S Techer

E Girard-Valenciennes

P Retailleau

J Neyts

F Guéritte

P Leyssen

M Litaudon

J Smadja

I Grondin

Tonantzitlolones from Stillingia lineata ssp. lineata as potential inhibitors of chikungunya virus

With the purpose of discovering new chemical classes of molecules, in particular those with selective antiviral activity, extracts from plants growing in Reunion Island were systematically evaluated in a chikungunya virus-cell-based assay. The entire ethyl acetate extract obtained from the stem bark of Stillingia lineata ssp. lineata (Euphorbiaceae) exhibited selective antiviral activity against the chikungunya virus with an EC 50 < 0.8 mg/mL, whereas only a weak cytotoxic effect was observed on the host cells. A phytochemical investigation of this extract led to the isolation of tonantzitlolone A and tonantzitlolone B (1 and 2), together with the new 4 0 -hydroxytonantzitlolone, named tonantzitlolone C (3), which has an uncommon C15-flexibilane skeleton, as well as in the new ent-12a-hydroxy-3,7dioxoisopimara-8,15-diene (4). Subsequent evaluation for inhibition of chikungunya virus replication in cellulo demonstrated that the 4 0 -acetoxytonantzitlolone (2) was endowed with antiviral activity against CHIKV.

Introduction

Chikungunya is an arboviral disease that is typically characterized by myalgia, polyarthralgia, fever, nausea, headaches and maculopapular rash. The chikungunya virus (CHIKV) is an Aedes mosquitoes-transmitted alphavirus that belongs to the Togaviridae family [START_REF] Kaur | Chikungunya virus: an update on antiviral development and challenges[END_REF]. Around a decade ago, there was a sudden reemergence of CHIKV infections with a previously unseen magnitude, with the virus moving from Africa throughout the Indian Ocean to India and Southeast Asia. Due to the steadily increasing number of cases, it became a serious concern and threat for public health [START_REF] Thiberville | Chikungunya fever: epidemiology clinical syndrome, pathogenesis and therapy[END_REF]. In 2005 and 2006, CHIKV caused a massive outbreak on Reunion Island, a French overseas territory, during which about 266,000 of the 775,000 inhabitants became infected [START_REF] Renault | A major epidemic of chikungunya virus infection on Reunion Island, France, 2005-2006[END_REF]. This was the dawn of a worldwide emergence of this virus, which has now even reached the Caribbean, the doorstep to the rest of the Americas. At present, no vaccine is yet available to prevent this infection, nor any antiviral drug(s) for treatment or prophylaxis.

The Reunion Island outbreak triggered our interest to explore the vast Indian Ocean plant biodiversity for novel natural molecules that selectively inhibit the replication of CHIKV. More than 1500 extracts from 972 endemic or indigenous plants from Madagascar, Mauritius and Reunion Island were evaluated for selective antiviral activity in a virus-cell-based assay for CHIKV [START_REF] Leyssen | Biodiversity as a source of potent and selective inhibitors of chikungunya virus replication[END_REF].

Among them, the species Croton mauritianus (Euphorbiaceae) from Reunion Island was found to elicit biological activity and was selected for a phytochemical investigation, leading to the isolation of tigliane-type diterpenoids [START_REF] Corlay | Tigliane diterpenes from Croton mauritianus as inhibitors of chikungunya virus replication[END_REF]. Tigliane-, daphnane-and jatrophane-type diterpenoids with potent and selective anti-CHIKV activities were also isolated from other Euphorbiaceae species: Trigonostemon cherrieri (Allard et al., 2012a,b;[START_REF] Bourjot | Trigocherrierin A, a potent inhibitor of chikungunya virus replication[END_REF], which is native to New Caledonia, T. howii [START_REF] Bourjot | Prostratin and 12-O-tetradecanoylphorbol 13-acetate are potent and selective inhibitors of chikungunya virus replication[END_REF], which is native to Vietnam, and the Corsican species Euphorbia amygdaloides ssp semiperfoliata [START_REF] Nothias-Scaglia | Jatrophane diterpenes as inhibitors of chikungunya virus replication: structure-activity relationship and discovery of a potent lead[END_REF]. These results prompted us to investigate another Euphorbiaceae species growing in Reunion Island, in casu Stillingia lineata ssp. lineata, of which the stem bark extract showed potent and selective anti-CHIK activity.

The genus Stillingia comprises 30 species that are mainly distributed in neotropical areas. In 1980, eight daphnane-and tigliane-type diterpenoids were isolated from roots of Stillingia sylvatica [START_REF] Adolf | New irritant diterpene-esters from roots of Stillingia sylvatica L. (Euphorbiaceae)[END_REF]. Cembrene A, two novel diterpenes with rare flexibilane skeletons (tonantzitlolone A and tonantzitlolone B), two new pimaranes, seven kauranes, three known atisanes, a new trachylobane, and the new pentacyclic diterpene sanguinolane with a novel skeleton have been reported from the roots of Stillingia sanguinolenta [START_REF] Dräger | Tonantzitlolone and other diterpenes from Stillingia sanguinolenta[END_REF].

Cytotoxic, proteolytic and antifungal activities have been reported for several species [START_REF] Adolf | New irritant diterpene-esters from roots of Stillingia sylvatica L. (Euphorbiaceae)[END_REF][START_REF] Chapuis | Screening for cytotoxic activity of plants used in traditional medecine[END_REF][START_REF] Cota | Biological potential of Stillingia oppositifolia[END_REF][START_REF] Sequeiros | Proteolytic activity in some Patagonian plants from Argentina[END_REF]. The in vitro cytotoxic activity of the species Stillingia lineata (Lam.) Müll. Arg. from Mauritius was evaluated on human tumor cells Co-115 (colon cancer), with petroleum ether and DCM leaf extracts that were highly cytotoxic [START_REF] Chapuis | Screening for cytotoxic activity of plants used in traditional medecine[END_REF]. This species has two subspecies: the ssp. pacifica which is located in Malaysia, and the ssp. lineata, which is endemic to Mauritius and to Reunion Island. The latter is a medicinal plant that has been traditionally used in Mauritius for diabetes [START_REF] Picot | Phytochemical profile and antioxidant properties of six medicinal plants traditionally used in the management of diabetes in Mauritius[END_REF].

This study reports the bio-assay-guided fractionation of the EtOAc bark extract from S. lineata ssp. lineata in a virus-cell-based assay for CHIKV on Vero cells (green monkey kidney cells Cercopithecus aethiops), as well as the characterization of the purified diterpenes-the most predominant secondary metabolite in this plant species-by detailed analysis of their spectral data, using 500 MHz 2D-NMR (COSY, NOESY, HSQC, HMBC) and mass spectrometry.

Results and discussion

A total of 320 EtOAc polyamide cartridge-filtered extracts were prepared from different parts of 171 Reunion Island plant species and were evaluated for selective antiviral activity in a CHIKV viruscell-based assay [START_REF] Leyssen | Biodiversity as a source of potent and selective inhibitors of chikungunya virus replication[END_REF]. The results of this screening indicated that the EtOAc extract that was obtained from eight species completely inhibited the virus from inducing cytopathic effects and without showing adverse effects on the host cell morphology and density compared to the untreated, uninfected cell control condition (EC 50 from <0.8 to 9.1 mg/mL).

Among them, the S. lineata ssp. lineata EtOAc bark extract (Euphorbiaceae) was selected for its potent anti-CHIKV activity (EC 50 < 0,8 mg/mL) and weak cytotoxicity on Vero cells (CC 50 = 60.9 mg/mL). This EtOAc extract was subjected to silica gel flash chromatography to give six fractions. The biologically active fractions F3, F4 and F5 (EC 50 = 7.23, 2.01 and 2.05 mg/mL, respectively) were further purified by preparative and semipreparative C18HPLC to yield three rare macrocycle C15-type diterpenes: tonantzitlolone A, tonantzilolone B and 4 0 -hydroxytonantzitolone (compounds 1-3), and also ent-12a-hydroxy-3,7dioxoisopimara-8,15-diene (4), a new pimarane Fig. 1.

The total assignment of 1 H and 13 C NMR data of tonantzitlolones 1, 2 and 3 and compound 4 are presented in Tables 1 and2.

