Patent Application
20240376092
The present invention relates to compounds capable of modulating the activity of the cellular prion protein (PrPC) and their use for the treatment of neurodegenerative and immune diseases. In particular the compounds of the invention are useful in the treatment of Alzheimer Disease, Prion Disease, Multiple Sclerosis, Autoimmune Encephalitis, Parkinson's Disease, Inflammatory Bowel Disease and Crohn's disease.
Aging is linked to a wide range of molecular, cellular and functional changes, which particularly affect the integrity of the nervous system. One fundamental process altered by aging is protein folding. When proteins misfold, they acquire alternative conformations capable of seeding a cascade of molecular events, ultimately resulting in neuronal dysfunction and death. Indeed, a wide range of age-related disorders is linked to protein misfolding and aggregation in the brain. Examples include common disorders such as Parkinson's and Alzheimer's diseases, as well as rarer disorders such as prion diseases1. Alzheimer's disease is the most common form of dementia in the elderly population, currently affecting almost 36 million individuals worldwide. The number will increase dramatically in the coming decades as the population ages, producing devastating medical and socio-economic consequences. According to the amyloid cascade hypothesis, Alzheimer's disease is a consequence of the accumulation in the brain of the 40-42 amino acid Aβ peptide, a cleavage product of the amyloid precursor protein (APP). The majority of Alzheimer's disease cases manifest as a late onset, sporadic form. However, approximately 5% of cases are inherited in an autosomal dominant fashion. These forms, collectively referred to as familial Alzheimer's diseases, are linked to at least 230 mutations in genes encoding for APP or the presenilins (PS1 or PS2)2. The mutations are thought to favor the accumulation of Aβ peptide in the brain, by increasing its production or reducing its clearance. The Aβ peptide spontaneously forms polymers ranging from small, soluble oligomers to large, insoluble fibrils. A great deal of evidence suggests that soluble Aβ oligomers, rather than fibrillar aggregates, are primarily responsible for the synaptic dysfunction underlying the cognitive decline in Alzheimer's disease3. Aβ oligomers are believed to act by binding to cell surface receptors that transduce their detrimental effects on synapses. The identification of such receptor sites has important therapeutic implications, as they represent potential targets for pharmacological intervention. Recently, a novel candidate has emerged as a receptor for Aβ oligomers: the cellular form of the prion protein (PrPC)4. PrPC, an endogenous, cell-surface glycoprotein of unknown function, plays a central role in transmissible neurodegenerative disorders commonly referred to as prion diseases. PrPC was originally discovered for its central role in transmissible spongiform encephalopathies (also called prion diseases) and has been claimed to participate in several other pathologies of the nervous system, including Alzheimer's and Parkinson's diseases, by acting as a toxicity-transducing receptor for different misfolded protein isoforms. Interestingly, PrPC has also been reported to exert important functions outside the nervous system as well, in particular in the immune system, and the protein has emerged as a key factor for myelin homeostasis. Consistent with these concepts, additional studies have revealed that the absence of PrPC exacerbates inflammatory damage in a variety of laboratory models of brain ischemia, brain trauma, experimental autoimmune encephalomyelitis (EAE), and experimental colitis.
Prion diseases, which can manifest in a sporadic, inherited or acquired fashion, are caused by the conformational conversion of PrPC into a misfolded isoform (called scrapie form of PrP, or PrPSc) that accumulates in the central nervous system of affected individuals. PrPSc is an infectious protein (prion) that propagates itself by binding to PrPC, triggering its conformational rearrangement into new PrPSc molecules5. A great deal of evidence indicates a distinction between prion infectivity and toxicity, and suggested that a physiological function of PrPC may be altered upon binding to PrPSc, to deliver neurotoxic signals. In fact, genetically depleting neuronal PrPC in prion-infected mice has been shown to reverse neuronal loss and clinical progression, despite the continuous production of PrPSc by surrounding astrocytes6. Thus, the presence of PrPC on the neuronal surface is critical not only for supporting PrPSc propagation, but also for transducing its neurotoxicity7,8. This conclusion recently found unexpected support from data involving Aβ oligomers. PrPC emerged from an expression cloning screen as a receptor capable of binding Aβ oligomers with nanomolar affinity. Importantly, PrPC was also found to be a mediator of Aβ-induced synaptotoxicity4. In support of this conclusion, hippocampal slices derived from PrP knockout (KO) mice were shown to be resistant to Aβ oligomer-induced suppression of long-term potentiation (LTP), an in vitro correlate of memory and synaptic function. Consistent with this, application of anti-PrP antibodies was shown to prevent Aβ-induced synaptic dysfunction in hippocampal slices9. Finally, PrPC was required for both the cognitive deficits and reduced survival observed in transgenic mouse models of Alzheimer's disease10. A number of subsequent studies have extended this observation by discovering that several Aβ assemblies, including neurotoxic Aβ oligomers, bind with high affinity to PrPC via two sites in the unstructured, N-terminal tail of the protein (residues 23-27 and 95-105)11. This interaction unleashes a toxic signalling involving the metabotropic glutamate receptor 5 (mGluR5), activation of the tyrosine kinase Fyn, and phosphorylation of the NR2B subunit of NMDA receptors, ultimately producing dysregulation of receptor function, excitoxicity and dendritic spine retraction2. Other recent studies provided evidence that PrPC could mediate the toxicity not only of Aβ oligomers, but also of other 3-sheet-rich protein conformers, including alpha synuclein, involved in Parkinson disease13-15. These results indicate that misfolded assemblies of several different pathogenic proteins could exert their effects by blocking, enhancing or altering the normal activity of PrPC8. The conclusion highlights a close connection between the role of PrPC in several neurodegenerative diseases and its physiological function. Several activities have been attributed to PrPC in the nervous system, mostly based on subtle abnormalities detected in mice or cells depleted for PrPC. These include roles in neuroprotection, synaptic integrity, neuronal excitability and memory formation16. Recently, PrPC has been also shown to play important functions outside the nervous system as well, in particular in the immune system17. PrPC appears to be protective in autoimmune colitis. Inflammatory bowel disease, induced by dextran sodium sulphate (DSS), is more severe in PrPO/O mice than in wild-type mice. Accordingly, overexpression of PrPC greatly attenuates DSS-induced colitis. Upon MHIC/peptide-driven interactions between T cells and dendritic cells (DCs), PrPC migrates to the immunological synapse and exerts differential effects on T cell proliferation and cytokine production, as revealed by ablation or antibody masking on the DCs or on the lymphocyte side of the synapse18. DCs are professional APCs and also very plastic cells that play an important role in T helper (Th) cells differentiation and thus are involved in the induction of both autoimmunity and tolerance19. Surprisingly, authors of the invention found that selected DC subsets express high level PrPC. Recent data have also shown that EAE is worsened in mice lacking PrPC, indicating that this protein may act as a regulatory molecule, and that cells lacking PrPC may become more inflammatory and behave more aggressively against the central nervous system. These results led us to hypothesize that targeting PrPC pharmacologically may activate protective immunoregulatory effects in MS.
A highly robust and quick assay to detect the spontaneous toxicity of mutant PrPC in cell cultures, named the cell-based drug assay (DBCA), has been previously described20. This novel assay was recently employed to identify small molecules capable of abrogating mutant PrPC activity21,22. Importantly, derivatives of one of such molecules (called SMs) arising from several cycles of chemical optimization rescued Aβ-induced synaptic dysfunction in primary hippocampal neurons, and rescued electrophysiological abnormalities induced by prions in mouse brain slices. Collectively, these data have led us to hypothesize that the pharmacological modulation of PrPC activity might confer therapeutic benefit in multiple sclerosis (MS), a neurodegenerative disorder characterized by progressive myelin loss. Moreover, in a mouse model of EAE, it was found that systemic administration of such PrPC modulators resulted in significant reduction of disease severity, compared to untreated controls. Within this conceptual framework, there is still the need for compounds capable of modulating the activity of PrPC.
In the present invention it was surprisingly found that a properly functionalized thiazine-dioxide scaffold provides a series of compounds capable of abrogating mutant PrPC activity. The results obtained within the present invention clearly demonstrate that compounds may represent new therapeutic options for several different pathologies, such as prion and Alzheimer's diseases, autoimmune encephalitis and MS.
It is an object of the present invention a compound of general Formula (I):
is not included.
It is a further object of the invention a compound of formula (I)
Preferably, in the compound of formula (I), A is benzene; and/or Y is SO2; and/or W is C(═O) or CH2; and/or Z is N; and/or X4 and X5 are H.
Still preferably the compound for use according to the invention has general formula (II):
Wherein X1, X2, X3, R1, R2, R2a, R3 and Q are as defined above.
In a preferred embodiment the compound for use according to the invention is selected form the list below:
It is a further object of the invention a compound of general Formula (III):
Preferably, in the compound as defined above A is benzene; and/or Y is SO2; and/or Z is N; and/or X4 and X5 are H.
In a preferred embodiment, the compound of formula (III) is a compound of formula (IIIA):
Wherein X1, X2, R1, R2, R2a, R3 and Q are as defined above for general formula (III).
Still preferably the compound of formula (III) is selected from:
In a preferred embodiment of formula (I) or (II), B is selected from:
In an embodiment of formula (I), ring B is
In a preferred embodiment of formula (III), B is selected from:
In a preferred embodiment the compound as defined above is for medical use, preferably for use in the treatment of a neurodegenerative disease or immune disease, even more preferably for use in the treatment of Prion Disease, Alzheimer Disease, Multiple Sclerosis, Autoimmune Encephalitis, Parkinson's Disease, Inflammatory Bowel Disease, Crohn's Disease.
Another aspect of the present invention relates to a method of treating a disease which benefit of modulation of the activity of PrPc, wherein said disease is Prion Disease, Alzheimer Disease, Multiple Sclerosis, Autoimmune Encephalitis, Parkinson's Disease, Inflammatory Bowel Disease, Crohn's Disease, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I), (II) or (III), with limitations and provisions set out above, including any pharmaceutically acceptable salt, solvate or stereoisomer thereof, as defined hereinabove.
It is a further object of the invention a pharmaceutical composition comprising at least one compound as above defined, alone or in combination with at least one further active compound, and at least one pharmaceutically acceptable excipient for use in the treatment of a neurodegenerative disease, preferably for use in the treatment of Alzheimer Disease, Prion Disease, Multiple Sclerosis and Autoimmune Encephalitis, even more preferably for use in the treatment of Multiple Sclerosis and Autoimmune Encephalitis.
It is a further object of the invention a process for the synthesis of a compound of general formula (III), wherein A is benzene and Y is SO2, comprising the following steps:
As used herein, “alkyl” is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, “C1-6alkyl” is defined to include groups having 1, 2, 3, 4, 5 or 6 carbons in a linear or branched arrangement and specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, hexyl, and so on. Preferably, “C1-6alkyl” refer to “C1-4alkyl” or “C1-3alkyl”. More preferably, “C1-6alkyl” or “C1-3alkyl” refer to methyl.
As used herein, “C1-4alkanediyl” includes methylene, 1,2-ethanediyl and the higher homologues thereof.
As used herein, “O-alkyl” or “alkoxy” represents an alkyl group of indicated number of carbon atoms attached through an oxygen bridge. “O-alkyl” therefore encompasses the definitions of alkyl above. Preferably, O-alkyl refers to a linear or branched OC1-6alkyl group, OC1-4alkyl group, OC1-3alkyl group, or OC1-2alkyl group, or OCH3. Examples of suitable O-alkyl groups include, but are not limited to methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy or t-butoxy. Preferred alkoxy groups include methoxy, ethoxy and t-butoxy.
As used herein, the terms “haloalkyl” and “O-haloalkyl” mean an alkyl or an alkoxy group in which one or more (in particular, 1 to 3) hydrogen atoms have been replaced by halogen atoms, especially fluorine or chlorine atoms. An haloalkoxy group is preferably a linear or branched haloalkoxy, more preferably a haloC1-3alkoxy group, still more preferably a haloC1-2alkoxy group, for example OCF3, OCHF2, OCH2F, OCH2CH2F, OCH2CHF2 or OCH2CF3, and most especially OCF3 or OCHF2. An haloalkyl group is preferably a linear or branched haloalkyl group, more preferably a haloC1-3alkyl group, still preferably a haloC1-2alkyl group, for example, CF3, CHF2, CH2F, CH2CH2F, CH2CHF2, CH2CF3 or CH(CH3)CF3. Still preferably, any one of haloalkyl, haloC1-6alkyl, haloC1-4alkyl group, haloC1-3alkyl group refers to: CF3, CHF2, CH(CH3)CF3, CH2CF3 or (CH3)2CF3.
As used herein the term “alkylamino” represents an alkyl group of indicated number of carbon atoms substituted by at least one amino group, wherein said amino group is —NH2 or is further substituted with one or two alkyl groups. For example, C1-4alkylamino indicates butylamine, isobutylamine, tert-butylamine, butyl-NH(CH3), isobutyl-NH(CH3), tert-butyl-NH(CH3), butyl-N(CH3)2, isobutyl-N(CH3)2, tert-butyl-N(CH3)2, butyl-NH(C2H5), isobutyl-NH(C2H5), tert-butyl-NH(C2H5), butyl-N(C2H5)2, isobutyl-N(C2H5)2, tert-butyl-N(C2H5)2, butyl-N(C2H5)(CH3), isobutyl-N(C2H5)(CH3), tert-butyl-N(C2H5)(CH3), and the like. As used herein “OC1-4alkylamino” represents the above C1-4alkylamino attached through an oxygen bridge.
As used herein, “NH-alkyl” represents an alkyl group of indicated number of carbon atoms attached through a NH bridge. Preferably, NH-alkyl refers to a linear or branched NHC1-6alkyl group, NHC1-4alkyl group, NHC1-3alkyl group, or NHC1-2alkyl group, or NHCH3. Similarly, “N(alkyl)2” represents two alkyl groups of indicated number of carbon atoms attached through a nitrogen bridge.
As used herein, “S-alkyl” represents an alkyl group of indicated number of carbon atoms attached through a sulphur bridge. “S-alkyl” therefore encompasses the definitions of alkyl above. Preferably, S-alkyl refers to a linear or branched SC1-6alkyl group, SC1-4alkyl group, SC1-3alkyl group, or SC1-2alkyl group, or SCH3. Examples of suitable S-alkyl groups include, but are not limited to thiomethyl, thioethyl, thiopropyl, thio-i-propyl, thio-n-butyl, thio-s-butyl or thio-t-butyl. Preferred S-alkyl groups include thiomethyl, thioethyl and thiopropyl.
As used herein, the term “aryl” means a monocyclic or polycyclic aromatic ring comprising carbon atoms and hydrogen atoms. If indicated, such aromatic ring may include one or more heteroatoms, then also referred to as “heteroaryl” or “heteroaromatic ring”. Illustrative examples of heteroaryl groups according to the invention include 5 or 6 membered heteroaryl such as thiophene, oxazole, oxadiazole, thiazole, thiadiazole, imidazole, pyrazole, pyrimidine, pyrazine and pyridine. A preferred aryl according to the present invention is phenyl. A preferred heteroaryl according to the present invention is pyridyl. Further preferred 5 membered heteroaryl rings are oxadiazole and oxazole. Said oxadiazole is preferably substituted with one methyl group.
As used herein, the term “cycloalkyl” means saturated cyclic hydrocarbon (cycloalkyl) with 3, 4, 5 or more carbon atoms and is generic to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and so on. The term “cycloalkyl” further refers to polycyclic saturated ring systems, such as decahydronaphthalene, octahydro-1H-indene, adamantane and the like. Said saturated ring optionally contains one or more heteroatoms (also referred to as “heterocyclyl” or “heterocyclic ring” or “heterocycloalkyl”), such that at least one carbon atom is replaced by a heteroatom selected from N, O and S, in particular from N and O. Preferably, said cycloalkyl is cyclohexyl, still preferably cyclopentyl. Preferably, said heterocycloalkyl is pyperidine, pyrrolidine, morpholine, piperazine and other cyclic amines. Still preferably said heterocycloalkyl is tetrahydrofurane or tetrahydropyrane. As used herein, the term “halogen” refers to fluorine, chlorine, bromine and iodine, of which fluorine, chlorine and bromine are preferred.
The compounds of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E. L. Eliel and S. H. Wilen, Stereochemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers and mixtures thereof, including optical isomers, all such stereoisomers being included in the present invention. Compounds described in this invention containing olefinic double bonds include E and Z geometric isomers, unless stated otherwise. Also included in this invention are all salt forms, polymorphs, hydrates and solvates.
The term “polymorphs” refers to the various crystalline structures of the compounds of the present invention. This may include, but is not limited to, crystal morphologies (and amorphous materials) and all crystal lattice forms. Salts of the present invention can be crystalline and may exist as more than one polymorph.
Solvates, hydrates as well as anhydrous forms of the salt or the free compound are also encompassed by the invention. The solvent included in the solvates is not particularly limited and can be any pharmaceutically acceptable solvent. Examples include water and C1-4 alcohols (such as methanol or ethanol).
“Pharmaceutically acceptable salts” are defined as derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as, but not limited to, hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as, but not limited to, acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Organic solvents include, but are not limited to, nonaqueous media like ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, the disclosure of which is hereby incorporated by reference.
“Pharmaceutically acceptable” is defined as those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
The compounds of the present invention find use in a variety of applications for human and animal health. The compounds of the present invention are small molecules capable of modulating or abrogating mutant PrPC activity. More specifically, the compounds of the invention suppress the spontaneous cytotoxicity of a mutant form of PrP (Δ105-125). Further to that, as the mutant PrP molecules sensitize cells to the cytotoxic effect of certain antibiotics, including G418 and Zeocin, the suppression of this antibiotic hypersensitivity phenotype was used as a cellular read-out for screening small molecules libraries in the DBCA assay. Using this approach, compounds of the invention have been found to display at least 30% of activity with respect to a reference compound in suppressing the neurodegenerative phenotype, preferably more than 60%, even more preferably more than 100% of the reference compound.
Compounds of the invention might potentially modulate in an indirect manner the Farnesoid X receptor (FXR)-mediated signaling pathway. Farnesoid X receptor (FXR) is a nuclear receptor for bile acids. Ligand activated-FXR regulates transcription of genes to allow feedback control of bile acid synthesis and secretion. Under physiological conditions, activation of FXR is the major mechanism to suppress bile-acid synthesis by directly inducing target genes in both the liver and intestine, including small heterodimer partner (SHP/Shp, encoded by the NROB2/Nr0b2 gene) and fibroblast growth factor (Fgf) 15 (FGF19 in humans), which in turn inhibits, or activates signaling pathways to inhibit, CYP7A1/Cyp7a1 and CYP8B1/Cyp8b1 gene transcription.36 Within the present invention it has been also discovered that similarly to SM231, the FXR agonist WAY-362450 potently rescues mutant PrP toxicity. In addition, SM231 promoted significant FXR transcriptional activity in mouse primary hepatocytes, although SM derivatives do not act as direct FXR receptor agonists (data not shown). These findings, open the hypothesis that an indirect modulation of the FXR activity is involved in the mechanism of action of the compounds of the invention.
The compounds of the invention find use in the treatment of immune diseases. As used herein, “immune disease” refers to autoimmune diseases or immune system disorders. In a preferred embodiment of the invention, immune disease refers to autoimmune colitis, Inflammatory Bowel Disease or Crohn's Disease.
The compounds of the invention can be administered orally or by parenteral administration, in a dosage range of 0.001 to 1000 mg/kg of mammal (e.g., human) body weight per day in a single dose or in divided doses. One dosage range is 0.01 to 500 mg/kg body weight per day orally in a single dose or in divided doses. Another dosage range is 0.1 to 100 mg/kg body weight per day orally in single or divided doses. For oral administration, the compositions can be provided in the form of tablets or capsules containing 1.0 to 500 milligrams of the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
Embodiments and experiments illustrating the principles of the invention will be discussed with reference to the following figures:
As used herein, the following abbreviations have the following meanings. If an abbreviation is not defined, it has its generally accepted meaning.
