131.4 (C-4'); 129.7 (C-5); 128.9 (C-6'); 126.4 (C-1'); 125.3 (C-3'); 124.4 (C-6); 119.1 (C-10); 118.6 (C-8); 97.1 (C-3); 79.8 (C-2). HRMS: Calcd for C 15 H 9 BrN 2 O 6 Na 414.9536, Found 414.9560. Compound 3b NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.15 (dd, J = 0.9, 8.1 Hz, 1H, H-3'); 8.07 (dd, J = 1.6, 7.9 Hz, 1H, H-5); 7.91 (dd, J = 1.3, 8.1 Hz, 1H, H-6'); 7.75 (td, J = 1.3, 7.8 Hz, 1H, H-4'); 7.70 (td, J = 1.7, 7.8 Hz, 1H, H-7); 7.66 (td, J = 1.3, 7.9 Hz, 1H, H-5'); 7.28 (td, J = 0.8, 7.6 Hz, 1H, H-6); 7.16 (br d, J = 8.3 Hz, 1H, H-8); 7.00 (s, 1H, H-2). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 178.3 (C=O); 159.8 (C-9); 149.5 (C-2'); 138.3 (C-7); 133.7 (C-4'); 131.6 (C-5'); 130.2 (C-3'); 129.6 (C-5); 125.4 (C-6'); 124.1 (C-1'); 124.3 (C-6); 118.6 (C-8); 116.9 (C-10); 97.1 (C-3); 76.2 (C-2). HRMS: Calcd for C 15 H 9 BrN 2 O 6 Na 414.9536, Found 414.9552.

2 (

 2 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 177.C=O); 163.4 (C-5'); 160.7 (C-9); 141.8 (C-2'); 138.0 (C-7); 129.7 (C-5); 129.1 (C-1'); 128.2 (C-3'); 124.4 (C-6); 119.1 (C-10); 118.6 (C-8); 115.8 (C-4'); 114.1 (C-6'); 97.2 (C-3); 79.7 (C-2); 56.4 (OCH 3 ). HRMS: Calcd for C 16 H 11 BrN 2 O 7 Na 444.9622, Found 444.9641. Compound 4b 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.08 (dd, J = 1.7, 8.0 Hz, 1H, H-5); 8.01 (d, J = 9.2 Hz, 1H, H-3'); 7.70 (td, J = 1.7, 7.8 Hz, 1H, H-7); 7.64 (d, J = 2.8 Hz, 1H, H-6'); 7.28 (td, J = 7.7 Hz, 1H, H-6); 7.17 (dd, J = 0.9, 8.4 Hz, 1H, H-8); 7.16 (s, 1H, H-2); 7.06 (dd, J = 2.7, 9.1 Hz, 1H, H-4'); 3.96 (s, 1H, OCH 3 ). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 178.4 (C=O); 162.6 (C-5'); 159.9 (C-9); 142.1 (C-2'); 138.2 (C-7); 129.7 (C-5); 128.2 (C3'); 127.5 (C-1'); 124.3 (C-6); 118.5 (C-8); 117.0 (C-10); 116.4 (C-6'); 115.2 (C-4'); 97.5 (C-3); 76.1 (C-2); 56.4 (OCH 3 ). HRMS: Calcd for C 16 H 11 BrN 2 O 7 Na 444.9644, Found 444.9641.

1 H

 1 NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.10 (dd, J = 1.7, 7.9 Hz, 1H, H-5); 7.94 (td, J = 1.7, 7.4 Hz, 1H, H-6'); 7.68 (td, J = 1.8, 7.8 Hz, 1H, H-7); 7.47 (m, 1H, H-4'); 7.31 (td, J = 1.0, 7.7 Hz, 1H, H-5'); 7.26 (td, J = 0.8, 7.6 Hz, 1H, H-6); 7.16 (dd, J = 0.6, 8.4 Hz, 1H, H-8); 7.94 (td, J = 1.1, 9.4 Hz, 1H, H-3'); 6.41 (s, 1H, H-2). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 179.1 (C=O); 160.1 (C-9); 160.0 (d, J = 252.2 Hz, C-2'); 138.1 (C-7); 132.5 (d, J = 9.8 Hz, C-4'); 130.5 (d, J = 2.0 Hz, C-6'); 129.5 (C-5); 124.4 (d, J = 3.7 Hz, C-5'); 124.1 (C-6); 119.2 (d, J = 12.1 Hz, C-1'); 118.6 (C-8); 117.1 (C-10); 116.0 (d, J = 19.9 Hz, C-3'); 98.3 (C-3); 76.7 (d, J = 2.8 Hz, C-2). HRMS: Calcd for C 15 H 9 BrFNO 4 Na 387.9591, Found 387.9637. Structure was confirmed by X-ray crystal structure.

13 C

 13 NMR (CDCl 3 , 125 MHz): δ (ppm) = 178.7 (d, J = 20.8 Hz, C=O); 161.3 (C-9); 138.6 (C-7); 130.7 (C-4'); 130.1 (d, J = 1.1 Hz, C-1'); 129.2 (2C, C-3', C-5'); 128.6 (d, J = 1.0 Hz, C-5), 127.0 (2C, C-2', C6'), 123.9 (C-6); 119.3 (d, J = 1.5 Hz, C-10); 119.0 (C-8); 107.5 (d, J = 247.6 Hz, C-3); 81.5 (d, J = 26.3 Hz, C-2). HRMS: Calcd for C 15 H 10 FNO 4 Na 310.0486, Found 310.0498.

general procedure 1 .

 1 The expected 3-fluoro-3-nitroflavanone 7 (73 mg) was obtained as a white solid with 63% yield. R f = 0.54 (cyclohexane/EtOAc 1:1). ESI-MS: m/z = 355 [M+Na] + . 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.13 (dd, J = 1.2, 8.3 Hz, 1H, H-3'); 8.09 (dd, J = 1.6, 7.9 Hz, 1H, H-5); 7.83 (dd, J = 1.2, 8.0 Hz, 1H, H-6'); 7.75 (m, 2H, H-7, H-5'); 7.68 (td, J = 1.3, 8.0 Hz, 1H, H-4'); 7.31 (br t, J = 7.6 Hz, 1H, H-6); 7.24 (br d, J = 8.3 Hz, 1H, H-8); 7.03 (d, J = 2.9 Hz, 1H, H-2). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 177.8 (d, J = 20.3 Hz, C=O); 160.8 (C-9); 148.2 (C-2'), 138.6 (C-7); 134.3 (C-5'); 131.5 (C-4); 128.8 (d, J = 1.2 Hz, C-5); 128.2 (C-6'); 125.8 (C-3'); 124.8 (d, J = 1.3 Hz, C-1'); 124.3 (C-6); 119.5 (d, J = 1.3 Hz, C-10); 118.7 (C-8); 108.0 (d, J = 252.2 Hz, C-3); 75.3 (d, J = 23.4 Hz, C-2). HRMS: Calcd for C 15 H 9 FN 2 O 6 Na 355.0337, Found 355.0350.

