1,3-diaza-4-oxa-[3.3.1]-bicyclic derivatives, their preparation and use as medicines, particularly for the treatment of obesity, diabetes and / or neurodegenerative diseases

Abstract

The present finding refers to compounds having structural formula (I) or an enantiomer, an enantiomeric mixture, or their pharmaceutically acceptable salt (I) wherein R1 and R2 are defined as reported in the description. Furthermore, the finding is directed to the compounds of formula (I) mentioned above, or an enantiomer, an enantiomeric mixture, or their pharmaceutically acceptable salt, for use in medicine, preferably in the treatment of obesity and conditions or diseases related to it, such as diabetes and / or neurodegenerative diseases.

Description

[0001] 1 ,3-DIAZA-4-OXA-[3.3.1]-BICYCLIC DERIVATIVES, THEIR PREPARATION AND USE AS A MEDICAMENT, PARTICULARLY FOR THE TREATMENT OF OBESITY, DIABETES, AND / OR NEURODEGENERATIVE DISEASES

[0002] ★★★★★★★

[0003] DESCRIPTION

[0004] FIELD OF INVENTION

[0005] The present invention relates to 1 ,3-diaza-4-oxa-[3.3.1 ]-bicyclic derivatives or an enantiomer, an enantiomeric mixture, or their pharmaceutically acceptable salt, and their use as a medicament, particularly in the treatment of obesity and of conditions or diseases related to it, such as diabetes and / or neurodegenerative diseases, thanks, inter alia, to the indirect increase in GLP-1 secretion.

[0006] STATE OF THE ART

[0007] The Diels-Alder reaction has long been widely employed in organic synthesis and involves a cycloaddition between a conjugated diene and a dienophile, leading to the formation of substituted cyclohexenes. The Diels-Alder reactions involving at least one heteroatom are collectively called "hetero-Diels-Alder" reactions; specifically, in the "nitroso-Diels-Alder" (NDA) reaction, the nitroso group (R-N=O) acts as the dienophile, and the cycloaddition introduces nitrogen and oxygen at positions 1 -4 of a 1 ,3-diene, forming an oxazine.

[0008] The use of 1 ,2-dihydropyridines as conjugated dienes in the acyl-nitroso-Diels- Alder reaction has not received much attention, despite the advantage of stereoselectively introducing a new carbon-oxygen bond and a new carbon- nitrogen bond into the pyridine framework, resulting in regioselective formation of the corresponding inverse nitroso cycloadduct of general formula (1 ), as shown in Scheme 1 below (PG = protecting group).

[0009] 1 ,2-DHPs can be obtained in an enantioenriched chiral form through the Mannich / Wittig / cycloisomerization reaction sequence (Bo-Shuai Mu et aL, Nature Communications (2021 ), vol. 12, Article number: 2219 https: / / doi.Org / 10.1038 / S41467-021 -22374-y).

[0010] In some studies (Knaus et al., J. Org. Chem, 1985; Streith et aL, Tetrahedron Lett., 1990), it was reported that the rearrangement of Cope [3,3]-hetero of benzoyl- nitroso cycloadducts ((1 ), where R1=H, R2=Ph, PG=COOMe) led to the corresponding 4a,7,8a-tetrahydropyrido[4,3-e]-1 ,4,2-dioxazine ((2), where R2=Ph, Scheme 1 ). However, in these studies, the reaction conditions for the rearrangement were poorly defined (Streith et al.) or required very long reaction times for the rearrangement to occur (up to 15 days, Knaus et aL). Due to the difficulty in their synthesis, despite their potential usefulness for chemical synthesis purposes, dioxazines (2) have not been extensively utilized as “building blocks.” Scheme 1 :

[0011] Incretins are a group of peptide hormones with a variety of physiological functions. They are produced by enteroendocrine cells located in the gastrointestinal tract (particularly in the ileum, colon, and duodenum) in response to nutrient ingestion and are released into the bloodstream. These hormones function to control blood glucose levels in several ways, including: (i) increasing insulin secretion from pancreatic beta cells; (ii) decreasing glucagon secretion from pancreatic alpha cells; (iii) slowing gastric motility and thus gastric emptying, and reducing appetite. The therapeutic relevance of incretins has rapidly increased in recent years, with one of the most studied incretins being GLP-1 (glucagon-like peptide-1 ), produced by L-type enteroendocrine cells (L-cells). GLP-1 integrates signals from nutrients to control food intake, energy absorption, and assimilation: it regulates plasma glucose levels and modulates body weight increase due to fat accumulation. Recently approved drugs that enhance incretin action provide new approaches for the physiological treatment of type 2 diabetes and related issues such as obesity associated with traditional antidiabetic drugs like insulin, sulfonylureas, or thiazolidinediones. Exenatide and liraglutide are first-generation GLP-1 receptor agonists that, being peptides, require parenteral administration once or twice daily. Obesity is a multifactorial disease characterized by excessive fat accumulation that contributes to the development of severe comorbidities, such as type 2 diabetes, cardiovascular diseases, and dyslipidemias. Due to the close correlation between obesity and diabetes (even referred to as an epidemic condition known as "diabesity"), new pharmacological approaches are needed to limit the development of these diseases.

[0012] Recent research has shown that TRP (Transient Receptor Potential) calcium channels, previously known for their role in pain perception, play a crucial role in regulating metabolism. In particular, activation of TRPA1 channels promotes insulin secretion and GLP-1 release.

[0013] Additionally, TRPA1 channels are involved in thermogenesis and the differentiation of brown adipose tissue, thus playing a fundamental role in the pathophysiology of diabetes and obesity.

[0014] Supporting the therapeutic potential of modulating the TRPA1 channel, it has been demonstrated that some activators of this channel, such as cinnamaldehyde, allylisothiocyanate, esperetin, hesperidin, naringenin, and gingerol, prevent weight gain and reduce insulin resistance, highlighting this channel as a promising target for the treatment of diabetes and obesity (Y. Wang ChemMedChem 2023, 18, e202200562).

[0015] Moreover, recently, the class of GLP-1 analog drugs has shown promising results in vitro and in animal models regarding neuroprotective properties and cardiovascular protection (A.F. Batista et al. CNS Drugs, 2019, 33, 209-223). In humans, some evidence suggests that chronic treatment with a GLP-1 analog could reduce the risk of progression from cognitive impairment to overt dementia (K. Seppa et al. Scientific Reports 2019, 9, 15742).

[0016] International patent application WO2018 / 220542 describes 1 ,3-diaza-4-oxa- [3.3.1 ]-bicyclic derivatives useful for treating diabetes, preferably type 2 diabetes, obesity, dyslipidemic syndromes, hepatic steatosis, and regulating satiety.

[0017] Despite this evidence, there is a need for innovative therapies capable of addressing the numerous factors underlying the pathophysiology of diabetes, obesity, and central nervous system disorders.

[0018] Therefore, there is a need for new compounds that act as activators of TRP calcium channels, particularly TRPA1 channels, which could be applied to develop a new therapeutic approach for treating obesity, diabetes, and / or neurodegenerative diseases. Moreover, it is desirable that these new compounds be easily bioavailable and orally administrable.

[0019] The present invention aims to provide new orally administrable compounds that are effective in the treatment and / or prevention of obesity, diabetes, or neurodegenerative diseases. The new compounds of the invention not only correct the main defects underlying diabetes but also offer neuroprotective activity through synergistic mechanisms, which can help prevent the onset of neurodegenerative diseases, such as those associated with diabetes and / or obesity, including Alzheimer’s or Parkinson’s diseases.

[0020] In particular, the invention aims to provide new compounds capable of indirectly increasing GLP-1 secretion, for example, by increasing cytosolic levels of intracellular calcium through the activation of TRPA1 channels.

[0021] Indeed, the compounds of this invention are not GLP-1 analogs but promote its release through indirect mechanisms.

[0022] Another objective of the invention is to provide compounds capable of indirectly increasing GLP-1 secretion, such as by increasing cytosolic levels of intracellular calcium through the activation of TRPA1 channels, that can be administered orally. Additionally, the invention aims to provide a process for preparing such compounds, particularly focusing on defining reaction conditions to achieve the process quickly, simply, and cost-effectively.

[0023] DEFINITIONS

[0024] Unless otherwise defined, all technical terms, notations, and other scientific terms used here are intended to have the meanings commonly understood by those skilled in the art to which this description pertains. In some cases, terms with commonly understood meanings are defined here for clarity and / or for quick reference; the inclusion of such definitions in this description should not be interpreted as representing a substantial difference from what is generally understood in the art.

[0025] The terms “comprising,” “having,” “including,” and “containing” are to be understood as open terms (i.e., meaning “comprising, but not limited to”) and are to be considered as supporting terms such as “to consist essentially of,” “consisting essentially of,” “to consist of,” or “consisting of.”

[0026] For all ranges indicated in the text, figures, and claims of this patent application, it is understood that the endpoints of these ranges are included.

[0027] The terms “obtainable,” “obtained,” “obtainable directly from,” “obtained directly from” are considered equivalent.

[0028] The term “pharmaceutically acceptable salt” refers to those salts that possess the biological efficacy and properties of the salified compound and that do not produce adverse reactions when administered to a mammal, preferably to a human. Pharmaceutically acceptable salts can be inorganic or organic salts; examples of pharmaceutically acceptable salts include, but are not limited to: carbonate, hydrochloride, bromide, sulfate, hydrogen sulfate, citrate, maleate, fumarate, trifluoroacetate, 2-naphthalenesulfonate, and para-toluenesulfonate. Additional information on pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, P. Stahl, C. Wermuth, WILEY- VCH, 127-133, 2008, incorporated herein by reference.

[0029] The acronym “1 ,2-DHP” refers to the compound 1 ,2-dihydropyridine.

[0030] The acronym “1 ,4-DHP” refers to the compound 1 ,4-dihydropyridine.

[0031] The acronym Cbz refers to carbobenzoxy.

[0032] The acronym “JT010” refers to a known calcium channel activator.

[0033] The acronym “A23187” refers to calcimycin, also known as a “calcium ionophore.” The acronym “M2” refers to the compound described in claim 10 of WO20 18220542 A1 , incorporated herein by reference:

[0034] The acronym “(-)-M2” refers to the compound 2-phenyl-1 -((1 S,5R,9S)-9-phenyl-4- oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3-yl)ethan-1 -one, which is not part of the present invention.

[0035] The acronym “(+)-M2” refers to the compound 2-phenyl-1 -((1 R,5S,9R)-9-phenyl- 4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3-yl)ethan-1 -one, which is not part of the present invention.

[0036] The acronym “M14” refers to the compound 2-(thiophen-2-yl)-1 -((1 S*,5S*,9R*)-9- (3-(trifluoromethyl)phenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6-en-3-yl)ethan-1 - one, which is the subject of the present invention whose structure is reported in the experimental part that follows.

[0037] The acronym “M21 ” refers to the compound 2-phenyl-1 -((1 S*,5S*,9R*)-9-(3- (trifluoromethyl)phenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3-yl)ethan-1 -one, which is the subject of the present invention whose structure is reported in the experimental part that follows. The acronym “M21 hydrochloride” or “M21 HCI” refers to the compound (1S*,5S*,9R*)-3-(2-phenylacetyl)-9-(3-(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-5-yl chloride, which is the subject of the present invention.

[0038] The acronym “M23” refers to the compound 1-((1 R*,5S*,9R*)-9-(4- (phenoxy)phenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6-en-3-yl)-2-phenylethan-1- one, which is not part of the present invention.

[0039] M23 has been prepared using a process analogue to that described in the experimental section for the compounds of the invention.

[0040] The acronym “M24” refers to the compound 1-((1S*,5S*,9R*)-9-(4-chlorophenyl)- 4-oxa-1 ,3-diazabicyclo[3.3.1]non-6-en-3-yl)-2-phenylethan-1-one, which is not part of the present invention:

[0041] M24 has been prepared for comparison of biological activity using a process analogue to that described in the experimental section for the compounds of the invention.

[0042] The acronym “M25” refers to the compound 1-((1 R*,5S*)-9-(4-(chloro)phenyl)-4- oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3-yl)-2-(thiophen-2-yl)ethan-1 -one, which is not part of the present invention:

[0043] M25 has been prepared using a process analogue to that described in the experimental section for the compounds of the invention. The acronym “M26” refers to the compound 2-(4-methoxyphenyl)-1 -((1 R*,5S*)-9- phenyl-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6-en-3-yl)ethan-1 -one, which is not part of the present invention

[0044] M26 has been prepared for comparison of biological activity using a process analogue to that described in the experimental section for the compounds of the invention.

[0045] The acronym “M27” refers to the compound 2-(4-hydroxyphenyl)-1 -((1 R*,5S*,9R*)- 9-phenyl-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6-en-3-yl)ethan-1 -one, which is not part of the present invention:

[0046] M27 has been prepared using a process analogue to that described in the experimental section for the compounds of the invention.

[0047] The acronym “M28” refers to the compound 2-phenyl-1 -((1 R*,5S*,9R*)-9-(4- (trifluoromethyl)phenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3-yl)ethan-1 -one, which is part of the present invention whose structure is reported in the experimental part that follows.

[0048] The acronym “M29” refers to the compound 1 -((1 R*,5S*,9R*)-9-(3,5- bis(trifluoromethyl)phenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3-yl)-2- phenylethan-1 -one, which is part of the present invention, whose structure is reported in the experimental part that follows.

[0049] The acronym “M30” refers to the compound 1 -(1 S*,5S*,9R*)-9-(3,5- bis(trifluoromethyl)phenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3-yl)-2- (thiophen-2-yl)ethan-1 -one, which is part of the present invention whose structure is reported in the experimental part that follows. The acronym “M31 ” refers to the compound 4'-((1 R*,5S*,9R*)-3-(2-phenylacetyl)- 4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-9-yl)-[1 ,1 '-biphenyl]-4-carbonitrile, which is not part of the present invention:

[0050] M31 has been prepared using a process analogue to that described in the experimental section for the compounds of the invention.

[0051] The acronym “M32” refers to the compound N-(((2R*,3S*)-3-hydroxy-2-phenyl-3,6- dihydropyridin-1 (2H)-yl)methyl)-2-phenylacetamide, which is not part of the present invention, whose structure is reported in the experimental part that follows. The acronym “M33” refers to the compound 1 -((1 S*,5S*,9R*)-9-(4- isopropylphenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6-en-3-yl)-2-phenylethan-1 - one, which is part of the present invention whose structure is reported in the experimental part that follows.

[0052] The acronym “M34” refers to the compound 2-methyl-1 -((1 S*,5S*,9R*)-9-phenyl- 4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3-yl)propan-1 -one, which is not part of the present invention:

[0053] M34 has been prepared using a process analogue to that described in the experimental section for the compounds of the invention.

[0054] The acronym “M35” refers to the compound 2-phenyl-1 -(1 S*,5S*,9R*)-9-(2- (trifluoromethyl)phenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3-yl)ethan-1 -one, which is part of the present invention whose structure is reported in the experimental part that follows. The acronym “M36” refers to the compound 1 -(1 S*,5S*,9R*)-9-(3-chlorophenyl)-4- oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3-yl)-2-phenylethan-1 -one, which is not part of the present invention:

[0055] M36 has been prepared for comparison of biological activity using a process analogue to that described in the experimental section for the compounds of the invention.

[0056] The acronym “M37” refers to the compound 1 -((1 R*,5S*,9R*)-9-(4-fluoro)-4-oxa- 1 ,3-diazabicyclo[3.3.1]non-6-en-3-yl)-2-phenylethan-1 -one, which is not part of the present invention:

[0057] M37 has been prepared for comparison of biological activity using a process analogue to that described in the experimental section for the compounds of the invention.

[0058] The acronym “M38” refers to the compound 1 -(1 S*,5S*,9R*)-9-(3- isopropylphenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6-en-3-yl)-2-phenylethan-1 - one, which is part of the present invention whose structure is reported in the experimental part that follows.

[0059] The acronym “M40” refers to the compound 3-Phenyl-1 -((5S*)-9-(3- (trifluoromethyl)phenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3-yl)propan-1 -one, which is part of the present invention whose structure is reported in the experimental part that follows. The acronym “M41 ” refers to the compound 1-((5S*)-9-(3-isopropylphenyl)-4-oxa- 1 ,3-diazabicyclo[3.3.1]non-6-en-3-yl)-3-phenylpropan-1 -one, which is part of the present invention whose structure is reported in the experimental part that follows. The acronym “PALM” refers to palmitate.

[0060] The acronym “MG” refers to methylglyoxal.

[0061] The term “physiologically acceptable excipient” refers to a substance with no inherent pharmacological effect that does not produce adverse reactions when administered to a mammal, preferably to a human. Physiologically acceptable excipients are well known in the art and are described, for example, in the Handbook of Pharmaceutical Excipients, sixth edition (2009), incorporated herein by reference.

[0062] The term “composition” as used in this document is intended to include a product comprising the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from the combination of the specified ingredients in the specified amounts. “Pharmaceutically acceptable” means that the carrier, diluent, or excipient must be compatible with the other components of the formulation and not deleterious to the recipient.

[0063] SUMMARY OF THE INVENTION

[0064] The present finding refers to compounds having structural formula (I) or an enantiomer, an enantiomeric mixture, or their pharmaceutically acceptable salt: wherein:

[0065] R1is selected from the group consisting of:

[0066] -a phenyl group substituted with at least one -CF3group, preferably with one or two -CF3groups;

[0067] -a phenyl group substituted with at least one alkyl group C1-C6, either linear or branched;

[0068] -a phenyl group substituted with at least one -CF3group, preferably with one or two -CF3groups, and at least one alkyl group C1-C6, either linear or branched; and R2is selected from the group consisting of:

[0069] (i) phenyl, benzyl, or 2-phenylethyl, optionally substituted with one or more functional groups independently selected from the group consisting of a halogen, a methoxy functional group, and -CF3;

[0070] (ii) isoxazole;

[0071] (iii) pyrrole; in which R’ is H or methyl;

[0072]

[0073] Furthermore, the invention concerns the compounds of formula (I) mentioned above, or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, for use as a medicinal product, particularly for the treatment of obesity, diabetes, and / or neurodegenerative diseases.

[0074] BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The invention will be described below with reference to some examples, provided by way of explanation and not limitation, and illustrated in the attached figures.

[0076] Figure 1 - Figure 1 shows graph 1 showing the increase in intracellular calcium by evaluation of the area under the curve obtained following the analysis of the fluorescence increase kinetics using the Fluo-4 NW assay after treatment with some known compounds and some compounds of the invention. The statistical analysis was carried out by means of a student t-test and the asterisks (*) indicate the degree of significance with respect to the vehicle. Figure 2 - Figure 2A shows graph 2A showing the amount of GLP-1 measured in pg / ml released by STC-1 cells following treatment with some known compounds and some compounds of the invention. Figure 2B shows graph 2B which describes the increase in fluorescence related to the entry of calcium into STC-1 cells following treatment with some known compounds and some compounds of the invention.

[0077] Figure 2C shows graph 2c that describes GLP-1 quantity measured in pg / ml, released by STC1 cells after M21 treatment in the presence or in the absence of TRPA1 channel blocker (A967079).

[0078] Figure 3 - Figure 3 shows graphs 3A, 3B, 3C and 3D, respectively in figures 3A, 3B, 3C and 3D, which show the increase in fluorescence related to the increase in intracellular calcium following treatments carried out on cells with some compounds according to the invention, reported in the presence or absence of the TRPA1 channel blocker (Fluo-4 NW assay).

[0079] Figure 3 also shows the graphs 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 30, 3P, 3Q, 3R, 3S, 3T, 3U, 3V, 3W e 3X, are reported respectively in figures 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 30, 3P, 3Q, 3R, 3S, 3T, 3U, 3V, 3W and 3X, which show the increase in fluorescence related to the increase in intracellular calcium following treatments carried out on cells with some compounds according to the invention and some compounds of reference, reported in the presence or absence of the TRPA1 channel blocker (Fluo-4 NW assay).

[0080] Graph 3Y of Figure 3Y shows the EC50 value of M21 , calculated using GraphPad Prism 8, by setting the Log(agonist) vs response function, to obtain the equation of the curve from which the EC50 is derived.

[0081] Figure 4 - Figure 4 shows the release of incretin in vivo after oral administration of the compound M21 at 40 mg / kg dose in "healthy mice":

[0082] Figure 5 - Figure 5 shows a graph from which the increase in glycated hemoglobin compared to the baseline can be appreciated, with reference to example 4.

[0083] Figure 6 - Figure 6 shows a graph from which the plasma triglyceride values at the end of chronic treatment can be appreciated, with reference to example 4.

[0084] Figure-7 - Figure 7 shows a graph from which the weight gain following chronic treatment can be appreciated compared to baseline, with reference to example 4. Figure 8 - Figure 8 shows the western blot of proteins analyzed with ImageJ in the example 5

[0085] Figure 9 - Figure 9 shows the graph related to the effect of compound M21 on Akt phosphorylation, compared to the effects on Akt caused by the control (basal cell culture without any pharmacological stimulus or stressor, i.e., without PALM or MG), palmitate alone, glyoxal alone, and the combination of palmitate and compound M21 , or methylglyoxal and compound M21 .

[0086] Figure 10 -Figure 10 shows a graph illustrating the effect of compound M21 on caspase-3, compared to the effects on caspase-3 caused by the control (basal cell culture without any pharmacological stimulus or stressor, i.e., without PALM or MG), palmitate alone, glyoxal alone, and the combination of palmitate and compound M21 , or methylglyoxal and compound M21

[0087] Figure 11 - Figure 11 shows a graph illustrating the effect of compound M21 on MAPK (ERK1 / 2) p42 / 44, compared to the effects on caspase-3 caused by the control (basal cell culture without any pharmacological stimulus or stressor, i.e., without PALM or MG), palmitate alone, glyoxal alone, and the combination of palmitate and compound M21 , or methylglyoxal and compound M21 .

[0088] Figure 12: Figure 12 shows the ratio between the nuclear area and the cytoplasmic area expressed as a percentage of the control in response to increasing concentrations of M21 as indicated in Example 6.

[0089] Figure 13: Figure 13 shows the ratio between the nuclear area and the cytoplasmic area expressed as a percentage compared to the control in response to increasing concentrations of glucose and in the presence of 10 μM M21. * p<0.05 compared to control, £ p<0.05 compared to HG30 mM.

[0090] Figure 14: Figure 14 shows the ratio between the nuclear area and the cytoplasmic area expressed as a percentage compared to the control under various experimental conditions. MG (methylglyoxal); P (palmitate); *p<0.05 compared to control, £ p<0.05 compared to MG, $ p<0.05 compared to MG+P.

[0091] Figure 15: Figure 15 shows the percentage change compared to control of intracellular P-Akt concentration normalized to T-Akt and p-Actin. * p<0.05 compared to control, £ p<0.05 compared to P, $ p<0.05 compared to MG. MG: methylglyoxal. P: palmitate.

[0092] Figure 16: Figure 16 shows the percentage change compared to control of intracellular P-Akt concentration normalized to T-Akt and p-Actin. +lns50 mM: cell exposure to insulin at 50 mM for 15 minutes. * p<0.05 compared to control, £ p<0.05 compared to P, $ p<0.05 compared to MG. MG: methylglyoxal. P: palmitate.