BOP: N-(Benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl)tyrosine sodium salt; DIPEA: N,N-Diisopropylethylamine; DMF: N,N-Dimethylformamide; DMSO: N,N-dimethylsulfoxide; EtOAc: ethyl acetate; MeOH: methanol; TBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate; Pyr: Pyridine;
Unless otherwise indicated, reagents and solvents were purchased from common commercial suppliers and were used as such. HPLC-grade solvents used for IPLC analysis were purchased by Sigma-Aldrich and all the employed mobile phases were degassed with 10 min sonication before use. Organic solutions were dried over anhydrous Na2SO4 and concentrated with a rotary evaporator at low pressure. All reactions were routinely checked by thin-layer chromatography (TLC) on silica gel 60F254 (Merck) and visualized by using UV or iodine. Microwave assisted reactions were carried out using the microwave reactor Biotage Initiator 2.0 and parameters were adjusted according to the reaction as indicated in the following examples. Flash chromatography on Merck silica gel 60 (mesh 230-400). Melting points were determined in capillary tubes (Büchi Electrotermal model 9100) and are uncorrected. 1H NMR spectra were recorded at 200 or 400 MHz (Bruker Avance DRX-200 or 400, respectively) while 13C NMR spectra were recorded at 100 MHz (Bruker Avance DRX-400) as well as 2D 1H NMR NOESY run in phase sensitive mode. Chemical shifts are given in ppm (δ) relative to TMS. Spectra were acquired at 298 K. Data processing was performed with standard Bruker software XwinNMR and the spectral data are consistent with the assigned structures. Yields were of purified products and were not optimized. All compounds were ≥95% pure as determined by LC/MS using an Agilent 1290 Infinity System machine equipped with DAD detector from 190 to 640 nm. The purity was revealed at 270.44 nm using a ZORBAX Eclipse Plus C18 (2.1×50 mm, 1.8 μM particle size column) reverse phase was used with gradient of 0-100% CH3CN with 0.1% formic acid (channel B) in water with 0.1% formic acid (channel A) for 20 min at 0.3 mL/min. Injection volume was 0.5 μL with a column temperature of 50° C. All compounds were ≥95% pure as determined by HPLC using a Waters System machine equipped with UV detector. The purity was revealed at 254 and 270 nm by using an X Terra C18 (x mm, μM particle size column) reverse phase was used with isocratic eluent 70:30 of CH3CN (channel C) and water with 0.1% formic acid (channel B) for 10 min at 1 mL/min. Injection volume was 20 μL with a column temperature of 25° C. Peak retention time is given in minutes. HRMS Detection was based on electrospray ionization (ESI) in negative polarity using Agilent 1290 Infinity System equipped with a MS detector Agilent 6540UHD Accurate Mass Q-TOF.
The compounds of the invention can be prepared while using a series of chemical reactions well known to those skilled in the art, altogether making up the process for preparing said compounds and exemplified further. The processes described further are only meant as examples and by no means are meant to limit the scope of the present invention. In particular, the compounds of the present invention may be prepared according to the general procedure outlined in the following Schemes 1, 2, 3 and 4. Alternative synthetic pathways and analogues structures will be apparent to those skilled in the art of organic chemistry.
Scheme 1 shows a procedure useful for making heterocyclic compounds of formula (I) having a dibenzo[c,e][1,2]thiazine 5,5 dioxide scaffold, i.e. wherein A is a phenyl, B is a phenyl, Y is a SO2 group, and wherein W is carbonyl, Z is nitrogen, X4 and X5 are hydrogen, n is as defined for general formula (I). The Q substituent can be selected from those described in general formula (I).
Coupling reaction of an appropriate 2-nitro-benzensulfonyl chloride of formula (1a) with unsubstituted or functionalized aniline, carried-out at 40° C. in dry pyridine, affords the corresponding aryl 2-nitrobenzensulfonamides of formula (2a), in high yields. The nitro group of intermediates of formula (2a) was reduced by using a catalytic reduction employing Raney-Ni and H2 flux or SnCl2·2H2O in acidic conditions, depending on the substrates, to afford amino compounds of formula (3a) which were subsequently diazotized using NaNO2 and HCl followed by addition of NaOH promoting in situ conversion of diazo compounds into unstable intermediates of formula (4a). These latter were immediately isolated as crude products and converted to intermediates of formula (5a) in moderate yields, employing Cu powder and DMSO as solvent at room temperature. Compounds of formula (5a) were reacted with ethyl 2-bromoacetate, under microwave irradiation at 50° C. for 15 min. in DMF and using DIPEA as scavenger to afford compounds of formula (6a) in good yields. Some intermediates of formula (5a) were alkylated exploiting a Mitsunobu reaction to give certain compounds of formula (6a). Some examples of intermediates of formula (6a) were treated with an excess of amines as defined by Q substituents, employing microwaves irradiation at 120° C. and neat conditions to give target compounds of formula (8a). In some cases, intermediates of formula (7a) were treated with a mixture of 10% aq. NaOH and EtOH (1:1 ratio) to afford the corresponding carboxylic acids of formula (7a) which were coupled with aryl amines or alkyl amines, as defined by the Q substituent, and exploiting two different methods that entails the use of condensing agents such as TBTU in CH2Cl2 and using DIPEA as scavenger or the use of SOCl2 as chlorinating agent followed by the addition of amines to give other examples of target compounds of formula (8a). In some examples, di-substituted anilines were used to prepare intermediates of formula (5a) as mixture of regioisomers that were used as it is for the next reaction steps to obtain certain compounds of formula (8a); regioisomers were then separated into final compounds by flash chromatography to afford each pure regioisomer.
Scheme 2 shows a procedure useful for making heterocyclic compounds of formula (I) having a dibenzo[c,e][1,2]thiazine 5,5 dioxide scaffold wherein A is a phenyl, B is a phenyl, Y is a SO2 group, W is absent, Z is nitrogen, X4 and X5 are hydrogen, n is as defined for general formula (I). The Q substituent can be selected from those described in general formula (I).
Certain compounds of formula (5a) were alkylated using bromo/chloroalkyls wherein Z was chosen between those substituents reported in formula (I) and with n=1, 2, 3, 4 by using the appropriate dihalide under microwave irradiation at 80° C. and using DIPEA as scavenger. Scheme 3 shows a procedure for synthesizing compounds SM226 and SM230 starting from a compound SM225 which was demethylated employing BBr3 in CH2Cl2 and added at −60° C. The reaction was then maintained at −30° C. to give the hydroxyl derivative SM226 used as intermediate for a successive O-alkylation using (2-chloroethyl)dimethylamine hydrochloride and Cs2CO3 in DMF at 80° C. to give the target compound SM230.
Scheme 4 depict an example of compound of formula (I) wherein A is a phenyl, B is a 3-methyl-pyrazole, Y is a SO2 group and W is carbonyl and the Q substituent can be selected from those indicated in the formula (I).
Compound 11a, reported in a Korean patent KR2011060653, was reacted with cyclohexylamine by using TBTU as condensing agent in presence of DIPEA affording intermediate 12a in good yield which was then condensed with hydrazine monohydrate in neat conditions at 60° C. giving the target product SM879.
The following examples are provided for the purpose of illustrating the present invention and by no means should be interpreted to limit the scope of the present invention.
The following examples are compounds purchased by AMBINTER and tested as it is for their biological activities. The synthetic procedures reported in schemes 1-3 can be easily adapted to prepare commercially available compounds whose synthesis has not been reported yet.
General procedure to obtain nitrobenzensulfonam ides of formula 2a (Scheme 1): To a solution of commercial or synthesized 2-nitrobenzenesulfonyl chloride (1 equiv.) and the appropriate aniline (2 equiv.) in CH2Cl2, pyridine (1 equiv.) was added at once and the mixture was maintained under magnetic stirring at 30° C. for 2 h. After concentration to one third volume, the mixture was poured into ice-water and acidified with 2N HCl (pH=3) and after digestion under magnetic stirring a precipitate was formed. After filtration the crude was then triturated with cyclohexane/EtOAc (8:2) and filtered again to give benzensulfonamides of formula 2a.
2-nitro-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide: Following the above general procedure and using 3-trifluoromethylaniline the compound was obtained in 93% yield as red solid: mp 132.6-132.7° C.; 1H NMR (200 MHz, acetone-d6): δ 9.40 (brs, 1H, NH), 8.10-7.75 (m, 4H, Ar—H), 7.60-7.30 (m, 4H, Ar—H).
N-(3-chloro-4-fluorophenyl)-2-nitrobenzenesulfonamide: Following the above general procedure and using 3-chloro-4-fluoroaniline the compound was obtained as red solid in 90% yield: mp 110.0-110.1° C.; 1H NMR (200 MHz, acetone-d6): δ 9.25 (brs, 1H, NH), 8.00-7.70 (m, 4H, Ar—H), 7.50-7.40 (m, 1H, Ar—H), 7.30-7.20 (m, 2H, Ar—H).
N-(3,5-dichlorophenyl)-2-nitrobenzenesulfonamide: Following the general procedure above reported and using 3,4-dichloroaniline, the compound was obtained as red solid in 95% yield: mp 128.0-129.0° C.; 1H NMR (400 MHz, acetone-d6): δ 9.55 (brs, 1H, NH), 8.20 (d, J=1.3 and 7.8 Hz, 1H, Ar—H), 8.10-7.80 (m, 3H, Ar—H), 7.40-7.35 (m, 2H, Ar—H), 7.28 (t, J=1.8 Hz, 1H, Ar—H).
5-Fluoro-2-nitro-N-[4-(trifluoromethyl)phenyl]benzenesulfonamide (2a(Int-1)): Following the general procedure reported above, intermediate 5-fluoro-2-nitrobenzenesulfonyl chloride (prepared as reported in Buhr, WO 212110603) was reacted with commercial 3-trifluoromethylaniline to give the compound as red solid in 86% yield: m.p 130-132° C.; 1H NMR (400 MHz, CDCl3): δ 7.30-7.40 (m, 3H, Ar—H), 7.50-7.60 (m, 3H, Ar—H and NH), 7.70 (dd, J=2.8 and 7.7 Hz, 1H, Ar—H), 7.90 (dd, J=4.5 and 8.8 Hz, 1H, Ar—H).
N-(4-bromophenyl)-2-nitrobenzenesulfonamide: the intermediate was prepared following the procedure reported by Kurkin, A. et al. in Tetrahedron: Asymmetry, 2009, 20, 1500-1505. Melting point and spectral data are in agreement with those reported in literature.
N-(3-bromophenyl)-2-nitrobenzenesulfonamide: the intermediate was prepared following the procedure reported by Abramovitch, R. A. et al. in J. Org. Chem. 1977, 42, 2914-2919. Melting point and spectral data are in agreement with those reported in literature.
2-nitro-N-[4-(trifluoromethyl)phenyl]benzenesulfonamide: the intermediate was prepared following the procedure reported by Kang, J. G. et al. in Biosci. Biotechnol. Biochem. 2002, 66, 2677-2682. Melting point and spectral data are in agreement with those reported in literature.
N-[4-(methylthio)phenyl]-2-nitrobenzenesulfonamide: the intermediate was prepared following the procedure reported in PCT WO 2007/003962 A2. Melting point and spectral data are in agreement with those reported in literature.
Scheme 5. Synthetic Procedure for the Preparation of Intermediate of Formula 2a(Int-2).
5-Methoxy-2-nitro-N-[4-(trifluoromethyl)phenyl]benzenesulfonamide (2a(Int-2)). A stirred mixture of 5-fluoro-2-nitro-N-[4-(trifluoromethyl)phenyl]benzenesulfonamide 2a(Int-1) (1.00 g, 2.86 mmol) in aqueous 10% NaOH (20 mL) and MeOH (40 mL) was kept at room temperature for 3 h. The reaction mixture was poured into ice/water and the formed precipitate was filtered off to give the title compound as a white solid in 99% yield: m.p. 142-144° C. 1H NMR (400 MHz, CDCl3): δ 3.89 (s, 3H, OCH3), 7.12 (dd, J=2.7 and 8.9 Hz, 1H, H-4), 7.37 (d, J=8.4 Hz, 2H, H-2′ and H-6′), 7.48 (d, J=2.7 Hz, 1H, H-6), 7.58 (d, J=8.5 Hz, 2H, H-3′ and H-5′), 8.02 (d, J=9.0 Hz, 1H, H-3).
General procedure to obtain aminobenzensulfonamides of formula 3a (Scheme 1): A stirred solution of nitro derivative of formula (2a) (1 equiv.) in EtOH (150 mL) was hydrogenated over a catalytic amount of Raney nickel at room temperature and atmospheric pressure for 2.5 h. The mixture was then filtered over Celite and the filtrate was evaporated to dryness to give the amino derivative pure by TLC (CHCl3/MeOH 98:2).
2-amino-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide: Following the general procedure reported above, the compound was obtained in 96% yield as a whitish solid: mp 88.1-88.2° C. (dec.); 1H NMR (200 MHz, acetone-d6): δ 9.50 (bs, 1H, NH), 7.60-7.25 (m, 5H, Ar—H), 7.25-7.15 (m, 1H, Ar—H), 6.75 (d, J=8.3 Hz, 1H, Ar—H), 6.50 (t, J=8.0 Hz, 1H, Ar—H), 6.75 (bs, 2H, NH2).
2-amino-N-(3-chloro-4-fluorophenyl)benzenesulfonamide: Following the procedure reported above, the compound was obtained as a grey solid, in 95% yield: mp 102.1-102.2° C.; 1H NMR (200 MHz, acetone-d6): δ 9.15 (bs, 1H, NH), 7.40 (dd, J=1.6 and 8.0 Hz, 1H, Ar—H), 7.25-7.00 (m, 4H, Ar—H), 6.75 (d, J=8.3 Hz, 1H, Ar—H), 6.55 (t, J=8.0 Hz, 1H, Ar—H), 5.60 (bs, 2H, NH2).
2-amino-N-(3,5-dichlorophenyl)benzenesulfonamide: Following the procedure reported above, the compound was obtained as a grey solid, in 92% yield: mp 103.0-105.0° C.; 1H NMR (400 MHz, acetone-d6): δ 9.50 (brs, 1H, NH), 7.59 (dd, J=1.5 and 7.9 Hz, 1H, Ar—H), 7.25 (dt, J=1.5 and 7.0 Hz, 1H, Ar—H), 7.18-7.12 (m, 2H, Ar—H), 7.10 (t, J=2.4 Hz, 1H, Ar—H), 6.85 (dd, J=0.9 and 7.9 Hz, 1H, Ar—H), 6.68 (dt, J=0.9 and 7.0 Hz, 1H, Ar—H), 5.65 (brs, 2H, NH2).
2-amino-N-[4-(trifluoromethyl)phenyl]benzenesulfonamide: Following the procedure reported above, the compound was obtained as a grey solid, in 85% yield (reaction time 1.5 h): mp 105.3-105.4° C.; 1H NMR (200 MHz, DMSO-d6): δ10.75 (bs, 1H, NH), 7.40-7.60 (m, 3H, Ar—H), 7.20-7.00 (m, 3H, Ar—H), 6.70 (d, J=8.0 Hz, 1H, Ar—H), 6.50 (t, J=8.0 Hz, 1H, Ar—H), 5.95 (bs, 2H, NH2).
2-amino-N-[4-(methylthio)phenyl]benzenesulfonamide: To a stirred suspension of the corresponding nitro derivative of formula 2a (0.20 g, 0.62 mmol) in 8N HCl (9.0 mL), SnCl2 2H2O (0.42 g, 1.85 mmol), dissolved in 8N HCl (2.0 mL), was added at once and the mixture was refluxed for 2 h. 10% NaOH was added to reach pH 6 and the precipitate so obtained was filtered and washed three time with CHCl3 (3×15 mL). The fractions were collected and the solvent was dried and evaporated to dryness to obtain the amino derivative (0.10 g, 50% yield) as a crude solid used as it is in the next reaction step: mp 94.1-94.3° C.; 1H NMR (200 MHz, CDCl3): δ 7.40 (dd, J=1.5 and 8.0 Hz, 1H, Ar—H), 7.27-7.20 (m, 1H, Ar—H), 6.75 (d, J=8.3 Hz, 1H, Ar—H), 7.10-6.90 (m, 2H, Ar—H), 6.80-6.90 (m, 2H, Ar—H), 6.75-6.55 (m, 3H, Ar—H and NH), 4.75 (bs, 2H, NH2).
2-Amino-5-fluoro-N-[4-(trifluoromethyl)phenyl]benzenesulfonamide: Following the procedure reported above, the compound was obtained as pale orange solid in 87% yield (reaction time 1 h, purification method: trituration by cyclohexane): m.p 115-117° C.; 1H NMR (400 MHz, CDCl3): δ 4.60 (bs, 2H, NH2), 6.70 (dd, J=4.3 and 8.8 Hz, 1H, Ar—H), 7.05 (dt, J=2.9 and 8.7 Hz, 1H, Ar—H), 7.15 (d, J=8.4 Hz, 2H, Ar—H), 7.30 (dd, J=2.9 and 7.9 Hz, 1H, Ar—H), 7.45 (d, J=8.4 Hz, 2H, Ar—H).
2-amino-N-(3-bromophenyl)benzenesulfonamide: the intermediate was prepared following the procedure reported by Abramovitch, R. A. et al. in J. Org. Chem. 1977, 42, 2914-2919. Melting point and spectral data are in agreement with those reported in literature.
2-amino-N-(4-methoxyphenyl)benzenesulfonamide: the intermediate was prepared following the procedure reported by Ramirez-Martinez, J. F. et al. in Molecules, 2013, 18, 894-913. Spectral data are in agreement with those reported in literature.
2-amino-N-(4-chlorophenyl)benzenesulfonamide: the intermediate was prepared following the procedure reported by Ramirez-Martinez, J. F. et al. in Molecules, 2013, 18, 894-913. Spectral data are in agreement with those reported in literature.
2-amino-N-(2-bromophenyl)benzenesulfonamide: the intermediate was prepared following the procedure reported by Giannotti, D. et al. in J. Med. Chem. 1991, 34, 1356-1362. Spectral data are in agreement with those reported in literature.
2-amino-N-(3-chlorophenyl)benzenesulfonamide: the intermediate was prepared following the procedure reported in PCT WO 96/05185. Melting point and spectral data are in agreement with those reported in literature.
2-amino-N-(4-bromophenyl)benzenesulfonamide: the intermediate was prepared following the procedure reported by Ramirez-Martinez, J. F. et al. in Molecules, 2013, 18, 894-913. Spectral data are in agreement with those reported in literature.
2-Amino-5-methoxy-N-[4-(trifluoromethyl)phenyl]benzenesulfonamide. Following the procedure reported above, the compound was obtained as a brown solid in 73% yield (reaction time 12 h, purification method: trituration by Et2O): m.p. 150-152° C. 1H NMR (400 MHz, CDCl3): δ 3.64 (s, 3H, OCH3), 5.38 (bs, 2H, NH2), 6.52 (d, J=8.4 Hz, 1H, H-3), 6.66-6.69 (m, 1H, H-4), 6.82 (d, J=8.0 Hz, 2H, H-2′ and H-6′), 7.15-7.21 (m, 3H, H-6, H-3′ and H-5′), 7.97 (s, 1H, NH).
General procedure to obtain 6H-dibenzo[c,e][1,2]thiazine 5,5-dioxides of formula 5a (Scheme 1): Aminobenzenesulfonamide of formula 3a (1 equiv.), NaOH (1.2 equiv.) and NaNO2 (1.2 equiv.) were mixed in water and the obtained solution was added dropwise to HCl 37% (6 equiv.) and kept to 0° C. The muddy mixture was mixed with a glass rod for 30 min verifying the formation of diazonium salt by the β-naphtol assay. The red mixture was than diluted with H2O and treated with AcONa powder till pH=5 and the orange solid so obtained was filtered and treated with cyclohexane to obtain instable crude solid of formula 4a. Due to its high instability, the solid was immediately added portion wise to a stirring suspension of Cu (5% of mass weight) powder in DMSO. After 30 min. the reaction mixture was filtered over Celite to remove the Cu powder and the filtrate was poured into ice/water acidifying with HCl 2N till pH=4 to afford a precipitate that was filtered under vacuum to give intermediates compounds of formula 5a.
9-bromo-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide: Following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a, the compound was obtained in 70% yield as brown solid and used as it is in the next reaction step: mp 229-231° C. 1H NMR (200 MHz, DMSO-d6):511.50 (bs, 1H, NH), 8.37 (d, J=2.3 Hz, 1H, Ar—H), 8.25 (d, J=7.6 Hz, 1H, Ar—H), 7.90 (dd, J=1.3 and 7.7 Hz, 1H, Ar—H), 7.76 (dt, J=1.4 and 7.5 Hz, 1H, Ar—H), 7.66 (dd, J=1.1 and 7.5 Hz, 1H, Ar—H), 7.59 (dd, J=2.1 and 8.6 Hz, 1H, Ar—H), 7.55-7.80 (m, 3H, H-2, H-3 and H-8), 7.10 (d, J=8.6 Hz, 1H, H-7).
9-chloro-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide: Following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a, the compound was obtained, after purification by flash column chromatography (CHCl3/MeOH 98:2), as a brown solid in 26% yield (reaction time 2 h): mp 231.4-231.5° C.; 1H NMR (200 MHz, DMSO-d6): δ11.50 (bs, 1H, NH), 8.29-8.25 (m, 2H, Ar—H), 7.89 (dd, J=1.5 and 7.5 Hz, 1H, Ar—H), 7.75 (dt, J=1.5 and 7.5 Hz, 1H, Ar—H), 7.62 (dt, J=1.0 and 9.0 Hz, 1H, Ar—H), 7.48 (dd, J=2.3 and 7.2 Hz, 1H, Ar—H).
