Abdulrahim A Alzain

Lucie Brisson

Pierre-Olivier Delaye

Mélanie Pénichon

Stéphanie Chadet

Pierre Besson

Stephan Chevalier

Hassan Allouchi

Magdi A Mohamed

Sébastien Roger
email: sebastien.roger@univ-tours.fr

Stéphan Chevalier

Cécile Enguehard-Gueiffier

Bioinspired imidazo[1,2-a:4,5-c']dipyridines with dual antiproliferative and anti-migrative properties in human cancer cells: The SAR investigation

Keywords: imidazo[1,2-a:4,5-c']dipyridines, antiproliferative activity, anti-migration activity, harmine analogues

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

Introduction

Breast cancer is the most commonly occurring cancer in women worldwide and the second most common cancer overall. From the World Health Organization, about 2.1 million new patients are diagnosed with breast cancer each year and about 627,000 died worldwide from the disease in 2018 [1]. According to the American Cancer Society, the 5-year survival rate for a localized breast cancer has improved significantly to 99%, while this rate drops to 26% if the cancer is diagnosed at a distant metastasis stage [2]. Metastasis development is a complex cascade that involves a first and necessary step of invasion, with the acquisition of the ability by malignant cells to degrade and migrate through extracellular matrices to distant tissues [3].

Inhibition of cancer cells migrative abilities constitutes a highly interesting approach. Indeed the biggest hurdle in controlling breast cancer associated mortality is the lack of effective antimetastatic therapy. Thus, anti-invasive drugs are urgently requested. They would be used in combination with other anticancer agents, not only for the treatment of localized tumors, but also in patients presenting with metastases at diagnosis. Indeed, a growing body of evidence indicates that the burden put on the primary tumor during radiotherapy or antiproliferative treatment may promote further metastatic dissemination and shorten survival time [4].

Natural product-oriented synthetic derivatives have played an important role in drug discovery [5], and the majority of chemotherapeutic agents currently used in clinical settings are derived from natural product scaffolds such as paclitaxel and vinblastine. In recent years, our laboratories have described an efficient one-pot sequence for the preparation of imidazo[1,2-a:4,5c']dipyridines [6], analogues of the β-carboline scaffold present in many naturally occurring alkaloids (Figure 1) [7]. β-Carbolines alkaloids such as harmine and its derivatives display several pharmacological activities [8], particularly antitumor effects. It is commonly reported that both natural and synthetic β-carboline alkaloids elicit their anticancer activities through multiple mechanisms such as DNA intercalation [9], topoisomerase I and II inhibition [10] or kinase inhibition (CDK, PLKs, IKK, DYRK1A) [11]. Imidazo[1,2-a:4,5-c']dipyridine scaffold was scarcely studied in the literature, and to the best of our knowledge only one publication mentioned an antimalarial activity for such tricyclic system [START_REF] Singh | Antimalarial pyrido[1,2-a] benzimidazoles: lead optimization, parasite life cycle stage profile, mechanistic evaluation, killing kinetics, and in vivo oral efficacy in a mouse model[END_REF]. However, imidazo[1,2-a:5,4-b′]dipyridines were lately presented as anticancer agents inhibiting human prostate cancer cell DU-145 proliferation with an IC50 of 1.6 μM for the most active compound [13].

Regarding the similarity between β-carboline and imidazo[1,2-a:4,5-c']dipyridine, we got interested in investigating the potential anticancer activity of our compounds, drawing on all the relevant literature. Indeed the β-carboline analogs were hugely studied from decades in the field of oncology [START_REF] Panicea | New 3-tetrazolyl-β-carbolines and β-carboline-3-carboxylates with anti-cancer activity[END_REF] and the structure-activity relationships of this scaffold against cancer cells was described in 2007 by Cao et al. [8a]. Six sites of modulation were characterized (Figure 2). Several harmine derivatives were developed on this model and studied in order to promote antiproliferative properties. Despite a high selectivity for cancer cells and an IC50 close to micromolar concentrations, these derivatives are generally difficult to develop as lead compounds for pre-clinical studies because of their poor solubility [15].

Imidazo[1,2-a:4,5-c']dipyridine structure present a supplementary nitrogen atom compared to harmine (Figure 1). This difference seems tenuous enough to conserve antitumoral properties, but could critically affect the pharmacokinetic (specially improving the aqueous solubility) and the reactivity of the scaffold to extend the scope of the functionalization. Based on the SAR of the β-carboline scaffold (Figure 2), we proposed in a first approach to explore positions 1, 2, 3, 7 and 10. Nevertheless, conversely to β-carboline, we rapidly noticed that position 2 of imidazo[1,2-a:4,5-c']dipyridines could not be studied. Indeed, N-2 was less nucleophilic than N-6 10 in our series of compounds, and the benzylation did not occurred on the 2 position but on the 10 position. Finally, we explored positions 1, 3, 7 and 10 (Figure 3). As antiproliferative activities seemed to be conserved with the preliminary screened 10-substituted compounds (8 and 10, Table 1) we concluded that the presence but not the position of this benzyl group is determinant. Moreover, we have never been confronted to solubility limitation for our new imidazo[1,2-a:4,5-c']dipyridines. In the present study, we explored the antiproliferative and anti-migrative activities of this new series of compounds against breast cancer cells as part of our on-going research, and attempted to further elucidate the mechanisms of action, providing a basis for future development of these compounds as human breast cancer therapy.

Results and discussion

Chemistry

The starting materials for the preparation of the attempted imidazo[1,2-a:4,5-c']dipyridines 6a-

p are the 3-alkynyl-2-cyano-imidazo[1,2-a]pyridines 5a-f.
The synthetic route to 5a-f is presented in Scheme 1 [6]. Esters 1a-b were transformed into amides 2a-b using aqueous solution of ammonia (30%) at room temperature overnight. Amides 2a-b were subjected to phosphorus oxychloride at reflux for 4h to generate nitriles 3a-b.

Iodination at 3 position of 3a was performed as previously described [6a], when 3b was iodinated in the presence of NIS overnight at reflux of acetonitrile. The Sonogashira coupling reaction with various alkynes was then applied to 4ab in the presence of Pd2dba3 and CuI in dioxane at room temperature for 3h. Scheme 2 displayed the preparation of the tricyclic 6a-h from 5a-d, by a Grignard reagent addition sequence to the nitrile group, followed by a 6-endo-dig cyclization on the triple bond at room temperature for 30 minutes to two hours [6]. In order to introduce an ester function in position 3 of the scaffold, an alternative protocol was achieved to avoid the reaction between ester and Grignard reagent. The starting material 5b with a THP protected hydroxyl group was first obtained and cyclized to 6b in the same conditions than described above. After removal of the protecting group in HCl 6M at room temperature for 1h, the alcohol was oxidized to carboxylic acid and esterified to 6i in non-optimized poor yield.

To extend the scope of the pharmacomodulation at position 1, a new alternative protocol was developed using sodium methoxide as nucleophile, in refluxing methanol overnight, in order to obtain the 1-methoxy substituted compounds 6j-l. Scheme 2. Synthetic routes to the compounds 6a-l. Reactions conditions for 6i: i : HCl 6M, CH2Cl2, r.t., 1h, 93%; ii : a) KMnO4, acetone, 40°C, overnight ; b) EtOH, H2SO4, reflux, 4h, 7%.

The same synthetic route was then applied to the 6-brominated starting materials 5e-f leading to compounds 6m-o via the Grignard procedure and compound 6p after sodium methoxide reaction (Scheme 3). All these compounds were converted into quaternary ammonium salts 8-30, after reaction with benzyl bromide or ethyl iodide in acetonitrile at 80°C for 24-48h (Scheme 5, Table 1). The structure of the salt 8 was confirmed by X-ray crystal structure analysis (Figure 4). This transformation not only aimed to improve the solubility [START_REF]For the cell viability and toxicity assays of our compounds, we needed to prepare 100 mM solutions in DMSO. Base compounds appear to be insoluble, salts were then synthetized and completely soluble at this concentration[END_REF] but also to introduce substituent on the 10-position, which has already been shown to be beneficial for antiproliferative activity against cancer cell lines in the β-carboline series (Figure 2) [START_REF] Cao | Synthesis and structure-activity relationships of harmine derivatives as potential antitumor agents[END_REF]. During this salt formation, compound 6j underwent a demethylation of the methoxy group leading to the 1-hydroxy derivative 22. To avoid this demethylation, the reaction time with benzyl bromide was limited to 24h leading to compound 18. Interestingly, with a pyridine at the C7 position (7c and 7e) the salt did not form selectively on N10 like for the other compounds but on pyridine only (30) or on both pyridine and N10 (29), respectively.

