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MD Malo Le Hanneur
email: malo.lehanneur@gmail.com

Adeline Cambon-Binder

Zoubir Belkheyar

Auteur Correspondant

C5/C6 brachial plexus palsy reconstruction using nerve surgery: long-term functional outcomes

Keywords: Intervalle), sauf indication contraire nombre de patients, lésions et/ou techniques (valeurs absolues), M: masculin, F: féminin, UBMB: ulnaire-biceps médian-brachial, MB: médian-brachial

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MANUSCRIPT

RESUME

Introduction : En cas de paralysie traumatique du plexus brachial proximal (i.e., C5/C6), une reconstruction des nerfs musculocutané, axillaire et suprascapulaire permet d'obtenir des résultats fonctionnels satisfaisants à court et moyen termes.

Hypothèse : Les résultats fonctionnels obtenus initialement se maintiennent à long terme.

Matériel et Méthodes : Une analyse rétrospective des dossiers de 29 patients présentant une paralysie C5/C6 du plexus brachial traitée par chirurgie nerveuse fut réalisée. L'amplitude et la force des mobilités actives du coude (i.e., flexion) et de l'épaule (i.e., flexion, abduction, rotations externes coude au corps et bras en abduction à 90°) furent évaluées cliniquement à l'aide d'un goniomètre et de l'échelle de cotation du British Medical Research Council, respectivement.

Résultats : Avec un recul moyen de 46 ± 15 mois (25;76), il fut observé une flexion active du coude d'une amplitude moyenne de 126 ± 18° (90;150) et d'une force moyenne de 3.8 ± 0.5 (2;4). A l'épaule, les amplitudes actives en flexion, abduction, rotations externes coude au corps et bras en abduction à 90° étaient de 109 ± 39° (0;180), 99 ± 38° (0;180°), 12 ± 34° (-80;70) et 3 ± 21° (-40;50), avec des forces moyennes évaluées à 3.6 ± 0.8 (0;4), 3.6 ± 0.8 (0;4), 3.4 ± 0.9 (0;4) et 2.5 ± 1.2 (0 ;4), respectivement.

Discussion : En cas de paralysie plexique C5/C6, la chirurgie nerveuse précoce permet d'obtenir des résultats fonctionnels satisfaisants et stables à long terme en terme de flexion du coude et d'élévation de l'épaule. Cependant, en l'absence de réinnervation du teres minor, la restauration d'une rotation externe d'épaule satisfaisante demeure un défi thérapeutique.

Niveau de preuve : IV, Séries de cas rétrospectives.

INTRODUCTION

En cas de lésion du plexus brachial par mécanisme de traction, les déficits neurologiques post-traumatiques sont limités aux territoires des deux racines cervicales les plus proximales, C5 et C6, dans environ 15% des cas [START_REF] Kim | Mechanisms of injury in operative brachial plexus lesions[END_REF]. Les limitations fonctionnelles observées chez ces patients intéressent l'abduction et la flexion d'épaule (i.e., muscles supraspinatus et deltoïde), la rotation externe d'épaule (i.e., muscles infraspinatus et teres minor) et la flexion du coude (i.e., muscles brachialis et biceps brachii) [START_REF] Tung | Brachial plexus injuries[END_REF].

Selon le type de lésion, le délai pré-opératoire, et les lésions associées, différentes options thérapeutiques peuvent être envisagées, avec notamment la chirurgie nerveuse, les transferts musculo-tendineux ou encore l'arthrodèse scapulo-humérale [START_REF] Spinner | Traumatic brachial plexus injury[END_REF]. Pour les patients pris en charge durant la première année suivant le traumatisme, en raison de l'étendue relativement modérée de ces paralysies C5/C6 (i.e., préservation des mobilités actives du coude en extension, du poignet et de la main), la restauration de l'ensemble des fonctions affectées est envisageable par chirurgie nerveuse seule, avec trois cibles principales que sont les nerfs musculocutané (NMC), suprascapulaire (NSS) et axillaire (NA) [START_REF] Bertelli | Reconstruction of C5 and C6 brachial plexus avulsion injury by multiple nerve transfers: spinal accessory to suprascapular, ulnar fascicles to biceps branch, and triceps long or lateral head branch to axillary nerve[END_REF]. Résultats fonctionnels. Au terme d'un suivi moyen de 46 ± 15 mois (intervalle, 25 à 76 mois), 28 patients avaient une force de flexion du coude satisfaisante (Figure 1) avec une moyenne évaluée 3.8 ± 0.5 (intervalle, de 2 à 4), associée à une mobilité moyenne de 126 ± 18° (intervalle, de 90 à 150°) (Tableau 2). Vingt-cinq patients présentaient une force de grade 4 selon l'échelle BMRC, leur permettant de soulever un poids moyen de 3.9 ± 1.7 kg (intervalle, de 1 à 7 kg). Un patient de 32 ans présentait une force de grade 2 à 18 mois de la chirurgie nerveuse (i.e., double transfert U-B M-B, réalisé un mois après le traumatisme); un transfert bipolaire du latissimus dorsi sur le biceps brachii fut réalisé, permettant d'obtenir une force de grade 4 et une flexion active à 120°.

Les forces de flexion et d'abduction de l'épaule furent de même considérées satisfaisantes chez 28 patients (Figure 2), avec des valeurs moyennes à 3.6 ± 0.8 (intervalle, de 0 à 4) pour les deux fonctions, associées à des mobilités actives moyennes de 109 ± 39° (intervalle, de 0 à 180°) et 99 ± 38° (intervalle, de 0 à 180°), respectivement. Un patient de 46 ans souffrant de lésions pré-ganglionnaires pour lesquelles avait été réalisés un transfert nerveux direct NSA-NSS ne présentait aucun signe clinique de récupération à 20 mois de suivi. Une arthrodèse glénohumérale fut réalisée secondairement, permettant d'obtenir de 80° de flexion et 70° d'abduction actives, cotées à 4 selon l'échelle BMRC.

La force en RE1 était satisfaisante chez 27 patients (Figure 3), avec une valeur moyenne de 3.4 ± 0.9 (intervalle, de 0 à 4) associée à une mobilité active moyenne de 12 ± 34° (intervalle, de -80 à 70°) alors que la force en RE2 était satisfaisante chez 18 patients, avec une valeur moyenne de 2.5 ± 1.2 (intervalle, de 0 à 4) et une mobilité active moyenne de

DISCUSSION

Une prise en charge précoce des lésions proximales du plexus brachial autorise en première intention la réalisation de chirurgie nerveuse de reconstruction, avec trois cibles principales que sont les nerfs suprascapulaire, axillaire et musculocutané [START_REF] Bertelli | Reconstruction of C5 and C6 brachial plexus avulsion injury by multiple nerve transfers: spinal accessory to suprascapular, ulnar fascicles to biceps branch, and triceps long or lateral head branch to axillary nerve[END_REF]. Avec un recul moyen de presque 4 ans, nous avons montré dans cette étude que les résultats fonctionnels à long terme d'une stratégie associant greffes et transferts nerveux étaient satisfaisants dans la majorité des cas. Cependant, restaurer la rotation externe d'épaule demeure un défi thérapeutique dans ce type de pathologie.

Stratégie chirurgicale. Si l'emploi de greffes nerveuses reste envisageable en cas de lésions post-ganglionnaires [START_REF] Spinner | Advances in the Repair of Peripheral Nerve Injury[END_REF], il est essentiel de tenir compte de la distance séparant le moignon radiculaire et le muscle ciblé. En effet, en raison de l'altération irréversible et progressive des plaques motrices au cours du temps, débutant aux alentours du 6 e mois post traumatique, les résultats fonctionnels de cette chirurgie sont directement impactés par le délai de réinnervation [START_REF] Tung | Nerve transfers: indications, techniques, and outcomes[END_REF]. C'est pourquoi nous privilégions les techniques de transferts nerveux depuis de nombreuses années pour la réinnervation des fléchisseurs du coude et du deltoïde, réservant l'usage des greffes à la seule reconstruction du NSS.

Par ailleurs, les résultats observés chez les patients opérés tardivement semblent être relativement similaires à ceux des patients dans un délai post-traumatique inférieur à 12 mois.

Compte tenu de la petitesse de l'échantillon des patients présentant ces caractéristiques dans cette série, une comparaison statistique aux résultats des patients opérés avant 12 mois ne nous semble pas pertinente. En revanche, contrairement à ce qui est habituellement indiqué dans la littérature [START_REF] Spinner | Traumatic brachial plexus injury[END_REF][START_REF] Tung | Nerve transfers: indications, techniques, and outcomes[END_REF], ces résultats nous apparaissent suffisamment encourageants pour recommander des chirurgies de réinnervation chez ces patients, généralement vus tardivement en consultation en raison d'une errance médicale, sous réserve que des potentiels de fibrillations mise en évidence dans les muscles « cibles » lors de l'électromyogramme préopératoire.

Le nerf musculocutané. Depuis la description originale d'Oberlin et al. d'un transfert

de fascicule ulnaire sur la branche motrice du biceps brachii [START_REF] Oberlin | Nerve transfer to biceps muscle using a part of ulnar nerve for C5-C6 avulsion of the brachial plexus: anatomical study and report of four cases[END_REF], de nombreuses équipes utilisant cette technique ont rapporté d'excellents résultats fonctionnels et démontré sa supériorité par rapport aux greffes [START_REF] Garg | Comparison of nerve transfers and nerve grafting for traumatic upper plexus palsy: a systematic review and analysis[END_REF]. Afin de maximiser les chances de récupération, certains auteurs recommandent la réinnervation conjointe de la deuxième branche motrice du NMC [START_REF] Tung | Nerve transfers to the biceps and brachialis branches to improve elbow flexion strength after brachial plexus injuries[END_REF][START_REF] Liverneaux | Preliminary results of double nerve transfer to restore elbow flexion in upper type brachial plexus palsies[END_REF]. Cette attitude est toujours l'objet de controverses, avec notamment Carlsen et al.

qui montrèrent lors d'une étude rétrospective l'absence de différence significative entre les résultats fonctionnel des simples et des doubles transferts lors d'une étude rétrospective chez des patients souffrant d'une paralysie plexique proximale [START_REF] Carlsen | Comparison of single versus double nerve transfers for elbow flexion after brachial plexus injury[END_REF]. Martins et al. confirmèrent ces résultats lors d'une étude prospective randomisée réalisée sur 40 patients [START_REF] Martins | A prospective study comparing single and double fascicular transfer to restore elbow flexion after brachial plexus injury[END_REF]; cependant, pour des raisons de faisabilité, les auteurs réduisirent leur échantillon à moins de 25% du nombre de patients estimés nécessaires lors de l'étude de puissance initiale, limitant ainsi la valeur de leurs résultats. Compte tenu de cette absence de preuve scientifique irréfutable en faveur d'un simple transfert, du caractère essentiel de la restauration de la flexion du coude pour la fonction globale du membre paralysé, ainsi que de la faible morbidité du prélèvement d'un second fascicule [START_REF] Hanneur | Postoperative motor deficits following elbow flexion reanimation by nerve transfer[END_REF], nous recommandons de réanimer les deux fléchisseurs du coude chez ces patients.