  R f = 0.55 (cyclohexane/EtOAc 1:1). ESI-MS: m/z = 385 [M+Na] + . 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.21 (d, J = 9.1 Hz, 1H, H-3'); 8.08 (dd, J = 1.6, 8.1 Hz, 1H, H-5); 7.75 (td, J = 1.5, 7.7 Hz, 1H, H-7); 7.30 (br t, J = 7.7 Hz, 1H, H-6); 7.25 (d, J = 2.8 Hz, 1H, H-6'); 7.24 (d, J = 8.7 Hz, 1H, H-8), 7.21 (d, J = 2.4 Hz, 1H, H-2); 7.06 (dd, J = 2.6, 6.4 Hz, 1H, H-4'); 3.94 (s, 3H, OCH 3 ). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 177.8 (d, J = 20.1 Hz, C=O); 163.8 (C-5'); 160.6 (C-9); 140.7 (C-2'); 138.4 (C-7); 128.6 (d, J = 1.0 Hz, C-5); 128.6 (C-3'), 127.6 (d, J = 1.3 Hz, C-1'); 124.1 (C-6); 119.5 (d, J = 1.3 Hz, C-10); 118.8 (C-8); 115.2 (C-4'), 113.5 (C-6'); 108.2 (d, J = 253 Hz, C-3); 75.2 (d, J = 26 Hz, C-2); 56.2 (OCH 3 ). HRMS: Calcd for C 16 H 11 FN 2 O 7 Na 385.0443, Found 385.0486.

  13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 178.5 (d, J = 20.0 Hz, C=O); 161.3 (C-9); 160.3 (d, J = 251.4 Hz, C-2'); 138.6 (C-7); 132.5 (d, J = 8.6 Hz, C-4'); 128.7 (d, J = 1.1 Hz, C-5); 127.7 (d, J = 2.3 Hz, C-6'); 125.2 (d, J = 3.6 Hz, C-5'); 124.0 (C-6); 119.5 (d, J = 1.4 Hz, C-1'); 119.0 (C-8); 118.0 (dd, J = 1.1, 12.7 Hz, C-10); 116.1 (d, J = 21.3 Hz, C-3'); 107.8 (d, J = 249.8 Hz, C-3); 75.1 (dd, J = 3.4, 28.8 Hz, C-2). HRMS: Calcd for C 15 H 9 F 2 NO 4 Na 328.0392, Found 328.0417. Structure was confirmed by X-ray crystal structure.

  13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 180.2 (C=O); 159.6 (C-9); 149.7 (C-2'); 138.1 (C-7); 132.5 (C-5'); 131.4 (C-4'); 129.7 (C-5); 129.5 (C-6'); 125.9 (C-1'); 125.5 (C-3'); 124.3 (C-6); 118.2 (C-8); 116.8 (C-10); 86.1 (C-3); 77.1 (C-2).

[

  M+Na] + . 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.09 (dd, J = 1.4, 8.0 Hz, 1H, H-5); 8.00 (d, J = 9.2 Hz, 1H, H-3'); 7.67 (td, J = 1.8, 7.8 Hz, 1H, H-7); 7.65 (d, J = 2.6 Hz, 1H, H-6'); 7.29 (m, 1H, H-6); 7.15 (br d, J = 8.3 Hz, 1H, H-8); 7.06 (dd, J = 2.7, 9.1 Hz, 1H, H-4'); 6.27 (s, 1H, H-2); 3.98 (OCH 3 ). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 180.3 (C=O); 162.4 (C-5'); 159.7 (C-9); 142.4 (C-2'); 138.0 (C-7); 129.7 (C-5); 128.5 (C-1'); 128.3 (C-3'); 124.3 (C-6); 118.3 (C-8); 116.8 (C-10); 115.8 (C-6'); 115.1 (C-4'); 86.8 (C-3); 77.4 (C-2), 56.4 (OCH 3 ). (E)-1-(2-methyl-2-nitroethenyl)-2-nitro-benzene (12)

(

  CDCl 3 , 500 MHz): δ (ppm) = 8.35 (s, 1H, CH=C); 8.26 (d, J = 8.0 Hz, 1H, H-3), 7.75 (t, J = 7.5 Hz, 1H, H-5), 7.63 (t, J = 7.5 Hz, 1H, H-4), 7.39 (d, J = 8.0 Hz, 1H, H-6); 2.25 (s, 3H, CH 3 ). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 148.6, 147.5 (2C-NO 2 ); 134.0 (C-5); 131.0 (CH=C); 130.9 (C-6); 130.3 (C-4); 128.7 (C-1); 125.4 (C-3); 13.7 (CH 3 ). E-stereochemistry was confirmed by 1 H-1 H-ROESY and 1 H-1 H-NOESY experiments. (Z)-(2-bromo-2-nitroethenyl)-benzene (13)

1 H

 1 NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.65 (s, 1H, CH=C); 7.89 (m, 2H) (H-2, H-6), 7.53-7.48 (m, 3H, H-3, H-4, H-5). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 136.6 (CH=C); 132.1 (C-4); 131.1 (2C, C-2, C-6); 130.5 (C-1); 129.1 (2C, C-3, C-5); 128.3 (CH=C). Structure was confirmed by crystal structure (Supporting Information) 3,4-Dihydro-4-hydroxy-3-methyl-3-nitro-2-(2'-nitrophenyl)-2H-1-benzopyran (14)

  R f = 0.35 (cyclohexane/EtOAc 5:2). ESI-MS: m/z = 353 [M+Na] + . 1 H NMR (CDCl 3 , 500 MHz) (N.B.: all NMR signals are doubled due to the presence of both diastereoisomers; only the major one is assigned here): δ (ppm) = 8.03 (dd, J = 1.3, 8.2 Hz, 1H, H-3'); 7.97 (dd, J = 1.2, 7.9 Hz, 1H, H-6'); 7.78 (td, J = 1.3, 7.6 Hz, 1H, H-5'); 7.63 (m, 1H, H-5); 7.61 (m, 1H, H-4'); 7.29 (m,

