Technical field

The present invention relates to produgs of vortioxetine and pharmaceutical compositions comprising said prodrugs which can be used in therapy.

Background

International patent applications including WO 03/029232 and WO 2007/144005 disclose the compound 1 -[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazine and pharmaceutically acceptable salts thereof. WHO has since published that vortioxetine is the recommended International Non-proprietary Name (INN) for 1 -[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]- piperazine. Vortioxetine was formerly referred to in the literature as Lu AA21004. In

September and December 2013 FDA and EMA, respectively, approved vortioxetine for the treatment of major depressive disorder/major depressive episode under the trade name Brintellix™. Of particular interest, vortioxetine has also shown effect in elderly suffering from recurrent major depressive disorder [Int. Clin. Psychopharm., 27, 215-227, 2012].

Vortioxetine is an antagonist on the 5-HT ₃ , 5-HT ₇ and 5-HT _{1 D} receptors, an agonist on the 5- HT _1A receptor and a partial agonist on the 5-HT _{1 B} receptor and an inhibitor of the serotonin transporter. Additionally, vortioxetine has demonstrated to enhance the levels of the neurotransmitters serotonin, noradrenalin, dopamine, acetylcholine and histamine in specific areas of the brain. All of these activities are considered to be of clinical relevance and potentially involved in the mechanism of action of the compound [J.Med.Chem., 54, 3206- 3221 , 201 1 ; Eur. Neuropshycopharmacol., 18(suppl 4), S321 , 2008; Eur.

Neuropshycopharmacol., 21 (suppl 4), S407-408, 201 1 ; Int. J. Psychiatry Clin Pract. 5, 47, 2012]. The pharmacological profile gives reason to believe that vortioxetine may have a pro- cognitive effect. This notion seems to be supported by clinical evidence where vortioxetine has been shown to have a direct beneficial effect on cognition independent of its

antidepressive effects [Int. Clin. Psychopharm., 27, 215-227, 2012; Int J neurophychopharm 17, 1557-1567, 2014; Neuropsychopharm 40, 2025-2037, 2015].

Vortioxetine is available on the market as film coated tablets containing 5, 10, 15 and

20 mg vortioxetine as the HBr salt and as an oral drop solution comprising 20 mg/ml vortioxetine as the DL lactate salt.

The present invention provides prodrugs of vortioxetine which may have improved properties compared to vortioxetine. The prodrugs of the present invention may e.g. improve uptake of vortioxetine, delay the release of vortioxetine, or lower the level of adverse events.

l Summary of the invention

In one embodiment, the present invention provides a prodrug of vortioxetine as shown in Example 1 - Example 10 or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention provides a prodrug as defined above or a pharmaceutically acceptable salt thereof for use in therapy.

In one embodiment, the invention provides a pharmaceutical composition comprising a prodrug of vortioxetine as defined above or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carrier or excipient.

In one embodiment, the present invention provides a vortioxetine prodrug as defined above or a pharmaceutically acceptable salt thereof for use in a method for the treatment of a CNS disease.

In one embodiment, the invention provides the use of a vortioxetine prodrug as defined above or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for the treatment of a CNS disease.

In one embodiment, the present invention provides a method for the treatment of a CNS disease, the method comprising the administration of a therapeutically effective amount of a vortioxetine prodrug as defined above or a pharmaceutically acceptable salt thereof to a patient in need thereof.

Detailed description of the invention

A prodrug in general is a compound which may not have any pharmacological activity itself, but which upon administration to a patient is metabolised to provide a pharmacologically active compound. More specifically, a vortioxetine prodrug of the present invention is a compound which upon administration to a patient is metabolised to provide vortioxetine.

Some of the vortioxetine prodrugs of the present invention may be provided as pharmaceutically acceptable acid addition salts. The term pharmaceutically acceptable salts includes salts formed with inorganic and/or organic acids such as hydrochloride acid, hydrobromide acid, phosphoric acid, nitrous acid, sulphuric acid, benzoic acid, citric acid, gluconic acid, lactic acid, maleic acid, succinic acid, tartaric acid, acetic acid, propionic acid, oxalic acid, maleic acid, fumaric acid, glutamic acid, pyroglutamic acid, salicylic acid, salicylic acid, saccharin and sulfonic acids, such as methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid and benzenesulfonic acid. Some of the acids listed above are di- or tri-acids, i.e. acids containing two or three acidic hydrogens, such as phosphoric acid, sulphuric acid, fumaric acid and maleic acid. Di- and tri-acids may form 1 :1 , 1 :2 or 1 :3 (tri- acids) salts, i.e. a salt formed between two or three molecules of the compound of the present invention and one molecule of the acid.

Additional examples of useful acids and bases to form pharmaceutically acceptable salts can be found e.g. in Stahl and Wermuth (Eds) "Handbook of Pharmaceutical salts. Properties, selection, and use", Wiley-VCH, 2008.

In the present context, the term "therapeutically effective amount" of a compound means an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and its complications in a therapeutic intervention comprising the

administration of said compound. An amount adequate to accomplish this is defined as "therapeutically effective amount". Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. A vortioxetine prodrug of the present invention is typically administered to achieve therapeutic effect comparable to the administration of 1 -60 mg vortioxetine, such as 1 -30 mg

vortioxetine, such as 5 mg, 10 mg, 15 mg or 20 mg calculated as the free base. This means e.g. that "20 mg vortioxetine" means 20 mg vortioxetine free base where the actual amount administered may have to be adjusted for the weight of a counter ion. The therapeutic effect achieved may e.g. be predicted based on the vortioxetine plasma concentrations achieved.

In the present context, "treatment" or "treating" is intended to indicate the

management and care of a patient for the purpose of alleviating, arresting, partly arresting or delaying progress of the clinical manifestation of the disease, or curing the disease. The patient to be treated is preferably a mammal, in particular a human being.

In the present context, "CNS disease" is intended to indicate a disease in the central nervous system.

Vortioxetine is approved by several health authorities for the treatment of major depression or major depressive episode. As disclosed in e.g. WO 03/029232 and WO 2007/144005 the pharmacological profile of vortioxetine is expected to also make the compound useful in the treatment of additional CNS diseases, such as general anxiety disorder; obsessive compulsive disorder (OCD), panic disorder; post-traumatic stress disorder; cognitive impairment; mild cognitive impairment (MCI); cognitive impairment associated with Alzheimer's disease, depression or schizophrenia (CIAS); and attention deficit hyperactivity disorder (ADHD).

Cognitive deficits, cognitive impairment or cognitive dysfunction include a decline in cognitive functions or cognitive domains, e.g. working memory, attention and vigilance, verbal learning and memory, visual learning and memory, reasoning and problem solving e.g. executive function, speed of processing and/or social cognition. In particular, cognitive deficits may indicate deficits in attention, disorganized thinking, slow thinking, difficulty in understanding, poor concentration, impairment of problem solving, poor memory, difficulties in expressing thoughts and/or difficulties in integrating thoughts, feelings and behaviour, or difficulties in extinction of irrelevant thoughts. The terms "cognitive deficits", "cognitive impairment" and "cognitive dysfunction" are intended to indicate the same and are used interchangeably.

The pharmaceutical compositions may be specifically formulated for administration by any suitable route such as the oral, rectal, nasal, buccal, sublingual, transdermal and parenteral (e.g. subcutaneous, intramuscular, and intravenous) route; the oral route being preferred.

It will be appreciated that the route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient.

In the present context, the term "excipient" or "pharmaceutically acceptable excipient" refers to pharmaceutical excipients including, but not limited to, fillers, antiadherents, binders, coatings, colours, disintegrants, flavours, glidants, lubricants, preservatives, sorbents, sweeteners, solvents, vehicles and adjuvants.

The present invention also provides a pharmaceutical composition comprising a vortioxetine prodrug of Example 1 - Example 10 or a pharmaceutically acceptable salt thereof. The pharmaceutical compositions according to the invention may be formulated with pharmaceutically acceptable excipients in accordance with conventional techniques such as those disclosed in Remington, The Science and Practice of Pharmacy, 22 ^th edition (2012), Edited by Allen, Loyd V., Jr.

Pharmaceutical compositions for oral administration include solid oral dosage forms such as tablets, capsules, powders and granules; and liquid oral dosage forms such as solutions, emulsions, suspensions and syrups as well as powders and granules to be dissolved or suspended in an appropriate liquid.

Solid oral dosage forms may be presented as discrete units (e.g. tablets or hard or soft capsules), each containing a predetermined amount of the active ingredient, and preferably one or more suitable excipients. Where appropriate, the solid dosage forms may be prepared with coatings such as enteric coatings or they may be formulated so as to provide modified release of the active ingredient such as delayed or extended release according to methods well known in the art. Where appropriate, the solid dosage form may be a dosage form disintegrating in the saliva, such as for example an orodispersible tablet.

Examples of excipients suitable for solid oral formulation include, but are not limited to, microcrystalline cellulose, corn starch, lactose, mannitol, povidone, croscarmellose sodium, sucrose, cyclodextrin, talcum, gelatin, pectin, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Similarly, the solid formulation may include excipients for delayed or extended release formulations known in the art, such as glyceryl monostearate or hypromellose.

If solid material is used for oral administration, the formulation may for example be prepared by mixing the active ingredient with solid excipients and subsequently compressing the mixture in a conventional tableting machine; or the formulation may for example be placed in a hard capsule e.g. in powder, pellet or mini tablet form. The amount of solid excipient will vary widely but will typically range from about 25 mg to about 1 g per dosage unit.