Le nerf axillaire. De même que pour les fléchisseurs du coude, nous recommandons de restaurer la fonction du deltoïde par transferts nerveux. En effet, si certains auteurs ne retrouvent pas de différence significative entre les greffes longues et les transferts nerveux pour la reconstruction du nerf axillaire [START_REF] Wolfe | Long-nerve grafts and nerve transfers demonstrate comparable outcomes for axillary nerve injuries[END_REF][START_REF] Baltzer | A Comparison of Outcomes of Triceps Motor Branch-to-Axillary Nerve Transfer or Sural Nerve Interpositional Grafting for Isolated Axillary Nerve Injury[END_REF], nous avons observés au cours de notre expérience une réelle amélioration du pronostic de récupération depuis que nous réalisons des transferts nerveux. A la différence de Leechavengvongs et al., nous prélevons une branche motrice destinée au chef latéral du triceps brachii [START_REF] Leechavengvongs | Nerve transfer to deltoid muscle using the nerve to the long head of the triceps, part II: a report of 7 cases[END_REF]. En effet, le chef long étant un muscle bi-articulaire, il participe à la stabilisation postéro-inférieure et à l'extension de l'articulation scapulo-humérale. Le chef latéral n'ayant pas d'action directe sur l'épaule, sa paralysie apparaît moins préjudiciable. Dans cette technique, seul le contingent antérieur du nerf axillaire est réinnervé, permettant ainsi d'éviter une déperdition de fibres motrices dans la branche sensitive du nerf axillaire et d'obtenir des résultats satisfaisants en termes d'élévations (i.e., abduction et flexion d'épaule) [START_REF] Leechavengvongs | Nerve transfer to deltoid muscle using the nerve to the long head of the triceps, part II: a report of 7 cases[END_REF]. En revanche, un tel transfert ne permet pas la réinnervation du teres minor. Par conséquent, la restauration de la rotation externe d'épaule repose uniquement sur la réinnervation de l'infraspinatus, via la reconstruction du NSS. Compte tenu des résultats observés dans notre étude, nous pensons que la réinnervation de la branche motrice à destinée du teres minor en addition de la branche antérieure du nerf axillaire, telle qu'originellement décrite par Bertelli et al. [START_REF] Bertelli | Axillary nerve repair by triceps motor branch transfer through an axillary access: anatomical basis and clinical results[END_REF], pourrait améliorer le pronostic fonctionnel des patients présentant ce type de paralysie en maximisant les chances de récupération de la rotation externe d'épaule.

Le nerf suprascapulaire. Compte tenu de sa simplicité technique et des résultats satisfaisants rapportés par différentes équipes, le transfert NSA-NSS est communément réalisé pour la réanimation des muscles spinati [START_REF] Tung | Nerve transfers: indications, techniques, and outcomes[END_REF][START_REF] Bertelli | Transfer of the accessory nerve to the suprascapular nerve in brachial plexus reconstruction[END_REF][START_REF] Leechavenvongs | Nerve Transfers to Restore Shoulder Function[END_REF]. Cependant, il n'a pas été montré de supériorité du transfert sur la greffe dans la reconstruction du nerf suprascapulaire [START_REF] Garg | Comparison of nerve transfers and nerve grafting for traumatic upper plexus palsy: a systematic review and analysis[END_REF] et ce transfert présente plusieurs désavantages notables, avec notamment une grande inadéquation du nombre moyen d'axones myélinisés entre le donneur et le receveur (e.g., 1603 ± 416 axones myélinisés pour le NSA contre 6004 ± 649 pour le NSS) [START_REF] Pruksakorn | Posterior approach technique for accessory-suprascapular nerve transfer: A cadaveric study of the anatomical landmarks and number of myelinated axons[END_REF]. De plus, Vathana et al. ont aussi montré que la distalisation du prélèvement du NSA majorait cette inadéquation, avec une moyenne de 1383 ± 291 d'axones myélinisés en proximal contre 863 ± 148 en distal [START_REF] Vathana | An anatomic study of the spinal accessory nerve: extended harvest permits direct nerve transfer to distal plexus targets[END_REF]. D'autre part, une importante différence de réinnervation entre les deux muscles spinati fut retrouvée par différentes équipes, la fonction du supraspinatus étant significativement supérieure à celle de l'infraspinatus [START_REF] Terzis | Suprascapular nerve reconstruction in 118 cases of adult posttraumatic brachial plexus[END_REF][START_REF] Baltzer | Evaluation of infraspinatus reinnervation and function following spinal accessory nerve to suprascapular nerve transfer in adult traumatic brachial plexus injuries[END_REF]. Enfin, le prélèvement du NSA n'est pas sans conséquence. En effet, le trapèze est un muscle péri-scapulaire primordial dans la stabilisation et la rétropulsion scapulaire, et sa paralysie, même partielle, peut être à l'origine de dyskinésies scapulaires [START_REF] Oberlin | Nerve transfers in brachial plexus palsies[END_REF]. De plus, chez les patients ayant une rotation externe d'épaule insuffisante à la suite de la chirurgie nerveuse, un tel prélèvement limitera les options de chirurgie palliative à l'arthrodèse scapulo-humérale, le transfert de trapèze inférieur étant de facto contre-indiqué. En raison de la brièveté de la greffe et d'un transfert de qualité hasardeuse associée à une morbidité non-négligeable, il nous semble justifiable de réaliser de

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LEGENDES DES FIGURES

L

  'objectif de cette étude rétrospective était d'évaluer les résultats fonctionnels à long terme des patients pour lesquels une reconstruction nerveuse microchirurgicale fut réalisée à la suite d'une paralysie plexique C5/C6. Population. De Janvier 2009 à Décembre 2013, l'ensemble des patients ayant bénéficié d'une reconstruction nerveuse microchirurgicale à la suite d'une paralysie plexique traumatique C5/C6 dont le suivi était supérieur ou égal à 2 ans furent inclus. Stratégie chirurgicale. Tous les patients furent opérés par le dernier auteur. Le premier temps consistait à explorer le plexus supraclaviculaire afin de reconstruire le NSS. Une évaluation électrophysiologique des racines C5 et C6 était tout d'abord réalisée à l'aide d'un neurostimulateur (Vari-Stim®, Medtronic, Minneapolis, MN, USA). En cas de contraction du serratus antérieur (i.e., stimulation du nerf thoracique long par conduction radiculaire rétrograde), la racine était sectionnée et sa tranche de section examinée sous microscope ; en cas de fascicules de qualité satisfaisante, une greffe surale était interposée entre les fascicules radiculaires antérieurs et le moignon du NSS, préalablement recoupé en zone saine; en l'absence de fascicule greffable, la branche latérale du nerf spinal accessoire (NSA) était alors identifiée par la même incision et transférée sur le NSS [4]. Le deuxième temps consistait à reconstruire les deux branches motrices du NMC par transferts nerveux au canal brachial ; en l'absence de réponse à la neurostimulation de ces dernières, un fascicule ulnaire destiné au fléchisseur ulnaire du carpe était transféré sur la branche motrice du biceps et un fascicule médian destiné au pronator teres et/ou au palmaris longus était transféré sur la branche motrice du brachialis [5,6]. Le troisième temps était la reconstruction de la branche antérieure du NA à l'aide d'un transfert direct de la branche motrice du chef latéral du triceps brachii. Collection des données et analyse statistique. Les recherches furent effectuées conformément aux standards éthiques de la Déclaration d'Helsinki de 1964 ainsi qu'aux normes de la méthodologie de référence MR-003 [7]; l'étude fut inscrite dans le registre des bases de données de la CNIL (No. 2204168 v 0) et chaque patient fut informé individuellement et consentit avant toute collection et/ou analyse de données. Une analyse rétrospective des dossiers des patients fut effectuée par un investigateur indépendant, permettant d'en extraire les données démographiques des patients, le type de procédure réalisée, le délai d'apparition des premières contractions musculaires, les mobilités actives du coude (i.e., flexion, extension) et de l'épaule (i.e., abduction, flexion, rotation interne, rotations externes coude au corps -RE1, et rotation externe bras en abduction à 90° -RE2), mesurées à l'aide d'un goniomètre, et la force musculaire, cotée selon l'échelle du British Medical Research Council (BMRC) ; une force supérieure ou égale à 3 était considérée comme satisfaisante. Chez les patients présentant une force de flexion du coude supérieure ou égale 4, le poids maximum qu'il pouvait soulever au poignet avec le coude fléchi à 90° était noté en kg. Outre l'analyse descriptive des résultats observés lors du dernier contrôle postopératoire (i.e., moyenne ± écart-type et intervalle), une analyse en sous-groupe des mobilités de l'épaule fut réalisée afin de comparer les résultats fonctionnels des patients ayant reçu une greffe à ceux des patients ayant reçu un transfert du NSA pour la réinnervation du NSS; le test de normalité de Shapiro-Wilk ayant démontré l'absence de distribution normale des données, le test de Mann-Whitney fut utilisé. Le seuil de significativité statistique était défini tel que p < .05 pour tous les tests. RESULTATS Caractéristiques de la cohorte. Vingt-neuf patients consécutifs furent inclus (Tableau 1). En raison d'une venue tardive en consultation, un délai pré-opératoire supérieur à 12 mois fut observé chez 3 patients ; lors de l'étude électrophysiologique de détection réalisée en préopératoire, tous présentaient des potentiels de fibrillation dans les différents muscles ciblés (i.e., persistance de plaques motrices), maintenant ainsi l'indication de chirurgie nerveuse. Une greffe nerveuse fut réalisée chez 6 patients, avec une longueur moyenne de greffon de 8.6 ± 2 .4 cm (intervalle, de 6 à 12 cm). Pour les 23 patients présentant des lésions pré-ganglionnaires, un transfert direct NSA-NSS fut réalisé, à l'exception d'un patient chez qui le NSS était inclus dans un volumineux névrome allant jusqu'au faisceau médial ; une fois le NSS sectionné distalement en zone saine, un greffon sural de 8 cm fut utilisé, permettant de conserver un diamètre suffisant au niveau du moignon distal du NSA. Vingt-sept patients reçurent un double transfert pour la réanimation de la flexion du coude, alors qu'un transfert simple médian-brachial fut réalisé chez 2 patients en raison d'une réponse satisfaisante du biceps brachii à la stimulation per-opératoire. Un transfert direct triceps-axillaire fut réalisé pour tous les patients.

