Conclusion et perpectives

L’étude réalisée sur ce réarrangement allylique 1,3 a permis de déterminer que cette réaction avait lieu selon un mécanisme de type SN1. Par conséquent, tout substrat énantiomériquement pur conduit à la formation d’un alcool rérrangé sous forme racémique. La géométrie de la double liaison formée est E quelle que soit celle de la double liaison initiale (Schéma 103).

E 29 30 OH R R OH * Schéma 103

L’étude sur l’utilisation d’un acide de Brønsted chiral de type BINOL n’a pas permis de réaliser le réarrangement de façon énantiosélective.

Une troisième étude peut être envisagée afin d’essayer d’effectuer un réarrangement diastéréosélectif. En particulier, il serait intéressant d’étudier le réarrangement de composés biaryliques tels que 2Cg (Schéma 104), par extension des résultats obtenus à partir de 2Bd et

2Cc (Schéma 85). La configuration de l’axe biarylique pourrait en effet influencer celle de

l’alcool dans le produit 13c, par différenciation des deux faces du carbocation allylique intermédiaire.

Le composé (±)-(S,aR)-13c pourrait ensuite conduire, après hydrogénation et cyclodéshydration, à la dibenzoxépine (±)-(S,aR)-1Cg.

MeO MeO OMe OTES OH H _MeO MeO OMe OTES MeO MeO OMe OTES OH (±)-(S,aR)-2Cg (±)-(S,aR)-1Cg (±)-(S,aR)-13c Acide de Brønsted 1. Hydrogénation 2. Cyclodéshydration H₂O -H₂O H₂O OH₂ O OMe MeO MeO Schéma 104

Conclusion générale

Les travaux décrits dans cette thèse ont consisté à synthétiser des dibenzoxépines analogues du N-acétylcolchinol. Cette famille de molécules est connue pour inhiber la polymérisation de la tubuline en microtubules en se liant au niveau du site de la colchicine.

Dans un premier temps, une nouvelle voie de synthèse plus adaptée à l’obtention d’analogues variés a été établie. Quatre séries de dibenzoxépines ont ainsi été obtenues. Ces molécules présentent des structures variées que ce soit au niveau de la chaîne latérale située sur le cycle médian ou encore sur le motif biphényle. Nous nous sommes attachés à déterminer la configuration des alcools benzyliques obtenus lors de l’étape d’introduction de la chaîne latérale et à proposer un mécanisme pour expliquer la diastéréosélectivité observée. La configuration relative des biaryles pontés a également été vérifiée ainsi que le mécanisme relatif à leur formation.

L’activité sur tubuline de ces dibenzoxépines a été évaluée et des relations structure-activité ont également été proposées. Plusieurs molécules ont montré une activité proche de celle calculée pour le N-acétylcolchinol voire légèrement supérieure pour un des analogues méthoxylé. Ces résultats sont encourageants pour l’évaluation de l’activité sur cellules cancéreuses de ces molécules. Ces tests, actuellement en cours, nous permettront de déterminer la cytotoxicité de nos dibenzoxépines par rapport à la colchicine et au

N-acétylcolchinol. Suivant ces résultats, d’autres analogues pourront être envisagés afin

d’améliorer les activités in vitro déjà obtenues.

Une partie méthodologie est également rapportée. Elle a consisté en l’étude de la réactivité d’alcools allyliques benzyliques en milieu acide. Les conditions réactionnelles ont tout d’abord été mises au point en présence d’acide trifluoroacétique. L’étude de la stéréospécificité de ce réarrangement allylique 1,3 au moyen d’alcools optiquement purs a permis de déterminer le mécanisme de cette réaction. Cette réaction a également été testée sur toute une gamme d’acides de Brønsted dont certains chiraux dérivés du BINOL mais aucune énantiosélectivité n’a été observée. Une approche diastéréosélective à partir de biaryles chiraux est envisagée afin de déterminer si la configuration de l’axe biarylique peut avoir une influence sur la stéréochimie des produits réarrangés formés.

Partie expérimentale

General: reactions were performed in oven-dried glassware under a positive pressure of

argon. They were magnetically stirred and employed rubber septa. Reactions were monitored by thin layer chromatography (TLC, Analtech 1000 μm or 250 μm), by NMR (ALS300, ALS400, DRX500 Brüker instruments) or GC/MS (Thermo-Finnigan Mat 95XL spectrometer). TLC plates were visualized by exposure to UV light (254 nm). All chemicals were purchased from commercial vendors and used as is, unless otherwise specified. Flash column chromatography was performed using normal phase silica gel (60 Å, particle size 60-200 μm). Chemical shifts (δ) are reported with respect to chloroform for ¹H NMR (δ = 7.26 ppm) and for ¹³C NMR (δ = 77.16 ppm) or methanol for ¹H NMR (δ = 3.31 ppm) and for ¹³C NMR (δ = 49.0 ppm). Data are reported as follows: chemical shift, apparent multiplicity (s = singulet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet), coupling constant J in Hz, integration. Assigments are based on 2D NMR by analogy between major diastereoisomers. NOESY spectrum were performed by Dr. B. Fenet and coll. High-resolution mass spectra were measured using chemical ionization (CI), electrospray ionization (ESI) or electronic impact (EI) mode by Dr. D. Bouchu and coll.. Flash column chromatography purifications were performed with GeduranP^® Si 60 from Merck (particle size 60-200 μm), and PTLC on Analtech 1000 μm or 250 μm. Enantiomeric excess were measured with Waters 1525 – 2487 HPLC using a CHIRACEL OD-H column. All reaction solvents were freshly distilled from appropriate drying agents (THF, dioxane: sodium / benzophenone ketyl / nitrogen (g), CH2Cl2: CaH2).