9-(Trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide: Following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a, the compound was obtained as a brown solid in 66% yield (reaction time 1 h): mp 235-237° C. 1H-NMR (200 MHz, CDCl3): δ 8.22 (brs, 1H, Ar—H), 8.03-7.98 (m, 2H, Ar—H), 7.80-7.74 (m, 2H, Ar.H), 7.64-7.57 (m, 2H, Ar—H), 7.20 (brs, 1H, NH).
9-(Methylthio)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide: Following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a the compound was obtained, after purification by flash column chromatography (CH2Cl2/MeOH 98: 2), as a pale brown solid in 25% yield (reaction time 2 h): mp 211.4-211.6° C.; 1H-NMR (200 M Hz, DMSO-d6): δ 11.29 (brs, 1H, NH), 8.25 (d, J=7.9 Hz, 1H, Ar—H), 8.00 (brs, 1H, Ar—H), 7.87 (d, J=7.7 Hz, 1H, Ar—H), 7.85 (t, J=7.3 Hz, 1H, Ar—H), 7.61 (t, J=7.4 Hz, 1H, Ar—H), 7.34 (d, J=8.5 Hz, 1H, Ar—H), 7.09 (d, J=8.5 Hz, 1H, Ar—H).
9-Methoxy-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide: Following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a the compound was obtained, after purification by flash column chromatography (CHCl3/MeOH 97:3), as pale brown solid in 41% yield (reaction time: 30 min.): mp 198-202° C. 1H-NMR (200 MHz, DMSO-d6) δ 10.97 (brs, 1H, NH), 8.25 (d, J=7.7 Hz, 1H, Ar—H), 7.85 (dd, J=1.4 and 7.7 Hz, 1H, Ar—H), 7.75 (dt, J=1.4 and 7.55 Hz, 1H, Ar—H), 7.68-7.55 (m, 2H, Ar—H), 7.13-6.95 (m, 2H, Ar—H), 3.80 (s, 3H, CH3).
7-Bromo-6H-dibenzo-[c,e][1,2]thiazine 5,5-dioxide: Following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a the compound was obtained as yellow solid in 78% yield: mp 188.2-188.4° C. (dec.). 1H-NMR (400 MHz, DMSO-d6): δ 10.80 (brs, 1H, NH), 8.25-8.30 (m, 2H, Ar—H), 7.95 (d, J=7.7 Hz, 1H, Ar—H), 7.85-7.80 (m, 2H, Ar—H), 7.72 (t, J=7.5 Hz, 1H, Ar—H), 7.36 (t, J=7.9, 1H, Ar—H).
8,10-Dichloro-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide: Following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a the compound was obtained as yellow solid in 77% yield: mp 190.0-191.0° C. (dec.); 1H-NMR (200 MHz, acetone-d6): δ10.25 (brs, 1H, NH), 8.58 (dd, J=1.7 and 7.8 Hz, 1H, Ar—H), 7.95 (dd, J=1.5 and 7.3 Hz, 1H, Ar—H), 7.83-7.65 (m, 2H, Ar—H), 7.48 (d, J=2.1 Hz, 1H, Ar—H), 7.32 (J=2.1 Hz, 1H, Ar—H).
3-Fluoro-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide: Following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a the compound was obtained as pale brown solid in 83% yield: m.p. 195-197° C. 1H NMR (400 MHz, DMSO-d6): δ 7.35 (d, J=8.4 Hz, 1H, H-7), 7.70 (dt, J=2.7 and 8.7 Hz, 1H, H-2), 7.80 (d, J=8.5 Hz, 1H, H-8), 7.85 (dd, J=2.6 and 7.6 Hz, 1H, H-4), 8.50 (dd, J=4.8 and 8.9 Hz, 1H, H-1), 8.55 (s, 1H, H-10), 12.10 (bs, 1H, NH).
8-bromo-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide and 10-bromo-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide: Following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a, a mixture of two regioisomers difficult to be separated was obtained and the crude was employed without further purification for the next reaction step.
8-chloro-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide and 10-chloro-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide: following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a, a mixture of two regioisomers difficult to be separated was obtained and the crude was employed without further purification for the next reaction step.
8-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide and 10-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide: following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a, a mixture of two regioisomers difficult to be separated was obtained and the crude was employed without further purification for the next reaction step.
8-Chloro-9-fluoro-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide and 10-chloro-9-fluoro-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide: following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a, a mixture of two regioisomers difficult to be separated was obtained and the crude was employed without further purification for the next reaction step.
3-Methoxy-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide: following the general procedure reported above and starting from the corresponding amino-benzensulfonamide of formula 3a, the compound was obtained in 40% yield as brown solid: m.p. 198-200° C. 1H NMR (400 MHz, DMSO-d6): δ 3.84 (s, 3H, OCH3), 7.30-7.43 (m, 3H, Ar—H), 7.75 (d, J=7.2 Hz, 1H, Ar—H), 8.37 (d, J=8.6 Hz, 1H, Ar—H), 8.49 (s, 1H, Ar—H), 11.78 (s, 1H, NH).
General procedure to obtain 6H-dibenzo[c,e][1,2]thiazine 5,5-dioxides N-6 ethyl acetates of formula 6a (Scheme 1): In a microwave reactor tube, a solution of the appropriate compound of formula 5a (1 equiv.), ethyl bromoacetate (1 equiv.), and DIPEA (3 equiv.) in dry DMF (5 mL) was irradiated at 50° C. for 15 min. by setting the following experimental parameters: pressure 5 bar, cooling off, FHT on, solvent absorption very high. The pitchy mixture was poured into ice-water and extracted three times with EtOAc. The combined organic layers were washed with brine, dried, and evaporated to dryness to give a crude slurry mass that was triturated with EtOH giving a precipitate that was filtered to afford the desired compound.
Ethyl 2-(9-bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetate: following the general procedure reported above and starting from the corresponding dibenzothiazine of formula 5a, the compound was obtained as pink solid in 75% yield: mp 89-91° C. 1H-NMR (200 MHz, DMSO-d6): δ 8.40 (d, J=2.3 Hz, 1H, Ar—H), 8.26 (d, J=7.6 Hz, 1H, Ar—H), 7.90-7.60 (m, 4H, Ar—H), 7.40 (d, J=8.6 Hz, 1H, Ar—H), 4.77 (s, 2H, NCH2), 3.90 (q, J=7.0 Hz, 2H, OCH2CH3), 0.80 (t, J=7.0 Hz, 3H, OCH2CH3).
Ethyl (9-chloro-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetate: following the general procedure reported above and starting from the corresponding dibenzothiazine of formula 5a, the compound was obtained as brown solid in 86% yield: mp 102.8-102.9° C.; 1H-NMR (200 MHz, CDCl3): δ 8.47-7.99 (m, 3H, Ar—H), 7.64 (dt, J=1.5 and 7.5 Hz, 1H, Ar—H), 7.53 (dt, J=1.2 and 7.7 Hz, 1H, Ar—H), 7.34 (dd, J=2.3 and 8.7 Hz, 1H, Ar—H), 7.17 (d, J=8.7 Hz, 1H, Ar—H), 4.59 (s, 2H, NCH2), 3.96 (q, J=7.2 Hz, 2H, OCH2CH3), 1.00 (t, J=7.2 Hz, 3H, OCH2CH3).
Ethyl [5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetate: following the general procedure reported above and starting from the corresponding dibenzothiazine of formula 5a, the compound was obtained as pale brown solid in 80% yield: mp 101-103° C. 1H-NMR (200 MHz, DMSO-d6): δ 8.60 (brs, 1H, Ar—H), 8.35 (d, J=8.0 Hz, 1H, Ar—H), 8.00-7.75 (m, 3H, Ar—H), 7.74-7.65 (m, 2H, Ar—H), 4.91 (s, 2H, NCH2), 3.92 (q, J=7.4 Hz, 2H, OCH2CH3), 0.92 (t, J=7.4 Hz, 3H, OCH2CH3).
Ethyl [9-(methylthio)-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetate: following the general procedure reported above and starting from the corresponding dibenzothiazine of formula 5a, the compound was obtained as yellowish solid in 73% yield: mp 103-105° C. 1H-NMR (200 MHz, DMSO-d6): δ 8.02-7.91 (m, 3H, Ar—H), 7.75 (t, J=7.9 Hz, 1H, Ar—H), 7.61 (t, J=7.4 Hz, 1H, Ar—H), 7.45-7.35 (m, 1H, Ar—H), 7.26-7.23 (m, 1H, Ar—H), 4.66 (s, 2H, NCH2), 4.05 (q, J=6.9 Hz, 2H, OCH2CH3), 2.57 (s, 3H, SCH3), 1.07 (t, J=6.9 Hz, 3H, OCH2CH3).
Ethyl (9-methoxy-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetate: following the general procedure reported above and starting from the corresponding dibenzothiazine of formula 5a, the compound was obtained as brown solid in 85% yield: mp 101-104° C. 1H-NMR (200 MHz, DMSO-d6): δ 8.26 (d, J=7.9 Hz, 1H, Ar—H), 7.88-7.74 (m, 2H, Ar—H), 7.70-7.62 (m, 2H, Ar—H), 7.49 (d, J=8.9 Hz, 1H, Ar—H), 7.12 (dd, J=2.7 and 8.9 Hz, 1H, Ar—H), 4.75 (s, 2H, NCH2), 3.94-3.77 (m, 5H, OCH3 and OCH2CH3), 0.90 (t, J=7.0 Hz, 3H, OCH2CH3).
Ethyl (7-bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetate: following the general procedure reported above and starting from the corresponding dibenzothiazine of formula 5a, the compound was obtained, after crystallization by EtOH, as pink solid in 50% yield: mp 169-171° C. 1H-NMR (400 MHz, CDCl3) δ 7.96-7.90 (m, 3H, Ar—H), 7.75-7.67 (m, 2H, Ar—H), 7.58 (t, J=7.8, 1H, Ar—H), 7.30 (t, J=7.9 Hz, 1H, Ar—H), 4.73 (s, 2H, NCH2), 3.80 (q, J=7.1 Hz, 2H, OCH2CH3), 1.00 (t, J=7.1 Hz, 3H, OCH2CH3).
Ethyl (8,10-dichloro-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetate: following the general procedure reported above and starting from the corresponding dibenzothiazine of formula 5a, was obtained as pink solid in 90% yield: mp 172-173° C. 1H-NMR (200 MHz, CDCl3) δ8.49 (dd, J=1.3 and 7.9 Hz, 1H, Ar—H), 7.90 (dd, J=1.8 and 7.6 Hz, 1H, Ar—H), 7.70-7.55 (m, 2H, Ar—H), 7.10 (d, J=2.1 Hz, 1H, Ar—H), 4.51 (s, 2H, NCH2), 4.02 (q, J=7.2 Hz, 2H, OCH2CH3), 1.05 (t, J=7.2 Hz, 3H, OCH2CH3).
Ethyl [3-fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetate: following the general procedure reported above and starting from the corresponding dibenzothiazine of formula 5a, was obtained as pale brown solid in 85% yield: m.p. 175-177° C.; 1H NMR (400 MHz, DMSO-d6): δ1.10 (t, J=7.2 Hz, 3H, OCH2CH3), 4.10 (q, J=7.2 Hz, 2H, OCH2CH3), 4.75 (s, 1H, NCH2), 7.35 (d, J=8.5 Hz, 1H, H-7), 7.45 (dt, J=2.7 and 8.3 Hz, 1H, H-2), 7.60-7.70 (m, 2H, H-4 and H-8), 8.00 (dd, J=4.6 and 8.8 Hz, 1H, H-1), 8.20 (s, 1H, H-10).
Ethyl [3-methoxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetate: following the general procedure reported above and starting from the corresponding dibenzothiazine of formula 5a, the compound was obtained as pink solid in 86% yield: m.p. 190-192° C. 1H NMR (400 MHz, DMSO-d6): δ 1.03 (t, J=7.0 Hz, 3H, OCH2CH3), 3.95 (s, 3H, OCH3), 3.98 (q, J=7.4 Hz, 2H, OCH2CH3), 4.93 (s, 2H, NCH2), 7.42-7.45 (m, 2H, H-2 and H-4), 7.73 (d, J=8.6 Hz, 1H, H-7), 7.85 (d, J=8.4 Hz, 1H, H-8), 8.38 (d, J=8.4 Hz, 1H, H-1), 8.54 (s, 1H, H-10).
N-[2-(9-bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)ethyl]cyclohexanamine (SM9). In a microwave reactor tube, a solution of the appropriate compound of formula 5a (0.6 g, 1.9 mmol), N-(2-chloroethyl)cyclohexanamine (0.31 g, 1.9 mmol), and DIPEA (0.66 mL, 3.8 mmol) in dry DMF (4 mL) was irradiated at 70° C. for 60 min. by setting the following experimental parameters: pressure 5 bar, cooling off, FHT on, solvent absorption very high. The residue was poured into ice-water acidified to pH 3 with 2N HCl and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine, dried, and evaporated to dryness to give a crude slurry mass that was purified by flash column chromatography eluting with CHCl3/MeOH (97:3) giving the target compound SM9 (0.65 g, 79%) as low melting pale brown solid: 1H NMR (400 MHz, CDCl3): δ 8.13 (d, J=2.2 Hz, 1H, Ar—H), 7.99 (dd, J=1.1 and 7.7 Hz, 1H, H-4), 7.92 (d, J=7.2 Hz, 1H, Ar—H), 7.73 (dt, J=1.3 and 7.8 Hz, 1H, Ar—H), 7.64-7.58 (m, 2H, Ar—H), 7.40 (d, J=8.7 Hz, 1H, Ar—H), 4.00 (t, J=6.7 Hz, 2H, SO2NCH2), 2.85 (t, J=6.7 Hz, 2H, NCH2), 2.40-2.25 (m, 1H, Cy-CH), 1.75-1.50 (m, 4H, Cy-CH2), 1.25-1.00 (m, 4H, Cy-CH2), 1.00-0.80 (m, 2H, Cy-CH2). 13C NMR (100 MHz, CDCl3): δ 137.58, 135.22, 133.05, 132.42, 131.16, 128.95, 128.45, 127.15, 125.68, 123.56, 122.51, 118.59, 56.17, 49.17, 44.52, 32.97, 25.87, 24.72. HRMS (ESI) calcd. for C20H23BrN2O2S[M++H]+: 435.0739, found: 435.0735; LC-MS: ret. time 4.075.
(R,S)-3-(9-Bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-1-cyclohexylpyrrolidin-2-one (SM1l). Following the procedure above described for compound SM9 and using 3-bromo-1-cyclohexyl-2-pyrrolidinone37, the title compound was purified by flash column chromatography, eluting with cyclohexane/EtOAc (6:4), and subsequent trituration with petroleum ether/Et2O, to give racemic target compound SM1l as a white solid in 70% yield: mp 177-179° C. 1H NMR (400 MHz, DMSO-d6): δ 8.42 (d, J=2.1 Hz, 1H, Ar—H), 8.28 (d, J=7.8 Hz, 1H, Ar—H), 7.91 (d, J=7.7 Hz, 1H, Ar—H), 7.85 (t, J=8.6 Hz, 1H, Ar—H), 7.75-7.70 (m, 2H, Ar—H), 7.30 (d, J=8.6 Hz, 1H, H-7), 7.37 (d, J=8.6 Hz, 1H, Ar—H), 4.90 (t, J=9.4 Hz, 2-Pyrrolidone-CH), 3.75-3.55 (m, 1H, Cy-CH), 3.20-3.00 (m, 2H, 2-Pyrrolidone-CH), 2.10-2.00 (m, 1H, 2-Pyrrolidone-CH), 1.75-1.48 (m, 6H, Cy-CH2 and 2-Pyrrolidone-CH), 1.40-1.00 (m, 5H, Cy-CH2). 13C NMR (100 MHz, DMSO-d6): δ 168.65, 136.83, 135.87, 133.56, 133.48, 131.05, 130.15, 129.38, 128.95, 127.40, 126.72, 122.36, 119.96, 62.56, 51.37, 25.45, 25.38, 25.33, 25.19, 22.83. HRMS (ESI) calcd for C22H23BrN2O3S: [M++H]+: 475.0692, found: 475.03691; LC-MS: ret. time 6.015.
Methyl 3-(9-bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)propanoate: To a solution the appropriate compound of formula 5a (0.300 g, 0.92 mmol) in dry THE (12 mL), commercial methyl 3-hydroxypropanoate (0.12 mL, 1.3 mmol) and PPh3 (0.33 g, 1.3 mmol), were added and the solution was sonicated at 25° C. for 7 min. DEAD (0.20 mL, 1.3 mmol) was then added drop-wise and the solution was sonicated for 18 h at 25° C. (approximately 70% of conversion followed by TLC). The mixture was concentrated under reduced pressure, poured into ice-water, basified with aqueous 10% NaOH to pH 10 in order to remove the residual starting material, and extracted with EtOAc (3×20 mL). The combined organic layers were washed brine, dried, and evaporated to dryness. The obtained brown oil was purified by column chromatography (petroleum ether/EtOAc 7:3) followed by trituration with Et2O to give the desired title compound of formula 6a (0.100 g, 30%) as a white solid: mp 101-103° C. 1H NMR (200 MHz, CDCl3): δ 8.10 (d, J=2.3 Hz, 1H, Ar—H), 7.94-7.83 (m, 2H, Ar—H), 7.70 (dt, J=1.5 and 7.4 Hz, 2H, Ar—H), 7.60-7.50 (m, 2H, Ar—H), 7.28 (d, J=8.8 Hz, 1H, Ar—H), 4.10 (t, J=7.4 Hz, 2H, NCH2), 3.50 (s, 3H, CH3), 2.50 (t, J=7.4 Hz, 2H, CH2).
General procedure of direct amidation of compounds of formula 6a with cyclohexylamine to obtain target compounds of formula 8a (Scheme 1): Using the microwave oven a tube containing a mixture of appropriate dibenzothiazine ethyl acetate or the intermediate in example 44 of general formula 6a (1 equiv.) and cyclohexylamine (4 equiv.) was irradiated at 120° C. for 4 h by setting the following experimental parameters: pressure 5 bar, cooling off, FHT on, solvent absorption normal. The residue was poured into ice-water and acidified with 2 N HCl to pH 3. The obtained precipitate was filtered off and crystallized by EtOH to give the target compound of formula 8a.
2-(9-Bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclohexylacetamide (SM3): following the general procedure above described the title compound was obtained as white solid in 62% yield: mp 211-213° C. 1H NMR (400 MHz, CDCl3): δ 8.20 (brs, 1H, Ar—H), 8.00 (d, J=7.8 Hz, 1H, Ar—H), 7.87 (d, J=7.8 Hz, 1H, Ar—H), 7.75 (t, J=7.5 Hz, 1H, Ar—H), 7.73 (t, J=7.4 Hz, 1H, Ar—H), 7.57 (d, J=7.4 Hz, 1H, Ar—H), 7.12 (d, J=8.7 Hz, 1H, H-7), 6.54 (d, J=7.0 Hz, 1H, NH), 4.42 (s, 2H, NCH2), 3.90-3.75 (m, 1H, Cy-CH), 1.95-1.75 (m, 2H, Cy-CH), 1.65-1.50 (m, 3H, Cy-CH), 1.45-1.25 (m, 2H, Cy-CH), 1.25-1.05 (m, 3H, Cy-CH). 13C NMR (100 MHz, DMSO-d6): δ 166.29, 137.11, 134.09, 133.49, 133.12, 131.05, 129.18, 128.57, 125.91, 125.59, 122.56, 121.09, 118.62, 51.64, 48.45, 32.45, 25.35, 24.35. HRMS (ESI) m/z [M+H]+ calcd. for C20H21BrN2O3S: 448.0535, found: 448.0456; LC-MS: ret. time 5.754.
2-(9-Bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclopentylacetamide (SM4): following the general procedure above described the title compound was obtained, after crystallization by EtOH, as white solid in 43% yield: mp 192-194° C. 1H NMR (400 MHz, CDCl3): δ 8.19 (d, J=2.1 Hz, 1H, Ar—H), 8.04 (d, J=7.9 Hz, 1H, Ar—H), 7.99 (d, J=7.9 Hz, 1H, Ar—H), 7.80 (dt, J=1.2 and 7.6 Hz, 1H, Ar—H), 7.69-7.61 (m, 2H, Ar—H), 7.15 (d, J=8.7 Hz, 1H, Ar—H), 6.62 (d, J=7.5 Hz, 1H, NH), 4.46 (s, 2H, NCH2), 4.31-4.24 (m, 1H, cyclopentyl-CH), 2.00-1.92 (m, 2H, cyclopentyl-CH2), 1.70-1.55 (m, 4H, cyclopentyl-CH2), 1.45-1.30 (m, 2H, cyclopentyl-CH2). 13C NMR (100 MHz, CDCl3): δ166.76, 137.03, 134.04, 133.49, 133.14, 131.00, 129.20, 128.57, 125.90, 125.53, 122.53, 121.04, 118.61, 51.60, 51.49, 32.80, 23.45. HRMS (ESI) m/z [M+H]+ calcd. for C19H19BrN2O3S: 435.0379, found: 435.03733; LC-MS: ret. time 5.434.