3 ±

 3 21° (intervalle, de -40 à 50°). Chez le patient ayant nécessité une arthrodèse d'épaule, un arc rotationnel de 70° fut observé (i.e., rotation interne à 80°, rotation externe à -10°). D'autre part, un patient de 59 ans présentant des lésions pré-ganglionnaires traitées par transferts nerveux directs n'avait pas récupéré de rotation externe satisfaisante, avec une force à 2 et une amplitude à -60° ; en raison d'élévations d'épaule tolérables (i.e., abduction à 60° et flexion à 70°) ainsi que d'une rotation interne satisfaisante (i.e., 120°), une ostéotomie de dérotation humérale fut proposée au patient, mais celui-ci refusa la chirurgie.Parmi les 3 patients opérés tardivement (i.e., plus de 12 mois après le traumatisme initial), tous récupèrent des forces satisfaisantes au coude comme à l'épaule, à l'exception de la RE2 chez un patient qui fut évaluée à 2 (Tableau 3).Analyse en sous-groupes. Si les valeurs moyennes de mobilité et de force tendaient à être supérieures chez les patients ayant bénéficié d'une greffe pour la reconstruction du NSS, aucune différence statistiquement significative ne fut mise en évidence (Tableau 4).

  manière systématique un examen per-opératoire des racines C5 et C6, électrophysiologique et visuel, afin d'évaluer la possibilité d'une greffe et d'épargner ainsi le NSA. Limitations. Les résultats de cette étude sont à considérer à la lumière de ses limitations méthodologiques. Tout d'abord, s'agissant d'une analyse de données recueillies par l'opérateur, un biais observationnel ne peut être exclu. De même, le caractère rétrospectif de l'étude et la petitesse des échantillons dans l'analyse en sous-groupe rendent impossible toute conclusion quant à la potentielle supériorité de la greffe radiculaire sur le transfert du nerf spinal accessoire dans la reconstruction du nerf suprascapulaire. Cependant, cette étude présente l'avantage de confirmer le maintien dans le temps des résultats obtenus à court et moyen termes, tout en mettant l'accent sur la difficulté de rétablir une rotation externe d'épaule satisfaisante. Conclusion. En cas de paralysie traumatique proximale du plexus brachial (i.e., C5/C6), une reconstruction précoce des nerfs suprascapulaire, axillaire et musculocutané permet d'obtenir des résultats fonctionnels globalement satisfaisants et stables dans le temps en ce qui concerne les élévations de l'épaule, antérieure et latérale, ainsi que la flexion du coude. Cependant, en l'absence de réinnervation du teres minor, la rotation externe d'épaule reste une fonction difficile à restaurer.

Figure 1 :

 1 Figure 1: Photographie d'un patient de 48 ans à 57 mois d'un double transfert nerveux ulnaire-biceps médian-brachial démontrant une flexion active du coude à 120°, de grade 4 selon l'échelle BMRC.

Figure 2 :

 2 Figure 2 : Photographie d'un patient de 21 ans à 30 mois d'un transfert nerveux tricepsaxillaire démontrant des élévations antérieures et latérales à 180°, de grade 4 selon l'échelle BMRC.

Figure 3 :

 3 Figure 3 : Photographie d'un patient de 21 ans à 54 mois d'un transfert spinal accessoiresuprascapulaire démontrant une rotation externe coude au corps à 20°, de grade 4 selon l'

  NSA-NSS: transfert direct du nerf spinal accessoire sur le nerf suprascapulaire; NTBL-NA: transfert direct de la branche motrice du chef latéral du triceps brachial sur la branche antérieure du nerf axillaire; U-B M-B: transferts directs de fascicules moteurs des nerfs ulnaire et médian sur les branches motrices du biceps brachial et du brachial antérieur; BMRC: échelle de cotation de force musculaire du British Medical Research Council; RE1: rotation externe coude au corps; RE2; rotation externe bras en abduction à 90° Tableau 4. Résultats fonctionnels à l'épaule selon la technique de réinnervation du nerf suprascapulaire. * Résultats présentés comme Moyenne ± Écart-type (Intervalle), sauf indication contraire.No: nombre de patients (valeurs absolues); C5: 5e racine cervicale; NSS: nerf suprascapulaire; NSA: nerf spinal accessoire: BMRC: échelle de cotation de force musculaire du British Medical Research Council; RE1: rotation externe coude au corps; RE2; rotation externe bras en abduction à 90°.

	Tableau 3. Détails des patients opérés après un délai post-traumatique supérieur ou égal Tableau 1. Données générales. Patients (No) Âge (années) Sexe (M / F, No) Etiologies Accident de la voie publique (motocyclette / automobile / bicyclette, No) Sports (ski, No) Lésions associées Fractures-luxations membre supérieur ispilatéral (épaule -bras / coude -avant-bras / poignet -main, No) Autres fractures (rachis / membre inférieur, No) Autres lésions (crâne / oeil, No) Délai pré-opératoire (mois) à 12 mois. Tableau 2. Résultats fonctionnels à long terme. * Articulations et mouvements Greffe C5-NSS Transfert NSA-NSS 34 ± 13 (21;59) 29 27 / 2 28 (22 / 5 / 1) 1 7 (5 / 1 / 1) 5 (4 / 1) 4 (3 / 1) 6 ± 5 (1-24) Patients Force Amplitude (degrés) p BMRC /5 Âge (années) 21 21 55 Patients (No) 6 23 BMRC ≤2 / 3 / ≥4 (No) Épaule Flexion 3.6 ± 0.8 (0;4) 1 / 8 / 20 109 ± 39 (0;180) Abduction 3.6 ± 0.8 (0;4) 1 / 8 / 20 Sexe (M / F) M M Âge (années) 29 ± 11 (21;51) 35 ± 13 (21;59) .27 M Délai pré-opératoire (mois) 18 24 12 Délai pré-opératoire (mois) 6.4 ± 5.7 (1;24) 5 ± 2.2 (2;8) .96 99 ± 38 (0;180) RE1 3.4 ± 0.9 (0;4) 2 / 12 / 15 Techniques de réinnervation Durée du suivi (mois) 46.6 ± 15 (25;76) 44.7 ± 16.8 (30;70) .87 12 ± 34 (-80;70) RE2 2.5 ± 1.2 (0;4) 11 / 13 / 5 3 ± 21 (-40;50) Coude Flexion 3.8 ± 0.5 (2;4) 1 / 3 / 25 Nerf suprascapulaire NSA-NSS NSA-NSS NSA-NSS Nerf axillaire NTBL-NA NTBL-NA Force (BMRC /5) NTBL-NA Nerf musculocutané U-B M-B U-B M-B Flexion 3.7 ± 0.5 (3;4) 3.6 ± 0.9 (0;4) .97 U-B M-B Abduction 3.7 ± 0.5 (3;4) 3.6 ± 0.9 (0;4) .97 126 ± 18 (90;150) RE1 3.8 ± 0.4 (3;4) 3.3 ± 0.9 (0;4) .09 Force (BMRC /5) RE2 3.2 ± 0.8 (2;4) 2.3 ± 1.3 (0;4) .14 Coude Techniques de réinnervation Nerf suprascapulaire (greffe radiculaire / transfert direct / transfert + greffe, No) 29 (6 / 22 / 1) Nerf axillaire (transfert direct) corps; Flexion 4 4 4 Amplitude (degrés) RE2; rotation externe bras en abduction à 90° Épaule Flexion 117 ± 39 (80;180) 107 ± 40 (0;180) .73 29 Nerf musculocutané (double transfert direct UBMB / simple transfert direct MB) 29 (27 / 2) Durée du suivi (mois) 46 ± 15 (25;76) Flexion 3 4 Abduction 108 ± 39 (70;180) 97 ± 38 (0;160) .59 4 Abduction 3 3 RE1 22 ± 26 (-10;60) 10 ± 36 (-80;70) .59 4 RE1 3 3 4 RE2 13 ± 12 (-10;20) 1 ± 22 (-40;50) .14
	Délai d'apparition des premières contractions musculaire (mois) RE2 3 3	2
	Spinati (supra-épineux / infra-épineux) Deltoïde Amplitude (degrés) Fléchisseurs du coude (biceps brachial / brachial antérieur) Coude Flexion 140	120	120	10 ± 2 (7;14) 9 ± 2 (5;13) 6 ± 1 (3;10)
	Épaule
	Flexion	80	90	120
	Abduction	60	70	90
	RE1	-10	0	0
	RE2	0	0	-10
	Durée du suivi (mois)	44	33	32

* Résultats présentés comme Moyenne ± Écart-type (Intervalle), sauf indication contraire. No: nombre de patients (valeurs absolues); BMRC: échelle de cotation de force musculaire du British Medical Research Council; RE1: rotation externe coude au M: masculin; F: féminin; *


Oleksandr Grytsai

Oksana Valiashko

Manon Penco-Campillo

Maeva Dufies

Anais Hagege

Luc Demange

Sonia Martial

Gilles Pagès

Cyril Ronco
email: cyril.ronco@univ-cotedazur.fr

Rachid Benhida
email: rachid.benhida@univ-cotedazur.fr

Synthesis and biological evaluation of 3-amino-1,2,4-triazole derivatives as potential anticancer compounds