Compound 1 was isolated as a white powder. The HR-ESIMS indicated a [M + Na] + ion peak at 487.2688, which suggested a molecular formula of C 26 H 40 O 7 Na (calc. 487.2672), thus requiring seven double-bond equivalents. Compound 2 was obtained as an amorphous powder. The HR-ESIMS of the [M + Na] + ion peak at m/z 545.2717 suggested that the molecular formula of compound 2 as C 28 H 42 O 9 (calculated for C 28 H 42 O 9 Na 545.2727), thus requiring eight double-bond equivalents. Considering the biological source, the 1 H and 13 C NMR spectroscopic data suggested that compounds 1 and 2 possessed the rare macrocycle C15-flexibilane skeleton and were identified through comparison with literature data as tonantzitlolone A and tonantzitlolone B (4 0 -acetoxytonantzitlolone) [START_REF] Dräger | Tonantzitlolone and other diterpenes from Stillingia sanguinolenta[END_REF][START_REF] Wittenberg | Towards the total synthesis of tonantzitlolone--preparation of key fragments and the complete carbon backbone[END_REF]. Both compounds possess characteristic NMR features of these flexibilane-type diterpenoids, in particular a tertiary and a secondary hydroxy group (OH-9 and OH-10) and two tertiary methyl groups (C-19 and C-20). Amongst the four quaternary carbons of the macrocycle were found a keto group (C-4), a non-functionalized carbon (C-15)-already mentioned for the flexibilene [START_REF] Kazlauskas | Cembranoid constituents from an australian collection of the soft coral Sinularia flexibilis[END_REF]-two deshielded carbons (C-9 and C-11), an acetal, and an oxygen-bearing carbon. The same deshielding was observed for the tertiary carbons of these oxygenated bridges (C-5 and C-14) as was found with other CH carbons. More particularly, the presence of the very deshielded C-9 flanked by two oxygen atoms was determined by chemical shift (dC 97) and by the 2 J HMBC correlation from OH-9. The location of this acetal C-9 was confirmed by multiple HMBC correlations (for example, chemical shifts of compound 1) from H-8 (dH 4.89), H-10 (dH 3.43), OH-10 (dH 3.10) and H-5 (dH 4.62), fixing the ether-ring position.

The X-ray diffraction analysis from crystals of compound 1 allowed us to confirm the absolute configuration of tonantzitlolone A that was previously established by Dräger and his colleagues as 3S,5R,7R,8R,9R,10S,11R,14S [START_REF] Dräger | Tonantzitlolone and other diterpenes from Stillingia sanguinolenta[END_REF] tonantzitlolone (1), 4 0 -acetoxytonantzitlolone (2), 4 0 -hydroxytonantzitlolone (3) andent-12a-hydroxy-3,7dioxoisopimara-8,15-diene (4). ORTEP plot is shown in Fig. 2. CCDC 1033736 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; or deposit@ccdc.cam.uk).

The three-dimensional structure of 4 0 -acetoxytonantzitlolone (tonantzitlolone B) (2) does not differ from that of tonantzitlolone A. Its absolute configuration is, however, known through the work of Busch and his colleagues, in which the R configuration was demonstrated [START_REF] Busch | Determination of the absolute configuration of the diterpene tonantzitlolone B[END_REF].

Compound 3 possessed the molecular formula C 26 H 40 O 8 , based on its [M + Cl] À ion peak at m/z 515.2410, obtained by HR-ESIMS (calculated for C 26 H 40 O 8 Cl 515.2412), thus implying seven doublebond equivalents. In agreement with the mass spectrum, the 13 C NMR spectrum indicated the presence of 26 carbon atoms. The chemical shifts and the DEPT135 spectrum allowed us to highlight the presence of six quaternary carbons (one ketone carbonyl, one carbonyl ester, one olefinic, one oxygenated and two nonfunctionalized), three methylenes, seven methyls, and ten methines (three olefinic and five oxygenated). The 1 H and 13 C NMR spectra (Table 1) of compound 3 were very similar to those of the known compounds 1 and 2, thus one could deduce that the structure of compound 3 was a tonantzitlolone derivative. All the 13 C NMR signals of compound 3 were reminiscent to those of compound 2, except for carbons C-2 0 -C-5 0 (dC 113.3, 162.8, 72.4 and 21.5, respectively for compound 3, and 115.2, 157.6, 73.8 and 19.1, respectively for compound 2), suggesting the modification of the side chain. In the 1 H NMR and HSQC spectra, the presence of a deshielded quartet of doublets at dH 4.62 (J = 6.5, 2.5 Hz) attributable to proton H-4 0 , attached to C-4 0 (dC 72.4), suggested the presence of the À ÀCH(OH)-CH 3 moiety. The HMBC correlation from this proton at dH 4.26 to the olefinic and methyl carbons at dC 113.3 (C-2 0 ) and 15.3 (C-6 0 ) confirmed that the À ÀCH(OH)-CH 3 moiety was located at C-3 0 . Consequently, the planar structure of compound 3 was elucidated as 4 0 -hydroxytonantzitlolone, named tonantzitlolone C. The relative configuration of compound 3, with the exception of the stereocenter C-4 0 , could be determined through the analysis of NOESY correlations, which are all comparable to those of compounds 1 and 2. In addition, since the specific rotation value of compound 3 ([a] 25 D + 76 [c 0.25, CHCl 3 ]) was found to have the same sign as the value found for tonantzitlonone A ([a] 25 D + 134 [c 0.25, CHCl 3 ]), it could be postulated that they possess the same absolute configuration [START_REF] Dräger | Tonantzitlolone and other diterpenes from Stillingia sanguinolenta[END_REF].

Compound 4 was obtained as an amorphous powder. The HR-ESIMS of the [M + H] + ion peak at m/z 317.2131 allowed us to establish its molecular formula as C 20 H 28 O 3 (calculated for C 20 H 29 O 3 317.2117), thus requiring seven double-bond equivalents. Its IR spectrum showed characteristic absorption bands at 3457 cm À1 for the hydroxy group, and 1707 and 1663 cm À1 for the carbonyl groups. The maximum absorption bands in the UV spectrum at 243 nm indicated the presence of an a,b-unsaturated carbonyl. The occurrence in the 1 H NMR spectrum (Table 2) of a vinyl group with three dd resonances at dH 5.74 (J = 17.5 and 10.8 Hz, H-15), dH 5.22 (J = 10.8 and 1.0 Hz, H-16cis), and dH 5.18 (J = 17.5 and 1.0 Hz, H-16trans) and the presence of four methyl group singlets suggested a pimarane-type skeleton. The analyses of 1 H, 13 C, DEPT135, HMBC and HSQC NMR data revealed that compound 4 contains 20 carbons, including four singlet methyls, six methylenes (one sp 2 and five sp 3 ), three methine (one sp 2 and two sp 3 ), seven quaternary carbons (one a,b-unsaturated carbonyl, and one oxygenated sp 2 , two sp 2 and three sp 3 ). The key 2D NMR correlations of compound 4 are presented in Fig. 3. The relative configuration of compound 4 was deduced from 1 H-1 H vicinal coupling constants and NOESY correlations. Cross peaks observed between Me-20 and Me-19, H-1a, H-2a and H-6a suggested that they are cofacial and, thus indicated that Me-20 is a-oriented as observed for an ent structure. The relative configuration of C-5 was established by the correlations between H-5 and Me-18 on the one hand, and between H-5 and H-1b, on the other hand, which is consistent with a b-orientation. This stereochemistry was also supported by the axial-axial coupling constants shown by both H-5 and H-6a (Table 2). The hydroxymethine proton H-12 resonated as a dd at dH 3.61 with coupling constants of 9.0 and 5.5 Hz. The magnitude of these coupling constants suggests the axial position for this proton. Moreover both H-12 and H14b are cross-correlated and both are also correlated with H-15 and H-11b.