Biological evaluation 2.2.1. In vitro antiproliferative activity against MDA-MB-468, MDA-MB-435, MDA-MB-231 human cancer cells

Determination of in vitro growth inhibitory activity was preliminary performed on three highly aggressive human cancer cell lines (MDA-MB-468, MDA-MB-435s, MDA-MB-231) using a colorimetric MTT assay. The best compounds were then tested against 4T1 murine mammary carcinoma in vitro to anticipate the evaluation of our compounds in the experimental mouse model. Cells were cultured during five days in presence (test) or absence (control) of compounds 8-30 or harmine, at concentrations from 10 nM to 100 µM. For each compound, the concentration required to reduce the global growth by 50% (IC50) was determined on each cell line (Table 1). Dimethylsulfoxyde (DMSO) was used in the control condition.

Compounds 8-10 from our initial screening, were preliminary tested for their cancer cell growth inhibitory activity on MDA-MB-468 and MDA-MB-435s cells (Table 1). Compound 8 exhibited the best activity with IC50 of 18.4 and 16.4 µM, respectively, compare to 9 (completely inactive) and 10 (30.7 and 43.9 µM). These first encouraging results gave a trend for the C3 position: t-butyl > c-propyl >> CO2Et. All the next newly synthetized compounds 11-25 were then tested on the three human breast cancer cell lines. To complete the study focusing on the C3 position, the phenyl analogue 11 (R1 = Methyl and R3 = Phenyl) was prepared and exhibited the best antiproliferative activity against MDA-MB-468. The superiority of the phenyl group at the C3 position was not recovered with compound 20 (R1 = OMe, R3 = Phenyl) compare to analogues 18 (R1 = OMe, R3 = t-Butyl) and 19 (R1 = OMe, R3 = c-Propyl). In the methoxylated series, the best compound 18 present a tertio-butyl in position 3.

Changing the benzyl group (compound 8) to an ethyl group (compound 12) at the N-10 position resulted in a complete loss of activity. This observation (benzyl >> Ethyl) was confirmed for compounds 17 and 21, compare to 16 and 19 respectively, and for compound 14, regardless of the nature of substituents in other positions.

We then explored the C1 position with compounds 13 (R1 = Ethyl) and 15 (R1 = Phenyl) compare to 8 (R1 = Methyl). These three compounds with a t-butyl group at the C3 position gave similar results, with a slightly higher activity of the ethyl group. We then moved to 1-methoxy substituted compounds 18-21. Besides compound 21 (R10 = Ethyl), they all exhibited strong antiproliferative effect with IC50 from 0.4 to 6.0 µM regardless of the cancer cell line, whereas harmine presented an IC50 of 17.1 µM against MDA-MB-435s cells. Compound 18 (R3 = t-Butyl) proved to be the most active derivative with the lowest IC50 on the three cell lines. On the opposite, introduction of a hydroxyl group at the C1 position led to a loss of activity (compound

22).

Finally, we introduced structural modifications at the C7 position. A bromine atom was well tolerated in this position if we compare the 7-brominated compounds 23 (R1 = Methyl) and 25 (R1 = Phenyl) to the corresponding analogues 8 (R1 = Methyl) and 15 (R1 = Phenyl) with almost no effect on the IC50 values. The following compounds 26-30 were then tested only on MDA-MB-231 and 4T1 murine mammary carcinoma. Introduction of a methyl or thienyl moiety drastically affected the antiproliferative activity (compounds 26 and 28) while a phenyl or benzylpyridinium group seems to be tolerated in this position (IC50 around 15 µM respectively for 27 and 29 on MDA-MB-231). We could again confirm the critical importance of the benzyl substituent in position 10, with the inactive compound 30.

From the in vitro growth inhibitory activity evaluation, 18 appeared as the most promising antiproliferative agent against the three highly aggressive human cancer cell lines tested (MDA-MB-468, MDA-MB-435s, MDA-MB-231) as well as against 4T1 murine mammary carcinoma cells. As in the SAR investigation of the β-carboline series (Figure 2), we can observe that the nature of the substituent in position 1 deeply influences the antiproliferative efficacy when position 3 is less decisive.

We also measured the cytotoxic activity of these compounds by assessing cell survival after oneday treatment to exclude any compound with acute toxic effect that could induce high toxicity in vivo. Among the best compounds, 11, 18-20 were chosen for further evaluation because of their low IC50 for antiproliferative activity and higher IC50 for cytotoxicity test.

Effects on MDA-MB-435s cells migration in vitro

Cell invasion and migration are critical processes in tumor metastasis. The inhibitory effect of compounds 8-30 and harmine on migration of the highly metastatic human cancer cell lines MDA-MB-435s was investigated using time-lapse videomicroscopy of in vitro wound healing.

These experiments were conducted at 1 µM, concentration for which no cytotoxicity at 24h can be observed (Figure 6).

In this assay, six compounds inhibited the migration of MDA-MB-435s cells by 41 to 50%

Conclusions

In conclusion, a series of bioinspired imidazo Furthermore, wound-healing experiments showed that this series of compounds present also very interesting anti-migration activity (conversely to harmine that proved to be inactive) with six compounds inhibiting MDA-MB-435s cell migration by 41 to 50% while four compounds (20, 25, 27, 30) inhibited the migration by 53 to 62%. SAR analysis showed that the nature of the substitution in position 1 and the presence of a 10-benzyl group are less discriminant for antimigration activity. The 7 position appeared to be important to explore further.

Interestingly, compound 20 may constitute a promising drug candidate with dual antiproliferative and anti-migration activity on aggressive human breast cancer cell lines.

Experimental section

General methods of chemistry

All reagents were used directly as obtained commercially. Harmine was purchased from Sigma-Aldrich (St Louis, MO, USA). Thin-layer chromatographies (TLC) were performed using Merck ® silica gel 60F254 plates. Column chromatographies were performed using Merck Assignment of carbons noted C* may be interchanged. Mass spectra were determined on a Hewlett Packard 5988A spectrometer or on a Shimadzu QP 2010 spectrometer by direct inlet at 70 eV. Compounds 1a, 2a, 3a, 4a, 5a, 5c, 5d, 6a, 6c, 6d, 6f and 6h were prepared according to literature [6].

Ethyl 6-bromoimidazo[1,2-a]pyridine-2-carboxylate (1b)
To a solution of 2-amino-5-bromopyridine (8.5 g, 49 mmol, 1 eq) in DME (300 mL) was added dropwise ethyl bromopyruvate (11 mL, 73.7 mmol, 1.5 eq). After stirring for 5h, the solid was filtered and washed with DME. The solid was then transfered in a second flask and heated in refluxing ethanol (500 mL) overnight. The ethanol was evaporated to dryness and the resulting solid was partitioned between water and CH2Cl2 (300 mL/300 mL). The aqueous phase was alkalinized with a saturated solution of Na2CO3 and extracted with twice 300 mL of CH2Cl2. The combined organic phases were dried over MgSO4 and evaporated to yield 11.

6-Bromoimidazo[1,2-a]pyridine-2-carbonitrile (3b)

2b (8.47 g, 35 mmol) was charged in a 250 mL round bottom flask, followed by POCl3 (66 mL, 705 mmol, 20 eq). The reaction mixture was heated at reflux for 4h. POCl3 was then directly evaporated from the flask, using vacuum pump equipped with ice-cooled trap. The resulting solid was then suspended in a mixture of water and CH2Cl2 (200 mL/200 mL), followed by alkalization with aqueous saturated solution of Na2CO3. The aqueous phase was then extracted with CH2Cl2 (3×200 mL). The combined organic phases were dried over MgSO4 and evaporated to yield 6.5 g (29 mmol, 83%) of crude product, as a brown solid. 3b was used directly for the next step without further purification.

m.p.: 223-227°C. 1 H NMR (300MHz, DMSO-d6) δ: 8.97 (dd, 4 J = 1.8Hz, 5 J = 0.9Hz, 1H, H-5), 8.69 (s, 1H, H-3), 7.66 (d, 3 J = 9.7Hz, 1H, H-8), 7.57 (dd, 3 J = 9.7Hz, 4 J = 1.9Hz, 1H, H-7). 13

6-Bromo-3-iodoimidazo[1,2-a]pyridine-2-carbonitrile (4b)

To a solution of 3b (8.4 g, 37.8 mmol) in CH3CN (170 mL) was added NIS (11.1 g, 49.2 mmol, 1.3 eq) in one portion. The reaction mixture was heated at reflux overnight. The CH3CN was then evaporated to dryness and the solid was washed several times with water, petroleum ether and diethyl ether. The crude mixture was purified by flash chromatography on alumina (CH2Cl2) to afford 10.5 g (30 mmol, 80%) of 4b as a white solid.

m.p.: 275-279°C. 1 H NMR (300MHz, DMSO-d6) δ: 8.56 (dd, 4 J = 1.7Hz, 5 J = 1.0Hz, 1H, H-5), 7.67 (dd, 3 J = 9.6Hz, 5 J = 0.9Hz, 1H, H-8), 7.61 (dd, 3 J = 9.6Hz, 4 J = 1.7Hz, 1H, H-7). 13 General method for Sonogashira cross-coupling (5b, 5e, 5f) 4 (5 mmol), Pd2dba3 (0.25 mmol, 5 mol%) and CuI (0.5 mmol, 10 mol%) were introduced in a sealed tube. Vacuum/refilling with argon cycles were repeated thrice and dioxane (5.5 mL), Et3N

(5.5 mL) and finally alkyne (1.2 eq) were added. The reaction mixture was stirred for 3h at room temperature. The reaction mixture was then filtered on celite ® with CH2Cl2. Organic phase was washed with a saturated solution of NH4Cl, dried over MgSO4 and evaporated. The crude mixture was purified by flash chromatography with silica or alumina with appropriate eluent.

