Jean Wilfried Fredy

Jérémy Scelle

Gregory Ramniceanu

Bich-Thuy Doan

Célia S Bonnet

Éva Tóth

Mickaël Ménand

Matthieu Sollogoub

Guillaume Vives

Bernold Hasenknopf

Mechanostereoselective One-Pot Synthesis of Functionalized Head-to-Head Cyclodextrin [3]Rotaxanes and Their Application as Magnetic Resonance Imaging Contrast Agents

Supramolecular chemistry offers modularity and fast synthesis of complex molecular objects. It is therefore well adapted to biological applications where multifunctional and modular devices are needed. Furthermore, in view of in vivo applications and eventual approval by regulatory agencies, the obtention of a single molecular species is preferable. We are particularly interested in the development of multimodal imaging agents using such a modular supramolecular approach. Our concept consists of preparing monofunctional molecular building units that self-assemble in a modular fashion to produce multifunctional imaging agents. A particularly appealing architecture for this are polyrotaxanes composed of different rings threaded on a common axle. 1 However, efficient threading of functionalized rings must be guaranteed, and different mechano-isomers, i.e. diastereomers that differ in the orientation of the mechanically bonded rings, 2 can be obtained using this strategy.

While a large variety of macrocycles such as cucurbituril, crown-ether, bis-paraquat or calixarene have been reported to produce rotaxanes, [2][3] cyclodextrins (CDs) present the particular advantages to be commercially available, cheap and easily functionalizable. 4 Moreover, CDs are biocompatible and water-soluble which enables the access to hydrosoluble rotaxanes with potential applications in biology and medicine such as imaging. 1,5 Native CDs have been widely used in rotaxanes or polyrotaxanes, 6 since the synthesis of first CD [2]rotaxane described by Ogino. 7 They are usually obtained by threading followed by end-capping reaction, with the hydrophobic effect as driving force for the self-assembly process. This requires forming the pseudo-rotaxane in water and, after isolation or in situ, performing the stoppering reaction also in aqueous solution to prevent the dethreading. These limitations have resulted in i) few CD rotaxanes described in the literature contain two CDs, and ii) polyrotaxanes assembled with already functionalized CDs remain less explored and form a mixture of orientational mechanoisomers. 8 Based on the polyammonium-CD polyrotaxanes described by Wenz,6a,9 we have developed polyrotaxanes with Gdfunctionalized CDs 1 that showed improved relaxivities compared to small molecules. It was also possible to thread bodipy and Gd-functionalized CDs simultaneously onto the polymer strand, proving the viability of the approach to obtain bimodal agents (fluorescence and MRI). However, they were obtained as a statistical mixture of orientational mechanoisomers and their high toxicity prevented their use in vivo.

In order to lower their toxicity, to test different functionalized CDs, to get single isomeric compounds, and to better understand the intrinsic role of the rotaxane architecture on the MRI properties, we decided to investigate smaller [3]rotaxanes as models for the above mentioned polyrotaxanes. While various synthetic strategies have been developed for multiple threading of other macrocycles 10 , in particular the 'activemetal template strategy', 11 only few methods yielding [3]rotaxanes of CDs have been described. Anderson showed that reaction of 2,6-dimethylphenol with bis-diazonioazobenzene in the presence of α-CD gave a tail-to tail [3]rotaxane. 12 Takata has recently reported another one-pot strategy to directly obtain head-to-head [3]rotaxanes of α-CD by using isocyanate stoppers. 13 A solid-phase synthesis yielded a head-to-tail [3]rotaxane, 14 and the coupling of two pseudo- [2]rotaxane gave a tail-to-tail [3]rotaxane. 15 Fort prepared polyrotaxanes with lactose-functionalized -CD. 8c Inspired by these results and the use of click chemistry for the stoppering reaction of CD-rotaxanes, 8c,10a,16 we aimed at the development of a new general mechanostereoselective synthetic strategy to produce functionalized CD [3]rotaxanes as single isomers. Herein, we report a versatile five-component one-pot synthesis, that was applied to α-CD mono or bi-functionalized by Gd(III)-complexes (Figure 1) to obtain rotaxane based MRI contrast agents. In vitro relaxivity of these [3]rotaxanes and their use as biocompatible contrast agents for in vivo imaging in mouse models are also reported.

Scheme 1. Synthesis of head-to-head [3]rotaxanes.

Rotaxanes were synthesized in a one-pot strategy using a hydrophobic C12 alkyl chain as thread and click chemistry for the stoppering reaction. The reaction conditions were optimized using native α-CD (Scheme 1). The diazidododecane axle 3 was, in a first step mixed with native α-CD in water to yield a pseudo-rotaxane by the hydrophobic effect. The formation of the inclusion complex can be observed by the appearance of turbidity in the reaction mixture. This turbidity is probably due to the formation of tubes of CD pseudorotaxanes and their aggregation via intermolecular hydrogen bonds as observed with other CD polyrotaxanes. 17 In a second step, reactants for CuAAC and the stoppers were added to perform the end-capping reaction. The dicarboxylic acid 5 was chosen according to literature, 16a as it is described to be large enough to prevent the dethreading of α-CD. Moreover, the carboxylate groups enable the solubility of the rotaxane in water at physiological pH, which is useful for potential in vivo studies.

The 1 H DOSY spectrum (Diffusion Ordered NMR) of the product 6 in DMSO showed the same diffusion coefficient for the protons of the CD, axle and stoppers (see Figure S1) indicating that they are part of the same supramolecular species. Moreover, the diffusion coefficient is lower than the one of each separate component, which is consistent with the larger molecular size of the assembly. Since DMSO is a solvent that prevents the formation of inclusion compounds with CD, 13 the interlocked structure forms a stable rotaxane. The integration of the 1 H NMR spectrum and mass spectrometry (see Figure S5) showed the formation of a single symmetrical [3]rotaxane with two threaded CDs. 2D T-ROESY spectrum (Transverse Rotating-frame Overhauser Enhancement Spectroscopy, Figure 1) provided further evidence for the formation of the interlocked structure with correlation peaks between the central axle protons and inner pointing CD protons H 3 and H 5 . Moreover, the correlation peak between H 5 and protons Ho, Ht and Hs as well as H 6 and Ho indicates that the CDs have their primary rim pointing to the stoppers and their secondary rim pointing to each other, hence adopting the head-to-head orientational mechanoisomery. This orientation can be justified by the preferred establishment of hydrogen bonds between the complementary secondary rims, 18 and is consistent with other [3]rotaxanes having an alkylene axle. 13, α-CD was then selectively functionalized by gadolinium complex of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid monoamide (Gd-DO3A-MA) complexes to act as MRI contrast agent. The synthesis is based on a flexible approach using a regioselective deprotection of perbenzylated CDs 19 and a Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) 20 between protected mono 21 and bis azido CDs 22 and propargylfunctionalized DO3A-MA followed by deprotection as described in our previous publication. 1 CDs selectively mono-or di-functionalized on diametrically opposed A,D positions on the primary rim by Gd-DO3A-MA complexes were obtained. As MRI is not a very sensitive imaging technique, the difunctionalized CD 8 is particularly interesting in order to increase local Gd(III) concentration. [START_REF]The Chemistry of Contrast Agents in Medical Magnetic Resonance Imaging[END_REF] Then, the same procedure was applied for the synthesis of the functionalized CD H 6 H 6 HO rotaxanes (Scheme 1) and the products were directly purified by size exclusion chromatography. The formation of the Gd-CD [3]rotaxanes RotGd2 and RotGd4 was confirmed by mass spectrometry (see Figure S6-7) with molecular ion peaks at m/z 1292.05 and 1706.14 respectively corresponding to [M-3H] 3-ions. The rotaxanes were obtained in 35-50% yield, which is suitable for a five-component one-pot reaction.

Lanthanum analogs (7a and 8a) were also synthesized to obtain further information on the interlocked structure by NMR studies. As observed with native CD, the same diffusion coefficient for the stoppers, functionalized CD and the axle in water was observed on the DOSY spectrum for RotLa2 1a and RotLa4 2a (Figure S2-3). For both rotaxanes, correlation peaks between the inner cavity CD H 3 and H 5 and central axle protons on the T-ROESY spectra (Figure S4) confirmed the interlocked structure. Moreover, correlation between H 5 and triazolyl proton Ht indicates that the primary rim of the functionalized CD is pointing to the stoppers. As observed with native CD, this head-to-head configuration enables intramolecular hydrogen bonding between the two secondary rims and seems inherently favored, even though in this case it also avoids the steric hindrance between the substituents on the primary rims. The effect of the supramolecular assembly on the longitudinal proton relaxivity of the Gd(III) complexes was then investigated by 1 H NMRD (Nuclear Magnetic Relaxation Dispersion) profiles by measuring T1 at different magnetic fields. The profiles (Figure 2) were recorded in water at 37°C for the two rotaxanes (1b and 2b) and compared to the corresponding CDs (7b and 8b). The relaxivities per Gd are considerably higher for RotGd2 1b and RotGd4 2b (12.30 and 15.70 mM -1 •s -1 at 20 MHz, respectively) than for the parent functionalized CDs (7.06 and 8.57 mM -1 •s -1 , respectively) or Gd-DOTA [START_REF] Powell | [END_REF] (3.83 mM -1 •s -1 ) The relaxivity values are comparable to multimeric Gd-DOTA functionalized CD systems of similar size ([Gd-DOTA]7-β-CD: 12.2 mM -1 •s -1 at 60 MHz; 25 Gd-DO3A-HP--CD: 7.82 mM -1 •s -1 at 60 MHz; 5 [Gd-DO3P]6.9-F0.1-β-CD: 21.6 mM -1 •s -1 at 20 MHz 26 ). The NMRD profile shows an enhancement at medium field (20-80 MHz) for the rotaxanes, which is not present for the functionalized CDs. This hump is characteristic of macromolecular contrast agents (such as polymers or liposomes) and it indicates that the interlocked architecture plays a crucial role in the rotational dynamics and subsequently in relaxivity properties. The temperature dependence (Table S1) showing an increased relaxivity with increasing temperature is also an indication of the rigidity of the system. Indeed, it proves that the relaxivity is limited by the water exchange rate of the complex and not by its rotation. It is most notable that our small rotaxanes feature also those properties which were previously observed with polyrotaxanes (CD-Gd/polyammonium PR: 16.4 mM -1 •s -1 ; 1 CD-Gd2/polyammonium PR: 19.6 mM -1 •s -1 ; 1 Gd-DO3A-HPCD/Pluronic PR: 22.8 mM -1 •s -1 5 at 60 MHz). The [3]rotaxanes behave indeed as a typical macromolecular contrast agent with promising relaxation properties at relevant body temperature. The biodistribution of rotaxanes RotGd2 and RotGd4 was then studied in vivo on BALB/c mice using adapted dynamic MRI sequences at 7T. Several regions of interest (ROI) were monitored (liver and kidney) and the MRI intensities of the regions were plotted pre-and post injection of the contrast agent at Gd concentration of 50 mol•kg -1 . Comparison with commercial Gd-DOTA as a reference was performed at the same concentration of Gd. Figure 3 shows MRI cross-sections where the kidney regions are highlighted as expected for the biodistribution of a molecular scaffold of a small size inferior to 5 nm. 27 Comparison of the pre-and post-contrast images clearly reveals the higher brightness of the kidney caused by RotGd2. For further images of the liver with this CA, and of kidney and liver with RotGd4 see SI (Figure S9-10). Contrast enhancements in both organs are expressed as percentage of enhancement compared to the pre-contrast image. The peaks of enhancement were observed at the same time, 6 minutes post-injection for all CA in the liver and the kidney followed by a phase of decay (Figure 3b andS11). The absolute values at 21 min post injection are highest for RotGd2 (16% in the liver, 67% in the cortical region of the kidney), followed by those of RotGd4 (9% and 28% respectively) and Gd-DOTA (6% and 17% respectively). After bloodstream circulation with a complementary transitory visualization through the vascular network in the liver, the CAs are excreted from the kidney cortex to the bladder. After 40 min, the levels of RotGd4 and Gd-DOTA came back to their pre-contrast values and to 10% signal enhancement for RotGd2 in the liver indicating a hepatic wash-out. However, a stronger retention in the cortical % signal enhancement region of the kidney was observed with enhancement of 46% and 19% for RotGd2 and RotGd4, respectively (11% for Gd-DOTA). Both rotaxanes behave similarly in the kidney glomerular and tubular clearance with slower elimination than the mononuclear complex Gd-DOTA. Due to the higher concentration of RotGd2 than that of RotGd4 injected both for an identical final Gd amount, RotGd2 takes longer to be eliminated. This imaging study evidences that both rotaxanes display higher contrast enhancement than the commercial CA Gd-DOTA over the full observation period, without the typical liver accumulation observed for macromolecular agents. Thus, they improve the imaging properties without undesired liver uptake. The relatively long circulation time opens the perspective for vectorization towards other tissues than the kidney.