Thus, the anti-position of H-15 compared to Me-20 suggests an isopimarane structure. In addition, the negative value of the optical rotation (À225, CHCl 3 , c = 0.20 mg/mL) of compound 4 is the same sign as those measured for other ent-pimarane derivatives [START_REF] Luo | ent-Pimarane derivatives from Dysoxylum hainanense[END_REF][START_REF] Rayanil | Ent-pimarane and enttrachylobane diterpenoids from Mitrephora alba and their cytotoxicity against three human cancer cell lines[END_REF]. Compound 4 was, therefore, elucidated as ent-12a-hydroxy-3,7-dioxoisopimara-8,15-diene.

It should be emphasized that the macrocycle-15C diterpene skeleton of compounds 1, 2 and 3 is particularly unusual in natural products. To the best of our knowledge, this constitutes the third example of tonantzitlolone-type diterpenes isolated from higher plants. The tonantzitlolone A (1) was isolated for the first time by [START_REF] Dräger | Tonantzitlolone and other diterpenes from Stillingia sanguinolenta[END_REF] from Stillingia sanguinolenta, an endemic species of Mexico, and later on from Sebastiania macrocarpa (Euphorbiaceae) [START_REF] Lima | Chemical constituents from Sebastiania macrocarpa Muell. Muell. Arg. (Euphorbiaceae)[END_REF]. These compounds-including tonantzitlolone B (2), which was also isolated from Stillingia sanguinolenta [START_REF] Dräger | Tonantzitlolone and other diterpenes from Stillingia sanguinolenta[END_REF]-have not yet been identified in other plants. The tonantzitlolone C (4'-hydroxytonantzitlolone (3)) represents the third natural compound of this chemical series that possesses a 15C-macrocycle flexibilen carbon skeleton. This original skeleton is also found in the structure of flexibilen, which was isolated from the soft coral Sinularia flexibilis [START_REF] Hérin | Chemical studies of marine invertebrates. XXV1. Flexibilene, an unprecedented fifteen-membered ring diterpene hydrocarbon from the soft coral Sinularia flexibilis (Coelenterata, Octocorallia, Alcyonacea)[END_REF][START_REF] Mcmurry | Synthesis of flexibilene: a naturally occurrring 15-membered-ring diterpene[END_REF]. Unlike tonantzitlolones, the pimaranes have been isolated from different genera and families. Two pimaranes were previously isolated from S. sanguinolenta: the ent-8b-hydroxy-3,12-dioxopimara-9(11), 15-diene and the ent-3b,12a-8-dihydroxypimara,15-diene, which belong to the enantiomeric series [START_REF] Dräger | Tonantzitlolone and other diterpenes from Stillingia sanguinolenta[END_REF].

The antiviral properties of the isolated compounds were evaluated in a CHIKV virus-cell-based assay, with chloroquine as a reference compound. Only the 4 0 -acetoxytonantzitlolone (2) selectively inhibited virus-induced cell death, with EC 50 and SI (selectivity index, calculated as CC 50 Vero/EC 50 CHIKV) values of 7 mM and 8.8, respectively, while 4 0 -hydroxytonantzitlolone (3) was not significantly selective (EC 50 :34 mM; SI:3.2). A comparison of the structures of these two bioactive compounds reveals that the presence of an acetyl group is possibly an important element in improving the inhibitory activity. In conclusion, the bioguided fractionation of the bark of Stillingia lineata ssp. lineata resulted in the isolation and characterisation of two new diterpenes (3 and 4), together with two known molecules (1 and 2). Their structures were elucidated mainly by 2D-NMR techniques and mass spectrometry. The isolated molecules were only slightly active against CHIKV in their purified state. The more pronounced antiviral activity of the extract could be due to additive or synergistic effects by other compounds that are present in low amounts. The correlation between the structure and biological activity of these tonantzitlolones is not yet complete. Therefore, further investigation of these active diterpenes in the context of CHIKV is required not only to elucidate the structure-activity relationship but also to enable the study of the molecular mechanism of action.

Experimental

General

Optical rotations were measured using a Jasco P1010 or an Anton Paar MCP200 polarimeter. UV spectra were acquired on a Varian Cary 100 Scan spectrophotometer. IR spectra were recorded on a PerkinElmer Spectrum FT-IR 100 spectrophotometer. NMR spectra were recorded on a Bruker Avance 300 MHz or 500 MHz instrument at 25 C. 1 H-and 13 C NMR chemical shifts refer to CHCl 3 at 7.26 ppm, and to CDCl 3 at 77.0 ppm, respectively. Coupling constants are in Hz. HR-ESIMS data were obtained on a Thermoquest TLM LCQ Deca ion-trap spectrometer. X-ray crystallographic data were collected using a Rigaku diffractometer constituted by a MM007HF rotating-anode generator, equipped with Osmic confocal optics, delivering filtered Cu-Ka radiation (l = 1.54187 Å) and a Rapid II curved Image Plate, at a cryogenic temperature (193 K).

Flash chromatographies were performed on a Sepacore system (Büchi) or a CombiFlash Companion apparatus (Teledyne Isco), using silica gel 60A or various prepacked normal-phase cartridges (Silicycle 1 15-40 mm, Redisep 1 15-60 mm, Redisep 1 35-70 mm, or Versapack 1 45-75 mm). All solvents were purchased from Carlo Erba (France). TLC separations were performed on Merck precoated silica gel 60G F254, Alugram 1 SIL G/UV254 (Macherey-Nagel) or Alugram 1 RP-18W/UV254 (Macherey-Nagel) analytical plates. Kromasil reversed-phase semi-preparative and preparative HPLC separations were carried out on Thermo Hypersil columns (250 Â 10 mm and 250 Â 21 mm ID, 5 mm), using Waters 1 apparatus (Alliance 2695 and Autopurification system Waters 1 -2767 Sample Manager) equipped with a binary pump (Waters 2525), a UV-vis diode array detector (190-600 nm, Waters 1 2996), and a PL-ELS 1000 ELSD detector (Polymer Laboratory).

Plant material

The whole plant of Stillingia lineata ssp. lineata was collected in March 2011 in Langevin (La Réunion, France) and was authenticated by the botanist Prof. Dominique Strasberg (University of La Réunion). A voucher specimen (No. REU09532) was deposited in the Herbarium of the University of La Réunion, France.

Extraction and isolation

Seven hundred sixty-nine grams of dried powder of bark of Stillingia lineata ssp. lineata were extracted with EtOAc, using a Dionex-accelerated solvent extractor equipped with 100 mL of stainless steel cells and 250 mL of collection bottles (ASE300, Voisins Le Bretonneux, France). Conditions were as follows: temperature, 40 C; pressure, 100 bars; 5 cycles with static extraction time of 6 min; and flush volume, 100%. The EtOAc crude extract was concentrated under reduced pressure to yield 33.9 g of residue (r = 4.4% w/w). This EtOAc extract was subjected to flash silica chromatography using a gradient of cyclohexane-AcOEt (95:5-0:100) and AcOEt-MeOH (100:0-50 :50) at 15 mL/min to produce six fractions, according to their TLC profiles. The biologically active fractions F3 (8.3 g, EC 50 = 7.23 mg/mL), F4 (4.7 g, EC 50 = 2.91 mg/mL), and F5 (3.5 g, EC 50 = 2.05 mg/mL) were further purified using successive preparative and semi-preparative chromatographies on reversed-phase silica gel in the C-18 column. Purifications of fraction F3 (8.3 g, cyclohexane-AcOEt 80:20) were carried out by preparative RP-18HPLC chromatography (Thermohypersil Kromasil, 5 mm, 250 Â 21.2 mm) to produce tonantzitlolone A (1), using MeOH-H 2 O + 0.1% formic acid (from 80:20 to 100:0 in 20 min at 21 mL/min ; t R 18.9 min). Fraction F4 (4.7 g, cyclohexane-AcOEt 80:20) was separated by preparative HPLC (Kromasil RP-18 using MeOH-H 2 O + 0.1% formic acid, from 70:30 to 100:0 in 30 min at 15 mL/min) to give the 4 0 -acetoxytonantzitlolone (2) (19.7 mg, t R 20.3 min). The fraction F5 (3.5 g, eluting from 70:30 to 50:50 cyclohexane-AcOEt) was subjected to preparative RP-18HPLC to yield compound 3 (9.3 mg, t R 17.2 min) and a sub-fraction 5-5 (20 mg). This sub-fraction 5-5 was further purified by a semipreparative HPLC (Thermo Hypersil Kromasil, MeOH-H 2 O + 0.1% formic acid, from 60:40 to 100:0 in 50 min at 4.7 mL/min) to get compound 4 (1.2 mg). (cm À1 ): 3457, 2924, 1707, 1663, 1380; 1 H and 13 C NMR (CHCl 3 ) data, see Table 2; HR-ESIMS (pos.) m/z 317.2131 (calcd. for C 20 H 29 O 3 , 317.2117).