3-(3-((tetrahydro-2H-pyran-2-yl)oxy)prop-1-yn-1-yl)imidazo[1,2-a]pyridine-2-carbonitrile (5b)

Compound 5b was obtained after purification as an inseparable mixture (80/20) with deiodinated side-product. It was used like this for the cyclisation.

6-Bromo-3-(3,3-dimethylbut-1-yn-1-yl)imidazo[1,2-a]pyridine-2-carbonitrile (5e)

Silica, PE/Et2O 80/20 50/50, yellow solid, m.p.: 137-141°C, 3.26 g. Yield: 94%. 1 H NMR (300MHz, CDCl3) δ: 8.26 (dd, 4 J = 1.8Hz, 5 J = 0.9Hz, 1H, H-5), 7.53 (dd, 3 J = 9.6Hz, 5 J = 0.8Hz, 1H, H-8), 7.42 (dd, 3 J = 9.6Hz, 4 J = 1.8Hz, 1H, H-7), 1.42 (s, 9H, t-Bu). 13

6-Bromo-3-(cyclopropylethynyl)imidazo[1,2-a]pyridine-2-carbonitrile (5f)

Alumina, CH2Cl2, yellow solid, m.p.: 161-165°C, 1.05 g. Yield: 74%. 1 H NMR (300MHz, CDCl3) δ: 8.33 (dd, 4 J = 1.8Hz, 5 J = 0.9Hz, 1H, H-5), 7.52 (dd, 3 J = 9.6Hz, 5 J = 0.9Hz, 1H, H-8), 7.41 (dd, 3 J = 9.6Hz, 4 J = 1.8Hz, 1H, H-7), 1.72-1.58 (m, 1H, CH c-Pr), 1.13-0.95 (m, 4H 2 CH2 c-Pr). 13

General method for cyclization with Grignard reagent (compounds 6e, 6g, 6i, 6m, 6n and 6o).

To a solution of 3-alkynylimidazo[1,2-a]pyridine-2-carbonitriles (e.g. 1 mmol) in CPME (10 mL) were added dropwise Grignard reagent (2 eq) at room temperature. After completion of the reaction (followed by TLC, ca. 30min-2h), 10 mL of NH4Cl aqueous saturated solution were added. After 30 minutes of hydrolysis the phases are separated and aqueous phase was extracted with thrice 15 mL of EtOAc. The crude mixture was purified by flash chromatography on silica gel (petroleum ether / diethyl ether mixture). ). 13

3-(tert-Butyl)-1-ethylimidazo[

]dipyridin-3-yl)methanol was dissolved in 22 mL of acetone and 403 mg (2.55 mmol, 2.5 eq) of KMnO4 were added in one portion. The reaction mixture was heated at 40°C overnight. The mixture was then filtrated, the solid washed with water and the filtrate was evaporated to dryness. The obtained residue was directly used for the next step and dissolved in 10 mL of EtOH. 250 µL of H2SO4 were added and the reaction mixture was heated to reflux for 4h. The solvent was evaporated to dryness and the residue was partitioned between 10 mL of a saturated aqueous solution of Na2CO3 and 10 mL of CHCl3. The aqueous solution was extracted two more times with 10 mL of CHCl3. The combined organic phases were dried over MgSO4 and evaporated. The crude mixture was purified on alumina column with a mixture CH2Cl2/AcOEt (9/1) as eluent. 17 mg (0.07 mmol, 7%) of 6i were obtained. Due to the small amount obtained, 6i was entirely engaged in the salt formation and only the proton NMR was recorded. 1 H NMR (300MHz, CDCl3) δ: 8.63 (s, 1H), 8.53 (d, 3 J = 6.9

Hz, 1H), 7.81 (d, 3 J = 9.4 Hz, 1H), 7.58 (ddd, 3 J = 9.3, 6.7Hz, 4 J = 1.2 Hz, 1H), 7.01 (td, 3 J = 6.8Hz, 4 J = 0.9 Hz, 1H), 4.53 (q, 3 J = 7.1 Hz, 2H), 3.08 (s, 3H), 1.48 (t, 3 J = 7.1 Hz, 3H). (2 eq) and boronic acid (1.1 eq) were introduced in a microwave reaction vial. DME (1 mL) and water (500 µL) were then added. The mixture was heated using microwave oven until completion (ca. 1-3h). The crude mixture was partitioned between 20 mL of water and 20 mL of CH2Cl2. Aqueous phase was then extracted with twice 20 mL of CH2Cl2. The combined organic phases were dried over MgSO4, filtered and evaporated to dryness. The crude mixture was purified by flash chromatography on silica gel with appropriate eluent.

7-Bromo-3-(tert-butyl)-1-methylimidazo[1,

3-(tert-Butyl)-

General method for salt formation (compounds 8-30)

To a solution of 6a, 6b-p or 7a-e (e.g. 0.5 mmol) in CH3CN (5 mL) was added benzyl bromide or ethyl iodide (8 eq for 6a, 6b-i, 6m-o and 7a-d or 1.5 eq for 6j-l, 6p and 7e). The tube was sealed and the reaction heated to 80°C for ca. 24-72h. After cooling in an ice bath the solid was filtered and washed with EtOAc. Test compounds were dissolved in DMSO at a stock solution of 100 mM and further diluted to 100 μM, 10µM, 1µM, 0.1µM and 0.01µM in culture medium with a final concentration of DMSO of 0.1% including the control condition.

Cell viability and toxicity assay

Cell viability and toxicity were evaluated in vitro using the MTT assay. This assay is based on that the yellow tetrazolium MTT can be reduced by living cells to colored formazan dye. The absorbance of the dye measured and is proportional to the cell viability. Cells were seeded in 24 well plates at a density of 5x10 3 -30x10 3 cells/well in 500 μL of medium containing FBS and allowed to attach overnight at 37°C and 5% CO2. The growth medium in each well was then replaced with culture medium containing compounds to be tested at 5 different concentrations and incubated for 1 day for toxicity or for 5 days for viability assay. After incubation, the medium was replaced by MTT reagent (5 mg/mL prepared in DMEM+5%FCS) and incubated for 30-40 min in the incubator at 37°C, 5% CO2. The formazan crystals were dissolved in DMSO and the absorbance was measured using a microplate spectrophotometer (Epoch, BioTek Instruments Inc, USA) at 570 nm. The obtained data were normalized to the DMSO treated cells and IC50, corresponding to the concentration of the test compound giving 50% inhibition of growth, were calculated by using nonlinear regression with variable slope using graph GraphPad Prism™ v.5 (GraphPad Software, Incorporation, California, USA).

Apoptosis assay

Apoptosis was measured using BD Biosciences -FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, France) according to manufacturer recommendation. Briefly cells treated with 30 µM of compounds for 48h were detached using accutase and stained with Annexin V and propidium iodide. Samples were analyzed on a flow cytometer Canto, BD Biosciences.

Western blotting

Pro-apoptotic and anti-apoptotic proteins were detected by standard western blot protocol. Cell lysate were loaded on polyacrylamide gel and then transferred on PVDF membranes. Primary antibodies against Bak, Bax, Bcl-2, Bcl-xL were from cell signaling (Ozyme, France) and against cleaved PARP were from Abcam, France.

Ki-67 staining

Cells were seeded in Labteck glass chambers and incubated with 30 µM of compounds for 48h. Then cells were fixed in 4% paraformaldehyde and permeabilized using 0.3% Triton-X-

Figure 1 .

 1 Figure 1. Chemical structures of harmine, as representative β-carboline derivative, and

Figure 2 .

 2 Figure 2. The structure-activity relationships of β-carboline derivatives against tumor cells by

Figure 3 .