In summary, we have successfully developed a one-pot fivecomponent mechanostereoselective synthesis of head-to-head CD [3]rotaxanes leading to the first [3]rotaxanes with Gd-DO3A-MA functionalized CDs. These rotaxanes have shown their ability to significantly improve the relaxivity properties compared to parent CDs indicating the interest of this supramolecular approach for MRI applications. Indeed the behavior of [3]rotaxanes is already comparable with those of much larger macromolecular agents while keeping the advantages of small molecules in terms of synthesis and characterization at the molecular level. These rotaxanes are non-toxic, and their biodistribution could be investigated by MR imaging with high temporal resolution. Both rotaxanes exhibit higher contrast enhancement and retention than the commercial Gd-DOTA, with a particular improvement for RotGd2. In conclusion, rotaxanation is a viable approach to obtain contrast agents with improved efficiency, even when the rotaxanes are relatively small. This simple supramolecular approach has multiple advantages over conventional covalent strategies such as modularity and versatility. Indeed, each component is synthesized and characterized separately before the assembly, and can be exchanged at will by other components.

Experimental procedures, compound characterization data, and NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

* bernold.hasenknopf@upmc.fr; guillaume.vives@upmc.fr § These authors contributed equally.

  Jean Wilfried Fredy, † § Jérémy Scelle, † § Gregory Ramniceanu, ‡ Bich-Thuy Doan, ‡ Célia S. Bonnet, ¶ Éva Tóth, ¶ Mickaël Ménand, † Matthieu Sollogoub, † Guillaume Vives, † * Bernold Hasenknopf † * † Sorbonne Universités, UPMC Univ Paris 06, CNRS, Institut Parisien de Chimie Moléculaire UMR 8232, 4 place Jussieu, 75005 Paris (France) ‡ Chimie ParisTech, CNRS, UMR8258 INSERM U1022 Unité de Technologies Chimiques et Biologiques pour la Santé, 11 rue Pierre & Marie Curie, 75005 Paris, (France) ¶ Centre de Biophysique Moléculaire, CNRS UPR4301, Université d'Orléans, Rue Charles Sadron, 45071 Orléans Cedex2 (France) Supporting Information Placeholder ABSTRACT: A versatile five-component one-pot synthesis of cyclodextrin (CD) [3]rotaxanes using Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) has been developed. Head-to-head [3]rotaxanes of α-CD selectively functionalized by one or two gadolinium 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid monoamide (Gd-DO3A-MA) complexes were obtained mechanostereoselectively. The Magnetic Resonance Imaging (MRI) efficiency, expressed by the longitudinal proton relaxivity of the rotaxanes was significantly improved as compared to the functionalized CD. In vitro and in vivo preclinical studies showed a higher contrast and retention in the kidney than gadolinium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex (Gd-DOTA) demonstrating the potential of these rotaxanes as MRI contrast agent.

Figure 1 .

 1 Figure 1. T-ROESY spectrum of CD [3]rotaxane 6 (600 MHz, DMSO-d6).

Figure 2 . 1 H

 21 Figure 2. 1 H NMRD profiles of aqueous solution of CD-Gd, CD-Gd2, RotGd2 and RotGd4 at 37°C.

Figure 3

 3 Figure 3. a) MRI cross-sections of the kidneys (red arrow) pre-(left) and post-injection (right, 40 min) of RotGd2 b) Dynamic MRI signal enhancement in renal cortex after CA injection.

We thank Agnès Pallier, Centre de Biophysique Moléculaire, CNRS UPR4301, Université d'Orléans, for performing ICP measurements on the Gd 3+ containing samples. Financial support from UPMC (programme Emergence) and the LabEx MiChem programme under reference ANR-11-IDEX-0004-02 and from the Ligue contre le Cancer are acknowledged. We thank also the Institut Weizmann Français for the postdoctoral fellowship to G. Ramniceanu and the IDEX Programme interdisciplinaire, Université Paris Descartes SINON and Université Paris Descartes for the MRI equipment.


Ruidy Nemausat
email: ruidy.nemausat@impmc.upmc.fr

Christel Gervais

Christian Brouder

Nicolas Trcera

Amélie Bordage

Cristina Coelho-Diogo

Pierre Florian

Aidar Rakhmatullin

Ion Errea

Lorenzo Paulatto

Michele Lazzeri

Delphine Cabaret

Temperature dependence of x-ray absorption and nuclear magnetic resonance spectra: probing quantum vibrations of light element in oxides †

A combined experimental-theoretical study on the temperature dependence of x-ray absorption near-edge structure (XANES) and nuclear magnetic resonance (NMR) spectra of periclase (MgO), spinel (MgAl 2 O 4 ), corundum (α-Al 2 O 3 ), berlinite (α-AlPO 4 ), stishovite and α-quartz (SiO 2 ) is reported. Predictive calculations are presented when experimental data are not available. For these light-element oxides, both experimental techniques detect systematic effects related to quantum thermal vibrations which are well reproduced by density-functional theory simulations. In calculations, thermal fluctuation of the nuclei are included by considering nonequilibrium configurations according to finite-temperature quantum statistics at the quasiharmonic level. Nuclear quantum fluctuations on XANES and NMR spectroscopies are particularly sensitive to the coordination number of the probed cation. Furthermore, the relative importance of nuclear dynamics and thermal expansion is quantified over a large range of temperatures.

Introduction

X-ray absorption near-edge structure (XANES) and solid-state nuclear-magnetic resonance (NMR) spectroscopies are versatile probes of condensed matter. 1,2 The combination of these two powerful techniques allows to thoroughly explore the electronic properties and local structure of materials. [3][4][5] Moreover, recent breakthroughs in methodology and instrumentation provide a qualitative enhancement of experimental data. [6][7][8][9][10][11] However, the use of accurate and appropriate theoretical tools is mandatory to fully interpret experiments. [12][13][14][15][16] Most state-of-the-art simulation methods neglect nuclear degrees of freedom by assuming that atoms are fixed at their equilibrium positions. Nonetheless, even at zero temperature the influence of quantum fluctuations on nuclear motion remains as the zero-point motion, especially in the presence of light elements. At room temperature, the nuclear dynamics generates specific discrepancies between standard calculations and measurements, such as an additional pre-edge peak in the K-edge region of XANES spectra in light-element oxides 17 and inaccurate assignment of NMR resonance peaks. 18 In organic compounds, several studies already investigated the influence of atomic vibrations on XANES and NMR measurements. [19][20][21][22][23] Besides, the impact of temperature is also noticeable in inorganic materials, in XANES preedge structure, [24][25][26] chemical shifts, 18,27 or electric-field gradient (EFG). 28,29 Some attempts to theoretically model thermal effects in both spectroscopies emerged in the literature. Empirical methods based on either small displacements of the probed atom, 30 or on averages of magnetic shielding tensors along specific orientations were proposed. 31,32 Nuclear motion may also be considered via averages along molecular dynamics trajectories. [33][34][35][36][37] This approach assumes that nuclei follow classical Newtonian trajectory of motion. Thus, it cannot describe phenomena associated to the quantum character of thermal fluctuations, which are especially relevant for light elements. Quantum thermal fluctuations may be taken into account by path integral molecular dynamics but the computational cost is an important limitation. 38,39 Finitetemperature effects occurring in molecular crystals were successfully included via fragment-based techniques. 40,41 In a recent paper, 26 we presented an ab-initio method, based on the density-functional theory (DFT), 42 to investigate the importance of quantum thermal vibrations in XANES and NMR spectroscopies. Following the work of Rossano et al., 43 in the quasiharmonic approximation (QHA), 44 thermal vibrations are modeled by constructing nonequilibrium structural configurations obeying the quantum statistics at finite temperature. This method was validated on MgO, 26 a highly symmetric crystal, providing results closer to experiments than previous approaches. 17,25,43 The current paper aims at generalizing this approach to several light-element oxides with various local cristallographic environments of the cations: periclase (MgO), spinel (MgAl 2 O 4 ), corundum (α-Al 2 O 3 ), berlinite (α-AlPO 4 ), stishovite and α-quartz (SiO 2 ). In these compounds, the probed atom is either 6-or 4fold coordinated. Temperature-dependent experimental data are presented for both techniques and are compared to calculations. In NMR, when experimental data are not available, predictive calculations are provided. Thus, the influence of quantum thermal vibrations on NMR and XANES spectroscopies is shown and the relative importance of nuclear dynamics and thermal expansion is quantified. Furthermore, the influence of vibrations in both techniques is found to be sensitive to the coordination number of the probed cations.

The paper is structured as follows. Section 2 recalls the theoretical model used thereafter and gives computational details for XANES and NMR calculations. In Sec. 3, experimental details are provided. In Sec. 4, the experimental and theoretical results are presented, firstly focusing on experimental-theoretical comparison, then discussing the sensitivity to the coordination number of the probed cation. Finally, the conclusions of this work are drawn in Sec. 5

Theoretical Approach

Quantum statistical mechanics

The influence of thermal fluctuations is described by generating nonequilibrium configurations accounting for quantum statistics at finite temperature. Given a crystal, let us consider a supercell of N nuclei, with R α i the position of the ith nucleus along the α Cartesian coordinate. For simplicity, the two indexes are contracted into a single one I such as R α i = R I , with I = {1, . . . , 3N}. Thereafter, the collective position vectors of the nuclei (R = R 1 , . . . , R 3N ) is used. Within the Born-Oppenheimer approximation the total energy E(R) of the cell is considered as a function of the nuclear positions. The lattice-dynamical properties of a crystal are determined by the dynamical matrix, which reads at the Γ point of the Brillouin zone

D IJ = 1 √ M I M J ∂ 2 E(R) ∂ u I ∂ u J u=0 , (1)
where M I is the mass of the Ith atom and u I is the displacement from the equilibrium position R I,(eq) (R I = R I,(eq) + u I ). The frequency Ω µ and polarization vector e I,µ of the phonon mode µ are obtained from

3N ∑ J=1 D IJ e Jµ = Ω 2 µ e Iµ , (2)
where ∑ I e I,µ 2 = 1. At a given temperature T , the probability P(R) of finding the crystal in the R configuration is

P(R) = A exp -∑ µ ∑ I √ M I e Iµ u I 2 2a µ ( 3
)
where A is a normalization constant, ∑ µ is the sum on the 3N -3 modes with Ω µ = 0 and

a µ = h 2Ω µ coth hΩ µ 2k B T (4)
is the normal length of the µth mode of vibration at the T temperature (k B is the Boltzmann constant). Nuclear configurations are generated by Monte-Carlo sampling of P(R).