[0093] Figure 17: Figure 17 shows the percentage change compared to control of intracellular P-p42 / 44 MAP kinase concentration normalized to T-p42 / 44 MAP kinase and p-Actin. +lns50 mM: cell exposure to insulin at 50 mM for 15 minutes.

[0094] * p<0.05 compared to control, £ p<0.05 compared to P, $ p<0.05 compared to MG. MG: methylglyoxal. P: palmitate.

[0095] Figure 18: Figure 18 shows the percentage change compared to control of intracellular P-p42 / 44 MAP kinase concentration normalized to T-p42 / 44 MAP kinase and p-Actin. +lns50 mM: cell exposure to insulin at 50 mM for 15 minutes.

[0096] * p<0.05 compared to control, £ p<0.05 compared to P, $ p<0.05 compared to MG. MG: methylglyoxal. P: palmitate.

[0097] Figure 19: Figure 19 shows the percentage change compared to control of intracellular Caspase-3 concentration normalized to p-Actin. * p<0.05 compared to control, £ p<0.05 compared to P, $ p<0.05 compared to MG. MG: methylglyoxal. P: palmitate.

[0098] Figure 20: Figure 20 shows the percentage variation compared to control of intracellular Caspase-3 and p-Actin concentrations. +lns50 mM: cell exposure to insulin at 50 mM for 15 minutes. * p<0.05 compared to control, £ p<0.05 compared to MG. MG: methylglyoxal. P: palmitate.

[0099] Figure 21 : Figure 21 shows the percentage change compared to control of neuronal glucose utilization under various experimental conditions. * p<0.05 compared to control; £ p<0.05 compared to MG; & p<0.05 compared to MG+Palmitate. MG: methylglyoxal.

[0100] Figure 22: A: Figure 22A shows cell viability obtained using the MTT reagent after exposure to increasing concentrations of M21 . B: Figure 22B shows cell viability obtained using the MTT reagent after exposure to AP25-35 and in combination with M21. *p<0.05, **p<0.01 , ***p<0.005.

[0101] Figure 23: A: Figure 23A shows IL-6 release, and B: Figure 23B shows IL-10 release after exposure to LPS / TNFa and LPS / TNFa in combination with M21. *p<0.05, **p<0.01 , ***p<0.005.

[0102] Figure 24: A: Figure 24A shows TNFa release, B: Figure 24B shows IL-6 release, and C: Figure 24C shows IL-10 release after exposure to AP25-35 and in AP25-35 in combination with M21. *p<0.05, **p<0.01 , ***p<0.005.

[0103] Figure 25: Figure 25 shows MTT expressed as a percentage variation compared to control after exposure to increasing concentrations of M21 .

[0104] Figure 26: A: Figure 26A shows insulin concentration in the culture medium with 2 mM glucose after exposure to M21 at increasing concentrations. B: Figure 26B shows the percentage change of insulin concentration in the culture medium (response to 2 mM glucose compared to 11 mM glucose) after exposure to increasing doses of M21 . *p<0.05 compared to control.

[0105] Figure 27: Figure 27 shows the percentage change of insulin concentration in the culture medium (response to 16.7 mM glucose compared to 3.3 mM glucose) after exposure to increasing doses of M21 . *p<0.05 compared to control.

[0106] DETAILED DESCRIPTION

[0107] From a chemical standpoint, the compounds of formula (I) according to the present invention are 1 ,3-diazo-4-oxa-[3.3.1 ]-bicycles derivatives. Hence, the present invention relates to a compound of formula (I), or an enantiomer, an enantiomeric mixture, or their pharmaceutically acceptable salt: in which R1is selected from the group consisting of:

[0108] -a phenyl substituted with at least one -CF3group, preferably one or two CF3groups;

[0109] -a phenyl substituted with at least one C1-C6linear or branched alkyl group;

[0110] -a phenyl substituted with at least one -CF3group, preferably one or two CF3groups, and at least one C1-C6linear or branched alkyl group; and in which R2is selected from a group consisting of:

[0111] (i) phenyl, benzyl or 2-phenylethyl optionally substituted by one or more functional groups independently selected from a group consisting of a halogen, a methoxy and -CF3;

[0112] (ii) isoxazole;

[0113] (iii) pyrrole; (xxi) .

[0114] In a preferred embodiment, R1is a phenyl substituted with a CF3group; according to a further preferred aspect, the CF3group is located in positions 2 or 3 (in ortho (o-) or meta (m-)positions of the phenyl respectively, with respect to the core of the molecule).

[0115] In another preferred embodiment, R1is a phenyl substituted with two -CF3groups; according to a further preferred aspect, the -CF3groups are located in position 3 and 5 (meta (m-) positions of the phenyl with respect to the core of the molecule). In a preferred embodiment, R1is a phenyl substituted with a -CF3group; according to a further preferred aspect, the -CF3group is located in position 2 or 3 (in ortho (o-) or meta (m-) positions of the phenyl with respect to the core of the molecule). In another embodiment, R1is a phenyl substituted with at least one C1-C6alkyl group, preferably isopropyl, sec-butyl, and / or tert-butyl, more preferably isopropyl. According to a further preferred aspect, R1is a phenyl substituted with at least one C1-C6alkyl group, preferably isopropyl, sec-butyl, and / or t-butyl, preferably located in position 3 or 4 (respectively in meta (m-) or para (p-) positions of the phenyl relative to the core of the molecule) more preferably isopropyl in position 3 or 4.

[0116] According to a further preferred aspect, R1is a phenyl substituted with at least one -CF3group, preferably one or two CF3groups, and at least one C1-C6linear or branched alkyl group, preferably isopropyl, sec-butyl, and / or t-butyl.

[0117] In a form of embodiment of compounds of formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof described herein, R2may preferably be a phenyl, benzyl, or 2-phenylethyl, and said phenyl, benzyl, or 2-phenylethyl may optionally be substituted with one or more functional groups independently selected from: a halogen, a methoxy group, and -CF3.

[0118] In a preferred embodiment, R2may be an unsubstituted phenyl, benzyl, or 2- phenylethyl.

[0119] In another preferred embodiment, R2may be a phenyl, benzyl, or 2-phenylethyl where said phenyl, benzyl, or 2-phenylethyl are substituted with a single functional group selected from: halogen, methoxy, and -CF3; more preferably, such functional group selected from: halogen, methoxy, and -CF3may be in the para (p-) or meta (m-) position of the aromatic ring.

[0120] In another preferred embodiment, when R2is a phenyl, benzyl, or 2-phenylethyl substituted with a halogen, said halogen may preferably be selected from chlorine and fluorine.

[0121] In another preferred embodiment, R2may be a phenyl, benzyl, or 2-phenylethyl wherein said phenyl, benzyl, or 2-phenylethyl are substituted with two -CF3functional groups.

[0122] In another preferred embodiment, R2may be a phenyl, benzyl, or 2-phenylethyl wherein said phenyl, benzyl, or 2-phenylethyl are substituted with a phenyl substituted with an alkyl chain, preferably substituted with a isopropyl.

[0123] In another embodiment, R2may have a structure formula selected from the group consisting of: in which R’ is H or methyl.

[0124] In another preferred embodiment, R2can have a structural formula selected from the group consisting of: The structural formulas herein indicated as (vii), (viii) and (ix) are referred to an indole, wherein the nitrogen is optionally substituted with a methyl group.

[0125] In a preferred embodiment, when R2has a structural formula selected from (vii), (viii), (ix) and (x) as defined above, R' can preferably be a hydrogen. More preferably, when R2has a structural formula selected from (vii), (ix) and (x) as defined above, R' can preferably be a hydrogen.

[0126] According to a particularly preferred aspect, R2is selected from the group consisting of: (i) phenyl, benzyl or 2-phenylethyl, optionally substituted by one or more functional groups independently selected from the group consisting of a halogen, a methoxy functional group and -CF3;

[0127] According to a further preferred embodiment, R1is selected from the group consisting of: phenyl replaced by at least one -CF3group, preferably by one or two CF3groups, or phenyl replaced by at least one linear or branched C1-C6alkyl group; and R2is selected from the group consisting of:

[0128] (i) phenyl, benzyl or 2-phenylethyl, optionally substituted by one or more functional groups independently selected from the group consisting of a halogen, a methoxy functional group and -CF3; (vi)

[0129] According to a particularly preferred embodiment, R1is phenyl replaced by one or two -CF3groups, and R2is benzyl or 2-phenylethyl or (xi)

[0130] According to a further particularly preferred embodiment, R1is phenyl replaced by at least one linear or branched C1-C6alkyl group, preferably isopropyl, and R2is benzyl or 2-phenyl-ethyl or (xi)

[0131] According to a further particularly preferred embodiment, R1is phenyl replaced by at least one linear or branched C1-C6alkyl group, preferably isopropyl, and R2is benzyl or 2-phenyl-ethyl or

[0132] In a particularly preferred embodiment, the compounds of formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof can be selected from those having the following formulas: Even more preferably, the compound of formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof can have the following formula:

[0133] M21

[0134] In this structure, the R1substituent is a phenyl substituted at position 3 with a -CF3group and the R2substituent is an unsubstituted benzyl; this compound corresponds to the structure indicated in this description as M21 .

[0135] Compounds of formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof described herein, are prepared with a procedure in which a 4a,7,8,8a-tetrahydropyrido[4,3-e]-1 ,4,2-dioxazine having formula (2): where R1and R2are as defined in reference to structure (I) and PG is a protective group comprising a carboxyl functional group (COO-), such as for example CO2Me, CO2Et or Cbz, is reacted with a hydride selected from the group consisting of: lithium triethyl borohydride (LiBHEt3), sodium triethyl borohydride (NaBHEt3), and lithium tetrahydroaluminate (LiAIH4), in the presence of tetrahydrofuran, at a temperature between -20°C and room temperature for a time between 15 minutes and 4 hours. In a preferred embodiment, the process can be carried out at a temperature of 0°C (± 1 °C). In another preferred embodiment the process can be carried out at room temperature: by standard room temperature (SAT, Standard Ambient Temperature) we mean the temperature of 25°C (± 1 °C), as it is known in the chemical sector.

[0136] In a preferred embodiment, the hydride that reacts with the dioxazine may be lithium triethyl borohydride (LiBHEt3or LiTEBH); lithium triethyl borohydride is a hydride available on the market and known by the trade name Super-hydride®.

[0137] In another preferred embodiment, the process of the invention can take place in the presence of a palladium-based catalyst and comprising at least one triphenylphosphine group. Advantageously, this catalyst can be selected from the group consisting of: tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) and bis(triphenylphosphine)palladium(ll) dichloride ((PPh3)2PdCl2). More preferably, the catalyst can be (PPh3)2PdCl2.

[0138] In one embodiment, the process of the synthesis can take place using the LiBHEt3hydride, in the absence of a catalyst and at a temperature of 0°C (± 1 °C). In another embodiment, the process can take place in the absence of a catalyst and at room temperature. In another embodiment, the process can take place in the presence of a catalyst and at a temperature of 0°C (± 1 °C).

[0139] The reaction time can vary between approximately 15 minutes and approximately 4 hours depending on the other reaction conditions (especially temperature and presence or absence of catalyst) and the hydride used. Preferably, the time employed can be between about 20 minutes and about 60 minutes when LiBHEt3is used, regardless of the presence of the catalyst. NaBHEt3is less effective and, when used in the process, longer reaction times are required, preferably between 2 hours 30 min and 3 hours 30 min, depending on whether the catalyst is present or not.

[0140] The nitrogen-bound PG protecting group of tetrahydropyridine plays an important role in the preparation of 1 ,3-diaza-4-oxa-[3.3.1 ]-bicyclic derivatives with the process according to the invention. The inventors have determined that the protecting group must be of a carbamate nature to give rise to the intramolecular formation of the bond with the nitrogen atom present on the oxazole ring of dioxazine, according to the mechanism shown in reaction scheme 3 of WO2018 / 220542, here incorporated by reference. For this to happen the protecting group must include a carboxyl group (COO-). As an example, some appropriate protecting groups may be -COOMe, -COOPh, -Cbz.

[0141] In one embodiment, the process of the synthesis can also include a phase, prior to the reaction of the dioxazine of formula (2) with the hydride, in which the dioxazine itself is obtained by rearrangement of the [3,3]-sigmatropic type starting from the corresponding inverse cycloadduct. The cycloadducts corresponding to the dioxazines of formula (2) which react in the process of the synthesis have the formula (1 ) shown in the following reaction scheme 2, where R1and R2have the meanings previously defined for the compounds of formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof and for the dioxazines of formula (2).

[0142] Schema 2:

[0143] Although this reaction is known to the state of the art, the inventors have been able to define the conditions that allow the reaction to take place effectively and with reasonable reaction times. Therefore, the rearrangement of the cycloadduct according to the present invention takes place in an organic solvent, preferably dichloroethane, at a temperature of 75°C (± 2°C) and in the presence of CuCI as a catalyst. Preferably, the amount of CuCI can be between 18 mol% and 22 mol%, more preferably it can be equal to 20 mol%.

[0144] The process of the present invention also allows the compounds of the invention to be obtained in their enantioenriched form.

[0145] The synthesis of the compounds of the invention in enantioenriched form is based on the proline-catalyzed synthesis of the corresponding chiral 1 ,2-DHPs.

[0146] Once the 1 ,2-DHP precursors of the compounds of the invention have been obtained in an enantioenriched form, the reactions described above of nitroso Diels-Alder cycloaddition, rearrangement to dioxazines, and reduction are carried out, as exemplified in the experimental part.

[0147] In another aspect, the invention concerns compounds having structural formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, where R1and R2are defined as above, for use as a medicament.

[0148] The inventors have in fact found that the compounds of structural formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, where R1and R2are defined as reported above, are capable of increasing the concentration of intracellular calcium, through the activation of TRPA1 channels, increasing the in vitro secretion of GLP-1. The compounds of the invention or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, in fact, act as activators of the TRP calcium channels, in particular of the TRPA1 channels: thanks to this action, they can be used in particular in the prevention and / or treatment of obesity, diabetes, preferably type 2 diabetes, in the management of appetite and / or the sense of satiety, dyslipidemia, metabolic diseases and hepatic steatosis. Furthermore, these compounds can also be used for the treatment and / or prevention of neurodegenerative diseases, such as those associated with diabetes, obesity, Alzheimer's and Parkinson's.

[0149] According to a preferred aspect, the compounds of the invention or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof are used in the treatment of diabetes, preferably type 2 diabetes.

[0150] Furthermore, the compounds of the invention or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof can be used in the treatment of some diabetes-related pathologies, such as dyslipidemic syndromes. The term "dyslipidemic syndromes" (or "dyslipidemias") refers generically to states in which the quantity of lipids (in particular cholesterol, triglycerides and / or phospholipids) in the blood circulation is altered compared to the physiological condition; typically in dyslipidemias the quantity of lipids is increased (hypercholesterolemia, hypertriglyceridemia, hyperphospholipidemia), but there are also dyslipidemias in which the quantity of lipids is lower than in physiological conditions. A typical complication of dyslipidemic syndromes is represented by hepatic steatosis (commonly called "fatty liver"), in which an intracellular accumulation of lipids occurs at the level of the liver tissue: hepatic steatosis can be treated using the compounds of the present invention or their salts pharmaceutically acceptable. Furthermore, the compounds of the invention or their pharmaceutically acceptable salts are also used to regulate the sense of satiety or appetite.

[0151] Another object of the invention is to provide a compound of formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, preferably the hydrochloride salt, which is capable of promoting the secretion of GLP-1 in an indirect manner, in order to allow oral administration, unlike current GLP-1 analogues which must be administered by injection. The oral administration is in fact far superior to the injective administration due to its simpler management and superior patients’ compliance.

[0152] A particularly preferred compound is M21 .

[0153] The invention also concerns a pharmaceutical composition comprising a compound of formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, preferably the hydrochloride salt, and at least one pharmaceutically acceptable excipient.

[0154] The pharmaceutical composition comprising a compound of formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, preferably the hydrochloride salt, described herein, may be prepared using a physiologically acceptable excipient which is considered safe and effective and may be administered to an individual without causing undesirable biological effects or unwanted interactions.

[0155] Furthermore, the invention provides a pharmaceutical composition comprising a compound of formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, preferably the hydrochloride salt, and at least one pharmaceutically acceptable excipient for use in the prevention and / or treatment of a pathology or condition selected from the group consisting of: obesity, diabetes, preferably type 2 diabetes, dyslipidemic syndromes, metabolic diseases, hepatic steatosis, regulation of the sense of satiety and / or appetite, neurodegenerative diseases, preferably those associated with diabetes, obesity, Alzheimer's and Parkinson's.

[0156] According to a further preferred aspect, the pharmaceutical composition comprising a compound of formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, preferably the hydrochloride salt, or a pharmaceutically acceptable salt and at least one pharmaceutically acceptable excipient can be administered orally and / or parenterally, preferably orally. A particularly preferred composition is a composition comprising the compound M21 and at least one pharmaceutically acceptable excipient.

[0157] Said composition can advantageously be used as a neuroprotector.

[0158] The compounds of formula (I) or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof according to the present invention, the process for their preparation, the pharmaceutical compositions comprising them and their use in the prevention and / or treatment of obesity, diabetes, preferably type 2 diabetes, dyslipidemic syndromes, hepatic steatosis, in the regulation of the sense of satiety and / or appetite, in the prevention and / or treatment of neurodegenerative diseases, preferably those associated with diabetes, obesity, Alzheimer's and Parkinson's, conceived as described herein, are susceptible to numerous modifications and variations, all falling within the scope of the inventive concept; furthermore, all the details may be replaced by other equivalent elements whose correspondence is known to the person skilled in the art.

[0159] Furthermore, it is to be understood that the characteristics of the embodiments described with reference to one aspect of the invention are to be considered valid also with regard to the other aspects of the invention, even if not explicitly repeated. The invention will now be illustrated by some examples whose purpose is absolutely not to be understood as limiting the scope of protection.

[0160] EXAMPLES

[0161] Experimental part:

[0162] Legend: 1 ,2-DHP = 1 ,2-dihydropyridine; 1 ,4-DHP = 1 ,4-dihydropyridine

[0163] Materials and methods:

[0164] All reactions carried out were monitored by thin layer chromatography (TLC) on Merck TLC silica gel 60 F254 aluminum foil. The detection of the compounds was performed with a UV lamp (254 nm) and / or by chemical detection by immersion in a solution consisting of: 48mL of EtOH, 1.16g of vanillin, 1.25mL of H2SO4cone. And 0.375mL of cone. AcOH. Purifications by flash chromatography were performed on a silica gel column (Kieselgel 40, 0.040-0.063 mm; Merk) or using the Biotage® isolara semi-automatic column. Reactions involving compounds sensitive to air or humidity were carried out in an inert atmosphere (Ar or N2), using glassware kept in an oven at 1 10°C, cooled under vacuum and with glass or silicone stoppers. Liquids and solutions sensitive to air or moisture were transferred via syringe / cannula. Magnesium sulfate (MgSO4) and sodium sulfate (Na2SO4) were used as dehydrating agents. All solvents and solutions used for the reaction treatments (NaCI s.s., NH4CI s.s., AcOEt, dichloromethane, etc) were purchased from Sigma-Aldrich. Unless otherwise specified, the necessary solvents and reagents were used as supplied by the manufacturer (Sigma-Aldrich, Fluka, Alfa Aesar, TCI) without further purification. In fact, anhydrous solvents and reagents such as tetrahydrofuran (THE), dimethylformamide (DMF) and pyridine are commercial and guaranteed by the manufacturers (Fluka and Sigma-Aldrich) and are packaged in bottles with "Sure-Seal" septa. All the deuterated solvents (CDCI3and CD3CN) were purchased from Sigma-Aldrich while the other anhydrous solvents or reagents necessary for the reactions were anhydrified by distillation or through the use of molecular sieves such as UOP 3A and were kept in atmosphere inert sealed. The THF was refluxed over Na / benzophenone in an argon atmosphere and the dichloromethane (DCM) was stored in a Schlenk containing molecular sieves activated via heat-gun vacuum heating / cooling cycles. All compounds were characterized using one-dimensional (1H and13C) and two- dimensional (HSQC, NOESY or COSY) NMR spectroscopic techniques. The NMR spectra were recorded on Bruker Avance II 400 spectrometers. Chemical Shift values are expressed in parts per million (ppm) and in reference to tetramethylsilane (TMS), while coupling constants (J) are expressed in Herz (Hz ). The abbreviations used for peak assignment are: s= singlet, bs = broad singlet, d= doublet, t= triplet, dd= double doublet, q= quartet, m= multiplet, bd= broad doublet. The melting points (mp) were determined on a kofler apparatus and have not be corrected. HRESIMS were acquired in positive ion mode with Orbitrap high- resolution mass spectrometer (Thermo, San Jose, CA, USA), equipped with an H- ESI source. Enantiomeric excesses (ee) were measured with a Waters 600E HPLC equipped with a Varian Prostar 325 detector using a Daicel® Chiralpak AD- H (250 X 4.6 mm).

[0165] Synthesis of 1 ,2-dihydropyridines:

[0166] General procedure: A solution of pyridine (1.5 eq) in anhydrous THF, in an inert atmosphere, is cooled to -20°C. The organometallic reagent (1 eq) is added, after which methyl chloroformate (1 eq) is slowly added dropwise. The reaction mixture is left under stirring for 20 minutes at -20°C then the cooling is interrupted and it is left under stirring for another 20 minutes at room temperature. Water is added, the organic portion is separated from the aqueous one and then the aqueous phase is extracted with three portions of diethyl ether. The combined organic portions are washed with saturated solution of Na2SO4, H2O, NaHCO3, NaCI. The organic phase is then dehydrated over MgSO4or Na2SO4, filtered and finally the solvents are evaporated under reduced pressure at 30°C. The reaction crude is then purified by separation on a chromatographic column.

[0167] Methyl- 2-(3-(trifluoromethyl)phenyl)pyridine-1(2H)-carboxylate (precursor of M14, M21 and M40):

[0168] According to the general procedure a three-necked flask was loaded with pyridine (37.5 mmol, 3.03 mL), (3-(trifluoromethyl)phenyl) magnesium bromide (25.0 mmol, 1 .0 M in THF, 25.0 mL), methyl chloroformate ( 25.0 mmol, 1 .9 mL) and anhydrous THF (37.5 mL). After the standard workup, the reaction crude was purified with flash chromatography (Hexane / Diethyl ether: 8.5 / 1 .5; Rf=0.32) giving the desired product as a yellow oil (3.53 g, yield: 50%).

[0169] 1 H NMR (400 MHz, CDCI3) δ 7.73-7.39 (m, 4H, 1 ,2+1 ,4-DHP), 7.04-6.97* and 6.89-6.82 (m, 1 H, 1 ,4-DHP), 6.95* and 6.75 (d, J = 7.9 Hz, 1 H, 1 ,2-DHP), 6.12- 5.99 (m, 1 H), 5.95* and 5.79 (d, J = 5.9 Hz, 1 H, 1 ,2-DHP), 5.71 -5.60 (m, 1 H, 1 ,2- DHP), 5.39-5.24 (m, 1 H, 1 ,2-DHP), 4.93 (d, J = 31 .7 Hz, 2H, 1 ,4-DHP), 4.30-4.22 (m, 1 H, 1 ,4-DHP), 3.84 (d, J = 1 .1 Hz, 3H, 1 ,4-DHP), 3.76 (d, J = 12.3 Hz, 3H, 1 ,2- DHP).