Liquid oral dosage forms may be presented as for example elixirs, syrups, oral drops or a liquid filled capsule. Liquid oral dosage forms may also be presented as powders for a solution or suspension in an aqueous or non-aqueous liquid. Examples of excipients suitable for liquid oral formulation include, but are not limited to, ethanol, propylene glycol, glycerol, polyethylenglycols, poloxamers, sorbitol, poly-sorbate, mono and di-glycerides,

cyclodextrins, coconut oil, palm oil, and water. Liquid oral dosage forms may for example be prepared by dissolving or suspending the active ingredient in an aqueous or non-aqueous liquid, or by incorporating the active ingredient into an oil-in-water or water-in-oil liquid emulsion.

Further excipients may be used in solid and liquid oral formulations, such as colourings, flavourings and preservatives etc.

Pharmaceutical compositions for parenteral administration include sterile aqueous and nonaqueous solutions, dispersions, suspensions or emulsions for injection or infusion, concentrates for injection or infusion as well as sterile powders to be reconstituted in sterile solutions or dispersions for injection or infusion prior to use. Examples of excipients suitable for parenteral formulation include, but are not limited to water, coconut oil, palm oil and solutions of cyclodextrins. Aqueous formulations should be suitably buffered if necessary and rendered isotonic with sufficient saline or glucose.

Other types of pharmaceutical compositions include suppositories, inhalants, creams, gels, dermal patches, implants and formulations for buccal or sublingual administration.

It is requisite that the excipients used for any pharmaceutical formulation comply with the intended route of administration and are compatible with the active ingredients.

Examples of vortioxetine prodrugs of the present invention, i.e. compounds as exemplified in Example 1 - Example 10 or a pharmaceutically acceptable salt thereof and their synthesis are described below. Reference to "Example X" is intended to include reference to the generic structure Example X and all specific examples provided i.e.

Example Xa, Example Xb, etc. Prodrugs of vortioxetine (1-(2-((2,4-dimethylphenyl)thio)phenyl)- piperazine)

Amides

Prodrugs of the general type Example 1 can undergo enzymatic hydrolysis as discussed in the literature [see for example 'Methods in Enzymology', Chapter 27 'Formation of Prodrugs of Amines, Amides, Ureides, and Imides' by H. Bundgaard in volume 1 12, 1985, pp. 347- 359 and H. Bundgaard 'Design of Prodrugs'; H. Bundgaard, Ed.; Elsevier: Amsterdam, 1985; pp. 1-92]. Amides of the general structure Example 1 can be prepared from vortioxetine and the appropriate acid [reagent 1] in the presence of a suitable peptide coupling agent

[reagent] or from vortioxetine and a pre-activated form of the appropriate acid [reagent 3]. The pre-activated acids [reagent 3] can be prepared from the parent acids [reagent 1].

These routes are exemplified in Scheme 1 .

Scheme 1 .

The peptide coupling reagent 2 can be chosen from the following list of commercially available reagents: BOP reagent ((benzotriazol-1 -yloxy)tris(dimethylamino)phosphonium hexafluorophosphate), DEPBT (3-(diethoxyphosphoryloxy)-1 ,2,3- benzotriazin-4(3H)-one), N,N'-dicyclohexylcarbodiimide, Ν,Ν'-diisopropylcarbodiimide, HATU (1 - [bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid

hexafluorophosphate), HBTU (3-[bis(dimethylamino)methyliumyl]-3H-benzotriazol-1 -oxide hexafluorophosphate), 1 -hydroxy-7-azabenzotriazole, hydroxybenzotriazole, PyAOP reagent ((7-azabenzotriazol-1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate), and PyBOP ((benzotriazol-1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate).

The activated acid reagent 3 can be chosen from acid chlorides, acid bromides, and acid anhydrides as well as from activated esters such as the ester derived from the appropriate acid and N-hydroxysuccinimide or pentafluorophenol. A common way to synthesize a N- hydroxysuccinimide acid is to mix N-hydroxysuccinimide with the desired carboxylic acid and a small amount of an organic base like triethyl amine, Ν,Ν-diisopropylethylamine (DIPEA), or pyridine in an solvent like dichloromethane, chloroform, N,N-dimethylformamide, tetrahydrofuran, or 2-methyltetrahydrofurane. A peptide coupling reagent 2 is then added to form a highly reactive activated acid intermediate. N-hydroxysuccinimide reacts to form a less labile activated acid. Using pentafluorophenol instead of N-hydroxysuccinimide affords an alternative version of reagent 3.

The base can be selected from both organic and inorganic bases such as these commercially available reagents: triethyl amine, Ν,Ν-diisopropylethylamine (DIPEA), potassium or cesium carbonate, potassium or cesium fluoride, and sodium or potassium or cesium hydroxide.

The solvent can be selected from dichloromethane, chloroform, N,N- dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofurane, and water as well as alcoholic solvents like methanol and ethanol. It is also possible to use monophasic or diphasic mixtures of these solvents for the reaction.

In cases such as Example 1 g where the acid [reagent 2] contains sensitive functional groups such as primary and secondary amines these should be protected with for example benzyl or para-methoxybenzyl groups or as carbamates by introduction of a benzyl-oxo- carbonyl or a te/t-butyl-oxy-carbonyl group. Methods for the introduction and removal of such protective groups are described in T.W. Greene and P.G.M. Wuts 'Protective Groups in Organic Synthesis', 3 ^rd Edition, John Wiley & Sons Inc.: New York, 1999.

In cases where the acid (or activated acid) contains two carboxyl groups (or activated carboxyl groups) the reaction can afford also bis-functionalized prodrugs that contain two molecules of vortioxetine per prodrug. In such cases the compounds can be prepared in one or two steps using unprotected acid precursors or appropriately mono-protected diacids, such as for example 5-methoxy-5-oxopentanoic acid to prepare Example 1 m (the coupling step is followed by a hydrolysis of the methyl ester under standard conditions as described by T.W. Greene and P.G.M. Wuts 'Protective Groups in Organic Synthesis', 3 ^rd Edition, John Wiley & Sons Inc.: New York, 1999). Example 1 m that can be further elaborated to Example 1 n by treatment with a peptide coupling agent as described above.

Example 1 a R ₁ - H

Example 1 b = CH ₃ Example i Ri ^_ 4-tetrahydropyranyl Example 1 c R-, = CH ₂ CH ₃ Example j -i = CH[CH ₃ ][CH ₂ ]4 ^{C H} 3 Example 1 d R ₁ = iso-propyl Example k R-i = CH[CH3]CH ₂ C[CH ₃ ]3 Example 1 e R ₁ = cyclopropyl Example I Ri = C[CH ₃ ][CH ₂ ] ₅ Example 1f F¾i = [CH ₂ ] ₃ OH Example m R ₁ = [CH ₂ ] ₃ COOH Example 1 g R-i = Example ° Ri ⁼ phenyl

Example 1 h

Example 1 a 4-(2-((2,4-dimethyl-phenyl)thio)phenyl)piperazine-1 -carbaldehyde

Example 1 b 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)ethan-1 -one

Example 1 c 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)propan-1 -one

Example 1 d 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)-2-methylpropan-1 -one Example 1e cyclopropyl(4-(2-((2,4-dimethylphenyl)thio)-phenyl)piperazin -1 -yl)methanone Example 1f 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)-4-hydroxybutan-1 -one Example 1 g (S)-2-amino-1 -(4-(2-((2,4-dimethylphenyl)thio)-phenyl)piperazin-1 -yl)-3-(1 H- indol-3-yl)propan-1 -one

Example 1 h N-(4-(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)-4-oxobutyl)- acetamide

Example 1 i (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)(tetrahydro-2H-pyran-4- yl)methanone

Example 1j racemic 1 -(4-(2-((2,4-di-methylphenyl)thio)phenyl)-piperazin-1 -yl)-2- methylheptan-1 -one and -(4-(2-((2,4-di-methylphenyl)thio)phenyl)-piperazin-1 -yl)-2- methylheptan-1 -one and (S)-1 -(4-(2-((2,4-di-methylphenyl)thio)phenyl)-piperazin-1 -yl)-2- methylheptan-1 -one

Example 1 k racemic 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)-2,4,4- trimethylpentan-1 -one and (R)- 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)- 2,4,4-trimethylpentan-1 -one and (S)- 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 - yl)-2,4,4-trimethylpentan-1 -one Example 11 (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)(1 -methylcyclohexyl)- methanone

Example 1 m 5-(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)-5-oxopentanoic acid Example 1 n 1 ,5-bis(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)pentane-1 ,5-dione Example 1 o (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)(phenyl)-methanone Example 1 p (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)(pyridin-2-yl)-methanone Example 1 q 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)-2-phenylethan-1 -one Example 1 r 2-(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)-2-oxoethyl methyl fumarate

Hydroxymethyl-esters

Prodrugs of amines of the type Example 2 have been reported in the patent literature [see for example WO 2004/089925 and WO 2013/035892].

Esters of the general structure Example 2 can be prepared from vortioxetine and an appropriately substituted alkylating agent [reagent 4] wherein X is a suitable leaving group such as F, CI, Br, I or tosylate or mesylate. This is shown in Scheme 2.

Scheme 2.