In practice, a set of configurations is generated as follows: for each µ mode, a set of N c random Gaussian numbers (with unit variance and zero expectation) {x i µ } i=1,N c is created. The nuclear positions collective vectors R i are then given by:

R i I = R i I,(eq) + ∑ µ 1 √ M I e Iµ a µ x i µ . (5)
As in several applications, 23,43 the quantum statistical average at temperature T of any observable O is computed via the following expression:

O(T ) = dR P(R)O(R) 1 N c N c ∑ i=1 O(R i ). (6)
In this paper, O is either the x-ray absorption cross section σ (hω), the NMR chemical shift δ or the quadrupolar coupling constant C Q . Henceforth, this model is referred to as the QHA model. For more details refer to Refs. 26,45.

Solid-state nuclear magnetic resonance

For each nucleus, the isotropic value of the magnetic shielding tensor σ is evaluated as σ iso = tr ( σ ) /3. To compare with NMR experiments, the isotropic chemical shift δ iso ≈ σ refσ iso is considered, where σ ref is the isotropic shielding of a standard reference. The chemical shift δ (T ) at finite temperature is obtained as in eqn (6):

δ iso (T ) = 1 N c N c ∑ i=1 δ iso (R i ). (7)
In the case of quadrupolar nuclei (nuclear spin > 1/2), the nuclear quadrupole electric moment Q interacts with the electricfield gradient (EFG) resulting in the quadrupolar interaction. 8 Ordering the principal values of the EFG tensors V such as |V zz | > |V xx | > V yy , the magnitude of the quadrupolar interaction is quantified by the quadrupolar coupling constant C Q = eQ |V zz | /h, with h the Planck constant and e the elementary charge.

X-ray absorption near-edge structure

For a given R configuration, in a single-electron approach, at the K edge, the XANES cross-section in the electric dipole approximation is usually given by 46 (8) where hω and ê are the energy and polarization vector of the incident x-ray beam, respectively, r the electron position and α 0 the fine structure constant. The final |ψ f and initial |ψ 1s monoelectronic states have ε f and ε 1s for respective energies. Eqn (8) is derived by assuming that the atoms are fixed in a generic R nuclear configuration, hence σ is parametrized by R.

σ (hω; R) = 4π 2 α 0 hω ∑ f ψ f | ê • r |ψ 1s 2 δ (ε f -ε 1s -hω),
The rigorous expression of the cross section accounting for the total system of nuclei and electrons reads

σ tot (hω) = 4π 2 α 0 hω ∑ f , j Ψ j f | ê • r |Ψ 0 g 2 δ (E j f -E 0 g -hω), (9)
where, r = {r 1 , . . . , r N e } is the collective position vector of the electrons, j indexes the vibrational states, the Ψ 0 g ground-state and Ψ j f final state of the electrons plus nuclei total system and E j f is the total energy in the f and j electronic and vibrational states, respectively.

In Ref. 26, we demonstrated that σ tot (hω) can be expressed in the Born-Oppenheimer approximation, in a single-electron framework, as an average at the cost of neglecting the nuclear kinetic energy, by

σ tot (hω) = dR P(R)σ (hω; R). (10)
In eqn (10), σ tot is the average of individual configuration cross sections σ (hω; R), given by eqn (8), using a probability distribution that takes into account the temperature and vibrations frequencies in a form given by eqn (6).

Computational details

Calculations are performed in the DFT framework on the six different oxides reported in Table 1. In all cases, the equilibrium structure (crystal-cell parameters and atomic positions) is the experimental one extracted from x-ray diffraction (XRD) analysis at various temperatures (see Refs. in Table 1). For each temperature, phonons are calculated for the corresponding experimental 3). To quantify the relative importance of lattice dynamics with respect to thermal expansion, calculations are also performed on the experimental XRD structures, referred as to the thermal expansion only (TEO) in the following.

DFT calculations are performed using the QUANTUM ESPRESSO plane-wave pseudopotential distribution, 55 particularly the PW, PH, 56,57 XSPECTRA 58,59 and GIPAW 60,61 packages. Stochastic nuclear configurations are generated in a large-enough supercell configuration (see ESI † ) with the SSCHA code. 62,63 Perdew-Burke-Ernzerhof implementation of the exchangecorrelation functional 64 and GIPAW reconstruction of coreelectron distributions 60,61 are used. NMR calculations are performed using norm-conserving pseudopotentials, 65 while ultrasoft ones are chosen for phonon and XANES calculations. 66 For XANES, the absorbing atom pseudopotential is generated from an electronic configuration, from which one 1s electron is removed. Other computational details (plane-wave cut-off, electronic sampling, pseudopotential details) are gathered in ESI. †

The NMR reference shieldings σ ref are chosen so that experimental and calculated values match at room temperature. To highlight the trends, we focus on the chemical shifts variations from the room-temperature: ∆δ iso = δ iso (T )δ iso (300 K). The C Q of 17 O, 25 Mg and 27 Al nuclei are calculated with Q values taken from Ref. 67, by averaging individual configuration values. 68 In each configuration, the chemical shift is computed for every atom sites. The number of configurations required to converge the averaged chemical shift of about 0.1 ppm is around 10.

In XANES simulations, for each temperature, the spectra are calculated in configurations including one 1s full core-hole in a random absorbing-atom site. The absorbing atom is isolated from its periodic images by constructing a large-enough supercell. For all compounds, the number of configurations necessary to converge the XANES calculation is between 30 and 40. A corelevel shift is applied before comparing and averaging theoretical spectra as, in the pseudopotential approximation, only valenceelectrons are considered, hence the energy scale loses its physical meaning. 69 3 Experimental Methods

Samples

Six mineral oxides samples are investigated, where the probed atom is either 4-or 6-fold coordinated (Table 1). The corundum sample was already used in Ref. 70. The stishovite polycrystal comes from a high-pressure synthesis. [START_REF]Two stishovite polycristalline samples were synthesized under 14 GPa and 1900 K conditions for 2 hours, using a multianvil press[END_REF] For NMR measurements, only powder samples are considered. For XANES, resorting to single-crystal samples for α-Al 2 O 3 and α-quartz allows polarized XANES measurements. For each compound, the maximum temperature explored remains below their respective phase-transition/fusion temperature.

Temperature-dependent experiments

Temperature-dependent NMR measurements are recorded at the CEMHTI (Orléans, France). The experimental acquisitions are carried out using a Bruker Avance spectrometer operating at 19.8 T (850 MHz) and a static NMR probe designed to handle high temperatures. [START_REF] Shakhovoy | [END_REF] The boron-nitride (BN) sample holder is heated by a CO 2 laser. Given the sensitivity of 27 Al nucleus and the expected variations of the chemical shifts compared to the static NMR peak width, 27 Al in α-AlPO 4 is the best suitable candidate. A magic-angle spinning NMR probe is required to increase the resolution in α-Al 2 O 3 and SiO 2 polymorphs. We also present the chemical shifts of 25 Mg and 17 O in MgO from Ref. 27.

X-ray absorption experiments are performed at the LUCIA beamline from SOLEIL french synchrotron facility (Saint-Aubin, France). 73 For each compound, the incident energy ranges are selected to include the K edge of either Mg, Al or Si. The experimental chamber pressure is set between 10 -7 and 10 -6 mbar. The temperature of the sample is set using a BN furnace and measured with a chromel-alumel thermocouple. The spectra are recorded in fluorescence with a four element silicon drift diode detector, protected from infrared and visible radiations by a beryllium window. To improve the signal to noise ratio, each point is obtained after an acquisition time of 5 s and several spectra are measured for each temperature. The normalization and self-absorption correction procedures are conducted as described in Ref. 25. For α-Al 2 O 3 and α-quartz samples, the two σ ⊥ and σ polarized spectra, corresponding respectively to ê parallel and perpendicular to the threefold symmetry axis of the crystals, were successively measured. Experimental Mg and Al K-edges XANES spectra in MgO and α-Al 2 O 3 , respectively, have already been published elsewhere. 25,26 4 Results and Discussion

Experimental and calculation comparison

Fig. 1 compares experimental and calculated temperaturedependent chemical shifts of 25 Mg and 17 O in MgO and 27 Al in α-AlPO 4 . From 300 to 1300 K, an increase of about 2 and 12 ppm is observed for experimental 25 Mg and 17 O δ iso values, respectively. On the other hand, experimental 27 Al chemical shift decreases with temperature, by about 4 ppm between 300 and 800 K. In both cases, simulations are in good agreement with experiments over the investigated range of temperatures. As detailed in Refs. 26,74, the remaining discrepancies between calculated and experimental slopes come mainly from deficiency of GGA. It is known that GGA slightly overestimates the interatomic distances of solids and underrates phonon frequencies. 75 In the present work, experimental lattice parameters are used to balance this effect. The use of GGA functionals more advanced than PBE might also enhance the agreement. 76,77 Usually, a linear thermal behavior of NMR parameters is assumed. 19,27 However, the temperature dependence of the chemical shift clearly shows two regimes. The linear correlation observed above a saturation temperature T s reflects a classical meanfield behavior of the Landau-type. 78 Between 0 K and T s a quantum saturation appears, where the chemical shift becomes inde- Experiment QHA 27 Alα-AlPO 4

Fig. 1 Experimental and calculated temperature dependence of 25 Mg and 17 O chemical shifts in MgO and of 27 Al chemical shifts in berlinite (α-AlPO 4 ). 25 Mg measurements come from Ref. 27, whereas 27 Al ones are original datasets. The red dashed line is the best fit using eqn (11).

pendent of the temperature. Therefore, the chemical shift is fitted using As observed in NMR, a saturation mechanism is also expected at low temperature. Fig. 3 displays the polarized Si K-edge XANES spectra calculated at 0 K, in stishovite and α-quartz, with all configuration spectra in background and compared to the equilibrium spectra. The latter are computed for atoms at their equilibrium positions in the 100-K XRD structure. The equilibrium calculation does not reproduce any pre-edge feature, unlike the QHA calculation. The presence of the P pre-edge peak at 0 K highlights the importance of accounting for quantum vibrations in XANES at low temperatures. Indeed, the zero-point motion, which is a pure quantum phenomenon, is sufficient to induce an sp hybridization resulting in the P peak occurrence. Fig. 3 also illustrates the angular dependance at 0 K. In either x-ray polarization, the P-A separation is larger in stishovite than in α-quartz. Figs. 2 and3 show that the P pre-edge-peak intensity is similar in both orientations. Thus, there is no preferential structural orientation for the hybridization from which P originates. It is therefore complicated to propose geometrical arguments explaining the hybridization process, as already exposed in Ref. 26. The P pre-edge peak arises from a hybridization between the 3s and 3p electronic states of the absorbing atom induced by quantum vibrations. The increase of the P peak is the sign of a stronger hybridization with temperature. The energy shift is related to the band gap narrowing as temperature rises. 26

δ (T ) = A + B coth 2T s T , (11)

Sensitivity to the local environment

The QHA model provides a satisfactory agreement between calculations and experiments that allows us to perform predictive computations when experimental data were not measured. Fig. 4 compares the calculated NMR chemical shifts for the cations in 4-fold (red triangles) and 6-fold coordinations, in the QHA model (full symbols) and by accounting for thermal expansion only (TEO, empty symbols). Opposite trends of QHA calculations are observed according to the coordination number of the probed cation. For 6-fold coordinated cations, the QHA corrected chemical shift increases with temperature, whereas it decreases for 4fold coordinated ones.