[0170] *Major Rotamer

[0171] Methyl 2-(4-(trifluoromethyl)phenyl)pyridine-1(2H)-carboxylate (precursor of M28):

[0172] F

[0173] According to the general procedure a three-necked flask was loaded with pyridine (25.8 mmol, 2.1 mL), anhydrous THF (37.5 mL), (4-trifluoromethyl)magnesium bromide (17.2 mmol), methyl chloroformate (17.2 mmol, 1.3 mL). After the standard workup, the reaction crude was purified with flash chromatography (Hex / Et2O: 9 / 1 , Rf=0.53) giving the desired product as a yellow oil (1.78 g, yield: 25%).1H NMR (400 MHz, CDCI3) δ 7.60-7.36 (m, 4H, 1 ,2-DHP + 1 ,4-DHP), 6.99- 6.96 (m, 1 H, 1 ,2-DHP), 6.85 (s, 1 H, 1 ,4-DHP), 6.76 (d, = 7.17 Hz, 1 ,2-DHP), 6.08-6.04 (m, 1 H, 1 ,2-DHP), 5.93 (d, J = 5.38 Hz, 1 H, 1 ,2-DHP), 5.79 (s, 2H, 1 ,4- DHP), 5.69-5.65 (m, 1 H, 1 ,2-DHP), 5.34-5.27 (m, 1 H, 1 ,2-DHP), 4.93 (d, J = 29.9 Hz, 2H, 1 ,4-DHP), 4.25 (m, 1 H, 1 ,4-DHP), 3.85 and 3.78* (s, 3H, 1 ,2-DHP), 3.73 (s, 3H, 1 ,4-DHP).

[0174] *Major Rotamer

[0175] Methyl 2-(3,5-bis(trifluoromethyl)phenyl)pyridine-1 (2H)-carboxylate

[0176] (precursor of M29 and M30):

[0177] According to the general procedure a three-necked flask was loaded with pyridine (30 mmol, 2.373 g, 2.43 mL), anhydrous THF (30 mL), (3,5- bis(trifluoromethyl)phenyl)magnesium bromide (20 mmol , 20 mL), methyl chloroformate (20 mmol, 1 .89 g, 1 .52 mL). After the standard workup, the reaction crude was purified via flash chromatography (Hexane / Et2O: 9 / 1 , Rf=0.5), giving the desired product as a yellow oil (5.51 g, 15.7 mmol, yield: 65.7%) .

[0178] 1H NMR (400 MHz, CDCI3) δ 7.95-7.71 (m, 3H), 6.94* (d, J = 7.8 Hz, 1 H), 6.76 (d, J = 7.8 Hz, 1 H), 6.15-5.98 (m, 2H), 5.88* (d, J = 5.7 Hz, 1 H), 5.71-5.59 (m, 1 H), 5.39-5.26 (m, 1 H), 3.82* (s, 3H), 3.77 (s, 3H).

[0179] *Major Rotamer

[0180] Methyl 2-(4-isopropylphenyl)pyridine-1 (2H)-carboxylate (Precursor of M33):

[0181] According to the general procedure a three-necked flask was loaded with pyridine (1.5 eq, 10.5 mmol, 830 mg, 1.0 mL), anhydrous THF (10.5 mL), (4- isopropylphenyl)magnesium bromide (7.0 mmol, 7.0 mL ), methyl chloroformate (1 .0 eq, 7.0 mmol, 661 .5 mg, 0.6 mL). After the standard workup, the reaction crude was purified via flash chromatography (Hexane / AcOEt: 9 / 1 ; Rf=0.3) giving the desired product as a yellow oil (751 .0 mg, yield: 41%)

[0182] 1H NMR (400 MHz, CDCI3) δ 7.44 (d, J = 7.2 Hz, 1 H), 7.37-7.30 (m, 1 H), 7.26- 7.19 (m, 2H), 6.98 (d, J= 7.9 Hz, 1 H), 6.77* (d, J= 7.8 Hz, 1 H), 6.10-5.99 (m, 1 H), 5.94-5.90* (m, 1 H), 5.78-5.66 (m, 1 H), 5.39-5.26 (m, 1 H), 3.78 (s, 3H), 2.93 (hept, J = 6.9 Hz, 1 H), 1 .28 (d, J = 7.0 Hz, 6H)

[0183] *Major Rotamer

[0184] Methyl 2-(2-(trifluoromethyl)phenyl)pyridine-1(2H)-carboxylate (precursor of M35):

[0185] According to the general procedure a three-necked flask was loaded with pyridine (1.5 eq; 37.5 mmol, 3.0 mL), anhydrous THE (25 mL), (2- (trifluoromethyl)phenyl)magnesium bromide (25.0 mmol, 25 mL ), methyl chloroformate (1 .0 eq, 25.0 mmol, 2.0 mL). After the standard workup, the reaction crude was purified via flash chromatography (Hexane / AcOEt: 7 / 3; Rf=0.57), giving the desired product as a yellow oil (3.3 g, 11 .77 mmol, yield: 47%).

[0186] 1H NMR (400 MHz, CDCI3) δ 7.61 -7.29 (m, 4H, 1 ,2-DHP+1 ,4-DHP), 7.13-7.1 1 (d, J=7.63 Hz, 1 H, 1 ,2-DHP), 6.84-6.86* e 6.99-7.01 (m, 1 H, 1 ,4-DHP), 6.19-6.10 (m, 1 H, 1 ,2-DHP), 5.83-5.79 (m, 1 H, 1 ,2-DHP), 5.66-5.59 (m, 1 H, 1 ,2-DHP), 5.26-5.19 (m, 1 H, 1 ,2-DHP), 4.91 (d, J= 27.3 Hz, 2H, 1 ,4-DHP), 4.58 (m,1 H, 1 ,4-DHP), 3.85 (d, J=1.1 Hz , 3H, 1 ,4-DHP), 3.60 (d, J=19.4 Hz, 3H, 1 ,2-DHP).

[0187] *Major rotamer

[0188] Methyl 2-(3-isopropylphenyl)pyridine-1 (2H)-carboxylate (precursor of M38 and M41): According to the general procedure a three-necked flask was loaded with pyridine (1.5 eq; 37.5 mmol, 3.0 mL), anhydrous THF (25 mL), (3- isopropylphenyl)magnesium bromide (25.0 mmol, 25 mL), methyl chloroformate (1.0 eq, 25.0 mmol, 2.0 mL). After the standard workup, the reaction crude was purified by flash chromatography (Hexane / Diethyl Ether: 9 / 1 ; Rf=0.44) giving the desired product as a yellow oil (2.77 g, 10.8 mmol, yield: 35%).

[0189] 1H NMR (400 MHz, CDCI3) δ7.15-7.32(m,4H, 1 ,2-DHP+1 ,4-DHP), 6.93 (d,1 H, 1 ,2- DHP), 6.73 (d, J=7.56 Hz, 1 H, 1 ,4-DHP), 5.99-6.06 (m, 1 H, 1 ,2-DHP), 5.87 (d, J= 6.06 Hz, 1 H, 1 ,2-DHP), 5.68-5.69 (m,1 H, 1 ,2-DHP), 5.26-5.33 (m,1 H, 1 ,2-DHP), 3.73-3.76* (s, 3H, 1 ,2-DHP), 2.89 (m, 1 H, 1 ,2-DHP), 1 .23 (d, J=6.98, 6H, 1 ,2-DHP). *Major rotamer

[0190] Synthesis of reverse cycloadducts of the Nitroso Diels-Alder (NDA) reaction: General procedure: In a three-necked flask in an inert atmosphere, NaIO4(1.1 eq, 1 .3 eq or 1 .5 eq) is slowly added in small portions at 0°C into a reaction mixture consisting of 1 ,2-DHP (1.0 eq) and hydroxamic acid (1.1 eq, 1.3 eq, or 1.5 eq) appropriate in MeOH (0.1 M). Water is then added (MeOH / H2O= 15:1 ) and the resulting reaction mixture is left under vigorous stirring at room temperature until the 1 ,2-DHP is completely converted. The reaction is stopped by adding water until a sticky precipitate is obtained, DCM is added to obtain a two-phase mixture. The organic portion is separated and then the aqueous portion is extracted with 3 portions of DCM, the combined organic portions are dehydrated over MgSO4, filtered and then the solvents are evaporated under reduced pressure at 30°C. The reaction raw material is subjected to chromatographic separation, crystallization or trituration.

[0191] Methyl ( 1S*,4H*,6H*)-3-(2-(thiophen-2-yl)acetyl)-6-(3-

[0192] (trifluoromethyl)phenyl)-2-oxa-3,5-diazabicyclo[2.2.2]oct-7-ene-5- carboxylate (precursor of M14):

[0193]

[0194] According to the general procedure a three-necked flask was loaded with methyl 2-(3-(trifluoromethyl)phenyl)pyridine-1 (2H)-carboxylate (4.38 mmol 1 ,2- DHP, 1.46 mg), N-hydroxy-2-(thiophen-2-yl)acetamide (1 .3 eq, 5.68 mmol, 892.8 mg), NaIO4(1.3 eq, 5.68 mmol, 1.214 g), MeOH (50 mL), H2O (3.0 mL). After the standard workup, the reaction crude was purified by flash chromatography (Hexane / AcOEt 7:3, Rf=0.24) providing the desired product as a yellow amorphous solid (1.3 g, yield: 67.6%); for the characterization of the product, a preparative TLC was performed (Hexane / AcOEt 7:3+1 % TEA).

[0195] 1H NMR (400 MHz, CD3CN) δ 7.62-7.57 (m, 1 H), 7.54-7.44 (m, 3H), 7.27 (dd, J = 5.2, 1.2 Hz, 1 H), 6.98-6.84 (m, 4H), 6.18-6.13 (m, 1 H), 5.27 (d, J = 3.6 Hz, 1 H), 5.14-5.10 (m, 1 H), 3.94-3.80 (m, 2H), 3.62 (s, 3H).

[0196] 13C NMR (101 MHz, CD3CN) δ 139.2, 136.2, 133.6, 131.5, 130.8 (q, JC-F = 31.83 Hz), 130.1 , 128.6, 128.1 , 127.6, 126.6, 126.1 , 125.5, 125.4, 124.4, 124.3, 123.9, 76.0, 60.5, 53.8, 34.8.

[0197] Methyl (1S*,4R*, 6R*)-3-(2-phenylacetyl)-6-(3- (trifluoromethyl)phenyl)-2-oxa- 3,5-diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (precursor of M21): According to the general procedure a three-necked flask was loaded with methyl 2-(3-(trifluoromethyl)phenyl)pyridine-1 (2H)-carboxylate (2.652 mmol 1 ,2-DHP, 880.2 mg), N-hydroxy -2-phenylacetamide (1 .3 eq, 3.44 mmol, 521 .37 mg), NaIO4(1.3 eq, 3.44 mmol, 735.6 mg), MeOH (30 mL), H2O (1.8 mL). After the standard workup, the reaction crude was purified via flash chromatography (Hexane / AcOEt: 8 / 2, Rf=0.23) providing the desired product as a yellow amorphous solid (756.7 mg, yield: 66.2%); for the characterization a preparative TLC was carried out on the product (Hexane / AcOEt 7:3+1 % TEA).

[0198] 1H NMR (400 MHz, CD3CN) δ 7.59 (d, J = 7.7 Hz, 1 H), 7.53-7.48 (m, 2H), 7.44 (d, J= 8.0 Hz, 1 H), 7.34-7.19 (m, 5H), 6.93-6.76 (m, 2H), 6.14-6.06 (m, 1 H), 5.23 (d, J = 3.7 Hz, 1 H), 5.13-5.08 (m, 1 H), 3.72-3.48 (m, 5H).

[0199] 13C NMR (101 MHz, CD3CN) δ 174.8, 151.4, 139.3, 135.4, 133.7, 131.6, 131.1 , 130.6, 130.2, 129.4, 129.3, 128.6, 127.7, 126.7, 125.5, 124.4, 124.0, 75.9, 60.6, 60.0, 53.8, 40.7, 30.3.

[0200] Methyl (1S*,4R*, 6R*)-3-(2-phenylacetyl)-6-(4-(trifluoromethyl)phenyl)-2-oxa- 3,5-diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (precursor of M28):

[0201] According to the general procedure a three-necked flask was loaded with methyl 2-(4-(trifluoromethyl)phenyl)pyridine-1 (2H)-carboxylate (1.47 g, 5.2 mmol), N- hydroxy-2-phenylacetamide ( 1 .0 g, 6.8 mmol), MeOH (40.0 mL), NaIO4(747.5 mg, 6.8 mmol), H2O (2.3 mL) (7h). After the standard workup, the reaction crude was purified via flash chromatography (Hexane / AcOEt: 7 / 3; Rf = 0.38) giving the desired product as a yellow amorphous solid (997.3 mg, yield: 44%).

[0202] 1H NMR (400 MHz, CD3CN) δ 7.63 (d, J = 8.13 Hz, 2H), 7.37 (d, J = 7.93 Hz, 2H), 7.33-7.21 (m, 5H), 6.86 (s, 2H), 6.10 (s, 1 H), 5.23 (m, 1 H), 5.11 -5.10 (m, 1 H), 3.71 - 3.53 (m, 5H).13C NMR (101 MHz, CD3CN) δ 175.1 , 142.8, 135.8, 134.0, 131.0, 130.5 (q, JCF = 32.07 Hz), 129.7, 129.0, 128.8, 128.1 , 127.1 , 126.5, 126.5, 124.4, 76.3, 61.0, 54.2, 41.1.

[0203] Methyl (1S*,4R*, 6R*)-6-(3,5-bis(trifluoromethyl)phenyl)-3-(2-phenylacetyl)-2- oxa-3,5-diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (precursor of M29):

[0204] According to the general procedure a three-necked flask was loaded with methyl 2-(3,5-bis(trifluoromethyl)phenyl)pyridine-1 (2H)-carboxylate (7.10 mmol, 2.5 g), MeOH (55.0 mL) , N-hydroxy-2-phenylacetamide (9.23 mmol, 1.4 g), NaIO4(9.23 mmol, 1.02 g), H2O (4.0 mL). After the standard workup, the reaction crude was purified via semi-automated chromatography with Biotage Isolera (Hexane / AcOEt: 7 / 3; Rf=0.4), giving the desired product as a light yellow solid (1 .77 g, 3.5 mmol , yield: 49.2%)(m.p. 145-148°C).1H NMR (400 MHz, CD3CN) δ 7.92 (s, 1 H), 7.79 (s, 2H), 7.34-7.18 (m, 5H), 6.95-6.76 (m, 2H), 6.12-6.04 (m, 1 H), 5.34-5.30 (m, 1 H), 5.18-5.12 (m, 1 H), 3.74-3.47 (m, 5H).

[0205] 13C NMR (101 MHz, CD3CN) δ 175.0, 157.4, 141.3, 135.3, 134.2, 132.1 (q, JCF=33.2 Hz), 130.7, 129.4, 128.5, 128.2, 127.8, 125.9, 123.2, 122.8, 122.7, 75.6, 60.3, 54.0, 40.8.

[0206] Methyl (1S*,4R*, 6R*)-6-(3,5-bis(trifluoromethyl)phenyl)-3-(2-(thiophen-2- il)acetyl)-2-oxa-3,5-diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (precursor of M30):

[0207] According to the general procedure a three-necked flask was loaded with methyl 2-(3,5-bis(trifluoromethyl)phenyl)pyridine-1 (2H)-carboxylate (2.68 g, 7.64 mmol), MeOH (60.0 mL) , N-hydroxy-2-(thiophen-2-yl)acetamide (1.51 g, 9.94 mmol), NaIO4(9.94 mmol, 1 .09 g), H2O (5.0 mL). After the standard workup, the reaction crude was purified via semi-automated chromatography with Biotage Isolera (Hexane / AcOEt: 7 / 3; Rf=0.5) giving the desired product as a yellow solid (1 .508 g, 3.0 mmol, yield: 39.2%)(m.p. 146-150°C).

[0208] 1H NMR (400 MHz, CD3CN) δ 7.93 (s, 1 H), 7.80 (s, 2H), 7.28 (d, J = 4.0 Hz, 1 H), 6.98-6.77 (m, 4H), 6.15-6.08 (m, 1 H), 5.36 (d, J= 3.7 Hz, 1 H), 5.22-5.16 (m, 1 H), 3.94-3.77 (m, 2H), 3.61 (d, J = 52.2 Hz, 3H).

[0209] 13C NMR (101 MHz, CD3CN) δ 173.2, 141.2, 136.2, 134.1 , 132.0 (q, CF =32.2 Hz), 128.5, 128.3, 128.2, 127.7, 126.1 , 125.8, 123.1 , 122.7, 122.7, 120.4, 75.7, 60.3, 53.9, 34.9.

[0210] Methyl (1S*,4R*, 6R*)-6-(4-isopropylphenyl)-3-(2-phenylacetyl)-2-oxa-3,5- diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (precursor of M33):

[0211] According to the general procedure a three-necked flask was loaded with methyl 2-(4-isopropylphenyl)pyridine-1 (2H)-carboxylate (1.0 eq, 2.63 mmol, 751 mg), MeOH (20 mL), N-hydroxy-2-phenylacetamide (3.4 mmol, 518.0 mg), NaIO4(3.4 mmol, 375.9 mg), H2O (1 .4 mL) (6h). After the standard workup, the reaction crude was purified by flash chromatography (Hexane / AcOEt: 8 / 2; Rf=0.17) giving the desired product as a yellow solid (391 .4 mg, yield: 37%) (m.p. 140-143 °C). 1H NMR (400 MHz, CD3CN) δ 7.39-7.10 (m, 9H), 6.87 (s, 2H), 6.09 (s, 1 H), 5.19 (d, J = 3.8 Hz, 1 H), 5.1 1-5.04 (m, 1 H), 3.82-3.45 (m, 5H), 2.93 (hept, J = 6.9 Hz, 1 H), 1.26 (d, J = 6.9 Hz, 6H).

[0212] 13C NMR (101 MHz, CD3CN) δ 174.5, 156.8, 155.4, 149.2, 135.4, 135.1 , 133.2, 130.7, 129.7, 129.5, 129.3, 129.1 , 127.7, 127.6, 127.3, 76.4, 61.0, 60.8, 60.0, 53.7, 40.8, 34.5, 24.4, 24.4.

[0213] Methyl (1S*,4R*, 6R*)-3-(2-phenylacetyl)-6-(2-(trifluoromethyl)phenyl)-2-oxa- 3,5-diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (precursor of M35):

[0214] According to the general procedure a three-necked flask was loaded with methyl 2-(2-(trifluoromethyl)phenyl)pyridine-1 (2H)-carboxylate (1.0 eq, 2.0 g, 9.07 mmol), MeOH (65 mL) , N-hydroxy-2-phenylacetamide (1.17 g, 3.99 mmol), NaIO4(1.30g, 11.76 mmol), H2O (4.5 mL) (5.5h). After the standard workup, the reaction crude was purified by flash chromatography (Hexanes / AcOEt: 7 / 3; Rf=0.4) giving the title product as a yellow amorphous solid (460.2 mg; yield: 29.3%).

[0215] 1H NMR (400 MHz, CD3CN) δ 7.33 (d, J= 7.91 Hz, 1 H), 7.54-7.45 (m, 2H), 7.33- 7.30 (m, 5H), 7.23-7.21 (d, J=7.22Hz, 1 H), 6.97-6.89 (m, 2H), 6.19-6.16 (m, 1 H), 5.43 (s, 1 H), 5.05 (s, 1 H), 3.78-3.45 (m, 5H).

[0216] 13C NMR (101 MHz, CD3CN) δ 174.8, 136.6, 134.1 , 133.6, 132.7, 132.3, 131.0, 130.8, 130.5, 129.7, 129.6, 129.0, 128.7, 128.3, 128.1 , 127.3, 124.7, 75.9, 60.2, 57.9, 54.1 , 41.0.

[0217] Methyl (1S*,4R*, 6R*)-6-(3-isopropylphenyl)-3-(2-phenylacetyl)-2-oxa-3,5- diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (precursor of M38):

[0218]

[0219] According to the general procedure, a three-necked flask was charged with methyl 2-(3-isopropylphenyl)pyridine-1 (2H)-carboxylate (1 .0 eq, 3.03g, 8.99mmol), MeOH (63.26 mL), N-hydroxy-2-phenylacetamide (1.423 g, 9.39 mmol), NaIO4(1.04g, 9.39 mmol), H2O (4.2 mL) (5h). After the standard workup, the reaction crude was purified by flash chromatography (Hexanes / AcOEt: 7 / 3; Rf=0.38), affording the title product as a yellow amorphous solid (1 .572 g, yield: 45.2%).

[0220] 1H NMR (400 MHz, CD3CN) δ 7.30-7.18 (m, 5H), 7.15-7.08 (m, 2H), 6.95-6.84 (m,3H), 6.08 (m, 1 H), 5.09 (d, J=29.8Hz, 2H), 3.51 -3.67 (m,5H), 2.88 (s,1 H), 1.94(s, 1 H), 1.21 (s,6H).

[0221] 13C NMR (101 MHz, CD3CN) δ 173.8, 154.6, 143.1 , 136.8, 134.6, 132.4, 128.8,

[0222] 128.4, 128.4, 128.1 , 126.8, 125.7, 124.9, 124.1 , 75.5, 60.2, 59.2, 52.8, 39.8, 33.9,

[0223] 23.4, 23.3.

[0224] Methyl (1S ,4R*, 6R*)-3-(3-phenylpropanoyl)-6-(3-(trifluoromethyl)phenyl)-2- oxa-3,5-diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (precursor of M40):

[0225] In accordance with the general procedure, a three-necked flask was filled with methyl-3-(trifluoromethylphenyl) pyridine-1 (2H)-carboxylate (8.0 mmol 1 ,2-DHP, 2.42 g), N-hydroxy-3-phenylpropanamide (1.3 eq, 10.4 mmol, 1.72 g), NaIO4(1.3 eq, 8.8 mmol, 970 mg), MeOH (59.2 mL), H2O (6 mL). After the standard work up, the reaction crude was purified by flash chromatography (Hex / AcOEt: 8 / 2, Rf=0,28), obtaining the desired compound as an amorphous yellow solid (1.67 g, yield: 50 %).

[0226] 1H NMR (400 MHz, CDCI3) δ 7.58 (d, J = 7.8 Hz, 1 H), 7.48 (m, 1 H), 7.44 - 7.29 (m, 4H), 7.25 (d, J = 7.1 Hz, 3H), 6.96 (s, 1 H), 6.17 (s, 1 H), 5.25 (s, 1 H), 4.90 (d, J = 5.3 Hz, 1 H), 3.72 (d, 3H), 2.97 (m, 2H), 2.74 (m,7.8 Hz, 1 H), 2.60 (d, J = 14.5 Hz, 1 H).13C NMR (101 MHz, CDCI3) δ 140.9, 130.0, 129.2, 128.5, 127.5, 126.3, 125.0, 123.4, 75.3, 60.2, 58.7, 53.5, 35.1 , 30.0.