 3 Figure 3. Proposed SAR explorations around imidazo[1,2-a:4,5-c']dipyridine core.

Scheme 1 .

 1 Scheme 1. Synthetic routes to the compounds 5a-f (THP = tetrahydropyrane, c-propyl =

Scheme 3 .

 3 Scheme 3. Synthetic route to the brominated compounds 6m-p.

Scheme 4 .

 4 Scheme 4. Synthetic route to the 7-substituted imidazo[1,2-a:4,5-c']dipyridines 7a-e

Scheme 5 .

 5 Scheme 5. Formation of quaternary ammonium salts 8-30.

Figure 4 .

 4 Figure 4. ORTEP diagram for 8[START_REF]Crystallographic data for the structure 4n have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 2043162[END_REF]

Figure 5 .

 5 Figure 5. Annexin V/PI staining for detection of apoptosis of MDA-MB-231 cells treated with

Figure 6 .

 6 Figure 6. Immunoblot analyses of levels of pro-apoptotic proteins Bax and Bak, anti-apoptotic

Figure 7 .

 7 Figure 7. Ki-67 staining for detection of cell proliferation of MDA-MB-231 cells treated with

(compounds 8 ,

 8 15, 18, 22-24) while four compounds inhibited the migration by 53 to 62% (20, 25, 27, 30). From these ten compounds, eight present a tertio-butyl group in position 3 and a structural diversity in position 1, which seems to be less decisive for the anti-migrative activity than for antiproliferative activity.Three closely related compounds 25, 27, and 30 deeply inhibited MDA-MB-435s cell migration at 1 µM by 62, 53 and 62%, respectively. Their structures present a phenyl group in position 1 and a tertio-butyl group in position 3. Diversity occurs in position 7 with either a bromine atom for compound 25, a phenyl group for 27 and a benzylpyridinium for 30. As the 10-benzyl part is absent in compound 30, this substituent did not appear to be essential for the anti-migration activity. Interestingly, compound 20 presented dual antiproliferative and anti-migration activity with an average IC50 of 3.2 µM for the inhibition of the MDA-MB-231, MDA-MB-468 and MDA-MB-435s cell proliferation, and 43% inhibition in the MDA-MB-435s migration assay at 1 µM (Figure6). Conversely, harmine had almost no effect on migration of MDA-MB-435s cells (MDA-MB-435s cells migration of 93%).

Figure 8 .

 8 Figure 8. (A) Effect of compound 20 on MDA-MB-435s cell migration in a wound healing

  [1,2-a:4,5-c']dipyridines, diversely substituted on positions 1, 3, 7 and 10, were synthetized and tested for their in vitro antitumor activity against three strains of highly aggressive human cancer cell lines (MDA-MB-468, MDA-MB-435s and MDA-MB-231). Among them, compounds 18-20 exhibited excellent cancer inhibitory activity in vitro with IC50 of 0.4-5 µM against the three cell lines, higher than harmine activity against MDA-MB-435s. Compound 11 was also selected for further exploration (IC50 of 1.2 and 2.8 µM against MDA-MB-468 and 231 respectively). SAR analysis showed that the 10-benzyl group played a critical role in the antitumor activity, as well as the 1-methoxy group that deeply improved the antiproliferative effect. On the opposite, the presence of an ester group on the 3 position or the substitution of the 7 position seems detrimental.Flow cytometry assay showed that compounds 11 and 18-20 induce apoptosis in MDA-MB-231 cells in a dose-dependent manner, compounds 18-20 being more potent than 11. Moreover, a different signaling pathway seems to be impacted by 11 compare to 1-methoxylated 18-20: 11 increased the expression of pro-apoptotic Bax while 18-20 reduced the level of anti-apoptotic Bcl-2 expression. The effect of these compounds on MDA-MB-231 cell proliferation using Ki-67 staining revealed that 18 and 19 also reduced cell proliferation.

  Geduran ® Si 60 (40-63µm) silica. Melting points were determined on a capillary apparatus (Stuart, Staffordshire, United Kingdom) and are uncorrected. Microwave heating was performed using CEM ® Explorer SP 12 S class apparatus (maw power 300 W). NMR experiments were performed at 300 MHz ( 1 H) and 75 MHz ( 13 C) on a Bruker-Avance 300 MHz spectrometer.

  C NMR (75MHz, DMSO-d6) δ: 143.3 (C-8a), 130.9 (C-7), 127.7 (C-5), 121.5 (C-3), 118.4 (C-8), 116.5 (C-2), 115.0 (CN), 108.3 (C-6). HRMS (ESI): m/z calc. for C8H4 79 BrN3 [M+I] + : 221.96614, found: 221.96547; m/z calc. for C8H4 81 BrN3 [M+I] + : 223.96409, found: 223.96336.

  C NMR (75MHz, CDCl3) δ: 143.0 (C-8a), 131.6 (C-7), 125.5 (C-5), 121.3 (C-2), 119.3 (C-8), 113.9 (C-3), 110.1 (CN*), 110.1 (C-6*), 59.6 (-C≡C-c-Pr), 9.9 (CH2 c-Pr), 0.7 (CH c-Pr).

  13 C NMR (75MHz, CDCl3) δ: 159.8 (C-3), 148.7 (C-9a), 147.7 (C-1), 138.8 (Ph-1), 137.0 (C-10a*), 135.2 (C-4a*), 130.2 (C-8), 129.5 (Ph-2,6), 128.9 (Ph-4), 128.5 (Ph-3,5), 125.5 (C-6), 119.1 (C-9), 111.0 (C-7), 98.8 (C-4), 38.0 (Cq t-Bu), 30.9 (CH3 t-Bu). HRMS (ESI): m/z calc. for C20H19N3 [M+I] + : 302.16517, found: 302.16415. Ethyl 1-methylimidazo[1,2-a:4,5-c']dipyridine-3-carboxylate (6i) Note: The sequence leading to compound 6i is an un-optimized sequence which was performed only once. Cyclization was performed on 5b (981 mg: mixture with de-iodinated side-product, 80/20) -3-(((tetrahydro-2H-pyran-2yl)oxy)methyl)imidazo[1,2-a:4,5-c']dipyridine (6b) were obtained. 1 H NMR (300MHz, CDCl3) δ: 8.41 (d, 3 J = 6.9Hz, 1H), 7.82 (s, 1H), 7.72 (d, 3 J = 9.4Hz, 1H), 7.45 (ddd, 3 J = 9.4, 6.6Hz, 4 J = 1.2Hz, 1H), 6.88 (td, 3 J = 6.9Hz, 4 J = 0.9Hz, 1H), 5.09 (d, 2 J = 13.3Hz, 1H), 4.86-4.81 (m, 2H), 3.98-3.91 (m, 1H), 3.60-3.54 (m, 1H), 3.00 (s, 3H), 1.94-1.55 (m, 6H

2 (

 2 13 C NMR (75MHz, CD3OD) δ: 166.1 (C-3), 147.6 (C-1), 144.9 (C-8), 144.8 (C-9a), 138.5 (1-Ph-1), 135.9 (C-4a), 135.1 (CH2Ph-1), 130.8 (1-Ph-4), 130.6 (1-Ph-2,6), 130.0 (1-Ph-3,5*), 129.8 (C-7), 129.5 (CH2Ph-3,5*), 129.CH2Ph-4), 129.0 (C-6), 126.9 (CH2Ph-2,6), 126.2 (C-10a), 111.9 (C-9), 103.5 (C-4), 50.

Table 1 .