1 H

 1 NMR (CDCl 3 , 500 MHz) (N.B.: all NMR signals are doubled due to the presence of both distereoisomers. Only the major one is assigned here): δ (ppm) 7.58 (m, 1H, H-5); 7.49 (m, 2H, H-2', H-6'); 7.42 (m, 3H, H-3', H-4', H-5'); 7.35 (m, 1H, H-7); 7.14 (td, J = 1.1, 7.7 Hz, 1H, H-6); 7.02 (dd, J = 1.1, 8.2 Hz, 1H, H-8); 5.90 (s, 1H, H-4); 5.65 (s, 1H, H-2); 2.59 (br s, 1H, OH). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 152.1 (C-9); 132.0 (C-1'); 130.6 (C-7); 130.4 (C-4'); 128.6 (2C) (C-3', C-5'); 128.0 (2C) (C-2', C-6'); 127.0 (C-5); 122.9 (C-6); 122.8 (C-10); 116.9 (C-8); 108.8 (C-3); 80.5 (C-2); 74.3 (C-4).

  R f = 0.31 (cyclohexane/EtOAc 1:1). ESI-MS: m/z = 204 [M+Na] + , 180 [M-H] -. 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 11.22 (s, 1H, OH); 7.60 (td, J = 1.5, 7.9 Hz, 1H, H-4'); 7.44 (dd, J = 1.5, 8.1 Hz, 1H, H-6'); 7.06 (dd, J = 0.8, 8.7 Hz, 1H, H-3'); 6.98 (td, J = 0.8, 7.6 Hz, 1H, H-5'); 5.91 (s, 2H, CH 2 ). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 190.8 (C=O); 162.9 (C-OH); 138.5 (C-4'); 128.8 (C-6'); 120.1 (C-5'); 119.4 (C-3'); 117.1 (C-1'); 80.7 (CH 2 ).

  yield. R f = 0.14 (EtOAc/cyclohexane 6:4). ESI-MS: m/z = 292 [M+Na] + . 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.01 (dd, J = 1.7, 8.0 Hz, 1H, H-5); 7.63 (td, J = 1.8, 7.8 Hz, 1H, H-7); 7.53 (m, 2H, H-2', H-6'); 7.48-7.45 (m, 3H, H-3', H-4', H-5'); 7.19 (td, J = 0.7, 7.6 Hz, 1H, H-6); 7.10 (br d, J = 8.5 Hz, 1H, H-8); 5.89 (d, J = 11.9 Hz, 1H, H-2); 5.80 (d, J = 11.8 Hz, 1H, H-3); 3.98 (OCH 3 ). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 181.6 (C=O); 161.0 (C-9);

17 - 20 +

 1720 138.0 (C-7); 133.7 (C-1'); 130.6 (C-4'); 129.4 (2C, C-3', C-5'); 128.3 (C-5); 127.6 (2C, C-2', C-6'); 123.3 (C-6); 119.0 (C-10); 118.5 (C-8); 91.7 (C-3); 81.3 (C-2). The title compound 18 was synthesised from the compound 16 (301 mg, 1.66 mmol) and 2nitrobenzaldehyde (255 mg, 1.69 mmol), following the general procedure 2. The title 3nitroflavanone 18 (354 mg, 1.13 mmol) was obtained after flash chromatography with 68% yield as a mixture of diastereoisomers and used as a mixture for the following step. R f = 0.05 (EtOAc/cyclohexane 6:4). ESI-MS: m/z = 313 [M-H] -, 337 [M+Na] + . The title compound 19 was synthesised from compound 16 (185 mg, 1.02 mmol) and 5methoxy-2-nitrobenzaldehyde (187 mg, 1.03 mmol), following the general procedure 1. The title 3-nitroflavanone 19 (167 mg, 0.49 mmol) was obtained after flash chromatography with 48% yield as a mixture of diastereoisomers and used as a mixture for the following step. R f = 0.20 (CH 2 Cl 2 /MeOH 95:5). ESI-MS: m/z = 367 [M+Na] + . The title compound 20 was synthesised from compound 16 (178 mg, 0.98 mmol) and 2fluorobenzaldehyde (185 µl, 1.76 mmol), following the general procedure 1. The title compound 20 (125 mg, 0.44 mmol) was obtained after flash chromatography with 44% yield as a mixture of diastereoisomers and used as a mixture for the following step. R f = 0.31 (CH 2 Cl 2 /MeOH 95:5).

x 4. 6 mm, 5

 65 µm) with 1 ml/min flow and the following gradient: H 2 O/acetonitrile 95:5 for 2 min then up to 0:100 in 10 min and maintained at 0:100 for 2 min with H 2 O and acetonitrile containing 0.1% formic acid. The tested compound was first injected in solution in 100% DMSO (10 mM concentration) to check its purity. Then 50 µl of a solution of 1 mM of tested compound was prepared by dilution in DNMT3A-c enzyme buffer (Hepes 20 mM pH 7.2, KCl 50 mM, EDTA 1 mM final concentration). The percentages of remaining compound were determined with the area of the corresponding HPLC peak. The mean of at least two experiments and the standard error is reported.

1-5, 10, 11 and 17-19 are reported in Table 1 and compared to lead compounds Flv69 and Flv880. Table 1. In vitro inhibition activity of hDNMT3A-c of compounds 1-5, 10, 11, 17-19. Methyl substitution on 3-position led to totally inactive compound 1, which illustrates the importance of the halide on this carbon. For all brominated compounds 2-5 the in vitro