The reaction to form Example 2 is typically performed in acetonitrile, tetrahydrofuran, 2- methyl-tetrahydrofuran, actetone, or dimethyl formamide. In cases where X is not I, the reaction can sometimes be promoted by the addition of sodium or potassium iodide or tetra n-butyl ammonium iodide. The liberated acid HX can be quenched by an added base such as potassium or cesium carbonate, sodium hydride (can also be used to deprotonate vortioxetine prior to addition of reagent 4), or organic bases such as triethyl amine or N,N- diisopropylethylamine (DIPEA).

In cases such as Examples 2o, 2u, and 2w where reagent 4 contains sensitive functional groups such as primary and/or secondary amines these should be protected with for example benzyl or para-methoxybenzyl groups or carbamates such as benzyl-oxo- carbonyl or te/t-butyl-oxy-carbonyl. Methods for the introduction and removal of such protective groups are described in T.W. Greene and P.G.M. Wuts 'Protective Groups in Organic Synthesis', 3 ^rd Edition, John Wiley & Sons Inc.: New York, 1999. The synthesis of Example 2d from 2c can be achieved by treatment with acid; further treatment of Example 2d with thionyl chloride and 2-propanol will afford Example 2e.

Example 2n can be treated with acid to afford Example 2o that can be treated with acetyl chloride or acetic acid anhydride to afford Example 2p under the conditions described for the synthesis of Example 1 . For a synthesis of a prodrug based on the ester moiety in Example 2w, see A.G. Sams et al., J. Med. Chem. 201 1 , 54, p. 751.

Example 2 wherein R ₄ is an unfunctionalized or a monofunctionalized alkyl group.

Example 2a R ₄ = CH ₃ Example 2h R ₄ = [CH ₂ ]io ^CH ₃

Example 2b R ₄ = C[CH ₃ ] ₃ Example 2i R ₄ = [CH ₂ ] ₆ ^CH ₃

Example 2c R ₄ = CH ₂ CH ₂ C0 ₂ c[CH ₃ ] ₃ Example 2j R ₄ = [CH ₂ ] ₄ ^CH ₃ Example 2d R ₄ = CH ₂ CH ₂ C0 ₂ H Example 2k R ₄ = C[CH ₃ ] ₂ ^OCH 2 ^CH ₃

Example 2e R ₄ = CH ₂ CH ₂ C0 ₂ CH[CH ₃ ] ₂ Example 2I R ₄ = [CH ₂ ] ₂ CN Example 2f R ₄ = cyclopropyl Example 2m R ₄ = [CH ₂ ] ₂ C0 ₂ CH ₃

Example 2g R ₄ = [CH ₂ ] ₈ CH ₃ Example 2r R ₄ = phenyl Example 2t R ₄ = CH ₂ NHCOCH ₃

Example 2a (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl acetate

Example 2b (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl pivalate

Example 2c te/t-butyl ((4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl) succinate

Example 2d 4-((4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methoxy)-4- oxobutanoic acid

Example 2e (4-(2-((2,4-dimethyl-phenyl)thio)phenyl)piperazin-1 -yl)methyl isopropyl succinate Example 2f (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl

cyclopropanecarboxylate

Example 2g (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl decanoate

Example 2h (4-(2-((2,4-dimethyl-phenyl)thio)phenyl)-piperazin-1 -yl)methyl dodecanoate Example 2i (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl octanoate

Example 2j (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl hexanoate

Example 2k (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl 2-ethoxy-2- methylpropanoate

Example 2I (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl 3-cyanopropanoate Example 2m (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl methyl succinate Example 2r (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl benzoate

Example 2t (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl acetyl-glycinate

Example 2n 1 -(ie/ -butyl) 4-((4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl) piperidine-1 ,4-dicarboxylate

Example 2o (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl piperidine-4- carboxylate

Example 2p 4-((4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl) 1 -methyl piperidine-1 ,4-dicarboxylate

Example 2s1 (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl thiophene-2- carboxylate

Example 2s2 (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl furan-2- carboxylate

Example 2s3 (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl 1 -methyl-1 H- pyrrole-2-carboxylate

Example 2u (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl acetyl-L- tryptophanate

Example 2v (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl 2,5,8,1 1 - tetraoxatetradecan- 14-oate

Example 2w (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl L-isoleucinate

Example 2a 1 R ₂ - CH ₃ , R ₃ - H

3, R ₃ = 2OCH ₃ , , R ₃ = H

Example 2a6 R ₂ = R ₃ = CH ₃

Example 2a1 racemic 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)ethyl acetate [(R)-1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)ethyl acetate and (fl)-1 -(4-(2- ((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)ethyl acetate]

Example 2a2 racemic 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)propyl acetate [(R)-1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)propyl acetate and (S)-1 -(4-(2- ((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)propyl acetate]

Example 2a3 racemic 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)ethane-1 ,2- diyl diacetate [(R)-1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)ethane-1 ,2-diyl diacetate and (S)-1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)ethane-1 ,2-diyl diacetate]

Example 2a4 racemic 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)-2- methoxyethyl acetate

[(R)-1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)-2-methoxyethyl acetate and (S)-1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)-2-methoxyethyl acetate] Example 2a5 racemic methyl 2-acetoxy-2-(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazi n- 1 -yl)acetate [(R)-methyl 2-acetoxy-2-(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazi n-1 - yl)acetate and (S)-methyl 2-acetoxy-2-(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazi n-1 - yl) acetate]

Example 2a6 2-(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)propan-2-yl acetate

In cases where reagent 4 contains two reactive handles [X-CR ₂ R ₃ -0-CO-] seprated by a linker L the reaction can afford bis-functionalized prodrugs that contain two molecules of vortioxetine per prodrug such as in Examples 2q1 - 2q5.

Example 2q1 L - Example 2q4 L- ·· ^**

Example 2q2 L- y. Example 2q5 L -

Example 2q3 L -

Example 2q1 bis((4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl) succinate Example 2q2 bis((4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl) 2,2- dimethylmalonate

Example 2q3 bis((4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl) octanedioate Example 2q4 bis((4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl)

decanedioate

Example 2q5 bis((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl) terephthalate

Acid-sensitive prodrugs I

This class of prodrugs may undergo acid-mediated cleavage, for example in the stomach. These compounds have the generic structure Example 3 and can be prepared from vortioxetine and an appropriately substituted alkylating agent [reagent 5] wherein X is a suitable leaving group such as F, CI, Br, I or tosylate or mesylate. This is illustrated in Scheme 3.

Scheme 3.

The reaction to form Example 3 is typically performed in acetonitrile, tetrahydrofuran, 2- methyl-tetrahydrofuran, acetone, or dimethyl formamide. In cases where X is not I, the reaction can sometimes be promoted by the addition of sodium or potassium iodide or tetra n-butyl ammonium iodide. The liberated acid HX can be quenched by an added base such as potassium or cesium carbonate, sodium hydride (can also be used to deprotonate vortioxetine prior to addition of reagent 5), or organic bases such as triethyl amine or N,N- diisopropylethylamine (DIPEA).

Example 31 R ₇

Example 3a 1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(methoxymethyl)-pipe razine

Example 3b 1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(ethoxymethyl)-piper azine

Example 3c 1 -(ie -butoxymethyl)-4-(2-((2,4-dimethylphenyl)thio)phenyl)-pipera zine

Example 3d 1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-((2-methoxyethoxy)me thyl)-piperazine Example 3e 1 -((allyloxy)methyl)-4-(2-((2,4-dimethylphenyl)thio)phenyl)-p iperazine

Example 3j racemic 1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(((tetrahydrofuran-2 - yl)oxy)methyl)piperazine [(R)-1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(((tetrahydrofuran-2 - yl)oxy)methyl)piperazine and (S)-1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(((tetrahydrofuran- 2-yl)oxy)methyl)piperazine]

Example 3k racemic 1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(((tetrahydro-2H-pyr an-2- yl)oxy)methyl)-piperazine [(R)-1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(((tetrahydro-2H- pyran-2-yl)oxy)methyl)-piperazine and (S)-1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4- (((tetrahydro-2H-pyran-2-yl)oxy)methyl)-piperazine]

Example 3I 1 -((benzyloxy)methyl)-4-(2-((2,4-dimethylphenyl)thio)phenyl)- piperazine

Example 3m 1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(((4-methoxybenzyl)o xy)methyl)- piperazine Example 3o 1 -(2-((2,4-dimethylphenyl)thio)phenyl) -4-((((1 R,2S,5S)-2-isopropyl-5- methylcyclohexyl)oxy)methyl)-piperazine

Example 3p 1 -(2-((2,4-dimethylphenyl)thio)phenyl) -4-((((1 S,2R,5R)-2-isopropyl-5- methylcyclohexyl)oxy)methyl)-piperazine

Example 3f R ₆ - H, R ₇ - CH ₃

Example 3g = H, R ₇ = CH ₂ CH ₃

Example 3h R ₆ = CH ₃ , R ₇ = OC[0]CH ₃

Example 3i R ₆ = H, R ₇ = vinyl

Example 3f racemic 1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(1 -methoxyethyl)piperazine [(R)-1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(1 -methoxyethyl)-piperazine and (S)-1 -(2-((2,4- dimethylphenyl)thio)phenyl)-4-(1 -methoxyethyl)-piperazine]

Example 3g racemic 1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(1 -ethoxyethyl)piperazine [(R)- 1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(1 -ethoxyethyl)piperazine and (S)-1 -(2-((2,4- dimethylphenyl)thio)phenyl)-4-(1 -ethoxyethyl)piperazine]