 1 Compounds 8-30 and their anti-proliferative (cell culture over five days) and cytotoxic (over 24 hours) activities (n=3 independent experiments). 48h at two different concentrations, 3 and 30 µM corresponding to about 1xIC50 and 10xIC50, respectively (Figure3). Dimethylsulfoxyde (DMSO) 0.1%, used as a vehicle for these compounds, was used in the control condition. The different parts of the graph

						Antiproliferative activity (IC50, µM)		Cytotox.
										(IC50, µM)
	Cpd R1	R3	R7	R10	MDA-MB-468 MDA-MB-435s MDA-MB-231	4T1	MDA-MB-435s
	8	Me	t-Bu	H	Bn	18.4±6.1	16.4±3.1	n.d.	n.d.	74.4±28.0
	9	Me CO2Et	H	Bn	>100	>100	n.d.	n.d.	>100
	10	Me	c-Pr	H	Bn	30.7±11.4	43.9±14.1	n.d.	n.d.	>100
	11	Me	Ph	H	Bn	1.2±0.9	22.4±1.5	2.8±0.2	>100	>100
	12	Me	t-Bu	H	Et	99.4±15.0	90.5±0.5	>100	n.d.	>100
	13	Et	t-Bu	H	Bn	8.7±1.7	4.8±0.3	10.7±2.6	n.d.	35.4±14.6
	14	Et	c-Pr	H	Et	>100	28.3±4.7	>100	n.d.	>100
	15	Ph	t-Bu	H	Bn	13.3±2.3	6.4±1.0	11.9±2.3	19.7±5.2	21.3±8.6
	16	Ph	c-Pr	H	Bn	31.1±16.1	13.1±6.5	32.1±4.5	n.d.	48.6±2.3
	17	Ph	c-Pr	H	Et	>100	47.6±20.4	>100	n.d.	>100
	18 OMe t-Bu	H	Bn	0.4±0.4	1.1±0.1	1.7±0.3	0.5±0.3	11.9±1.7

n.d. stands for not determinated

2.2.2. Effect on apoptosis in MDA-MB-231 Cells

A double staining flow cytometric assay using annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI) was carried out to evaluate the potential of compounds 11 and 18-20 to induce apoptosis in MDA-MB-231 cancer cells. MDA-MB-231 cells were treated with compounds 11 and 18-20 for represented the percentage of living, apoptotic, and necrotic cell populations. Compared to the control group with a total apoptosis rate of 2.38%, almost no effect was observed at 3 µM with apoptosis of 3.30, 3.34, 3.50 and 4.11 % for compounds 11 and 18-20, respectively. Nevertheless, at 30 µM the total apoptosis rate strongly increased for compounds 11, 18-20 to 7.78, 6.72, 12.5 and 6.03 % respectively. Late apoptotic and necrotic cell population also dramatically increased with 30 µM of 11 (from 2.94% in control group to 13.4%) and 18-20 (from 2.94% to around 30%).

  Deprotection of the THP was performed by adding 10 mL of HCl (6M) to a solution of 6b (529 mg, 1.78 mmol) in CH2Cl2 (40 mL). The reaction mixture was stirred at room temperature for 1h. The aqueous phase was alkalinized with Na2CO3 aqueous saturated solution and extracted with CH2Cl2 (twice 40 mL). The organic phase was dried over MgSO4 and then evaporated to give 141 mg of a yellow solid. The aqueous phase was cooled in an ice-bath for 2h. After filtration, 214 mg more of yellow solid was obtained. All the solids were combined to yield 355

	mg of the free alcohol (1-methylimidazo[1,2-a:4,5-c']dipyridin-3-yl)methanol (1.66 mmol,
	93%). 1 H NMR (300MHz, DMSO-d6) δ: 8.84 (d, 3 J = 7.0Hz, 1H), 8.08 (s, 1H), 7.70-7.62 (m, H),
	7.08-7.03 (m, 1H), 4.92 (s, 2H), 2.89 (s, 3H). 13 C NMR (75MHz, DMSO-d6) δ: 152.9, 151.8,
	150.3, 138.8, 135.2, 133.4, 128.2, 118.4, 113.3, 102.5, 65.7, 19.7.
	218 mg (1.02 mmol) of (
	C NMR (75MHz,
	CDCl3) δ: 151.8, 148.5, 148.3, 138.7, 133.1, 130.3, 125.7, 118.7, 111.2, 101.1, 98.6, 70.2, 62.5,
	30.6, 25.4, 20.3, 19.6.

  CH2 c-Pr). HRMS (ESI): m/z calc. for C22H21 79 BrN3 [M] + : 406.09134, found: 406.09033; m/z calc. for C22H21 81 BrN3 [M] + : 408.08929, found: 408.08815. ), 8.62 (s, 1H, H-4), 8.30 (dd, 3 J = 9.4Hz, 4 J = 1.5Hz, 1H, H-8), 8.10 (d, 3 J = 9.4Hz, 1H, H-9), 7.57-7.40 (m, 5H, 1-Ph), 7.25-7.13 (m, 3H, CH2Ph-3,4,5), 6.69-6.66 (m, 2H, CH2Ph-2,6), 5.61 (s, 2H, CH2Ph), 2.65 (s, 3H, Me), 1.53 (s, 9H, t-Bu).

	NMR (75MHz, CD3OD) δ: 158.6 (C-3), 146.9 (C-1*), 146.0 (C-9a*), 143.7 (C-8), 137.2 (Ph-1),
	135.7 (C-4a), 131.5 (C-6), 130.6 (Ph-3,5), 129.7 (Ph-4), 127.9 (C-10a), 126.8 (Ph-2,6), 119.2 (C-
	7), 112.5 (C-9), 105.1 (C-4), 50.3 (CH2Ph), 20.6 (Me), 16.8 (CH c-Pr), 11.6 (CH2 c-Pr). HRMS 10-Benzyl-7-bromo-3-(tert-butyl)-1-phenylimidazo[1,2-a:4,5-c']dipyridin-10-ium bromide (25)
	(ESI): m/z calc. for C21H20N3 [M] + : 314.16517, found: 314.16443.
	Yellow solid, m.p.: 156-160°C, 112 mg. Yield: 33%. 1 H NMR (300MHz, CDCl3) δ: 10.67 (d, 4 J
	10-Benzyl-1-methyl-3-phenylimidazo[1,2-a:4,5-c']dipyridin-10-ium bromide (11) = 0.9Hz, 1H, H-6), 9.05 (s, 1H, H-4), 8.50 (d, 3 J = 9.7Hz, 1H, H-9), 8.15 (dd, 3 J = 9.6Hz, 4 J =
	Brown solid, m.p: 238-242°C, 35 mg. Yield: 30%. 1 H NMR (300MHz, DMSO-d6) δ: 9.85 (d, 3 J 1.1Hz, 1H, H-8), 7.57-7.41 (m, 5H, 1-Ph), 7.18-7.10 (m, 3H, CH2Ph-3,4,5), 6.75-6.72 (m, 2H,
	= 6.9Hz, 1H, H-6), 9.37 (s, 1H, H-4), 8.54-8.47 (m, 2H, H-8 & H-9), 8.26 (d, 3 J = 7.2Hz, 2H, CH2Ph-2,6), 5.92 (s, 2H, CH2Ph), 1.48 (s, 9H, t-Bu). 13 C NMR (75MHz, CDCl3) δ: 166.3 (C-3),
	Ph-2,6), 7.91 (td, 3 J = 6.9Hz, 4 J = 0.9Hz, 1H, H-7), 7.61-7.47 (m, 3H, Ph-3,4,5), 7.42-7.26 (m, 146.3 (C-1), 143.2 (C-8), 142.5 (C-9a), 136.7 (1-Ph-1), 133.9 (CH2Ph-1*), 133.4 (C-4a*), 130.7
	5H, CH2Ph), 6.23 (s, 2H, CH2Ph); 2.84 (s, 3H, CH3). 13 C NMR (75MHz, DMSO-d6) δ: 150.4 (C-(C-6), 130.1 (1-Ph-4), 129.7 (1-Ph-2,6), 129.2 (1-Ph-3,5*), 128.8 (CH2Ph-3,5*), 128.7 (CH2Ph-
	3), 144.8 (C-1), 144.2 (C-9a), 141.5 (C-8), 137.5 (3-Ph-1), 135.7 (CH2Ph-1*), 134.7 (C-4a*), 4), 126.4 (CH2Ph-2,6), 124.7 (C10a), 114.6 (C-9), 112.1 (C-7), 104.1 (C-4), 50.4 (CH2Ph), 38.8
	130.4 (C-6), 129.5 (3-Ph-4), 129.1 (CH2Ph-3,5*), 129.0 (3-Ph-3,5*), 128.1 (CH2Ph-4), 126.8 (C-
	10a), 126.7 (3-Ph-2,6), 125.7 (CH2Ph-2,6), 117.4 (C-7), 111.4 (C-9), 103.2 (C-4), 48.3 (CH2Ph),
	22.4 (Me). HRMS (ESI): m/z calc. for C24H20N3 [M] + : 350.16517, found: 350.16432. 10-Benzyl-3-(tert-butyl)-7-methyl-1-phenylimidazo[1,2-a:4,5-c']dipyridin-10-ium bromide (26)
	3-(tert-Butyl)-10-ethyl-1-methylimidazo[1,2-a:4,5-c']dipyridin-10-ium iodide (12) White solid, m.p.: 237-241°C, 57 mg. Yield: 53%. 1 H NMR (300MHz, CD3OD) δ: 9.56 (s, 1H,
	Brown solid, m.p: 244-248°C, 52 mg. Yield: 63%. 1 H NMR (300MHz, DMSO-d6) δ: 9.72 (d, 3 J H-6
	= 6.9Hz, 1H, H-6), 8.61 (s, 1H, H-4), 8.50-8.40 (m, 2H, H-9 & H-8), 7.76 (td, 3 J = 6.7Hz, 4 J =
	1.2Hz, 1H, H-7), 4.88 (q, 3 J = 7.1Hz, 2H, CH2CH3), 3.08 (s, 3H, Me), 1.49 (t, 3 J = 7.2Hz, 3H,
	CH2CH3), 1.44 (s, 9H, t-Bu). 13 C NMR (75MHz, DMSO-d6) δ: 162.9 (C-3), 143.6 (C-1*), 142.8
	(C-9a*), 140.3 (C-8), 133.4 (C-4a), 129.9 (C-6), 125.2 (C-10a), 116.6 (C-7), 111.4 (C-9), 101.8
	10-Benzyl-3-(tert-butyl)-1-methylimidazo[1,2-a:4,5-c']dipyridin-10-ium bromide (8) (C-4), 40.8 (CH2CH3), 37.6 (Cq t-Bu), 30.2 (CH3 t-Bu), 22.7 (Me), 15.2 (CH2CH3). HRMS
	(ESI): m/z calc. for C17H22N3 [M] + : 268.18082, found: 268.17990.