2-(9-Bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cycloheptylacetamide (SM5): following the general procedure above described the title compound was obtained, after crystallization by EtOH, as white solid in 51% yield: mp 208-210° C. 1H NMR (400 MHz, CDCl3): δ 8.18 (d, J=1.8 Hz, 1H, Ar—H), 8.03 (d, J=7.4 Hz, 1H, Ar—H), 7.98 (d, J=7.9 Hz, 1H, Ar—H), 7.80 (t, J=7.6 Hz, 1H, H-2), 7.70-7.60 (m, 2H, Ar—H), 7.15 (d, J=8.7 Hz, 1H, Ar—H), 6.60 (d, J=7.5 Hz, 1H, NH), 4.45 (s, 2H, NCH2), 4.20-3.95 (m, 1H, cycloheptyl-CH), 1.90-177 (m, 2H, cycloheptyl-CH2), 1.70-1.30 (m, 10H, cycloheptyl-CH2). 13C NMR (100 MHz, CDCl3): δ 166.01, 137.08, 134.08, 133.47, 133.13, 131.04, 129.18, 128.57, 125.92, 125.55, 122.55, 121.04, 118.60, 51.60, 50.63, 34.59, 27.84, 23.72. HRMS (ESI) m/z [M+H]+ calcd. for C21H23BrN2O3S: 463.0689, found: 463.0688; LC-MS: ret. time 6.018.
3-(9-Bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclohexylpropanamide (SM10). following the general procedure above described the title compound was obtained, after purification by flash column chromatography (cyclohexane/EtOAc 7:3), as white solid in 34% yield: mp 114-116° C. 1H NMR (400 MHz, CDCl3): δ 8.13 (brs, 1H, Ar—H), 8.00 (d, J=7.6 Hz, 1H, Ar—H), 7.90 (d, J=7.7 Hz, 1H, Ar—H), 7.75 (t, J=7.7 Hz, 1H, Ar—H), 7.65-7.60 (m, 2H, Ar—H), 7.42 (d, J=8.7 Hz, 1H, Ar—H), 5.55 (d, J=6.4 Hz, 1H, NH), 4.15 (t, J=6.6 Hz, 2H, NCH2), 3.75-3.60 (m, 1H, Cy-CH), 2.60 (t, J=6.6 Hz, 2H, CH2), 1.85-1.53 (m, 5H, Cy-CH2), 1.50-1.10 (m, 5H, Cy-CH2). 13C NMR (100 MHz, CDCl3): δ 168.33, 137.37, 134.19, 132.91, 132.21, 130.81, 128.46, 127.79, 126.07, 125.26, 123.13, 122.13, 118.23, 47.96, 45.98, 36.49, 32.36, 24.90, 24.18. HRMS (ESI) calcd for C21H23BrN2O3S [M++H]+: 463.0689, found: 463.0693; LC-MS: ret. time 4.772
2-(9-chloro-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclohexylacetamide (SM254): following the general procedure above described the title compound was obtained, after crystallization by EtOH, as white solid in 60% yield: mp 218-220° C. 1H NMR (400 MHz, CDCl3): δ 8.02-7.95 (m, 2H, Ar—H), 7.92 (d, J=8.1 Hz, 1H, Ar—H), 7.75 (dt, J=1.2 and 7.7 Hz, 1H, Ar—H), 7.63 (dt, J=0.8 and 8.0 Hz, 1H, Ar—H), 7.43 (dd, J=2.2 and 8.7 Hz, 1H, Ar—H), 7.18 (d, J=8.7 Hz, 1H, H-7), 6.53 (d, J=7.0 Hz, 1H, NH), 4.42 (s, 2H, NCH2), 3.90-3.75 (m, 1H, Cy-CH), 1.90-1.75 (m, 2H, Cy-CH), 1.65-1.50 (m, 4H, Cy-CH), 1.40-1.25 (m, 2H, Cy-CH), 1.25-1.05 (m, 2H, Cy-CH). 13C NMR (100 MHz, CDCl3): δ166.29, 136.69, 134.16, 133.08, 131.17, 131.11, 130.58, 129.16, 125.88, 125.61, 125.32, 122.58, 120.97, 51.75, 48.45, 32.45, 25.34, 24.32; HRMS (ESI) m/z [M+H]+ calcd. for C20H21ClN2O3S: 405.1039, found: 404.1032. LC-MS: ret. time 6.492 min.
N-cyclohexyl-2-[5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (SM231): following the general procedure above described the title compound was obtained, after purification by flash column chromatography (cyclohexane/EtOAc 7:3), as white solid in 70% yield: mp 225-226° C. 1H-NMR (200 MHz, CDCl3): δ 8.29 (brs, 1H, Ar—H), 8.03 (d, J=7.5 Hz, 1H, Ar—H), 7.81 (d, J=7.8 Hz, 1H, Ar—H), 7.75-7.65 (m, 2H, Ar—H), 7.35 (d, J=8.4 Hz, 1H, Ar—H), 6.57 (brs, 1H, NH), 4.52 (s, 2H, NCH2), 3.85-3.75 (m, 1H, Cy-CH), 1.85-1.75 (m, 2H, Cy-CH), 1.65-1.48 (m, 3H, Cy-CH), 1.40-1.25 (m, 2H, Cy-CH), 1.20-1.10 (m, 3H, Cy-CH). 13C NMR (100 MHz, CDCl3): δ 166.04, 140.60, 134.01, 133.29, 131.16, 129.36, 127.33 (q, JC-F=33.1 Hz, C-9), 127.32 (d, JC-F=3.5 Hz, C-10), 126.02, 123.86, 123.63 (q, JC-F=270.7 Hz, CF3), 122.96 (d, JC-F=3.8 Hz, C-8), 119.49, 51.25, 48.55, 32.43, 25.32, 24.33. HRMS (ESI) m/z [M+Na]+ calcd. for C21H21F3N2O3S: 461.1118, found: 461.1124. LC-MS: ret. time 4.688 min.
N-cyclohexyl-2-[9-(methylthio)-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (SM340): following the general procedure above described the title compound was obtained, after crystallization by EtOH, as white solid in 63% yield: mp 178-180° C. 1H-NMR (400 MHz, CDCl3): δ 8.03-7.98 (m, 2H, Ar—H), 7.92 (d, J=1.6 Hz, 1H, Ar—H), 7.78 (t, J=7.6 Hz, 1H, Ar—H), 7.64 (t, J=7.4 Hz, 1H, Ar—H), 7.39 (dd, J=1.9 and 8.6 Hz, 1H, Ar—H), 7.20 (d, J=8.6 Hz, 1H, Ar—H), 6.60 (d, J=7.5 Hz, 1H, NH), 4.44 (s, 2H, NCH2), 3.90-3.75 (m, 1H, Cy-CH), 2.58 (s, 3H, SCH3), 1.90-1.80 (m, 2H, Cy-CH), 1.75-1.50 (m, 3H, Cy-CH), 1.40-1.25 (m, 2H, Cy-CH), 1.20-1.10 (m, 3H, Cy-CH). 13C-NMR (100 MHz, CDCl3): δ166.60, 135.92, 135.61, 134.20, 132.99, 131.79, 129.14, 128.79, 125.81, 124.44, 124.07, 122.59, 120.25, 51.84, 48.39, 32.46, 25.35, 24.35, 16.44. HRMS (ESI) m/z [M+H]+ calcd for C21H24N2O3S2: 417.1309, found: 417.1305; LC-MS: ret. time 5.403 min.
N-Cyclohexyl-2-(9-methoxy-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetamide (SM225): following the general procedure above described the title compound was obtained, after purification by flash column chromatography (cyclohexane/EtOAc 7:3), as white solid in 45% yield: mp 216-217° C. 1H-NMR (400 MHz, CDCl3): δ 7.99-7.93 (m, 2H, Ar—H), 7.74 (t, J=7.6 Hz, 1H, Ar—H), 7.59 (t, J=7.6 Hz, 1H, Ar—H), 7.49 (d, J=2.6 Hz, 1H, Ar—H), 7.20 (d, J=8.9 Hz, 1H, Ar—H), 7.02 (dd, J=2.7 and 8.9 Hz, 1H, Ar—H), 6.59 (d, J=7.8 Hz, 1H, NH), 4.32 (s, 2H, NCH2), 3.88-3.75 (m, 4H, OCH3, and Cy-CH), 1.85-1.75 (m, 2H, Cy-CH), 1.65-1.50 (m, 3H, Cy-CH), 1.35-1.25 (m, 2H, Cy-CH), 1.20-1.10 (m, 3H, Cy-CH). 13C-NMR (100 MHz, CDCl3): δ166.82, 157.36, 134.29, 132.96, 132.22, 131.86, 128.76, 125.87, 125.50, 122.84, 121.68, 116.50, 110.77, 55.78, 52.75, 48.27, 32.52, 25.39, 24.43. HRMS (ESI) m/z [M+H]+ calcd for C21H24N2O4S: 401.1539, found: 401.1533; LC-MS: ret. time 5.544 min.
2-(7-Bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclohexylacetamide (SM227): following the general procedure above described the title compound was obtained, after purification by flash column chromatography (cyclohexane/EtOAc 6:4), as white solid in 40% yield:mp 159-160° C. 1H-NMR (200 MHz, DMSO-d6) δ 8.15 (d, 2H, H-4 and H-8), 7.70-7.85 (m, 4H, H-1, H-9, H-10 and NH), 7.55-7.70 (t, J=7.4 Hz, 1H, H-2), 7.40 (t, J=7.9 Hz, 1H, H-3), 4.40 (brs, 2H, NCH2), 3.00-3.10 (m, 1H, cyclohexyl CH), 0.60-1.60 (m, 10H, cyclohexyl CH2). 13C-NMR (100 MHz, CDCl3): δ 165.51, 139.33, 134.86, 133.55, 133.17, 132.58, 130.40, 129.51, 129.50, 126.35, 125.40, 125.09, 121.45, 54.69, 48.36, 32.70, 25.52, 25.40. HRMS (ESI) m/z [M+H]+ calcd for C20H21BrN2O3S: 448.0539, found: 448.0267; LC-MS: ret. time 4.10 min.
2-(8-Bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclohexylacetamide (SM228) and 2-(10-bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclohexylacetamide (SM229): following the general procedure above described a mixture of the two regioisomers of formula 6a was reacted with cyclohexylamine obtaining the two regioisomers of formula 8a that were separated by flash column chromatography (CH2Cl2/acetone 98:2) and each compound was further purified by crystallization with EtOH to afford target compounds SM228 (Rf>by TLC) and SM229 (Rf<by TLC).
SM228: 8% yield: mp 184-185° C. 1H-NMR (400 MHz, CDCl3): δ 8.10-7.80 (m, 3H, Ar—H), 7.82 (t, J=7.4 Hz, 1H, Ar—H), 7.65 (t, J=7.6 Hz, 1H, Ar—H), 7.52 (dd, J=1.5 and 8.5 1H, Ar—H), 7.40 (brs, 1H, Ar—H), 6.55 (d, J=8.0 Hz, 1H, NH), 4.50 (s, 2H, NCH2), 4.00-3.75 (m, 1H, Cy-CH), 2.00-1.80 (m, 2H, Cy-CH2), 1.75-1.50 (m, 2H, Cy-CH2), 1.45-1.00 (m, 6H, Cy-CH2). 13C NMR (100 MHz, DMSO-d6): δ 165.60, 140.02, 134.99, 133.22, 131.50, 129.45, 128.16, 127.94, 126.66, 123.86, 123.82, 123.30, 121.76, 50.06, 48.12, 32.67, 25.53, 24.76. HRMS (ESI) m/z [M+Na]+ calcd for C20H21BrN2O3S: 471.0354, found: 471.041; LC-MS: ret. time 12.592 min.
SM229: 21% yield: mp 211-212° C. 1H-NMR (400 MHz, CDCl3): δ 8.62 (d, J=8.3 Hz, 1H, Ar—H), 8.00 (dd, J=1.4 and 7.8 Hz, 1H, Ar—H), 7.75-7.65 (m, 4H, Ar—H), 7.28-7.24 (m, 2H, Ar—H), 6.50 (d, J=8.1 Hz, 1H, NH), 4.40 (s, 2H, NCH2), 3.80-3.70 (m, 1H, Cy-CH), 1.90-1.80 (m, 2H, Cy-CH2), 1.75-1.50 (m, 2H, Cy-CH2), 1.45-1.00 (m, 6H, Cy-CH2). NMR COSY spectrum showed two relevant NOE cross-peaks: H-9 (δ 7.70, dd)→H-8 (δ 7.32, t), H-9→H-7 (δ 7.28, dd). NMR NOESY spectrum showed one relevant NOE cross-peak: H-8→NCH2. 13C NMR (100 MHz, DMSO-d6): δ 165.49, 140.92, 134.85, 131.81, 131.48, 131.19, 131.13, 130.37, 129.41, 125.44, 121.64, 121.38, 120.59, 50.77, 48.11, 32.66, 25.52, 24.76. HRMS (ESI) m/z [M+Na]+ calcd for C20H21BrN2O3S: 471.0354, found: 471.0407; LC-MS: ret. time 12.893 min.
2-(8-chloro-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclohexylacetamide (SM586) and 2-(10-chloro-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclohexylacetamide (SM585): following the general procedure above described a mixture of the two appropriate regioisomers of formula 6a was reacted with cyclohexylamine obtaining the two regioisomers of formula 8a that were separated by flash column chromatography (CH2Cl2/acetone 98:2) and each compound was further purified by crystallization with EtOH to afford target compounds SM586 (Rf>by TLC) and SM585 (Rf<by TLC).
SM586: 18% yield: mp 193-195° C. 1H-NMR (400 MHz, CDCl3): δ8.02-7.90 (m, 3H, Ar—H), 7.74 (dt, J=1.2 and 7.7 Hz, 1H, Ar—H), 7.60 (dt, J=0.7 and 8.0 Hz, 1H, Ar—H), 7.33 (dd, J=2.0 and 8.2 Hz, 1H, Ar—H), 7.28 (d, J=2.0 Hz, 1H, Ar—H), 6.50 (d, J=7.5 Hz, 1H, NH), 4.49 (s, 2H, NCH2), 3.90-3.75 (m, 1H, Cy-CH), 1.90-1.80 (m, 2H, Cy-CH2), 1.75-1.50 (m, 4H, Cy-CH2), 1.45-1.35 (m, 2H, Cy-CH2), 1.20-1.05 (m, 2H, Cy-CH2); 13C-NMR (100 MHz, CDCl3): δ 166.05, 139.07, 136.51, 133.87, 133.07, 131.56, 128.74, 126.80, 125.71, 125.68, 122.52, 122.41, 119.71, 51.57, 48.41, 32.41, 25.36, 24.30; HRMS (ESI) m/z [M+H]+ calcd. for C20H21ClN2O3S: 405.1039, found:405.1037; LC-MS: ret. time 5.628 min.
SM585: 15% yield: mp 200-202° C. 1H-NMR (400 MHz, CDCl3): δ 8.60 (d, J=8.0 Hz, 1H, Ar—H), 7.95 (dd, J=1.2 and 8.5 Hz, 1H, Ar—H), 7.70 (dt, J=1.3 and 8.5 Hz, 1H, Ar—H), 7.59 (dd, J=1.2 and 8.0 Hz, 1H, Ar—H), 7.43 (dd, J=1.1 and 8.1 Hz, 1H, Ar—H), 7.34 (t, J=8.1 Hz, 1H, Ar—H), 7.20 (dd, J=1.1 and 8.1 Hz, 1H, Ar—H), 6.35 (d, J=7.0 Hz, 1H, NH), 4.48 (s, 2H, NCH2), 3.90-3.75 (m, 1H, Cy-CH), 1.90-1.80 (m, 2H, Cy-CH2), 1.75-1.50 (m, 4H, Cy-CH2), 1.45-1.35 (m, 2H, Cy-CH2), 1.20-1.05 (m, 2H, Cy-CH2); 13C-NMR (100 MHz, CDCl3): δ 166.25, 139.99, 135.69, 132.59, 131.57, 130.35, 130.16, 130.02, 128.78, 128.70, 123.48, 122.29, 118.69, 52.15, 48.38, 32.45, 25.34, 24.33; HRMS (ESI) m/z [M+H]+ calcd. for C20H21ClN2O3S: 405.1039, found: 405.1037; LC-MS: ret. time 5.631 min.
N-cyclohexyl-2-[5,5-dioxido-8-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (SM338) and N-cyclohexyl-2-[5,5-dioxido-10-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (SM339): following the general procedure above described a mixture of the two appropriate regioisomers of formula 6a was reacted with cyclohexylamine obtaining the two regioisomers of formula 8a that were separated by flash column chromatography(CH2Cl2/acetone 99:1) and each compound was further purified by crystallization with EtOH to afford target compounds SM338 (Rf>by TLC) and SM339 (Rf<by TLC).
SM338: 40% yield: mp 230-232° C. 1H-NMR (400 MHz, CDCl3): δ 8.20-8.10 (d, J=8.2 Hz, 1H, Ar—H), 8.05-7.98 (m, 2H, Ar—H), 7.78 (dt, J=1.5 and 7.5 Hz, 1H, Ar—H), 7.78 (dt, J=1.5 and 7.5 Hz, 1H, Ar—H), 7.69 (dt, J=1.3 and 7.7 Hz, 1H, Ar—H), 7.65 (d, J=8.0 Hz, 1H, Ar—H), 7.50 (brs, 1H, Ar—H), 6.50 (d, J=6.9 Hz, 1H, NH), 4.48 (s, 2H, NCH2), 3.90-3.75 (m, 1H, Cy-CH), 1.90-1.80 (m, 2H, Cy-CH2), 1.75-1.50 (m, 4H, Cy-CH2), 1.45-1.35 (m, 2H, Cy-CH2), 1.20-1.05 (m, 2H, Cy-CH2); 13C-NMR (100 MHz, CDCl3): δ 165.86, 138.57, 134.66, 133.16, 132.50 (q, JC-F=33.1 Hz, C-8), 131.11, 129.58, 127.08, 126.42, 126.27, 123.79 (q, JC-F=271.0 Hz, CF3), 122.59, 121.89 (q, JC-F=5 Hz, C-9), 116.83 (q, JC-F=6 Hz, C-7), 51.71, 48.44, 32.40, 25.33, 24.33; HRMS (ESI) m/z [M+H]+ calcd for C21H21F3N2O3S: 439.1303, found: 439.1296; LC-MS: ret. time 6.892 min.
SM339: 26% yield: mp 211-212° C.; 1H-NMR (400 MHz, CDCl3): δ 8.03-7.95 (m, 2H, Ar—H), 7.82-7.58 (m, 4H, Ar—H), 7.51 (d, J=8.3 Hz, 1H, Ar—H), 6.35 (d, J=8.0 Hz, 1H, NH), 4.40 (s, 2H, NCH2), 4.00-3.75 (m, 1H, Cy-CH), 1.90-1.80 (m, 2H, Cy-CH2), 1.75-1.50 (m, 4H, Cy-CH2), 1.45-1.05 (m, 4H, Cy-CH2); 13C-NMR (100 MHz, CDCl3): δ 168.08, 140.01 (brs, C-6a), 139.80, 134.90, 134.32, 132.30, 130.56, 127.7 (d, JC-F=2.0 Hz, C-8), 124.57, 122.30 (q, JC-F=29.0 Hz, C-10), 119.60 (q, JC-F=270.1 Hz, CF3), 117.80 (q, JC-F=3.1 Hz, C-9), 115.85 (q, JC-F 2.0 Hz, C-10a), 115.09, 52.02, 49.50, 32.40, 26.28, 24.12; HRMS (ESI) m/z [M+H]+ calcd for C21H21F3N2O3S: 439.1303, found: 439.1298; LC-MS: ret. time 6.598 min.
2-(8-chloro-9-fluoro-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclohexylacetamide (SM336) and 2-(10-chloro-9-fluoro-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclohexylacetamide (SM337): following the general procedure above described a mixture of the two appropriate regioisomers of formula 6a was reacted with cyclohexylamine obtaining the two regioisomers of formula 8a that were separated by flash column chromatography (cyclohexane/EtOAc 7:3) followed by crystallization with EtOH to afford target compounds SM336 (Rf>by TLC) and SM337 (Rf<by TLC).