Example 3h 2-(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)propan-2-yl acetate Example 3i racemic 1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(1 -(vinyloxy)ethyl)piperazine [(R)-1 -(2-((2,4-dimethylphenyl)thio)phenyl)-4-(1 -(vinyloxy)ethyl)piperazine and (S)-1 -(2-((2,4- dimethylphenyl)thio)phenyl)-4-(1 -(vinyloxy)ethyl)piperazine]

Acid-sensitive prodrugs II

This class of prodrugs may undergo acid-mediated cleavage, for example in the stomach. These compounds have the generic structure Example 4 and can be prepared from vortioxetine and reagent 6 [di-te/t-butyl carbonate 6a, vinyl carbonochloridate 6b, or S- phenyl O-vinyl carbonothioate 6c depending on the nature of the unit/atom Z]. These reactions can be performed in organic solvents like dichloromethane, chloroform, N,N- dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofurane, and water as well as alcoholic solvents like methanol and ethanol. It is also possible to use monophasic or diphasic mixtures of these solvents for the reaction. A organic or inorganic base such as pyridine, triethyl amine, Ν,Ν-diisopropylethylamine (DIPEA), potassium or cesium carbonate, potassium or cesium fluoride, and sodium or potassium or cesium hydroxide, or magnesium oxide can be added to reaction. This is illustrated in Scheme 4.

vortioxetine Example reagent 6a [ _Z = OC0 ₂ C[CH ₃ ]3l. ^R 8 = C[CH ₃ ] ₃ ]

/^^"e reagent 6b [z = CI, R ₈ ⁼ vinyl]

reagent 8c [z = SPh, R ₈ = vinyl]

Scheme 4.

Example 4a R ₈ = C[CH ₃ ] ₃

Example 4b R ₈ = vinyl

Example 4a te/t-butyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazine-1 -carboxylate Example 4b vinyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine-1 -carboxylate

Carbohydrate-based prodrugs

This class of prodrugs may undergo acid-mediated cleavage, for example in the stomach. They may also be converted to vortioxetine by enzymatic processes. These compounds have the generic structure Example 5 and can be prepared from vortioxetine under a number of conditions for example as described in the literature [see for example, R.W. Gantt et al., Nature Chemical Biology 201 1 , vol. 7, p. 685 and V. Zsoldos-Madya ^* et al., J.

Carbohydrate Chem. 2005, 24, p. 19]. This is shown in Scheme 5.

Scheme 5.

Example 5. These compounds may be subjects to active transport by one of the glucose transporters across the intestinal gut wall or across the blood-brain-barrier to increase bioavailability and/or brain disposition.

Example 5a (2S,3R,4S,5S,6R)-2-(4-(2-((2,4-dimethylphenyl)thio)phenyl)pi perazin-1 -yl)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

Example 5b (2R,3R,4S,5S,6R)-2-(4-(2-((2,4-dimethylphenyl)thio)phenyl)pi perazin-1 -yl)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

Phosphate-ester-based prodrugs

Phosphate prodrugs can be used to enhance the aqueous solubility of parent drugs in some of the cases where oral absorption is limited by the thermodynamic and/or kinetic aqueous solubility of the parent drug [see for example D. Fleisher, R. Bong, B.H. Stewart Adv. Drug Deliv. Rev. 1996, 19, p. 1 15 and J. P. Krise, V.J. Stella Adv. Drug Deliv. Rev. 1996, 19, p. 287]. Some phosphonooxymethyl prodrugs have been shown to be enzymatically labile [see for example J. Golik et al., Bioorg. Med. Chem. Lett. 1996, 6, p. 1837 and J. P. Krise et al. J. Med. Chem. 1999, 42, p. 3094]. The formal 'double hydrolysis' of prodrugs like Example 6 and Example 7 occurs via a two-step reaction; enzymatic cleavage of the ester group followed by decomposition of the hydroxymethyl intermediate (sometimes this is a spontaneous or at least relatively fast reaction), for example at the gut wall after oral dosing. It is important to point out that the hydroxymethyl intermediate can release the parent drug by spontaneous chemical hydrolysis [for a general discussion see: H. Bundgaard 'Design of Prodrugs'; H. Bundgaard, Ed.; Elsevier: Amsterdam, 1985; pp. 1-92].

These compounds have the generic structure Example 6 and Example 7 and can be prepared from vortioxetine and an appropriately substituted alkylating agent [reagent 7] wherein X is a suitable leaving group such as F, CI, Br, I or tosylate or mesylate. This is illustrated in Scheme 6. vort oxet ne

Example 1

Scheme 6.

Examples of reagents 7 include di-ieri-butyl (iodomethyl) phosphate, di-te/t-butyl

(chloromethyl) phosphate, di-te/t-butyl (bromomethyl) phosphate [for which the te/t-butyl groups can be removed with acid directly after the reaction or as a separate deprotection step], dibenzyl (chloromethyl) phosphate [for which the benzyl groups can be removed by hydrogenolysis directly after the reaction or in a separate deprotection step], diallyl

(chloromethyl) phosphate [for which deprotection methods are described in T.W. Greene and P.G.M. Wuts 'Protective Groups in Organic Synthesis', 3 ^rd Edition, John Wiley & Sons Inc.: New York, 1999]. Methods for the synthesis of these and related reagents are described in the literature [c.f. , A. Mantyla et al., Tetrahedron Letters 2002, 43, p. 3793]. The synthesis of a prodrug using these reagents to from compounds related to Example 6a and Example 7a is discussed in the literature [c.f. , A.G. Sams et al., J. Med. Chem. 201 1 , 54, p. 751 ].

The reaction to form Example 6 is typically performed in acetonitrile, tetrahydrofuran,

2-methyl-tetrahydrofuran, or dimethyl formamide. In cases where X is not I, the reaction can sometimes be promoted by the addition of sodium or potassium iodide or tetra n-butyl ammonium iodide. The liberated acid HX can be quenched by an added base such as potassium or cesium carbonate, sodium hydride (can also be used to deprotonate vortioxetine prior to addition of reagent 7), or organic bases such as triethyl amine or N,N- diisopropylethylamine (DIPEA)., Examples 6 and 7.

Example 6a R ',9 H, R ₁₀ = C[CH ₃ ] ₃

Example 6b R ',9 ^CH 3 . ^R io = C[CH ₃ ] ₃

Example 6c R ',9 H, R-io ⁼ benzyl

Example 6d R 'i9 H, R ₁₀ = allyl

Example 7a R ',9

Example 7b R ',9 Example 6a di-te/t-butyl ((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl) phosphate

Example 6b racemic di-ieri-butyl (1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 - yl)ethyl) phosphate [(R)-di-te/t-butyl (1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 - yl)ethyl) phosphate and (S)-di-ieri-butyl (1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin- 1 -yl)ethyl) phosphate]

Example 6c dibenzyl ((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl) phosphate

Example 6d diallyl ((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl) phosphate Example 7a (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)methyl dihydrogen phosphate

Example 7b racemic 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)ethyl dihydrogen phosphate [(R)-1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)ethyl dihydrogen phosphate and (S)-1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)ethyl dihydrogen phosphate].

Carbonate-amides

Prodrugs of amines of this type are envisioned to function, at least in part, due to

anchiemeric assistance. An initial enzymatic or non-enzymatic step cleavage of the amide bond in Example 8 releases an Intermediate 9 that cyclizes spontaneously or in a enzymatic transformation forming Intermediate 10 that would subsequently hydrolyse to unmask vortioxetine. Compounds like example 8 may also hydrolyse directly at the carbonate unit to release vortioxetine in one or two formal steps. This is illustrated in Scheme 7.

Scheme 7.

Compounds of the generic structure Example 8 can be prepared by alkylation of vortioxetine with an appropriately substituted alkylating agent [reagent 8; examples of this are shown in Scheme 8 wherein LG is a suitable leaving group such as a CI, Br, or I; for a reference on the use of this type of reagent, see for example: E.T. Binderup, P.-J.V. Hjarnaa WO

2002042265. Subsequent cleavage of the nitrogen protective group (PG; typically a carbamate as in reagent 8d, 8e, and 8f) under the conditions described in the literature [see for example T.W. Greene and P.G.M. Wuts 'Protective Groups in Organic Synthesis', 3 ^rd Edition, John Wiley & Sons Inc.: New York, 1999] followed by acylation under the conditions described for the synthesis of prodrugs of the generic structure Example 1. The sequence is outlined in Scheme 8.

The reactions to form Example 8 are typically performed in acetonitrile,

tetrahydrofuran, 2-methyl-tetrahydrofuran, methylene chloride, chloroform or dimethyl formamide. In cases where LG is not I, the reaction can sometimes be promoted by the addition of sodium or potassium iodide or tetra n-butyl ammonium iodide. The liberated acid HX can be quenched by an added base such as potassium or cesium carbonate, sodium hydride (can also be used to deprotonate vortioxetine prior to addition of reagent 8), or organic bases such as triethyl amine or Ν,Ν-diisopropylethylamine (DIPEA). t 8a or 8c ent 8b

Scheme 8.

Examples 8.