10-Benzyl-3-(tert-butyl)-1-ethylimidazo[

1,2-a:4,5-c']dipyridin-10-ium bromide (13) (Cq t-Bu), 30.7 (CH3 t-Bu). HRMS (ESI): m/z calc. for C27H25 79 BrN3 [M] + : 470.12264, found: 470.12126; m/z calc. for C27H25 81 BrN3 [M] + : 472.12059, found: 472.11915.

  1 (CH2Ph), 39.2 (Cq t-Bu), 30.7 (CH3 t-Bu), 17.9 (Me). HRMS (ESI): m/z calc. for C28H28N3 [M] + : 406.22777, found: 406.22663. 1), 130.2 (C-6), 129.4 (C-8), 129.3 (1-Ph-4*), 129.2 (1-Ph-3,5*), 129.1 (CH2Ph-4*), 128.8 (1-Ph-2,6), 128.6 (CH2Ph-2,6), 128.3 (CH2Ph-3,5*), 124.1 (pyr-3,5), 118.2 (C-7), 117.7 (C-9), 101.7 (C-4), 62.4 (CH2Ph), 37.7 (Cq t-Bu), 30.6 (CH3 t-Bu). HRMS (ESI): m/z calc. for C32H29N4 [M] + : 469.23867, found: 469.23716.4.2. Cell cultureHuman cancer MDA-MB-435s, MDA-MB-231, MDA-MB-468 cells and murine mammary cancer 4T1 lines were obtained from the American Type Culture Collection (LGC Promochem, France). MDA-MB-435s, MDA-MB-231 and MDA-MB-468 cell lines were cultivated in plastic culture flasks in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS). 4T1 cells were grown in RPMI supplemented with 10% FBS. All cells were cultured at 37°C under a humidified atmosphere containing 5% CO2.

Acknowledgements

We gratefully acknowledge the generous support provided by the Ligue Nationale Contre le Cancer -Interrégion Grand-Ouest. Abdulrahim Alzain research was supported by a grant from the Ministry of Higher Education and University of Gezira, Sudan.

100. After saturation in 3% BSA, Ki-67 were stained using primary antibody from BD Bioscience (France) and secondary AlexaFluor488 antibody (Invitrogen, France). Slides were mounted with Vectashield Vibrance with Dapi (Eurobio, France) and pictures were taken with Nikon Eclipse (Nikon Corp., Tokyo, Japan) equipped with NIS Elements software.

Wound healing assay

Migration studies were performed using 2-well culture inserts (ibidi, Regensburg, Germany) which were transferred into 24-well culture plate to form a well-defined gap without scratching the cell monolayer. Cells were cultivated in 70 µL of growth medium in a density of 40x10 3 per chamber of the culture insert and incubated overnight. After removal of inserts using a sterile forceps, different subtoxic concentrations of each test compound in 500 μL culture medium were added to each well plate. Then cells were monitored and photographed every 15 minutes for 24 hours for wound closure using Nikon Eclipse (Nikon Corp., Tokyo, Japan) equipped with NIS Elements software. The open area was analyzed with Tscratch software (CSElab, Zurich, Switzerland).

Author information

The authors declare no competing financial interest.

Supplementary data

Supplementary data to this article can be found online at


Dr Antoine Léotard
email: antoine.leotard@aphp.fr

Marius Lebret

Najeh Daabek

Hélène Prigent

Christelle Marie Destors

Saint- Amélie Sagniez

Karl Leroux

Renaud Tamisier

Frédéric Lofaso

Christel Saint-Raymond

Jean Louis Pépin

Jean Christian Borel

Richard Yvinec

Vincent Delord

Mickael Berlusconi

Steffy Masucco

Galina Penkov

Sebastien Bailly

Marie Peeters

Mélanie Lopez

Pierre Ansay

la eficacia de la ventilación no invasiva en pacientes con enfermedad neuromuscular: un ensayo clínico aleatorizado cruzado

Keywords: Neuromuscular diseases, non-invasive ventilation, nasal mask, oronasal mask

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Impacto del tipo de mascarilla en la eficacia de la ventilación no invasiva en pacientes con enfermedad neuromuscular: un ensayo clínico aleatorizado cruzado RESUMEN Antecedentes y objetivo: Alrededor del 25% de los pacientes con enfermedades neuromusculares (ENM) son tratados mediante ventilación no invasiva (VNI) a través de una máscara oronasal. Sin embargo, existen crecientes indicios de que las máscaras nasales requieren presiones de VNI más bajas y resultan en menos eventos obstructivos residuales.

Nuestra hipótesis fue que las máscaras nasales mejorarían la eficacia y reducirían los efectos secundarios en comparación con las máscaras oronasales en esta población.

Métodos: Estudio abierto, cruzado, aleatorizado, en dos hospitales de atención terciaria. Los pacientes con ENM tratados mediante VNI domiciliaria fueron aleatorizados durante períodos de una semana de duración a las mascarillas nasales y oronasales, alternativamente (cruzado). Al final de cada período, se realizó una polisomnografía nocturna (con monitorización de la apertura bucal) con VNI, sincronizada con la medición transcutánea de la presión parcial de CO2 (tcCO2). Los datos se recopilaron utilizando el software integrado en la VNI y se recogieron los efectos secundarios de la VNI. Se realizaron análisis por intención de tratar y por protocolo. El criterio de valoración principal fue la SpO2 nocturna media. Los criterios secundarios fueron: porcentaje de sueño con SpO2 <90%, índice de desaturación de oxígeno (IDO), tcCO2 media, duración media de la apertura bucal durante el sueño, nivel de fugas no intencionales y efectos secundarios.

Resultados: Se incluyeron treinta pacientes con ENM. No hubo diferencias entre los grupos para los resultados primarios o secundarios. Las comparaciones a posteriori mostraron que cambiar mascarillas reducía la eficacia de la VNI: SpO2 nocturna media (p = 0,04), IDO (p = 0,01), tcCO2 media (p = 0,048), efectos secundarios (p = 0,008).

Conclusión:

Las máscaras nasales no mejoraron la eficacia de la VNI ni redujeron los efectos secundarios en comparación con las máscaras oronasales en pacientes con ENM tratados con VNI domiciliaria. La eficacia de VNI se reduce durante la transición a otra mascara, lo que requiere una estrecha vigilancia.

Palabras clave:

Enfermedades neuromusculares, ventilación no invasiva, mascarilla nasal, mascarilla oronasal

INTRODUCTION

Home Noninvasive Ventilation (NIV) is the reference treatment for chronic alveolar hypoventilation in patients with neuro-muscular diseases (NMD) [START_REF] Annane | Nocturnal mechanical ventilation for chronic hypoventilation in patients with neuromuscular and chest wall disorders[END_REF][START_REF] Leger | Nasal intermittent positive pressure ventilation. Long-term follow-up in patients with severe chronic respiratory insufficiency[END_REF]. NIV can be provided through different types of interfaces, the most common of which are nasal masks and oronasal masks. The choice of interface is very important as it affects both the efficiency of the NIV and patient adherence, which is essential for effective treatment. Prior to initiation of NIV, clinicians must therefore determine which interface will provide the most efficient treatment but will also be comfortable for the patient so that they do not abandon the treatment.

Nasal masks are the most commonly used interface for home NIV in patients with NMD(3).

However, persistent unintentional leaks, especially through the mouth trigger nasal congestion and/or nasopharyngeal and mouth dryness. Mouth leaks can also reduce NIV efficacy and impair sleep quality [START_REF] Teschler | Effect of mouth leak on effectiveness of nasal bilevel ventilatory assistance and sleep architecture[END_REF]. Oronasal masks covering the nose and mouth are thus commonly proposed to patients in these situations [START_REF] Mckim | Home mechanical ventilation: a Canadian Thoracic Society clinical practice guideline[END_REF].