[0227] Methyl (1S*,4R*, 6R*)-6-(3-isopropylphenyl)-3-(3-phenylpropanoyl)-2-oxa-3,5- diazabicyclo[2.2.2]ott-7-ene-5-carboxylate (precursor of M41)

[0228] In accordance with the general procedure, a three-necked flask charged with methyl-2-(3-isopropylphenyl) pyridine-1 (2H)-carboxylate (6.8 mmol 1 ,2-DHP, 1.76 g), N-hydroxy-3-phenylpropanamide (1 .3 eq, 8.8 mmol, 1 .45 g), NaIO4(1 .3 eq, 7.5 mmol, 830 mg), MeOH (50.3 mL), H2O (5.1 mL). After standard workup, the crude mixture was purified by flash chromatography (hexanes / AcOEt: 8 / 2, Rf=0.29), to afford the title product as a yellow amorphous solid (1.16 g, yield: 40 %).

[0229] 1H NMR (400 MHz, CDCI3) δ 7.25 - 7.19 (m, 3H), 7.18 - 7.10 (m, 4H), 7.06 (m, J = 7.8, 1 .5 Hz, 1 H), 6.96 - 6.82 (m, 3H), 6.08 (s, 1 H), 5.08 (s, 1 H), 4.77 (s, 1 H), 3.87 - 3.34 (m, 3H), 2.83 (m, 6.7 Hz, 3H), 2.63 (m, 1 H), 2.58 - 2.38 (m, 1 H), 1.16 (m, 6 H).13C NMR (101 MHz, CDCI3) δ 174.0, 148.8, 140.7, 132.0, 128.2, 128.2, 125.9, 125.6, 124.5, 123.7, 75.41 , 60.2, 58.9, 53.0, 34.7, 33.8, 31.7, 29.6, 28.8, 23.8, 23.7, 22.5, 14.0.

[0230] Synthesis of tetrahydropyrido[4,3-e]-1 ,4,2-dioxazines:

[0231] General Procedure: In a vacuum-dried three-necked flask, a solution of the corresponding inverse cycloadduct of the NDA reaction in DCE (0.15M) is degassed (with argon or nitrogen). CuCI (0.2 eq, purity 99.99%) is added in inert atmosphere. The resulting reaction mixture is heated to 75°C and left under stirring until complete conversion of the bicycle. The reaction is interrupted by adding water and the biphasic mixture is extracted with 3 portions of DCM. The organic phase is dehydrated on MgSO4, The solvent is then evaporated under pressure reduced at 30°C. The raw material from the reaction is purified by flash chromatography or crystallization.

[0232] Methyl (4aR*,8S*,8aS*)-3-(thiophen-2-ylmethyl)-8-(3-(trifluoromethyl)phenyl)- 8,8a-dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (precursor of

[0233] In accordance with the general procedure, a three-necked flask was charged with methyl (1 S*,4R*,6R*)-3-(2-(thiophen-2-yl)acetyl)-6-(3-(trifluoromethyl)phenyl)-2- oxa-3,5-diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (2.965 mmol, 1.3 g), CuCI (1.0 eq, 2.965 mmol, 293.42 mg), DCE (20 mL). The mixture was allowed to stir at 75°C for 28h. After standard workup, the reaction crude has been purified by flash chromatography (Hexane / AcOEt: 7 / 3+ Et3N 1 %, Rf=0.37) providing the desired product as a yellow amorphous solid (868.1 mg, 66.9%).

[0234] 1H NMR (400 MHz, CD3CN) δ 7.60-7.42 (m, 4H), 7.20-7.05 (m, 2H), 6.86 (s, 2H), 5.49 (s, 1 H), 4.78-4.72 (m, 1 H), 4.51 (s, 1 H), 4.17-4.12 (m, 1 H), 3.62 (d, J = 5.9 Hz, 5H).

[0235] 13C NMR (101 MHz, CD3CN) δ 155.9, 154.4, 138.4, 138.3, 131.6 (q, JCF = 32.03 Hz), 131.0, 130.6, 128.0, 127.9, 127.4, 126.0, 125.9, 123.7, 123.7, 102.5, 68.6, 66.4, 58.3, 54.3, 33.1.

[0236] Methyl (4aR*,8S*,8aS*)-3-benzyl-8-(3-(trifluoromethyl)phenyl)-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (precursor of M21):

[0237] In accordance with the general procedure, a three-necked flask was charged with methyl (1S*,4R*, 6R*)-3-(2-phenylacetyl)-6-(3-(trifluoromethyl)phenyl)-2-oxa-3, 5- diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (1.76 mmol, 759 mg), CuCI (1.0 eq, 1.76 mmol, 174.2 mg), DCE (0.15 M, 11.67 mL). The mixture was allowed to stir at 75°C for 24 h. After standard workup, the reaction crude has been purified by flash chromatography (Hexanes / AcOEt: 8 / 2+Et3N 1%; Rf=0.31) to afford the title product as a yellow-green amorphous solid fornendo (473.9 mg, yield: 62.3%).

[0238] 1H NMR (400 MHz, CD3CN) δ 7.67-7.62 (m, 1 H), 7.60-7.53 (m, 2H), 7.49 (d, J = 8.0 Hz, 1 H), 7.34-7.22 (m, 5H), 7.14 (d, J = 8.6 Hz, 1 H), 5.56 (s, 1 H), 4.75 (d, J = 8.6 Hz, 1 H), 4.53-4.48 (m, 1 H), 4.23-4.18 (m, 1 H), 3.69 (s, 3H), 3.47 (s, 2H).

[0239] 13C NMR (101 MHz, CD3CN) δ 156.5, 154.5, 151.4, 138.3, 136.7, 131.5 (q, CF = 32.11 Hz), 130.9, 130.5, 129.6, 129.4, 127.9, 127.8, 126.0, 126.0, 123.7, 123.7, 102.6, 68.6, 66.2, 58.3, 54.3, 38.5.

[0240] Methyl (4aR*,8S*,8aS*)-3-benzyl-8-(4-(trifluoromethyl)phenyl)-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (precursor of M28):

[0241] In accordance with the general procedure, a three-necked flask was charged with methyl (1S*,4R*, 6R*)-3-(2-phenylacetyl)-6-(4-(trifluoromethyl)phenyl)-2-oxa-3,5- diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (997.3 mg, 2.3 mmol), DCE (15.4 mL, 0.15 M), CuCI (45.7 mg, 0.5 mmol). The mixture was allowed to stir at 75°C for 6 h. After standard workup, the reaction crude has been purified by flash chromatography (Hexanes / AcOEt: 7 / 3, Rf= 0.55) to afford the title product as a yellow amorphous solid (700 mg, yield: 70%).1H NMR (400 MHz, CD3CN) δ 7.68 (d, J = 8.25 Hz, 2H), 7.44 (d, J = 7.93 Hz, 2H), 7.30-7.33 (m, 2H), 7.26-7.23 (m, 3H), 7.12 (m, 1 H), 5.54 (s, 1 H), 4.73 (s, 1 H), 4.51 - 4.50 (m, 1 H), 4.21 -4.20 (m, 1 H), 3.69 (s, 3H), 3.47 (s, 2H).

[0242] 13C NMR (101 MHz, CD3CN) δ 157.0, 154.9, 141.9, 137.1 , 131.0 (q, JC-F = 32.1 Hz), 130.0, 129.9, 128.3, 128.3, 128.0, 127.2, 103.0, 69.0, 66.6, 58.8, 54.7, 38.9.

[0243] Methyl (4aR*,8S*,8aS*)-3-benzyl-8-(3,5-bis(trifluoromethyl)phenyl)-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (precursor of M29):

[0244] In accordance with the general procedure, a three-necked flask was charged with methyl ( 1S*,4R*,6R*)-6-(3,5-bis(trifluoromethyl)phenyl)-3-(2-phenylacetyl)-2-oxa- 3,5-diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (3.44 mmol, 1.72 g), DCE (0.15 M, 22.9 mL), and CuCI (1 .0 eq, 3.44 mmol, 340.5 mg). The mixture was allowed to stir at 75°C for 3 h. After standard workup, the reaction crude has been purified by flash chromatography (hexanes / AcOEt:8 / 2; R =0.56) to afford the title product as a yellow amorphous solid (882.4 mg, 1 .76 mmol, yield: 51 .2%).

[0245] 1H NMR (400 MHz, CD3CN) δ 7.97 (s, 1 H), 7.81 (s, 2H), 7.34 - 7.22 (m, 5H), 7.14 (d, J = 8.5 Hz, 1 H), 5.64 (s, 1 H), 4.77 (d, J = 8.6 Hz, 1 H), 4.54 - 4.50 (m, 1 H), 4.27 - 4.22 (m, 1 H), 3.70 (s, 3H), 3.47 (s, 2H).

[0246] 13C NMR (101 MHz, CD3CN) δ 156.6, 154.4, 140.1 , 136.7, 132.7 (q, JC-F =33.2 Hz) 129.6, 129.5, 127.9, 127.7, 125.7, 123.4, 123.4, 123.3, 123.0, 102.9, 68.2, 66.0, 58.1 , 54.4, 38.5.

[0247] Methyl (4aR*,8S*,8aS*)-8-(3,5-bis(trifluoromethyl)phenyl)-3-(thiophen-2- iylmethyl)-8,8a-dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (precursor of M30):

[0248]

[0249] In accordance with the general procedure, a three-necked flask was charged with methyl (1S*,4R*, 6R*)-6-(3,5-bis(trifluoromethyl)phenyl)-3-(2-(thiophen-2- yl)acetyl)-2-oxa-3,5-diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (1.46 g, 2.87 mmol), DCE (0.15 M, 19.0 mL), CuCI (1.0 eq. 2.87 mmol, 294.0 mg). The mixture was allowed to stir at 75°C for 4 h. After standard workup, the reaction crude has been purified by flash chromatography (hexanes / AcOEt:7 / 3; Rf=0.54) to afford the title product as a yellow amorphous solid (975 mg, 1 .9 mmol, yield 67.1%).

[0250] 1H NMR (400 MHz, CD3CN) δ 7.99 (s, 1 H), 7.84 (s, 2H), 7.26 (dd, J = 4.8, 1.6 Hz, 1 H), 7.20-7.13 (m, 1 H), 6.97 - 6.91 (m, 2H), 5.67 (d, J = 3.3 Hz, 1 H), 4.88-4.81 (m, 1 H), 4.62-4.57 (m, 1 H), 4.30-4.25 (m, 1 H), 3.69 (s, 5H).

[0251] 13C NMR (101 MHz, CD3CN) δ 156.0, 154.4, 140.1 , 138.4, 132.8 (q, JC-F =33.4 Hz), 128.0, 127.9, 127.7, 127.5, 125.9, 125.7, 123.40, 123.0, 120.3, 102.8, 68.3, 66.2, 58.0, 54.4, 33.1.

[0252] Methyl (4aR*,8S*,8aS*)- 3-benzyl-8-(4-isopropylphenyl)-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (precursor of M33):

[0253] In accordance with the general procedure, a three-necked flask was charged with methyl ( 1S*,4R*, 6R*)-6-(4-isopropylphenyl)-3-(2-phenylacetyl)-2-oxa-3, 5- diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (1.0 eq, 391 mg, 0.96 mmol), DCE (0.15 M, 6.5 mL), CuCI (20 mol%, 19.0 mg, 0.192 mmol). The mixture was allowed to stir at 75°C for 5 h. After standard workup, the reaction crude has been purified by flash chromatography (hexanes / AcOEt:7 / 3; Rf=0.53) to afford the title product as a yellow amorphous solid (305 mg, yield: 78%).

[0254] 1H NMR (400 MHz, CD3CN) δ 7.34-7.09 (m, 10H), 5.42 (s, 1 H), 4.70 (s, 1 H), 4.47 (s, 1 H), 4.14-4.10 (m, 1 H), 3.78-3.58 (m, 3H), 3.46 (s, 2H), 2.89 (hept, 1 H), 1.20 (d, J = 6.9 Hz, 6H).

[0255] 13C NMR (101 MHz, CD3CN) δ 156.5, 149.9, 136.8, 134.3, 129.6, 129.5, 128.1 , 128.0, 127.8, 126.6, 102.3, 69.0, 66.6, 58.5, 54.2, 38.5, 34.4, 24.1.

[0256] Methyl (4aR*,8S*,8aS*)-3-benzyl-8-(2-(trifluoromethyl)phenyl)-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (precursor of M35):

[0257] In accordance with the general procedure, a three-necked flask was charged with methyl (1S*,4R*, 6R*)-3-(2-phenylacetyl)-6-(2-(trifluoromethyl)phenyl)-2-oxa-3, 5- diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (1.0 eq, 412 mg, 0.95 mmol), DCE (0.15 M, 6.34 mL), CuCI (2.4 eq 219.04 mg, 2.21 mmol). The mixture was allowed to stir at 75°C for 18 h. After standard workup, the reaction crude has been purified by flash chromatography (Hexanes / AcOEt:7 / 3; Rf=0.41 ) to afford the title product as a yellow amorphous solid (92.1 mg, yield: 22.4%).

[0258] 1H NMR (400MHz, CD3CN) δ7.77 (d, J=7.36HZ, 1 H), 7.61 -7.57 (m, 1 H), 7.52-7.48 (m, 1 H), 7.37 (d, J=7.64 Hz, 1 H), 7.38-7.22 (m, 6H), 5.63 (s, 1 H), 4.80 (m, 2H), 4.07 (s, 1 H), 3.69-3.54 (m, 3H), 3.47 (s, 2H).

[0259] 13C NMR (101 MHz, CD3CN) δ 156.7, 154.7, 154.6, 137.2, 137.1 , 134.6, 130.2, 130.0, 129.9, 129.2, 128.4, 128.3, 128.1 , 127.2, 124.5, 102.7, 102.3, 66.7, 66.5, 56.7, 54.7, 38.8.

[0260] Methyl (4aR*,8S*,8aS*)-3-benzyl-8-(3-isopropylphenyl)-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (precursor of M38):

[0261]

[0262] In accordance with the general procedure, a three-necked flask was charged with methyl (1S*,4R*, 6R*)-6-(3-isopropylphenyl)-3-(2-phenylacetyl)-2-oxa-3,5- diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (1.0 eq, 1.522 g, 3.74mmol), DCE (0.15 M, 24.9 mL), CuCI (0.2 eq; 81 .5 mg, 0.82 mmol). The mixture was allowed to stir at 75°C for 5 h. After standard workup, the reaction crude has been purified by flash chromatography (Hexanes / AcOEt:7 / 3; Rf=0.38)to afford the title product as a light-yellow amorphous solid (1.158 g, yield: 76.1 %).

[0263] 1H NMR (400 MHz, CD3CN) δ.19-7.30 (m, 8H), 7.15 (s,1 H), 7.01 (d, J=7.63, 1 H), 5.49 (s, 1 H), 4.70(s, 1 H), 4.48 (s, 1 H), 4.14-4.16 (m, 1 H), 3.63-3.71 (m, 3H), 3.47 (s,2H), 2.85-2.92 (m, 1 H), 1 .21 (d, J= 6.9, 6H).

[0264] 13C NMR (101 MHz, CD3CN) δ 156.5, 154.6, 150.8, 137.1 , 136.8, 130.0, 129.7, 129.5, 128.2, 127.9, 127.2, 125.0, 123.9, 102.2, 69.1 , 66.6, 58.9, 54.2, 38.6, 34.8, 24.3, 24.2.

[0265] Methyl (4aR*,8S*,8aS*) 3-phenylethyl-8-(3-(trifluoromethyl)phenyl)-8,8a- dihydropyrido-4H-pyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (precursor of M40)

[0266] In accordance with the general procedure, a three-necked flask was charged with methyl (1S*,4R*, 6R*)-3-(3-phenylpropanoyl)-6-(3-(trifluoromethyl)phenyl)-2-oxa 3,5-diazabicyclo[2.2.2]ott-7-ene-5-carboxylate (3.70 mmol, 1.65 g), CuCI (1.48 mmol, 146.51 mg), DCE (0.15 M, 24.79 mL). The mixture was allowed to stir at 75°C for 24 h. After standard workup, the reaction crude has been purified by flash chromatography (Hex / AcOEt: 8 / 2; Rf=0.42), to afford the title product as a yellow- green amorphous solid (1.00 g, yield: 70%).

[0267] 1H NMR (400 MHz, CDCI3) δ 7.59 (d, J = 7.8 Hz, 1 H), 7.51 (t, J = 7.8 Hz, 1 H), 7.42 (d, J = 20.1 Hz, 2H), 7.30 - 7.25 (m, 3H), 7.24 - 7.15 (m, 3H), 5.54 (d, J = 39.5 Hz, 1 H), 4.82 (d, J = 22.2 Hz, 1 H), 4.54 (s, 1 H), 4.00 (s, 1 H), 3.73 (d, J = 40.6 Hz, 3H), 2.87 (td, J = 7.4, 1 .9 Hz, 2H), 2.60 - 2.43 (m, 2H).

[0268] 13C NMR (101 MHz, CDCI3) 156.0, 140.3, 137.4, 130.0, 129,2, 128,6, 128,5, 127.7, 126.5, 125.5, 122.6, 68.9, 65.3, 57.7, 53.9, 33.3, 32.1.

[0269] Methyl (4aR*,8S*,8aS*)-8-(3-isopropylphenyl)-3-phenylethyl-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (precursor of M41)

[0270] In accordance with the general procedure, a three-necked flask was charged with methyl (1S*,4R*, 6R*)-6-(3-isopropyl)-3-(3-phenylpropanoyl)-2-oxa-3,5- diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (2.71 mmol, 1.14 g), CuCI (1.08 mmol, 106.90 mg), DCE (0.15 M, 18.16 mL). The mixture was allowed to stir at 75°C for 24 h. After standard workup, the reaction crude has been purified by flash chromatography (Hexane / AcOEt: 8 / 2; Rf=0.42), to afford the title product as a yellow-green amorphous solid (851.3 mg, yield: 75%).

[0271] 1H NMR (400 MHz, CDCI3) δ 726.21 - 725.69 (m, 10H), 724.31 (d, J = 44.9 Hz, 1 H), 723.76 - 723.51 (m, 1 H), 723.40 (d, J = 15.1 Hz, 1 H), 722.83 (s, 1 H), 722.49 (d, J = 48.6 Hz, 3H), 721.75 - 721.56 (m, 3H), 721 .38 - 721 .16 (m, 2H), 720.24 - 719.96 (m, 7H).13C NMR (101 MHz, CDCI3) δ 156.0, 149.7, 140.1 , 135.9, 128.9, 128.3, 128.2, 127.4, 127.1 , 126.1 , 126.1 , 123.8, 123.5, 122.7, 101.3, 100.8, 68.9, 65.4, 57.8, 53.3, 33.9, 33.1 , 31.8, 23.9, 23.7.

[0272] Synthesis of biologicaly active compounds having 4-oxa-1 ,3- diazabicyclic[3.3.1 ]non-6-ene scaffold.

[0273] General Procedure: The corresponding dioxazine derivative and anhydrous THF are placed in a three-necked flask dried under vacuum, under an inert atmosphere. The resulting solution is cooled to -20°C or 0°C and a commercial solution of LiBEt3H (1 .0 M in THF, 8.0 equiv.) is added slowly dropwise under vigorous stirring. The reaction is maintained at the same temperature until complete conversion of the dioxazine and then the reaction is slowly quenched by dropwise water. The biphasic mixture is extracted with 3 portions of DCM, the combined organic portions are dehydrated over MgSO4, filtered and finally the solvents are evaporated under reduced pressure at 30°C. The reaction crude is purified by flash chromatography, crystallization or trituration.

[0274] 2-(Thiophen-2-yl)-1-(( 1S*,5S*, 9R*)-9-(3-(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)etan-1-one (M14):

[0275] In accordance with the general procedure, a three-necked flask was charged with methyl (4aR*,8S*,8aS*-)3-(thiophen-2-ylmethyl)-8-(3-(trifluoromethyl)phenyl)-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (1.98 mmol, 870.0 mg), THF (0.21 M, 10 mL), LiBEt3H (15.88 mmol, 15.88 mL), at 0°C. After standard workup, the reaction crude has been purified by flash chromatography (Hexanes / AcOEt 7:3, Rf=0.24) to afford the title product as a white solid (307.6 mg, 39.4%) (mp. 102-105°C).

[0276] 1H NMR (400 MHz, CDCI3) δ 7.62-7.59 (m, 1 H), 7.56-7.43 (m, 3H), 7.20 (dd, J = 4.8, 1.6 Hz, 1 H), 6.97-6.93 (m, 2H), 6.09-5.99 (m, 2H), 5.64-5.58 (m, 1 H), 5.00- 4.96 (m, 1 H), 4.59 (s, 1 H), 4.49 (d, J= 13.2 Hz, 1 H), 3.97 (d, J= 16.0 Hz, 1 H), 3.81 (dd, J = 15.9, 0.8 Hz, 1 H), 3.41-3.34 (m, 1 H), 3.24-3.17 (m, 1 H).

[0277] 13C NMR (101 MHz, CDCI3) δ 171 .2, 137.9, 137.8, 136.0, 130.9 (q, JC-F= 32.2 Hz), 130.0, 128.9, 126.8, 126.7, 125.0, 124.6, 123.7, 118.9, 72.1 , 65.8, 59.1 , 47.6, 33.7. 2-Phenyl-1-(( 1S*,5S*, 9R*)-9-(3-(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1 ]non-6-en-3-yl)etan-1 -one (M21 ) :

[0278] In accordance with the general procedure, a three-necked flask was charged with methyl (4aR*,8S*,8aS*)-3-benzyl-8-(3-(trifluoromethyl)phenyl)-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (1.09 mmol, 474.0 mg), THF (0.21 M, 5.22 mL), LiBEt3H (8.77 mmol, 8.77 mL, very slow addition), at -20°C for 3h. After standard workup, the reaction crude has been purified by flash chromatography (Hexanes / AcOEt: 8 / 2+ Et3N 1%; Rf=0.28) to give the desired product as a white solid (160.0 mg, yield: 37.6%) (mp. 1 11 -1 16°C).

[0279] 1H NMR (400 MHz, CDCI3) δ 7.57 (s, 1 H), 7.53-7.38 (m, 3H), 7.32-7.18 (m, 5H), 5.97 (d, J = 3.4 Hz, 2H), 5.60-5.55 (m, 1 H), 4.94-4.90 (m, 1 H), 4.53 (s, 1 H), 4.43 (d, J = 13.1 Hz, 1 H), 3.77 (d, J = 14.8 Hz, 1 H), 3.51 (d, J = 14.8 Hz, 1 H), 3.33 (d, J = 18.4 Hz, 1 H), 3.16 (d, J = 19.6 Hz, 1 H).

[0280] 13C NMR (101 MHz, CDCI3) δ 172.2, 138.0, 137.4, 135.0, 130.8 (q, JC-F = 31 .9 Hz), 130.0, 129.6, 128.8, 128.5, 126.9, 124.5, 123.7, 119.0, 71 .9, 65.7, 59.2, 47.6, 39.8. ( 1S*,5S*, 9R*)-3-(2-Phenylacetyl)-9-(3-(trifluoromethyl)phenyl)-4-oxa-1 l4,3- diazabicyclo[3.3.1]non-6-en-1-ylium chloride (M21 -hydrochloride):

[0281] To a solution of 2-phenyl-1 -(( 1S*,5S*, 9R*)-9-(3-(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)etan-1 -one (20 mg, 0.05 mmol) in Et3O (3 mL) Et2O-HCI (4 mL) has been added at 0°C under vigorous stirring. When the dripping is stopped, the stirring is also stopped and a white precipitate is obtained which is filtered under vacuum, washed with Et3O and dried under vacuum (21 mg, 0.049 mmol, yield: 98%).