H, R-|2 ^~~ CH ₃

R ₁₂ = CH ₃

H, R ₁₂ = H

H, R ₁₂ = cyclopropyl

H, R-12 ⁼ 4-tetrahydropyranyl

H, Ri2 ⁼ phenyl

H, R-12 ⁼ benzyl

H, R-12 ⁼ 2-pyridyl

Example 8i R^ = H, R ₁₂ = [CH ₂ ]3 ^OH

Example 8a 2-acetamidoethyl ((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl) carbonate

Example 8b 2-acetamidoethyl (1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)ethyl) carbonate

Example 8c (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl (2-formamidoethyl) carbonate

Example 8d 2-(cyclopropanecarboxamido)ethyl ((4-(2-((2,4- dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl) carbonate Example 8e (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl (2-(tetrahydro-2H- pyran-4-carboxamido)ethyl) carbonate

Example 8f 2-benzamidoethyl ((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl) carbonate

Example 8g (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl (2-(2- phenylacetamido)ethyl) carbonate

Example 8h (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl (2- (picolinamido)ethyl) carbonate

Example 8i (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl (2-(4- hydroxybutanamido)ethyl) carbonate

Example 8j R12 ^~ H

Example 8k R ₁₂ = CH ₃

Example 8I ₁₂ = [CH ₂ ]2 ^OCH 3

Example 8m R ₁₂ = [CH ₂ ]2 ^CN

Example 8n R ₁₂ = phenyl

Example 80 R ₁₂ = benzyl

Example 8p R12 ⁼ 4-pyridyl

Example 8q R ₁₂ = [CH ₂ ]2 ^OH

Example 8j (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl (3-formamidopropyl) carbonate

Example 8k 3-acetamidopropyl ((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 - yl)methyl) carbonate

Example 8I (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl (3-(3- methoxypropanamido)propyl) carbonate

Example 8m 3-(3-cyanopropanamido)propyl ((4-(2-((2,4- dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl) carbonate

Example 8n 3-benzamidopropyl ((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 - yl)methyl) carbonate

Example 8o (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl (3-(2- phenylacetamido)propyl) carbonate

Example 8p (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl (3- (isonicotinamido)propyl) carbonate

Example 8q (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl (3-(3- hydroxypropanamido)propyl) carbonate Carbamate-amides

Prodrugs of amines of this type are envisioned to function, at least in part, due to anchiemeric assistance. An initial enzymatic or non-enzymatic step cleavage of the amide bond in Example 9 releases an Intermediate 11 that cyclizes spontaneously or in a enzymatic transformation to unmask vortioxetine. Compounds like Example 9 may also hydrolyse directly at the carbamate unit to release vortioxetine in one or two formal steps. This is illustrated in Scheme 9.

Scheme 9.

Compounds of the generic structure Example 9 can be prepared by reaction of vortioxetine with an appropriately substituted reagent [reagent 9; examples of this are shown in Scheme 10 wherein Q is a suitable leaving group and L an alkyl or alkoxy group]. Subsequent cleavage of the nitrogen protective group under the conditions described in the literature [see for example T.W. Greene and P.G.M. Wuts 'Protective Groups in Organic Synthesis', 3 ^rd Edition, John Wiley & Sons Inc.: New York, 1999] followed by acylation under the conditions described for the synthesis of prodrugs of the generic structure Example 1 . The sequence is outlined in Scheme 10.

The base can be selected from both organic and inorganic bases such as these commercially available reagents: triethyl amine, Ν,Ν-diisopropylethylamine (DIPEA), potassium or cesium carbonate, potassium or cesium fluoride, and sodium or potassium or cesium hydroxide.

The solvent can be selected from dichloromethane, chloroform, N,N- dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofurane, methylene chloride, and chloroform. a

Example 9

Scheme 10.

Examples 9.

Example 9a R-13 ⁼ H

Example 9b R-13 ⁼ CH ₃

Example 9c R ₁₃ = cyclopropyl

Example 9d R ₁₃ = 4-tetrahydropyranyl

Example 9e R ₁₃ = phenyl

Example 9f R ₁₃ = benzyl

Example 9g ^R i ₃ ⁼ 2-pyridyl

Example 9h R ₁₃ = [CH ₂ ] ₃ ^OH

Example 9a 2-formamidoethyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine-1

carboxylate

Example 9b 2-acetamidoethyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine-1

carboxylate

Example 9c 2-(cyclopropanecarboxamido)ethyl 4-(2-((2,4- dimethylphenyl)thio)phenyl)piperazine-1 -carboxylate

Example 9d 2-(tetrahydro-2H-pyran-4-carboxamido)ethyl 4-(2-((2,4- dimethylphenyl)thio)phenyl)piperazine-1 -carboxylate Example 9e 2-benzamidoethyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine-1 - carboxylate

Example 9f 2-(2-phenylacetamido)ethyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine-1 - carboxylate

Example 9g 2-(picolinamido)ethyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine-1 - carboxylate

Example 9h 2-(4-hydroxybutanamido)ethyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine- 1 -carboxylate

Example 9i R ₁₃ = H

Example 9j R-o ⁼ CH ₃

Example 9k R-i3 ⁼ 3-oxetanyl

Example 9I R ₁₃ = [CH ₂ ]2 ^NCOCH 3

Example 9m R-o ⁼ phenyl

Example 9n R ₁₃ = benzyl

Example 9o R ₁₃ = 3-pyridyl

Example 9p R ₁₃ = [ΟΗ ₂ ] ₂ ^ΟΗ

Example 9i 3-formamidopropyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine-1 - carboxylate

Example 9j 3-acetamidopropyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine-1 - carboxylate

Example 9k 3-(oxetane-3-carboxamido)propyl 4-(2-((2,4- dimethylphenyl)thio)phenyl)piperazine-1 -carboxylate

Example 9I 3-(3-acetamidopropanamido)propyl 4-(2-((2,4- dimethylphenyl)thio)phenyl)piperazine-1 -carboxylate

Example 9m 3-benzamidopropyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine-1 - carboxylate

Example 9n 3-(2-phenylacetamido)propyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine- 1 -carboxylate

Example 9o 3-(nicotinamido)propyl 4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazine-1 - carboxylate

Example 9p 3-(3-hydroxypropanamido)propyl 4-(2-((2,4- dimethylphenyl)thio)phenyl)piperazine-1 -carboxylate

N-Mannich Bases

Prodrugs of this type are known undergo non-enymatic hydrolysis at physiologically relevant pH values (between 2 and 9). They may also potentially undergo enzymatic cleavage. Example 10 prodrugs can be prepared from vortioxetine, formaldehyde, and an appropriate NH acidic compound [reagent 10] as outlined in Scheme 1 1 . The reaction typically occurs in water, ethanol, methanol, or etheral solvents like 1 ,4-dioxane or tetrahydrofuran [for a discussion on the synthesis and profile of N-Mannich prodrugs, see for example: 'Methods in Enzymology', Chapter 27 'Formation of Prodrugs of Amines, Amides, Ureides, and Imides'

-355].

vortioxetine

Scheme 10. In cases where reagent 10 contains two acidic NH groups the reaction can afford also bis- functionalized prodrugs that contain two molecules of vortioxetine per prodrug such as Example 10j. In such cases the compounds can be prepared in one or two steps using unprotected or appropriately mono-protected variants of reagent 10 leading to compounds like for example 10i and 10j.

Examples 10.

Example 0a R-| ₄ ⁼ H

Example 0b R ₁₄ = CH ₃

Example 10c R ₁₄ = cyclopropyl

Example 10d R ₁₄ = 4-tetrahydropyranyl

Example 10e R ₁₄ = phenyl

Example 10f R ₁₄ = benzyl

Example 10g R-| ₄ ⁼ 2-pyridyl

Example 10h R ₁₄ = [CH ₂ ] ₃ OH

Example 10i R ₁₄ = [CH ₂ ]CONH ₂

Example 10a N-((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl)formamide Example 10b N-((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl)acetamide Example 10c N-((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 - yl)methyl)cyclopropanecarboxamide Example 10d N-((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl)tetrahydro-2H- pyran-4-carboxamide

Example 10e N-((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl)benzamide Example 10f N-((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl)-2- phenylacetamide

Example 10g N-((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl)picolinamide Example 10h N-((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl)-4- hydroxybutanamide

Example 10i N1 -((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)methyl)malonamide Example 10j N1 ,N3-bis((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1 - yl)methyl)malonamide.

The metabolic transformation of the compound of the present invention may be tested in plasma or hepatocyte assays as describes in the examples part. Alternatively, the metabolic transformation may be tested in an assay mimicking metabolic transformation in the intestines, e.g. the stomach.

Examples

Example 1 a 4-(2-((2,4-dimethyl-phenyl)thio)phenyl)piperazine-1-carbalde hyde

Acetic acid anhydride (63 microL) and formic acid (24 mg) were mixed at ambient temperature. The mixture was heated to 60 °C for 30 min before a solution of vortioxetine (100 mg) in dimethyl formamide (5.0 mL) was added. The resulting mixture was stirred at 80 °C overnight. The mixture was cooled to ambient temperature and concentrated. The residue was purified by prep-HPLC (using a Gilson 281 semi-preparative HPLC system fitted with a YMC-Actus Triart C18 150x30mm, 5 microm column and with mixtures of two mobile phases (A: 10mM ammonuim carbonate in water; B: acetonitrile) at a flow rate: 25mL/min (0 min 60% B; 12 min, 80% B; 12.1 min, 80% B; 12.2 min, 100% B; 14.2 min, 100% B; 14.3 min, 60% B; 15.5 min, 60% B) and monitor wavelengths of 220 nm and 254 nm to afford Example 1 a (40 mg) as a white solid. ¹ H NMR (400 MHz, CDCI ₃ ), δ 8.12 (s, 1 H), 7.37 (d, J = 7.6 Hz, 1 H), 7.17 (s, 1 H), 7.01 -7.1 1 (m, 3H), 6.89-6.93 (m, 1 H), 6.54-6.57 (m, 1 H), 3.75 (d, J = 4.8 Hz, 2H), 3.56-3.58 (m, 2H), 3.03-3.10 (m, 4H), 2.38 (s, 3H), 2.33 (s, 3H).