SM336: 21% yield: mp 211-213° C. 1H-NMR (400 MHz, DMSO-d6): δ 7.96 (dd, J=1.5 and 7.0 Hz, 1H, Ar—H), 7.8 (d, J=8.2 Hz, 1H, Ar—H), 7.78-7.70 (m, 2H, Ar—H), 7.61 (dt, J=1.4 and 7.7 Hz, 1H, Ar—H), 7.33 (d, J=6.4 Hz, 1H, Ar—H), 6.4 (d, J=9.0 Hz, 1H, Ar—H), 4.35 (s, 2H, NCH2), 3.80-3.70 (m, 1H, Cy-CH), 1.85-1.75 (m, 2H, Cy-CH2), 1.75-1.45 (m, 2H, Cy-CH2), 1.45-1.00 (m, 6H, Cy-CH2). 13C NMR (100 MHz, DMSO-d6): δ 165.59, 154.86 (d, JC-F=243.0 Hz, C-9), 136.05 (d, JC-F=2.6 Hz, C-6a), 135.17, 133.19, 130.80, 130.05, 127.16, 125.69 (d, JC-F=8.0 Hz, C-10a), 123.97, 121.84, 121.29 (d, JC-F=20.0 Hz, C-8), 113.96 (d, JC-F=24.0 Hz, C-10), 50.97, 48.12, 32.62, 25.51, 24.73. HRMS (ESI) m/z [M+H]+ calcd. for C20H2OClFN2O3S: 423.0946, found: 423.0938; LC-MS: ret. time 6.560 min.
SM337: 53% yield: mp 216-217° C. 1H-NMR (400 MHz, CDCl3): δ 8.51 (d, J=8.2 Hz, 1H, Ar—H), 8.00 (d, J=7.7 Hz, 1H, Ar—H), 7.73 (dt, J=1.2 and 7.5 Hz, 1H, Ar—H), 7.64 (t, J=7.5 Hz, 1H, Ar—H), 7.35-7.20 (m, 2H, Ar—H), 6.25 (d, J=7.1 Hz, 1H, NH), 4.25 (s, 2H, NCH2), 3.80-3.70 (m, 1H, Cy-CH), 1.90-1.80 (m, 2H, Cy-CH2), 1.75-1.50 (m, 4H, Cy-CH2), 1.40-1.00 (m, 4H, Cy-CH2). 13C NMR (100 MHz, CDCl3): δ 166.08, 156.77 (d, JC-F=246.1 Hz, C-9), 135.85 (brs, C-6a), 135.81, 131.72, 129.99, 129.81 (d, JC-F=2.8 Hz, C-10a), 129.39, 125.44, 122.69, 120.41 (JC-F=8.1 Hz, C-7), 119.5 (JC-F=20.1 Hz, C-10), 117.43 (JC-F=24.0 Hz, C-8), 52.73, 48.45, 32.53, 25.33, 24.39; HRMS (ESI) m/z [M+H]+ calcd. for C20H2OClFN2O3S: 423.0946, found: 423.0938; LC-MS: ret. time 6.520 min.
N-cyclohexyl-2-(8,10-dichloro-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetamide (SM587): following the general procedure above described the title compound was obtained, after crystallization by EtOH, as white solid in 50% yield: mp 212.0-213.0° C.; H-NMR (200 MHz, DMSO-d6) δ 8.50 (d, J=8.0 Hz, 1H, Ar—H), 7.97 (dd, J=1.3 and 7.8, Hz, 1H, Ar—H), 7.71 (dt, J=1.4 and 8.0 Hz, 1H, Ar—H), 7.61 (dt, J=0.9 and 7.6, Hz, 1H, Ar—H), 7.45 (d, J=2.0 Hz, 1H, Ar—H), 7.22 (d, J=2.0 Hz, 1H, Ar—H), 6.28 (d, J=7.6 Hz, 1H, NH), 4.31 (s, 2H, NCH2), 3.80-3.70 (m, 1H, Cy-CH), 1.90-1.80 (m, 2H, Cy-CH2), 1.75-1.50 (m, 4H, Cy-CH2), 1.40-1.00 (m, 4H, Cy-CH2); 13C NMR (100 MHz, CDCl3): δ 165.74, 140.50, 135.53, 135.44, 133.58, 131.78, 129.81, 129.75, 129.03, 128.46, 122.36, 122.06, 119.02, 52.00, 48.47, 32.46, 25.32, 24.37; HRMS (ESI) m/z [M+H]+ calcd. for C20H20Cl2N2O3S: 439.0649, found: 439.0646; LC-MS: ret. time 1.920 min.
2-(5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclohexylacetamide (SM7): to a suspension of LiAlH4 (0.021 g, 0.55 mmol) in dry THE (1 mL) cooled to 0° C., a solution of SM3 (0.100 g, 0.22 mmol) in dry THE (4 mL) was added drop-wise under N2 and then the mixture was stirred at 50° C. for 2 h. After cooling and quenching with EtOAc followed MeOH, the mixture was then poured into ice-water and extracted with EtOAc (3×20 mL). The combined organic layers were washed with brine, dried, and evaporated to dryness. The crude colorless oil obtained was purified by flash column chromatography, eluting with cyclohexane/EtOAc (7:3), to give SM7 (0.040 g, 49%) as a white solid: mp 176-178° C. 1H NMR (400 MHz, CDCl3): δ 8.25-8.00 (m, 3H, Ar—H), 7.77 (dt, J=1.2 and 8.4 Hz, 1H, Ar—H), 7.62 (t, J=7.7 Hz, 1H, Ar—H), 7.51 (dt, J=1.3 and 8.6 Hz, 1H, Ar—H), 7.39 (dt, J=1.0 and 8.4 Hz, 1H, Ar—H), 7.25 (dd, J=1.8 and 7.2 Hz, 1H, Ar—H), 6.60 (brs, 1H, NH), 4.48 (s, 2H, N—CH2), 3.91-3.83 (m, 1H, Cy-CH), 1.90-1.80 (m, 2H, Cy-CH2), 1.60-1.50 (m, 3H, Cy-CH), 1.40-1.25 (m, 2H, Cy-CH2), 1.20-1.00 (m, 3H, Cy-CH). 13C NMR (100 MHz, CDCl3): δ 166.18, 137.66, 133.57, 132.43, 131.81, 130.31, 127.98, 125.31, 125.21, 124.88, 123.36, 121.96, 118.99, 51.19, 47.85, 31.92, 24.87, 23.81. HRMS (ESI) calcd for C20H22N2O3S [M++H]+: 371.1429, found: 371.1397. LC-MS: ret. time 5.212.
N-Cyclohexyl-2-(9-hydroxy-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6yl)acetamide (SM226): to a solution of target compound SM225 (0.22 g. 0.55 mmol) in dry CH2Cl2 (12 mL) and under N2 flux, 1M BBr3 in CH2Cl2 (2.75 g, 2.75 mmol) was added dropwise at −60° C. and then the solution was stirred at −30° C. for 12 h. After quenching of the excess of BBr3 with MeOH, H2O, and saturated solution of NaHCO3, the mixture was acidified with 2N HCl to pH and extracted with CH2Cl2 (3×30 mL). The combined organic layers were washed with brine, dried, and evaporated to dryness and the residue which was purified by flash column chromatography (CHCl3/MeOH 95:5), to give compound SM226, as white solid in 88% yield: mp 216-217° C. 1H-NMR (400 MHz, DMSO-d6): δ 9.77 (s, 1H, OH), 8.15 (d, J=8.9 Hz, 1H, Ar—H), 7.90-7.74 (m, 3H, Ar—H and NH), 7.62 (t, J=7.6 Hz, 1H, Ar—H), 7.45 (d, J=2.5 Hz, 1H, Ar—H), 7.25 (d, J=8.8 Hz, 1H, Ar—H), 6.85 (dd, J=2.6 and 8.7 Hz, 1H, Ar—H), 4.30 (s, 2H, NCH2), 3.40-3.30 (m, 1H, Cy-CH), 1.75-1.40 (m, 5H, Cy-CH2), 1.30-0.90 (m, 5H, Cy-CH2). 13C-NMR (100 MHz, DMSO-d6): δ 166.19, 154.95, 143.08, 135.01, 133.28, 132.00, 129.29, 126.41, 126.31, 123.94, 121.99, 118.10, 111.45, 51.74, 48.13, 32.38, 25.31, 24.59. HRMS (ESI) m/z [M+H]+ calcd for C20H22N2O4S: 387.1380, found: 387.1372; LC-MS: ret. time 4.691 min.
N-Cyclohexyl-2-[9-[2-(dimethylamino)ethoxy]-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (SM230): to a solution of target compound SM226 (0.18 g. 0.47 mmol) in dry DMF (7 mL), Cs2CO3 (0.23 g. 0.70 mmol) and commercial 1-chloro-N,N-dimethylethanamine hydrochloride (0.07 g. 0.47 mmol) were added and the mixture was maintained under magnetic stirring at 85° C. for 2 h. The mixture was poured into ice-water, extracted with EtOAc (3×20 mL) and the combined organic layers were washed with brine, dried and evaporated to dryness to give an oil which was purified by flash column chromatography (CHCl3/MeOH 9:1), to afford SM230 as low melting solid in 57% yield: mp 66-67° C. 1H-NMR (200 MHz, DMSO-d6): δ 8.23 (d, J=8.1 Hz, 1H, Ar—H), 7.90-7.65 (m, 3H, Ar—H and NH), 7.60-7.55 (m, 2H, Ar—H), 7.28 (d, J=8.9 Hz, 1H, Ar—H), 7.10 (dd, J=2.5 and 9.0 Hz, 1H, Ar—H), 4.40 (s, 2H, SO2N—CH2), 4.20 (t, J=5.3 Hz, 2H, OCH2), 3.90-3.80 (m, 1H, Cy-CH), 2.80 (t, J=5.3 Hz, 2H, NCH2), 2.40 (s, 6H, NCH3), 1.75-1.40 (m, 5H, Cy-CH2), 1.30-0.90 (m, 5H, Cy-CH2). 13C-NMR (100 MHz, DMSO-d6): δ 165.84, 156.34, 135.42, 132.86, 132.29, 132.26, 129.15, 126.89, 126.26, 123.65, 121.87, 117.63, 110.75, 66.68, 51.40, 48.04, 45.99, 32.66, 25.53, 24.77. HRMS (ESI) m/z [M+H]+ calcd for C24H31N3O4S: 458.2200, found: 458.2200; LC-MS: ret. time 6.360 min.
Experimental procedure for making compounds of formula 7a in Scheme 1 are described below.
2-(9-Bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetic acid of formula 7a: A stirred mixture of ethyl 2-(9-bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetate of formula 6a (Example 36; 0.600 g, 1.5 mmol) in aqueous 10% NaOH (7 mL) and EtOH (7 mL) was refluxed for 30 min, then cooled, concentrated under reduced pressure, poured into ice-water and acidified with 2N HCl to pH 2. The formed precipitate was filtered off to give the compound (0.540 g, 96%) as a white solid that was used as is in the next reaction step: mp 207-209° C. 1H NMR (400 MHz, DMSO-d6): δ 8.40 (d, J=2.2 Hz, 1H, Ar—H), 8.31 (d, J=8.0 Hz, 1H, Ar—H), 7.91 (dd, J=1.1 and 7.7 Hz, 1H, Ar—H), 7.84 (dt, J=1.3 and 7.7 Hz, 1H, Ar—H), 7.75-7.69 (m, 2H, Ar—H), 7.45 (d, J=8.7 Hz, 1H, H-7), 4.80 (s, 2H, NCH2).
3-Fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetic acid of formula 7a: to a solution of ethyl [3-fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetate of formula 6a (Example 43; 1.25 g, 3.09 mmol) in dioxane (25 mL), a solution of 1N LiOH monohydrate (2.47 mL) was added. The reaction mixture was stirred at room temperature for 10 min. and then poured into ice-water and acidified with 2N HCl (pH=2). The precipitate formed was filtered and dried to give the desired compound as white solid (1.16 g, 96%). 1H NMR (400 MHz, CDCl3): δ 4.70 (s, 1H, NCH2), 7.35 (d, J=8.5 Hz, 1H, H-7), 7.40-7.50 (m, 1H, H-2), 7.60-7.65 (m, 1H, H-4), 7.70 (d, J=8.5 Hz, 1H, H-8), 8.00 (dd, J=4.5 and 8.8 Hz, 1H, H-1), 8.20 (s, 1H, H-10).
2-(9-bromo-5,5-dioxo-6H-dibenzo[c,e][1,2]thiazin-6 (5H)-yl)-N-phenylacetamide (SM6). A mixture of 2-(9-Bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetic acid of general formula 7a (Example 65) (0.530 g, 1.44 mmol) and SOCl2 (2 mL) was refluxed under magnetic stirring for 1 h, then the excess of SOCl2 was removed by distillation and the residue was washed 3 times with dry toluene. The obtained acyl chloride was solubilized in dry DMF (7 mL) and added drop-wise, under N2 atmosphere, to a stirred solution of aniline (0.264 mL, 2.88 mmol) and Et3N (0.401 mL, 2.88 mmol) in dry DMF (3 mL) at room temperature. The mixture was left under magnetic stirring overnight then poured into ice-water and acidified with 2N HCl to pH 3. The precipitate was filtered and purified by flash column chromatography, eluting with CHCl3, to give target compound SM6 (0.150 g, 25%) as a white solid: mp 128-130° C. 1H NMR (400 MHz, CDCl3): (8.37 (bs, 1H, NH), 8.20 (d, J=2.1 Hz, 1H, Ar—H), 8.10 (d, J=7.7 Hz, 1H, Ar—H), 8.00 (d, J=7.7 Hz, 1H, Ar—H), 7.80 (t, J=7.7 Hz, 1H, Ar—H), 7.70 (t, J=7.7 Hz, 1H, Ar—H), 7.60 (dd, J=2.2 and 7.8 Hz, 1H, Ar—H), 7.55-7.48 (m, 2H, Ar—H), 7.35-7.30 (m, 2H, Ar—H), 7.20 (d, J=7.8 Hz, 1H, Ar—H), 7.10 (t, J=7.4 Hz, 1H, Ar—H), 4.52 (s, 2H, CH2). 13C NMR (100 MHz, CDCl3): δ 165.47, 137.03, 136.86, 133.88, 133.75, 133.33, 131.09, 129.30, 129.06, 128.73, 126.02, 125.66, 125.09, 122.76, 121.21, 120.04, 118.98, 52.02. HRMS (ESI) m/z [M+H]+ calcd. for C20H15BrN2O3S: 443.0069, found: 443.0057; LC-MS: ret. time 5.571.
2-(9-Bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)-N-cyclohexyl-N-methylacetamide. The appropriate compound of general formula 7a (2-(9-Bromo-5,5-dioxido-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetic acid; Example 65) (0.59 g, 1.6 mmol) was chlorinated as above reported and the corresponding acyl chloride, solubilized in dry DMF (8 mL), was added drop-wise, under N2 atmosphere, to a solution of N-methylcyclohexylamine (0.83 mL, 6.4 mmol) in dry DMF (2 mL) at rt. The mixture was heated to 40° C. for 1.5 h, then poured into ice-water, and acidified with 2N HCl to pH 3. The precipitate was filtered and purified by flash column chromatography, eluting with CHCl3, and subsequent trituration with petroleum ether/Et2O to give target compound SM8 (0.197 g, 30%) as a white solid: mp 170-172° C. 1H NMR (400 MHz, DMSO-d6): (mixture of rotamers) δ 8.42 (d, J=1.7 Hz, 1H, Ar—H), 8.30 (d, J=8.0 Hz, 1H, Ar—H), 7.87 (d, J=7.8 Hz, 1H, Ar—H), 7.82 (t, J=7.6 Hz, 1H, Ar—H), 7.75-7.65 (m, 2H, Ar—H), 7.43 (t, J=8.9 Hz, 1H, H-7), 4.97 (s, 0.88H, NCH2), 4.90 (s, 1.12H, NCH2), 4.00-3.90 (m, 0.54H, Cy-CH), 3.60-3.50 (m, 0.46H, Cy-CH), 2.80 (s, 1.68H, NCH3), 2.60 (s, 1.32H, NCH3), 1.75-0.95 (m, 10H, Cy-CH2). 13C NMR (100 MHz, DMSO-d6): (mixture of rotamers) δ 165.91, 165.87, 138.45, 138.32, 135.56, 135.52, 133.15, 133.11, 132.94, 132.91, 131.09, 129.59, 128.36, 126.96, 126.92, 123.76, 123.73, 121.54, 121.48, 117.71, 117.65, 55.11, 52.84, 50.10, 30.59, 29.48, 28.68, 27.38, 25.61, 25.45, 25.30, 25.15. HRMS (ESI) calcd for C21H23BrN2O3S [M++H]+: 463.0692, found: 463.0678. LC-MS: ret. time 6.034. The two rotamers collapsed to one molecule after recording the NMR spectrum at 50° C.
2-(3-fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetic acid (7a(Int-1)) and 2-(3-ethoxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetic acid (7a(Int-2)): A stirred mixture of compound of formula 6a(Int-1) (0.40 g, 0.99 mmol) in aqueous 10% NaOH (3 mL) and EtOH (3 mL) was refluxed for 30 min. After cooling, the organic solvent was evaporated under reduced pressure and the residue was poured into ice-water and acidified with 2N HCl (pH=2). The formed precipitate was filtered off to give a mixture of two compounds (7a(Int-1) and 7a(Int-2) in a 1:1 ratio as highlighted by the presence of two spots in TLC (CHCl3:MeOH 8:2) and also confirmed by 1H-NMR spectrum. 1H NMR (400 MHz, CDCl3): δ 8.20 (bs, 0.5H, H-10), 8.17 (bs, 0.5H, H-10), 7.90 (dd, J=5 and 9 Hz, 0.5H, H-1), 7.85 (d, J=9 Hz, 0.5H, H-1), 7.73-7.60 (m, 1H, H-4 and H-8), 7.60 (d, J=8.5 Hz, 0.5H, H-8), 7.45 (m, 0.5H, H-2), 7.40 (s, 0.5H, H-4), 7.35 (d, J=8 Hz, 0.5H, H-7), 7.20-7.30 (m, 1H, H-2 and H-7), 4.67 (s, 1H, N—CH2), 4.65 (s, 1H, N—CH2), 4.10 (q, J=7.0 Hz, 1H, OCH2), 1.45 (t, J=7.0 Hz, 1.5H, CH3). The compounds 7a(Int-1) and 7a(Int-2) were obtained as an orange solid that was used as such in the successive amidation step.
Scheme 6: Preparation of target compounds deriving from intermediates of general formula 7a not included in Scheme 1.
N-cyclohexyl-2-(3-fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetamide (SM882) and N-cyclohexyl-2-(3-ethoxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetamide (SM883): A stirred mixture of 7a(Int-1) and 7a(Int-2) (0.50 g, 1.33 mmol), cyclohexylamine (0.18 mL, 1.6 mmol), TBTU (0.55 g, 1.7 mmol), and DIPEA (0.93 mL, 5.33 mmol) in dry CH2Cl2 (3 mL) was reacted at room temperature for 1 h. The solvent was then evaporated to dryness and the residue was poured in ice-water obtaining a precipitate that was filtered and the crude was purified by flash chromatography eluting with CH2Cl2 obtaining SM882 (Rf>) and SM883 (Rf<) respectively.
Each compound was further purified by crystallization with EtOH to give: SM882: white solid (0.064 g, 14%), mp 232-233° C. 1H NMR (400 MHz, CDCl3): δ1.10-1.20 and 1.30-1.40 (m, each 2H, cyclohexyl CH2), 1.50-1.70 (m, 4H, cyclohexyl CH2), 1.80-1.90 (m, 2H, cyclohexyl CH2), 3.85-3.95 (m, 1H, cyclohexyl CH), 4.55 (s, 1H, NCH2), 6.45 (d, J=7.5 Hz, 1H, CONH), 7.40 (d, J=8.5 Hz, 1H, Ar—H), 7.55 (dt, J=2.6 and 8.1 Hz, 1H, Ar—H), 7.75-7.85 (m, 2H, Ar—H), 8.10 (dd, J=4.6 and 8.9 Hz, 1H, Ar—H), 8.30 (s, 1H, Ar—H); 13C NMR (101 MHz, CDCl3): δ 24.2, 25.2, 32.4, 48.5, 51.4, 109.8 (d, JC-F=25.4 Hz), 119.8, 120.8 (d, JC-F=22.2 Hz), 122.7 (d, JC-F=3.6 Hz), 123.5, 128.8 (q, JC-F=273.3 Hz), 127.1 (d, JC-F=3.3 Hz), 127.4, 127.7, 128.5 (d, JC-F=8.1 Hz), 135.4 (d, JC-F=7.3 Hz), 140.1, 162.2 (d, JC-F=256.9 Hz), 165.6. HRMS (ESI) m/z [M+H]+ calcd. for C21H20F4N2O3S: 457.1210, found: 457.1207.