Keywords: 1, 2, 4-Triazole Anticancer agent 3-Amino-1, 2, 4-triazole

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

Introduction

Cancer is the second leading cause of death globally and the most prevalent one in developed countries. In 2018, it was responsible for an estimated 9.6 million deaths and over 18 million new cases were reported. Moreover, this prevalence is expected to increase in the upcoming years [START_REF] Bray | GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF] . Despite sustained efforts in cancer research and indisputable results for some types of cancers [START_REF] Bray | GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF][START_REF] Smith | Cancer screening in the United States, 2019: A review of current American Cancer Society guidelines and current issues in cancer screening[END_REF] , the main goal of durable cure remains unachieved for the majority of cancer types. One of the challenges on the way to durable anticancer cure is drug resistance phenomena exhibited by cancer cells to escape drug pressure, which leads in many cases to transient responses to the treatments [START_REF] Ramos | Mechanism-based cancer therapy: resistance to therapy, therapy for resistance[END_REF][START_REF] Millet | Metastatic melanoma: insights into the evolution of the treatments and future challenges[END_REF] . One strategy to prevent or attenuate the appearance of strong resistance mechanisms, relies on distributing the drug pressure rather than concentrating it on a single target [START_REF] Millet | Discovery and Optimization of N-(4-(3-Aminophenyl) thiazol-2-yl) acetamide as a Novel Scaffold Active against Sensitive and Resistant Cancer Cells[END_REF][START_REF] Ronco | Structure activity relationship and optimization of N-(3-(2-aminothiazol-4yl)aryl)benzenesulfonamides as anti-cancer compounds against sensitive and resistant cells[END_REF][START_REF] Benhida | HA15, une nouvelle molécule qui pousse les cellules cancéreuses au suicide : du diabète au mélanome[END_REF] . In line with this, it is appealing to utilize dual-target therapy, which consists of creating drugs blocking simultaneously two or more essential mechanisms.

Two main cancer hallmarks are proliferation and angiogenesis [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF] . Proliferation is characterized by the ability of cancer cells to sustain uncontrolled replication that leads to tumor growth. While, angiogenesis is the process of new blood vessels formation developed by cancer cells, to sustain high metabolism, hypoxia and fast replication. The newly formed neo-vasculature facilitates the intake of increased amounts of nutrients and oxygen by the tumor and the evacuation of metabolic wastes and carbon dioxide. Therefore, drugs targeting two essential mechanisms like tumor proliferation and angiogenesis would have a real potential to display enhanced and lasting anticancer efficiency.

Over the last decades, increased research has been devoted to triazole drugs [START_REF] Ronco | New Huprine Derivatives Functionalized at Position 9 as Highly Potent Acetylcholinesterase Inhibitors[END_REF][START_REF] Ronco | Synthesis and structure-activity relationship of Huprine derivatives as human acetylcholinesterase inhibitors[END_REF][START_REF] Ronco | Huprine Derivatives as Sub-Nanomolar Human Acetylcholinesterase Inhibitors: From Rational Design to Validation by X-ray Crystallography[END_REF] . Among them, 3-amino-1,2,4-triazoles derivatives have attracted special attention as they demonstrated a broad spectrum of bioactivities, including potential applications against thrombotic disorders [START_REF] Short | Multisubstituted aromatic compounds as inhibitors of thrombin[END_REF] , fibrotic [START_REF] Jones | Heterocyclylamino-substituted triazoles as modulators of rho-associated protein kinase[END_REF] , and auto-immune diseases [START_REF] Jones | Heterocyclylamino-substituted triazoles as modulators of rho-associated protein kinase[END_REF] , central nervous system disorders [START_REF] Boyle | Derivatives of 3-amino-1[END_REF] , obesity [START_REF] Murali | Modulators of the glucocorticoid receptor, AP-1, and/or NF-KB activity and use thereof[END_REF] , diabetes [START_REF] Murali | Modulators of the glucocorticoid receptor, AP-1, and/or NF-KB activity and use thereof[END_REF][START_REF] Bebbington | Triazole compounds useful as protein kinase inhibitors[END_REF] , Alzheimer's disease [START_REF] Bebbington | Triazole compounds useful as protein kinase inhibitors[END_REF] , microbial infections [START_REF] Boyle | Derivatives of 3-amino-1[END_REF][17][START_REF] Ram | Chemotherapeutic Agents. Part 13. Synthesis of 2-Pyridyl-1, 2, 4-triazolo (1, 5-a) pyrimidines as Antimicrobial Agent[END_REF][START_REF] Beyzaei | A green one-pot synthesis of 3 (5)-substituted 1, 2, 4-triazol-5 (3)-amines as potential antimicrobial agents[END_REF] or cancer [START_REF] Jones | Heterocyclylamino-substituted triazoles as modulators of rho-associated protein kinase[END_REF][START_REF] Bebbington | Triazole compounds useful as protein kinase inhibitors[END_REF][START_REF] Romagnoli | 3-Aryl/Heteroaryl-5-amino-1-(3′, 4′, 5′-trimethoxybenzoyl)-1, 2, 4-triazoles as antimicrotubule agents. Design, synthesis, antiproliferative activity and inhibition of tubulin polymerization[END_REF][START_REF] El-Sherief | Novel 1, 2, 4-triazole derivatives as potential anticancer agents: Design, synthesis, molecular docking and mechanistic studies[END_REF][START_REF] Min | Optimization of a novel series of ataxiatelangiectasia mutated kinase inhibitors as potential radiosensitizing agents[END_REF][START_REF] Ouyang | Synthesis and structure-activity relationships of 1, 2, 4-triazoles as a novel class of potent tubulin polymerization inhibitors[END_REF][START_REF] Ouyang | Heterocyclic compounds and their use as anticancer agents[END_REF] . Substitution of 3-amino-1,2,4-triazole (Figure 1a) by aryl groups leads to particularly promising anticancer scaffolds. For example, the 3-pyridotriazole core I is found in several antiangiogenic compounds [START_REF] Romagnoli | 3-Aryl/Heteroaryl-5-amino-1-(3′, 4′, 5′-trimethoxybenzoyl)-1, 2, 4-triazoles as antimicrotubule agents. Design, synthesis, antiproliferative activity and inhibition of tubulin polymerization[END_REF][START_REF] El-Sherief | Novel 1, 2, 4-triazole derivatives as potential anticancer agents: Design, synthesis, molecular docking and mechanistic studies[END_REF][START_REF] Ouyang | Synthesis and structure-activity relationships of 1, 2, 4-triazoles as a novel class of potent tubulin polymerization inhibitors[END_REF][START_REF] Ouyang | Heterocyclic compounds and their use as anticancer agents[END_REF] , as well as the phenylamino triazoles derivatives II [START_REF] Jones | Heterocyclylamino-substituted triazoles as modulators of rho-associated protein kinase[END_REF][START_REF] Bebbington | Triazole compounds useful as protein kinase inhibitors[END_REF][START_REF] Ouyang | Synthesis and structure-activity relationships of 1, 2, 4-triazoles as a novel class of potent tubulin polymerization inhibitors[END_REF][START_REF] Ouyang | Heterocyclic compounds and their use as anticancer agents[END_REF] . A representative example of aryl-substituted 3-amino-1,2,4-triazole is compound III (Figure 1a) that shows potent antiproliferative and anti-tubulin polymerization activities [START_REF] Ouyang | Synthesis and structure-activity relationships of 1, 2, 4-triazoles as a novel class of potent tubulin polymerization inhibitors[END_REF] . In addition to frequent potent activities, the 3-amino-1,2,4-triazole motif displays valuable inherent physicochemical-properties that improve overall solubility, bioavailability, or chemical stability of a molecule [START_REF] El-Sherief | Novel 1, 2, 4-triazole derivatives as potential anticancer agents: Design, synthesis, molecular docking and mechanistic studies[END_REF][START_REF] Eastwood | Indolin-2-one p38α inhibitors III: Bioisosteric amide replacement[END_REF][START_REF] Lipinski | Bioisosteric design of conformationally restricted pyridyltriazole histamine H2-receptor antagonists[END_REF] . Moreover, the structural features of 3-amino-1,2,4triazole enable it to mimic different functional groups, e.g. they areare often considered as urea bioisostere, with enhanced solubility and decreased trend to form aggregates [START_REF] Lipinski | Bioisosteric design of conformationally restricted pyridyltriazole histamine H2-receptor antagonists[END_REF] . Besides, everal clinically approved drugs with potent antiproliferative and antiangiogenic properties contain the diarylurea function [START_REF] Dufies | New CXCR1/CXCR2 inhibitors represent an effective treatment for kidney or head and neck cancers sensitive or refractory to reference treatments[END_REF] , making,the 1,2,4-amino-triazole core an interesting and understudied pharmacophore.

In line with these observations, we investigated the anticancer potential of arylamino-3-pyrido-1,2,4-triazoles, which appears to be a particularly promising scaffold. Indeed, the fusion of structures I and II leads to a core with highly attractive steric and electronic features (Figure 1b). This hybrid scaffold could display both antiproliferative and antiangiogenic activities, along with particularly favorable physicochemical properties for a drug. The molecular weight (237 g/mol), the partition coefficient (cLogP = 2.91), the number of hydrogen-bonds accepting sites (3-4), donating sites (2-3) and rotatable bonds (3) perfectly complies with the Lipinski rules of three standards and should lead to beneficial pharmacological properties for its derivatives. This arylamino-3-pyrido-1,2,4-triazole scaffold is also innovative as only one such series of compounds has been identified so far [START_REF] Ouyang | Synthesis and structure-activity relationships of 1, 2, 4-triazoles as a novel class of potent tubulin polymerization inhibitors[END_REF] . In this study, we revisited the synthesis of these 1,2,4-triazole compounds and prepared a series of analogs intending to further evaluate the synergism of the fusion of the two identified antiproliferative and antiangiogenic cores (Figure 1c). In total, 25 new diaryl-1,2,4-triazole derivatives have been synthesized and fully characterized, as well as 14 benzoylthiourea intermediates. All compounds have been biologically tested to assess their activity on a panel of cancer cell lines, as well as on an endothelial cell line.