LC/MS (method 1 ): RT 3.24 min, 98.7% UV-purity, mass observed 327 (MW+1 ).

Example 1 d 1-(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1 -yl)-2-methylpropan-

1-one

To a mixture of vortioxetine (100 mg) in dichloromethane (5.0 mL) was added triethyl amine (93 microL, 2.00 eq) and isobutyryl chloride (39 microL) at 0 °C. The mixture was stirred at ambient temperature overnight. The mixture was diluted with water, and the product was extracted into dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by prep-TLC (eluent: petroleum ether/ethyl aceate 3:1 ) and lyophilized to give Example 1 d (50 mg) as a white solid.

¹ H NMR (400 MHz, CDCI ₃ ), δ 7.38 (d, J = 7.6 Hz, 1 H), 7.17 (s, 1 H), 7.01 -7.1 1 (m, 3H), 6.88- 6.92 (m, 1 H), 6.55 (d, J = 6.4 Hz, 1 H), 3.83 (broad s, 2H), 3.71 (broad s, 2H), 3.15-3.00 (m, 4H), 2.83-2.88 (m, 1 H), 2.37 (s, 3H), 2.33 (s, 3H), 1 .18 (d, J = 6.4 Hz, 6H).

LC/MS (method 2): RT 3.03 min, 99.8% UV-purity, mass observed 369 (MW+1 ). Example 1 e cyclopropyl(4-(2-((2,4-dimethylphenyl)thio)-phenyl)piperazin -1- yl)methanone

Prepared in a similar manner as Example 1 d from vortioxetine (100 mg) and cyclopropanecarbonyl chloride (34 microL) and with petroleum ether/ethyl acetate 2:1 as eluent for the prep-TLC to afford Example 1e (48 mg) as a white solid.

¹ H NMR (400 MHz, CDCI ₃ ), δ 7.38 (d, J= 8.4 Hz, 1 H), 7.17 (s, 1 H), 7.01 -7.1 1 (m, 3H), 6.88- 6.92 (m, 1 H), 6.55-6.57 (m, 1 H), 3.88 (broad s, 4H), 3.1 1 -3.05 (m, 4H), 2.37 (s, 3H), 2.34 (s, 3H), 1 .26 (broad s, 1 H), 1 .03-1 .05 (m, 2H), 0.78-0.82 (m, 2H).

LC/MS (method 2): RT 2.92 min, 100% UV-purity, mass observed 367 (MW+1 ).

Example 1f 1-(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1-yl)-4-h ydroxybutan-1 - one

To a mixture of gamma-butyrolactone (5.0 g) in ethanol (20 mL) was added sodium hydroxide (2.79 g) in water (70 mL). The mixture was stirred at ambient temperature overnight before it was concentrated. The residue was poured into acetic acid anhydride (20 mL) and the mixture was stirred at 60 °C for 6 hours. The mixture was cooled to ambient temperature and concentrated to afford 4-acetoxybutanoic acid (6.0 g) as an oil sufficiently pure for the next step. To a mixture of a portion of this material (49 mg) in dichloromethane (5.0 mL) was added triethyl amine (93 microL) and HATU (191 mg) at ambient temperature. The mixture was stirred for 30 min before vortioxetine (100 mg) was added. Then the mixture was stirred at ambient temperature overnight before it was concentrated. The residue was dissolved in methanol (5.0 mL) and treated with potassium carbonate (32.4 mg) at ambient temperature for 1 hour. The mixture was concentrated. The residue was purified by prep-TLC (eluent: petroleum ether/ethyl aceate 1 :1 ) to afford Example 1 f (15.0 mg) as a white solid. ¹ H NMR (400 MHz, CDCI ₃ ), δ 7.37 (d, J = 8.4 Hz, 1 H), 7.16 (s, 1 H), 7.01 -7.09 (m, 3H), 6.88- 6.91 (m, 1 H), 6.55 (d, J = 8.0 Hz, 1 H), 3.79-3.84 (m, 2H), 3.69-3.75 (m, 2H), 3.60-3.69 (m, 2H), 3.05-3.06 (m, 4H), 2.56-2.60 (m, 2H), 2.37 (s, 3H), 2.33 (s, 3H), 1 .95-1 .98 (m, 2H), 2.56-2.60(m, 2H, 2.37(s, 3H), 2.33(s, 3H), 1.95-1 .98 (m, 2H). LC/MS (method 3): RT 2.49 min, 98.5% UV-purity, mass observed 385 (MW+1 ).

Example 1 h N-(4-(4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1-yl) -4-oxobutyl)- acetamide

A mixture of 4-acetamidobutanoic acid (48.6 mg), triethyl amine (93 microL), and HATU (191 mg) in dichloromethane (5.0 ml_) was stirred at ambient temperature for 30 min. Then vortioxetine (100 mg) was added, and the mixture was stirred overnight. The mixture was concentrated. The residue was purified by prep-HPLC (using a Gilson 281 semi-preparative HPLC system fitted with a YMC-Actus Triart C18 150x30 mm, 5 microm column and with mixtures of two mobile phases: (A: 0.075% trifluoroacetic acid in water; B: acetonitrile) at a flow rate of 25mL/min (0 min 45% B; 12 min, 75%; 12.1 min, 75% B; 12.2 min, 100% B; 14.2 min, 100% B; 14.3 min, 45% B; 15.5 min, 45% B) and monitor wavelengths of 220 nm and 254nm). The fractions containing the product were pooled and treated with Amberlyst A-21 ion exchange resin (pH 8), filtered, and concentrated to afford Example 1 h (42.0 mg) as a white solid.

¹ H NMR (400 MHz, CDCI ₃ ), δ 7.36 (d, J = 7.6 Hz, 1 H), 7.16 (s, 1 H), 7.01 -7.1 1 (m, 3H), 6.88- 6.92 (m, 1 H), 6.53-6.56 (m, 1 H), 6.44 (s, 1 H), 3.81 (broad s, 2H), 3.65 (broad s, 2H), 3.33 (d, J = 4.8 Hz, 2H), 3.04-3.07 (m, 4H), 2.46-2.49 (m, 2H), 2.37 (s, 3H), 2.32 (s, 3H), 1 .99 (s, 3H), 1 .90-1 .93 (m, 2H).

LC/MS (method 1 ): RT 3.01 min, 97.7% UV-purity, mass observed 426 (MW+1 ).

Example 1 i (4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1-yl)(tetra hydro-2H-pyran- 4-yl)methanone

A mixture of tetrahydro-2H-pyran-4-carboxylic acid (43.6 mg), HATU (140 mg), and triethyl amine (93 microL) in dichloromethane (5.0 mL) was stirred at 0 °C for 30 min. Vortioxetine (100 mg) was added and the mixture was stirred at ambient temperature overnight. The mixture was diluted with water, and the product was extracted into dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by prep-HPLC as described for Example 1 o to give Example 1 i (64.0 mg) as a white solid.

¹ H NMR (400 MHz, CDCI ₃ ), δ 7.37 (d, J = 8.0 Hz, 1 H), 7.17 (s, 1 H), 7.01 -7.1 1 (m, 3H), 6.88- 6.93 (m, 1 H), 6.54-6.56 (m, 1 H), 4.06 (d, J= 10.0 Hz, 2H), 3.83 (broad s, 2H), 3.71 (broad s, 2H), 3.45-3.51 (m, 2H), 3.05-3.08 (m, 4H), 2.76-2.83 (m, 1 H), 2.37 (s, 3H), 2.33 (s, 3H), 1 .93-2.03 (m,2H), 1 .68-1 .64 (m, 2H).

LC/MS (method 4): RT 3.41 min, 99.3% UV-purity, mass observed 41 1 (MW+1 ). Example 1 n 1 ,5-bis(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1-yl) pentane-1 ,5- dione

To a mixture of vortioxetine (100 mg) in dichloromethane (5.0 mL) was added triethyl amine (47 microL) and glutaroyi dichloride (22 microL) at 0 °C. The mixture was stirred at ambient temperature overnight. The mixture was diluted with water, and the product was extracted into dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by prep-HPLC (using a Gilson 281 semi-preparative HPLC system fitted with a Boston Green ODS 150x30 mm, 5 microm column and with mixtures of two mobile phases: (A: 0.075% trifluoroacetic acid in water; B: acetonitrile) at a flow rate of 25mL/min (0 min 95% B; 1 1 .5 min, 100%; 1 1 .6 min, 100% B; 1 1 .7 min, 100% B; 14.7 min, 100% B; 14.8 min, 95% B; 16 min, 95% B) and monitor wavelengths of 220 nm and 254nm). The fractions containing the product were pooled and treated with Amberlyst A-21 ion exchange resin (pH 8), filtered, and concentrated to afford Example 1 n (34.0 mg) as a white solid. ¹ H NMR (400 MHz, CDCI ₃ ), δ 7.37 (d, J = 8.0 Hz, 2H), 7.16 (s, 2H), 7.01 -7.10 (m, 6H), 6.88- 6.92 (m, 2H), 6.54-6.56 (m, 2H), 3.82 (broad s, 4H), 3.70-3.72 (m, 4H), 3.03-3.09 (m, 8H), 2.51 -2.55 (m, 4H), 2.37 (s, 6H), 2.33 (s, 6H), 2.02-2.08 (m, 2H).