	DNMT inhibition activity is similar ranging from EC 50 = 3.2 ± 1.1 (3b) to EC 50 = 6.7 ± 1.1 µM (2). These activities are slightly lower than for the chlorinated analogues Flv69 and Flv880. Interestingly, compound 3, for which both diastereoisomers (3a and 3b) were obtained, showed similar inhibition activities with EC 50 = 5.1 ± 2.0 µM for 3a and EC 50 = 3.2 ± 1.1 µM for 3b. This similarity of diastereoisomers inhibition activity was also measured for X R 1 R 2 EC 50 (µM) Flv69 Cl -NO 2 H 4.4 ± 2.2 Flv880 Cl -NO 2 -OMe 4.4 ± 0.8 1 -CH 3 -NO 2 H > 40 2 Br H H 6.7 ± 1.1 3a Br -NO 2 H 5.1 ± 2.0 3b Br -NO 2 H 3.2 ± 1.1 4a Br -NO 2 -OMe 4.7 ± 1.6 4b Br -NO 2 -OMe 5.3 ± 1.0 5 Br F H 5.7 ± 1.9 10 I -NO 17 H H H inactive* 18 H -NO 2 H inactive* diastereoisomers 4a (EC Cpd 19 H -NO

50 = 4.7 ± 1.6 µM) and 4b (EC 50 = 5.3 ± 1.0 µM). This highlights the 2 H 2.8 ± 1.6 11 I -NO 2 -OMe 2.0 ± 1.4 2 -OMe inactive* EC 50 values were determined against the human DNMT3A-c in at least four independent experiments. * 0% inhibition at 32 µM

1 H

 1 NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.07 (dd, J = 1.7, 8.0 Hz, 1H, H-5); 7.87 (d, J = 9.2 Hz, 1H, H-3'); 7.71 (td, J = 1.8, 7.9 Hz, 1H, H-7); 7.58 (d, J = 2.7 Hz, 1H, H-6'); 7.42 (s, 1H, H-2); 7.28 (td, J = 0.9, 7.7 Hz, 1H, H-6); 7.17 (m, 1H, H-8); 7.06 (dd, J = 2.8, 9.1 Hz, 1H, H-4').

  1, starting from compound 15. After purification by flash chromatography, the title

	(C-4'); 129.4 (C-5); 128.8 (2C) (C-3', C-5'); 128.0 (2C) (C-2', C-6'); 123.8 (C-6); 118.6 (C-
	8); 116.9 (C-10); 99.7 (C-3); 82.9 (C-2); 15.3 (CH 3 ). HRMS: Calcd for C 15 H 10 BrNO 4 Na
	369.9685, Found 369.9718.
	compound 2 (26 mg, 0.07 mmol, 34% over 2 steps) was obtained exclusively as the syn
	diastereoisomer as white solid. R f = 0.55 (cyclohexane/EtOAc 3:2). ESI-MS: m/z = 370
	[M+Na] + . 1 H NMR (CDCl 3 , 500 MHz): δ (ppm) = 8.08 (dd, J = 1.7, 8.0 Hz, 1H, H-5); 7.69

(td, J = 1.9, 7.7 Hz, 1H, H-7); 7.52 (m, 2H, H-2', H6'); 7.47 (m, 1H, H-4'); 7.46 (m, 2H, H-3', H-5'); 7.25 (m, 1H, H-6); 7.20 (br d, J = 8.4 Hz, 1H, H-8); 6.08 (s, 1H, H-2). 13 C NMR (CDCl 3 , 125 MHz): δ (ppm) = 179.6 (C=O); 160.1 (C-9); 138.2 (C-7); 131.2 (C-1'); 130.6

Acknowledgements

We thank Dr Laure Vendier (LCC, UPR 8241 CNRS, Toulouse) for crystallography structure determinations and Dr Jacques Fahy for helpful scientific discussion about synthesis pathways. This work was supported to PBA and ML PlanCancer2014 (N°EPIG201401) and to DP by Pierre Fabre and the Centre National de la Recherche Scientifique (CNRS).

Supplementary Information

Crystallographical data, 1 H and 13 C NMR spectra of compound Flv69, Flv880, 1-13, 16 and 17 are available in supplementary information.

Declaration of interest

The authors do not have any conflicts of interest to declare.

Graphical abstract


Anu Rani

Matt D Johansen

Françoise Roquet-Banères

Laurent Kremer

Paul Awolade

Oluwakemi Ebenezer

Parvesh Singh

Sumanjit Sumanjit

Dr Vipan Kumar

Dr Vipan

Rani Anu

; Oluwakemi

Ebenezer ; Parvesh

Singh ; Sumanjit

Arun Sharma
email: raghusharma4@gmail.com

Raghu Raj

Gaurav Bhargava

Yogesh Kumar
email: yogesh.kumar@durham.ac.uk

Matthias Dhooghe
email: matthias.dhooghe@ugent.be

Kelly Chibale
email: kelly.chibale@uct.ac.za

Sumanjit

Design and synthesis of 4-Aminoquinoline-isoindoline-dione-isoniazid triads as potential anti-mycobacterials

Keywords: 4-aminoquinoline-isoindoline-dione-isoniazid, Antimycobacterial activity, Cytotoxicity, Selectivity index, Molecular docking 4-aminoquinoline-isoindoline-dione-isoniazid, Anti-mycobacterial activity, Cytotoxicity, Selectivity index, Molecular docking

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Tuberculosis (TB), an infectious disease caused by the bacterium, Mycobacterium tuberculosis, has resulted in chronic granulomatous lesions in the lungs, which can be accompanied by extrapulmonary infection foci in the skin, brain and lymph nodes [1]. The disease is the major cause of death after the Human Immune-deficiency Virus (HIV), accounting for 1945 deaths every day [2]. World Health Organization (WHO) estimated that at least 10 million people developed TB with 1.3 million deaths in 2017 [3]. Despite the availability of an important variety of drugs, TB remains a significant global health threat [4][5]. The foremost obstacle in the total eradication of TB is the development of resistance of M. tuberculosis to many existing drugs, which has led to the appearance of multidrug-resistance (MDR) and extensively drug-resistant (XDR) strains [6][7][8][9][10][11]. Also, TB and HIV co-infection is a major concern with nearly two-thirds *Manuscript Click here to view linked References of the patients diagnosed with TB also being HIV-1 seropositive [12]. The re-designing and repositioning of known anti-TB scaffolds is an effective strategy for the development of new frameworks with favorable pharmacological profiles such as high efficacy and low toxicity [13]. Moreover, this strategy can enable the rapid synthesis of highly effective compounds that are already clinically approved, ultimately shortening the drug commercialization pipeline. Isoniazid (INH), a first-line anti-TB drug, is the most powerful agent used to treat M. tuberculosis infection since 1952 [14]. It is a pro-drug requiring activation via enzyme catalaseperoxidase encoded by katG to its active form [15]. The activated metabolite of INH inhibits the synthesis of mycolic acid, a vital component of the mycobacterial cell wall, by targeting the enoyl-ACP reductase InhA of the type II fatty acid synthase [16][17][18]. The decrease in catalaseperoxidase activity as a result of katG mutations is considered as the most common mechanism associated with INH resistance [19]. Among all the genes involved in INH resistance, excluding katG, mutations of InhA confer clinically relevant levels of resistance to INH.