Material and methods

Chemistry

All the starting materials have been purchased from chemical providers Aldrich, Merck or Alfa Aesar and used without additional purification. All the solvents have been purchased from Sigma Aldrich and used without precursive distillation. Thin layer chromatography (TLC) have been performed on pre-coated silica gel plates (Kieselgel 60 F254, E. Merck, Germany) and visualized by UV lamp (wavelength 254 nm). NMR spectra have been recorded in DMSO-d6 ( 1 H and [START_REF] Jones | Heterocyclylamino-substituted triazoles as modulators of rho-associated protein kinase[END_REF] C NMR) and TFA-d1 (13C NMR) on a Bruker AC 200 at 200 MHz for 1 H NMR and 50 MHz for [START_REF] Jones | Heterocyclylamino-substituted triazoles as modulators of rho-associated protein kinase[END_REF] C NMR, or on a Bruker AC 400 spectrometers at 400 MHz for 1H NMR and 101 MHz for [START_REF] Jones | Heterocyclylamino-substituted triazoles as modulators of rho-associated protein kinase[END_REF] C NMR; δ is expressed in ppm related to TMS (0 ppm) as an internal standard. Splitting patterns are named as following: s (singlet), d (doublet), t (triplet), m (multiplet). Coupling constants (J) values are expressed in Hertz (Hz). Mass spectra (ESI-MS) have been recorded on a Bruker Daltonics Esquire 3000+, all the solid samples have been dissolved in methanol and pre-filtrated. The purity of compounds has been assayed by HPLC analysis on JASCO PU-2089, with Supelco analytical column Ascentis Express C18, 100 mm × 46 mm 5 μM. Eluent A: 99.9% water with 0.1% formic acid. Eluent B: 99.9% acetonitrile with 0.1% formic acid. Method 1: 25% B to 100% B for 13 min, 100% B for 5 min, from 100% B to 5% B for 1 min (19 min total).

Synthetic procedures and characterizations:

General Procedure A: To the solution of substituted N-(phenylcarbamothioyl)benzamide (1 equiv) in 1,4-dioxane (10 mL/1 mmol) at 0 °C, hydrazine hydrate (3 eq) was added. The reaction was stirred for 24-30 h at room temperature, the conversion was monitored by TLC and LC-MS until the equilibrium was reached. The crude compound was purified by using column chromatography General Procedure B: A mixture of substituted 1-phenylisothiouronium iodide (1 equiv) and nicotynohydrazide (1 equiv) in pyridine (10 mL/1 mmol) was refluxed overnight. After the reaction was completed (TLC monitoring) the solution was poured into the water (50 mL/1 mmol) and extracted with ethyl acetate (3 x 25 ml). Organic layer was collected, washed with brine, dried with MgSO4 and the solvent was evaporated under reduced pressure. The crude compound was purified by using column chromatography.

Compounds 1.1-32 were synthesized by following the previously described procedures [START_REF] Baltabaev | Synthesis of nicotinoylthiourea derivatives[END_REF][START_REF] Aydin | Synthesis of two novel aroyl thioureas and their use as anion binding receptors[END_REF][START_REF] Tong | Microwave assisted synthesis and biological activity of N-aryl-N'-nicotinoyl thiourea[END_REF][START_REF] Rödl | Multi-dimensional target profiling of N,4-diaryl-1,3-thiazole-2-amines as potent inhibitors of eicosanoid metabolism[END_REF][START_REF] Ma | Design and Synthesis of N-Aryl Isothioureas as a Novel Class of Gastric H+/K+-ATPase Inhibitors[END_REF][START_REF] Mahboobi | Design of Chimeric Histone Deacetylase-and Tyrosine Kinase-Inhibitors: A Series of Imatinib Hybrides as Potent Inhibitors of Wild-Type and Mutant BCR-ABL, PDGF-Rβ, and Histone Deacetylases[END_REF][START_REF] Ye | Synthesis and Cytotoxicity in Vitro of N-Aryl-4-(tert-butyl)-5-(1H-1,2,4-triazol-1-yl)thiazol-2-amine[END_REF][START_REF] Rasmussen | Improved Procedures for the Preparation of Cycloalkyl-, Arylalkyl-, and Arylthioureas[END_REF][36][START_REF] Jian | Application of N-substituted aryl(arylformamido)-N'-substituted aryl formyl thiourea compound in inhibition of Cyanobacteria growth[END_REF][38][START_REF] Rauf | Solution-phase microwave assisted parallel synthesis of N,N'-disubstituted thioureas derived from benzoic acid: biological evaluation and molecular docking studies[END_REF] . The NMR spectral data of compounds 1.1-32 were in accordance with those reported in the literature N,5-diphenyl-1H-1,2,4-triazol-3-amine [START_REF] Kodomari | A convenient and efficient method for the synthesis of monoand N,N-disubstituted thioureas[END_REF] (2. 1.092 min (method 1). [START_REF] Kodomari | A convenient and efficient method for the synthesis of monoand N,N-disubstituted thioureas[END_REF] (2.7) Synthesized following the general procedure A using N-(4tolylcarbamothioyl)benzamide (0.270 g, 1 mmol) and hydrazine hydrate (0.146 mL, 3 mmol) to afford the title compound as a white powder (173 mg, 69%). 1.325 min (method 1).

5-phenyl-N-(p-tolyl)-1H-1,2,4-triazol-3-amine

N-(3,5-dichlorophenyl)-5-(pyridin-3-yl

)-1,2,4-triazol-3-amine (4.12) Synthesized following the general procedure B using 1-( [START_REF] Ladani | Novel 1,3,4-oxadiazole motifs bearing a quinoline nucleus: synthesis, characterization and biological evaluation of their antimicrobial, antitubercular, antimalarial and cytotoxic activities[END_REF] (5) A solution of 2-nicotinoyl-N-phenylhydrazinecarboxamide (1.19 g, 5 mmol) in POCl3 (5 ml) was refluxed for 2 h. After cooling to room temperature, the reaction mixture was poured into a cold water (200 mL).

N-phenyl-5-(pyridin-3-yl)-1,3,4-oxadiazol-2-amine

After stirring for 10 min the precipitated product was collected by filtration, washed with water and Et2O, and dried under reduced pressure yielding to a pure product as a white powder (0.74 g, 62%). [START_REF] Bray | GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF] [START_REF] Shanmugasundaram | Synthesis,characterization and molecular docking studies of novel 2, 5-disubstituted thiadiazole and oxadiazole derivatives[END_REF] (6) To a dry 2-nicotinoyl-N-phenylhydrazinecarbothioamide (0.408 g, 1.5 mmol) H2SO4 (15 ml, 30 mmol) was added slowly with syringe at 0 °C and the stirring solution was left overnight at room temperature under argon atmosphere. After the reaction was completed as monitored by TLC the reaction mixture was poured into a cold water (200 mL)

N-phenyl-5-(pyridin-3-yl)-1,3,4-thiadiazol-2-amine

and the pH was adjusted to 7 with NaOH solution. White precipitate was separated by filtration and purified by column chromatography yielding to a pure product as a white powder (130 mg, 51%). [START_REF] Bray | GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF]

N-phenyl-5-(pyridin-3-yl)-1H-pyrazol-3-amine 44 (7) To a solution of 3-(methylthio)-3-(phenylamino)-1-(pyridin-3-yl)propan-1-one

(1.18 g, 5 mmol) in isopropanol (30 ml) hydrazine hydrate (1.25 g, 25 mmol) was added and the reaction mixture was refluxed overnight. The solvent was evaporated under reduced pressure to yield pure product as a light pink powder (1.01 g, 86%). [START_REF] Bray | GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF]

Cytotoxicity measurement (XTT)

Principle of the measurement: The assay is based on the cleavage of the tetrazolium salt 2,3-Bis-(2-méthoxy-4-nitro-5sulfophényle)-2H-tétrazolium-5-carboxanilide (XTT, Sigma-Aldrich®, Saint-Quentin-Fallavier, FRANCE), in the presence of an electron-coupling reagent, producing a soluble formazan salt. This conversion only occurs in viable (metabolically active) cells. The number of viable cells is directly correlated with the amount of orange formazan by measuring the absorbance at 450 nm of the dye on a spectrophotometer.

Medulloblastoma and endothelial cells.

5,000 DAOY cells and 50,000 HD-MB03 cells (for tumor cells), 20,000 TIME cells (for endothelial cells) were incubated in a 96well plate in triplicate with two different concentrations of inhibitors (5 and 20 µM), for 48 hours, in a total volume of 100 µl. A blank without cells was produced. At the indicated time, 50 µl of XTT reagent were added to each well. After incubation at 37°C for 30 min -1h30 (depending on the cell type), the absorbance of the produced formazan was measured at 450 nm using the Promega GLOMAX®-Multi + detection spectrophotometer. The gross relative effect of each new compound on each cell line was assessed by normalizing the mean optical density (3 determinations of a product-containing well), at each concentration, by the mean optical density obtained for the same cell line without any added agent.

Head and neck and kidney cancer cells.

Cells (5x10 3 cells/100 μl) were incubated in a 96-well plate with different effectors for 48h. 50 μl of XTT reagent were added to each well. The assay is based on the cleavage of the yellow tetrazolium salt XTT to form an orange formazan dye by metabolically active cells. Absorbance of the formazan product, reflecting cell viability, was measured at 490 nm. Each assay was performed in triplicate.

Results and discussion

Chemistry

The compounds synthesized can be divided into two groups depending on the nature of the cycle in position 5 of the triazole: 3pyridyl (Series A) or phenyl (Series B) (Figure 1c). The initial retrosynthetic analysis envisaged the synthesis of triazole compounds trough the double condensation of hydrazine on N-(benzylcarbamothioyl)arylamide precursors 1 (Scheme 1). These precursors 1 could be obtained by the addition of diversely substituted anilines on arylcarbonyl isothiocyanates, which can be prepared from commercially available nicotinic acid or benzoyl chloride by adapting described procedures. [START_REF] Rödl | Multi-dimensional target profiling of N,4-diaryl-1,3-thiazole-2-amines as potent inhibitors of eicosanoid metabolism[END_REF] Thus, chlorination and treatment with ammonium isothiocyanate proceeded in quantitative yields [START_REF] Wang | Homochiral crystallization of helical coordination chains bridged by achiral ligands: can it be controlled by the ligand structure[END_REF] , then the addition of variously substituted provided N- The formation of the 1,2,4-triazole by hydrazine condensation required a preliminary conditions screening (Table 1). We conducted a study of the effect of the solvent, the number of hydrazine equivalents, reaction temperature, and time on the model substrate 1.11.