SM883: white solid (0.069 g, 15%), mp 201-202° C. 1H NMR (400 MHz, CDCl3): δ1.10-1.20 (m, 4H, cyclohexyl CH2), 1.30-1.40 (m, 2H, cyclohexyl CH2), 1.50 (t, J=6.9 Hz, 3H, OCH2CH3), 1.60-1.70 and 1.80-1.90 (m, each 2H, cyclohexyl CH2), 3.80-3.90 (m, 1H, cyclohexyl CH), 4.20 (q, J=6.9 Hz, 2H, OCH2CH3), 4.55 (s, 1H, NCH2), 6.55 (d, J=7.7 Hz, 1H, CONH), 7.30-7.40 (m, 2H, Ar—H), 7.50 (d, J=2.1 Hz, 1H, Ar—H), 7.70 (d, J=8.6 Hz, 1H, Ar—H), 7.95 (d, J=8.9 Hz, 1H, Ar—H), 8.25 (s, 1H, Ar—H); 13C NMR (101 MHz, CDCl3): δ 14.4, 24.2, 25.2, 32.3, 48.4, 51.2, 64.4, 106.1, 119.3, 121.1, 122.1 (d, JC-F=3.6 Hz), 123.9, 126.1 (d, JC-F=3.3 Hz), 127.5, 134.9, 139.6, 159.6, 166.0. HRMS (ESI) m/z [M+H]+ calcd. for C23H25F3N2O4S: 483.1566, found: 483.1565.
N-(1-Ethylpropyl)-2-[3-fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (SM884): A stirred mixture of 3-Fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetic acid of formula 7a (Example 63; 0.30 g, 0.8 mmol), 3-aminopentane (0.084 g, 0.96 mmol), TBTU (0.33 g, 1.04 mmol), DIPEA (0.56 mL, 3.2 mmol) in CH2Cl2 (6 mL) was kept at room temperature for 3 h.
The organic solvent was evaporated and the residue was poured into ice/water and the mixture was acidified with 2N HCl (pH=4) maintaining the mixture under stirring for 40 min. until a precipitated was observed. The precipitate was filtered, dried and crystallized by cyclohexane/EtOAc (3:1 ratio) to obtain SM884 as pinkish solid in 34%: mp 184-185° C. 1H NMR (400 MHz, CDCl3): δ 0.80 (t, J=7.4 Hz, 6H, pentyl CH3), 1.30-1.40 and 1.45-1.55 (m, each 2H, pentyl CH3), 3.75-3.80 (m, 1H, pentyl CH), 4.50 (s, 1H, NCH2), 6.25 (d, J=8.6 Hz, 1H, CONH), 7.40 (d, J=8.6 Hz, 1H, Ar—H), 7.50 (dt, J=2.6 and 8.3 Hz, 1H, Ar—H), 7.70-7.75 (m, 2H, Ar—H), 8.10 (dd, J=4.6 and 8.9 Hz, 1H, Ar—H), 8.25 (s, 1H, Ar—H); 13C NMR (100 MHz, CDCl3): δ 9.9, 27.0, 51.4, 52.7, 109.8 (d, JC-F=25.5 Hz), 119.8, 120.9 (d, JC-F=22.3 Hz), 122.7 (d, JC-F=3.5 Hz), 123.4, 126.1 (q, JC-F=273.0 Hz), 127.1 (d, JC-F=3.2 Hz), 127.7, 128.6 (d, JC-F=8.1 Hz), 135.3 (d, JC-F=7.3 Hz), 140.1, 162.2 (d, JC-F=257.0 Hz), 166.4. HRMS (ESI) m/z [M+H]+ calcd. for C20H20F4N2O3S: 445.1210, found: 445.1207.
2-[3-Fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]-N-(tetrahydro-2H-pyran-4-yl)acetamide (SM885): following the procedure reported above for compound SM884 and using tetrahydro-2H-pyran-4-amine, the target compound was obtained after crystallization by cyclohexane/EtOAc, in 34% yield as pale pink solid: mp 241-242° C. 1H NMR (400 MHz, CDCl3): δ 1.40-1.50, 1.80-1.90, 3.40-3.50, and 3.75-3.85 (m, each 2H, pyran CH2), 4.00-4.10 (m, 1H, pyran CH), 4.50 (s, 1H, NCH2), 6.45 (d, J=7.4 Hz, 1H, CONH), 7.40 (d, J=8.6 Hz, 1H, Ar—H), 7.50 (dt, J=2.7 and 8.5 Hz, 1H, Ar—H), 7.65-7.75 (m, 2H, Ar—H), 8.05 (dd, J=4.6 and 8.9 Hz, 1H, Ar—H), 8.25 (s, 1H, Ar—H); 13C NMR (100 MHz, CDCl3): δ 32.4, 46.0, 51.3, 66.2, 109.8 (d, JC-F=25.5 Hz), 119.7, 121.0 (d, JC-F=22.2 Hz), 122.7 (d, JC-F=3.5 Hz), 123.4, 126.1 (q, JC-F=273.0 Hz), 127.2, 127.4 (d, JC-F=3.2 Hz), 127.8, 128.6 (d, JC-F=8.1 Hz), 135.3 (d, JC-F=7.3 Hz), 140.0, 162.2 (d, JC-F=257.2 Hz), 166.0. HRMS (ESI) m/z [M+H]+ calcd. for C20H18F4N2O4S: 459.1002, found: 459.1002.
2-[3-Fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]-N-morpholin-4-ylacetamide (SM881): following the procedure reported for compound SM884 and using morpholin-4-amine, the target compound was obtained after crystallization by EtOH, in 34% yield as pale pink solid: mp 276-278° C. Two rotamers were identified by 1H-NMR and they collapsed to one molecule carrying out experiments at 60° C. 1H NMR (400 MHz, DMSO-d6, 25° C.): δ 2.50-2.60, 2.75-2.95, 3.40-3.50, and 3.60-3.80 (m, each 2H, morpholine CH2), 4.50 and 5.00 (s, each 1H, NCH2), 7.60-7.75 (m, 2H, Ar—H), 7.75-7.80 and 7.80-7.90 (m, each 1H, Ar—H), 8.45 (dd, J=4.6 and 8.6 Hz, 1H, Ar—H), 8.55 (s, 1H, Ar—H), 8.80 and 9.25 (s, each 0.5H, CONH); 13C NMR (100 MHz, DMSO-d6): δ 32.4, 46.0, 66.2, 110.1 (d, JC-F=25.5 Hz), 119.7, 122.0 (d, JC-F=22.2 Hz), 123.7 (d, JC-F=3.5 Hz), 123.4, 125.1 (q, JC-F=273.0 Hz), 127.2, 127.4 (d, JC-F=3.2 Hz), 127.8, 129.2 (d, JC-F=8.1 Hz), 134.2 (d, JC-F=7.3 Hz), 140.0, 161.2 (d, JC-F=257.2 Hz), 166.0. HRMS (ESI) m/z [M+H]+ calcd. for C19H17F4N3O4S: 460.0955, found: 460.0954.
N-(2-chloropyridin-4-yl)-2-[5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (SM880): The title compound was prepared starting from 2-(5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetic acid of formula 7a (Example 75) and following the procedure reported for compound SM884 and using 2-chloro-4-pyridineamine. Title compound was obtained after crystallization by cyclohexane/EtOAc, as pale pink solid in 34% yield: mp 184-185° C. 1H NMR (400 MHz, CDCl3): δ 0.80 (t, J=7.4 Hz, 6H, pentyl CH3), 1.30-1.40 and 1.45-1.55 (m, each 2H, pentyl CH3), 3.75-3.80 (m, 1H, pentyl CH), 4.50 (s, 1H, NCH2), 6.25 (d, J=8.6 Hz, 1H, CONH), 7.40 (d, J=8.6 Hz, 1H, Ar—H), 7.50 (dt, J=2.6 and 8.3 Hz, 1H, Ar—H), 7.70-7.75 (m, 2H, Ar—H), 8.10 (dd, J=4.6 and 8.9 Hz, 1H, Ar—H), 8.25 (s, 1H, Ar—H); 13C NMR (101 MHz, CDCl3): δ 9.9, 27.0, 51.4, 52.7, 109.8 (d, JC-F=25.5 Hz), 119.8, 120.9 (d, JC-F=22.3 Hz), 122.7 (d, JC-F=3.5 Hz), 123.4, 126.1 (q, JC-F=273.0 Hz), 127.1 (d, JC-F=3.2 Hz), 127.7, 128.6 (d, JC-F=8.1 Hz), 135.3 (d, JC-F=7.3 Hz), 140.1, 162.2 (d, JC-F=257.0 Hz), 166.4. HRMS (ESI) m/z [M+H]+ calcd. for C20H20F4N2O3S: 445.1210, found: 445.1207.
Scheme 7: Preparation of target compounds deriving from intermediates of general formula 7a, not included in Scheme 1.
2-(5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetic acid (7a(Int-3)): compound of formula 7a(Int-3) was prepared from ethyl [5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetate according to the procedure reported for a similar compound described in Example 65. The intermediate was obtained as brown solid in 81% yield: 1H NMR (400 MHz, DMSO-d6): δ 8.55 (d, J=2.2 Hz, 1H, Ar—H), 8.27 (d, J=8.0 Hz, 1H, Ar—H), 8.00-7.75 (m, 4H, Ar—H), 7.50-7.50 (m, 2H, Ar—H), 4.75 (s, 2H, NCH2).
N-(1-benzylpiperidin-4-yl)-2-[5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide of formula 8a(Int-3): to a solution of 2-(5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl)acetic acid of formula 7a(Int-3) (Example 75; 0.280 g, 0.78 mmol) in dry CH2Cl2 (10 mL), N-benzyl-4-aminopiperidine (0.180 g, 0.94 mmol), TBTU (0.376 g, 0.12 mmol), and DIPEA (0.510 mL, 0.31 mmol) were added. The reaction mixture was stirred at room temperature for 4 h and then poured into ice-water and acidified with 2N HCl (pH=2). The mixture was extracted with CH2Cl2 (3×30 mL) and the combined organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness to give a brown oil which was purified by flash column chromatography (CHCl3/MeOH 95:5), to afford N-(1-benzylpiperidin-4-yl)-2-[5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide as solid in 45% yield: mp ° C. 1H-NMR (200 MHz, CDCl3): δ 8.27 (s, 1H, Ar—H), 8.03-7.99 (d, J=7.9 Hz, 2H, Ar—H), 7.82-7.60 (m, 3H, Ar—H), 7.32-7.15 (m, 6H, Ar—H), 6.53 (d, J=7.3 Hz, 1H, NH), 4.54 (s, 2H, benzylic-CH2), 4.83-4.71 (m, 1H, piperidine-CH), 3.45 (s, 2H, CH2), 2.67-2.62 (m, 2H, piperidine-CH2), 2.15-1.80 (m, 6H, piperidine-CH2×2), 1.47-1.32 (m, 2H, piperidine-CH2).
2-[5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]-N-piperidin-4-ylacetamide of general formula 8a (SM655): to a solution of the appropriate compound of formula 8a (0.180 g, 0.34 mmol) in EtOH (20 mL), Pd/C (20% w/w, 0.036 g) was added. The reaction mixture was stirred at room temperature for 7 h under H2 bubbling. The mixture was filtered over Celite® and the filtrate was evaporated to dryness to give a brown solid which was crystallized by EtOH to afford SM665 as white solid in 27% yield. 1H NMR (400 MHz, DMSO-d6): δ 8.55 (s, 1H, Ar—H), 8.38-8.35 (m, 2H, Ar—H), 7.91-7.81 (m, 3H, Ar—H), 7.71 (t, J=7.6 Hz, 1H, Ar—H), 7.62 (d, J=8.6 Hz, 1H, Ar—H), 4.63 (s, 2H, benzylic-CH2), 3.70-3.59 (m, 1H, piperidine-CH), 3.16-3.13 (m, 2H, piperidine-CH2), 2.82 (t, J=10.7 Hz, 2H, piperidine-CH2), 1.77-1.74 (m, 2H, piperidine-CH2), 1.49-1.37 (m, 2H, piperidine-CH2).
Scheme 8: Preparation of target compounds deriving from intermediates of general formula 7a, not included in scheme 1.
2-[5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]-N-(trans-4-hydroxycyclohexyl)acetamide of formula 8a(Int-4): to a solution of compound of formula 7a(Int-3) (Example 75; 0.100 g, 0.30 mmol) in dry CH2Cl2 (4 mL), trans-4-aminocyclohexanol (0.041 g, 0.36 mmol), BOP (0.199 g, 0.45 mmol), and DIPEA (0.200 mL, 1.2 mmol) were added at 0° C. The reaction mixture was stirred at room temperature for 12 h, then it was concentrated under vacuum and poured into ice-water and acidified with 2N HCl (pH=4). The mixture was extracted with EtOAc (3×20 mL) and the combined organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness to give a brown solid which was crystallized by EtOH to afford SM588 8a(Int-4) as a white solid in 88% yield: mp 212-213° C. 1H-NMR (400 MHz, CDCl3): δ 8.29 (d, J=1.3 Hz, 1H, Ar—H), 8.03 (d, J=7.3 Hz, 2H, Ar—H), 7.81 (td, J=1.3 and 7.4 Hz, 1H, Ar—H), 7.72 (dd, J=1.6 and 6.3 Hz, 1H, Ar—H), 6.67 (t, J=7.5 Hz, 1H, Ar—H), 7.33 (d, J=7.9 Hz, 1H, Ar—H), 6.52 (d, J=7.6 Hz, 1H, NH), 4.51 (s, 2H, benzyl-CH2), 3.85-3.77 (m, 1H, cyclohexyl-CH), 3.61-3.46 (m, 1H, cyclohexyl-CH), 1.99-1.89 (m, 4H, cyclohexyl-CH2×2), 1.46 (s, 1H, OH), 1.42-1.34 (m, 2H, cyclohexyl-CH2), 1.23-1.16 (m, 2H, cyclohexyl-CH2).
trans-4-({2-[5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetyl}amino)cyclohexyl 4-nitrobenzenesulfonate of general formula 8a (SM589): to a solution of an appropriate compound SM588 of formula 8a(Int-4) (0.350 g, 0.77 mmol) in dry CH2Cl2 (6 mL), 4-nitrobenzenesulfonyl chloride (0.355 g, 1.60 mmol), DMAP (0.094 g, 0.77 mmol), and ET3N (0.320 mL, 2.30 mmol) were added at 0° C. The reaction mixture was stirred at room temperature for 2 h, then it was concentrated under vacuum and poured into ice-water and acidified with 2N HCl (pH=4). The mixture was extracted with CH2Cl2 (3×20 mL) and the combined organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness to give a white solid which was crystallized by EtOH to afford SM589 as a white solid in 38% yield: mp 151-152° C. 1H-NMR (400 MHz, CDCl3): δ 8.35 (d, J=8.5 Hz, 2H, Ar—H), 8.29 (s, 1H, Ar—H), 8.12-7.93 (m, 4H, Ar—H), 7.81 (t, J=7.0 Hz, 1H, Ar—H), 7.72-7.61 (m, 2H, Ar—H), 7.26 (d, J=8.6 Hz, 1H, Ar—H), 6.64 (d, J=7.5 Hz, 1H, NH), 4.60-4.51 (m, 1H, cyclohexyl-CH), 4.49 (s, 2H, CH2), 3.91-3.85 (m, 1H, cyclohexyl-CH), 2.01-1.88 (m, 4H, each 2H cyclohexyl-CH2), 1.68-1.59 (m, 2H, cyclohexyl-CH2), 1.52 (s, 1H, OH), 1.32-1.10 (m, 2H, cyclohexyl-CH2).
trans-4-({2-[5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetyl}amino)cyclohexyl 4-aminobenzenesulfonate of formula 8a (SM656): to a solution of compound SM589 (0.400 g, 0.63 mmol) in DMF (30 mL), Raney/Ni (10% w/w, 0.046 g) was added. The reaction mixture was stirred at room temperature for 2 h under H2 bubbling. The mixture was filtered over Celite©and the filtrate was evaporated to dryness to give a brown solid which was crystallized by EtOH to afford SM656 as brownish solid in 58% yield. 1H NMR (400 MHz, DMSO-d6): δ 8.45 (s, 1H, Ar—H), 8.37 (d, J=7.9 Hz, 1H, Ar—H), 8.05 (d, J=7.2 Hz, 1H, Ar—H), 7.90-7.80 (m, 3H, Ar—H and NH), 7.70 (t, J=7.6 Hz, 1H, Ar—H), 7.57 (d, J=8.4 Hz, 1H, Ar—H), 7.44 (d, J=8.8 Hz, 2H, Ar—H), 6.59 (d, J=8.6 Hz, 2H, Ar—H), 6.19 (s, 2H, NH2), 4.58 (s, 2H, CH2), 4.11-4.23 (m, 1H, cyclohexyl-CH), 1.69-1.61 (m, 4H, each 2H, cyclohexyl-CH2), 1.39-1.31 (m, 2H, cyclohexyl-CH2), 1.17-1.08 (m, 2H, cyclohexyl-CH2).
2-(3-acetyl-4-hydroxy-1,1-dioxido-2H-1,2-benzothiazin-2-yl)-N-cyclohexylacetamide (12a) (Scheme 4). A mixture of 11a, prepared according to literature, (0.63 g, 2.11 mmol), cyclohexylamine (0.53 mL, 4.66 mmol), TBTU (1.63 g, 5.08 mmol), and Et3N (4 equiv.) in dry THE was reacted at room temperature for 2 h. The reaction mixture was then poured in ice-water and acidified with 2N HCl (pH=4) obtaining a precipitate that was filtered and dried to give 12a (0.75 g, 94%) as pale-yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 0.80-1.20 and 1.40-1.60 (m, each 5H, cyclohexyl CH2), 2.40 (s, 3H, CH3), 4.00 (s, 2H, NCH2), 7.75-7.85 (m, 4H, Ar—H and CONH), 7.95-8.10 (m, 1H, Ar—H), 15.20 (bs, 1H, OH).
N-cyclohexyl-2-(3-methyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-4 (1H)-yl)acetamide (SM879). The mixture of 12a (0.30 g, 0.79 mmol) and hydrazine monohydrate (0.19 mL, 3.96 mmol) was reacted at 60° C. for 1 h. After cooling, the reaction mixture was poured in ice-water and acidified with 2N HCl (pH=4), yielding a precipitate that was filtered and purified by flash chromatography eluting with CH2Cl2:MeOH 97:3 followed by crystallization by EtOH to afford SM879 (0.08 g, 54%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 1.10-1.20 and 1.25-1.45 (m, each 2H, cyclohexyl CH2), 1.50-1.75 (m, 4H, cyclohexyl CH2), 2.80-2.90 (m, 2H, cyclohexyl CH2), 2.30 (s, 1H, CH3), 3.75 (m, 1H, cyclohexyl CH), 4.05 (s, 2H, NCH2), 6.50 (d, J=8.1 Hz, 1H, NH), 7.55 (dt, J=1.2 and 7.8 Hz, 1H, Ar—CH), 7.70 (dt, J=1.2 and 7.7 Hz, 1H, Ar—CH), 7.80 (dd, J=0.9 and 7.8 Hz, 1H, Ar—CH), 7.95 (d, J=7.3 Hz, 1H, Ar—CH), 10.50 (bs, 1H, CONH). HRMS (ESI) m/z [M+H]+ calcd for C24H31N3O4S: 375.1460, found: 375.1485; LC-MS: ret. time 4.109 min.
N-(4-aminocyclohexyl)-2-[5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (SM886). A stirred mixture of [5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetic acid of formula 7a(Int-3) (0.10 g, 0.28 mmol), trans-1,4-diaminocyclohexane (0.32 g, 2.80 mmol), TBTU (0.12 g, 0.36 mmol), DIPEA (0.19 mL, 1.12 mmol) in dry DMF (3 mL) was kept at room temperature for 3 h. The reaction mixture was poured into ice/water and extracted with CH2Cl2 (×3). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness to give a brown oil. After purification by trituration with Et2O, the title compound was obtained as a yellow solid in 16% yield: m.p. 212-214° C. 1H NMR (400 MHz, MeOD): δ 1.16-1.29 (m, 4H, CH2×2), 1.87-1.89 (m, 4H, CH2×2), 2.60-2.63 (m, 1H, CH), 3.49-3.54 (m, 1H, CH), 4.66 (s, 2H, NCH2), 7.57 (d, J=8.6 Hz, 1H, Ar—H), 7.73 (t, J=7.5 Hz, 1H, Ar—H), 7.82-7.89 (m, 2H, Ar—H), 7.99 (d, J=7.8 Hz, 1H, Ar—H), 8.24 (d, J=8.0 Hz, 1H, Ar—H), 8.49 (s, 1H, Ar—H). HRMS (ESI) m/z [M+H]+ calcd. for C21H22F3N3O3S: 454.1412, found: 454.14162.