X-ray crystallographic analysis of 1

A large colourless crystal of tonantzitlolone A (1), recrystallized from isopropyl oxide, was mounted at cryogenic temperature (193 K) on a Rigaku diffractometer constituted by a MM007HF rotating-anode generator, equipped with Osmic confocal optics, delivering filtered Cu-Ka radiation (l = 1.54187 Å) and a Rapid II curved Image Plate. The crystal-to-detector distance was 127.40 mm. The X-ray data were collected up to a maximum 2u value of 136.5 . Four images with one degree v oscillation spanning a 60 v angular space were used to derive cell constants and matrix orientation using d*trek [START_REF] Pflugrath | The finer things in X-ray diffraction data collection[END_REF]. According to a subsequent strategy in mmm symmetry, a total of 108 oscillation images were collected from three sweeps of data using v oscillations in 5.0 steps. The exposure rate was 60.0 s per degree.

Data reduction and scaling were carried out using Fs_Process. 2 . The structure was solved by Direct methods (SHELXS-97) and by subsequent difference Fourier syntheses, and refined by full matrix least squares on F 2 using the SHELXL-2014/6 [START_REF] Sheldrick | A short history of SHELX[END_REF]. Anisotropic thermal parameters were used for all non-hydrogen atoms whereas hydrogen atoms, located from difference Fourier maps, were refined as a riding model with U iso = 1.2U eq of the parent atom (1.5 for the methyl hydrogen atoms and the hydroxyl hydrogen ones). The C28 methyl carbon atom went to nonpositive-definite and was further restrained with a soft ISOR (esd 0.04) restraint to be approximately isotropic. Despite the absence of strong anomalous scatterers (Friedif value of 34 [START_REF] Flack | The mean-square Friedel intensity difference in P1 with a centrosymmetric substructure[END_REF]), the absolute configuration was attributed with some confidence by the use of the Hooft y parameter [START_REF] Hooft | Determination of absolute structure using Bayesian statistics on Bijvoet differences[END_REF], calculated as 0.03 (5), the P2(true) and P3(true) values being both 1.000, from a total of 1979 Bijvoet pairs (97% coverage). This Bayesian analysis on the Bijvoet pairs implemented in Platon [START_REF] Spek | Structure validation in chemical crystallography[END_REF] enhanced the weak significance of the Flack x parameter [START_REF] Flack | On enantiomorph-polarity estimation[END_REF], which was determined to be x = 0.1 (3) by the use of 1519 quotients [(I+) À (IÀ)]/[(I+) + (IÀ)] from the Parsons' method [START_REF] Parsons | Use of intensity quotients and differences in absolute structure refinement[END_REF] The crystallographic data are summarized as follows: a colourless tab with dimensions 0.60 Â 0.48 Â 0.12 mm, C 26 H 40 O 7 , Mr = 464.58, an orthorhombic system, space group P 2 1 2 1 2 1 , Z = 2, a = 10.1348(2) Å, b = 10.2248(2) Å, c = 25.1440(18) Å, V = 2605.6

(2) Å 3 , D calcd. = 1.184 g/cm 3 , F(000) = 1,008, m = 0.690 mm À1 , 17,654 collected reflections (3.516 u 68.228 , À9 h 12, À9 k 12, À30 l 30), 4681 independent reflections (R int = 0.0340), goodness of fit on F 2 : S = 1.138, R 1 = 0.0594 and wR 2 = 0.1561 for all 4681 reflections, R 1 = 0.0508 and wR 2 = 0.1350 for 4020 observed reflections [I > 2s(I)], refining 308 parameters and 6 restraints, semi-empirical absorption corrections from multi-scans (T min = 0.77, T max = 0.92), and a final electron density between À0.186 and 0.258 e.Å À3 .

Virus-cell-based antiviral assay

Throughout the experiments, Vero (African green monkey kidney) cells and chikungunya virus strain 899 were used [START_REF] Bourjot | Prostratin and 12-O-tetradecanoylphorbol 13-acetate are potent and selective inhibitors of chikungunya virus replication[END_REF]. Serial dilutions of extract, fractions or pure compounds, as well as the reference compound chloroquine (positive control), were prepared in an assay medium [MEM] Rega3 (Cat. N 19993013; Invitrogen), 2% FCS (Integro), 5 mL of 200 mM L-glutamine, and 5 mL of 7.5% sodium bicarbonate that was added to empty wells of a 96-well microtiter plate (Falcon, BD) on a liquid handling platform (Freedom EVO200, Tecan).

Subsequently, 50 mL of a 4Â virus dilution in assay medium were added, followed by 50 mL of a cell suspension. This suspension, with a cell density of 25,000 cells/50 mL, was prepared from a Vero cell line that was sub-cultured in a cell growth medium (MEM Rega3 supplemented with 10% FCS, 5 mL of L-glutamine and 5 mL of sodium bicarbonate) at a ratio of 1:4 and grown for seven days in 150 cm 2 tissue culture flasks (Techno Plastic Products). The assay plates were returned to the incubator for six to seven days (37 C, 5% CO 2 , 95-99% relative humidity), a length of time in which maximal virus-induced cell death or cytopathic effect (CPE) can be observed in untreated, infected controls. Subsequently, the assay medium was aspirated, replaced with 75 mL of a 5% MTS (Promega) solution in a phenol red-free medium and incubated for 1.5 h. Absorbance was measured at a wavelength of 498 nm (Safire2, Tecan); optical densities (OD values) reached 0.6-0.8 for the untreated, uninfected controls. Raw data were converted to a percentage of the controls and the EC 50 (50% effective concentration or concentration, which is calculated to inhibit virus-induced cell death by 50%) and CC 50 values (50% anti-metabolic concentration or a concentration that is calculated to inhibit the overall cell metabolism by 50%) were derived from the dose-response curves. All assay conditions producing an antiviral effect exceeding 50% were checked microscopically for minor signs of CPE or adverse effects on the host cell (i.e., altered cell morphology). A compound is only considered to elicit a selective antiviral effect on virus replication when following microscopic quality control, at least at one concentration of compound, and when no CPE nor any adverse effect is observed (image resembling untreated, uninfected cells). Multiple, independent experiments were performed.

Fig. 1 .

 1 Fig. 1. Compounds isolated from S. lineata ssp. lineata stem bark: tonantzitlolone(1), 4 0 -acetoxytonantzitlolone (2), 4 0 -hydroxytonantzitlolone (3) and ent-12a-hydroxy-3,7dioxoisopimara-8,15-diene (4).

Fig. 2 .

 2 Fig.2. An ORTEP-3 view of the tonantzitlolone A (1). Ellipsoids are drawn at the 30% probability level.

  4 0 -Hydroxytonantzitlolone (tonantzitlolone C) (3) White, amorphous powder; [a] 23 D + 76 (c 0.25, CHCl 3 ); UV (CHCl 3 )

Fig. 3 .