LC/MS (method 5): RT 2.69 min, 98.6% UV-purity, mass observed 693 (MW+1 ).

Example 1 o (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1-yl)(phen yl)-methanone

Prepared in a similar manner as Example 1 d from vortioxetine (100 mg) and benzoyl chloride (43 microL). The product was purified by prep-HPLC (using a Gilson 281 semi- preparative HPLC system fitted with a Boston Green ODS 150x30mm, 5 microm column and with mixtures of two mobile phases (A: 0.075% trifluoroacetic acid in water; B: acetonitrile) at a flow rate of 25ml_/min (0 min 60% B; 1 1 .5 min, 90%; 1 1 .6 min, 90% B; 1 1 .7 min, 100% B; 14.7 min, 100% B; 14.8 min, 60% B; 16 min, 60% B) and monitor wavelengths of 220 nm and 254nm). The fractions containing the product were pooled and treated with Amberlyst A- 21 ion exchange resin (pH 8), filtered, and concentrated to afford Example 1 o (65 mg) as a white solid.

¹ H NMR (400 MHz, CDCI ₃ ), δ 7.43-7.48 (m, 5H), 7.36 (d, J= 8.0 Hz, 1 H), 7.16 (s, 1 H), 7.01 - 7.12 (m, 3H), 6.88-6.92 (m, 1 H), 6.53-6.56 (m, 1 H), 4.00 (broad s, 2H), 3.63 (broad s, 2H), 3.16-3.01 (m, 4H), 2.37 (s, 3H), 2.32 (s, 3H). LC/MS (method 2): RT 3.21 min, 100% UV-purity, mass observed 403 (MW+1 ).

Example 1 p (4-(2-((2,4-dimethylphenyl)thio)phenyl)-piperazin-1-yl)(pyri din-2-yl)- methanone

To a mixture of picolinic acid (41 .3) in dichloromethane (5.0 ml_) was added HATU (191 mg) at 0 °C. Vortioxetine (100 mg) was added and the mixture was stirred at ambient temperature overnight. The mixture was diluted with water, and the product was extracted into dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by prep-HPLC as described for Example 1 o to afford Example 1 p (1 1 .7 mg) as a white solid.

¹ H NMR (400 MHz, CDCI ₃ ), δ 8.63 (d, J = 4.0 Hz ,1 H), 7.81 -7.85 (m, 1 H), 7.69 (d, J = 8.0 Hz ,1 H), 7.35-7.38 (m, 2H), 7.16 (s, 1 H), 7.03-7.12 (m, 3H), 6.88-6.92 (m, 1 H), 6.54-6.56 (m, 1 H), 4.02 (broad s, 2H), 3.76-3.79 (m, 2H), 3.18-3.21 (m, 2H), 3.08-3.10 (m, 2H), 2.37 (s, 3H), 2.32 (s, 3H).

LC/MS (method 2): RT 2.71 min, 98.8% UV-purity, mass observed 404 (MW+1 ).

Example 1 r 2-(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1-yl)-2-o xoethyl methyl fumarate

To a mixture of vortioxetine (150 mg) and triethyl amine (174 microL) in dichloromethane (5.0 ml_) was added chloroacetyl chloride (52 microL) at 0 °C. The mixture was stirred at ambient temperature overnight. The mixture was concentrated to afford 2-chloro-1 -(4-(2- ((2,4-dimethylphenyl)thio)phenyl)piperazin-1 -yl)ethan-1 -one (150 mg) as a yellow oil sufficiently pure for the next step. To a mixture of this material and (£)-4-ethoxy-4-oxobut-2- enoic acid (52.1 mg) in dimethyl formamide (10 ml_) was added potassium carbonate (82.9 mg). The mixture was stirred at 60 °C overnight before it was concentrated. The residue was purified by prep-HPLC (using a Gilson 281 semi-preparative HPLC system fitted with a YMC-Actus Triart C18 150x30mm, 5 microm column and with mixtures of two mobile phases (A: 10mM ammonium carbonate in water; B: acetonitrile) at a flow rate of 25mL/min (0 min, 70% B; 12.0 min, 90% B; 12.1 min, 90% B, 12.2 min, 100% B, 14.2 min, 100% B, 14.3 min, 70% B, 15.5 min, 70% B) with monitor wavelengths of 220 nm and 254 nm to afford

Example 1 r (22.0 mg) as a colorless oil. ¹ H NMR (400 MHz, CDCI ₃ ), δ 7.36 (d, J = 7.6 Hz, 1 H), 7.16 (s, 1 H), 7.01 -7.12 (m, 3H), 6.99 (d, J= 2.0 Hz, 2H), 6.89-6.95 (m, 1 H), 6.55-6.57 (m, 1 H), 4.92 (s, 2H), 3.83 (broad s, 5H), 3.58-3.60 (m, 2H), 3.07-3.1 1 (m, 4H), 2.37 (s, 3H), 2.33 (s, 3H).

LC/MS (method 6): RT 2.06 min, 99.0% UV-purity, mass observed 469 (MW+1 ).

Example 1 q 1 -(4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1-yl)-2-ph enylethan-1 - one

Prepared in a similar manner as Example 1 o from vortioxetine (100 mg) and 2-phenylacetyl chloride (49 microL) to afford Example 1 q (73 mg) as a yellow solid.

¹ H NMR (400 MHz, CDCI ₃ ), δ 7.36-7.41 (m, 3H), 7.29-7.33 (m, 3H), 7.18 (s, 1 H), 7.05-7.1 1 (m, 2H), 6.97-6.99 (m, 1 H), 6.88-6.93 (m, 1 H), 6.36-6.56 (m, 1 H), 3.87 (s, 2H), 3.83 (s, 2H), 3.64-3.67 (m, 2H), 3.04-3.06 (m, 2H), 2.89-2.91 (m, 2H), 2.39 (s, 3H), 2.33 (s, 3H). LC/MS (method 2): RT 3.22 min, 99.0% UV-purity, mass observed 417 (MW+1 ).

Example 10b N-((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1-yl)met hyl)- acetamide

A mixture of acetamide (59 mg), paraformaldehyde (30 mg), potassium carbonate (276 mg), and vortioxetine (328 mg) in 1 ,4-dioxane (10 mL) was stirred at 85 °C overnight. The mixture was concentrated. The residue was purified by prep-HPLC (Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Gemini 150x25mm, 5 microm; Mobile phase A: water with 0.05% ammonia in water; Mobile phase B: acetonitrile; Gradient: B from 50% to 80% in 10 min, hold 100% B for 2 min; Flow Rate: 25 mL/min; Column Temperature: 30 °C; Wavelength: 220 nm, 254 nm) to afford Example 10b (196 mg) as a white solid.

¹ H NMR (CDCI ₃ , 400MHz): δ 7.38-7.36 (m, 1 H), 7.15 (s, 1 H), 7.06-7.01 (m, 3H), 6.87-6.83 (m, 1 H), 6.50-6.49 (m, 1 H), 5.91 (broad s, 1 H), 4.20 (d, J = 6.4 Hz, 2H), 3.10 (broad s, 4H), 2.78 (broad s, 4H), 2.36 (s, 3H), 2.31 (s, 3H), 2.05 (s, 3H).

LC/MS (method 7): R = 2.00 min, 99.8% UV purity, 100% ELS purity, mass observed 370 (MW+1 ).

Example 10c N-((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin

cyclopropanecarboxamide

Prepared in a similar manner to Example 10b from cyclopropanecarboxamide (85.0 mg), paraformaldehyde (30 mg), and vortioxetine (328 mg) to afford Example 10c (196 mg) as a white solid using a different HPLC method (Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Gemini 150x25 mm, 5 microm; Mobile phase A: water with 0.05% ammonia in water; Mobile phase B: acetonitrile; Gradient: B from 42% to 72% in 10 min, hold 100% B for 2 min; Flow Rate: 25 mL/min; Column Temperature: 30 °C; Wavelength: 220 nm, 254 nm).

¹ H NMR (CDCI ₃ , 400MHz): δ 7.40-7.36 (m, 1 H), 7.15 (s, 1 H), 7.07-6.99 (m, 3H), 6.89-6.83 (m, 1 H), 6.50-6.49 (m, 1 H), 6.03 (broad s, 1 H), 4.23 (d, J = 6.4 Hz, 2H), 3.10 (broad s, 4H), 2.78 (broad s, 4H), 2.36 (s, 3H), 2.31 (s, 3H), 1 .37-1 .35 (m, 1 H), 1 .02-0.99 (m, 2H), 0.8-0.77 (m, 2H).

LC/MS (method 8): RT 2.18 min, 96.5% UV purity, mass observed 31 1 (MW+1 ).

Example 10e N-((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1-yl)met hyl)- benzamide

Prepared in a similar manner to Example 10b from benzamide (121 mg), paraformaldehyde (30 mg), and vortioxetine (328 mg) to afford Example 10e (183 mg) as a white solid.