Multiple other genes such as nat coding for N-acetyltransferase (NAT) contribute to resistance to INH in M. tuberculosis before KatG activation. Furthermore, both NAT and KatG enzymes interact with the pro-drug directly [20]. Following INH metabolism in the liver, hydrazine metabolites (nitrogen-centered free radicals) are produced which generate highly reactive oxygen species and act as a stimulator of lipid peroxidation, resulting in cell death and hepatic necrosis [21][22]. As INH is the major therapeutic arsenal in the treatment of TB infection, continuous efforts are being made to develop new INH derivatives with greater activity, low cytotoxicity, and fewer side effects [23][24]. Several current reports show that the amalgamation of hydrophobic moieties into the basic structure of INH can enhance the penetration of the drug into the lipophilic cell wall of the bacterium [25]. Further, the N-acetyltrans-2 (NAT2) promoted inactivation of INH could also be avoided by functionalizing its hydrazine group [26].

Quinoline moiety is present in a variety of natural products and has diverse biological properties. Notably, quinoline appears as a central core in the recently developed anti-TB drugs such as TMC 207 or bedaquiline and some fluoroquinolones viz. gatifloxacin, and moxifloxacin (Figure 1) [27].

Recently, we introduced a functionalized isoindoline-1,3-diones into the alkyl chain of 4aminoquinolines to access their anti-mycobacterial potency. The compounds were promising candidates with a MIC of 5.9-6.25 µg/mL against the mc 2 6230 strain of M. tuberculosis (Figure 2) [28][29]. Motivated by these results and in continuation [30], the present work is a logical extension and involves the synthesis and anti-mycobacterial evaluation of 4aminoquinoline-isoniazid hybrids linked via isoindoline-1,3-diones. The length of the alkyl chain spacer between the pharmacophores along with the position of INH and 4aminoquinoline around the isoindoline-1,3-dione were meticulously altered to study their influence on Structure-Activity Relationship.

Results and Discussion

Synthetic Chemistry:

For the synthesis of first series of desired 4-aminoquinoline-isoindoline-dione-isoniazid triads, 6a-e, the reaction of substituted phthalic anhydride 1 with quinoline based diamines 2 was carried out in DMSO at 150  C for 5 min, in a microwave synthesizer to result in 4aminoquinoline-isoindoline-diones 5a-e. EDC-HOBt promoted amide coupling between 5a-e and isoniazid afforded the corresponding conjugates 6a-e in moderate to good yields (Scheme

1).

Another set of 4-aminoquinoline-isoindoline-dione-isoniazid triads, 8a-g were prepared via an initial microwave heating of substituted phthalic anhydride 1 with isoniazid 3 to result in isoniazid-isoindoline-dione 7. Amide/Ester coupling between 7 and quinoline based diamine 2/quinoline based alcohol 2' afforded 8a-g in good yields. A cycloalkyl viz. piperazinyl analogue 11 was also synthesized to determine the influence of alkyl chain on antimycobacterial activities. Further, in order to rationalize the anti-mycobacterial effect of quinoline core in the present series of triads, amide-tethered isoniazid-isoindoline-dione conjugates 13a-b were synthesized via an initial treatment of 1 with amino acids 4 to afford 12 with subsequent treatment with isoniazid using standard coupling procedure. The structures of the synthesized 4-aminoquinoline-isoindoline-isoniazid conjugates were assigned based on the spectral and analytical evidence. For example, the compound 6a showed a molecular ion peak at 515.1177 in its high-resolution mass spectrum (HRMS). The significant features of its 1 H NMR spectrum involved the presence of a triplet at δ 3.82 because of methylene (N-CH 2 -); two doublets at 6.62 (J=5. 3 Hz) and 7.76 (J=2.2 Hz) corresponding to quinoline protons along with a multiplet of isoindoline ring protons at 8. 27-8.31. The presence of signals in its 13 C NMR spectrum at δ 164. 6, 164.7, 167.7, and 167.8 corresponding to imide-and amide carbonyls, along with methylene carbons at 36.5 and 40.7 further validated the assigned structure.

In vitro anti-mycobacterial and cytotoxic evaluation:

The synthesized 4-aminoquinoline-isoniazid conjugates were assessed for their antimycobacterial activities against avirulent M. tuberculosis mc 2 6230 strain and the results are enlisted in Table 1. The cytotoxicity of all derivatives to mammalian Vero cells was also evaluated to establish whether the observed activities were due to their inherent antimycobacterial ability or due to cytotoxicity (Table 1). As evident, the compounds showed promising anti-mycobacterial activities, although they were not as active as INH. A closer examination of the results showed the activity dependence on both the length of the alkyl chain between the pharmacophores as well as the position of INH and 4-aminoquinoline around isoindoline core. Among the 4-aminoquinoline-isoindoline-1,3-diones 5a-e (without INH-core), the compounds lack any anti-mycobacterial activity except 5c and 5d with weak activity but cytotoxicity. Inclusion of INH among these conjugates not only improved their antimycobacterial activity substantially but also decreased their cytotoxicity as seen in triads, 6a-e.

The improvement in anti-mycobacterial and cytotoxicity profile is apparent at longer chain lengths, as displayed by 6d and 6e with MIC 99 of 12.5 and 3.1 µg/mL and IC 50s of 73 and 77 µg/mL, respectively. Furthermore, interchanging the position of INH with quinoline diamines around isoindoline core in compound 8a-e improved the anti-mycobacterial activities without much dependence on the alkyl chain length. The triad 8e with an octyl spacer not only retained the antimycobacterial efficacy (MIC 99 =3.1 µg/mL), it is also non-cytotoxic (IC 50 ≥ 100 µg/mL). The inclusion of a cyclic amine viz. piperazine instead of alkyl amine as in triad 11 also resulted in the good anti-mycobacterial activity.

The conjugates, 13a and 13b without a quinoline core, did not show any substantial antimycobacterial activity; thus, justifying the importance of the proposed design of 4aminoquinoline-isoindoline-dione-isoniazid triads to elicit anti-mycobacterial activity and reduce cytotoxicity.