 3 Fig. 3. Selected COSY (bold), HMBC (blue arrows) and NOESY (red arrows) correlations for structural elucidation of ent-12a-hydroxy-3,7-dioxoisopimara-8,15-diene (4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1 1

 1 H and 13 C NMR spectroscopic data for tonantzitlolones (1-3) (in CDCl 3 , at 300 and 75 MHz, respectively ; d in ppm).

	Position	1		2		3
		d C	d H , mult, J (Hz)	d C	d H , mult, J (Hz)	d C	d H , mult, J (Hz)
	1	140,1	5,85 (d, 1H, 15.5)	140,1	5,85 (d, 1H, 15.0)	140,0	5,85 (d, 1H, 15.5)
	2	126,8	5,23 (dd, 1H, 15.5, 9.5)	126,8	5,24 (dd, 1H, 15.5, 9.5)	126,8	5,23 (dd, 1H, 15.5, 9.5)
	3	49,5	3,33 (dq, 1H, 9.5, 7.0)	49,5	3,32 (dq, 1H, 9.5, 7.0)	49,5	3,34 (dq, 1H, 9.5, 7.0)
	4	211,3	-	211,2	-	211,2	-
	5	74,1	4,62 (dd, 1H, 12.0, 3.0)	74,1	4,62 (dd, 1H, 12.0, 3.0)	74,1	4,62 (dd, 1H, 12.0, 3.0)
	6a	28,8	1,84 (dt, 1H, 13.5, 3.5)	28,7	1,84 (dt, 1H, 13.5, 3.5)	28,8	1,85 (ddd, 1H, 13.0, 3.0, 2.5)
	6b		1,40 (ddd, 1H, 13.5, 12.5, 3.0)		1,40 (ddd, 1H, 13.5, 12.5, 3.0)		1,39 (ddd, 1H, 13.5, 12.5, 3.0)
	7	29,0	2,33 (dddq, 1H, 12.5, 7.0, 4.0, 2.5)	29,0	2,32 (dddq, 1H, 12.5, 7.0, 4.0, 2.5)	29,0	2,33 (dddq, 1H, 11.5, 7.0, 4.0, 2.5)
	8	73,2	4,89 (d, 1H, 2.5)	73,7	4,90 (d, 1H, 2.5)	73,5	4,90 (d, 1H, 2.5)
	9	97,1	-	97,1	-	97,0	-
	10	78,2	3,43 (d, 1H, 6.0)	78,1	3,40 (br.d, 1H, 6.0)	78,2	3,41 (br.d, 1H, 2.5)
	11	87,6	-	87,7		87,6	-
	12a	37,3	1,53 (ddd, 1H, 12.5, 12.5, 7.5)	37,3	1,53 (ddd, 3H, 12.5, 12.5, 7.5)	37,3	1,53 (ddd, 1H, 12.5, 12.5, 7.5)
	12b		2,44 (dd, 1H, 12.5, 7.5)		2,43 (dd, 1H, 12.5, 7.5)		2,41 (dd, 1H, 12.5, 7.5)
	13a	28,0	1,76 (ddd, 1H, 12.5, 7.5, 5.0)	28,0	1,76 (ddd, 1H, 12.5, 7.5, 5.0)	28,0	1,76 (ddd, 1H, 12.5, 7.5, 5.0)
	13b		2,04 (dddd, 1H, 12.5, 12.5, 12.5, 7.5)		2,03 (dddd, 1H, 13.0, 13.0, 11.5, 7.5)		2,03 (dddd, 1H, 11.5, 11.5, 11.5, 7.5)
	14	88,9	3,76 (dd, 1H, 11.5, 5.0)	88,9	3,76 (dd, 1H, 11.5, 5.0)	88,9	3,76 (dd, 1H, 11.5, 5.0)
	15	38,7	-	38,7	-	38,7	-
	16	25,4	0,91 (s, 3H)	25,4	0,91 (s, 3H)	25,4	0,91 (s, 3H)
	17	25,1	1,14 (s, 3H)	25,1	1,13 (s, 3H)	25,1	1,14 (s, 3H)
	18	28,1	1,37 (s, 3H)	28,1	1,37 (s, 3H)	28,1	1,37 (s, 3H)
	19	17,0	0,84 (d, 3H, 7.0)	17,0	0,84 (d, 3H, 7.0)	17,0	0,85 (d, 3H, 7.0)
	20	16,0	1,11 (d, 3H, 7.0)	16,0	1,12 (d, 3H, 7.0)	16,0	1,12 (d, 3H, 7.0)
	1 0	166,6	-	166,3	-	166,5	-
	2 0	113,9	5,69 (q, 1H, 1.5)	115,2	5,90 (q, 1H, 1.2)	113,3	6,00 (s, 1H)
	3 0	163,0	-	157,6		162,8	-
	4 0	33,9	2,17 (q, 2H, 7.0)	73,8	5,27 (q, 1H, 6.5)	72,4	4,62 (dq, 1H, 6.5, 2.5)
	5 0	11,8	1,07 (t, 3H, 7.5)	19,1	1,35 (d, 3H, 7.0)	21,5	1,32 (d, 1H, 6.5)
	6 0	19,0	2,15 (d, 3H, 1.5)	15,2	2,13 (d, 3H, 1.5)	15,3	2,12 (s, 1H)
	1 00			170,1	-
	2 00			21,2	2,09 (s, 3H)
	OH-4 0						Not resolved
	OH-9		5,65 (s, 1H)		5,70 (s, 1H)		5,63 (s, 1H)
	OH-10

3,10 (d, 1H, 6.0) 3,05 (br.d, 1H, 6.5)

3,15 (d, 1H, 5.5)

Table 2 1

 2 H and 13 C NMR spectroscopic data for ent-12a-hydroxy-3,7-dioxoisopimara-8,15-diene (4) (in CDCl 3 , at 500 and 75 MHz, respectively).

	Position	d C	d H , mult., (J en Hz)	NOESY a
	1a	34,5	2,17 (ddd, 1H, 13.0, 6.0, 3.0)	2a,20
	1b		1,78 (td, 1H, 13.0, 6.0)	2b, 5
	2a	34,6	2,74 (ddd, 1H, 16.0,13.0, 6.0)	1a, 19, 20
	2b		2,48 (ddd, 1H, 16.0, 6.0, 3.0)	1b
	3	218,8	-	-
	4	46,5	-	-
	5	49,8	2,20 (dd, 1H, 14.0, 4.0)	1b11b, 18
	6a	35,4	2,53 (dd, 1H, 17.5, 14.0)	19, 20
	6b		2,46 (dd, 1H, 17.5, 4.0)	18
	7	204,8	-	-
	8	133,0	-	-
	9	160,8	-	-
	10	38,8	-	-
	11a	30,3	2,22 (dd, 1H, 17.5, 9.0)	17
	11b		2,58 (dd, 1H, 17.5, 5.5)	5, 12b, 14b
	12b	71,0	3,61 (dd, 1H, 9.0, 5.5)	11b, 14b, 15
	13	40,0	-	-
	14a	34,2	2,35 (d, 1H, 17.0)	17
	14b		2,23 (d, 1H, 17.0)	11b, 12b, 15
	15	144,7	5,74 (dd, 1H, 17.5, 10.8)	12b, 14b
	16cis	115,5	5,22 (dd, 1H, 10.8, 1.0)	-
	16trans		5,18 (dd, 1H, 17.5, 1.0)	17
	17	15,0	0,93 (s, 3H)	11a, 14a, 16trans
	18	25,2	1,11 (s, 3H)	5, 6b
	19	21,4	1,14 (s, 3H)	2a, 6a, 20
	20	17,3	1,30 (s, 3H)	1a, 2a, 6a, 19
	OH		2,24 (br.s, 1H)	-

  l max (log e) 240 (2.46) nm; IR n max (cm À1 ): 3372, 2967, 2933, 1712; 1 H and 13 C NMR (CHCl 3 ) data, see Table 1; HR-ESIMS (neg.) m/z 515.2410 (calculated for C 26 H 40 O 8 Cl, 515.2412).

	Ent-12a-hydroxy-3,7-dioxoisopimara-8,15-diene (4)
	White, amorphous powder; [a] 23	D À 225 (c 0.20, CHCl 3 ); UV
	(CHCl 3

) l max (log e

) 201 (1.83) 227 (2.01) 243 (2.36

) nm; IR n max

a Key NOESY cross peaks.

Acknowledgements

This work was supported by the Centre de Recherche et de Veille des maladies émergentes dans l'Océan Indien (CRVOI), by the Regional Council of La Réunion and by the European Regional Development Fund (ERDF). The authors are very grateful to E. Boyer and D. Strasberg (UMR C_53 PVBMT) for their contribution to plant material identification and species collection, to M.-T. Martin for her help and advice in NMR studies, to A. Marvilliers and B. Illien for their helpul discussions. We would like to acknowledge C. Collard, S. Delmotte and T. Bellon for their assistance with the collection of the biological data.