Inhibition of microtubule assembly: The molecule was dissolved in DMSO at various

concentrations and pre-incubated with a solution of tubulin at 37°C for 10 min. The solution was then cooled to 0°C for 5 min to achieve complete tubulin depolymerization. The solution was placed in a temperature-controlled cell at 37°C (microtubule assembly), and the increase in optical density was monitored in a UV spectrophotometer at 350 nm for 1 min. The maximum rate of assembly was recorded and compared with a sample without drug. The IC50

value is the concentration of compound required to inhibit 50% of the rate of microtubule assembly. It was calculated from the effect of several concentrations, and was compared with the IC50 value of colchicine obtained within the same day with the same tubulin preparation. The reported values are an average of three experiments.

General procedures

A - General reduction procedure:

Diisobutylaluminum hydride (4eq) was added to a solution of benzoic ester (1eq) in THF at -78°C, under argon. The mixture was stirred at -78°C then MeOH was added à 0°C. Solvents were removed under vacuum and the residue was dissolved in AcOEt. The organic layer was washed with saturated aq. solution of NaHCO3 then dried over MgSO4, filtered, and evaporated under vacuum. The residue was purified by flash chromatography using cyclohexane and ethyl acetate as the eluents.

B - General iodination procedure:

Silver trifluoroacetate (1.08eq) and iodine (1.05eq) were added in one portion to a solution of the alcohol (1eq) in chloroform at 0°C. After stirring for 15 min at 0°C, the mixture was filtered through celite and washed with a saturated aq. solution of Na2SO3. The organic layer was dried over MgSO4, filtered, and evaporated under vacuum. The residue was purified by flash chromatography using cyclohexane and ethyl acetate as the eluents.

C - General Suzuki-Miyaura coupling procedure:

A sealed tube was charged with the iodoarene (1eq), aryl boronate (1.5eq), Pd(OAc)2

(5%mol), S-Phos (10%mol), Ba(OH)2·8H2O (1.1eq) and dioxane/water (9:1; [aryl halide] = 0.5 to 0.75 M). The tube was sealed and placed in an oil bath preheated at 100°C and stirred for 2.5h to 6h. After cooling to 20°C, the reaction mixture was filtered through celite and MgSO4. The residue was purified by flash chromatography using cyclohexane and ethyl acetate as the eluents.

D - General MnO2 oxidation procedure:

To a solution of benzylic alcohol (1eq) in dichloromethane was added manganese dioxyde (12eq) at 20°C under argon. The mixture was then stirred for 24h.The reaction mixture was filtered through silica gel. The solvent was removed under pressure. The residue was purified by flash chromatography using cyclohexane and ethyl acetate as the eluents.

E - General TPAP/NMO oxidation procedure:

Tetrapropylammonium perruthenate and N-methylmorpholine-N-oxide were added to a solution of alcohol in CH2Cl2 at 20°C, under argon. The mixture was stirred at 20°C during 2h. Water was added then the aqueous layer was extracted with EtOAc. The combined organic layers were washed with a solution of HCl 1N and dried over MgSO4, filtered, and

evaporated under vacuum. The residue was purified by flash chromatography using cyclohexane and ethyl acetate as the eluents.

F - General Grignard addition procedure:

A solution of Grignard reagent (3eq) was added dropwise to a solution of aldehyde in THF at -78°C, under argon. The mixture was stirred at -78°C and was then allowed to warm up to 20°C for 1 to 5h. A saturated aq. solution of NH4Cl was added, the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine and dried over MgSO4, filtered, and evaporated under vacuum. The resulting mixture was purified by flash chromatography using cyclohexane and ethyl acetate as the eluents.

G - General cyclodehydratation procedure:

A solution of HF (48 to 51% aq) was added to a solution of compound in CH3CN at 20°C, and the mixture was stirred for 48h. It was cautiously poured into a saturated aq. solution of NaHCO3 and the aqueous layer was extracted with EtOAc. The combined organic layers were dried over MgSO4 and evaporated under vacuum. The resulting mixture was purified by flash chromatography using cyclohexane and ethyl acetate as the eluents.

H - General procedure for ammonium salts synthesis:

Hydrogen chloride (1M in ether) was added to a solution of dibenzoxepine in ether at 20°C and the mixture was stirred for 1h. The solvent was removed under pressure then the solid was washed with cyclohexane.

I - General procedure for 1,3-allylic rearrangement with TFA:

To a solution of benzylic alcohol in dichloromethane (0.5 M) à 0°C under inert atmosphere, a solution of trifluoroacetic acid (2eq) in dichloromethane (1 M) was added. The mixture was stirred at 0°C for 15-30min. An aq. solution of NaOH 15% was added, the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine and dried over MgSO4, filtered, and evaporated under vacuum.

J - General procedure for 1,3-allylic rearrangement with chiral Brønsted acid:

To a solution of benzylic alcohol in solvent à 0°C under inert atmosphere, phosphonate acid was added. The mixture was stirred at 0°C for 4-48h. An aq. solution of NaOH 15% was added, the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine and dried over MgSO4, filtered, and evaporated under vacuum.