(
Suitable conversion was found with the following conditions: addition of 3 equivalents of hydrazine hydrate at 0 °C in 1,4-dioxane and stirring at 20 °C for 24 h (Table 1, Entry 7). Using these conditions, N,3-diphenyl-1H-1,2,4-triazol-5-amines 2.1-10 were synthesized in good to moderate yields (Scheme 2). Unfortunately, the amide bond of nicotinamide precursors 1.1-10 revealed to be fragile when subjected to the high nucleophilicity of hydrazine. Despite all our efforts, cyclization attempts with hydrazine on our model substrate 1.1 always led to the cleavage of this bond yielding 1-phenylthioureas 3 as a major product (Scheme 2). 3.2 Biological evaluation

Antiproliferative activity on cancer cells

The new synthesized 3-(3-pyrido)-azole compounds of series A (4.1-12, 5, 6, and 7) were subjected to cell viability assay on three aggressive cancer models for which no efficient and longstanding treatment is currently available, i.e. medulloblastoma, head and neck squamous cell carcinoma, and kidney cancer. Precisely, the 12 pyridotriazoles 4.1-12 as well as the imidazolo-, oxadiazolo-and thiadiazolo analogs 5-7 were screened against 6 cancer cell lines: Daoy and HD-MB03 human medulloblastoma cell lines; CAL27 and CAL33 head and neck squamous cell carcinoma cell lines; and A498 and 786-O kidney cancer cell lines. All the defined cells were treated with the new synthetic compounds for two days and XTT assays were used to measure cell metabolism (cell proliferative ability, by extension). The IC50 values (μM) are listed in Table 2. One of the tested compounds, 4.6, bearing a 3-bromophenyl moiety, showed significant antiproliferative effects with IC50 values < 15 μM on head and neck squamous head and neck and kidney cancers cells. On the other hand, the replacement of the 1,2,4-triazole by an imidazole, oxadiazole, or thiadiazole proved to be unfavorable. 3. Globally, the same structure-activity relationships were observed for the 3,5-diarylamino-1,2,4-triazole derivatives 2.1-2.10 with the presence of apolar substituents like a halogen or a nitro group that seemed to be favored. In particular, the 3-bromo substitution again provided the best compound of this series (2.6). Moreover, this clear beneficial effect of the 3-bromophenyl moiety was noted on all cell lines tested, with a slightly lower activity on medulloblastoma. This effect on cell lines of multiple origins predicts a successful use of this compound as a basis for a generic anti-tumoral therapeutic strategy, which remains to be thought of with the specificities of each pathology. Concerning the N-benzoylthioureas 1.11-29, significant antiproliferative activity was only noticed for derivatives 1.13 and 1.25 with 2-hydroxy substituents on the HD-MB03 medulloblastoma cell line (IC50 = 5 µM) and for the 2-chloro-5-nitro derivative 1.28 on kidney cell lines A498 and 786-O. HD-MB03 cell line, derived from a highly aggressive form of medulloblastoma, seems to be much more sensitive to N-benzoylthioureas than its Daoy counterpart (less aggressive). This suggests that HD-MB03 cells are richer in N-benzoylthiourea targets than Daoy cells. This difference in sensitiveness was also demonstrated with the main reference treatment of this pathology, i.e. etoposide. Indeed, HD-MB03 cells were twice as sensitive to this compound as Daoy cells (Table 3), thus suggesting that HD-MB03 cells are more sensitive to therapeutic agents, as a whole, than Daoy cells. This confirms that medulloblastoma must be considered as a heterogeneous group of pathologies rather than one unique disease [START_REF] Northcott | Subgroup-specific structural variation across 1,000 medulloblastoma genomes[END_REF] and that a targeted therapeutic strategy might be the most path to follow. Overall, the most compelling structures are the triazole compounds with a 3-bromophenyl moiety (4.6 and 2.6) since they show efficiency on all the tested cell lines. The N-benzoylthioureas with a 2-hydroxyphenyl moiety (1.13 and 1.25) also showed promising activities but further studies on these compounds are needed to understand the selectivity towards one specific cell line.

Antiangiogenic activity

The antimetabolic activity on non-cancer cells from the tumor microenvironment was tested in preliminary experiments. Tumor vasculature plays a capital part in tumor development, as blood vessels bring oxygen and nutrients, which contributes to tumor growth.

Homeostasis of endothelial cell proliferation and permeability is then crucial. Affecting one or the other may lead to resorption of the tumor. Thus, we studied the antimetabolic activity of the newly prepared compounds of series B on an immortalized endothelial cell line (TIME), with the same XTT assay as a reflect of the antiproliferative, antiangiogenic activity of these compounds. The results, presented in Table 3, show that the triazole compound with a 3-bromophenyl moiety (2.6), one of the most efficient compounds on tumor cells, is also the most efficient in reducing the proliferation of their companion endothelial cells. It is noteworthy to mention the specific activity of compound 1.25 on the TIME endothelial cells and on two different types of medulloblastoma, but not on the other studied types of cancers. Similarly, product 1.28 is the only one which seems to act on two different models of renal clear cell carcinoma, a highly vascularized tumor, as well as on the TIME endothelial cells. 3D experiments of tumor cell proliferation and migration, vascular angiogenesis (spheroids) are likely to confirm these preliminary positive results. Besides, a highly favorable selectivity was observed for compounds 1.13 and 1.25 towards HD-MB03 medulloblastoma cells compared to TIME cells representative of healthy cells.

Conclusions

Two series of new synthetic azole compounds were synthesized and evaluated for their in vitro antiproliferative activity against different aggressive cancer models (two medulloblastoma, two head and neck squamous cells and two kidney cancer cell lines) for which no efficient and longstanding treatment is currently available. These derivatives consist of arylamino-1,2,4-triazoles, substituted in position 5 of the triazole either by a 3-pyridyl (series A) or a phenyl (series B). To the series B were also added some Nbenzoylthiourea precursors. The synthetic pathway for series A derivatives was adapted by taking advantage of the amide bond weakness of the corresponding nicotinamide thiourea precursors. The structure-activity relationship allowed a first stepwise optimization. The comparison of the corresponding IC50 values showed i) the necessity to keep the 1,2,4-triazole core towards its replacement by other azoles, ii) a clear beneficial effect of the 3-bromophenylamino moiety in position 3 of the triazole for both Series A (compound 4.6) and Series B (compound 2.6), on all cell lines tested. Moreover, preliminary results on the antimetabolic activity of the compounds of series B were obtained on an immortalized endothelial cell line (TIME), demonstrating the antiangiogenic activity of these compounds. Altogether, these results show that the 5-aryl-3-phenylamino-1,2,4-triazole structure possesses fair anticancer activities and that derivatives with a 3-bromophenyl moiety seem to be the most promising ones.

Figure 1 .

 1 Figure 1. a. Structure of cores I, II, and anticancer compound III; b. Design of the arylamino-3-pyrido-1,2,4-triazole fusion scaffold c. Structure of the targeted analogs.

47 Scheme 1 .

 471 Scheme 1. Synthesis of 1,2,4-triazole precursors N-(phenylcarbamothioyl)nicotinamides 1.1-l0 and N-(phenylcarbamothioyl) benzamides 1.11-32.

Scheme 2 .Scheme 3 .

 23 Scheme 2. Synthesis of targeted N,3-diphenyl-1H-1,2,4-triazol-5-amines 2.1-10; and cleavage of the amide bond to form 3-pyridyl derivatives 3.1-12.Consequently, an alternative synthetic pathway was developed for nicotinamide derivatives, which takes advantage of this weak amide bond, (Scheme 3). By using harsh reaction conditions, N-(phenylcarbamothioyl)benzamide precursors 1.11-32 could be hydrolyzed in hot alkaline solution (aq. 2M NaOH, 85 °C) leading to substituted 1-phenylthioureas 3.1-12. S-methylation of these compounds, followed by condensation with previously prepared nicotinohydrazide[START_REF] Goher | Development of novel liver X receptor modulators based on a 1,2,4-triazole scaffold[END_REF] allowed efficient triazole cyclization leading to the desired N-phenyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-amines 4.1-12 (Scheme 4).

Scheme 4 . 7 . 1 - 12 , 5 , 6 ,

 4711256 Scheme 4. Synthesis of targeted 1,2,4-triazole analogues 1,3,4-oxadiazole 5, 1,3,4-thiadiazole 6 and 1H-pyrazole 7. In summary, 3-pyridyl-containing analogs 4.1-12, 5, 6, and 7 constitute Series A, aimed at studying both the influence of the modulations on the phenylamino moiety and of the 1,2,4-triazole. The phenyl-based triazole derivatives 2.1-10 and N-(phenylcarbamothioyl)benzamide precursors 1.11-32 are gathered in Series B which was produced to evaluate the replacement of the 3pyridyl ring by phenyl and assess the activity potential of the (phenylcarbamothioyl)benzamide precursors. The related N-(phenylcarbamothioyl)nicotinamides 1.1-10 have not been evaluated due to their fragility towards nucleophiles (namely the instability of the amide bond), which suggests poor pharmacological properties for these molecules in cellulo.

3 3 . 5 ± 0. 3 triazoles 2 . 1 - 2 .