The decision to choose an oronasal mask is most often based on self-reported symptoms (especially leaks and side effects). Data collected from the NIV built-in software may also lead clinicians and home care providers to suspect leakage due to mouth opening. However, the proportion of real sleep time spent with an open mouth is usually not objectively documented [START_REF] Lebret | Nasal obstruction and male gender contribute to the persistence of mouth opening during sleep in CPAP-treated obstructive sleep apnoea[END_REF] and once an oronasal mask has been prescribed, a shift back to a nasal mask is almost never considered during follow-up as mouth leaks are considered to be a permanent problem. About 25% of patients with NMD who are treated with nocturnal NIV at home use an oronasal mask [START_REF] Lofaso | Long-term mechanical ventilation equipment for neuromuscular patients: meeting the expectations of patients and prescribers[END_REF][START_REF] Rabec | Evaluating noninvasive ventilation using a monitoring system coupled to a ventilator: a bench-to-bedside study[END_REF][START_REF] Pasquina | What does built-in software of home ventilators tell us? An observational study of 150 patients on home ventilation[END_REF] but clinical reality shows that there is great heterogeneity of use between different countries, regions or units. A recent analysis showed that NMD patients who used oronasal interfaces were older and had higher BMI than patients who used nasal mask; two factors which are linked to obstructive apneas. Accordingly, positive expiratory pressures (PEP) set on the NIV were also higher with oro-nasal masks [START_REF] Leotard | Factors associated with the type of mask used during nocturnal NIV in patients with neuromuscular disorders[END_REF]. This type of interface have been reported to increase upper-airway (UA) resistance by pushing the mandible backward, which in turn facilitates occurrence of upper airway obstruction [START_REF] Vrijsen | Upper airway obstruction during noninvasive ventilation induced by the use of an oronasal mask[END_REF][START_REF] Schellhas | Upper airway obstruction induced by non-invasive ventilation using an oronasal interface[END_REF].

Very few studies have compared, using appropriate methodology, the efficacy of nasal mask versus oronasal mask in patients treated by long term nocturnal NIV [START_REF] Fernandez | Nasal versus oronasal mask in home mechanical ventilation: the preference of patients as a strategy for choosing the interface[END_REF][START_REF] Willson | Nasal versus full face mask for noninvasive ventilation in chronic respiratory failure[END_REF]. The aim of this study was to compare the efficacy and rate of side effects between nasal and oronasal masks in patients with NMD. We hypothesized that the application of an oronasal mask might compromise nocturnal mechanical ventilation and gas exchange by favoring pharyngeal closure or increased resistance in patients already prone to upper airway obstruction due to their underlying disease or to their maxillofacial profile. Thus, the use of a nasal mask (that does not reduce upper airway stability) would provide more effective NIV with fewer side effects compared to an oronasal mask.

METHODS (see details in online supplement) Trial design

We performed an open label, cross-over, randomized, study in 2 tertiary care hospitals in France.

Both hospitals had substantial experience in the management of NMD and treatment with NIV. The study was approved by our ethics committee (CPP Nord-ouest II) following French legislation, and written informed consent was obtained from all patients. The trial was registered in ClinicalTrials.Gov (number NCT03458507).

Participants

Patients with NMD treated with home NIV who were followed by the participating centers were screened for eligibility between May 2018 and May 2019. The inclusion criteria were: adults (>18 years old) treated with nocturnal non-invasive ventilation <15 hours/day (to exclude patient with severe respiratory insufficiency in which transition period should have been at risk) and diagnosed with a NMD (Becker muscular dystrophy, facio-scapulo-humeral dystrophy, limb-girdle dystrophy, myotonic dystrophy, Duchenne muscular dystrophy). Exclusion criteria were: occurrence of an acute cardiorespiratory or ear-nose-throat event during the month prior to inclusion, rapidly progressive NMD (such as ALS), severe nasal obstruction and maxillofacial deformities or previous upper airway surgery preventing the usage of one type of mask (nasal or oronasal).

Interventions

Inclusion visit. Patients tested the interface they did not usually use (i.e. nasal mask for those treated with oronasal masks or oronasal mask for those treated with nasal masks) during a one-hour diurnal NIV session. The most appropriate alternative mask (in terms of brand and size) was chosen for each patient in order to minimize leakage and maximize comfort. Follow up. At the end of the inclusion visit, patients were randomized to start either with their usual or alternative interface. During the week with the alternative interface, patients were asked to use this new interface with the aim to sleep overnight with it. During the week with the usual interface, patients were asked to pursue their treatment as usual. The number and nature of interface-related problems reported by patients and/or the need for a home care provider visit were collected during both weeks.

At the end of each week, an unattended type III nocturnal polygraphy under NIV was performed at home using a Somnolter® (Nomics, Liege, Belgium) device synchronized with transcutaneous partial pressure in CO2 (PtcCO2) monitoring by SenTec V-Sign™ System. This polygraph also records mandibular mouth opening.

Assessment of NIV side-effects was performed with the Modified SECI questionnaire( 14) (for each commonly reported side effect, the patient is asked to rate the frequency, magnitude and perceived impact on adherence on a five-point Likert-type scale).

Outcomes

Patients were evaluated on two occasions, at the end of each week with either the usual or the alternative interface (cross-over). The primary objective was to compare mean nocturnal oxygen saturation between oronasal masks and nasal masks (Mean nocturnal Sp02, measured by polygraphy, Somnolter®). We also assessed the following exploratory secondary outcomes: percentage of sleep with SpO2<90% (polygraphy, Somnolter®); oxygen desaturation index (polygraphy, Somnolter®); mean nocturnal transcutaneous partial pressure in CO2 (PtcCO2, SenTec V-Sign™); mean mouth opening during sleep (polygraphy, Somnolter®); level of non-intentional leaks (percentage of recording time spend with leaks assessed by SomnoHolter®); the side-effects for each type of mask (Modified SECI).

Randomization

Participants were randomized (computer-generated random numbers list) to either begin the trial with their usual interface (for one week) or the alternative interface (for one week) in order to allow a balance between the numbers of patients who would begin with their usual interface versus with an alternative interface, the randomization was stratified for the type of interface usually used by the patient.

Sample size

Very few studies have compared nasal and oronasal masks for noninvasive ventilation in chronic respiratory failure [START_REF] Vrijsen | Upper airway obstruction during noninvasive ventilation induced by the use of an oronasal mask[END_REF][START_REF] Fernandez | Nasal versus oronasal mask in home mechanical ventilation: the preference of patients as a strategy for choosing the interface[END_REF][START_REF] Willson | Nasal versus full face mask for noninvasive ventilation in chronic respiratory failure[END_REF]. Therefore, in this pilot study, the sample size estimation was based on our inclusion capability and was based on the following: 1) at least 100 eligible patients were followed in the two participating centers, 2) 25% to 30% of patients with NMD use oronasal masks and we wanted to recruit equal numbers of patients who usually used oronasal masks and nasal masks and 3) we estimated that about 50% of eligible patients would accept to participate. Therefore, we planned to 30 patients (15 with nasal mask and 15 patients with oronasal mask). This sample size was sufficiently powered to detect a mean difference of mean nocturnal Spo2 of 2 ± 2% (considered as clinically significant) between oronasal versus nasal mask (alpha=0.05, power=80%).

Statistical analysis

A treatment effect test, a period effect test, and a test for the interaction between treatment and period were used to assess respective effect of treatment (nasal versus oronasal), treatment sequence, and the first-order carryover risk. Paired student t-tests were conducted to assess differences between the two interfaces for normally distributed outcomes. Wilcoxon signed-rank tests for paired values were used to compare nonnormally distributed outcomes. Significance was set at p <0.05. Missing data were not imputated.

All randomized patients were analyzed in intention to treat (ITT): if a patient did not tolerate the alternative interface, polygraphy was performed with their usual interface and data collected during this evaluation was used in the analysis. A per-protocol (PP) was secondarily performed using data from patients who completed both polygraphies with the appropriate masks without any protocol deviation. Finally, a post-hoc comparison using the same statistical models was performed to compare the usual and alternative interfaces to assess the impact of mask change among those patients.

RESULTS

Recruitment

The study flow is depicted in figure 1, 30 patients with NMD were included. Among them, 13 used an oronasal mask at home. During the week with the alternative interface, 5 patients had major intolerances (2 with the nasal mask and 3 with the oro-nasal) despite home care provider interventions and thus resumed the use of their usual interface (Figure 1). Table 1 presents the anthropometric characteristics of patients and ventilator settings. Patients were middle aged and equally distributed in terms of sex. Myotonic dystrophy was the most represented neuromuscular disorder (36.7%). Pressure support mode was used by 86.7% of patients with no differences in IPAP and EPAP settings between those who usually used nasal and those who used oronasal masks. There was no difference in mean NIV adherence, residual AHI or tidal volume estimated by NIV built-in software between patients who used nasal and those who used oronasal interfaces.