Scheme 10: Synthetic procedure for the preparation of target compound SM887.
Dimethyl 3-({[5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetyl}amino)pentanedioate (8a(Int-5)). A stirred mixture of [5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetic acid of formula 7a(Int3) (0.33 g, 0.92 mmol), dimethyl 3-aminopentanedioate (0.19 g, 1.11 mmol), TBTU (0.38 g, 1.19 mmol), DIPEA (0.64 mL, 3.68 mmol) in CH2Cl2 (10 mL) was kept at room temperature for 2 h. The organic solvent was evaporated, and the residue was poured into ice/water and extracted with EtOAc (×3). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness to give a brown oil. After purification by flash column chromatography, eluting with CHCl3/MeOH (98:2), the title compound was obtained as a white solid in 25% yield: m.p. 126-128° C. 1H NMR (400 MHz, CDCl3): δ 2.37-2.44 (m, 4H, CH2×2), 3.58 (s, 6H, OCH3×2), 4.52 (s, 2H, NCH2), 4.59-4.64 (m, 1H, CH), 7.17 (d, J=8.4 Hz, 1H, NH), 7.40 (d, J=8.5 Hz, 1H, H-7), 7.65 (t, J=7.8 Hz, 1H, H-3), 7.72 (d, J=8.5 Hz, 1H, H-8), 7.79 (td, J=1.0 and 7.4 Hz, 1H, H-2), 8.02 (d, J=8.1 Hz, 2H, H-1 and H-4), 8.27 (s, 1H, H-10).
2-[5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]-N-[3-hydroxy-1-(2-hydroxyethyl)propyl]acetamide (SM887). A stirred mixture of dimethyl 3-({[5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetyl}amino)pentanedioate of formula 8a(Int-5) (0.45 g, 0.87 mmol) and NaBH4 (1.32 g, 35.97 mmol) in dry THE (20 mL) was stirred at reflux for 16 h. Then, the reaction mixture was cooled up to 0° C. and MeOH (15 mL) was added to quench the excess of NaBH4. The organic solvent was evaporated and the residue was poured into ice/water and extracted with EtOAc (×3). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness to give a yellow oil. After purification by flash column chromatography, eluting with CHCl3/MeOH (98:2), the title compound was obtained as a white solid in 28% yield: m.p. 136-138° C. 1H NMR (400 MHz, DMSO-d6): δ 1.45-1.58 (m, 4H, CH2×2), 3.29-3.39 (m, 4H, CH2×2), 3.76-3.78 (m, 1H, CH), 4.30 (t, J=5.1 Hz, 2H, OH×2), 4.66 (s, 2H, NCH2), 7.65 (d, J=8.5 Hz, 1H, H-7), 7.76 (t, J=7.5 Hz, 1H, H-3), 7.86-7.93 (m, 2H, Ar—H), 7.97 (d, J=7.2 Hz, 1H, Ar—H), 8.01 (d, J=8.6 Hz, 1H, NH), 8.43 (d, J=7.9 Hz, 1H, Ar—H), 8.60 (s, 1H, H-10). 13C NMR (101 MHz, DMSO-d6): δ 38.0, 44.1, 49.7, 58.2, 121.6, 121.7, 123.3, 124.4 (q, JC-F=268.4 Hz), 124.7, 125.6 (q, JC-F=32.6 Hz), 127.1, 127.2, 129.9, 130.9, 133.2, 135.0, 141.9, 166.2. HRMS (ESI) m/z [M+K]+ calcd. for C20H21F3N205S: 497.0760, found: 497.0756.
Alternative procedure for the synthesis of [3-Fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetic acid (7a(Int-1)). A stirred mixture of compound of formula 6a(Int-1) (1.25 g, 3.10 mmol) in aqueous 1N LiOH (15.5 mL, 15.5 mmol) and dioxane (30 mL) was kept at room temperature for 30 min. The reaction mixture was poured into ice-water and acidified with 2N HCl (pH=2). The formed precipitate was filtered off and dried to give the title compound in 98% yield: m.p. 100-102° C. 1H NMR (400 MHz, CDCl3): δ 4.72 (s, 2H, NCH2), 7.33 (d, J=8.4 Hz, 1H, Ar—H), 7.45 (td, J=2.5 and 8.1 Hz, 1H, H-2), 7.65-7.72 (m, 2H, Ar—H), 7.98 (dd, J=4.4 and 8.6 Hz, 1H, H-1), 8.21 (s, 1H, H-10).
Dimethyl 3-({[3-fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetyl}amino)pentanedioate (8a(Int-6)). A stirred mixture of [3-fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetic acid of formula 7a(Int-1) (0.71 g, 1.9 mmol), dimethyl 3-aminopentanedioate (0.40 g, 2.28 mmol), TBTU (0.79 g, 2.47 mmol), DIPEA (1.32 mL, 7.6 mmol) in CH2Cl2 (30 mL) was kept at room temperature for 2 h. The organic solvent was evaporated and the residue was poured into ice/water and the mixture was acidified with 2N HCl (pH=4) maintaining the mixture under stirring for 10 min. until a precipitated was observed. The precipitate was filtered to give the title compound as a white solid in 87%: m.p. 153-155° C. 1H NMR (400 MHz, CDCl3): δ 2.60-2.69 (m, 4H, CH2×2), 3.89 (s, 6H, OCH3×2), 4.57 (s, 2H, NCH2), 4.64-4.66 (m, 1H, CH), 7.14 (d, J=8.5 Hz, 1H, NH), 7.48 (d, J=8.4 Hz, 1H, Ar—H), 7.54 (td, J=2.3 and 8.4 Hz, 1H, Ar—H), 7.75-7.77 (m, 2H, Ar—H), 8.07 (dd, J=4.7 and 8.9 Hz, 1H, Ar—H), 8.26 (s, 1H, Ar—H). 2-[3-Fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]-N-[3-hydroxy-1-(2-hydroxyethyl)propyl]acetamide (SM888). A stirred mixture of dimethyl 3-({[3-fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetyl}amino)pentanedioate of formula 8a(Int-6) (0.45 g, 0.85 mmol) and NaBH4 (1.28 g, 33.81 mmol) in dry THE (15 mL) was stirred at reflux for 30 h. Then, the reaction mixture was cooled up to 0° C. and MeOH (15 mL) was added to quench the excess of NaBH4. The organic solvent was evaporated and the residue was poured into ice/water and extracted with EtOAc (×3). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness to give a yellow oil. After purification by flash column chromatography, eluting with cyclohexane/EtOAc (70:30), the title compound was obtained as a white solid in 9% yield: m.p. 136-138° C. 1H NMR (400 MHz, DMSO-d6): δ 1.43-1.58 (m, 4H, CH2×2), 3.27-3.34 (m, 4H, CH2×2), 3.74-3.76 (m, 1H, CH), 4.30 (t, J=5.1 Hz, 2H, OH×2), 4.66 (s, 2H, NCH2), 7.69 (d, J=8.5 Hz, 1H, H-7), 7.76 (td, J=2.7 and 8.7 Hz, 1H, H-2), 7.84 (dd, J=2.7 and 8.6 Hz, 1H, H-4), 7.91 (dd, J=1.6 and 8.5 Hz, 1H, H-8), 8.02 (d, J=8.6 Hz, 1H, NH), 8.51 (dd, J=4.4 and 8.3 Hz, 1H, H-1), 8.60 (s, 1H, H-10). 13C NMR (101 MHz, DMSO-d6): δ 38.2, 44.3, 50.7, 58.3, 109.1 (d, JC-F=25.5 Hz), 120.8 (d, JC-F=22.1 Hz), 122.5, 123.5, 124.5 (q, JC-F=274.0 Hz), 124.7, 126.1 (q, JC-F=33.2 Hz), 127.2, 127.9, 130.7 (d, JC-F=8.4 Hz), 136.7 (d, JC-F=7.5 Hz), 141.7, 162.3 (d, JC-F=252.8 Hz), 166.4. HRMS (ESI) m/z [M+Na]+ calcd. for C20H20F4N2O5S: 499.09267, found: 499.09354.
N-{1-[(dimethylamino)methyl]propyl}-2-[3-fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (SM889). A stirred mixture of [3-fluoro-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetic acid of formula 7a(Int-1) (0.30 g, 0.8 mmol), (2-aminobutyl)dimethylamine (0.13 mL, 0.96 mmol), TBTU (0.33 g, 1.04 mmol), DIPEA (0.56 mL, 3.2 mmol) in CH2Cl2 (30 mL) was kept at room temperature for 1 h. The organic solvent was evaporated, and the residue was poured into ice/water and extracted with EtOAc (×3). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness to give a brown oil. After purification by flash column chromatography, eluting with CHCl3/MeOH (95:5), the title compound was obtained as a light brown solid in 17% yield: m.p. 167-169° C. 1H NMR (400 MHz, CDCl3): δ 0.88 (t, J=7.4 Hz, 3H, CH2CH3), 1.46-1.49 (m, 1H, CHCH2CH3×1/2), 1.60-1.62 (m, 1H, CHCH2CH3×1/2), 2.21-2.23 (m, 1H, CHCH2N×1/2), 2.26-2.29 (m, 1H, CHCH2N×1/2), 3.89-3.93 (m, CH, 1H), 4.46 (d, J=17.5 Hz, 1H, NCH2×1/2), 4.70 (d, J=17.5 Hz, 1H, NCH2×1/2), 6.46 (d, J=6.0 Hz, 1H, NH), 7.51-7.56 (m, 1H, H-2), 7.60 (d, J=8.6 Hz, 1H, H-7), 7.73-7.77 (m, 2H, H-4 and H-8), 8.07 (dd, J=4.5 and 8.8 Hz, 1H, H-1), 8.26 (s, 1H, H-10). 13C NMR (101 MHz, CDCl3): δ 9.9, 25.9, 45.7, 49.3, 51.5, 62.5, 110.0 (d, JC-F=25.3 Hz), 120.8, 120.9 (d, JC-F=21.2 Hz), 122.7 (d, JC-F=3.0 Hz), 123.7 (q, JC-F=273.7 Hz), 123.9, 127.2, 127.7 (d, JC-F=3.0 Hz), 127.8 (q, JC-F=33.3 Hz), 128.7 (d, JC-F=8.0 Hz), 135.9 (d, JC-F=7.1 Hz), 140.5, 162.4 (d, JC-F=256.5 Hz), 166.8. HRMS (ESI) m/z [M+H]+ calcd. for C21H23F4N3O3S: 474.1474, found: 474.14908.
Scheme 12: Synthetic Procedure for the Preparation of the Intermediate of Formula 7a(Int-4).
[3-Methoxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetic acid (7a(Int-4)). A stirred mixture of compound of formula 6a(Int-2) (0.34 g, 0.76 mmol) in aqueous 10% NaOH (3 mL) and MeOH (3 mL) was stirred at reflux for 1 h. The reaction mixture was poured into ice/water and acidified with 2N HCl (pH=2). The formed precipitate was filtered off and dried to give the title compound in 46% yield; m.p. 184-186° C. 1H NMR (400 MHz, DMSO-d6): δ 3.47 (s, 3H, OCH3), 4.27 (s, 2H, NCH2), 7.38-7.41 (m, 2H, Ar—H), 7.55-7.58 (m, 1H, Ar—H), 7.76 (d, J=7.3 Hz, 1H, Ar—H), 8.31 (d, J=8.7 Hz, 1H, Ar—H), 8.45 (s, 1H, Ar—H).
Scheme 13: Synthetic Procedure for the Preparation of the Intermediates of Formula 8a(Int-7), 8a(Int-8) and 8a(Int-9).
N-cyclohexyl-2-[3-methoxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (8a(Int-7)). A stirred mixture of 3-methoxy-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide of formula 7a(Int-4) (0.19 g, 0.48 mmol), cyclohexylamine (0.07 mL, 0.57 mmol), TBTU (0.20 g, 0.62 mmol), DIPEA (0.25 mL, 1.91 mmol) in dry CH2Cl2 (10 mL) was kept at room temperature for 2 h. The organic solvent was evaporated, and the residue was poured into ice/water. The obtained precipitate was filtered to give the title compound as a white solid in 58% yield: m.p. 184-185° C. 1H NMR (400 MHz, CDCl3): δ 1.14-1.22 (m, 4H, CH2×2), 1.32-1.41 (m, 2H, CH2), 1.63-1.66 (m, 2H, CH2), 1.86-1.89 (m, 2H, CH2), 3.87-3.89 (m, 1H, CH), 3.98 (s, 3H, OCH3), 4.55 (s, 2H, NCH2), 6.58 (d, J=7.3 Hz, 1H, NH), 7.34-7.38 (m, 2H, H-1 and H-2), 7.52 (d, J=2.4 Hz, 1H, H-4), 7.70 (d, J=8.6 Hz, 1H, H-8), 7.97 (d, J=8.8 Hz, 1H, H-7), 8.24 (s, 1H, H-10).
2-[3-Methoxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]-N-(tetrahydro-2H-pyran-4-yl)acetamide (8a(Int-8)). A stirred mixture of 3-methoxy-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide of formula 7a(Int-4) (0.45 g, 0.96 mmol), 4-aminotetrahydropyran (0.12 mL, 1.15 mmol), TBTU (0.40 g, 1.25 mmol), DIPEA (0.67 mL, 3.84 mmol) in dry CH2Cl2 (10 mL) was kept at room temperature for 2 h. The organic solvent was evaporated, and the residue was poured into ice/water. The obtained precipitate was filtered to give the title compound as a white solid in 55% yield: m.p. 118-120° C. 1H NMR (400 MHz, CDCl3): δ 1.07-1.24 (m, 2H, CH2), 1.29-1.40 (m, 1H, CH2×1/2), 1.59-1.65 (m, 1H, CH2×1/2), 3.23-3.42 (m, 3H, CH2×1/2 and CH2), 3.60-3.69 (m, 1H, CH), 3.75-3.80 (m, 1H, CH2×1/2), 3.93 (s, 3H, OCH3), 4.63 (s, 2H, NCH2), 7.38-7.40 (m, 2H, Ar—H and CONH), 7.56-7.63 (m, 1H, Ar—H), 7.78-7.84 (m, 1H, Ar—H), 8.21-8.25 (m, 1H, Ar—H), 8.35-8.33 (m, 1H, Ar—H), 8.50 (s, 1H, Ar—H).
N-(1-ethylpropyl)-2-[3-methoxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (8a(Int-9)). A stirred mixture of 3-methoxy-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazine 5,5-dioxide of formula 7a(Int-4) (0.45 g, 0.96 mmol), 3-aminopentane (0.13 mL, 1.15 mmol), TBTU (0.40 g, 1.25 mmol), DIPEA (0.67 mL, 3.84 mmol) in dry CH2Cl2 (10 mL) was kept at room temperature for 2 h. The organic solvent was evaporated, and the residue was poured into ice/water. The obtained precipitate was filtered to give the title compound as a white solid in 55% yield: m.p. 151-153° C. 1H NMR (400 MHz, CDCl3): δ 0.73 (t, J=6.7 Hz, 6H, CH3×2), 1.24-1.29 (m, 2H, CH2), 1.38-1.44 (m, 2H, CH2), 3.39-3.43 (m, 1H, CH), 3.93 (s, 3H, OCH3), 4.65 (s, 2H, NCH2), 7.39-7.42 (m, 2H, Ar—H and CONH), 7.63 (d, J=8.1 Hz, 1H, Ar—H), 7.83 (d, J=8.2 Hz, 1H, Ar—H), 7.89 (d, J=8.2 Hz, 1H, Ar—H), 8.35 (d, J=8.3 Hz, 1H, Ar—H), 8.50 (s, 1H, Ar—H).
N-cyclohexyl-2-[3-hydroxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (SM890). To a solution of N-cyclohexyl-2-[3-methoxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide 8a(Int-7) (0.13 g, 0.28 mmol) in dry CH2Cl2 (8 mL), 1M BBr3 in dry CH2Cl2 (0.84 mL, 0.84 mmol) was added dropwise at 0° C. and then, the reaction mixture was kept at 10° C. for 2 h.
The mixture was poured into ice/water and extracted in EtOAc (×3). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness to give a brown solid. After purification by flash column chromatography, eluting with CHCl3/MeOH (99:1), the title compound was obtained as a little brown solid in 24% yield: m.p. 236-240° C. 1H NMR (400 MHz, DMSO-d6): δ 1.13-1.20 (m, 6H, CH2×3), 1.51-1.56 (m, 1H, CH), 1.65-1.67 (m, 4H, CH2×2), 4.60 (s, 2H, NCH2), 7.22-7.25 (m, 2H, Ar—H), 7.57 (d, J=8.3 Hz, 1H, Ar—H), 7.80 (d, J=7.9 Hz, 1H, NH), 8.06-8.09 (m, 1H, Ar—H), 8.22 (d, J=8.3 Hz, 1H, Ar—H), 8.43 (s, 1H, Ar—H), 10.78 (bs, 1H, OH). 13C NMR (101 MHz, DMSO-d6): δ 24.7, 25.5, 32.6, 48.1, 49.8, 107.1, 120.7, 121.6, 121.7, 122.1 (2C), 124.5 (q, JC-F=272.9 Hz), 125.4 (q, JC-F=32.8 Hz), 125.6, 129.2, 136.2, 140.8, 158.9, 165.5. HRMS (ESI) m/z [M+Na]+ calcd. for C21H21F3N204S: 477.1071, found: 477.10749.
2-[3-Hydroxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]-N-(tetrahydro-2H-pyran-4-yl)acetamide (SM891). To a solution of 2-[3-methoxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]-N-(tetrahydro-2H-pyran-4-yl)acetamide 8a(Int-8) (0.25 g, 0.52 mmol) in dry CH2Cl2 (6 mL), 1M BBr3 in dry CH2Cl2 (2.34 mL, 2.34 mmol) was added dropwise at 0° C. and then, the reaction mixture was kept at 10° C. for 24 h. The mixture was poured into ice/water and extracted in EtOAc (×3). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness to give a brown solid. After purification by flash column chromatography, eluting with CHCl3/MeOH (98:2), the title compound was obtained as a little brown solid in 6% yield: m.p. 226-228° C. 1H NMR (400 MHz, DMSO-d6): δ 1.30-1.39 (m, 2H, CH2), 1.62-1.65 (m, 2H, CH2), 3.26-3.32 (m, 2H, CH2O), 3.63-3.67 (m, 1H, CH), 3.77-3.80 (m, 2H, CH2O), 4.61 (s, 2H, NCH2), 7.21-7.24 (m, 2H, Ar—H), 7.58 (d, J=8.3 Hz, 1H, Ar—H), 7.81 (d, J=8.6 Hz, 1H, Ar—H), 8.21-8.24 (m, 2H, Ar—H and NH), 8.44 (s, 1H, Ar—H), 10.70 (bs, 1H, OH).
N-(1-ethylpropyl)-2-[3-hydroxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide (SM892). To a solution of N-(1-ethylpropyl)-2-[3-methoxy-5,5-dioxido-9-(trifluoromethyl)-6H-dibenzo[c,e][1,2]thiazin-6-yl]acetamide 8a(Int-9) (0.23 g, 0.72 mmol) in dry CH2Cl2 (6 mL), 1M BBr3 in dry CH2Cl2 (2.16 mL, 2.16 mmol) was added dropwise at 0° C. and then, the reaction mixture was kept at 10° C. for 2 h. The mixture was poured into ice/water and extracted in EtOAc (×3). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness to give a white solid. After purification by flash column chromatography, eluting with CHCl3/MeOH (98:2), the title compound was obtained as a white solid in 35% yield: m.p. 216-218° C. 1H NMR (400 MHz, CDCl3): δ 0.74 (t, J=7.2 Hz, 6H, CH3×2), 1.20-1.28 (m, 2H, CH2), 1.36-1.43 (m, 2H, CH2), 3.37-3.43 (m, 1H, CH), 4.62 (s, 2H, NCH2), 7.19-7.24 (m, 2H, Ar—H and CONH), 7.59 (d, J=8.4 Hz, 1H, Ar—H), 7.79 (d, J=7.5 Hz, 1H, Ar—H), 7.87 (d, J=8.6 Hz, 1H, Ar—H), 8.22 (d, J=8.8 Hz, 1H, Ar—H), 8.43 (s, 1H, Ar—H), 10.69 (s, 1H, OH).