QHA and TEO calculations present distinct trends according to the cation coordination. For tetrahedral sites, QHA and TEO calculations give a decreasing chemical shift with temperature, with very similar slopes in the linear regime. On the other hand, in octahedral sites, there is an important deviation between QHA and TEO calculations, resulting in an increase of the QHA-calculated chemical shifts with temperature. These observations illustrate the competition between the nuclear dynamics and the thermal expansion. Nuclear dynamics is negligible compared to thermal expansion for 4-fold coordinated cations, whereas nuclear dynamics is the major component for 6-fold coordinated cations, and induces an increase of δ iso experimentally observed in MgO. This phenomenon probably originates from the local symmetry differences. Indeed, the bond lengths are longer in an octahedron than in a tetrahedron, giving more freedom to the 6-fold coordinated cations. For example, an analysis of the configurations at 300 K shows an average 0.07 % off-center displacement in octahedra, whereas it reduces to 0.04 % in tetrahedra. No significant variation in angles is observed.

It has already been observed that the chemical shift decreases when the cation-O bond length i.e., the tetrahedral volume increase. [80][81][82] TEO calculations endorse this matter of fact. For Mg in MgAl 2 O 4 and Al in α-AlPO 4 , the chemical shift decrease is about 3 ppm K -1 , while it is less important for Si in α-quartz about 0.5 ppm K -1 . These differences are not directly related to the expansion of tetrahedra in each materials, since, from 300 to 800 K, their volumes increase of about 1.55 % for MgO 4 , 2.91 % for AlO 4 and 2.53 % for SiO 4 units.

The calculated temperature-dependent NMR chemical shifts of 17 O in oxides are presented in Fig. 5, including both QHA and TEO calculations. Overall, the chemical shift varies significantly with temperature and the quantum nuclear dynamics influence is more important than in the TEO case. Interestingly, the most important variation occurs in ternary oxides (MgAl 2 O 4 and α-AlPO 4 ), possibly due to the fact that oxygen atoms are sur- α-Quartz -Si(4) Stishovite -Si(6) 29

Si

Fig. 4 Calculated temperature-dependent NMR chemical shifts of the studied cations in oxides, where the absorbing atom is either 4-or 6-fold coordinated. Black squares and blue diamonds are used for cations in octahedral environment. Red triangles represent the cation in tetrahedral environments, the full and empty symbols correspond to the QHA and TEO (thermal expansion only) calculations, respectively. The dashed lines are the best fits using eqn (11). Saturation temperatures, T s , are given in ESI. † rounded by different atom-type neighbors.

In XANES spectroscopy, the temperature-dependence is also sensitive to the probed cation coordination number. For octahedral environments, earlier studies demonstrated that the P peak observable at the K pre-edge of light-cation XANES spectra orig- inates from 1s to 3s-like transitions allowed by sp hybridizations induced by quantum vibrations. 17,25,26 The appearance of the P pre-edge peak at the K edges of atoms in tetrahedral environments shows that a similar mechanism occurs, but some distinctions are observed between the 4-and 6-fold cases. Fig. 2 shows that at the K edge of 4-fold coordinated atoms, the A peak energy position is systematically located at lower energies than for 6-fold coordinated cations. This well-known phenomenon is commonly used to identify chemical environments in unknown species. [83][84][85] The P pre-edge peak is less discernable in tetrahedral symmetry given that the P-A energy distance is only about 1.8 eV while in octahedral symmetry it is about 2.5 eV. The experimental P energy variations as a function of temperature are displayed in Fig. 6. When the probed atom lies in an octahedral site, the P peak shifts towards lower energies are larger than in tetrahedral sites. These shifts can be related to variations of elec- tronic band gaps with temperature. Using optical measurements, the electronic band gap was found to decrease by 0.91 eV in MgO, 1.0 eV in Al 2 O 3 and only 0.78 eV in MgAl 2 O 4 , where cations are both 4 and 6-fold coordinated. [86][87][88]

Quadrupolar coupling constant in temperature

In quadrupolar nuclei, quadrupolar interaction between Q nuclear quadrupole moment and the electric-field gradient at the nucleus generated by the other charges in the system must be taken into account. Hence, there is much structural information to be obtained from the C Q quadrupolar coupling parameters. The computed C Q for quadrupolar nuclei ( 25 Mg, 27 Al and 17 O) are reported in Fig. 7. In the equilibrium configuration of MgAl 2 O 4 and MgO, the 25 Mg C Q vanishes due to symmetry arguments. 89 In MgO, the dynamic disorder induced by quantum thermal vibrations does not produce any quadrupolar coupling, whereas in MgAl 2 O 4 the C Q varies with temperature, as observed for all other nuclei. This observation provides an explanation of the overestimation of the 17 O-C Q in equilibrium calculations. 90 The temperature-dependence of quadrupole resonance frequencies was investigated in the 1950s by nuclear quadrupolar resonance measurements of 35 Cl in KClO 3 and 63 Cu in Cu 2 O. 91 The observed trends were explained by a dependence to the volume variation and strain deformations induced by thermal vibrations. By describing vibrations at the quantum harmonic level with analytical expressions, Wang 92 showed that when the vibration amplitude increases, the quadrupolar coupling decreases. However, the present calculations prove that theses trends are not systematic. Moreover, the computation of the quadrupolar coupling of deuterium in hydrogen-bounded compounds using Car-Parrinello molecular dynamics 93 demonstrated that, in some cases, the unexpected augmentation of the quadrupolar coupling arises from anharmonic vibrations. 68

Conclusions

The combination of experimental and theoretical investigations permits to investigate the effect of temperature on NMR and XANES spectra of light-element oxides. The presence of the preedge peak in the K-edge XANES spectra and the variations of NMR resonance values are relevant signatures of the influence of quantum thermal vibrations. These observations result from both contributions of thermal expansion and quantum nuclear vibrations. In both cases, the experiment-calculation agreement is very good.

In NMR, linear extrapolations to 0 K are inappropriate to predict temperature-dependent NMR parameters. Thus, thermal effect must be considered using a relevant theoretical methodology even at low temperatures. This is a crucial issue as with the extension of variable-temperature XANES and NMR measurements, such as in the field of catalyst, it is mandatory to discriminate features coming from thermal vibration effects than from the reaction itself.

In XANES, an unexpected pre-edge peak is observed at the K edge of light cations. This feature originates from electronic transitions allowed by sp hybridizations induced by quantum vibrations. The presence of such feature starting from 0 K high-lights the importance of quantum vibration effects in XANES spectroscopy.

The influence of the local symmetry of the cation on the occurrence of thermal effects is brought to light. In both techniques, the impact of vibrations is more important when the probed atom lies in an octahedral environment. In XANES, the additional pre-edge features and energy shifts are stronger and in NMR, the chemical shifts deviate greatly from the thermal expansion scheme in 6-fold coordinated compounds. On the other hand, experimental data are dominated by lattice thermal expansion in tetrahedral environments.

Further experimental measurements are expected to assess the NMR and nuclear quadrupolar resonance predictive calculations provided in this paper. As it is, this methodology is perfectly suitable to investigate more complicated systems such as zeolites or hybrid materials, of interest for physicists, chemists and Earth scientists.

Fig. 2 Fig. 3

 23 Fig. 2 Experimental and calculated temperature-dependent XANES spectra at the K edges of Mg, Al and Si in the mineral oxides, where the probed cation is either 6 (leftside) or 4-fold coordinated (rightside) to oxygen. The isotropic spectra σ iso are shown for MgO, stishovite, MgAl 2 O 4 and α-AlPO 4 . When available, polarized spectra, σ ⊥ and σ , are displayed. The vertical dashed lines point to P and A peak energy positions.

4 Fig. 5

 45 Fig. 5 Calculated temperature-dependent NMR chemical shifts of 17 O in oxides. Full and empty symbols dedicates to the QHA and TEO calculations, respectively.

Fig. 6

 6 Fig.6 Experimental energy positions of the P pre-edge peak as a function of temperature. The energy positions are given with respect to the A peak position at 300 K.

Fig. 7

 7 Fig. 7 Temperature dependence of C Q for 25 Mg, 27 Al and 17 O. Red triangles represent tetrahedral environments. Dashed lines are displayed as a guide for eyes.

Table 1

 1 List of the studied compounds and temperature-dependent diffraction data references. The coordination number of the probed atom is indicated between parentheses the generation of a set of various random nuclear configurations according to quantum-statistical mechanics rules (see Sec. 2.1). XANES spectra and NMR parameters are then calculated and averaged on these configurations (see Sec. 2.2 and 2.

Mineral (Formula)

Sample type Probed atom Refs. Periclase (MgO) single crystal Mg (6) 47 Spinel (MgAl 2 O 4 ) powder Mg (4), Al (6) 48,49 Corundum (α-Al 2 O 3 ) single crystal Al (6) 49,50 Berlinite (α-AlPO 4 ) powder Al (4) 51 Stishovite (SiO 2 ) polycrystal Si (6) 52 α-Quartz (SiO 2 ) single crystal Si (4) 53,54 structure, allowing

Acknowledgements

This work was supported by French state funds managed by the ANR within the Investissements d'Avenir programme under reference ANR-11-IDEX-0004-02, and more specifically within the framework of the Cluster of Excellence MATISSE led by Sorbonne Universités. Computer facilities were provided by GENCI-IDRIS (Project No. 2015100172). We acknowledge SOLEIL for provision of synchrotron radiation facilities and we would like to thank the staff of the LUCIA beamline (Proposal 20141057) and "IR RMN THC FR 3050". The authors thank L. Verger for synthesizing the spinel powder sample, A. Shatskiy for the high-pressure synthesis of the stishovite sample and E. Balan for fruitful discussions.