Dorine Petit

Jean-Philippe Georges

Thierry Divoux

Bruno Regnier

Philippe Miramont

Freshness analysis of functional sequences in launchers ⋆

Keywords: Observability, freshness, switched network, tele-monitoring, space vehicles

Nowadays, many embedded systems use specific data buses to guarantee the exchange of data. The next generation of space applications is moving to components off-theshelf (COTS) technology as switched Ethernet network in order to reduce the financial cost, being attentive to the mass. However, performance should be gained without sacrificing the reliability and the properties that enabled a tele-monitoring. This paper focuses on how to obtain a single traffic capture file that will be sent over ground-board communications and how to test later the freshness requirement satisfaction. A freshness formalization is proposed as well as a freshness verification algorithm based on the analysis of single trace captured using multicast communications. An experimental evaluation has been leaded in nominal and link failure cases.

INTRODUCTION

In space applications (aircrafts, satellites, launchers), conventional communication technologies are evolving from specific buses and federative control strategies to switched topologies that will support modular and distributed architecture [START_REF] Monchaux | Avionicx: A demonstrator for the next generation launcher avionics[END_REF]. Component off-theshelf (COTS) technology is aimed at replacing the current MIL-STD-1553B (Department of Defense (1978)) for control traffic and (TDMA based) Pulse-Coded Modulation buses for telemetry traffic, buses embedded in the european (unmanned) launchers. Switched Ethernet network are hence expected to be embedded into the nextgeneration of the space launchers [START_REF] Robert | Toward self-reconfiguration of switched ethernet architectures in the next generation of space launchers[END_REF]). Candidates protocols may be AFDX (ARINC 664 P7 (2003)) or TTEthernet (SAE AS 6802 (2011)).

Traffic monitoring can be the cornerstone for understanding such communication networks. The monitoring activity aims at collecting from the various network devices a set of relevant data. This enables to characterize the network state and therefore to identify unusual network behavior. According to the application domain, the purposes of the monitoring can also be different like network management, network security, network performance analysis, etc. The monitoring mechanisms depend directly on the intended application and also on the nature of the observed system.

In launchers, the on-board controller manages the control state by sending specific data from/to the sen- sors/actuators. It corresponds to traffic command (noted TC) for mission control, on-board operations scheduling and automated procedures. Additionally, the network supports telemetry traffic (noted TM) for low rate mission control and housekeeping data [START_REF] Notebaert | Future on-board networks in space systems[END_REF]. By analyzing the content of the packets, it should be possible to retrieve the values measured by the sensors and those sent by the controller to the actuators. Hence, the network acts as an observer of the control state. This is the applicable approach for launchers. Indeed, the whole traffic is monitored on-board and both TC and TM exchanges are sent over a ground-board link. It enables tele-diagnosis of launchers by post-analysis studies and replaying a network in order to improve the performances of a given architecture. The Ethernet protocols used in launcher applications may be different from those used for industrial factory automation, but the demands on end-to-end delays and other real-time requirements remain in the same order of magnitude. The main difference comes from the fact that the diagnosis of the devices and applications states relies here on the packet captured on the network. There is no additional supervision traffic, such performance metrics like freshness have to be recovered from the traces sent by air-ground data link.

The objective of this paper is to propose a method to validate end-to-end freshness inside a network as part of an observability analysis. The main novelty consists in achieving such validation for launchers just based on a single frame capture file that will be monitored through a ground-board communication. Section 2 summarizes the related work concerning network monitoring. Section 3 details the challenges and restrictions of such method in which the network state is observed from a specific machine. It shows ambiguous situations to be taken into account for both freshness and ordering properties with regards to a sequence of frames. Section 4 explains how the method might be implemented while section 5 illustrates it on an experimental platform. It should be helpful for engineers to discuss about the performance of the avionics during a past launch and provide capacity to detect, isolate and report faults.

The processing time of each task will not be taken into account in this study. It is not assumed for the traffic to be periodic and it should be robust to dynamic change of the topology (for instance a booster separation) or a different mission of the flight.

RELATED WORK AND BACKGROUND

This paper aims at providing a framework to analyse the traffic capture. It will be helpful for trade-off of quality-ofservice and to detect, isolate and report faults. For such critical real-time constrained application, numerous works have already focused on the performance evaluation of the network end-to-end delays (network calculus, trajectory approach, real-time calculus). However, avionics requires to verify different properties that encompass not a single, but a sequence of frames mapping a functional command. An important property is to test if the end-to-end freshness is satisfied as defined in [START_REF] Lauer | Latency and freshness analysis on ima systems[END_REF]. Consider the periodic chain T 1 d1 -→ . . . T n dn -→ T n+1 (task T i regularly produces data d i for T i+1 ; each task is computed on a given device). A δ-freshness requirement may be defined such that a data d n is "globally" produced from data d i themselves produced not earlier than δ time units before. From a traffic capture point of view, a functional sequence would be observed according to the frames F 1 . . . F n (that respectively corresponds to the transportation of data d 1 . . . d n ). The verification of the freshness requires then to evaluate the difference between the timestamp of the frame related to the event at the end of a functional sequence and the timestamp of the frame that corresponds to the earliest dependent event at the beginning of the sequence. The paper will show the challenges to retrieve such analysis: how to obtain a single traffic capture file that will be sent over ground-board communications and how to test later the freshness requirement satisfaction.

The first issue deals with the acquisition of a unique traffic capture file. There exist several different techniques to capture network traffic. A point-to-point link can be split with a special device, named network Test Access Point (TAP) which enables to connect a monitor on this particular link in a passive way. A second method, called port mirroring, consists of using a special switches function (available on the most of commercial switches), which enables to copy all traffic coming from all or part of ports to a dedicated port.

Contrary to conventional topologies based on a bus, many monitors have to be implemented to obtain a real picture of the communications in switched networks whatever is the solution retained for traffic monitoring [START_REF] Robert | On the observability in switched ethernet networks in the next generation of space launchers: Problem, challenges and recommendations[END_REF]. It means that all local capture files should be sent to a common device that will merge the files in order to generate a new file that will represent the load all along the network (file that has to be transmitted to the ground via the telemetry channel). To merge all the local traces, it needs a global reference time with synchronisation offsets have to be as small as possible since the clocks in each monitor are initially running asynchronously and may produce significant offsets. The underlying question is therefore the time synchronization method that may be addressed by using a synchronisation protocol as Network Time Protocol (NTP) or IEEE1588 -Precision Time Protocol (PTP). Some work (mainly, in an operating system tracing) suggest to rely on offline synchronisation by using a post-processing algorithm. These algorithms are mainly based on regression analysis (linear, leastsquares, convex hull, etc.) or linear programming. [START_REF] Robert | On the observability in switched ethernet networks in the next generation of space launchers: Problem, challenges and recommendations[END_REF] discuss the challenges and the limits of those methods.

The verification of properties has been already studied. QoS performance of switched Ethernet networks, and in particular, end-to-end delays have been estimated by determinist methods as noticed by [START_REF] Bauer | Improving the worst-case delay analysis of an afdx network using an optimized trajectory approach[END_REF]: network calculus, real-time calculus, trajectory approach. End-to-end properties have been also estimated, especially for Integrated Modular Avionics (IMA). [START_REF] Lauer | Latency and freshness analysis on ima systems[END_REF] propose hence computation of upper-bounds of end-to-end properties computed as optimal solutions of Integer Linear Programming. Results integrate here the computation time required by each function. [START_REF] Badache | Managing temporal allocation in integrated modular avionics[END_REF] propose then criteria enabling to quantify hence the quality of valid temporal allocations. These works are strongly linked to the notion of virtual links valid for AFDX networks. Furthermore, these estimations consider the knowledge of the traffic.

OBSERVABILITY

Assume that each message is sent over the network in multicast. The destination address corresponds to a group that gathers the original destination and the address of a PC that will be called the observer o. This station has then the availability to capture each frame that will be sent over the network.

A functional sequence is defined as a set of frames F 1 . . . F n sent by m devices (m ≤ n). In launchers, three kind of devices can be distinguished: the sensors, the actuators and the controller. It leads that avionics requirements are usually linked to a set of exchanges between those three kind of devices. To simplify, the functional sequence s X -→ c Y -→ a is now considered. It corresponds to the periodic transportation of a measure X produced by a sensor s and of an action Y computed by the (on-board) controller c for an actuator a. Fig. 1 details the exchanges, with the copy to the observer o.