Cell lines used in this paper have been cultured in Dulbecco's Minimal Essential Medium (DMEM, Gibco, #11960-044), 10% heat-inactivated fetal bovine serum (A56-FBS), Penicillin/Streptomycin (Pen/Strep, Corning #20-002-C1), non-essential amino acids (NEAA, Gibco, #11140-035) and L-Glutamine (Gibco, #25030-024), unless specified differently. HEK293 cells were obtained from ATCC (ATCC CRL-1573). We used a subclone (A23) of HEK293 stably expressing a mouse WT, ΔCR, or EGFP-tagged PrP. Cells were passaged in T25 flasks or 100 mm Petri dishes in media containing 200 μg/ml of Hygromycin and split every 3-4 days. Cells have not been passaged more than 20 times from the original stock. Compounds used in the experiments were resuspended at 30 or 50 mM in DMSO, and diluted to make a 1000× stock solution, which was then used for serial dilutions. A 1 μl aliquot of each compound dilution point was then added to cells plated in 1 mL of media with no selection antibiotics. Cloning strategies used to generate cDNAs encoding for WT, ΔCR or EGFP-tagged PrP have been described previously20,31,32. The EGFP-PrP construct contains a monomerized version of EGFP inserted after codon 34 of mouse PrP. The identity of all constructs was confirmed by sequencing the entire coding region. All constructs were cloned into the pcDNA3.1(+)/hygro expression plasmid (Invitrogen). All plasmids were transfected using Lipofectamine 2000 (Life Technologies), following manufacturer's instructions.
The DBCA was performed as described previously24, with minor modifications. Briefly, HEK293 cells expressing ΔCR PrP were cultured at ˜60% confluence in 24-well plates on day 1. On day 2, cells were treated with 500 μg/mL of Zeocinfor. Medium (containing fresh Zeocin and/or compound or vehicle) was replaced every 24 hr. On day 5, cell medium was removed and cells were incubated with 1 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Aldrich, St. Louis, MO) in PBS for 30 min at 37° C. MTT was carefully removed, and cells were re-suspended in 500 μL of DMSO. Values for each well were obtained by measuring at 570 nm, using a plate spectrophotometer (Biotek).
Field Excitatory Post-Synaptic Potential (EPSP) mouse hippocampal slices of 11 weeks old C57BL/6 mice was measured with a Multi Electrode Array (MEA) system. Slices were recorded for a 30 minutes baseline, LTP was then induced with a tetanic stimulation (3 trains, 500 MHz each) and recorded for additional 30 minutes. Prion synaptotoxicity was induced by incubating the slices for 5 minutes during the baseline with a 4% w/v lysate of MoRK13 cells chronically infected with M1000 prion strain. In order to evaluate the potential rescuing activity of SM884, the molecule was continuously perfused during the whole recording. The percentage of LTP was calculated considering the average EPSP amplitude of the last 10 minutes of recording, over the average EPSP amplitude of the last five minutes before the tetanic stimulation.
Cells expressing EGFP-PrP were plated on CellCarrier-384 Ultra microplates (Perkin Elmer) at a concentration of 12,000 cells/well and grown for approximately 24 h, to obtain a semi-confluent layer (60%). Vehicle (0.1% DMSO, volume equivalent) was used as a negative control. Cells were treated for 24 h and then fixed for 12 min at RT by adding methanol-free paraformaldehyde (Thermo Fisher Scientific) to a final concentration of 4%. Plates were then washed twice with PBS and counterstained with Hoechst 33342. The cell localization of EGFP-PrP was monitored using an Operetta High-Content Imaging System (Perkin Elmer). Imaging was performed in a widefield mode using a 20× High NA objective (0.75). Five fields were acquired in each well over two channels (380-445 Excitation-Emission for Hoechst and 475-525 for EGFP and Alexa 488). Image analysis was performed using the Harmony software version 4.1 (Perkin Elmer).
Samples were diluted 1:1 in 2× Laemli sample buffer (2% SDS, 10% glycerol, 100 mM Tris-HCl pH 6.8, 0.002% bromophenol blue, 100 mM DTT), heated at 95° C. for 10 min, then analyzed by SDS-PAGE. Proteins were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes, which were then blocked for 20 min in 5% (w/v) non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20. After incubation with appropriate primary and secondary antibodies, signals were revealed using enhanced chemiluminescence (Luminata, BioRad), and visualized by a Bio-Rad XRS Chemidoc image scanner (Bio-Rad).
Synthetic Aβ (1-42) peptide (Cat. Number KP2107, Karebay Biochem., Rochester, NY) was dissolved in hexafluoro-2-propanol, incubated for 10 min in a bath sonicator at maximum power, centrifuged at 15.000×g for 1 min, aliquoted, dried, and stored at −80° C. Before use, the dried film was dissolved using DMSO and diluted to 100 μM in F12 Medium (Invitrogen, Waltham, MA). Oligomers were obtained by incubating the peptide for 16 h at 25° C. This preparation routinely produces oligomers that elute near the void volume of a Superdex 75 10/300 size exclusion column (GE Healthcare, Little Chalfont, UK), and that react with oligomer-specific antibody A11. Final Aβ oligomer concentrations were considered as monomer equivalents, since the size of the oligomers is heterogeneous.
Primary neuronal cultures were derived from the hippocampi of 2-day-old postnatal mice, and cultured as described previously11. Neurons were plated on 35-mm dishes (500,000 cells/dish) pre-coated with 25 μg/mL poly-D-lysine (Sigma P6407) in B27/Neurobasal-A medium supplemented with 0.5 mM glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (all from Invitrogen). Experiments were performed 12 days after plating. Neurons were pre-treated for 20 min with each candidate compound or controls and then exposed for 20 mins or 3 hr to Aβ oligomers (3 μM). Triton-insoluble fractions (TIF) were analyzed by immunoblot with antibodies against phospho-SFK (Tyr 416) or Fyn. The phospho-SFK antibody detects pY416 in several SFKs, but previous studies showed that PrPC-dependent activation of kinases is specific for Fyn. Actin was used as loading control. Subcellular fractionation was performed as reported previously, with minor modifications. Neurons were homogenized using a Potter-Elvehjem homogenizer in 0.32 M ice-cold sucrose buffer (pH 7.4) containing 1 mM HEPES, 1 mM MgCl2, 10 mM NAF, 1 mM NaHCO3, and 0.1 mM PMSF in the presence of protease inhibitors (Complete mini, Roche Applied Science, Penzberg, Germany) and phosphatase inhibitors (PhosSTOP, Roche Applied Science). Samples were centrifuged at 13.000×g for 15 min to obtain a crude membrane fraction. The pellet was re-suspended in buffer containing 150 mM KCl and 0.5% Triton X-100 and centrifuged at 100,000×g for 1 hr. The final pellet, referred to as the Triton-insoluble fraction, was re-homogenized in 20 mM HEPES supplemented with protease and phosphatase inhibitors and then stored at −80° C. or directly used in further experiments. Protein concentration in each sample was quantified using the Bradford assay (Bio-Rad), and proteins (5 μg) were then analyzed by Western blotting. Primary antibodies were as follow: anti-GluN2A and anti-GluN2B (both 1:2000; Invitrogen), anti-GluA1 and anti-GluA2 (both 1:1000; Millipore, Billerica, MA), anti-PSD-95 (post-synaptic density protein 95; 1:2000; Cayman Chemical, Ann Arbor, MI), and anti-actin (1:5000; Millipore). Western blots were analyzed by densitometry using Quantity One software (Bio-Rad). All experiments were repeated on at least 4 independent culture preparations (n≥4).
RecHuPrP23-231 was expressed by competent E. coli Rosetta (DE3) bacteria harboring pOPIN E expression vector containing a wild type human Prnp construct (N-KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHGGGWG QPHGGGWGQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGL GGYMLGSAMSRPIIHFGSDYEDRYYRENMIHRYPNQVYYRPMDEYSNQNNFVHDC VNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQRGSS-C; SEQ ID No. 1). Bacteria from a glycerolate maintained at −80° C. were grown in a 250 ml Erlenmeyer flask containing 50 ml of LB broth overnight. The culture was then transferred to two 2 L Erlenmeyer flasks containing each 500 ml of minimal medium supplemented with 3 g/L glucose, 1 g/L NH4Cl, 1M MgSO4, 0.1 M CaCl2), 10 mg/mL thiamine and 10 mg/mL biotin. When the culture reached an OD600 of 0.9-1.2 AU, Isopropyl 3-D-1-thiogalactopyranoside (IPTG) was added to induce expression of PrP overnight under the same temperature and agitation conditions. Bacteria were then pelleted, lysed, inclusion bodies collected by centrifugation, and solubilized in 20 mM Tris-HCl, 0.5M NaCl, 6M Gnd/HCl, pH=8. Although the protein does not contain a His-tag, purification of the protein was performed with a histidine affinity column (HisTrap FF crude 5 ml, GE Healthcare Amersham) taking advantage of the natural His present in the octapeptide repeat region of PrP. After elution with buffer containing 20 mM Tris-HCl, 0.5M NaCl, 500 mM imidazole and 2 M guanidine-HCl, pH=8, the quality and purity of protein batches was assessed by BlueSafe (NZYTech, Lisbon) staining after electrophoresis in SDS-PAGE gels. The protein was folded to the PrPC conformation by dialysis against 20 mM sodium acetate buffer, pH=5. Aggregated material was removed by centrifugation. Correct folding was confirmed by CD and protein concentration, by measurement of absorbance at 280 nm. The protein was concentrated using Amicon centrifugal devices and the concentrated solution stored at −80° C. until used.
The EnSight Multimode Plate Reader (Perkin Elmer, Waltham, MA) was used to carry out DMR analyses. Immobilization of full-length (residues 23-230), human recombinant PrPC (15 μL/well of a 2.5 μM PrPC solution in 10 mM sodium acetate buffer, pH 5) on label-free microplates (EnSpire-LFB high sensitivity microplates, Perkin Elmer) was obtained by amine-coupling chemistry. The interaction between each molecule, diluted to different concentrations in assay buffer (10 mM P04, pH 7.5, 2.4 mM KCl, 138 mM NaCl, 0.05% Tween-20) and PrPC, was monitored after a 30 min incubation at room temperature. All the steps were executed by employing a Zephyr Compact Liquid Handling Workstation (Perkin Elmer). The Kaleido software (Perkin Elmer) was used to acquire and process the data.
All the data were collected and analyzed blindly by two different operators. Statistical analyses, performed with the Prism software version 7.0 (GraphPad), included all the data points obtained, with the exception of experiments in which negative and/or positive controls did not give the expected outcome, which were discarded. No test for outliers was employed. The Kolmogorov-Smirnov normality test was applied (when possible, n≥5). Results were expressed as the mean±standard errors, unless specified. In some case, the dose-response experiments were fitted with a 4-parameter logistic (4PL) non-linear regression model, and fitting was estimated by calculating the R2. All the data were analyzed with the one-way ANOVA test, including an assessment of the normality of data, and corrected by the Dunnet post-hoc test. Probability (p) values <0.05 were considered as significant (*<0.05, **<0.01, ***<0.001).
Bone marrow cells were isolated from C57BL/6 mice as previously describe (DOI: 10.1073/pnas.1619863114). BM was harvested from femur, tibia and pelvis using mortar and pestle in 1×PBS supplemented with 0.5% BSA and 2 mM EDTA (MACS buffer), passed through a 70 μm cell strainer and centrifuged at 1400 r.p.m for 5 minutes. Red blood cells were lysed with ACK lysis buffer (Ammonium Chloride 0.15 M, Potassium Carbonate 10 mM) and debris were removed by a gradient centrifugation using Histopaquel 119 (#11191, Sigma-Aldrich) prior to culture. Cells were resuspend at 2×106 cells/ml in Iscove's Modified Dulbecco's Media (IMDM, #12440053, Thermo Fisher) supplemented with 0.1 Non-essential Amminoacids (#11140-035 Thermo Fisher), 1 mM Sodium Pyruvate (#11360-070, Thermo Fisher), 5 mM glutamine (#25030-024, Thermo Fisher), 50 μM 2-Mercaptoethanol (#31350-010, Thermo Fisher), 100 U/ml penicillin, 100 g/ml streptomycin (#15140-122, Thermo Fisher) and 10% FBS (#10270-106, Thermo Fisher) (complete IMDM) containing 5% murine Flt3-L and were seed 5 ml/well in 6-plate tissue culture plates at 37° C. for 8-10 days. For all culture experiments, loosely adherent and suspension cells were harvested by gentle pipetting at the indicated time point.
cDC1 and cDC2 were sorted into complete IMDM were sorted by FACSAria Fusion as pDC B220+Bst2+, cDC1 B220-CD11c+MHC-II+CD24+CD172α−, cDC2 as B220-CD11c+MHCII+CD24-CD172α+. Sort purity of >95% was confirmed by post-sort analysis before cells were used for further experiments.
All mice used were 12 weeks animals on the C57BL/6 background. EAE was induced with 200 μg of myelin oligodendrocyte glycoprotein fragment MEVGWYRSPFSRVVHLYRNGK (SEQ ID No. 2; MOG35-55 peptide; #crb1000205n Cambridge Research Biochemicals) mixed with incomplete Freund's Adjuvant (#263910, BD) containing 4 mg/ml Mycobacterium tuberculosis TB H37 Ra (#231141 BD), at a ratio of 1:1 (v/v). Mice received 2 subcutaneous injections of 100 μl each of the MOG/CFA mix. Mice then received a single intraperitoneal injection of pertussis toxin (#180, List Biological Laboratories) at a concentration of 1 ng/μL in 200 μL of PBS. Mice received a second injection of pertussis toxin at the same concentration two days after the initial EAE induction. Mice were orally treated with different doses of SM231 dissolved in 1×PBS on alternating days starting at day 10 post-EAE induction. Mice were monitored and scored daily thereafter. EAE clinical scores were defined as follows: 0—no signs, 1—fully limp tail, 2—hindlimb weakness, 3—hindlimb paralysis, 4—forelimb paralysis, 5—moribund, as described previously (Mayo et al., 2014; Rothhammer et al., 2016). Sex differences were not analyzed but only a single sex was used within any set of EAE experiments. Mice were randomly assigned to treatment groups.
Identification, characterization and optimization of SM3. Mutations in the central region of PrPC, including artificial deletions or disease-associated point mutations, induce a toxic ion channel activity that can be detected in transfected cells by patch-clamping techniques23,24. Cells expressing PrP mutants are also hypersensitive to several cationic drugs commonly used for selection of transfected cell lines, including aminoglycosides and phleomycin analogues20. The latter effect was used to establish a novel cellular assay for studying mutant PrPC-related toxicity, called the “drug-based cell assay”, or DBCA25. Importantly, co-expression of wild type (WT) PrPC suppresses both channel activity and citoxicity, likely indicating that mutant PrP forms aberrantly activate a signaling pathway normally regulated by PrPC. Thus, the DBCA represents a unique tool to identify compounds capable of modulating PrPC activity. We have developed an optimized and scaled-up format of the DBCA in 384-well plates, which was later employed to screen tens of thousands of small molecules21,22. Several compounds were found to suppress the toxicity of mutant PrP, with no detectable toxicity in WT cells. We focused efforts on one of these compounds (named SM3 [dibenzo [3,4][c,e]thiazine 5,5-dioxide], shown in
SM231 inhibits the synaptotoxic effects of Aβ oligomers. Recent studies identified a role for PrPC into the toxicity of various misfolded oligomers of diseases-associated proteins, such as the amyloid B, whose accumulation underline the cognitive decline occurring in Alzheimer's disease2,4. The interaction between PrPC and Aβ oligomers unleashes a rapid, toxic signaling pathway involving the metabotropic glutamate receptor 5 (mGluR5), activation of the tyrosine kinase Fyn, and phosphorylation of the NR2B subunit of NMDA receptor, ultimately producing dysregulation of receptor function, excitotoxicity and dendritic spine retraction12. In order to evaluate the effect of SM231 on Aβ-induced activation of Fyn, we exposed primary hippocampal neurons to different concentrations of Aβ oligomers for short times (10, 20 or 60 minutes). We confirmed that the oligomers induce a quick phosphorylation of the Fyn kinase (results at the 20 min time point are shown in
Chemical optimization of SM231 to more metabolically stable derivatives. Within the present invention it was carried-out a further chemical optimization cycle functionalizing positions predicted to positively improve the metabolic stability (
SM884 rescues the synaptotoxic effects of prions in mouse brain slices. To test whether SM884 is able to inhibit prion-induced toxicity in a disease-relevant context, we turned to a recently developed ex vivo toxicity model27,28. This assay is based on mouse brain slices acutely exposed to either brain homogenates of terminally ill mice infected with lysates of cell lines chronically infected with the mouse-adapted M1000 human prion strain. We found that SM884 administration at a concentration of 0.1-0.03 μM induces a significant (34% and 71%, respectively) rescue of long-term potentiation (LTP;
Mouse DC1 and DC2 subsets express PrPC, and DC2 treated with SM231 promotes Treg cells expansion in DC-T cell co-cultures. Bone marrow derived dendritic cells were analyzed for expression of PrPC after stimulation with two different concentrations of SM231 or Fe(III)-TMPyP or vehicle. For this analysis, PrPC expression in each DC subsets was determined by western blot using specific anti-PrPC antibody. The authors of the present invention found that DC1 and DC2 expressed a baseline level of PrPC that slightly increases upon SM231 treatment, especially in DC2 (
Compounds SM888 and SM889, like SM231, promote tolerogenic activity in cDC2. cDC2 cells have been reported to trans-present IL-6 indispensable for priming myelin peptide specific encephalitogenic pathogenic TH17 in a model of EAE. To assess whether additional derivates (i.e SM887, SM888, SM889) were able to induce regulatory functions in DCs subsets we performed in vitro co-cultures of cDC2 cells with naïve ovalbumin (OVA)-specific transgenic CD4+ T cells in the presence of different concentrations of OVA. T cell proliferation was analyzed. We found that priming of cDC2 was significantly affected by cDC treatment with SM derivatives and more significantly by SM888 and SM889. Specifically, these cells were able to suppress antigen-specific CD4+ T cell proliferation and this effect was more pronounced when the molecules were used at the concentration of 10 uM (
Administration SM231 ameliorates EAE and suppresses inflammatory cytokines in vivo. The authors of the invention investigated whether PrPC modulators could have a protective role in this experimental model. Groups of WT female C57BL/6 mice were immunized with the MOG35-55 peptide and injected intraperitoneally (i.p.) with Fe(III)-TMPyP or SM231 at two doses every other day from day 3 until day 24 after vaccination. Control mice received vehicle alone. EAE clinical scores were recorded daily over this timeframe (
SM231 does not act by directly targeting PrPC. In light of the promising ability of SM compounds to modulate the activity of PrPC in several experimental contexts, the hypothesis that these molecules act by directly targeting the protein was tested. First, it was hypothesized that the compound may promote the re-localization of PrPC from the cell surface, a mode of action recently observed for an anti-prion phenothiazine derivative (chlorpromazine, CPZ)29,30. HEK293 cells stably expressing an EGFP-tagged version of PrPC were treated with different concentration of SM231, CPZ or vehicle control, and PrPC localization at the cell surface was monitored by imaging techniques (
An FXR-inhibitor suppresses mutant PrP cytotoxicity. The two FXR agonists, WAY-362450 and Fexaramine, whose structure is reported below, were tested using the DBCA assay.
HEK293 cells expressing ΔCR PrP were cultured at ˜60% confluence in 24-well plates on day 1. On day 2, cells were treated with 500 μg/mL of Zeocin and/or individual FXR agonists at different concentrations (0.03-30 μM) for 72 hr. Medium (containing fresh Zeocin and/or FXR agonists) was replaced every 24 hr. On day 5, cell medium was removed and cells were incubated with 1 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS for 30 min at 37° C. to evaluate cell viability. Interestingly, the FXR agonist WAY-362450 rescued ΔCR PrP-dependent citotoxicity in a dose-dependent fashion, with an inhibitory concentration at 50% (IC50) value in the sub-micromolar range (
SM231 mediates FXR gene transcriptional activity in murine hepatocytes. Mouse primary hepatocytes were isolated from 6-8-week-old C57Bl6/J wild-type male mice (from Charles River). 3×106 prymary hepatocytes were stimulated with increasing concentrations of SM231 or WAY-362450, a potent and selective Farnesoid X receptor (FXR) agonist for 4 or 12 hours. The expression of FXR (nr1 h4) and the FXR target gene Nr0b2, was evaluated by RT-qPCR using specific primers.
In this experiment, similarly to the reference agonist WAY-362450, SM231 promoted significant FXR transcriptional activity in these cells, specifically three hours after treatment (
| Number | Date | Country | Kind |
|---|---|---|---|
| 102021000013244 | May 2021 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/063806 | 5/20/2022 | WO |