Gael Le Douaron

Laurent Ferrié

Julia E Sepulveda-Diaz

Blandine Séon-Méniel

Rita Raisman-Vozari

Patrick P Michel

Bruno Figadère

Identification of a Novel 1,4,8-Triazaphenanthrene Derivative as a Neuroprotectant for Dopamine Neurons Vulnerable in Parkinson's Disease

Keywords: Suzuki coupling reaction, primary neuronal cultures, neurodegenerative diseases, phenotypic screening, medicinal chemistry

Parkinson's disease (PD) is a chronic degenerative disorder characterized by typical motor symptoms caused by the death of dopamine (DA) neurons in the midbrain and ensuing shortage of DA in the striatum, at the level of nerve terminals. No curative treatment is presently available for PD in clinical practice. In our search for neuroprotectants in PD, we generated new 1,4,8-triazaphenanthrenes by combining 6-endo-dig-cycloisomerisation of propargylquinoxalines and Suzuki or Sonogashira cross-coupling reactions. Neuroprotection assessment of newly synthesized 1,4,8-triazaphenanthrenes in a PD cellular model resulted in the discovery of a new hit compound PPQ (5m). Neuroprotection by 5m was concentrationdependent and the result of a combined effect on intracellular calcium release channels and astroglial cells. Of interest, 5m also counteracted DA cell loss in a mouse model of PD, making this molecule a promising candidate for PD treatment.

INTRODUCTION

Parkinson's disease (PD) is a chronic neurodegenerative disorder of aging, which is frequent and characterized by a diminished ability for voluntary movements. [START_REF] Michel | Understanding Dopaminergic Cell Death Pathways in Parkinson Disease[END_REF] Motor symptoms are caused by the progressive disappearance of substantia nigra (SN) dopamine (DA) neurons and the ensuing reduction of DA levels in the striatum, the projection area of these neurons. [START_REF] Michel | Understanding Dopaminergic Cell Death Pathways in Parkinson Disease[END_REF] The nature of the molecular mechanisms causing disease onset and progression is not yet fully understood, but a number of hypotheses, which are not mutually exclusive, currently prevail. Most notably, deficits in mitochondrial function, disturbances in protein or calcium homeostasis, oxidative stress and neuroinflammation are pathological events likely to be involved in PD neurodegeneration. [START_REF] Michel | Understanding Dopaminergic Cell Death Pathways in Parkinson Disease[END_REF][START_REF] Kincses | Pharmacological Therapy in Parkinson's Disease: Focus on Neuroprotection[END_REF]3,[START_REF] Michel | Role of activity-dependent mechanisms in the control of dopaminergic neuron survival[END_REF] At the clinical level, treatments currently available are essentially symptomatic, as they restore motor control by transiently enhancing DA levels in the striatum. [START_REF] Connolly | Pharmacological treatment of Parkinson disease: a review[END_REF] Thus, neuroprotective agents, with the potential to halt or delay disease progression, are urgently required. A number of molecules developed for this purpose have demonstrated great potential in preclinical models, but none of them has led to a therapy for PD patients until now. [START_REF] Athauda | The ongoing pursuit of neuroprotective therapies in Parkinson disease[END_REF][START_REF] Stocchi | Obstacles to the development of a neuroprotective therapy for Parkinson's disease[END_REF] Thus, the quest for neuroprotective drugs is still ongoing. [START_REF] Zhang | Emerging targets and new small molecule therapies in Parkinson's disease treatment[END_REF] To this aim, we have synthesized small non-peptide compounds that were then selected for their neuroprotective/neuritogenic potential through structure-activity relationship (SAR) studies. 9,[START_REF] Schmidt | Tryptamine-derived alkaloids from Annonaceae exerting neurotrophin-like properties on primary dopaminergic neurons[END_REF] We initially designed hybrids of melatonin and long chain fatty acids, i.e. compounds related to natural products reported previously for having antioxidative, neuroprotective or neurotrophic effects. [START_REF] Mayo | Melatonin and Parkinson's disease[END_REF][START_REF] Cao | Effects of docosahexaenoic acid on the survival and neurite outgrowth of rat cortical neurons in primary cultures[END_REF] More specifically, we synthesized N-acylated tryptamine derivatives and screened these compounds using a cell culture system that models DA cell degeneration in PD. This resulted in the discovery of compounds having interesting anti-oxidative and neuroprotective/neuritogenic properties with, however, poor blood-brain barrier (BBB) permeability. [START_REF] Schmidt | Tryptamine-derived alkaloids from Annonaceae exerting neurotrophin-like properties on primary dopaminergic neurons[END_REF] To obtain compounds with better BBB permeability, we synthesized 6-aminoquinoxaline derivatives. This led us to find a hit compound MPAQ which was neuroprotective for DA neurons and exerted neuritogenic effects on these neurons as well. 13 This compound operated indirectly through a repressive effect on astroglial cells. 13 New 6-aminoquinoxalines and their N-propargyl derivatives were then designed and synthesized, and the most active one PAQ was selected on the basis of its protective activity in vitro. PAQ appeared to work via a direct effect on DA neurons through a mechanism that partly relied on ryanodine receptor activation. Finally, we also established that when given orally, PAQ was providing protection to DA neurons and their striatal terminals in a rodent model of PD. [START_REF] Le Douaron | New 6-aminoquinoxaline derivatives with neuroprotective effects on dopaminergic neurons in cellular and animal Parkinson disease models[END_REF] At this stage, we decided to explore the neuroprotective potential of 1,4,8triazaphenanthrenes derived synthetically from 6-aminoquinoxalines. Indeed, we recently reported that the 6-endo-dig cycloisomerization of N-propargyl-6-aminoquinoxalines leads to the expected 1,4,8-triazaphenanthrenes in a highly regioselective manner. [START_REF] Vallerotto | 6-Endo-digcycloisomerisation of N-propargylaminoquinoxalines: a new route to 1,4,8triazaphenanthrenes[END_REF]

RESULTS & DISCUSSION

Several 2,3-substituted 1,4,8-triazaphenanthrenes 3a-h were prepared from the amino quinoxalines 1a-h that were described by us previously (Scheme 1). [START_REF] Le Douaron | New 6-aminoquinoxaline derivatives with neuroprotective effects on dopaminergic neurons in cellular and animal Parkinson disease models[END_REF] We reported before that the phenyl ring located at position 3 of the quinoxaline core was essential for neuroprotection by 6-aminoquinoxalines 13 and that the lack of substituent at position 2 was apparently crucial to optimize this activity. [START_REF] Le Douaron | New 6-aminoquinoxaline derivatives with neuroprotective effects on dopaminergic neurons in cellular and animal Parkinson disease models[END_REF] Therefore, we designed a specific convergent route for the preparation of 3-aryl-1,4,8-triazaphenanthrenes 5c, 5f, 5g, 5k-o, based on a palladiumcatalyzed Suzuki coupling reaction of aryl-boronic acids or boronate esters with 3-chloro-1,4,8-triazaphenanthrene 5i. Compound 5j was prepared from a Sonogashira cross-coupling with TMS-acetylene followed by methanolysis (Scheme 1). All synthesized 1,4,8-triazaphenanthrenes (for 3a-h, see ref. 15, and see Scheme 1) were then assessed for neuroprotection using a PD culture model that we used before and in which DA neurons degenerate selectively and spontaneously as a consequence of a mechanism involving proliferating astrocytes and calcium dyshomeostasis. [START_REF] Le Douaron | New 6-aminoquinoxaline derivatives with neuroprotective effects on dopaminergic neurons in cellular and animal Parkinson disease models[END_REF][START_REF] Rousseau | The iron-binding protein lactoferrin protects vulnerable dopamine neurons from degeneration by preserving mitochondrial calcium homeostasis[END_REF][START_REF] Toulorge | Neuroprotection of midbrain dopamine neurons by nicotine is gated by cytoplasmic Ca2+[END_REF] Briefly, cultures were exposed or not for 10 days to various test molecules and neuronal rescue was assessed by counting the number of tyrosine hydroxylase (TH) positive neurons surviving at that time.

In agreement with earlier studies, we found that more than 60% of TH + neurons initially present in these cultures had died over a 10 day period in the absence of any treatment. [START_REF] Rousseau | The iron-binding protein lactoferrin protects vulnerable dopamine neurons from degeneration by preserving mitochondrial calcium homeostasis[END_REF][START_REF] Toulorge | Neuroprotection of midbrain dopamine neurons by nicotine is gated by cytoplasmic Ca2+[END_REF] N 6 , 2'-O-dibutyryladenosine 3':5' cyclic monophosphate (dbcAMP), a lipophilic derivative of cAMP, used as reference neuroprotectant in this model system, [START_REF] Michel | Chronic activation of the cyclic AMP signaling pathway promotes development and long-term survival of mesencephalic dopaminergic neurons[END_REF] provided strong protection to vulnerable DA neurons (Figure 1). Among a series of 19 1,4,8-triazaphenanthrene derivatives, 8 of them led to significant rescue of DA neurons at 10 µM (3g, 5c, 5f, 5g, 5k, 5m, 5n, 5o). In particular, the protection afforded by compounds 3g, 5c, 5g, 5k, 5m, 5n, 5o at 10 µM was either greater than, or at least comparable to the first generation molecule PAQ, at the same concentration (see compounds whose protective activity is above red line in Figure 1). The efficacy of 5g, 5k, 5m, 5n and 5o at 10 µM was not statistically different from the reference compound dbcAMP, used at 1 mM.

Of interest, almost all molecules with a 2-hydrogen-3-aryl-1,4,8-triazaphenanthrene core structure (5c, 5f, 5g, 5k, 5m, 5n, 5o) afforded neuroprotection to DA neurons, indicating that lack of substitution at position 2 of the triazaphenanthrene group was beneficial for this activity. The only 2-hydrogen-3-aryl-1,4,8-triazaphenanthrene, which was not neuroprotective at 10 µM, i.e., compound 5l, had, however, a substituent in position 3 susceptible to sterically hinder the 2-position. At this stage, we selected the 5 most active 1,4,8-triazaphenanthrene derivatives (5g, 5k, 5m, 5n, 5o) whose protective activity at 10 μM was in the range of that of dbcAMP at 1 mM (5g: 268.6 ± 9.8 %; 5k: 275.5 ± 10.2 %; 5m: 257.7 ± 10.5 %; 5n: 281.2 ± 12.6 % ; 5o: 273.5 ± 12.7 %; dbcAMP: 283.2 ± 11.7 % TH + neurons). To refine the screening, the protective activity of these 5 compounds was assessed at a 10-fold lower concentration, i.e., 1 μM, using the same experimental paradigm (see Figure 2). All test compounds remained neuroprotective at 1 µM, although their efficacy was lower than at 10 µM. Compound 5m (also named PPQ) appeared to be the most effective of these compounds with a EC50 around 1.1 µM and it possessed similar efficacy as PAQ, at 10 µM (compound 5m 1 µM: 193.4 ± 8.6 % vs PAQ 10 µM: 182.3 ± 7.4 %). Interestingly compound 5m is also structurally closest to PAQ. Figure 3 further illustrates the neuroprotective activity of compound 5m on DA neurons. Note that the fiber network of TH + neurons appeared also to be denser in 5m (10 µM)-treated cultures, suggesting a possible trophic effect of this compound on rescued DA neurons. Scale bar = 60 μm. Note that the fiber network of TH + neurons appears to be denser in 5m (10 µM)-treated cultures.