¹ H NMR (CDCI ₃ , 400MHz): δ 7.82-7.80 (m, 2H), 7.54-7.43 (m, 3H), 7.35-7.33 (m, 1 H), 7.1 1 (s, 1 H), 7.06-7.04 (m, 2H), 6.99-6.83 (m, 1 H), (6.56 (s, 1 H), 6.49 (s, 1 H), 4.40 (d, J = 6.4 Hz, 2H), 3.12 (broad s, 4H), 2.86 (broad s, 4H), 2.34 (s, 3H), 2.29 (s, 3H).

LC/MS (method 9): R 1 .19 min, 98.6% UV purity, 100% ELS purity, mass observed 432

(MW+1 ).

Example 10f N-((4-(2-((2,4-dimethylphenyl)thio)phenyl)piperazin-1-yl)met hyl)-2- phenylacetamide

Prepared in a similar manner to Example 10b from 2-phenylacetamide (124 mg), paraformaldehyde (25 mg), and vortioxetine (273 mg) to afford Example 10f (201 mg) as a white solid.

¹ H NMR (CDCI ₃ , 400MHz): δ 7.38-7.25 (m, 6H), 7.15 (s, 1 H), 7.15-7.02 (m, 3H), 6.88-6.82 (m, 1 H), 6.49-6.47 (m, 1 H), 5.80 (broad s, 1 H), 4.16 (d, J = 6.4 Hz, 2H), 3.63 (s, 2H), 3.05 (broad s, 4H), 2.68 (broad s, 4H), 2.36 (s, 3H), 2.31 (s, 3H).

LC/MS (method 9): R 1 .89 min, 98.9% UV purity, 100% ELS purity, mass observed 468 (MW+1 ).

Analytical LC/MS methods:

Method 1 : Performed on a Agilent 1200 LCMS system fitted with a Xbridge ShieldRP18

50x2.1 mm, 5 microm column at 40 °C and a mixture of two mobile phases (A: 10 mM ammonium carbonate in water; B: acetonitrile) at a flow rate of 0.8 mL/min (0 min, 15% B; 0.4 min, 15% B; 3.4 min, 90% B; 3.85 min, 100% B; 3.86 min, 15% B; 4.5 min, 15% B) and monitor wavelength of 254 nm.

Method 2: Performed on a Agilent 1200 & 61 10A LCMS system fitted with a Phenomenex Luna-C18 50x2 mm, 5 microm column operated at 40 °C and a mixture of two mobile phases (A: 0.037% trifluoroacetic acid in water; B: 0.018% trifluoroacetic acid in acetonitrile) at a flow rate of 0.8 mL/min (0 min, 40% B; 0.4 min, 40% B; 3.4 min, 100% B; 3.85 min, 100% B; 3.86 min, 40% B; 4.5 min, 40% B) and monitor wavelength of 220 nm.

Method 3: Performed on a Agilent 1200 & 6120 LCMS system fitted with a Xbridge

ShieldRPI 8 50x2.1 mm, 5 microm column operated at 40 °C and a mixture of two mobile phases (A: 10 mM ammonium carbonate in water; B: acetonitrile) at a flow rate of 0.8 mL/min (0 min, 30% B; 0.4 min, 30% B; 3.4 min, 100% B; 3.85 min, 100% B; 3.86 min, 30% B; 4.5 min, 30% B) and monitor wavelength of 220 nm.

Method 4: Performed on a Agilent 1200 & 61 10A LCMS system fitted with a Phenomenex Luna-C18 50x2 mm, 5 microm column operated at 40 °C and a mixture of two mobile phases (A: 0.037% trifluoroacetic acid in water; B: 0.018% trifluoroacetic acid in acetonitrile) at a flow rate of 0.8 mL/min (0 min, 10% B; 0.4 min, 10% B; 3.4 min, 100% B; 3.85 min, 100% B; 3.86 min, 10% B; 4.5 min, 10% B) and monitor wavelength of 220 nm.

Method 5: Performed on a Agilent 1200 & 1956A LCMS system fitted with a Phenomenex Luna-C18 50x2 mm, 5 microm column operated at 40 °C and a mixture of two mobile phases (A: 0.037% trifluoroacetic acid in water; B: 0.018% trifluoroacetic acid in acetonitrile) at a flow rate of 0.8 mL/min (0 min, 60% B; 0.4 min, 60%; 3.4 min, 100%; 3.85 min, 100% B; 3.86 min, 60% B; 4.5 min, 60% B) and monitor wavelength of 220 nm.

Method 6: Performed on a Agilent 1200 & 6120 LCMS system fitted with a Xbridge

ShieldRPI 8 50x2.1 mm, 5 microm column operated at 40 °C and a mixture of two mobile phases (A: 10 mM ammonium carbonate in water; B: acetonitrile) at a flow rate of 0.8 mL/min (0 min, 50% B; 0.4 min, 50%; 3.4 min, 100%; 3.85 min, 100% B; 3.86 min, 50% B; 4.5 min, 50% B) and monitor wavelength of 220 nm.

Method 7: Performed on a Agilent 1200 LCMS system fitted with a Phenomenex Luna-C18 50x2 mm, 5 microm column operated at 50 °C and a mixture of two mobile phases (A: 0.1 % trifluoroacetic acid in water; B: 0.05% trifluoroacetic acid in acetonitrile) at a flow rate of 0.8 mL/min (0 min, 10% B; 3.4 min, 100% B; 4 min, 100% B; 4.01 min, 10% B; 4.5 min, 10% B) and monitor wavelength of 254 nm. Method 8: Performed on a Agilent 1200 LCMS system fitted with a Venusil XBP-C18 50x2.1 mm, 12 microm column operated at 50 °C and a mixture of two mobile phases (A: 1 .5 ml_ trifluoroacetic acid in 4L water; B: 0.75 ml_ trifluoroacetic acid in 4 L acetonitrile) at a flow

rate of 0.8 mL/min (0 min, 10% B; 0.4 min, 10% B; 3.4 min, 100% B; 3.85 min, 100% B; 3.86 min, 10% B; 4.5 min, 10% B) and monitor wavelength of 254 nm.

Method 9: Performed on a Agilent 1200 LCMS system fitted with a Phenomenex Luna-C18

50x2 mm, 5 microm column operated at 50 °C and a mixture of two mobile phases (A: 0.1 % trifluoroacetic acid in water; B: 0.05% trifluoroacetic acid in acetonitrile) at a flow rate of 0.8 mL/min (0 min, 25% B; 3.4 min, 100% B; 4 min, 100% B; 4.01 min, 25% B; 4.5 min, 25% B) and monitor wavelength of 254 nm.

Metabolism in human plasma

The metabolism of test compounds and the formation of vortioxetine in human plasma were tested in the following assay.

Pooled frozen plasma was thawed in water bath at 37 °C prior to experi centrifuged at 4000 rpm for 5 min. The pH was adjusted to 7.4 ± 0.1 if needed.

Test compound solutions (50 μΜ in DMSO) and positive control solution (100 μΜ

propantheline in DMSO) were prepared. For each time point, 2 μί test solution or 2 μ

positive control solution was mixed with 98 μΙ blank plasma to afford of 1 μΜ test compound and 2 μΜ positive control solutions. Samples were incubated at (0, 0.5, 1 , 2, 4 and 6 hr in duplicate at 37 °C in wat comprising 200 ng/mL tolbutamide and 20 ng/mL buspirone in 50% MeCN/MeOH.

Following addition of the stop solution, each plate was centrifuged at 4,000 rpm for

10 min. 100 μΙ supernatant from each well was transferred mixed with 200 μί ultra-pure

water. The plate was shaked at 800 rpm for about 10 min before submitting to LC-MS/MS

analysis.

Data Analysis:

The % remaining test compound after incubation in plasma was calculated using following equation: % Remaining= 100 x (PAR at appointed incubation time / PAR at TO time), where

PAR is the peak area ratio of analyte versus internal standard. The % Formation =1 00 x (PAR at appointed incubation time of test compound / PAR at TO time of vortioxetine), where PAR is the peak area ratio of vortioxetine versus internal standard. Metabolism in human hepatocytes

The metabolism of test compounds and the formation of vortioxetine in human hepatocytes were tested in the following assay.

Preparation of cell suspension: Cryopreserved human hepatocytes were thawed, isolated and suspended in Williams' Medium E and further diluted with pre-warmed Williams' Medium E to 0.625 ^χ 1 0 ⁶ cells/mL.

Williams' Medium E was used to obtain solutions comprising 5 μΜ test compound or 15 μΜ positive control (7-hydroxycoumarine).

40 μΙ cell suspension and 1 0 μΙ test compound/positive control solution were mixed in wells in 96-well plates and incubated for 0, 1 5, 30, and 90 minutes at 37 °C and 5% C0 ₂ . The reaction was quenched by addition of MeCN followed by vortexing and centrifugation at 3220 x g for 20 minutes to isolate the supernatant. He supernatant was subsequently analysed for vortioxetine.

Data Analysis:

The % remaining of test compound was calculated using following equation: % Remaining = 100 x (PAR at appointed incubation time / PAR at TO time), where PAR is the peak area ratio of analyte versus internal standard.

The % Formation = 1 00 x (PAR at appointed incubation time of test compound / PAR at TO time of metabolite), where PAR is the peak area ratio of analyte (vortioxetine) versus internal standard.