[0282] 1H NMR (400 MHz, CDCI3) δ 8.06 (d, J = 7.7 Hz, 1 H), 7.71-7.62 (m, 2H), 7.57 (t,

[0283] J = 7.7 Hz, 1 H), 7.40-7.21 (m, 6H), 6.09 (s, 2H), 5.88 (d, J = 12.9 Hz, 1 H), 5.16- 5.03 (m, 2H), 4.81 (d, J= 13.0 Hz, 1 H), 3.80 (d, J= 14.9 Hz, 1 H), 3.65 (d, J = 19.4 Hz, 1 H), 3.59 (d, J = 14.9 Hz, 1 H), 3.47 (d, J = 18.8 Hz, 1 H).

[0284] 2-Phenyl-1-((1R*,5S*,9R*)-9-(4-(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)etan-1-one (M28):

[0285] In accordance with the general procedure, a three-necked flask was charged with methyl (4aR*,8S*,8aS*) -3-benzyl-8-(4-(trifluoromethyl)phenyl)-8,8a- dihydropyrido[4,3- e][1 ,4,2]dioxazine-7(4aH)-carboxylate (704 mg, 1 .6 mmol), THF (7.6 mL), LiBEt3H (1 .0 M in THF, 8.0 equiv.) at 0°C for 3h. After standard workup, the reaction crude has been purified by flash chromatography (Hexanes / AcOEt: 8 / 2; Rf=0.38), to afford the title product as a white solid (265.7 mg, yield: 42%)(mp. 140 °C).

[0286] 1H NMR (400 MHz, CDCI3) δ 7.59 (d, J = 8.35 Hz, 2H), 7.45 (d, J= 8.35, 2H), 7.33- 7.22 (m, 5H), 5.99 (d, J = 3.62 Hz, 2H), 5.61 (d, J = 13.4 Hz, 1 H), 4.94 (m, 1 H), 4.57 (s, 1 H), 4.46 (d, J = 13.11 Hz, 1 H), 3.80 (d, J = 14.73 Hz, 1 H), 3.54 (d, J = 14.73 Hz, 1 H), 3.39-3.19 (m, 2H).

[0287] 13C NMR (101 MHz, CDCI3) δ 173.5, 142.3, 138.6, 136.2, 130.9, 130.1 (q, JC-F= 32.41 Hz) 129.8, 128.4, 128.1 , 127.8, 126.6, 124.1 , 120.4, 73.2, 67.0, 60.5, 48.9, 41.0.

[0288] 1-((1R*,5S*,9R*)-9-(3,5-bis(Trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)-2-phenyletan-1-one (M29):

[0289] In accordance with the general procedure, a three-necked flask was charged with methyl (4aR*,8S*,8aS-3*)-benzyl-8-(3,5-bis(trifluoromethyl)phenyl)-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (0.164 mmol, 82 mg), THF (1 mL), LiBEt3H (8.0 eq., 1.32 mmol, 1.3 mL) at -20°C for 30 min. After standard workup, the reaction crude has been purified by flash chromatography (Hexanes / AcOEt:7 / 3; Rf=0.43), to afford the title product as a light-yellow solid (12.2 mg, 0.03 mmol, yield: 17%) (mp. 112°C).

[0290] 1H NMR (400 MHz, CDCI3) δ 7.84-7.75 (m, 3H), 7.37-7.21 (m, 5H), 6.02 (s, 2H), 5.62 (d, J = 13.0 Hz, 1 H), 4.99 (s, 1 H), 4.59 (s, 1 H), 4.47 (d, J = 13.0 Hz, 1 H), 3.80 (d, J = 14.7 Hz, 1 H), 3.56 (d, J = 14.7 Hz, 1 H), 3.46-3.10 (m, 2H).

[0291] 13C NMR (101 MHz, CDCI3) δ 171.8, 143.1 , 139.4, 137.4, 135.3, 133.07 (q, JC- F=33.3 HZ) 131.6, 129.3, 128.3, 126.8, 126.7, 125.1 , 121.5, 118.5, 11 1.7, 71.1 , 65.3, 58.6, 47.3, 39.5.

[0292] 1-(1R*,5S*,9R*)-9-(3,5-bis(Trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)-2-(thiophen-2-yl)etan-1-one (M30):

[0293] In accordance with the general procedure, a three-necked flask was charged with methyl (4aR*,8S*,8aS*)-8-(3,5-bis(trifluoromethyl)phenyl)-3-(thiophen-2-ylmethyl)- 8,8a-dihydropyrido[4,3-e][1.4.2]dioxazine-7(4aH)-carboxylate (910.0 mg, 1.8 mmol), THF (8.6 mL), LiBHEt3(1 .0 M, 14.4 mL), at -20°C for 40 min. After standard workup, the reaction crude has been purified by flash chromatography (DCM / Hexanes: 8 / 2; Rf=0.49), to afford the title product as a yellow solid (103.6 mg, 0.224 mmol, yield: 12.5%)(mp. 108-1 11 °C).

[0294] 1H NMR (400 MHz, CDCI3) δ 7.83-7.76 (m, 3H), 7.20 (d, J= 4.9 Hz, 1 H), 6.97-6.92 (m, 2H), 6.11-6.00 (m, 2H), 5.62 (d, J = 13.2 Hz, 1 H), 5.01 (d, J = 4.9 Hz, 1 H), 4.61 (s, 1 H), 4.49 (d, J = 13.2 Hz, 1 H), 3.99-3.78 (m, 2H), 3.46 - 3.10 (m, 2H).

[0295] 13C NMR (101 MHz, CDCI3) δ 171.1 , 139.6, 138.0, 135.8, 131.8 (q, JC-F=33.4 HZ), 127.0, 126.9, 126.7, 125.0, 124.7, 122.0, 121 .8, 118.7, 77.5, 77.4, 77.2, 76.8, 71 .5, 65.7, 58.9, 47.6, 33.7.

[0296] 1-((1R*,5S*,9R*)-9-(4-lsopropylphenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6- en-3-yl)-2-phenyletan-1-one (M33):

[0297]

[0298] In accordance with the general procedure, a three-necked flask was charged with methyl (4R*,8S*,8S*)-3-benzyl-8-(4-isopropylphenyl)-8,8a-dihydropyrido[4,3- e][ 1 ,4,2]dioxazine-7(4aH)-carboxylate (1 .0 eq, 270 mg, 0.66 mmol), THF (4.0 mL), LiBHEt3(8,0 eq, 5.3 mL, 5.3 mmol), at a 0°C for 2h. After standard workup, the reaction crude has been purified by crystallization with MeOH to afford the title product as a white solid (226 mg, yield: 94%)(mp. 1 13-115°C).

[0299] 1H NMR (400 MHz, CDCI3) δ 7.36-7.14 (m, 9H), 6.00 (d, J = 3.4 Hz, 2H), 5.60 (d, J = 13.1 Hz, 1 H), 4.90 (d, J = 2.6 Hz, 1 H), 4.53 (s, 1 H), 4.46 (d, J = 13.1 Hz, 1 H), 3.68 (dd, J = 1 10.7, 14.7 Hz, 2H), 3.38-3.24 (m, 2H), 2.90 (hept, J = 6.9 Hz, 1 H), 1.25 (d, J = 6.9 Hz, 6H).

[0300] 13C NMR (101 MHz, CDCI3) δ 172.2, 148.2, 137.1 , 135.1 , 134.2, 129.6, 128.4, 126.8, 126.6, 126.3, 1 19.3, 72.4, 65.6, 59.4, 47.6, 39.7, 33.8, 24.0.

[0301] 2-Phenyl-1-(1S*,5R*,9R*)-9-(2-(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)etan-1-one (M35):

[0302] In accordance with the general procedure, a three-necked flask was charged with methyl (4R*,8S*,8S* )-benzyl-8-(2-(trifluoromethyl)phenyl)-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (1.0 eq, 92 mg, 0.21 mmol), THF (1.3 mL), LiBHEt3(8.0 eq, 1.7 mL, 1.7 mmol), at -20°C for 5 h. After standard workup, the reaction crude has been purified by flash chromatography (Hexanes / AcOEt:7 / 3; Rf=0.48) to afford the title product as a light-yellow solid (15.8 mg, yield: 19.4%)(mp. 1 11 °C).

[0303] 1H NMR (400MHz, CDCI3) δ 7.75-7.72 (m, 1 H), 7.37-7.26 (m, 5H), 7.45-7.41 (m, 2H), 7.44-7.24 (m, 1 H), 6.16-6.12 (m, 2H), 5.53 (d, J =12.9 Hz, 1 H), 4.96 (s, 1 H), 4.74 (d, J =5.6, 1 H), 4.47 (d, J =13.2, 1 H), 3.80 (d, J =14.1 Hz, 1 H), 3.54 (d, J = 17.1 Hz, 1 H), 3.37-3.31 (m, 1 H), 3.06 (d, J =19.9 Hz, 1 H)

[0304] 13C NMR (101 MHz, CDCI3) δ 172.2, 137.0, 135.9, 135.6, 131.4, 129.8, 129.5, 129.2, 128.7, 128.5, 128.3, 127.8 (qC-F= 33.06 Hz) 126.6, 120.3, 72.0, 65.2, 57.4, 47.5, 39.1. 1-(1S*,5R*,9R*)-9-(3-lsopropylphenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en- 3-yl)-2-phenyletan-1-one (M38):

[0305] In accordance with the general procedure, a three-necked flask was charged with methyl (4R*,8S*,8S*)-3-benzyl-8-(3-isopropylphenyl)-8,8a-dihydropyrido[4,3- e][1 ,4,2]dioxazine-7(4aH)-carboxylate (1.108g, 2.72 mmol), THF (16.6 mL), LiBHEt3(8.0 eq, 21.8 mL, 21.8 mmol), at 0°C for 1 h. After standard workup, the reaction crude has been purified by flash chromatography (Hexanes / AcOEt:7 / 3; Rf=0.47) to afford the title product as a light-pink solid (320 mg, yield: 32.4%)(mp. 103-104°C).

[0306] 1H NMR (400 MHz, CDCI3) δ 7.31 -7.35 (m, 7H), 7.16-7.06 (m, 2H), 6.1 (d, J =4.42, 2H), 5.63 (m, 1 H), 4.94 (m, 1 H), 4.57(s, 1 H), 4.48 (m, 1 H), 3.83 (d, J =14.5, 1 H), 3.56 (d, J =14.4, 1 H), 3.32 (m, 1 H), 2.91 (m, 1 H), 1.25 (d, J =7.0 Hz 6H).

[0307] 13C NMR (101 Hz, CDCI3) δ 173.1 , 149.9, 138.0, 137.7, 136.0, 130.5, 129.4, 129.1 , 127.7, 126.5, 126.0, 124.8, 120.2, 73.3, 66.6, 60.5, 48.6, 40.6, 35.1 , 25.1 , 25.0.

[0308] 3-Phenyl-1-((5S*)-9-(3-(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-yl)propan-1-one (M40)

[0309] In accordance with the general procedure, a three-necked flask was charged with methyl 4aR,8S,8aR) 3-phenylethyl-8-(3-(trifluoromethyl)phenyl)-8,8a- dihydropyrido-4H-pyrido[4,3-e][1 ,2]oxazine-7(4aH)-carboxylate (1.0 mmol, 440.0 mg), LiBEt3H (4 mmol, 4 mL) THF (6 mL) at -20°C. After standard workup, the reaction crude has been purified by flash chromatography (Hex / AcOEt: 7:3, Rf: 0.3) to afford the title product as a white solid (138.01 mg, yield: 35%).

[0310] 1H NMR (400 MHZ,CDCI3) δ 7.57 - 7.52 (s, 1 H), 7.50 - 7.31 (m, 3H), 7.28 - 7.07 (m, 5H), 6.03 - 5.86 (m, 1 H), 5.81 (m,1 H), 5.56 (m, 1 H), 4.81 (m, 1 .0 Hz, 1 H), 4.49 (d, J = 2.3 Hz, 1 H), 4.38 (d, J = 13.1 Hz, 1 H), 3.27 (m,1 H), 3.11 (m, 2H), 2.95 - 2.80 (m, 2H), 2.66 (m, 1 H), 2.59 - 2.44 (m, 1 H).

[0311] 13C NMR (101 MHz, CDCI3) δ 173.6, 141.5, 138.1 , 137.3, 130.0, 128.8, 128.6, 128.5, 126.2, 124.5, 123.7, 1 19.0, 71 .8, 65.5, 59.2, 47.6, 34.4, 30.5.

[0312] 1-((5S*)-9-(3-lsopropylphenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6-en-3-yl)-3- phenyl propan-1 -one (M41)

[0313] In accordance with the general procedure, a three-necked flask was charged with methyl (4aF?,8S,8aS)-8-(3-isopropylphenyl)-3-phenylethyl-8,8a-dihydropyrido[4,3- e][1 ,4,2]dioxazine-7(4aH)carboxylate (1.24 mmol, 521 mg), LiBEt3H (4.96 mmol, 4.96 mL), THF anidro (7.44 mL) at -20°C. After standard workup, the reaction crude has been purified by flash chromatography (Hex / AcOEt: 7 / 3, Rf: 0.48) to afford the title product as a white solid (250.0 mg, yield: 50%).

[0314] 1H NMR (400 MHz, CDCI3) δ 7.32 - 7.27 (m, 2H), 7.25 - 7.16 (m, 5H), 7.13 (d, J = 7.7 Hz, 1 H), 7.07 (m,1 H), 6.00 - 5.92 (m, 1 H), 5.87 (m,1 H), 5.62 (m,1 H), 4.85 (m, 1 H), 4.55 - 4.51 (m, 1 H), 4.46 (d, J = 13.1 Hz, 1 H), 3.28 (m, 2H), 2.96 - 2.86 (m, 3H), 2.72 (m,1 H), 2.63 - 2.51 (m, 1 H), 1.59 (s, 1 H), 1.23 (d, J = 6.9 Hz, 6H).

[0315] 13C NMR (101 MHz, CDCI3) δ 172.6, 148.0, 140.6, 136.0, 127.6, 127.5, 127.2, 125.2, 124.6, 124.2, 122., 118.3, 71.3, 64.6, 58.6, 46.6, 33.4, 33.3, 29.5, 23.1 , 23.0.

[0316] N-(((2R* ,3S*)-3-idrossi-2-fenil-3,6-diidropiridin-1 (2H)-il)metil)-2- fenilacetammide (M32):

[0317] In a vacuum dried Schlenk flask was charged, under inert atmosphere, with Cp2TiCl2(127 mg, 0.496 mmol), anhydrous THF (2.5 mL), and zinc powder (66 mg, 0.99 mmol) and the reaction was kept under stirring for 50 minutes. A solution of 2-phenyl-1 -(( 1R*, 5S*,9R*)-9-phenyl-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6-en-3- yl)etan-1 one (64 mg, 0.2 mmol) in THF (0.8 mL) is added to the reaction mixture at -30°C followed MeOH (2.0 mL). The reaction is maintained under stirring at - 30°C for 4h, then the temperature is allowed to warm up to 0°C and left to react for further 2h. The reaction is quenched with H2O (6.5 mL) at room temperature, followed by a saturated aqueous solution of NaHCO3(2.0 mL). The organic phase The aqueous phase was extracted with AcOEt (3x8 mL) and the combined organic phases were dehydrated over MgSO4, and then solvents were evaporated under reduced pressure. The crude was purified by flash chromatography (DCM / MeOH: 98 / 2, Rf= 0.34), affording the title product as a yellow oil (20 mg, yield: 32%).

[0318] 1H NMR (400 MHz, CDCI3) 5 7.45-7.39 (m, 3H), 7.30-7.21 (m, 5H), 6.99-6.97 (m, 2H), 5.90-5.87 (m, 1 H), 5.83-5.79 (m, 1 H), 5.30 (s, 1 H), 4.36 (dd, J = 8.5 Hz, 13.7 Hz, 1 H), 4.29 (d, J= 7.2 Hz, 1 H), 3.64-3.46 (m, 3H), 3.30 (m, 2H), 3.06 (d, J = 7.3, 1 H).

[0319] 13C NMR (101 MHz, CDCI3) δ 173.2, 136.0, 131.1 , 130.7, 130.6, 130.0, 129.8, 129.2, 129.0, 72.8, 69.5, 58.26, 51.1 , 45.2.

[0320] The synthesis of enantioenriched M-type compounds is based on the organocatalyzed proline synthesis of the corresponding 1 ,2-DHP. Once these are obtained in enantiopure form, the reactions described above are carried out. (nitroso Diels-Alder, rearrangement to dioxazines, reduction and intramolecular amination promoted by Super-H).

[0321] For the synthesis of 1 ,2-DHP we refer to the previously cited article Bo-Shuai Mu et al. (htt ps: / / doi.org / 10.1038 / s41467-021 -22374-y). With respect to this paper we made some modifications as detailed below:

[0322] Synthesis of a-amido sulfones: General Procedure: Benzyl carbamate (1.0 eq.) and sodium benzenesulfinate dihydrate (2.0 eq.) are placed in a three-necked flask, then MeOH and water (2:1 ) are added. The appropriate freshly distilled aldehyde (1 .25-1 .5 eq.) is added in one portion and finally formic acid (4.0 eq.). The mixture is stirred vigorously for 72 h and then filtered under vacuum. The resulting white solid is washed with diethyl ether and water and dried under vacuum, removing the remaining water by forming an azeotropic mixture with chloroform. The desired product is used without further purification.

[0323] Benzyl (phenyl(phenylsulfonyl)methyl)carbamate:

[0324] According to the general procedure a three-neck flask is charged with benzyl carbamate (39.2 mmol, 5.92 g, 1.0 eq.), sodium benzenesulfinate dihydrate (39.2 mmol, 7.85 g, 2.0 eq.), MeOH (38 mL), water (76 mL) (2:1 ), freshly distilled benzaldehyde (5 mL, 1 .25 eq.), formic acid (5.91 mL, 4.0 eq.). The mixture is stirred vigorously for 72 h and then filtered under vacuum. After appropriate workup, the desired product is obtained as a white solid (7.7 g, 20.2 mmol, yield = 52%).

[0325] 1H NMR (400 MHz, CDCI3) δ 7.91-7.79 (m, 2H), 7.65-7.55 (m, 1 H), 7.48-7.32 (m, 10H), 7.27-7.19 (m, 2H), 6.10-5.91 (m, 2H), 5.00-4.84 (m, 2H).

[0326] Benzyl ((phenylsulfonyl)(3-(trifluoromethyl)phenyl)methyl)carbamate:

[0327] According to the general procedure, a three-neck flask is charged with benzyl carbamate (60 mmol, 9.07 g, 1.0 eq.), sodium benzenesulfinate dihydrate (90 mmol, 18.0 g, 1.5 eq.), MeOH (58 mL), water (117 mL) (2:1 ), freshly distilled 3- (trifluoromethyl)benzaldehyde (8.42 mL, 1.25 eq.), formic acid (9.06 mL, 4.0 eq.). The mixture is left under vigorous stirring for 72 h then filtered under vacuum. After appropriate workup, the desired product is obtained as a white solid (7.0 g, 15.6 mmol, yield = 26%). 1H NMR (400 MHz, CDCI3) δ 7.84 (d, J = 7.8 Hz, 2H), 7.70-7.54 (m, 5H), 7.53- 7.42 (m, 4H), 7.40-7.31 (m, 6H), 7.27-7.16 (m, 2H), 6.35 (d, J= 10.9 Hz, 1 H), 6.05 (d, J = 10.8 Hz, 1 H), 4.94 (d, J = 3.7 Hz, 2H).

[0328] 13C NMR (101 MHz, CDCI3) δ 154.8, 136.2, 136.0, 134.5, 132.8, 132.4, 131.4, 131.1 , 130.9, 129.8, 129.5, 129.4, 129.2, 128.7, 128.4, 128.2, 126.8, 125.7, 74.1 , 68.0, 67.2.

[0329] Synthesis of N-Cbz Imines:

[0330] General Procedure

[0331] Method A: A three-neck flask is charged with K2CO3(6.0 eq.) and Na2SO4(7.0 eq.). The solids are dried under hot vacuum, once cooled and under inert atmosphere, the corresponding a-amido sulfone (1.0 eq.) and anhydrous THF (0.1 M) are added. The flask is equipped with a water condenser and the reaction mixture is refluxed under vigorous stirring for 18h. The reaction mixture is then cooled to room temperature and filtered under vacuum in an inert atmosphere. The filtrate is concentrated under reduced pressure to give the desired product as a solid which is used in the next step without further purification.

[0332] Method B: In a reaction flask a solution of a-amido sulfone in DCM is formed and an aqueous solution of K2CO3(1 .4 M) is added. It is left under vigorous stirring for 18 h, after which the biphasic mixture is extracted 3 times with DCM, the combined organic phases are dehydrated on MgSO4, the solvent is then evaporated under reduced pressure. The desired product is obtained as a white solid which is used without further purification.

[0333] Benzyl benzylidenecarbamate:

[0334] According to the general procedure (method A) a three-neck flask is charged with K2CO3(18 g, 6.0 eq.) and Na2SO4(21.6 g, 7.0 eq.), benzyl (phenyl(phenylsulfonyl)methyl)carbamate (8.28, 1.0 eq.) and anhydrous THF (215 mL, 0.1 M). The reaction is refluxed for 18h. After standard work up the desired product is obtained as a white solid.

[0335] 1H NMR (400 MHz, CDCI3) δ 9.00 (s, 1 H), 7.98 (d, J = 7.1 Hz, 2H), 7.65-7.34 (m, 8H), 5.39 (s, 2H).13C NMR (101 MHz, CDCI3) δ 171.2, 163.7, 135.4, 133.9, 133.8, 130.4, 128.9, 128.6, 128.6, 128.5, 128.1 , 68.9.

[0336] Benzyl (3-(trifluoromethyl)benzylidene)carbamate:

[0337] In accordance with the general procedure (method B), a reaction flask was charged with benzyl ((phenylsulfonyl)(3-(trifluoromethyl)phenyl)methyl)carbamate (14.2 mmol, 6.4 g), DCM (222 mL) and an aqueous solution of K2CO3(227 mL, 1 .4 M). After vigorous stirring during 18h, standard workup afforded the desired product as a white solid (4.3 g, 13.9 mmol, yield: 97%).

[0338] 1H NMR (400 MHz, CDCI3) δ 8.95 (s, 1 H), 8.21 (d, J = 1.9 Hz, 1 H), 8.08 (d, J = 7.8 Hz, 1 H), 7.86-7.77 (m, 1 H), 7.63 (t, J = 7.9 Hz, 1 H), 7.52-7.42 (m, 2H), 7.44-7.32 (m, 4H), 5.33 (s, 2H).

[0339] 13C NMR (101 MHz, CDCI3) δ 169.4, 163.3, 135.2, 134.7, 133.6, 130.3, 129.8, 128.8, 126.9, 126.7, 69.3.