In the cellular model we used here for screening purposes, we previously reported that the rescue of DA neurons could possibly result from the inhibition of proliferating glial cells, in particular, astrocytes and their progenitor cells, which represent the vast majority of cells that proliferate in present experimental conditions. [START_REF] Michel | Rescue of mesencephalic dopamine neurons by anticancer drug cytosine arabinoside[END_REF][START_REF] Mourlevat | Prevention of dopaminergic neuronal death by cyclic AMP in mixed neuronal/Glial mesencephalic cultures requires the repression of presumptive astrocytes[END_REF] For instance, we have shown that the reference neuroprotectant dbcAMP and also one of the previous hit compounds MPAQ were protective via a mechanism of this kind. 13,[START_REF] Mourlevat | Prevention of dopaminergic neuronal death by cyclic AMP in mixed neuronal/Glial mesencephalic cultures requires the repression of presumptive astrocytes[END_REF] For that reason, we wished to determine whether 5m had a similar effect. Thus, we used a DNA proliferation assay that consists in measuring the incorporation of thymidine, [methyl-3 H], a tritiated analog of the nucleoside thymidine. [START_REF] Mourlevat | Prevention of dopaminergic neuronal death by cyclic AMP in mixed neuronal/Glial mesencephalic cultures requires the repression of presumptive astrocytes[END_REF] We established that 5m had no impact on glial cell proliferation, at 1 µM, a concentration already providing significant protection to DA neurons. However, glial cell proliferation was significantly reduced at concentrations of 5m that provide better protection to DA neurons, i.e., 3 and 10 µM. This suggests that the effects of 5m were probably partly indirect in this range of concentrations.

To better characterize the survival promoting action of 5m towards DA neurons, we tested whether compounds that block endoplasmic reticulum (ER) calcium release channels had an impact on the protective action of this compound. The rationale was that previous studies have shown that intracellular calcium pools released from the ER are crucially involved in the control of DA cell survival. [START_REF] Michel | Understanding Dopaminergic Cell Death Pathways in Parkinson Disease[END_REF][START_REF] Rousseau | The iron-binding protein lactoferrin protects vulnerable dopamine neurons from degeneration by preserving mitochondrial calcium homeostasis[END_REF] Consistent with this hypothesis, we established that the protective effect of 5m (10 µM) was partially antagonized by dantrolene (20 µM), a skeletal muscle relaxant drug that operates as a blocker of ER ryanodine receptor channels. This is reminiscent of earlier findings showing that neuroprotection by our previous hit compound PAQ, which is structurally related to 5m, was also partially reversed by dantrolene. [START_REF] Le Douaron | New 6-aminoquinoxaline derivatives with neuroprotective effects on dopaminergic neurons in cellular and animal Parkinson disease models[END_REF] Remarkably, 2-aminoethoxydiphenyl borate (2-APB; 10 µM), a blocker of IP3 receptors, another type of calcium release channel located on the ER, was more effective in blocking the protective effects of 5m. Overall, these data indicate that 5m-mediated neuroprotection required intracellular calcium mobilization. We then studied early pharmacokinetics of 5m. For that, compound 5m was solubilized at 0.5 mg/mL using a 0.9% saline solution containing 10% of the formulation vehicle, cremophore EL. Then the preparation was injected i.v. at a dose of 1 mg/kg in two groups of 3 CD-1 mice, which were sacrificed either 5 min or 30 min after the injection. The purpose of the present protocol was two-fold: (i) to evaluate BBB permeability to 5m and (ii) to estimate this compound lifetime in the brain and plasma. After euthanasia, aliquots of plasma and brain extracts were purified and analysed by HPLC. Data summarized in Figure 5 show (i) that compound 5m crosses the BBB, its accumulation peaking 5 min after the injection and (ii) that it is also rapidly eliminated from the brain over a period of time of 30 min. A similar elimination profile was observed in the plasma. In a next step, we used a mouse model of PD where DA cell death is induced by repeated injections of the neurotoxin MPTP. More specifically, C57BL/6 mice were given MPTP in a sub-chronic manner so as to generate a progressive loss of SN DA neurons and cause concurrent deficits in DA and its metabolites in the striatum. [START_REF] Bové | Toxin-induced models of Parkinson's disease[END_REF][START_REF] Smeyne | The MPTP model of Parkinson's disease[END_REF][START_REF] Kuroiwa | Biochemical alterations of the striatum in an MPTP-treated mouse model of Parkinson's disease[END_REF] This experimental paradigm is of interest as it is thought to replicate some of the key events that contribute to PD progression. [START_REF] Bové | Toxin-induced models of Parkinson's disease[END_REF][START_REF] Smeyne | The MPTP model of Parkinson's disease[END_REF] SN DA neurons were identified by immunohistochemistry of the TH enzyme, using 3,3diaminobenzidine (DAB) as a chromogen. As shown earlier, [START_REF] Kuroiwa | Biochemical alterations of the striatum in an MPTP-treated mouse model of Parkinson's disease[END_REF][START_REF] Dehmer | Protection by pioglitazone in the MPTP model of Parkinson's disease correlates with IkBα induction and block of NFB and iNOS activation[END_REF] the sub-chronic administration of MPTP in C57BL/6 mice induced a substantial loss (-39%) of TH + neurons.

After 11 days of a treatment regimen with 5m, at a dose of 2x25 mg/kg/day (per os), we observed an almost total rescue of TH + neurons as the survival rate of these neurons in MPTP/5m-treated mice was estimated to be 93 ± 4% of controls vs 61 ± 5% in MPTP/vehicle-treated mice. At a higher dosage (2x50 mg/kg/day), 5m was slightly less effective as DA cell survival reached 81 ± 4% of controls. These results suggested that a dose of 2x25 mg/kg/day (per os) was sufficient to combat efficiently the neurotoxic action of MPTP for DA neurons. The neuroprotective effect of 5m for SN DA neurons in MPTPtreated mice is described in detail in Figure 6.

Note that we also established that 5m was a poor inhibitor of monoamine oxidase-B (MAO-B), an astrocytic enzyme that transforms MPTP into a toxic metabolite MPP + , which kills DA neurons, selectively. [START_REF] Bové | Toxin-induced models of Parkinson's disease[END_REF][START_REF] Smeyne | The MPTP model of Parkinson's disease[END_REF] More precisely, IC50 values for selegiline (a prototypical inhibitor for MAO-B) and for 5m were found to be 1.86 and 116 µM, respectively. This means that neuroprotection by 5m against MPTP was most probably not caused by a reduction in the availability of MPP + for target neurons. Finally, we also assessed DA and its metabolites in the striatum of control and MPTPtreated mice (Figure 7). In mice receiving MPTP and the vehicle, DA levels were reduced to 28% of control values. Following 11 days of treatment with 5m (2x25 mg/kg/day, p.o.), DA levels were substantially restored in the striatum of mice receiving MPTP (46 ± 3 %). In the same group of mice, the levels of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were found to be slightly increased, although not significantly in the case of DOPAC, in comparison to mice receiving MPTP and the vehicle.

Note, however that 5m notably reduced the rise of 3,4-dihydroxyphenylacetic acid (DOPAC) + homovanillic acid (HVA) / DA ratios. This means that 5m limited the increase in DA turnover produced by MPTP under degenerative conditions, suggesting that this compound also partially preserved the function of the rescued neurons (see Figure 7). At the highest dosage (2x50 mg/kg/day), 5m produced comparable effects on striatal DA levels and DA turnover. To recapitulate, we synthesized 1,4,8-triazaphenanthrene derivatives through an effective strategy depending on palladium-catalyzed Suzuki cross-coupling of arylboronic acids with 3chloro-1,4,8-triazaphenanthrene. The desired compounds were obtained in moderate to good yields. The newly synthesized compounds were evaluated using a cell culture system that models DA cell death in PD. [START_REF] Rousseau | The iron-binding protein lactoferrin protects vulnerable dopamine neurons from degeneration by preserving mitochondrial calcium homeostasis[END_REF][START_REF] Toulorge | KATP channel blockade protects midbrain dopamine neurons by repressing a glia-to-neuron signaling cascade that ultimately disrupts mitochondrial calcium homeostasis[END_REF] A number of molecules provided efficient protection to DA neurons. Note, however, that among 1,4,8-triazaphenanthrenes, the presence of a methyl group at position 2 of the heterocyclic scaffold gave rise to compounds that were much less active, compared to the unsubstituted ones. Overall, 5 compounds exerted strong neuroprotective effects at 10 µM, and lower but still significant effects at 1 µM, (5g, 5k, 5m, 5n, 5o). Because 5m retained better neuroprotective efficacy in the low concentration range (i.e., at 1 µM) than its congeners (5g, 5k, 5n, 5o), we selected 5m for further investigations.

Since in the present culture model, dividing astroglial cells exert deleterious effects onto DA neurons, [START_REF] Rousseau | The iron-binding protein lactoferrin protects vulnerable dopamine neurons from degeneration by preserving mitochondrial calcium homeostasis[END_REF][START_REF] Toulorge | KATP channel blockade protects midbrain dopamine neurons by repressing a glia-to-neuron signaling cascade that ultimately disrupts mitochondrial calcium homeostasis[END_REF] we evaluated the potential of 5m to act via a repressive effect on these cells. 5m had no impact on glial cell proliferation at 1 µM, a low effective concentration of this compound. However, glial cell proliferation was moderately but significantly reduced when 5m was used at concentrations of 3 and 10 µM, which provide better protection to DA neurons. Thus, we may assume that neuroprotection by 5m resulted from a direct effect on DA neurons, at low effective concentrations of this compound whereas at higher concentrations, DA cell rescue was improved through the repression of dividing astroglial cells.

Previous studies also suggested that intracellular calcium stored in the ER may enable DA neurons to control their own survival. [START_REF] Michel | Understanding Dopaminergic Cell Death Pathways in Parkinson Disease[END_REF][START_REF] Toulorge | KATP channel blockade protects midbrain dopamine neurons by repressing a glia-to-neuron signaling cascade that ultimately disrupts mitochondrial calcium homeostasis[END_REF][START_REF] Guerreiro | the primary metabolite of caffeine, provides protection against dopaminergic cell death via stimulation of ryanodine receptor channels[END_REF] In relation to that, we established that the blockade of ER calcium release channels caused a significant reduction in 5m-mediated DA cell rescue.

In this context, the antagonist of IP3 receptors, 2-APB was more effective than dantrolene a blocker of ryanodine receptors channels. It appears, therefore, that ER calcium mobilization was involved in 5m-mediated neuroprotection. Whether the calcium release effect of 5m was related in some way to that exerted on proliferating astrocytes remains, however, to be determined.

Through pharmacokinetics studies, we showed that 5m reaches the brain after i.v.

administration (1 mg/kg) to mice. Even if eliminated relatively rapidly from the brain, the strong neuroprotective potential of 5m observed in vitro led us to further investigate the efficacy of this compound after oral administration in an animal model of PD.