Relating the activity and cytotoxicity data of currently synthesized compounds (6e, 8e, 11) with our earlier report (I-IV) [28][29][30][31], the introduction of INH at C-5 of isoindoline ring not only improved anti-mycobacterial activity but also resulted in the reduction of cytotoxicity (Figure 2). The general anti-TB SAR/cytotoxicity of the synthesized series of 4-aminoquinolineisoindoline-isoniazid triads is elucidated in Figure 3.

Molecular docking studies and ADME properties prediction

Molecular docking studies were conducted using AutoDock to explore the potential binding interactions of selected compounds with M. tuberculosis enoyl-acyl carrier protein reductase (InhA; PDB ID: 4TZK). InhA is a prominent enzyme involved in the biosynthesis of mycolic acid for mycobacterial cell wall and the molecular target of isoniazid. Six inhibitors were docked into the active site of InhA, as shown in Figures 4 and5, while the docking results are summarized in Table 2.

In consonant with the experimental results, the binding energies and receptor interactions of compounds 6e, 8e and 11 were superior to compounds I, II and III (Table 2). For instance, in compound I (Figure 4a), the chlorine atom was sandwiched between residues Phe149 and Met199 via hydrophobic interactions while the amino group of quinoline core formed a hydrogen bond interaction with the hydroxyl oxygen of Gly192. The isoindoline core also afforded alkyl, π-π stacking, π-alkyl, and π-σ interactions with Ile95, Phe41, Val65, Ile122, Phe149, and Met199. A similar binding profile was observed for the isoindoline core of compound II (Figure 2), i.e. π-π stacking with residue Phe41 as well as π-σ, π-alkyl, and alkyl interactions with Ile21, Val65, Ile122, Phe149, Ala198, Met147 and Ile95. The unit also featured a hydrogen bond interaction between the isoindoline and Ile95, while the fluorine substituent formed a halogen bond with Asp64. The docked complex of III (Figure 3) was established in the hydrophobic pocket created by hydrophobic interaction with the side chains of ten amino acid residues. Surprisingly, the oxygen atom of the isoindoline core formed a strong hydrogen bond with Tyr168. The interaction was absent in compounds I and II; hence, a potential platform for further structural modifications to enhance the anti-mycobacterial activity.

Furthermore, compound 6e, 8e and 11 binds in essentially the same manner within the active site of InhA. The compound 6e-InhA complex is characterized by six hydrogen bond interactions between the amino acid residues of side chain and both the oxygen atom of isoniazid moiety and the amine linker. Besides, the quinoline core was anchored in the hydrophobic pocket containing Val65, Ile95, Phe41, and Ile122, which suggested that the twofold increase in potency might stem from additional interactions between the isoniazid moiety and the side chains of the active site residues. Furthermore, the isoniazid moiety in compound 8e interacts with Gly192, Ala191 and Lys165 via hydrophobic and H-bonding interactions while the aminoquinoline group formed hydrophobic interactions with residue Val65, Leu63, Gly14 and Gly40, respectively. Other residues that made H-bond interactions include, Gly96, Ile95, Ile165, which also serve as motivators for the stabilization of the inhibitor within the catalytic pocket of the receptor. Moreover, the docked complex of compound 11 showed additional hydrogen bond interactions between the piperazine ring and the carbonyl oxygen of Gly96, which was accompanied by hydrophobic interaction with Ile16.

On the other hand, the molecular descriptors ADME properties were calculated for potent compounds using web-based software pkCSM (http://biosig.unimelb.edu.au/pkcsm/prediction) and SwissADME (http://www.swissadme.ch/). The results are presented in Table 2. The drug candidates with TPSA values of 140 Å or lower are expected to have good absorption.

Accordingly, all the compounds were within this limit, i.e., <140 Å, which implies that these compounds fulfilled the optimal requirement for drug absorption.

Conclusion

In conclusion, a series of 4-aminoquinoline-isoindoline-dione-isoniazid triads have been synthesized with the target of reviewing their SAR against M. tuberculosis. Nearly all of the synthesized conjugates presented promising anti-TB activity and useful selectivity index. The conjugates without the quinoline core 13a/13b and the INH nucleus 5a-e failed to inhibit the growth of M. tuberculosis as was observed in our previous report on 1,8-naphthalimideisoniazid hybrids [31], further validating the design of triads in the present case. Electronic Supplementary Information: Material and methods, Synthesis and spectral data of all the compounds along with scanned 1 H and 13

Table 1:

In vitro anti-mycobacterial activity of synthesized compounds against M. tuberculosis mc 2 6230 and cytotoxicity evaluation on mammalian Vero cells and their selectivity index (SI). and 11 with previously reported scaffolds I-IV [28][29]31] Scheme 1: Synthesis of 4-aminoquinoline-isoindoline-dione-isoniazid conjugates. Bruker-micrOTOF-Q II spectrometer using ESI as the ion source.

Supplementary Material

General Procedure for the synthesis of 4-aminoquinoline-isoindoline-isoniazid triad:

Synthesis of 2-(((7-chloroquinolin-4-yl)amino)alkyl)-1,3-dioxoisoindoline-5-carboxylic acid (5a-e):

An equimolar mixture of phthalic anhydride 1 (1 eq.) and 7-chloro-4-aminoquinolinediamines 2 (1 eq.) was dissolved in a 2 mL of DMSO in a microwave glass vial. The reaction vial was irradiated to 150 o C for 5 min in microwave synthesizer. After completion of the reaction as evident from TLC, the vessel contents were poured in water (20 mL) and solid precipitates obtained were filtered and washed with cold water and recrystallized using absolute ethanol.

Synthesis of 2-(((7-chloroquinolin-4-yl)

amino)alkyl)-N'-isonicotinoyl-1,3-dioxoisoindoline-5- carbohydrazide (6a-e):
1.0 equivalent of 4-aminoquinoline-isoindoline acids 5, N-ethyl-N-dimethylaminopropyl carbodiimide (EDC) (1.1 eq.), hydroxybenzotriazole (HOBt) (1.2 eq.) and 4diisopropylethylamine (DIPEA) (2 eq.) were mixed in minimum DMF, and the obtained mixture was stirred for 5 min. Isoniazid (1.1 eq.) was subsequently added to the reaction mixture, and the stirring was continued for 3 h at room temperature. The progress was monitored by thin-layer chromatography (TLC). DMF was then evaporated under reduced pressure using a rotary evaporator, and cold water (15 mL) was added, and solid precipitates obtained were filtered and washed with cold water. The crude product was recrystallized in absolute ethanol.