[0340] Proline-catalyzed Mannich reactions:

[0341] General Procedure: In a three-neck flask under inert atmosphere add N-Cbz Imine (1 .0 eq.) and (R)- or (S)-proline (30 mol %), then anhydrous CH3CN (0.1 M). The resulting solution is cooled to 0°C and under vigorous stirring a first portion of freshly distilled acetaldehyde (12.5 eq.) is added. After 2 h at the same temperature a second portion of freshly distilled acetaldehyde (12.5 eq.) is added. The reaction is kept under stirring at the same temperature for a further 2 h then quenched with water. The solvent is partially evaporated under reduced pressure and then the resulting suspension is extracted with 3 portions of AcOEt. The combined organic moieties are dehydrated over MgSO4, filtered and then the solvents are evaporated under reduced pressure. The desired product is then isolated by flash chromatography.

[0342] Benzyl (R)-(3-oxo-1-phenylpropyl)carbamate: According to the general procedure, a three-neck flask is charged with benzyl benzylidenecarbamate (1 .86 g, 7.76 mmol, 1 .0 eq.), R-proline (268 mg, 30 mol %), anhydrous CH3CN (80 mL, 0.1 M), freshly distilled acetaldehyde (10.8 mL, 12.5 eq.). After 2 h, freshly distilled acetaldehyde (10.8 mL, 12.5 eq.) is added. After 2 h, the reaction is quenched with water. After standard workup, the desired product is isolated by flash chromatography (Hexanes / AcOEt:8 / 2), giving the desired product as a white solid (61 1 mg, 4.6 mmol, yield: 60%) ([α]D=+8, 18.8 mg in CHCI3).

[0343] 1H NMR (400 MHz, CDCI3) δ 9.69 (s, 1 H), 7.41-7.18 (m, 10H), 5.29-4.99 (m, 3H), 3.04-2.75 (m, 2H).

[0344] 13C NMR (101 MHz, CDCI3) δ 200.1 , 155.7, 136.2, 134.6, 129.9, 129.0, 128.9, 128.7, 128.4, 128.3, 128.0, 126.4, 125.9, 67.2, 67.1 , 50.7, 49.6.

[0345] Benzyl (S)-(3-oxo-1 -phenylpropyl)carbamate:

[0346] According to the general procedure, a three-neck flask is charged with benzyl benzylidenecarbamate (1.7 g, 7.1 mmol, 1.0 eq.), S-proline (245 mg, 30 mol %), anhydrous CH3CN (71 mL, 0.1 M). After 2h freshly distilled acetaldehyde (10.5 mL, 12.5 eq.) is added. After 2 h, the reaction is quenched with water. After standard workup, the desired product is isolated by flash chromatography (Hexanes / AcOEt:8 / 2) to afford the title product as a white solid (1 .45 g, 5.14 mmol, yield: 72%) ([α]D=-8, 18.8 mg in CHCI3).

[0347] 1H NMR (400 MHz, CDCI3) δ 9.69 (s, 1 H), 7.41-7.18 (m, 10H), 5.29-4.99 (m, 3H), 3.04-2.75 (m, 2H).

[0348] 13C NMR (101 MHz, CDCI3) δ 200.1 , 155.7, 136.2, 134.6, 129.9, 129.0, 128.9,

[0349] 128.7, 128.4, 128.3, 128.0, 126.4, 125.9, 67.2, 67.1 , 50.7, 49.6.

[0350] Benzyl (S)-(3-oxo-1-(3-(trifluoromethyl)phenyl)propyl)carbamate: According to the general procedure, a three-neck flask is charged with benzyl (3- (trifluoromethyl)benzylidene)carbamate (2.15 g, 7.0 mmol, 1.0 eq.), S-proline (241.6 mg, 30 mol %), anhydrous CH3CN (70 mL, 0.1 M). After 2h freshly distilled acetaldehyde (9.78 mL, 12.5 eq.) is added to the mixture. After 2 h, the reaction is quenched with water. After standard workup, the desired product is isolated by flash chromatography (Hexanes / AcOEt :7 / 3) to afford the title product as a yellow amorphous solid (1 .63 g, yield: 66%) ([α]D= -5°, 20.1 mg in 2 mL CHCI3).

[0351] 1H NMR (400 MHz, CDCI3) δ 9.71 (s, 1 H), 7.54-7.33 (m, 9H), 5.68 (s, 1 H), 5.29 (s, 1 H), 5.19-5.03 (m, 2H), 3.07-2.90 (m, 2H).

[0352] 13C NMR (101 MHz, CDCI3) δ 199.5, 155.7, 136.2, 131.3 (q, JC-F = 32.8 HZ) 130.0, 129.5, 128.7, 128.5, 128.3, 124.9, 123.2, 67.4, 50.3, 49.3.

[0353] Benzyl (R)-(3-oxo-1-(3-(trifluoromethyl)phenyl)propyl)carbamate:

[0354] According to the general procedure, a three-neck flask is charged with benzyl (3- (trifluoromethyl)benzylidene)carbamate (2.15 g, 7.0 mmol, 1.0 eq.), S-prolina (241.6 mg, 30 mol %), anhydrous CH3CN (70 mL, 0.1 M). After 2h freshly distilled acetaldehyde (9.78 mL, 12.5 eq) is added. After 2 h, the reaction is quenched with water. After standard workup, the desired product is isolated by flash chromatography (Hexanes / AcOEt:2 / 1 , Rf: 0.34), to afford the title product as a yellow amorphous solid (1.70 g, 4.8 mmol, yield: 69%) ([α]D= -5°, 20.1 in 2 mL CHCI3).

[0355] 1H NMR (400 MHz, CDCI3) δ 9.71 (s, 1 H), 7.54-7.33 (m, 9H), 5.68 (s, 1 H), 5.29 (s, 1 H), 5.19-5.03 (m, 2H), 3.07-2.90 (m, 2H).

[0356] 13C NMR (101 MHz, CDCI3) δ 199.5, 155.7, 136.2, 131.3 (q, JC-F = 32.8 HZ) 130.0, 129.5, 128.7, 128.5, 128.3, 124.9, 123.2, 67.4, 50.3, 49.3.

[0357] Synthesis of phosphorus yilides:

[0358] 2-(triphenyl-λ5-phosphaneylidene)acetaldehyde: In a three-neck flask under inert atmosphere, a suspension of methyltriphenylphosphonium bromide (20.0 mmol, 7.19 g) and t-BuOK (24 mmol, 2.7 g) in anhydrous Et2O (24 mmol, 2.7 g) is prepared and stirred at room temperature for 1.5 h. A solution of ethyl formate (28 mmol, 2.07 g) in Et2O (40.0 mL) is added and the resulting suspension is stirred for 1 h. At 0°C, 100 mL of 0.1 M HCI and then 12 mL of 1 M HCI are added. The organic phase is extracted with 3 portions of 0.1 M HCI. The combined aqueous portions are basified using 10% NaOH to form a precipitate. After 2h the solid obtained is filtered under vacuum, washed with water and Et2O, and then dried under vacuum to give the desired product as a yellow solid (3.992 g, 13.1 mmol, yield: 65.5%).

[0359] 1H NMR (400 MHz, CDCI3) δ 8.98 (s, 1 H), 8.89 (s, 1 H, Z), 7.77-7.38 (m, 30H).

[0360] Synthesis of enantioenriched 1 ,2-dihydropyridine

[0361] General Procedure: in a three-neck flask under inert atmosphere, the phosphorus ylide (1 .0 equiv.) are added, then anhydrous AcOEt (0.22 M) is added. The resulting mixture is heated to 80°C with stirring for 15 h, then allowed to cool to room temperature. Trifluoroacetic acid (1.0 eq.) is added to the reaction dropwise and the mixture is again heated to 80°C for two h. After that, the mixture is allowed to cool to room temperature and is dropped into a saturated NaHCO3solution at 0°C. The resulting biphasic mixture is extracted with 3 portions of AcOEt, the combined organic portions are dehydrated over MgSO4and the solvents are evaporated under reduced pressure at 30°C. The desired product is isolated by flash chromatography.

[0362] Benzyl (R)-2-phenylpyridine-1(2H)-carboxylate (Precursor of (-)-M2):

[0363] In accordance with the general procedure, a flask was charged with benzyl (R)-(3- oxo-1 -phenylpropyl)carbamate (1.33 g, 4.7 mmol, 1.0 equiv.), triphenyl-λ5- phosphaneylidene)acetaldehyde (1.48 g, 1.0 equiv.), AcOEt (22.4 mL, 0.22 M). After 15h at 80°C, trifluoroacetic acid is added (0.36 mL, 1.0 eq.) at r.t. and the mixture was again warmed at 80°C for 2h. After standard workup, the desired product is isolated by flash chromatography (Hexanes / Et2O :9 / 1 ), to give an light yellow oil (175 mg, yield: 30%) ([α]D= +560°, 19.5 mg in CHCI3).1H NMR (400 MHz, CDCI3) δ 7.50-7.25 (m, 5H), 6.97 (d, J = 8.6 Hz, 1 H), 6.79* (d, J = 7.7 Hz, 1 H), 6.07-5.99 (m, 1 H), 5.99-5.93 (m, 1 H), 5.91 (d, J = 6.0 Hz, 1 H), 5.77-5.66 (m, 2H), 5.63 (d, J = 7.9 Hz, 1 H), 5.35-5.20 (m, 3H), 5.14 (s, 2H).

[0364] *Major rotamer

[0365] Benzyl (S)-2-phenylpyridine-1(2H)-carboxylate (Precursor of (+)-M2):

[0366] In accordance with the general procedure, a flask was charged with benzyl (S)-(3- oxo-1 -phenylpropyl)carbamate (1.08 g, 3.81 mmol, 1.0 equiv.), triphenyl-λ5- phosphaneylidene)acetaldehyde (1.16 g, 1.0 equiv.), AcOEt (18 mL, 0.22 M). After 15h at 80°C, trifluoroacetic acid is added (0.23 mL, 1 .0 eq.) at r.t. and the mixture was again warmed at 80°C for 2h. After standard workup, the desired product is isolated by flash chromatography (Hexanes / Et2O:9 / 1 ), to give an light yellow oil (319 mg, 1.09 mmol, yield: 29%.) ([α]D= -558°, 19.1 mg in CHCI3). The enantiomeric ratio has been determined to be 99:1 by HPLC analysis eluting with heptane / i-PrOH 95 / 5, 0.5 ml / min, λ = 220 nm), 10.04 min (major) and 10.98 min (minor).

[0367] 1H NMR (400 MHz, CDCI3) δ 7.50-7.25 (m, 5H), 6.97 (d, J = 8.6 Hz, 1 H), 6.79* (d, J = 7.7 Hz, 1 H), 6.07-5.99 (m, 1 H), 5.99-5.93 (m, 1 H), 5.91 (d, J = 6.0 Hz, 1 H), 5.77-5.66 (m, 2H), 5.63 (d, J = 7.9 Hz, 1 H), 5.35-5.20 (m, 3H), 5.14 (s, 2H).

[0368] Benzyl (S)-2-(3-(trifluoromethyl)phenyl)pyridine-1(2H)-carboxylate

[0369] (Precursor of (+)-M21):

[0370] In accordance with the general procedure, a flask was charged with benzyl (S)-(3- oxo-1 -(3-(trifluoromethyl)phenyl)propyl)carbamate (1.57 g, 4.4 mmol, 1.0 equiv.), triphenyl-λ5-phosphaneylidene)acetaldehyde (1.36 g, 1.0 equiv.), AcOEt (18 mL, 0.22 M). After 15h at 80°C, trifluoroacetic acid is added (2.5 eq.) at r.t. and the mixture was again warmed at 80°C for 5h. After standard workup, the desired product is isolated by flash chromatography (Hexanes / Et2O:9 / 1 ), to give a light yellow oil (500 mg, 1 .03 mmol, yield: 31%.) ([α]D=-420°, 19.7 mg in 2 mL di CHCI3). The enantiomeric ratio has been determined to be 93:7 by HPLC analysis eluting with heptane / i-PrOH 95 / 5, 0.5 ml / min, λ = 220 nm), 7.04 min (major) and 7.73 min (minor).

[0371] 1H NMR (400 MHz, CDCI3) δ 7.81-7.10 (m, 9H), 6.93 (dd, J = 71.6, 7.8 Hz, 1 H), 6.13-5.97 (m, 2H), 5.83* (d, J = 5.7 Hz, 1 H), 5.74-5.59 (m, 1 H), 5.42-5.09 (m, 3H).

[0372] *Minor rotamer

[0373] Benzyl (R)-2-(3-(trifluoromethyl)phenyl)pyridine-1(2H)-carboxylate

[0374] (Precursor of (-)-M21):

[0375] In accordance with the general procedure, a flask was charged with benzyl (R)-(3- oxo-1 -(3-(trifluoromethyl)phenyl)propyl)carbamate 1.7 g, 4.8 mmol, 1.0 equiv.),triphenyl-λ5-phosphaneylidene)acetaldehyde (1.48 g, 1.0 equiv.), AcOEt (23 mL, 0.22 M). After 15h at 80°C, trifluoroacetic acid is added (1 .39 mL, 2.5 eq.) at r.t. and the mixture was again warmed at 80°C for 5h. After standard workup, the desired product is isolated by flash chromatography (Hexanes / Et2O:9 / 1 , Rf:0.37), to give a light yellow oil (425 mg, 1.18 mmol, yield: 25%.) ([α]D=+420°, 19.7 mg in 2 mL di CHCI3).

[0376] 1H NMR (400 MHz, CDCI3) δ 7.81-7.10 (m, 9H), 6.93 (dd, J = 71.6, 7.8 Hz, 1 H), 6.13-5.97 (m, 2H), 5.83* (d, J = 5.7 Hz, 1 H), 5.74-5.59 (m, 1 H), 5.42-5.09 (m, 3H).

[0377] *Minor rotamer

[0378] Deriving from (R) 1 ,2-DHP

[0379] Benzyl ( 7R,4S,6R)-6-phenyl-3-(2-phenylacetyl)-2-oxa-3,5- diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (Precursor of (-)-M2):

[0380]

[0381] In accordance with the general procedure, a three-neck flask was charged with benzyl (R)-2-phenylpyridine-1 (2H)-carboxylate (0.6 mmol 1 ,2-DHP, 175 mg), N- hydroxy-2-phenylacetamide (1.3 eq, 0.78 mmol, 1 18.3 mg), NaIO4(1.3 eq, 0.54 mmol, 1 16.12 mg), MeOH (5 mL), H2O (0.33 mL). After usual workup, the crude mixture was purified by means of flash chromatography (Hexanes / AcOEt: 7 / 3, Rf: 0.35) to give the title compound as an amorphous yellow solid (103 mg, yield: 47.1 %) ([α]D= +47°, 1 1 .2 mg in 2 mL CHCI3).

[0382] 1H NMR (400 MHz, CD3CN) δ 7.48-7.07 (m, 14H), 6.88 (d, J = 18.5 Hz, 3H), 6.20- 6.06 (m, 1 H), 5.27-4.87 (m, 4H), 3.76-3.50 (m, 2H).

[0383] 13C NMR (101 MHz, CD3CN) δ 173.6, 155.1 , 137.0, 136.7, 136.4, 134.6, 132.4, 129.8, 128.5, 128.5, 127.9, 126.9, 75.50, 67.3, 60.2, 58.8, 39.9.

[0384] Benzyl (4aR, 8S,8aS)-3-benzyl-8-phenyl-8,8a-dihydropyrido[4, 3- e][1 ,4,2]dioxazine-7(4aH)-carboxylate (Precursor of (-)-M2):

[0385] In accordance with the general procedure, a three-neck flask was charged with benzyl ( 1R,4S, 6R)-6-phenyl-3-(2-phenylacetyl)-2-oxa-3,5-diazabicyclo[2.2.2]oct- 7-ene-5-carboxylate (1.0 eq, 103 mg, 0.234 mmol), DCE (0.15 M, 1.56 mL), CuCI (0.2 eq; 4.63 mg, 0.1 mmol). The reaction mixture was allowed to react under stirring at 75°C for 5h. After usual workup, the crude mixture was purified by means of flash chromatography (Hexanes / AcOEt:7 / 3, Rf: 0.4) to give the title compound as an amorphous light yellow solid (54 mg, yield: 61 %) ([α]D= -153°, 22.1 mg in 2 mL CHCI3).

[0386] 1H NMR (400 MHz, CDCI3) δ 7.49-7.11 (m, 15H), 6.96 (d, J = 7.0 Hz, 1 H), 5.60 (d, J = 47.0 Hz, 1 H), 5.26-5.01 (m, 2H), 4.88-4.68 (m, 1 H), 4.60 (s, 1 H), 4.11 (d, J = 15.8 Hz, 1 H), 3.53 (s, 2H).

[0387] 13C NMR (101 MHz, CDCI3) δ 156.0, 153.4*, 152.8, 136.6*, 136.1 , 135.7, 135.6, 129.4, 129.0, 128.8, 128.8, 128.6, 128.5, 128.2, 127.8, 127.5, 127.3, 125.9, 101.7*, 101 .2, 68.7, 68.7, 68.0, 65.7, 58.4*, 58.2, 38.4.

[0388] *Major rotamer

[0389] 2-Phenyl-1 -(( 1S,5R, 9S)-9-phenyl-4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3- yl)etan-1-one [(-)-M2] correspond to GT64:

[0390] In accordance with the general procedure, a three-neck flask was charged with benzyl (4aR, 8S,8aS)-3-benzyl-8-phenyl-8,8a-dihydropyrido[4, 3- e][1 ,4,2]dioxazine-7(4aH)-carboxylate (53 mg, 0.12 mmol), THF (0.8 mL), LiBHEt3(8.0 eq, 0.36 mL) stirring at 0°C for 3 h. After standard workup the crude mixture has been purified by trituration with MeOH and n-pentane, to afford the title product as a white solid (5.2 mg, yield: 15%) ([α]D= -128°, 16.3 mg in 2 mL CHCI3).

[0391] Deriving from (S) 1 ,2-DHP

[0392] Benzyl 2-oxo-2-((fS,4H,6S)-6-phenyl-3-(2-phenylacetyl)-2-oxa-3,5- diazabicyclo[2.2.2]oct-7-en-5-il)acetate (Precursor of (+)-M2): In accordance with the general procedure, a three-necked flask was loaded with benzyl (S)-2-phenylpyridine-1 (2H)-carboxylate (1.1 mmol 1 ,2-DHP, 320 mg), N- hydroxy-2-phenylacetamide (1.23 eq, 1.35 mmol, 210 mg), NaIO4(1.23 eq, 1.35 mmol, 210 mg), MeOH (9 mL), H2O (0.67 mL). After the standard workup, the reaction crude was purified by flash chromatography (Hexane / AcOEt: 7 / 3, Rf: 0.35) yielding the desired product as a yellow amorphous solid (206 mg, yield: 42.6%) ([α]D= -47°, 11 mg in 2 mL CHCI3).

[0393] 1H NMR (400 MHz, CD3CN) δ 7.48-7.07 (m, 14H), 6.88 (d, J = 18.5 Hz, 3H), 6.20- 6.06 (m, 1 H), 5.27-4.87 (m, 4H), 3.76-3.50 (m, 2H).

[0394] 13C NMR (101 MHz, CD3CN) δ 173.6, 155.1 , 137.0, 136.7, 136.4, 134.6, 132.4, 129.8, 128.5, 128.5, 127.9, 126.9, 75.50, 67.3, 60.2, 58.8, 39.9.

[0395] Benzyl (4aS, 8aR) 3-benzyl-8-phenyl-8,8a-dihydropyrido[4, 3- e][1 ,4,2]dioxazine-7(4aH)-carboxy-late (Precursor of (+)-M2):

[0396] According to the general procedure a three-necked flask was loaded with benzyl 2-oxo-2-(( 1S,4R,6S)-6-phenyl-3-(2-phenylacetyl)-2-oxa-3,5- diazabicyclo[2.2.2]oct-7-en-5-yl)acetate (1.0 eq, 206 mg, 0.47 mmol), DCE (0.15 M, 3.2 mL), CuCI (0.2 eq; 9.31 mg, 0.1 mmol). The mixture is allowed to stir at 75°C for 5 h. After usual workup, the crude mixture was purified by means of flash chromatography (Hexanes / AcOEt:7 / 3, Rf: 0.4) to afford the desired product as a light yellow amorphous solid (110 mg, yield: 53.2%) ([α]D= +150°, 21.1 mg in 2 mL CHCI3).

[0397] 1H NMR (400 MHz, CDCI3) δ 7.49-7.11 (m, 15H), 6.96 (d, J = 7.0 Hz, 1 H), 5.60 (d, J = 47.0 Hz, 1 H), 5.26-5.01 (m, 2H), 4.88-4.68 (m, 1 H), 4.60 (s, 1 H), 4.11 (d, J = 15.8 Hz, 1 H), 3.53 (s, 2H).

[0398] 13C NMR (101 MHz, CDCI3) δ 156.0, 153.4*, 152.8, 136.6*, 136.1 , 135.7, 135.6, 129.4, 129.0, 128.8, 128.8, 128.6, 128.5, 128.2, 127.8, 127.5, 127.3, 125.9, 101.7*, 101 .2, 68.7, 68.7, 68.0, 65.7, 58.4*, 58.2, 38.4. *Major rotamer

[0399] 2-Phenyl-1-((f / ?,5S,9fi)-9-phenyl-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6-en-3- yl)ethan-1-one [(+)-M2) correspond to GT49:

[0400] According to the general procedure a three-necked flask was loaded with benzyl (4aS,8R, 8aR)-3-benzyl-8-phenyl-8,8a-dihydropyrido[4,3-e][1 ,4,2]dioxazine- 7(4aH)-carboxylate (1 10 mg, 0.25 mmol), THF (1 .2 mL), LiBHEt3(8,0 eq, 2.0 mL); The mixture is allowed to stir at 0°C for 3 h. After standard workup the crude mixture has been purified by trituration with MeOH and n-pentane, to afford the title product as a white solid (9.8 mg, yield: 1 1%). ([α]D= +130°, 16.8 mg in 2 mL CHCI3).

[0401] Deriving from (S) 1 ,2-DHP with CF3:

[0402] Benzyl 2-oxo-2-((1S,4R,6S)-3-(2-phenylacetyl)-6-(3-(trifluoromethyl)phenyl)-

[0403] 2-oxa-3,5-diazabicyclo[2.2.2]oct-7-en-5-yl)acetate (Precursor of (+)-M21):

[0404] According to the general procedure a three-necked flask was loaded with benzyl (S)-2-(3-(trifluoromethyl)phenyl)pyridine-1 (2H)-carboxylate (1.39 mmol, 498 mg), N-hydroxy-2-phenylacetammide (1.3 eq, 274 mg), NaIO4(1.3 eq, 385 mg), MeOH (11 .6 mL), H2O (0.77 mL). After usual workup, the crude mixture was purified by means of flash chromatography (Esano / AcOEt: 7 / 3, Rf: 0.28) to afford the desired product as a light yellow amorphous solid (401 mg, resa: 53.7%) ([α]D= -18.2°,

[0405] 20.8 mg in 2 mL CHCI3).

[0406] 1H NMR (400 MHz, CDCI3) δ 7.62-7.01 (m, 15H), 6.90-6.62 (m, 2H), 6.04 (t, J =

[0407] 6.8 Hz, 1 H), 5.43-5.08 (m, 2H), 4.88 (s, 1 H), 3.93-3.54 (m, 2H).13C NMR (101 MHz, CD3CN) δ 134.6, 130.8, 129.8, 129.4, 128.5, 127.9, 126.9, 124.7, 123.7, 75.1 , 67.4, 59.8, 39.9, 19.4.