We note x (k) the date at which the sensor is sending the k-value of the measure and y (l) the date at which the controller is sending the l-value of the command. An endto-end freshness requirement means here that the value X, that has served to compute the value Y sent at y (l), has to be received before but not δ time units before (x (k) ≥ y (l)-δ). Assume now that the delay of the frame sent at x (k) is noted ∆ (x (k)). End-to-end freshness for a command Y (l) will be satisfied if it can be found a measure X (k) such that:

s c a t o x (k -1) x (k) y (l) y (l) + ∆ o (y (l)) = y o (l)
Fig. 1. Functional sequence and freshness

∀l ∈ N, ∃k ∈ N | y (l) -δ ≤ x (k) + ∆ (x (k)) ≤ y (l) (1)
The existence enables also to take into account potential frames losses and oversampling.

(k) = x (k) + ∆ o (x (k)) and y o (l) = y (l) + ∆ o (y (l))
where ∆ o (z) corresponds to the delay to send frame z to the observer. y o (l) represents the date at which the command Y (l) has been captured by the observer (the timestamp in the trace). And since the path between the sensor and the controller is not the same as between the sensor and the observer, delays may be different

(∆ (x (k)) = ∆ o (x (k))).
Since only the dates in the capture file are known, a new rule is proposed to check if the end-to-end freshness is satisfied for a functional sequence s

X -→ c Y -→ a.
It consists in verifying if the observer receives an instance k of a measure at a time compatible with the reception of a command l that will be based on this measure (Y (l) = f (X (k))). It is hence suggested the following proposition. Proposition 1. The end-to-end freshness for a command Y (l) (l ∈ N) will be satisfied if it can be found a measure X (k) such that:

∃k ∈ N | y o (l) -δ + ∆ max ≤ x o (k) ≤ y o (l) -2∆ max (2)
where ∆ max corresponds to the maximum delay of any frame in the network. The proposition is valid for δ ≥ 3∆ max .

Proof. Starting from (1):

∃k ∈ N | y (l) -δ ≤ x (k) + ∆ (x (k)) ≤ y (l)
First, by introducing ∆ o (y (l)) and then ∆ o (x (k)), it can be written that:

y (l) + ∆ o (y (l)) ≥ x (k) + ∆ (x (k)) + ∆ o (y (l)) y o (l) ≥ x (k) + ∆ (x (k)) + ∆ o (y (l)) y o (l) + ∆ o (x (k)) ≥ x o (k) + ∆ (x (k)) + ∆ o (y (l))
And hence:

y o (l) + ∆ o (x (k)) -∆ (x (k)) -∆ o (y (l)) ≥ x o (k) (3)
Secondly, on the left side of the inequality,

x (k) + ∆ (x (k)) + ∆ o (y (l)) ≥ y (l) -δ + ∆ o (y (l)) x (k) + ∆ (x (k)) + ∆ o (y (l)) ≥ y o (l) -δ x o (k) + ∆ (x (k)) + ∆ o (y (l)) ≥ y o (l) -δ + ∆ o (x (k)) And hence: x o (k) ≥ y o (l) -δ + ∆ o (x (k)) -∆ (x (k)) -∆ o (y (l)) (4)
Based on (3) and ( 4), the end-to-end freshness property is satisfied if:

y o (l) + ∆ o (x (k)) -∆ (x (k)) -∆ o (y (l)) ≥ x o (k) ≥ y o (l) + ∆ o (x (k)) -∆ (x (k)) -∆ o (y (l)) -δ (5)
Assuming that delays are bounded by ∆ max , then:

-2∆ max ≤ ∆ o (x (k))-∆ (x (k))-∆ o (y (l)) ≤ ∆ max (6) It enables to write that any k ′ ∈ N that verifies: y o (l) -2∆ max ≥ x o (k ′ ) ≥ y o (l)
δ + ∆ max would also verifies (5) (due to (6)). It implies also that it verifies (1). ( 6) =⇒ (5

) ⇐⇒ y (l) ≥ x (k ′ ) + ∆ (x (k ′ )) ≥ y (l) -δ
It may be noticed that the proposition given in ( 2) is more restrictive since it rules out two time windows. It corresponds in fact to ambiguous time windows. Fig. 2 shows that it may contain measures not relevant for a given command Y (l).

s c o x (k) x o (k) y y o (l) y o (l) -2∆ max (a) Late measure δ x (k) y o (l) y o (l) -δ (b) Early measure Fig. 2. Ambiguous situations (Y (l) = f (X (k))
Fig. 2a highlights a situation in which a frame X is captured before a frame Y (l) whereas this command was not computed from this measure since it has been received after on the controller. In Fig. 2b, a frame X (k) seems to be not earlier than δ time units before the frame Y (k) but it was received before on the controller such that it was not considered to produce the command. That's the reason why time windows [y o (l) -2∆ max , y o (l)] and [y o (l)δ, y o (l)δ + ∆ max ] are rejected (even if they may contain relevant situations). Moreover, other situations may happen outside those intervals. For instance, a measure observed just after a command might have been received before on the controller and then used to produce the command. Moreover, a measure observed before the freshness requirement might have been in fact received on the controller later, and might be hence valid. Fig. 3 summarizes the validity domains of the measures that will refresh the command.

y o ( l ) -δ -2 ∆ m a x y o ( l ) -δ y o ( l ) -δ + ∆ m a x y o ( l ) -2 ∆ m a x y o ( l ) y o ( l ) + ∆ m a x
valid measures

Fig. 3. Excluded time windows

Hatched areas in Fig. 3 correspond to measures out of the scope of a command Y (l), while the white area contains valid (useful) measures. Gray areas correspond to domains that need to be excluded too since they are ambiguous (might contain both valid and not valid measures). Considering periodic measurements and commands, it means from there that measurement period has to be less than δ -3∆ max (regardless clocks synchronisation).

It is important to note here that the width of the ambiguous domains is related to the maximum delay ∆ max . For high bandwidth networks, it means that the valid domain will be close to the interval given in (1). The accuracy may also be improved by considering separately the three delays mentioned in (6) rather than a common maximum value. By interconnecting the observer with a higher throughput link, it is also possible to decrease ∆ o (z).

This study was focussing on end-to-end freshness. It is not limited to periodic communications between a sensor, a controller and an actuator. A similar analysis may be however achieved regarding the end-to-end latency defined in [START_REF] Lauer | Latency and freshness analysis on ima systems[END_REF]: the time elapsed between an event at the beginning of a functional chain and the first event depending on it at the end of the chain. This paper shows furthermore that if someone wants to check the frame arrival order between two measures or commands, it is necessary as shown, in Fig. 2a, that the second value received at the observer at least after 2∆ max time units (if the sending inter-arrival time is larger than ∆ max ).

METHODOLOGY

Data preparation

At this point, it is assumed that a trace (captured by the on-board observer with a tool like tcpdump and sent to the ground) is available. The trace corresponds to a Wireshark pcap file as shown in Fig. 4. As explained previously, the multicast functionality is used to capture the traffic in order to be free of the synchronisation and multiple observability points. It is important to note however that it may introduced issues that need to be taken into account in the following data processing (several frames might be simultaneously forwarded on a switched network and the frame order perception might be different from one point compared to another).

In the trace, frames are timestamped (with their arrival date on the observer) and sorted chronologically. We assumed also that each frame is encoded according to a common application protocol and contains a data type (might be a measure, a command or even a telemetry information) and a sequence number fields. It means that is possible to associate for each frame i the type T i , its sequence number K i and its arrival date on the observer D i . The capture file might be seen here as a matrix F where each row corresponds to the set

F i = {T i , D i , K i } with i ∈ {1, . . . , n}.
In addition, it can be retrieved from the specification of the mission of the flight a list of functional sequences for which the end-to-end freshness (and the ordering) property has to be satisfied. For a given sequence S j , it corresponds both to the ordered sets of the related type in the related chain Fig. 4. Extract of a pcap Wireshark file C j = {C j,1 , . . . , C j,m } and of the freshness requirements R j = {δ j,1 , . . . , δ j,m }. δ j,m means that it is required that a frame with the type C j,m-1 arrived not earlier than δ j,m time-units before a frame with the type C j,m . It means that specifications might be gathered in a set S where each item corresponds to the set S j = (C j |R j ). We consider in this paper that a task only requires a single fresh data frame T j-1 to produce a data T j .