Synthesis of 2-(isonicotinamido)-1,3-dioxoisoindoline-5-carboxylic acid (7):

A mixture of phthalic anhydride 1 (1 eq.) and isoniazid 3 (1.1 eq.) was dissolved in a 2 mL of DMSO in a microwave glass vial. The reaction vial was then irradiated to 150 o C for 10 min in microwave synthesizer. After completion of the reaction as evident from TLC, the vessel contents were poured in water (20 mL) and solid precipitates obtained were filtered and washed with cold water and recrystallized using absolute ethanol.

Synthesis of N-(((7-chloroquinolin-4-yl) eq.) and 4-diisopropylethylamine (DIPEA) (2 eq.) were dissolved in minimum DMF and was stirred for 5 min at room temperature. Then, quinoline-diamines 2/quinoline-ethanolamines 2' (1.1 eq.) was added to the reaction mixture, and the stirring was continued for 5 h. On completion, DMF was evaporated under vacuum using a rotary evaporator and cold water ( 15mL) was added. The solid precipitates obtained were filtered and washed with cold water with subsequently re-crystallization in absolute ethanol.

Synthesis of N-(5-(4-(7-chloroquinolin-4-yl)piperazin-1-yl) -1,3-dioxoisoindolin-2yl)isonicotinamide 11:

To a microwave reaction vial was added a solution of 4-fluoro-phthalic anhydride 1 (1.0 eq.)

and Isoniazid (1.0 eq.) in 2mL of DMSO. After sealing, the vial was heated to 150  C for 10 min in the microwave reactor. After the completion of the first step, as clear from TLC, 7chloro-4-(piperazin-1-yl)quinoline 10 (1.0 mmol) was added in the same reaction vial. The reaction mixture was again heated at 180  C for 15 min in the microwave, and the completion was determined using TLC. When all the reactants consumed, the contents were poured in water (20 mL) resulting in the precipitation of the desired product. The obtained product was filtered and recrystallized using absolute ethanol.

Synthesis of N-phthaloylamino acids 12:

A mixture of phthalic anhydride 1 (1 eq.) and amino acids 4 (1 eq.) in a small aliquot of glacial acetic acid was placed in a 10 mL microwave glass vial and irradiated for 10 min. The reaction mixture on completion was treated with cold water to result in a white powder (12), which was filtered, dried and recrystallized from ethanol.

Synthesis of N'-(2-(1,3-dioxoisoindolin-2-yl)acetyl)isonicotinohydrazide/N'- (4-(1,3dioxoisoindolin-2-yl)benzoyl)isonicotinohydrazide (13a-b):

1.0 equivalent of N-phthaloylamino acids, N-ethyl-N-dimethylaminopropyl carbodiimide (EDC) (1.1 eq.), hydroxybenzotriazole (HOBt) (1.2 eq.) and 4-Dimethylaminopyridine (DMAP) (0.2 eq.) were mixed in minimum DMF and the obtained mixture, was stirred for 5 min. Isoniazid (1.1 eq.) was added to the reaction mixture and the stirring was continued for 3 h at room temperature. DMF was evaporated using a rotary evaporator, and cold water (15 mL) was added. The solid precipitates thus obtained were filtered, washed with cold water and recrystallized in absolute ethanol.

Methods for assessment of the anti-tubercular activity of test compounds:

Bacterial strains and growth conditions: M. tuberculosis mc 2 6230 [1] was grown at 37°C

in Middlebrook 7H9 supplemented with oleic-albumin-dextrose-catalase enrichment (OADC)

and 109 µM of pantothenic acid (complete 7H9).

Drug susceptibility testing:

The vulnerability of M. tuberculosis mc 2 6230 to the synthesized compounds was determined as reported previously [2]. Briefly, an exponentially growing culture was diluted in complete 7H9 to OD 600 = 0.01. The bacteria were then seeded in 100 µL volumes in all the wells of a 96-well plate except the first column of wells which contained 200 µL of the bacterial suspension. Compounds (stock concentration 10 mg/mL) were directly added to the wells of the first column to achieve a concentration of 200 µg/mL. Two-fold serial dilutions were then carried out by transferring 100 µL of bacterial suspension from the first column of wells to the second column, mixing, and repeating this procedure for each consecutive column. The plates were then placed in sealed plastic bags and incubated at 37°C. After 7 days of incubation, plates were visually inspected to determine the MIC, which was defined as the minimal concentration of compound at which no growth of bacteria was observed. In addition, 10 µL of resazurin (0.025% w/v) (Sigma, Germany) was added to each well and left to incubate for 24 h to determine metabolic activity of remaining bacteria. If the colour remains blue, bacteria are dead/metabolically inactive, while if there is a colour shift to bright pink, bacteria are alive and metabolically active (Graphical abstract).

Cytotoxicity assay

Cell viability was determined using Vero cells (ATCC, Sigma, Germany) grown in RPMI medium (Gibco, USA), supplemented with 10% decomplemented fetal calf serum, under a 5% CO 2 atmosphere. Cells were seeded in 96-well plates at a density of 2 × 10 4 cells/well in 160 µL RPMI medium and incubated overnight at 37°C to allow cells to adhere. Compounds (dissolved in DMSO) were freshly diluted to appropriate concentrations in RPMI, to allow the addition of 20 µL volumes of the diluted compounds to the cells that resulted in serial dilutions ranging from 100 µg/mL to 0.78 µg/mL. The maximum final concentration of DMSO was 1 % (v/v), and no cytotoxic effect of DMSO was observed at this concentration.

After 24 h incubation at 37°C, 10 µL of resazurin (0.025% w/v) (Sigma, Germany) was added to each well, and the cells were incubated for an additional 3 h at 37°C. Fluorescence was measured in a Polarstar Omega fluorometer using appropriate filters (540 nm excitation and 590 nm emission wavelength). Percentage survival was determined by dividing fluorescence values obtained in the compound containing wells by values obtained for control wells containing cells incubated with a dilution series of DMSO and multiplying this value by 100. The IC 50 is defined as the lowest concentration of compound tested at which at least 50% cell viability was observed.