[0408] Benzyl (4aS,8R,8aR)-3-benzyl-8-(3-(trifluoromethyl)phenyl)-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (Precursor of (+)-

[0409] According to the general procedure a three-necked flask was loaded with benzyl 2-oxo-2-(( 1S,4R,6S)-3-(2-phenylacetyl)-6-(3-(trifluoromethyl)phenyl)-2-oxa-3,5- diazabicyclo[2.2.2]oct-7-en-5-yl)acetate (1.0 eq, 380 mg, 0.74 mmol), DCE (0.15 M, 4.93 mL), CuCI (0.2 eq; 14.7 mg). The mixture is allowed to stir at 75°C for 5 h. After usual workup, the crude mixture was purified by means of flash chromatography (Hexanes / AcOEt:7 / 3, Rf= 0.4) to afford the desired product as a light yellow amorphous solid (110 mg, yield: 53.2%) ([α]D= +98°, 27 mg in 2 mL CHCI3).

[0410] 1H NMR (400 MHz, CD3CN) δ 7.69-6.93 (m, 14H), 5.62 (d, J = 3.2 Hz, 1 H), 5.16 (d, J = 10.0 Hz, 2H), 4.78 (s, 1 H), 4.52 (s, 1 H), 4.21 (s, 1 H), 3.48 (s, 2H).

[0411] 13C NMR (101 MHz, CD3CN) δ 156.5, 153.8, 134.0, 136.7, 131.6 (q, JCF = 32.3 Hz), 131.0, 130.7, 130.6, 129.6, 129.5, 129.4, 129.1 , 128.2, 127.9, 127.8, 126.5, 126.0, 126.0, 123.8, 123.7, 102.9, 68.6, 66.2, 58.5, 38.5.

[0412] 2-Phenyl-1-((1S,5S,9S)-9-(3-(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)ethan-1-one [(+)-M21) correspond to GT78]:

[0413]

[0414] According to the general procedure a three-necked flask was loaded with benzyl (1-(4aS,8R,8aR)-3benzyl-8-(3-(trifluoromethyl)phenyl)-8,8a-dihydropyrido[4,3- e][1 ,4,2]dioxazine-7(4aH)-carboxylate (21 1 mg, 0.42 mmol), THF (2.77 mL), LiBHEt3(8,0 eq, 3.3 mL); the mixture is allowed to stir at 0°C for 2 h. After standard workup the crude mixture has been purified by flash chromatography (Hexanes / Et2O : 1 / 1 , Rf: 0.2) to afford the title compound as an amorphous white solid (10 mg, yield: 5.9%)([«]D = +83°, 6 mg in 2 mL CHCI3), (mp. 11 1 -1 16°C). The enantiomeric ratio has been determined to be 93:7 by HPLC analysis eluting with heptane / i-PrOH 90 / 10, 0.5 ml / min, λ = 220 nm), 14.03 min (major) and 15.17 min (minor).

[0415] Deriving from (R) 1 ,2-DHP with CF3

[0416] Benzyl (1R,4S,6R)-3-(2-phenylacetyl)-6-(3-(trifluoromethyl)phenyl)-2-oxa-3,5- diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (Precursor of (-)-M21):

[0417] According to the general procedure a three-necked flask was loaded with benzyl R)-2-(3-(trifluoromethyl)phenyl)pyridine-1 (2H)-carboxylate (1.18 mmol, 425 mg), N-hydroxy-2-phenylacetamide (1.3 eq, 233 mg), NaIO4(1.3 eq, 327 mg), MeOH (10 mL), H2O (0.66 mL). After usual workup, the crude mixture was purified by means of flash chromatography (Hexanes / AcOEt: 2 / 1 , Rf: 0.3) to give the desired title compound as a yellow amorphous solid (343 mg, yield: 57.2%) ([α]D= +18.3°, 21 mg in 2 mL CHCl3).

[0418] Benzyl (4aH,8S,8aS)-3-benzyl-8-(3-(trifluoromethyl)phenyl)-8,8a- dihydropyrido[4,3-e][1 ,4,2]dioxazine-7(4aH)-carboxylate (Precursor of (-)-

[0419] According to the general procedure a three-necked flask was loaded with benzyl ( 7R,4S,6 / :?)-3-(2-phenylacetyl)-6-(3-(trifluoromethyl)phenyl)-2-oxa-3,5- diazabicyclo[2.2.2]oct-7-ene-5-carboxylate (1.0 eq, 343 mg, 0.68 mmol), DCE (0.15 M, 4.53 mL), CuCI (0.2 eq; 13.5 mg). The mixture is allowed to stir at 75°C for 15 h. After usual workup, the crude mixture was purified by means of flash chromatography (Hexanes / AcOEt:7 / 3, Rf: 0.4) to give the desired title compound as a yellow amorphous solid (240 mg, yield: 69.4%)([α]D= -98°, 28 mg in 2 mL CHCl3).

[0420] 2-Phenyl-1-((1S,5R,9S)-9-(3-(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)ethan-1-one [(-)-M21) correspond to GT79]:

[0421] According to the general procedure a three-necked flask was loaded with benzyl (4aR,8S,8aS)-3-benzyl-8-(3-(trifluoromethyl)phenyl)-8,8a-dihydropyrido[4,3- e][1 ,4,2]dioxazine-7(4aH)-carboxylate (240 mg, 0.62 mmol), THE (4.13 mL), LiBHEt3(8,0 eq, 4.96 mL).The mixture is allowed to stir at 0°C for 2 h. After usual workup, the crude mixture was purified by means of flash chromatography (Hexanes / Et2O : 1 / 1 , Rf: 0.2) to give the desired title compound as a white amorphous solid (1 1 mg, yield: 5.9%)([α]D= -85°, 8 mg in 2 mL CHCl3), (mp. 1 11 - 116°C). II rapporto enantiomerico di 90 / 10 e stato determinato tramite HPLC con colonna Daicel AD-H (heptane / i-PrOH 90 / 10, 0.5 ml / min, λ = 220 nm), 14.03 min (minor) and 15.17 min (major).

[0422] Example 2: in vitro test

[0423] In vitro assay of intracellular calcium increase. The increase in intracellular calcium was evaluated by Fluo-4 NW assay (ThermoFischer). This test allows in particular to highlight whether a certain compound promotes the increase in intracellular calcium concentration.

[0424] STC1 cells were incubated with the probe provided by the kit. Following the baseline reading, the compounds were added and the increase in fluorescence was monitored, which is directly proportional to the increase in intracellular calcium.

[0425] The results are shown in figure 1 . The graph shown here (Figure 1 ) shows the area under the curve obtained following the analysis of the kinetics of increase in fluorescence, and therefore increase of intracellular calcium, following the reported treatment (with DMSO 1%, JT010, A23187, M2, M21 , M21 HCI, M23, M24, M25, M26, M27, M28, M29, M30, M31 , M32, M33, M34, M35, M36, M37, M38, GT49, GT64, GT78, GT79).

[0426] It is specified that:

[0427] - the DMSO compound represents the reference vehicle;

[0428] - compound JT010 is a potent selective activator of TRPA1 channel;

[0429] - A23187 is a compound capable of forming stable complexes with calcium ions, (calcium chromophore)

[0430] - compounds M21 , M21 HCI, M28, M29, M30, M33, M35 and M38 are compounds in accordance with the invention;

[0431] - compounds M2, M24, M26 and M36 are compounds covered by the previous international patent WO2018 / 220542 and included in the present experimentation for comparison;

[0432] - compounds, M23, M25, M27, M31 , M32, M37 and M34 are alternative compounds, not included in the invention nor in accordance with the previous patent WO2018 / 220542; - compounds GT64, GT49, GT78 e GT79, are enantioenriched compounds of, respectively, M2 (GT64, GT49) and M21 (GT78, GT79).

[0433] The statistical analysis was carried out by One way ANOVA, followed by Dunnet post-test and the asterisks (*) indicate the degree of significance with respect to the vehicle.

[0434] As expected, the known activator of TRPA1 channel and calcium chromophore A23187, show a significant increase of intracellular calcium. In particular, as regard compounds in accordance with the invention M30, M33, M29, M21 (also in its chlorohydrate form), M35 and M38, and also enantioenriched compounds GT78 and GT79, of which, in particular, the levorotatory isomer (-)-M21 (GT79) is the more active form, have shown a marked significant activity with respect to the vehicle. Actually, compounds M21 (also in its hydrochloride form), M29, M35 and M38 have shown an activity superior to that of potent reference compounds JT010 and A23187.

[0435] From this screening test, among all the compounds tested, compound M21 was the best performer, both in base form and in hydrochloride salt form, and its activity was still higher or comparable to that of the reference compounds, even at less than a third of the doses tested (3μm vs 10μm).

[0436] Evaluation of the increase of GLP-1

[0437] The increase in GLP-1 measured in pg / ml produced by STC-1 cells following treatments with known compounds (JT010 and M23) and with compounds in accordance with the invention (M33, M29, M30 and M21 ).

[0438] The results of this test have been reported in graph 2A of figure 2A.

[0439] Figure 2B shows the area under the curve obtained following the analysis of the kinetics of fluorescence increase, and therefore intracellular calcium increase, following the treatments with compounds (M23, M33, M30, M29, M21 ) covered by the invention that showed greater activity compared to the vehicle and hence have been the object of further pharmacological investigation.

[0440] The statistical analysis was carried out by means of a student t-test and the asterisks (*) indicate the degree of significance with respect to the vehicle.

[0441] The compound JT010, a known activator of TRPA1 channels, induced a significant increase in the amount of GLP-1 compared to the vehicle. Surprisingly, given the considerable structural difference compared to JT010, a similar effect was obtained following treatment with the compound M29. Indeed, the compounds M21 and M30 promoted a higher release of GLP-1 than the reference compound.

[0442] Figure 2C shows the amount of GLP-1 measured in pg / ml produced by STC-1 cells as a result of treatments with known compounds and with compound M21 according to the invention, selected as the best performer also in term of secretion of GLP-1.

[0443] The model set-up was carried out using the well-known activator JT010: the pre- incubation of the A967079 blocker as shown in graph 2C of figure 2C, completely eliminated the increase in GLP-1 production, indicating the involvement of the TRPA1 channel in the release of GLP-1 . In addition, the pre-incubation of the A967079 blocker has zeroed, bringing back to the values obtained with the vehicle, the production of GLP-1 following the incubation of M21 , the compound object of the invention.

[0444] To have information about the mechanism of action, the most active compounds (M21 , M29 and M30) were tested in the presence of the blocker. The results are shown in Figure 3 (3A, 3B, 3C and 3D), in which the increase in intracellular calcium kinetics can be appreciated.

[0445] The set up of the model was carried out using the well-known activator JT010: the pre-incubation of the A967079 blocker as shown in graph A of figure 3A, completely eliminated the increase in fluorescence and therefore the increase in intracellular calcium, indicating the failure to open the channel. A similar condition was recorded when the blocker was added before the compounds M21 , M29 and M30. As can be seen from graphs B, C and D in figures 3B, 3C and 3D respectively, the compounds tested in the presence of the blocker showed a significantly reduced ability to induce calcium ingress. Surprisingly, the compound M21 (graph C in figure 3C) in the presence of the blocker still shows an activity comparable to the known reference activator JT010. This response will be the investigation of subsequent experiments.

[0446] Figure 3 also shows the graphs showing the increase in fluorescence relative to the increase in intracellular calcium following treatments carried out on cells with some compounds according to the invention and some comparison compounds, reported in the presence or absence of the TRPA1 channel blocker (Fluo-4 NW assay). See the graphs 3E, 3F, 3G, 3H, 31, 3J, 3K, 3L, 3M, 3N, 30, 3P, 3Q, 3R, 3S, 3T, 3U, 3V, 3W and 3X, respectively in figure 3E, 3F, 3G, 3H, 31, 3J, 3K, 3L, 3M, 3N, 30, 3P, 3Q, 3R, 3S, 3T, 3U, 3V, 3W and 3X.

[0447] The graphs show the trend of intracellular calcium increase using STC1 cells following treatment with the patented compounds.

[0448] In particular in graph:

[0449] 3E, it can be appreciated how compounds M2, GT49 and GT64 show a similar trend to that of vehicle;

[0450] 3F, it can be appreciated how compound M21 maintain a similar activity to potent JT010, also in the presence of a blocker of TRPA1 channels;

[0451] 3G, it can be appreciated how compound M23 have a similar activity to that of JT010 despite the considerable structural differences;

[0452] 3H, it can be appreciated how compound M24 shows a similar and comparable trend to the vehicle;

[0453] 3I, it can be appreciated how compound M25 shows a similar and comparable trend to the vehicle;

[0454] 3J, it can be appreciated how compound M26 shows a similar and comparable trend to the vehicle;

[0455] 3K, it can be appreciated how compound M27 shows a similar and comparable trend to the vehicle;

[0456] 3L, it can be appreciated how compound M28 shows a similar and comparable trend to the vehicle;

[0457] 3M, it can be appreciated how compound M29 shows a superior activity with respect to the potent JT010 and how in the presence of TRPA1 channel blocker the increase of calcium has zeroed;

[0458] 3N, it can be appreciated how compound M29 shows a superior activity with respect to the potent JT010 and how in the presence of TRPA1 channel blocker the increase of calcium has zeroed;

[0459] 30, it can be appreciated how compound M32 shows a similar and comparable trend to the vehicle;

[0460] 3P, it can be appreciated how compound M33 shows a superior activity with respect to the potent JT010;

[0461] 3Q, it can be appreciated how compound M35 shows a marked superior activity with respect to the potent JT010; 3R, it can be appreciated how compound M36 shows a marked superior activity with respect to the potent JT010;

[0462] 3S, it can be appreciated how compound M37 shows a similar and comparable trend to the vehicle;

[0463] 3T, it can be appreciated how compound M38 shows a marked superior activity with respect to the potent JT010;

[0464] 3U, it can be appreciated how the compound M21 shows a concentration- dependent trend with regards to the increase in intracellular calcium;

[0465] 3V, it can be appreciated how compound M21 , GT78 and GT79 show a marked superior activity and camparable between them, with respect to the potent JT010;

[0466] - 3W, it can be appreciated how compound M40 shows a superior activity with respect to the potent JT010;

[0467] - 3X, it can be appreciated how compound M41 shows a superior activity with respect to the potent JT010.

[0468] Graph 3Y of figure 3Y shows the EC50 value of M21 , calculated using the GraphPad Prism 8 program, interpolating the data using the function that describes the response obtained as a function of the Log of the concentration of M21 , a compound covered by the patent. The EC50 value ca. 2.18 |iM obtained for M21 denotes an interesting pharmacological potency.

[0469] Example 3 - in vivo test

[0470] Effect of M21 (40 mg / kg) on incretin release in healthy mice with intraperitoneal glucose load

[0471] Method

[0472] "Healthy C57BL6 / j mice" of 8-9 weeks were acclimatized over a period of one week. On the day of the experiment, the mice fasting for 6 hours are divided into two groups, one having the function of placebo and the other of treatment with 6 animals per group (n = 6). All animals received oral gavage. Briefly, the placebo group was treated with 0.25% CMC (carboxymethylcellulose) while the treatment group was given a formulation of M21 prepared with 0.25% CMC at 10 mL / kg volume dose. To verify the impact of M21 on incretin secretions (GLP-1 and GIP), all mice were subjected to intraperitoneal glucose loading (2 mg / kg) 30 minutes after treatment. One hour after the glucose injection, blood samples are taken from each mouse in the retromandibular cheek pocket with a sterile needle. To prevent degradation of GLP-1 , a protease inhibitor cocktail (EMD-Millipore) is added to the sample along with K3EDTA. The plasma contained in the samples is immediately separated by centrifugation at 1 ,500 G for 10 minutes at 4°C. The separated plasma samples were immediately refrigerated to -80 °C until GLP-1 and GIP were quantified using commercial ELISA kits (EMD-Millipore).

[0473] The data was expressed as MEAN ± SEM. Statistical significance was calculated using student t-test in GraphPad Prism (version 7).

[0474] Results

[0475] Oral administration of M21 at a 40 mg / kg dose in healthy mice significantly elevated GLP-1 concentrations after 1 hr of glucose load compared to placebo control (see Figure 4). As for the plasma GIP values, they were found to be similar to the placebo control group.

[0476] Example 4 - Chronic essay

[0477] Effect of chronic administration of M21 at a dosage of 10 mg / Kg.

[0478] Method

[0479] The experiments conducted in chronic use involve the use of 4 treatment groups: healthy control group:

[0480] -5 C57BL6 / mice;

[0481] -diabetic control group: 5 db / db mice;

[0482] -reference drug treatment group: 5 db / db mice treated with Metformin 100mg / Kg; -M21 treatment group: 5 db / db mice treated with M21 10mg / Kg.

[0483] The 6-week-old mice wererandomized according to starting weight and housed according to regulations. The experiments were carried out following ministerial authorization.

[0484] The molecules were administered dissolved in drinking water for a duration of 12 weeks. During treatment, water and food intake, body weight and blood glucose were monitored. The glycated hemoglobin parameter was measured at baseline and at the end of treatment. At the end of treatment, plasma cholesterol and triglyceride levels were assessed. The mice were sacrificed following an overdose with urethane. The blood was protected through the eye, centrifuged in a tube containing EDTA at 1.500 G for 10 minutes at 4°C. The organs (right and left cortex, hippocampus, hypothalamus, retinas, heart, aorta, liver, ileum, colon, visceral and perineal adipose tissue, skeletal muscle) were explanted and stored at -80°C in paraformaldehyde or OCT for further histological analysis. Results:

[0485] The group of db / db mice showed a higher blood glucose level than the healthy group, as evidenced by both basal blood glucose and glycated hemoglobin levels. The weekly blood glucose measurements, being carried out on conscious mice, underwent fluctuations probably due to exogenous factors such as stress and pain during the incision of the tail. This leads to an increase in adrenergic tone and therefore unexpected changes in blood sugar. Measurement of the change in glycated hemoglobin from baseline showed promising results. In fact, calculating the increase in glycated hemoglobin, the groups treated with metformin showed a lower increase than the control group db / db. The compound M21 , despite its complete structural diversity, shows results comparable to the drug already on the market (see Fig. 5).

[0486] At the end of the treatment, plasma triglyceride values were also assessed. As shown, Metoformin halved the triglyceride value compared to db / db mice. The M21 compound even brought circulating triglyceride values back to the levels of the healthy control group (see Figure 6).

[0487] Weight gain since the start of treatment has shown promising results. In fact, when comparing weight gain curves, the compound M21 showed even greater weight reduction activity than metformin (see Figure 7).

[0488] Example 5

[0489] Evaluation of the neuroprotective effects of compound M21

[0490] To evaluate the neuroprotective effects of M21 , an in vitro model was used with SH-SY5Y cells (ATTC CRL-2266™) derived from human neuroblastoma, originating from a bone marrow biopsy. The SH-SY5Y cells were maintained in culture according to certified protocols and differentiated with retinoic acid for 48 hours. After the 48 hours, the cells were subjected to treatment according to the following scheme:

[0491] - Control (basal cell culture without any pharmacological stimulus or stressor, meaning without PALM or MG);

[0492] - Palmitate 400 μM for 4 hours;

[0493] - Methylglyoxal 400 μM for 4 hours;

[0494] - M21 (pre-treatment 10 μM for 48 hours) + / - palmitate (treatment 400 μM for 4 hours); - M21 (pre-treatment 10 μM for 48 hours) + / - methylglyoxal (treatment 400 μM for 4 hours).

[0495] Methylglyoxal and palmitate were used to simulate glucotoxic and lipotoxic damage, respectively, which can be observed in humans with diabetes and / or obesity. Western Blot was used to evaluate the modulation of key signaling pathways underlying multiple cellular activities. Specific proteins such as P-Akt, T- Akt, Caspase-3, and [3-Actin were quantified and normalized using ImageJ software (Figure 8).

[0496] The results shown in Figure 9 demonstrate that M21 increases Akt phosphorylation compared to the control.

[0497] This signaling pathway is fundamental for multiple cellular processes, including cell survival, growth, proliferation, migration, and angiogenesis. As expected, the activity of this pathway is significantly reduced in the presence of methylglyoxal and palmitate, but is fully restored in the presence of M21 .

[0498] Figure 10 shows a graph illustrating the effect of compound M21 on Caspase-3. Caspase-3 belongs to the cysteine-aspartate-specific protease family and serves as a central regulator of cell apoptosis. Recent studies in rats have also shown that Caspase-3 is involved in the regulation of neurogenesis and synaptic activity. In the experimental model performed, it was observed that M21 significantly reduces Caspase-3 compared to the control, as well as in the presence of methylglyoxal and palmitate, confirming the potential anti-apoptotic effect of M21 .

[0499] Figure 1 1 shows a graph illustrating the effect of compound M21 on MARK (ERK1 / 2) p42 / 44. The MARK (ERK1 / 2) p42 / 44 signaling pathway is crucial for the regulation of several transcription factors activated by a wide range of extracellular stimuli with mitogenic properties, growth factors, cytokines, and some evidence suggests the involvement of this pathway in certain forms of neuronal plasticity. In the experimental model, a significant effect of M21 on the phosphorylation of this signaling pathway was observed, suggesting a strong activation that is consistently maintained even in the presence of methylglyoxal and palmitate, which act by reducing the activity of this pathway. Preliminary data based on a limited number of experiments also confirm that the profile of M21 is superior to that of Liraglutide, which, although still preliminary, only protects against damage caused by methylglyoxal and palmitate but does not alter these signaling pathways compared to the control. These aspects reinforce the hypothesis that the mechanism of action of M21 differs from that of GLP-1 analogs in the context of signaling pathways involved in neuroprotection.

[0500] Example 6

[0501] In-depth Assessment of the Neuroprotective Activity of Compound M21

[0502] The evaluation of the neuroprotective potential of M21 was performed using an in vitro model based on human neuroblastoma-derived SH-SY5Y cells (ATTC® CRL- 2266™). These cells were cultured following certified protocols and differentiated with retinoic acid for 48 hours to acquire characteristics similar to human brain neurons. Following the establishment of these experimental conditions, the effects of M21 on various aspects of SH-SY5Y cell biology were assessed under both normal conditions and conditions mimicking the metabolic disturbances typical of diabetes and obesity. The following parameters were evaluated across at least three independent experiments:

[0503] - cell morphology and viability: High-content analysis (HCA) using confocal microscopy (Operetta CLS) was employed to examine the impact of M21 and the simulated diabetic / obesity conditions on cellular structure;

[0504] - intracellular signaling pathways: Western blot analysis was used to measure key proteins involved in neuronal processes: o AKT Pathway: Crucial for cell survival, growth, proliferation, and insulin signaling (which is disrupted in diabetes and obesity). Phosphorylated AKT (P-AKT) represents the active form and was quantified relative to total AKT (T-AKT); o P42 / 44 MAPK Pathway: Involved in synaptic transmission and plasticity, influencing cognitive processes like memory and learning. Phosphorylated p42 / 44 (P-p42 / 44) was assessed relative to total p42 / 44 (T-p42 / 44); o Caspase-3: Caspase-3 belongs to a group of proteases and functions to carry out apoptosis and programmed cell death. Caspase-3 plays a crucial role in the loss of synaptic spines in hippocampal neurons, a brain region essential for memory. Several studies show that inhibiting the action of this protein is associated with an improvement in cognitive functions; o p-Actin: fundamental component of neuronal cytoskeleton and primarily used to normalize the results obtained with the other analyzed proteins; - neuronal glucose utilization was assessed by incubating cells with a glucose analog (2-NBDG), which is indistinguishable from native glucose for the cell and emits fluorescence, allowing the calculation of the amount and location of glucose internalized within the cell. Glucose is the main energy substrate for neurons, but exposure to high glucose levels or advanced glycation end-products (such as in diabetes) causes cellular damage. Therefore, this parameter is particularly important for studying the potential neuroprotective effects of M21 .