More specifically, we studied the in vivo effects of 5m in the MPTP mouse model of PD. [START_REF] Bové | Toxin-induced models of Parkinson's disease[END_REF][START_REF] Smeyne | The MPTP model of Parkinson's disease[END_REF] When C57BL/6 mice were given MPTP according to a sub-chronic regimen of administration, we found that the loss nigrostriatal DA neurons was quite substantial, at the time of sacrifice, i.e., 3 days after the last MPTP injection. The loss of DA cell bodies in the SN resulted in a profound reduction of DA levels in the striatum and in a significant increase in DA turnover as revealed by higher (DOPAC+HVA)/DA ratios. After forced oral feeding of mice twice daily with 5m for 11 days (starting 3 days before and finishing 3 days after initiation and termination of MPTP treatment, respectively), we observed that SN DA cell bodies were robustly protected from degeneration. Note that the integrity of the dendritic network was also well preserved by 5m in these neurons. The treatment with 5m only partially restored DA levels in the striatum and limited the rise of the (DOPAC+HVA)/DA ratios, at both dosage regimen (2x25 and 2x50 mg/kg/day), which suggests that the function of rescued DA neurons was partly preserved under these conditions of treatment. The observation that 5m was more efficacious to protect DA cell bodies in the SN than to preserve DA levels in the striatum may be attributable to the fact that axonal nerve endings are generally more vulnerable than corresponding cell bodies in the process of neurodegeneration. [START_REF] Lassus | Alterations of mitochondrial dynamics allow retrograde propagation of locally initiated axonal insults[END_REF] Because the bioconversion of MPTP into its toxic metabolite MPP + occurs through the enzyme MAO-B located within brain astrocytes, [START_REF] Bové | Toxin-induced models of Parkinson's disease[END_REF][START_REF] Smeyne | The MPTP model of Parkinson's disease[END_REF] we wished to determine whether MAO-B inhibition could account for 5m-mediated neuroprotection against MPTP. In fact, 5m proved to be a poor MAO-B inhibitor in contrast to the reference molecule selegiline, thus indicating that 5m was unlikely to be protective by decreasing MPP + availability for DA neurons in MPTP-intoxicated mice. This confirmed the view that 5m was acting as a true neuroprotectant for vulnerable DA neurons.

In conclusion, we have synthesized 1,4,8-triazaphenanthrene derivatives, and after evaluating their effects in a cell culture system of spontaneous and selective DA cell death, we selected the most active of them, 5m. This compound seemed to operate through a dual mechanism involving intracellular calcium release channels and astroglial cells. Besides, we established that an oral treatment regimen with 5m was effective in reducing lesions of the nigrostriatal pathway in a mouse model of PD, thus confirming the ability of 5m to act as a neuroprotectant for DA neurons. Additional preclinical studies will be needed, however, to confirm that 5m is of potential interest for PD treatment.

EXPERIMENTAL SECTION

Chemistry. General. Tetrahydrofuran was distilled over sodium/benzophenone and under nitrogen atmosphere prior to use. Reactions involving moisture-sensitive reactants were performed under nitrogen atmosphere using oven-dried glassware. Routine monitoring of reactions was performed by thin-layer chromatography, using Riedel-deHaën, S, 0.063 mm, 0.032 silica gel plates, using a UV-lamp at 254 nm to see UV active compounds.

Complementary TLC analysis was performed with a dying reagent by dipping the plate into a 10 % sulfomolybdic acid solution or o-vanillin in EtOH/H2SO4 (95:5) and then heated to stain or dipped a few min in a mixture of silica and iodide. Silica gel flash chromatography was performed using Riedel-deHaën, S, 0.063 mm, 0.032 silica under moderate pressure with the appropriate solvent giving us a migrating front (Rf) in the range of 0.2-0.

Compound 5i

To a mixture of -6-aminoquinoxaline 4 (6.0g, 33.4 mmol, 1 equiv.), K2CO3 (4.6g, 33.4 mmol, 1 equiv.) in anhydrous DMF (20 mL) under inert atmosphere was added propargyl bromide (5.6 mL, 1.5 equiv., 80% in toluene) and the reaction mixture was heated at 100°C for 24 h.

After cooling to room temperature, the mixture was diluted with water, extracted with ethyl acetate, washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The residue was purified by silica gel flash chromatography (CH2Cl2: EtOAc, 90:10 to 80:20) to afford mono-propargyl derivative (2.7 g, 12.4 mmol, 37%) as a brownish yellow solid.

To a solution N-propargyl derivative (1.28 g, 5.89 mmol, 1 equiv.) in DMSO (20 mL) was added CuCl (583 mg, 5.89 mmol, 1 equiv.) and the reaction mixture was heated at 120 °C for 3h. The reaction mixture was quenched at rt with 10% NH3 in water (50 mL), stirred 10 min, filtered over celite®, washed with EtOAc. The aqueous layer was extracted with EtOAc and the combined organics phases were washed with brine (2x40 mL), dried over MgSO4, filtered and concentrated. Purification on silica gel chromatography, (CH2Cl2: EtOAc, 80:20) afforded compound 5i, 1.03g, 74%). [START_REF] Michel | Understanding Dopaminergic Cell Death Pathways in Parkinson Disease[END_REF] H NMR (300 MHz, CDCl3) δ 9.40 (ddd, J = 8.3, 1.7, 0.6 Hz, 1H), 9.12 (dd, J = 4.4, 1.7 Hz, 1H), 8.91 (s, 1H), 8.31 (dd, J = 9.3, 0.6 Hz, 1H), 8.20 (d, J = 9.3 Hz, 1H), 7.69 (dd, J = 8.3, 4.4 Hz, 1H). 13 C NMR (75 MHz, CDCl3) δ 152. 52, 149.36, 147.42, 144.95, 140.28, 140.13, 133.10, 132.92, 129.73, 125.43, 122.53. IR (cm -1 ): 3049, 1493, 1207, 1146, 900, 841. General procedure for the synthesis of compounds 5c, 5g, 5k-o. To a suspension of compound 5i (1 equiv.), K2CO3 (2.6 equiv.), boronic acid or MIDA boronate (1.3 equiv.), PdCl2(PPh3)2 (0.03 equiv.) in a dioxane : H2O mixture (8 : 2 mL/mmol of 5i) is refluxed for 1h. After cooling, the reaction mixture was extracted three times with EtOAc, washed once with 10% aqueous K2CO3. The organic solution was dried over MgSO4, filtered and concentrated. The residue was then purified on a silica gel column to give compound 5c, 5g or 5k-o as a beige solid.

Compound 5c (155.2 mg, 96%). [START_REF] Michel | Understanding Dopaminergic Cell Death Pathways in Parkinson Disease[END_REF] H NMR (300 MHz, CDCl3): δ 9.59 (ddd, J = 8.3, 1.7, 0.5 Hz, 1H), 9.39 (s, 1H), 9.12 (dd, J = 4.4, 1.7 Hz, 1H), 8.29 (dd, J = 9.4, 0.5 Hz, 1H), 8.23 (d, J 1H), 8.20 (d, J = 9.4 Hz, 1H), 7.70 (dd, J = 8.3, 4.3 Hz, 1H), 3.50 (s, 1H). 13 C NMR (75 MHz, CDCl3) δ ppm : 152. 3, 149.3, 147.7, 140.8, 140.2, 137.4, 133.9, 133.0, 130.1, 125.9, 122.6, 81.6, 81.0. HRMS (ESI), calcd m/z for [M+H] + C13H8N3: 206.0718, found: 276.0715.

BIOLOGICAL ASSAYS

Ethic statement. Animals were housed, handled, and cared for in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication no. 85-23, revised 1996) and with the European Union Council Directives (2010/63/EU). The protocol was approved by the Committee on the Ethics of Animal Experiments Charles Darwin n°5 (reference number N° 5012099-A). Animals were manipulated according to protocols that have been validated by the scientific community and all efforts were made to minimize suffering. Animals were housed and maintained at a constant temperature (22 ± 1 °C) and in a humidity-controlled (55 ± 20 %) environment. A 12 h/12 h light-dark cycle was kept constant, with lights turned on at 08:00 a.m. During the acclimatization period (3 days) and throughout all the study, the animals had free access to food and water.

Midbrain cultures. At gestational day 15.5, embryos were collected from the uterine horns of Sprague-Dawley female rats (Janvier LABS, Le Genest St. Isle, France), which had been previously deeply anesthetized with pentobarbital and sacrificed by cervical dislocation. The ventral mesencephalon was dissected out and cultures were prepared using N5 medium and 1 mg/mL polyethylenimine as a coating, as previously described. 13,[START_REF] Michel | Chronic activation of the cyclic AMP signaling pathway promotes development and long-term survival of mesencephalic dopaminergic neurons[END_REF] Under these conditions, the loss of DA neurons is spontaneous and progressive as a function of time. [START_REF] Rousseau | The iron-binding protein lactoferrin protects vulnerable dopamine neurons from degeneration by preserving mitochondrial calcium homeostasis[END_REF][START_REF] Toulorge | Neuroprotection of midbrain dopamine neurons by nicotine is gated by cytoplasmic Ca2+[END_REF] Treatments of the cultures. Test treatments were applied after culture plating and then daily after the first 3 DIV and until completion of the cultures. Treatments were renewed by exchanging 70% of the culture medium. Note that vehicles used to solubilize test compounds had no impact on DA cell survival.

Immunolabelling of TH + neurons in culture. After fixation with a 4% formaldehyde solution in Dulbecco's phosphate buffered saline medium (PBS) for 12 min and two rapid washes with PBS, the cultures were processed for TH immunodetection. Briefly, the cultures were incubated overnight at 4°C with a mouse anti-TH monoclonal antibody (1/5000 in PBS, Euromedex, Souffelweyersheim, France). Subsequent incubations were performed at rt, with a secondary anti-mouse IgG Alexa Fluor 488 (1/500 in PBS, Life Technologies, Paisley, UK). TH + cells were uniquely DA neurons in our culture conditions [START_REF] Traver | The phenotypic differentiation of locus coeruleus noradrenergic neurons mediated by brain derived neurotrophic factor is enhanced by corticotropin releasing factor through the activation of a cAMP-dependent signaling pathway[END_REF] and they represented ~2-3% of the total number of neurons present in these cultures, at the time of plating. [START_REF] Guerreiro | the primary metabolite of caffeine, provides protection against dopaminergic cell death via stimulation of ryanodine receptor channels[END_REF] In the phase of screening, image acquisition was performed with the HCI software (Hamamatsu Corp., Bridgewater, NJ) using an inverted fluorescent microscope (TE-300, Nikon, Tokyo, Japan) coupled to an ORCA-ER digital camera (Hamamatsu, Japan). DA cell survival was performed by visual counting of TH + cells at a 200x magnification using a 20x objective matched with a 10x ocular. More precisely, the number of TH + neurons was estimated after counting all visual fields distributed along the X-and Y-axes of each culture well. For post-screening experiments, TH + cell counting was performed with an Arrayscan XTi automated fluorescence microscope (ThermoScientific, Courtaboeuf, France) equipped with the HCStudio software for image acquisition and quantification (Thermoscientific). In this case, data analysis was performed over 60% of the whole surface area of each culture well.

Quantification of glial cell proliferation.

Thymidine-[methyl-3 H], a marker of DNA synthesis, was used to quantify glial cell proliferation as described previously. [START_REF] Mourlevat | Prevention of dopaminergic neuronal death by cyclic AMP in mixed neuronal/Glial mesencephalic cultures requires the repression of presumptive astrocytes[END_REF][START_REF] Toulorge | KATP channel blockade protects midbrain dopamine neurons by repressing a glia-to-neuron signaling cascade that ultimately disrupts mitochondrial calcium homeostasis[END_REF] Briefly, midbrain cultures maintained up to 7 DIV in the presence of test treatments were exposed to 1μCi of thymidine-[methyl-3H] (70-90Ci/mmol; Perkin Elmer, Waltham, MA, USA) for 2 h at 37°C, in serum-free N5 medium supplemented with 5 mM glucose. After two rapid washes, the cells were allowed to recover for an additional 2 h in the same culture medium for eliminating the fraction of radioactivity not incorporated into DNA. After a last wash with PBS, the cells were scraped off culture wells in distilled water and samples assessed by liquid scintillation spectrometry.