 353212 All values are the mean of 3 experiments ± SD.The overall modest results observed prompted us to study another promising related fusion scaffold: N,3-diphenyl-1,2,4-triazol-5-amine (Scheme 2, X = CH), as well as some synthetic intermediates 1.11-29. Only intermediates bearing a phenyl moiety were chosen for evaluation because of the lability of the amide bond observed for the pyridine analogues. Thus, these 10 new 3,5-diarylamino-1,2,4-10 and the 14 N-benzoylthioureas 1.11-29-consisting of compounds of series B -were subjected to biological evaluation of their antiproliferative potential under the same assay conditions on the six previously described cancer cell lines. The results for the 25 compounds of series B are displayed in Table

  carbamothioyl)benzamide (0.335 g, 1 mmol) and hydrazine hydrate (0.146 mL, 3 mmol) to afford the title compound as a yellow powder (227 mg, 72%).[START_REF] Bray | GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF] H NMR (400 MHz, TFA-d1): δ 8.98 (d, J = 2.5 Hz, 1H), 8.07 (dd, J = 8.8, 2.6 Hz, 1H), 7.89 -7.83 (m, 2H), 7.70 (dd, J = 14.1, 8.3 Hz, 2H), 7.62 (t, J = 7.7 Hz, 2H). 13 C NMR (101 MHz, TFA-d1): δ 153.31, 152.88, 149.02, 137.08, 136.64, 134.81, 133.16, 132.25 (2C), 129.17 (2C), 122.89, 120.97, 118.39. LCMS (m/z): [M+H] + calc. for C14H11ClN5O2 .40, 153.77, 149.53, 146.72, 146.50, 132.81, 129.44, 127.52, 123.82, 121.22, 120.72, 116.69, 111.76, 63.91, 14.71. LC-MS (m/z): [M+H] + calc. for C15H16N5O + , 282.13; Found: 282.27. HPLC (λ280): Purity 99.5%; tR: 1.258 min (method 1). .90 (s, 0.5H), 12.85 (s, 0.5H), 9.40 (s, 0.5H), 9.17 (d, J = 6.6 Hz, 1.5H), 8.75 -8.51 (m, 1H), 8.29 (d, J = 8.5 Hz, 1H), 7.53 (dt, J = 36.4, 6.5 Hz, 1H), 7.19 (d, J = 35.4 Hz, 2H), 6.50 (d, J = 46.8 Hz, 1H), 2.23 (d, J = 8.0 Hz, 6H). 13 C NMR (50 MHz, TFA-d1): δ 154.15 , 147.09 , 146.28 , 145.58 , 144.05 , 142.12 , 135.06 , 133.72 , 130.97 (2C), 128.33, 124.23 (2C), 21.76 (2C). LC-MS (m/z): [M+H] + calc. for C15H16N5 + , 266.14; Found: 266.27. HPLC (λ280): Purity 99.7%; tR: 1.250 min (method 1).

	N-(2-chlorophenyl)-5-(pyridin-3-yl)-1,2,4-triazol-3-amine (4.4) Synthesized following the general procedure B using 1-(2-
	chlorophenyl)-2-methylisothiouronium iodide (0.328 g, 1 mmol) and nicotynohydrazide (0.137 g, 1 mmol) to afford the title compound
	as a mixture of 1-H triazole and 2-H triazole tautomers (1:1). White powder (171 mg, 63%). 1 H NMR (400 MHz, DMSO-d6): δ 14.12 N-(2,5-dichlorophenyl)-5-(pyridin-3-yl)-1,2,4-triazol-3-amine (4.10) Synthesized following the general procedure B using 1-(2,5-
	(s, 0.5H), 12.61 (s, 0.5H), 9.19 (d, J = 2.1 Hz, 1H), 8.89 (s, 0,5H), 8.62 (d, J = 4.8 Hz, 1H), 8.31 (dt, J = 7.9, 2.0 Hz, 2,5H), 7.61 -7.27 dichlorophenyl)-2-methylisothiouronium iodide (0.363 g, 1 mmol) and nicotynohydrazide (0.137 g, 1 mmol) to afford the title
	(m, 3H), 6.97 (t, J = 7.6 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6): δ 156.45, 153.32, 149.91, 146.84, 137.16, 133.02, 129.41, 127.94, compound as a mixture of 1-H triazole and 2-H triazole tautomers (1:1). Beige powder (189 mg, 62%). 1 H NMR (200 MHz, DMSO-
	127.45, 123.91, 122.25, 120.95, 118.83. LCMS (m/z): [M+H] + calc. for C13H11ClN5 + , 272.07; Found: 272.27. HPLC (λ280): Purity d6): δ 14.25 (s, 0.5H), 12.66 (s, 0.5H), 9.16 (dd, J = 2.3, 0.9 Hz, 1H), 8.77 -8.17 (m, 4H), 7.50 (dd, J = 20.2, 7.8 Hz, 2H), 6.99 (d, J =
	98.9%; tR: 1.275 min (method 1). 8.0 Hz, 1H). 13 C NMR (50 MHz, DMSO-d6): δ 150.65, 150.28, 146.84, 138.51 (2C), 133.16, 132.29, 130.69, 124.08, 121.29 (2C),
	119.04, 117.51. LC-MS (m/z): [M+H] + calc. for C13H10Cl2N5 + , 306.03; Found: 306.27. HPLC (λ280): Purity 95.6%; tR: 1.358 min
	N-(2-bromophenyl)-5-(pyridin-3-yl)-1,2,4-triazol-3-amine (4.5) Synthesized following the general procedure B using 1-(2-(method 1).
	bromophenyl)-2-methylisothiouronium iodide (0.372 g, 1 mmol) and nicotynohydrazide (0.137 g, 1 mmol) to afford the title compound
	as a mixture of 1-H triazole and 2-H triazole tautomers (1:1). White powder (211 mg, 67%). 1 H NMR (400 MHz, DMSO-d6): δ 14.11 N-(3,4-dichlorophenyl)-5-(pyridin-3-yl)-1,2,4-triazol-3-amine (4.11) Synthesized following the general procedure B using 1-(3,4-
	(s, 0.5H), 12.69 (s, 0.5H), 9.18 (d, J = 2.2 Hz, 1H), 8.88 -8.47 (m, 2H), 8.36 -8.14 (m, 2H), 7.61 (dd, J = 8.0, 1.5 Hz, 1H), 7.57 -7.45 dichlorophenyl)-2-methylisothiouronium iodide (0.363 g, 1 mmol) and nicotynohydrazide (0.137 g, 1 mmol) to afford the title
	compound as a mixture of 1-H triazole and 2-H triazole tautomers (7:3). White powder (180 mg, 59%). 1 H NMR (400 MHz, DMSO-
	d6): δ 14.10 (s, 0.7H), 13.13 (s, 0.3H), 9.87 (d, J = 60.4 Hz, 1H), 9.15 (s, 1H), 8.77 -8.55 (m, 1H), 8.29 (d, J = 7.9 Hz, 1H), 7.94 (s,
	1H), 7.67 -7.39 (m, 3H).
	N-(4-chlorophenyl)-5-(pyridin-3-yl)-1,2,4-triazol-3-amine (4.8) Synthesized following the general procedure B using 1-(4-
	Hz, 1H), 8.46 (ddd, J = 8.4, 3.6, 2.2 Hz, 2H), 7.99 (dtd, J = 15.5, 8.2, 1.5 Hz, 2H), 7.71 -7.64 (m, 1H). 13 C NMR (400 MHz, TFA-d1): chlorophenyl)-2-methylisothiouronium iodide (0.329 g, 1 mmol) and nicotynohydrazide (0.137 g, 1 mmol) to afford the title compound
	δ 153.64 , 149.13 , 147.23 , 145.44 , 142.87 , 142.25 , 138.96 , 132.70 , 130.89 , 130.25 , 129.20 , 129.00 , 125.90 . LC-MS (m/z): as a mixture of 1-H triazole and 2-H triazole tautomers (1:1). Beige powder (192 mg, 71%). 1 H NMR (400 MHz, DMSO-d6): δ 13.99
	[M+H] + calc. for C13H11N6O2 + , 283.09; Found: 283.13. HPLC (λ280): Purity 100.0%; tR: 1.192 min (method 1). (s, 0.5H), 12.95 (s, 0.5H), 9.62 (d, J = 67.9 Hz, 1H), 9.15 (dd, J = 8.0, 2.3 Hz, 1H), 8.65 (s, 1H), 8.29 (dt, J = 7.9, 2.0 Hz, 1H), 7.70 -
	7.43 (m, 3H), 7.32 (d, J = 13.3 Hz, 2H). 13 C NMR (101 MHz, TFA-d1): δ 153.91, 147.05, 146.54, 145.50, 142.11, 138.59, 133.71,
	N-(2-ethoxyphenyl)-5-(pyridin-3-yl)-1,2,4-triazol-3-amine (4.3) Synthesized following the general procedure B using 1-(2-133.14 (2C), 130.85, 128.16 (2C). LC-MS (m/z): [M+H] + calc. for C13H11ClN5 + , 272.07; Found: 272.33. HPLC (λ280): Purity 99.4%; tR:
	ethoxyphenyl)-2-methylisothiouronium iodide (0.338 g, 1 mmol) and nicotynohydrazide (0.137 g, 1 mmol) to afford the title compound 1.233 min (method 1).

[START_REF] Bray | GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF]

H NMR (400 MHz, TFA-d1): δ 7.70 (d, J = 7.6 Hz, 2H), 7.57 (t, J = 7.4 Hz, 1H), 7.47 (t, J = 7.5 Hz, 2H), 7.32 -7.10 (m, 4H), 2.31 (s, 3H).

[START_REF] Jones | Heterocyclylamino-substituted triazoles as modulators of rho-associated protein kinase[END_REF]

C NMR (101 MHz, TFA-d1): δ 153.68, 153.

16

, 142.97, 135.73, 133.48 (2C), 133.06, 131.92 (2C), 128.96 (2C), 126.95 (2C), 124.08, 21.74. LCMS (m/z): [M+H] + calc. for C15H15N4 + , 251.13; Found: 251.27. HPLC (λ280): Purity 99.1%; tR: 1.108 min (method 1).

N-(2-methoxy-5-nitrophenyl)-5-phenyl-1H-1,2,4-triazol-3-amine

(2.8)

Synthesized following the general procedure A using N-((2methoxy-5-nitrophenyl)carbamothioyl)benzamide (0.331 g, 1 mmol) and hydrazine hydrate (0.146 mL, 3 mmol) to afford the title compound as a yellow powder (205 mg, 66%).