[0505] To determine the optimal dose for evaluating M21 neuroprotective effects, a toxicity test was first conducted. Neurons were exposed to increasing concentrations of M21 (0.1 uM, 3 uM, 10 uM, 50 uM and 100 uM), and cellular morphology was analyzed using high-content confocal microscopy. Nuclear morphology was assessed with DAPI (4',6-diamidino-2-phenylindole) staining, which is a fluorescent organic dye that binds strongly DNA region rich in A-T sequences. The morphology of cellular membranes was visualized using fluorescent CellMask647. Concentrations of 50 and 100 μM resulted in significant nuclear and membrane alterations, which were quantified by calculating the nucleus-to-cytoplasm area ratio, a marker of cellular damage (Fig. 12). Therefore, the higher the ratio between the areas, the greater the cellular damage. The results were expressed as a percentage, with the control considered as 100%. It can be observed that the concentration of 10 μM is associated with a slight reduction in the area ratio, which then progressively increases up to the dose of 100 μM, which is clearly cytotoxic. The 10 μM dose was selected for subsequent experiments based on its minimal impact on cell morphology.

[0506] The neuroprotective properties of M21 were evaluated by exploring the effects of the molecule under experimental conditions designed to simulate what happens to neurons in the presence of diabetes and obesity. Specifically, to simulate the effects of acute hyperglycemia, neurons were exposed for 48 hours to glucose concentrations of 30 mM (HG30mM) and 70 mM (HG70mM), comparing these effects to those associated with a physiological glucose concentration of 5 mM. As expected, the results show a progressive and severe cellular deterioration (Fig. 13), which is completely prevented by exposure to M21 in the presence of 30 mM glucose, a condition that can occur in humans when blood glucose levels exceed 500 mg / dL during diabetic ketoacidosis or a hyperosmolar hyperglycemic state. The 70 mM glucose concentration was used to demonstrate the experimental model's ability to capture cellular deterioration with increasing glucose levels; however, M21 was not evaluated in the presence of 70 mM glucose since such a concentration is extremely rare in humans.

[0507] To simulate the effects of chronic exposure of neurons to high glucose concentrations, methylglyoxal was used, a reactive dicarbonyl glucose metabolite with cytotoxic properties. Methylglyoxal is also involved in the formation of advanced glycation end products (AGEs), which are responsible for chronic diabetes complications, including neurodegeneration. To simulate damage associated with obesity, palmitate was used, which increases in high-fat diets, accumulates in the brain, disrupts neurotransmission, and promotes neurodegeneration.

[0508] In these experimental conditions, SH-SY5Y cells were exposed to damage by treating them with 400 μM methylglyoxal (MG) for 4 hours, 400 μM palmitate for 4 hours (P), and their combination (MG + P). M21 was added to MG, P, and MG + P treatments to explore its ability to prevent cellular damage (Fig. 14), which was first evaluated using confocal microscopy to study the effects on cellular morphology and then by western blot analysis to assess intracellular signaling pathways.

[0509] The results show that M21 and palmitate do not alter the ratio between nuclear and cytoplasmic area. Methylglyoxal significantly increases the nuclear-to-cytoplasmic area ratio, even in combination with palmitate. Exposure to M21 provides significant protection against damage from both methylglyoxal alone and in combination with palmitate, confirming its ability to mitigate toxic damage caused by molecules directly involved in the chronic cellular damage underlying diabetes and obesity complications. M21 has also shown the ability to modulate key intracellular signaling pathways, as indicated by data obtained through western blot analysis (Fig. 15). It is evident that palmitate and methylglyoxal significantly reduce Akt activation. M21 significantly increases Akt activation both on its own and in the presence of methylglyoxal and palmitate, confirming its neuroprotective effects. Furthermore, the activation of this signaling pathway is approximately 20% higher than that observed with Liraglutide, suggesting the existence of alternative signaling pathways to those activated by GLP-1 receptor activation that contribute to neuroprotection mechanisms.

[0510] The effect of M21 was also evaluated in response to insulin stimulation (Fig. 16). As expected, insulin significantly increases Akt activation, while methylglyoxal (MG) mitigates this effect, confirming MG role in insulin resistance. In the presence of insulin alone, M21 significantly enhances Akt activation, and interestingly, this effect is maintained even in the presence of MG. These results suggest that M21 can enhance insulin effect on neurons and protect against the reduction typically present in diabetes. Similarly, the activation of P42 / 44 MAP kinase in response to M21 and exposure to MG and palmitate was assessed (Fig. 17). The results show that palmitate does not significantly alter this signaling pathway, while MG significantly reduces it. M21 significantly increases the activation of this pathway, even after exposure to palmitate and MG. The P42 / 44 MAP kinase pathway was also evaluated in response to insulin stimulation (Fig. 18). For this pathway as well, M21 significantly increases insulin ability to activate it and protects against the reduction caused by MG exposure. Finally, M21 effect on caspase-3 was evaluated to explore its anti-apoptotic effects under baseline conditions and in response to MG and palmitate exposure (Fig. 19).

[0511] Both palmitate and MG significantly increase caspase-3 levels, confirming the apoptotic effects of these compounds. M21 significantly reduces caspase-3 levels both alone and in the presence of palmitate and MG, confirming its anti-apoptotic properties.

[0512] In response to insulin stimulation, caspase-3 levels increase significantly, indicating heightened cell proliferation / programmed apoptosis, which is not further exacerbated by MG. However, M21 significantly reduces caspase-3 levels both alone and in the presence of MG, again confirming its neuroprotective effect under these conditions (Fig. 20).

[0513] Next, M21 effect on neuronal glucose utilization was assessed. For this purpose, a model was used under similar experimental conditions with the addition of a glucose analog (2-NBDG), which cells cannot distinguish from native glucose. The unique feature of 2-NBDG is that once internalized in neurons, it is phosphorylated at the C-6 site into 2-NBDG-6-phosphate by the same enzymatic pathway that phosphorylates native glucose. However, 2-NBDG-6-phosphate acquires a chemical conformation that prevents it from continuing through normal metabolic pathways as an energy substrate, making it well-retained in the cell. It emits fluorescence (475-550 nm), whose intensity is proportional to the cell glucose uptake activity. Fluorescence can be evaluated using high-throughput confocal microscopy for High-Content Analysis (HCA), allowing quantification and localization of 2-NBDG-6-phosphate.

[0514] Using a similar experimental design as for the intracellular signaling studies, the effect of M21 was evaluated, both alone and in combination with MG and palmitate. M21 does not significantly alter neuronal glucose utilization, but quantification shows a reduction of about 7% (p=0.009) compared to the control (Fig. 21 ). In contrast, MG significantly reduces neuronal glucose utilization (-14%; p=0.001), and unlike with M21 , this effect is associated with significant alterations in cell morphology. This difference suggests that with M21 , the slight reduction in glucose utilization could limit neuronal exposure to glucose, resulting in neuroprotection, whereas with MG, the reduction in glucose utilization is a direct consequence of cell damage, as shown by previous results on MG effects on cell morphology and viability. However, co-incubation of M21 with MG restores both neuronal glucose utilization and cell morphology (Fig. 21 ). Palmitate and the combination of M21 with palmitate do not significantly alter neuronal glucose utilization (Fig. 21 ). The combination of MG and palmitate enhances the reduction in neuronal glucose utilization, but M21 exposure can still restore glucose utilization.

[0515] In a subsequent series of experiments, M21 neuroprotective effect was also evaluated in astrocytes. Studying astrocytes role in neurodegenerative diseases and diabetes is crucial for better understanding the shared and distinct pathogenic mechanisms contributing to these complex conditions. Astrocytes, glial cells in the central nervous system, play key roles in maintaining neuronal homeostasis, regulating the blood-brain barrier, and modulating the inflammatory response.

[0516] In neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis (ALS), astrocytes exhibit functional alterations that contribute to neuronal dysfunction and disease progression. For example, astrocytes can become reactive, releasing pro-inflammatory cytokines and reactive oxygen species, thereby amplifying inflammation and neuronal damage. Similarly, in the context of diabetes, astrocytes play a key role in regulating cerebral glucose metabolism and the inflammatory response. Diabetes-associated neuroinflammation can contribute to cognitive dysfunction and increase the risk of developing neurodegenerative diseases. Additionally, diabetes can affect astrocyte function, leading to a reduced ability to support neurons and maintain cerebral homeostasis. For this purpose, a microglial cell line (ATCC® CRL-3304™), also known as the HMC3 cell line, was used, representing human astrocytes in research contexts related to neurodegeneration and diabetes. The cells were cultured according to standard protocols to evaluate M21 effects on cell viability and the release of pro-inflammatory and anti-inflammatory cytokines under specific experimental conditions.

[0517] Cell viability was assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] reagent. Briefly, HMC3 cells were exposed to increasing concentrations of M21 (1 - 100 μM) and incubated at 37°C for 24 hours. The MTT reagent was then added to each well at a concentration of 5 mg / mL. After 2 hours, the culture medium was removed from the wells, and 50 pL of DMSO was added. After a 10-minute incubation at 37°C, absorbance at 540 nm was measured using an automatic microplate reader (BIO-TEK, Winooski, VT, USA). The percentage of cell viability was calculated relative to vehicle-treated control cells. The same procedure was followed to detect the cytotoxic effect in HMC3 cells after incubation with AP25-35 (10 μM for 24 hours) in the absence and presence of M21 (Fig. 22).

[0518] AP25-35 is a fragment of the beta-amyloid peptide (Ap), a protein that tends to aggregate, forming amyloid plaques in the brains of patients with Alzheimer's disease. These plaques are considered one of the main pathological markers of the disease and are associated with neuroinflammation and neurodegeneration. Exposing HMC3 cells to AP25-35 allows for the simulation of neuroinflammation, induces cytotoxicity, and alters numerous intracellular signaling pathways that regulate the production of inflammatory cytokines and chemokines.

[0519] The results show that cell viability, as assessed by MTT, is not affected by high M21 concentrations. Exposure to AP25-35 significantly reduces cell viability, which is partially restored by combined exposure to M21 at a dose of 10 μM (the same used in neuron experiments). It has been evaluated the ability of M21 to modulate the release of pro-inflammatory and anti-inflammatory cytokines was also evaluated in response to a pro-inflammatory stimulus with LPS (10 pg / mL) / TNFa (50 ng / mL) (Fig. 23). The results show that exposure to LPS / TNFa increases IL-6 release without significantly altering IL-10 release. M21 treatment significantly reduces IL-6 release while increasing IL-10 release, confirming an anti- inflammatory effect. The impact of M21 on exposure to AP25-35 was also evaluated (Fig. 24). Also in this set of experiments, as expected, AP25-35 causes a significant increase in TNFa and IL-6 release while only marginally altering IL-10 levels. In all conditions, M21 is associated with a reduction in pro-inflammatory cytokine release and an increase in IL-10, confirming M21 protective effects against cell damage associated with AP25-35 exposure.

[0520] In conclusion, the results indicate that M21 is associated with neuroprotective effects, likely due to its pleiotropic actions. In the neuronal model, M21 has demonstrated the ability to protect cellular structure from damage induced by compounds capable of simulating glucotoxicity and lipotoxicity typical in diabetes and obesity. This neuroprotective effect is also observable in intracellular signaling pathways directly involved in cellular functions underlying proliferation, viability, and neuronal apoptosis. These effects are also associated with M21 ability to actively modulate neuronal glucose utilization. In an independent experimental model using glial cells, M21 has shown the ability to reduce the pro-inflammatory effects typically associated with diabetes, obesity, and neurodegenerative diseases like Alzheimer's. Overall, M21 has demonstrated the capacity to effectively modulate various cellular functions underlying neuroprotection, making it a candidate for further exploration as a potential neuroprotective drug in metabolic and neurodegenerative diseases.

[0521] In another cell model, the effect of M21 on other aspects of carbohydrate metabolism, specifically its ability to modulate insulin release from pancreatic p- cells, was evaluated. For this purpose, INS-1 cells, a cell line derived from a rat insulinoma, were used. These cells are widely utilized as an in vitro model for studying insulin secretion. This cell model provides a practical and controllable platform to investigate the mechanisms of insulin secretion, which is essential for understanding the dysfunctions characteristic of diabetes and obesity. INS-1 cells are extensively used to test the efficacy and safety of new antidiabetic drugs and also to perform studies on the toxicity of chemicals and drugs on beta cells. In our experimental model, INS-1 832 / 13 cells were cultured with RPMI 1640 medium supplemented with 10% FBS. The cells were initially cultured in 24-well plates (500,000 cells / well) for 48 hours and then treated with increasing concentrations of M21 : 0.05 μM , 0.1 μM, 0.2 μM , 4 μM, and 10 μM for 1 hour to assess cytotoxicity using the MTT assay. The results show that despite the increase in M21 concentration, there is only a slight reduction in cell viability at the 10 μM dose (Fig. 25). Subsequently, the ability of M21 to modulate insulin release was evaluated under low glucose concentrations (2 mM) and in response to high glucose concentrations (11 mM). The insulin concentration released into the culture medium was measured using the ELISA method, which is one of the primary techniques for measuring insulin concentration (Fig. 26). The results show that increasing concentrations of M21 do not significantly alter insulin release when the glucose concentration is 2 mM (Fig. 26 A). M21 at 10 μM is associated with a slight but not significant increase in insulin release, possibly related to cellular damage as indicated by the trend towards reduced cell viability measured by the MTT assay. Evaluation of insulin release with higher glucose concentrations (11 mM) (Fig. 26 B) shows that INS-1 cells adequately respond to high glucose concentrations (+60% increase in insulin release) and M21 enhances this glucose response up to a concentration of 4 μM. At higher concentrations of M21 , there is a sharp reduction in insulin release, likely due to potential damage induced by M21 concentrations of 10 μM .

[0522] To assess the effect of M21 on the enhancement of insulin release, an experiment was also conducted using human pancreatic islets. The compound M21 was tested on a batch of human pancreatic islets (batch number HP-24067-01 ) obtained from Tebubio. The donor was a 55-year-old Caucasian male, height: 170 cm, weight:87 kg, BMI of 29.9 kg / m2, who had died from a traumatic event; his HbA1C was 5.6% and he was negative for the COVID test.

[0523] The islets were treated by transferring them from the transport vial containing the transport medium (Transport Solution) to an untreated T25 flask containing approximately 6 ml of complete PIM(S) culture medium, gently pipetting the cell suspension to break up any islet clusters. The islets were allowed to recover for 48 hours in a humidified incubator (37°C + 5% CO2). The experiment was conducted by stimulating the islets at a basal glucose concentration (3.3 mM glucose) and at a high glucose concentration (16.7 mM glucose) under various conditions: control (DMSO, diluted 1 :1000 from stock) and M21 , which was tested separately at four final concentrations (0.1 μM, 0.2 μM, 4 μM, 10 μM). The islets were stimulated sequentially, then the sample in each well was first incubated in basal glucose (+M21 or +DMSO) and then at high glucose concentrations (+M21 or +DMSO). After 1 hour, the plate was briefly centrifuged (200 g x 2 min) and the supernatants were collected on ice into 1 .5 ml tubes. All islets in the wells were gently resuspended in KRH + 16.7 mM glucose + / - M21 for the second stimulation period (1 hour at 37°C + 5% CO2). The supernatant collection procedure was repeated as described after the second incubation (high glucose time point). Experimental procedures were replicated at least three times (CTRL, n=3; 0.1 μM, n=4; 0.2 μM, n=4; 4 μM, n=3; 10 μM, n=3). All collected supernatants were then assayed for insulin content using an ELISA assay within a week of the experiment. Preliminary results from this experiment are shown in Figure 27 as %AHG-LG, where HG stands for high glucose (16.7 mM) and LG stands for low glucose (basal, 3.3 mM); the bars represent mean values±SEM. As shown in the graph, M21 at 0.1 μM appears to be the most suitable concentration for enhancing insulin secretion at high glucose levels, whereas 0.2 μM does not seem to have any significant effect compared to the control. In contrast, 10 μM of M21 appears to strongly impair insulin secretion at high glucose concentrations compared to the control. These data also demonstrate the pleiotropic properties of M21 and, although these results are preliminary, they show that in two independent cell models (rat cells and human islets), M21 enhances insulin release from pancreatic beta cells only in the presence of high glucose concentrations. This characteristic suggests that the effect of M21 might be glucose-dependent rather than glucose-independent, as observed with sulfonylureas in pancreatic beta cell exposure.

Claims

CLAIMS1 . A compound having structural formula (I):or an enantiomer, an enantiomeric mixture, or their pharmaceutically acceptable salt, wherein:R1is selected from the group consisting of-a phenyl group substituted with at least one -CF3group, preferably with one or two -CF3groups;-a phenyl group substituted with at least one alkyl group C1-C6, either linear or branched;-a phenyl group substituted with at least one -CF3group, preferably with one or two -CF3groups, and at least one alkyl group C1-C6, either linear or branched; and R2is selected from the group consisting of:(i) phenyl, benzyl, or 2-phenylethyl, optionally substituted with one or more functional groups independently selected from the group consisting of a halogen, a methoxy functional group, and -CF3;(ii) isoxazole;(iii) pyrrole;2. The compound of formula (I) according to claim 1 or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, wherein R1is a phenyl substituted by a -CF3group, preferably in position 2 or 3.

3. The compound of formula (I) according to claim 1 or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, wherein R1is a phenyl substituted by two -CF3groups, preferably in positions 3 and 5.

4. The compound of formula (I) according to claim 1 or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, wherein R1is a phenyl substituted with at least one alkyl group C1-C6, preferably isopropyl, sec- butyl and / or t-butyl.

5. The compound of formula (I) according to claim 4 or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, wherein R1is a phenyl substituted with at least one alkyl group C1-C6, preferably isopropyl, preferably in position 3 or 4.

6. The compound of formula (I) according to any one of the preceding claims or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, wherein R2is selected from the group consisting of:(i) phenyl, benzyl, or 2-phenylethyl, optionally substituted with one or more functional groups independently selected from the group consisting of a halogen, a methoxy functional group, and -CF3;7. The compound of formula (I) according to any one of preceding claims or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, wherein R1is selected from a group consisting of: a phenyl substituted by at least a -CF3group, preferably by one or two -CF3groups, or a phenyl substituted with at least one branched or linear alkyl group C1-C6; and R2is selected from a group consisting of:(i) phenyl, benzyl, or 2-phenylethyl, optionally substituted with one or more functional groups independently selected from the group consisting of a halogen, a methoxy functional group, and -CF3;8. The compound of formula (I) according to any one of claims 1 -3, 6-7 or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, wherein R1is a phenyl substituted with one or two -CF3groups, and R2is benzyl, 2-phenylethyl or(xi)9. The compound of formula (I) according to any one of claims 1 , 4-7 or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, wherein R1is a phenyl substituted with at least one linear or branched alkyl group C1-C6, preferably isopropyl, and R2is benzyl, 2-phenylethyl or10. The compound of formula (I) according to claim 1 or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, selected from a group consisting of:M30 M2911. The compound of formula (I) according to claim 1 or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, having the following structure:M2112. The compound of formula (I) according to claim 1 or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, selected from: - 2-(Thiophen-2-yl)-1 -(( 1S*,5S*,9R*)-9-(3- (trifluoromethyl)phenyl-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)ethan-1 -one;2-Phenyl-1 -(( 1S*,5S*,9R*)-9-(3-(trifluoromethyl)phenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6-en-3-yl)ethan-1 -one;(1 S*,5S*,9R*)-3-(2-Phenylacetyl)-9-(3-(trifluoromethyl)phenyl)-4-oxa-114,3- diazabicyclo[3.3.1]non-6-en-5- ylium chloride;2-Phenyl-1 -((1 R*,5S*,9R*)-9-(4-(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)ethan-1 -one;1 -((1 R*,5S*,9R*)-9-(3,5-bis(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)-2-phenylethan-1 -one;1 -(1 S*,5S*,9R*)-9-(3,5-bis(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)-2-(thiophen-2-yl)ethan-1 -one;- 1 -(( 1S*,5S*,9R*)-9-(4-lsopropylphenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6-en-3- yl)-2-phenylethan-1 -one;2-Phenyl-1 -(1 S*,5S*,9R*)-9-(2-(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-3-yl)ethan-1 -one;- 1 -(1 S*,5S*,9R*)-9-(3-lsopropylphenyl-4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3- yl)-2-phenylethan-1 -one;3-Phenyl-1 -((5S’*)-9-(3-(trifluoromethyl)phenyl)-4-oxa-1 ,3- diazabicyclo[3.3.1]non-6-en-yl)propan-1 -one; and1 -((5S*)-9-(3-lsopropylphenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1 ]non-6-en-3-yl)-3- phenylpropan-1 -one.

13. The compound according to any one of claims 1 -12 or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, for use as a medicament.

14. The compound according to claim 13, wherein the pharmaceutically acceptable salt is the hydrochloride salt.15 The compound according to claim 14, wherein the compound is 2-phenyl-1 - ((1 S*,5S*, 9R>9-(3-(trifluoromethyl)phenyl)-4-oxa-1 ,3-diazabicyclo[3.3.1]non-6- en-3-yl)ethan-1 -one or ( 1S*,5S*,9R* )-3-(2-phenylacetyl)-9-(3-(trifluoromethyl)phenyl)-4-oxa-1 l4,3-diazabicyclo[3.3.1]non-6-en-5-ylium chloride.

16. The compound according to any one of claims 13-15, wherein said use is in the prevention and / or treatment of a pathology or condition selected from a groupconsisting of: obesity, diabetes, preferably type 2 diabetes, dyslipidemic syndromes, metabolic diseases, hepatic steatosis, regulation of sense of satiety and / or appetite, neurodegenerative diseases, preferably those associated with diabetes, obesity, Alzheimer or Parkinson diseases.

17. A pharmaceutical composition comprising a compound according to any one of claims 1 -12 or an enantiomer, an enantiomeric mixture, or a pharmaceutically acceptable salt thereof, preferably the hydrochloride salt, and at least a pharmaceutically acceptable excipient.

18. The pharmaceutical composition according to claim 17, wherein said use is for oral and / or parenteral administration.

19. The pharmaceutical composition according to claim 17 or 18, wherein said use is in the prevention and / or treatment of a pathology or condition selected from a group consisting of: obesity, diabetes, preferably type 2 diabetes, dyslipidemic syndromes, metabolic diseases, hepatic steatosis, regulation of sense of satiety and / or appetite, neurodegenerative diseases, preferably those associated with diabetes, obesity, Alzheimer or Parkinson diseases.

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