Primary outcome

There were no significant differences in mean nocturnal SpO2 between the interfaces (ITT, p=0.73; PP, p=0.59) (Figure 2).

Secondary outcomes

There were no differences in secondary outcomes between the interfaces (Figure 2): percentage of sleep recording spent with SpO2<90% (ITT, p=0.33; PP, p=0.57); oxygen desaturation index (ITT, p=0.74; PP, p=0.59); mean nocturnal transcutaneous partial pressure in CO2 (ITT, p=0.13; PP, p=0.11); mean mouth opening during sleep (ITT, p=0.1931; PP, p=0.054); level of non-intentional leaks (ITT, p=0.58; PP, p=0.80) or side-effects according to each type of mask (ITT, p=0.46; PP, p=0.98). Additionally, no differences were found between masks in terms of mean NIV adherence, residual AHI, nadir SpO2, time spent with leaks or time spent with tcCO2 > 55mmHg.

There were no differences in the ratio of patients who reported interface problems or the number of home care provider interventions between interfaces (Table 2).

Post hoc analysis of interface transitions (Figure 3) Post-hoc comparisons showed that changing from one interface to another reduced NIV efficacy: oxygen desaturation index (ITT, p=0.01; PP, p=0.01); mean nocturnal transcutaneous partial pressure in CO2 (ITT, p=0.048; PP, p=0.09); side-effects (ITT, p=0.008; PP, p=0.02).

During the week with the alternative interface, mask problems ratio and home care provider intervention numbers were higher (p < 0.01) and mean adherence was lower (p=0.02) (Table 2). Accordingly, patients perceived an impact on adherence during the week with the alternative interface (Table 3).

Complaints reported by participants about the alternative interface were predominantly mask intolerance, leaks, mouth dryness and cutaneous lesions. Problems relating to the underlying condition and impaired autonomy were reported especially with the use of an oronasal interface (impaired communication leading to the incapacity to call for help if needed, compromised vocal command device use, compromised hydration autonomy at night, etc. )

DISCUSSION

This original randomized cross-over study found no differences between nasal and oronasal masks in terms of nocturnal NIV efficacy and NIV tolerance. However, during the transition between the interfaces mean nocturnal SpO2 and adherence decreased and oxygen desaturation, mean nocturnal tcCO2 and side effects increased.

In a previous RCT that included a heterogeneous group of patients with chronic respiratory failure, Willson et al. reported poorer quality of sleep, impaired tolerance (less comfort, more perceived leaks) with the use of oronasal masks compared to nasal masks [START_REF] Willson | Nasal versus full face mask for noninvasive ventilation in chronic respiratory failure[END_REF]. However, similarly to the present study, they also found no differences in terms of nocturnal gas exchange or sleep disordered breathing between the two interfaces. In Willson et al.'s study, all subjects were treated with nocturnal NIV at home via a nasal mask [START_REF] Willson | Nasal versus full face mask for noninvasive ventilation in chronic respiratory failure[END_REF]. Therefore, the reduced tolerance reported with oronasal mask may simply reflect difficulty switching from the usual interface to another. This hypothesis is supported by the results of the post-hoc analyses in the present study.

We hypothesised that the oronasal mask might increase upper airway obstruction and jeopardize NIV efficacy. This was based on a recent case report in a patient with ALS (without bulbar involvement) that showed that use of an oronasal mask with NIV caused persistent obstructive respiratory events, with oxygen desaturations, sleep fragmentation and persistent sleep hypercapnia [START_REF] Vrijsen | Upper airway obstruction during noninvasive ventilation induced by the use of an oronasal mask[END_REF]. In the field of obstructive sleep apnea, the impact of the mask type has been more extensively assessed in patients treated with continuous positive airway pressure (CPAP). Similar reports of persistent obstructive respiratory events with the use of oronasal masks have been published [START_REF] Fleck | An adverse effect of positive airway pressure on the upper airway documented with magnetic resonance imaging[END_REF][START_REF] Nascimento | Body Position May Influence Oronasal CPAP Effectiveness to Treat OSA[END_REF][START_REF] Schorr | Continuous positive airway pressure delivered by oronasal mask may not be effective for obstructive sleep apnoea[END_REF]. Although, the exact mechanisms have not been completely elucidated, two main hypotheses have been put forward: i) mechanical constraint of the chin induced by the oronasal mask and traction of straps may push the mandible posteriorly during sleep [START_REF] Borel | Impact of CPAP interface and mandibular advancement device on upper airway mechanical properties assessed with phrenic nerve stimulation in sleep apnea patients[END_REF]; ii) air pressure blowing into the mouth and oral breathing may push the tongue backward and reduce pharyngeal patency [START_REF] Fleck | An adverse effect of positive airway pressure on the upper airway documented with magnetic resonance imaging[END_REF][START_REF] Madeiro | Transmission of Oral Pressure Compromises Oronasal CPAP Efficacy in the Treatment of OSA[END_REF][START_REF] Andrade | Nasal vs Oronasal CPAP for OSA Treatment: A Meta-Analysis[END_REF]. The results of the present study did not suggest that oronasal masks increased upper airway obstruction, despite the fact that this is a common occurrence in patients with neuromuscular diseases. Although mask-related upper airway obstruction has been shown to be highly prevalent in patients with neuromuscular disorders, it may be more prevalent in patients with ALS not included in the present study [START_REF] Schellhas | Upper airway obstruction induced by non-invasive ventilation using an oronasal interface[END_REF]. Indeed, the level of EPAP was relatively low both in patients with nasal and oronasal mask suggesting that the patients included may be unlikely to have severe upper airway collapsibility. Finally, our specialized nurses are strongly aware of this issue and thus teach patients and primary caregivers to avoid any over tightening of mask straps during the positioning.

The patients included in this study, were all treated with home nocturnal NIV and the median adherence was greater than 7 hours/night. The post hoc analysis showed that during the transition to a new interface, NIV efficacy was reduced and side effects increased. This information is important for clinicians since in practice, interfaces are often changed by home care providers or on the patient's own initiative with no systematic medical supervision. In the present study, despite close follow up during the week of change (and in-hospital initial mask choice and adjustment), mean nocturnal SpO2 decreased and mean tcCO2 increased as did the oxygen desaturation index and the number of side effects occurs. To our knowledge, no study has previously addressed the acute consequences and risks related to mask change in patients with neuromuscular diseases with nocturnal NIV. Our results suggest that information regarding changing masks should be provided during patient and caregiver education to ensure that appropriate medical supervision is sought, should they decide to change interface.

Limitations

Although our study was powered to detect a mean difference of mean nocturnal Spo2 of 2 ± 2% between oronasal and nasal masks, it is possible that the study was under powered due to the fact 5 patients did not tolerate the alternative interface.

The patients were not naive to NIV and the heterogeneity of the underlying diseases and level of autonomy could also have impacted the results. Yet, this recruitment reflects daily practice in tertiary care hospitals who deals with respiratory care in NMD which remain rare diseases (therefore it would have not been possible to complete this study in a reasonable time-frame with naive patients).

In our study, oronasal mask patients appeared to not have significant enough issues with upper airway resistance. We should have focused on more carefully selected patients with high upper airway resistance, residual events or low efficacy of oronasal NIV and assess the benefits to switch interface in those patients.

Finally, the period of adaptation to the alternative interface might have been too short.

Because of the high specificity of the sample included, the results of this study should not be extrapolated to other populations such as people with obesity hypoventilation syndrome and chronic obstructive pulmonary disease.

CONCLUSION

This study found no difference between oronasal and nasal masks in patients with neuromuscular diseases under long term nocturnal NIV in terms of treatment tolerance and efficacy. The results showed that treatment efficacy may be reduced during transition between interfaces, thus changes in masks should be carried out under close medical supervision.

Figure 2 :

 2 Figure 2: Effect of mask type on SpO2 and PtcCO2 parameters in patients with neuromuscular diseases

Figure 3 :

 3 Figure 3: Effect of usual vs alternative mask on SpO2 and PtcCO2 parameters in patients with neuromuscular diseases

This study received a grant from "Association Française contre les Myopathies -AFM".

Individual participant data that underlie the results reported in this article after deidentification (including data dictionaries) will be shared beginning 3 months and ending 5 years following article

APPENDICES

p-value are given for the differences at baseline between patients using nasal compared to those using oronasal interface.

# reported by built-in NIV softwares