[START_REF] Bray | GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF]

H NMR (400 MHz, TFA-d1): δ 8.79 (d, J = 2.6 Hz, 1H), 8.23 (dd, J = 9.2, 2.6 Hz, 1H), 7.95 -7.89 (m, 2H), 7.73 (t, J = 7.5 Hz, 1H), 7.62 (t, J = 7.7 Hz, 2H), 7.16 (d, J = 9.2 Hz, 1H), 4.09 (s, 3H).

[START_REF] Jones | Heterocyclylamino-substituted triazoles as modulators of rho-associated protein kinase[END_REF]

C NMR (101 MHz, TFA-d1): δ 158.

30, 152.90, 152.69, 142.96, 136.74 , 132.18 (2C)

, 129.16 (2C), 128.04, 125.

21, 121.66, 118.23, 112.93, 58.38

. LCMS (m/z):

[M+H] + calc. for C15H14N5O3 + , 312.11; Found: 312.13. HPLC (λ280): Purity 99.7%; tR: 1.100 min (method 1).

N-(2-chloro-5-nitrophenyl)-5-phenyl-1H-1,2,4-triazol-3-amine (2.9) Synthesized following the general procedure A using N-((2-chloro-5-nitrophenyl)+ , 316.06; Found: 316.13. HPLC (λ280): Purity 99.1%; tR: 1.158 min (method 1). N-(2,4-dichlorophenyl)-5-phenyl-1H-1,2,4-triazol-3-amine (2.10) Synthesized following the general procedure A using N-((2,4dichlorophenyl)carbamothioyl)benzamide (0325 g, 1 mmol) and hydrazine hydrate (0.146 mL, 3 mmol) to afford the title compound as a white powder (247 mg, 81%).

[START_REF] Bray | GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF]

H NMR (400 MHz, TFA-d1): δ 7.77 -7.70 (m, 2H), 7.61 (t, J = 7.5 Hz, 1H), 7.55 -7.44 (m, 4H), 7.33 (dd, J = 8.6, 2.3 Hz, 1H).

[START_REF] Jones | Heterocyclylamino-substituted triazoles as modulators of rho-associated protein kinase[END_REF]

C NMR (101 MHz, TFA-d1): δ 153.78, 152.98, 138.21, 136.10, 133.07, 132.99, 132.01, 131.94 (2C), 131.08, 129.06, 128.83 (2C), 122.98. LCMS (m/z): [M+H] + calc. for C14H11Cl2N4 + , 305.04; Found: 305.20. HPLC (λ280): Purity 99.7%%; tR: 1.150 min (method 1). N-phenyl-5-(pyridin-3-yl)-1,2,4-triazol-3-amine (4.1) Synthesized following the general procedure B using 2-methyl-1phenylisothiouronium iodide (0.294 g, 1 mmol) and nicotynohydrazide (0.137 g, 1 mmol) to afford the title compound as a mixture of 1-H triazole and 2-H triazole tautomers (1:1). Beige powder (130 mg, 55%). 1 H NMR (400 MHz, DMSO-d6): δ 13.93 (s, 0.5H), 12.86 (s, 0.5H), 9.43 (d, J = 83.3 Hz, 1H), 9.16 (s, 1H), 8.64 (s, 1H), 8.30 (dt, J = 8.0, 2.0 Hz, 1H), 7.59 (m, J = 8.1 Hz, 3H), 7.27 (s, 2H), 6.86 (d, J = 30.5 Hz, 1H). 13 C NMR (101 MHz, TFA-d1): δ 154.09, 147.08, 146.36, 145.56, 142.11, 135.31, 133.035 (2C), 132.10, 130.92, 128.24, 126.865 (2C) . LC-MS (m/z): [M+H] + calc. for C13H11N5 + , 238.11; Found: 238.27. HPLC (λ280): Purity 99.5%; tR: 1.475 min (method 1). N-(2-nitrophenyl)-5-(pyridin-3-yl)-1,2,4-triazol-3-amine (4.2) Synthesized following the general procedure B using 2-methyl-1-(2nitrophenyl)isothiouronium iodide (0.339 g, 1 mmol) and nicotynohydrazide (0.137 g, 1 mmol) to afford the title compound as an orange powder (144 mg, 51%). 1 H NMR (400 MHz, TFA-d1): δ 9.71 (d, J = 1.9 Hz, 1H), 9.38 (dt, J = 8.4, 1.6 Hz, 1H), 9.15 (d, J = 5.7 as a mixture of 1-H triazole and 2-H triazole tautomers (3:7). Beige powder (135 mg, 48%). 1 H NMR (400 MHz, DMSO-d6): δ 14.03 (s, 0.3H), 12.44 (s, 0.7H), 9.17 (d, J = 2.2 Hz, 1H), 8.64 (d, J = 30.6 Hz, 1H), 8.42 (s, 0.7H), 8.31 (dt, J = 8.1, 2.0 Hz, 1.7H), 8.16 (s, 0.3H), 7.50 (s, 1.3H), 6.98 (dd, J = 31.1, 9.0 Hz, 3H), 4.14 (q, J = 6.4 Hz, 2H), 1.41 (t, J = 6.9 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6): δ 156(m, 1H), 7.44 -7.33 (m, 1H), 6.92 (t, J = 7.7 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6): δ 156.53, 153.52, 149.90, 146.82, 138.32, 133.02, 132.69, 128.55, 127.27, 123.91, 123.18, 119.36, 111.84. LC-MS (m/z): [M+H] + calc. for C13H11BrN5 + , 316.02; Found: 316.13. HPLC (λ280): Purity 98.0%; tR: 1.275 min (method 1). N-(3-bromophenyl)-5-(pyridin-3-yl)-1,2,4-triazol-3-amine (4.6) Synthesized following the general procedure B using 1-(3bromophenyl)-2-methylisothiouronium iodide (0.372 g, 1 mmol) and nicotynohydrazide (0.137 g, 1 mmol) to afford the title compound as a mixture of 1-H triazole and 2-H triazole tautomers (1:1). Beige powder (227 mg, 72%). 1 H NMR (400 MHz, DMSO-d6): δ 14.04 (s, 0.5H), 13.06 (s, 0.5H), 9.65 (s, 1H), 9.15 (dd, J = 5.6, 2.2 Hz, 1H), 8.66 (d, J = 8.4 Hz, 1H), 8.29 (dt, J = 8.0, 2.0 Hz, 1H), 7.91 (t, J = 2.0 Hz, 1H), 7.67 -7.41 (m, 2H), 7.21 (t, J = 8.2 Hz, 1H), 7.00 (s, 1H). 13 C NMR (101 MHz, DMSO-d6): δ 160.46, 150.79, 150.27, 146.85, 143.46, 133.22, 130.69, 124.05, 123.35, 121.89, 121.63, 117.97, 114.81. LC-MS (m/z): [M+H] + calc. for C13H11BrN5 + , 316.02; Found: 316.33. HPLC (λ280): Purity 96.2%; tR: 1.233 min (method 1). 5-(pyridin-3-yl)-N-(p-tolyl)-1,2,4-triazol-3-amine (4.7) Synthesized following the general procedure B using 2-methyl-1-(ptolyl)isothiouronium iodide (0.308 g, 1 mmol) and nicotynohydrazide (0.137 g, 1 mmol) to afford the title compound as a mixture of 1-H triazole and 2-H triazole tautomers (6:4). White powder (110 mg, 44%). 1 H NMR (400 MHz, DMSO-d6): δ 14.15 (s, 0.4H), 12.70 (s, 0.6H), 9.18 (m, J = 2.1 Hz, 1.6H), 8.63 (s, 1H), 8.52 -8.02 (m, 2.4H), 7.74 -7.24 (m, 3H). 13 C NMR (101 MHz, DMSO-d6): δ 153.81, 147.08, 146.89, 145.48, 142.13, 139.72, 134.50, 133.39, 131.38, 131.16, 130.85, 130.42, 128.19. LC-MS (m/z): [M+H] + calc. for C13H10Cl2N5 + , 306.03; Found: 306.27. HPLC (λ280): Purity 98.2%; tR: 1.383 min (method 1). N-(3,5-dimethylphenyl)-5-(pyridin-3-yl)-1,2,4-triazol-3-amine (4.9) Synthesized following the general procedure B using 1-(3,5dimethylphenyl)-2-methylisothiouronium iodide (0.322 g, 1 mmol) and nicotynohydrazide (0.137 g, 1 mmol) to afford the title compound as a mixture of 1-H triazole and 2-H triazole tautomers (1:1). White powder (180 mg, 68%). 1 H NMR (400 MHz, DMSO-d6): δ 1313 C NMR (101 MHz, DMSO-d6): δ 160.21, 150.89, 150.40, 146.87, 142.08, 133.28, 131.03, 130.54, 124.09, 123.28, 120.14, 116.67, 116.12. LC-MS (m/z): [M+H] + calc. for C13H10Cl2N5 + , 306.03; Found: 306.27. HPLC (λ280): Purity 98.5%; tR:

Table 1 .

 1 Conditions survey for the 1,2,4-triazole formation.

	Entry	Equiv. of N2H4•H2O	Solvent	Temperature (°C)	Time (h)	Conversion (%)
	1	5	EtOH	reflux	24	<5
	2	5	EtOH	0 to r.t.	24	13
	3	5	CHCl3	0 to r.t.	24	28
	4	5	1,4-dioxane	0 to r.t.	24	35
	5	-	No solvent	0 to r.t.	24	19
	6	3	CHCl3	0 to r.t.	24	61
	7	3	1,4-dioxane	0 to r.t.	24	73
	8	3	1,4-dioxane	0 to r.t.	48	75
	9	3	1,4-dioxane	30	24	30
	10	5	1,4-dioxane	30	24	12

Table 2 .

 2 Antiproliferative activities of synthetic compounds 4.1-12, 5, 6, and 7 of series A.

	IC50 (µM)

Table 3 .

 3 Antiproliferative activities of synthetic compounds of series B (compounds 2.1-10 and some of 1.11-29).All values are the mean of 3 experiments ± SD.

	IC50 (µM)

Acknowledgments

This work was supported by the French government, managed by the French National Research Agency under the project Investissements d'Avenir UCAJEDI (# ANR-15-IDEX-01), CNRS, and Cancéropôle PACA. This study was also partly conducted in the Centre Scientifique de Monaco Research Program, funded by the Government of the Principality of Monaco.