Subchronic MPTP mouse model of PD. C57BL/6 male mice (Janvier labs, Le Genest-Saint-Isle, France, 22-28 g) were divided into 4 groups (n=12/group) for testing the effects of compound 5m in vivo. One group received MPTP hydrochloride (20 mg/kg, i.p.; once daily) injected for 5 consecutive days 29 , a treatment, which was followed by a washout period of 3 days. Two groups received MPTP together with 5m hydrochloride, administrated per os in 1% carboxymethylcellulose and 0.5% of tween 80 (2x25 or 2x50 mg/kg/day;10 mL/kg), the administration starting 3 days before initiation of MPTP treatment and continuing until sacrifice time. The control group received vehicles used for MPTP and 5m administration and the MPTP group, the vehicle used for 5m treatment, only. Six hours after the last administration of 5m or vehicle, all mice were sacrificed by cervical dislocation. The striatums were dissected out, weighted and snap frozen for the measurement of DA and its metabolites. Tissues samples were stored at -80 °C and used for HPLC analysis of DA and its metabolites. The caudal part of the brain (containing the SN) was placed in 4% formaldehyde (in 0.1 M PBS, pH 7.4) for 5 days and then, transferred to 20% sucrose (20% in 0.1 M PBS) for cryoprotection. Tissues were stored at -80 °C until processing for TH + immunohistochemistry.

HPLC measurement of striatal DA and its metabolites. The striatums from the left hemispheres were homogenized using a Bandelin UW70 ultrasonic probe and 10 volumes (w/v) of 0.1 N perchloric acid aqueous solution. Homogenates were then centrifuged at 15,000 rpm for 7 min at 0 °C before 10 μL of supernatant (or calibration standard) was injected into an HPLC with an electrochemical detection system for the assessment of DA, DOPAC and HVA. The system consisted of a MD 150 x 3.2mm 3μm C18 column (ESA), a dual electrode 5014B micro-dialysis cell (ESA) with the electrode potentials set at -150 mV and +375 mV. DA and its metabolites were detected at the second electrode, combined with a CoulArray detector (ESA). A 5020 guard cell (ESA) with a potential of +350 mV was placed before the auto-sampler. The mobile phase contained 90 mM sodium dihydrogen phosphate, 50 mM citric acid, 1.7 mM 1-octane sulphonic acid, 50 μM EDTA, 10% acetonitrile, pH 3.0 at a flow rate of 0.5 mL/min. The calibration standards contained DA (Sigma-Aldrich, Saint Quentin Fallavier, France), DOPAC (Sigma-Aldrich, Saint Quentin Fallavier, France) or HVA (Sigma-Aldrich, Saint Quentin Fallavier, France). A calibration curve was created from a range of standards (24.4 -1562.5 ng/mL).

HPLC measurement of 5m in plasma and brain. 5m was injected i.v. at 1 mg/kg to CD1 mice (Charles River) and the levels of this compound were determined in plasma/brain using UHPLC with a triple quadrupole detection system (Shimadzu LC-MS 8030). Briefly, plasma aliquots (400 µL) were poured in acetonitrile (1 mL), stirred for 3 min and sonicated for 3 more min (ultrasonic probe) prior to centrifugation at 4 °C or 5 min, at 15,000 rpm. After removal from the skull, brains were homogenized in a saline solution (0.9 % aqueous solution, 400 µL) before being processed as plasma samples. The supernatants from brain or plasma samples or the calibration standards containing 5m, at concentrations comprised between 0.05 and 5 nM, were then injected into the UHPLC-MS/MS system for quantification.

Detection of SN TH + neurons in tissue sections. Twenty micrometer coronal sections of the mesencephalon obtained with a freezing microtome were collected in 10 regularly spaced series. All sections were stored in PBS/0.4% sodium azide solution, at 4 °C until use. They were rinsed in PBS 0.1M, treated with 0.3% H2O2/PBS for 30 min to neutralize endogenous peroxidase activity, and incubated with 5% goat serum (Sigma-Aldrich, Saint Quentin Fallavier, France) in PBS/0.15% Triton X-100 for 1 h. Sections were then incubated with a mouse anti-TH (1/500 in PBS; US Biologicals, Salem, USA) antibody for 24 hours. After washing with PBS and incubating for 1 h with a biotinylated anti-mouse antibody diluted 1/250 in PBS (Vector Laboratories, Burlingame, USA), the staining was revealed with a streptavidin-biotin horse radish peroxidase preformed complex (Vector Laboratories, Burlingame, USA) and DAB (Sigma-Aldrich, Saint Quentin Fallavier, France) as the chromogen.

Quantification of SN DA neurons in tissue sections. Quantification of DA cell loss was done stereologically on regularly spaced DAB-stained sections covering the whole mesencephalon from the rostral pole of the SN to the caudal part of the structure, as already reported. [START_REF] Höglinger | Chronic systemic complex I inhibition induces a hypokinetic multisystem degeneration in rats[END_REF] High-resolution bright-field digital images of labelled sections were acquired using a NanoZoomer Digital Scanner (Hamamatsu Photonics France, Massy, France. objective × 40) equipped with the NDP software (Hamamatsu Photonics France, Massy, France.). One SN was delineated at ×4 magnification for each section. TH + cell bodies were subsequently quantified stereologically in magnified images (×20) from selected regions using the ImageJ software (W.S. Rasband, ImageJ, US National Institutes of Health, Bethesda, MD). Total cell numbers were assessed by integration along the rostrocaudal extent of the SN. [START_REF] Höglinger | Chronic systemic complex I inhibition induces a hypokinetic multisystem degeneration in rats[END_REF] The quantification was performed by an experimenter blinded to the treatment groups. The total number of SN TH + neurons in one hemisphere varied from 4850 to 5535 in saline-treated mice.

Assessment of MAO-B inhibition.

The potential of 5m to inhibit MAO-B activity, was assessed using a modification of a protocol published by Scorza et al. [START_REF] Scorza | Monoamine oxidase inhibitory properties of some methoxylated and alkylthioamphetamine derivatives: structure-activity relationships[END_REF]

Scheme 1 .

 1 Scheme 1. Synthesis of 1,4,8-triazaphenanthrenes. a

Figure 1 .Chart 1 .

 11 Figure 1. Screening of 1,4,8-triazaphenanthrene derivatives in a cellular system that models DA cell death in PD. Number of TH + neurons surviving after 10 days in vitro (DIV) in

Figure 2 .

 2 Figure 2. Evaluation of previously selected 1,4,8-triazaphenanthrene derivatives. Selected

Figure 3 .

 3 Figure 3. Neuroprotective effects of 5m onto DA neurons in midbrain cultures. Digitized images showing the rescuing effects of 5m (1 and 10 μM) and dbcAMP (1mM) for TH +

Figure 4 .

 4 Figure 4. Neuroprotection by 5m results from combined effects on astroglial cells and

Figure 5 .

 5 Figure 5. HPLC analysis of 5m in brain and plasma, after acute i.v. administration (1 mg/kg)

Figure 6 .

 6 Figure 6. Neuroprotective effects of 5m for SN DA neurons in MPTP-treated mice. (A) Number of SN TH + neurons in vehicle-treated mice and in MPTP-treated mice receiving or

Figure 7 .

 7 Figure 7. Effects of treatment regimens with 5m (2x25, 2x50 mg/kg/day; p.o.) on levels of

  3. 1 H NMR spectra were recorded as CDCl3 solutions using BRUKER AR 200 P (200 MHz), BRUKER AVANCE 300 (300 MHz) and AM-400 (400 MHz) spectrometers. Chemical shifts were expressed in parts per million (ppm, δ), downfield from tetramethylsilane (TMS) and were referenced to CHCl3 (7.26 ppm) as internal standard. 13 C NMR spectra were recorded as CDCl3 solutions with BRUKER AR 200 P (50 MHz) and AM-400 (100 MHz) spectrometers. Chemical shifts were expressed in parts per million (ppm, δ), downfield from tetramethylsilane (TMS) and were referenced to CDCl3 (77.0 ppm) as internal standard. Mass spectra (MS) were measured on a Nermag-Sidar R10-10C spectrometer with a quadrupole filter. High Resolution Mass Spectra (HRMS) were obtained from a Xevo Q-Tof Waters ® spectrometer. Compounds 1a-h, 4, 6 and PAQ were described in ref.14 whereas compounds 2a-h, 3a-h, 7 were reported in ref. 15.

  HRMS (ESI), calcd m/z for [M+H] + C11H7N3 35 Cl: 216.0329, found: 216.0323.

  Briefly, 5m or the reference MAO-B inhibitor selegiline, were added to an ammonium acetate buffer solution G.L.D. synthesized and characterized compounds and performed biological tests. L.F. synthesized and characterized compounds, and wrote the paper. J.S.-D. performed biological tests. B.S.-M. synthesized compound 5m in bulk quantity. R.R.-V. co-supervised the biological assays. P.P.M. supervised biological assays and wrote the manuscript. B.F. designed the study, supervised the project and wrote the manuscript.

ACKNOWLEDGMENTS

Motac (Manchester, UK) performed the in vivo treatments protocols with MPTP and the measurements of DA and its metabolites whereas TechMedIll (Strasbourg, France) carried out the pharmacokinetics experiments. This work benefited from equipment and services of the Celis and Histomics core facilities, at ICM. We thank Alexis Genin, director of research applications at ICM for his help in the valorisation process of our compounds. We also thank Karine Leblanc (Univ. Paris-Sud, BioCIS) who performed analyses of purity and HRMS and Dr. A. Harfouche (Univ. Paris-Sud, BioCIS) for MAO-B experiments. We are grateful to L. Acuña (ICM, Paris) for performing some of the post-screening experiments in vitro and to Prof. T. Morton (UCR, Riverside, USA) for fruitful discussions and proof reading our manuscript.

Funding

The research leading to these results was supported by program Investissements d'Avenir ANR-10-IAIHU-06 and ANR-11-INBS-0011-NeurATRIS: Translational Research Infrastructure for Biotherapies in Neurosciences, by Institut Carnot and ANR-10-labx-33.

Notes

The authors declare no competing financial interest.

) containing a crude rat brain mitochondrial extract, the MAO-B specific substrate 4dimethylaminophenethylamine, aldehyde dehydrogenase and the pyridine nucleotide cofactor β-NAD. After 30 min of incubation at 37°C, HClO4 was added to stop the reaction and the supernatant recovered by centrifugation for the dosage of the reaction product 4dimethylaminophenylacetic acid, using HPLC-HRMS.

Data analysis. All statistical analyses were performed using SigmaPlot 11.0. For in vitro experiments, experimental values expressed as mean ± S.E.M were derived from at least triplicates of 3 independent experiments. For in vivo experiments, the number of samples in each group of treatment is given in the text or the figure caption. Multiple comparisons against a single reference group were made by one-way analysis of variance (ANOVA) followed by the Dunnett's post-hoc test. When all pairwise comparisons were required, we used the Student-Newman-Keuls post-hoc test. A p value of less than 0.05 was considered significant.

ASSOCIATED CONTENTS

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI:

Copy of 1 H and 13 C NMR spectra (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Phone: +33 146835598. E-mail: bruno.figadere@u-psud.fr; Phone:+33 157274534. E-mail: patrick-pierre.michel@upmc.fr.

Author Contributions