  C NMR spectra of few representative compounds viz. 5a, 6a, 6b, 7, 8a, 9, 13a and 11 are provided in the electronic supplementary information. » (ANR-10-LABX-12-01). LK acknowledges the support by the Fondation pour la Recherche Médicale (FRM) (DEQ20150331719). PS acknowledges to the National Research Foundation (NRF), South Africa for a Competitive Grant for unrated Researchers (Grant No. 121276), and the Centre for High-Performance Computing (CHPC), Cape Town, for the supercomputing facilities.

Figure 1 :

 1 Figure 1: Chemical structures of standard drug isoniazid (INH) and quinoline based anti-tubercular agents.

Figure 2 :Scheme 1 :

 21 Figure 2: Comparison of activity against mc 2 6230 and cytotoxicity against Vero cells of 6e, 8e and 11 with previously reported scaffolds I, II and III. Scheme 1: Synthesis of 4-aminoquinoline-isoindoline-dione-INH conjugates (6a-e), (8a-e), 11 and (13ab).

Figure 3 :

 3 Figure 3: Structure-Activity Relationship (SAR) of both series of synthesized 4-aminoquinolineisoindoline-dione-INH triads.

Figure 4 :Figure 5 : 11 Table 2 :

 45112 Figure 4: Binding interactions of compounds (a) I (b) II (c) III Figure 5: Binding interactions of compounds (a) 6e (b) 8e (c) 11 Table 2: Molecular descriptors and the binding energy of the potent compounds

Figure 1 :

 1 Figure 1: Chemical structures of standard drug isoniazid (INH) and quinoline based antitubercular agents.

Figure 2 :

 2 Figure 2: Comparison of activity against mc 2 6230 and cytotoxicity against Vero cells of 6e, 8e

Figure 3 :

 3 Figure 3: Structure-Activity Relationship (SAR) of both series of synthesized 4aminoquinoline-isoindoline-dione-INH triads.

Figure 4 :Figure 5 :

 45 Figure 4: Binding interactions of compounds (a) I (b) II (c) III

3 .

 3 and spectral data of 5a-e, 6a-e, 8a-g, 11 and 13a-b Copies of 1 H and13 C NMR spectra of 5a, 6aand reagents were purchased from Sigma-Aldrich and Merck. Thinlayer chromatography was performed on a film of silica gel that contained a fluorescent indicator F 254 supported on an aluminum sheet (Merck). Melting points were determined by open capillary using a Stuart Digital Melting Point apparatus (SMP10) and are uncorrected.1 H NMR spectra were recorded in DMSO-d 6 Jeol 400 (400 MHz) spectrometers while TMS is used as the internal standard. Microwave reactions were performed in an Anton Paar Monowave Initiator+ instrument using a sealed 10 mL process vials. Reaction times refer to irradiation time at the target temperature, not the total irradiation time. The temperature was measured with an IR sensor. Chemical shift values are specified as parts per million (ppm) downfield from TMS while coupling constant (J) values are in Hertz. Patterns of Splitting are designated as s: singlet, d: doublet, t: triplet, dd: double doublet, m: multiplet, ddd: doublet of doublet of doublet, and br: broad peak.13 C NMR spectra were recorded on Jeol 100 MHz spectrometers in DMSO-d 6 . High-Resolution Mass Spectra (HRMS) were recorded on a

  amino)alkyl)-2-(isonicotinamido)-1,3dioxoisoindoline-5-carboxamide (8a-g):A mixture of 2-(isonicotinamido)-1,3-dioxoisoindoline-5-carboxylic acid 7 (1.0 eq.), N-ethyl-N-dimethylaminopropyl carbodiimide (EDC) (1.1 eq.), hydroxybenzotriazole (HOBt)(1.2

Table 1 :

 1 In vitro anti-mycobacterial activity (MIC 99 ) of synthesized compounds against mc 2 6230 strain of M. tuberculosis and cytotoxicity (IC 50 ) evaluation on mammalian Vero cells and their selectivity index (SI).

	Code	Structure	%
			Yield

MIC 99 (µg/mL) IC 50 (µg/mL) SI value

	5e 13a	79 83	200 100	>100 >100	>0.5 >1
	6a 13b	81 85	200 50	>100 >100	>0.5 >2
	6b INH	76 -	25 0.01	63	2.5
	6c	82	12.5	9	0.7
	6d	78	12.5	73	5.8
	6e	79	3.1	77	25
	8a	79	3.1	33	10.6
	8b	77	3.1	85	27
	8c	74	12.5	8	0.6
	8d	76	6.3	19	3
	8e	72	3.1	>100	>31
	5a	81	>200	>100	>0.5
	8f	85	>200	>100	>0.5
	5b	76	200	99	0.5
	8g	84	6.3	>100	>16
	5c	77	100	17	0.2
	11	83	3.1	>100	>31
	5d	76	100	11	0.1

Table 2 :

 2 Molecular descriptors and the binding energy of the potent compounds

		B.E
	Compound		TPSA	logP	HBD	HBA	logS	Caco2	log Kp	logBB
		(kcal/mol)
	I	-9.7	62.30	5.05	1	3	-5.5	0.465	-2.773	0.133
	II	-9.9	91.40	2.77	2	5	-3.716	0.645	-2.816	-1.407
	III	-10.2	65.54	4.35	3	3	-4.945	0.76	-2.877	0.231
	6e	-10.4	133.39	4.42	3	6	-4.094	0.768	-2.735	-1.745
	8e	-10.3	133.39	4.40	3	6	-4.324	1.65	-2.735	-1.544
	11	-11.7	98.74	2.27	1	5	-4.044	0.692	-2.767	-1.129
	TPSA -Topological Polar Surface Area (60 -140); log BB -logarithm of predicted blood/brain barrier partition coefficient
	(-3.0-1.2); Caco-2 -cell membrane permeability (> 0.9 is high); HBA -number of hydrogen bond acceptors; HBD number of
	hydrogen bond donors; log K p -predicted skin permeability(SP) > -2.5 is low.

H NMR of N-(5-fluoro-1,3-dioxoisoindolin-2-yl)benzamide (9):

Acknowledgments

: The authors thank the Council of Scientific and Industrial Research, New Delhi, India for funding (AR Ref. No. 09/254(0269)/2017-EMR-I). MDJ received a postdoctoral fellowship from Labex EpiGenMed under the program « Investissements d'avenir

Conflict of interest

The authors have declared no conflict of interest.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

*Declaration of Interest Statement