Data processing

The next step, following the data selection, is data processing. The Algorithm 1 is used to parse the trace file and hence verify if the freshness is respected for all types defined in all iterations of all sequences. In fact, it provides all frames collections that successfully satisfy the sequences specifications.

Algorithm 1: Parsing trace Data: The frames captured F = (T |D|K) and the sequences specifications S = (C|R) Result: V the successful frames collections

1 forall S j ∈ S do 2 F ′ ←-{F i ∈ F | T i ∈ C j }; 3 l ←-|C j |; 4 foreach F ′ i | T i = C j,l do 5 P ←-{F ′ i }; 6 forall P z ∈ P | P z NEq C j do 7 k ←-|P z |; 8 p ←-x | F ′ x = P z,k ; 9 V ← FindInputs(F ′ , p, C j,l-k , R j,l-k+1 ); 10 P ←-P ∪ {P z × V}; 11 P ←-P\P z ; 12 if P = ∅ then 13 V j ←-V j ∪ P;
Line 2 filters the trace (according to the sequence) in order to speed up the algorithm. If a sequence refers several times to the same type, line 4 might lead to consider a frame as the final end of a sequence while it was just in the middle. In line 6, all candidates chains that do not correspond to the sequence chain specification (function NEq checks that all types regardful of the ordering are contained) are taken into consideration. The core of the algorithm consists then in retrieving a tree with all sets (P) of frames that match the sequence specifications. The roots of the trees correspond to the different iterations of the ending type of the sequence. The function FindInputs detailed in Algorithm 2 is used to retrieve the list of frames that are relevant as input of the one considered in the sequence.

Algorithm 2: FindInputs(F , p, τ, δ) Input: The trace F = (T |D|K), the initial position p, the searched type τ and the δ-freshness property Output: The list V of matching frames

1 V ←-∅; 2 i ←-p -1; 3 while i ≥ 0 and D i > D p -2∆ max do 4 i ←-i -1;
/* skip ambiguous frames */

5 while i ≥ 0 and D i ≥ D p -δ + ∆ max do 6 if T i = τ then 7 V ←-V ∪ {F i }; 8 i ←-i -1; 9 return V;
The validity is analyzed in Algorithm 2 according to (2) (see lines 3 and 5). ∆ max is a pre-processing constant that will have been computed (for instance through Network Calculus). Several frames are possible when the interarrival time of the input is lower than the freshness property. These frames will generate the branches of the trees in Algorithm 1. If no frames are valid, the branch will be pruned at line 11. At the end, if the condition in line 12 is false, the sequence was not here satisfied. Otherwise, Algorithm 1 gives all frames collections that may be used to print the freshness (like in the following section) or the number of inputs that satisfied any specific type. It also possible to compute the whole latency of a sequence (the difference between the arrival date of the last and the first elements of the sequence). Finally, it may be noticed that a similar algorithm might be defined to check the ordering property of the frames of a given type. Such algorithm will check that the sequence number is increasing all along the trace and that the time difference between two consecutive numbers is at least 2∆ max (to avoid ambiguous situations).

CASE STUDIES

The results presented in this section were obtained by applying the previous algorithms on a real benchmark detailed in (Robert et al., 2015, Fig. 5).

The benchmark

Two scenarios are experimented in the upper stage of a launcher when the separation of lower stage and boosters already happened. The topology (Fig. 5 Several sequences are defined. Each frame requires the end-to-end freshness δ to be not greater than 10 ms. By consequence, the δ > 3 * ∆ max hypothesis is verified and the Algorithm 1 is used to verify the freshness.

Results: nominal case

This section presents the results in the nominal case without any failures. The 10 ms-freshness property is verified for a functional sequence like in section 3, i.e. based on a command computed from a measure. The measure is periodic with a period of 5 ms, such that 1 or 2 measures (X (k) and X (k + 1)) might be used to compute a command Y (l). The verification of the freshness is done by evaluating the time difference between a command Y (l) and each valid measure X (k). Fig. 6a shows each time difference that respects the freshness requirement.

Fig. 6a shows that at least one fresh measure is available to compute a command in the framework of (2). The variation of the freshness is due to the non synchronisation of the two flows and to the jitters. The clocks deviation between machines and observer remained however limited during the experiments, such that X (k) freshness values remained similar and did not vary so much between 0 to 5 ms as it could be the case for another experiment. Sometimes, two measures are even available (when it is not the case, one of the two measure is rejected due to the non synchronisation issue or the ambiguous domains).

We observe also the ordering in a second experiment. Here the tested sequence consists of 22 aperiodic frames (of three types). Table 1 gives additional information that might be derived from the proposed algorithms. In nominal Indeed, the minimum inter-arrival time is always higher than the 2∆ max criteria, such that frames are received according to the predefined order.

Results: link failure case

This case is experimented by unplugging the controller of the network in solid in Fig. 5 during about 10 s (the whole experiment lasts 18 s). Table 1 shows that, in this case, losts occur and the inter-arrival time decreases.

Regarding the freshness analysis (see Fig. 6b), no measure X (k) is received and hence enables to satisfy the freshness requirement during the link failure (for l ∈ [30,123]). In the launcher case, this issue is however taken into consideration by using a redundancy in the nodes strategy. As shown by Fig. 5, a second sensor and a second network (the dashed lines) will sent the same type of data, such that the controller might use it to compute a new command. The proposed algorithms enable to verify that this redundancy will also be able to satisfy freshness requirements since a measure X ′ (k) will be at least received in time.

CONCLUSION

This paper addresses an important topic for embedded systems like launchers (or even distributed network satellites): the tele-monitoring. It focus especially on how advanced properties may be verified simply based on a trace of all exchanges through the network during a flight. The analysis of the single capture is complex since the timestamps in the trace do not correspond to the exact sending or receiving time of the frame from the source to the destination. This paper proposes methods to verify the end-to-end freshness and arrival ordering requirements based on a single trace.

Future works aim at adding pre-processing of the trace into the observer (embedded in the launcher) through a Software Defined Network architecture. Here the frames capture might be substituted by OpenFlow communications.

⋆

  Co-founded study by CNES and CRAN in the frame of CNES Launchers' Research and Technology program.

Fig. 6 .

 6 Fig.6. Freshness of each measure valid for a command. Abscissa gives the sequence number l of the command case, no packet is lost and the inter-arrival time at the observer is very close in average to the theoretical value. Indeed, the minimum inter-arrival time is always higher than the 2∆ max criteria, such that frames are received according to the predefined order.

  The problem is that both x (k) and y (l) are local values, based on different clocks. From the point of view of the observer (that will receive each frame), available timestamps are slightly different. Dates are modified according the Quality of Service offered by the network, such that timestamps available are x o

  ) is a 100 M b/s switched Ethernet network architecture. Experiments are achieved in a nominal and a link failure cases.During the experiments, all traffic going through the network is scheduled and known. 66 flows are configured among the machines connected to a same network. The length of each flow is 72 bytes except for six of them that have a length of 200 bytes. The traffic load is around 2 M b/s and the maximum delay, if all the flows are transmitted at the same time is 0.4416 ms. Traffic might be periodic, aperiodic or event-triggered.

	Observer
	Sensors
	Controller
	Actuators
	Fig. 5. Upper stage topology
	2 switches. The observer is connected to both networks
	in order to get a unique trace gathering all traffics. The
	switching plane is implemented in a static way in order to
	not generate additional traffic on the network.
	The architecture is divided in two networks (line and
	dashed) and is composed of 17 terminal machines and

Table 1 .

 1 Order results

	Criteria		Nominal case	Link failure case
	Packet lost ratio		0 %	29 %
	Inter-arrival time	avg	0.705 s	0.340 s
		min	0.009 s	0.009 s
		max	4.010 s	1.990 s