NMDA antagonist prodrugs

Abstract

The present application provides a prodrug of the NMDA antagonist (S)-1-phenyl-2-(pyridin-2-yl)ethylamine, which is used to treat depression (especially major depression) or pain; a composition containing them and a preparation method thereof.

Description

[0001] The present invention application is a divisional application based on a patent application with application date of October 30, 2014, application number 201480060416.0 (international application number PCT / GB2014 / 053236), and invention name “NMDA antagonist prodrug”.

[0002] The present invention relates to a prodrug of the NMDA antagonist (S)-1-phenyl-2-(pyridin-2-yl)ethylamine and its use in treating depression and depressive disorders (especially major depressive disorder (MDD)) and for treating pain (such as neuropathic pain). The prodrug of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine can be used to treat Rett syndrome, suicidal ideation, bipolar disorder (including bipolar depression), obsessive-compulsive disorder, sarin poisoning and status epilepticus. The present invention also relates to a pharmaceutical composition comprising the prodrug and a method for preparing the same.

[0003] Pain in various forms is ubiquitous in human life. Pain from injuries and postoperative pain is often temporary, but can also be very severe and persistent. Neuropathic pain such as diabetic neuropathy and postherpetic neuralgia seriously affects patients. Every year, tens of millions of people around the world, including patients at the end of their lives, suffer from pain without adequate treatment.

[0004] Depression affects about 120 million people worldwide. The symptoms of depression include, but are not limited to, low emotions, loss of interest or pleasure, guilt or reduced self-worth, sleep or appetite disorders, lack of energy and inattention, or any combination thereof. These problems can be long-term or recurrent and can cause the ability of individuals to take care of their own daily duties to be significantly impaired. Measured by Years Lived with a Disability (YLD), depression is the main cause of disability and in 2000, measured by Disability Adjusted Life Years (DALY; the sum of the years of potential life loss due to premature death and the years of effective life loss due to disability), depression is the fourth major factor in the global burden of disease. By 2020, it is estimated that depression will become the second place in the DALY ranking calculated for all ages, in both men and women. At present, depression has become the second cause of DALY in the 15-44 years old category for both sexes of mixed ages.

[0005] (S)-1-phenyl-2-(pyridin-2-yl)ethylamine dihydrochloride has been disclosed for use in treating MDD via intravenous infusion therapy (Gerard Sanacora et al., poster presented at the Annual Meeting of the American College of Neuropsychopharmacology, Hollywood, Florida, USA, December 6, 2012). Other relevant disclosures include WO1993 / 020052, WO2000 / 056324, and WO2000 / 63175. For convenience, it is of interest to be able to administer the drug as an oral dosage form. However, a problem with this oral dosage form of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine dihydrochloride would be that it could lead to intravenous abuse, for example, by fragmenting the oral dosage form of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine dihydrochloride and then immediately injecting the resulting pulverized oral dosage form of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine dihydrochloride. It is predicted that the prodrug of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine decomposes in the human body to provide (S)-1-phenyl-2-(pyridin-2-yl)ethanamine, and thus, when the prodrug of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine is orally administered, it will decompose to release a therapeutically effective dose of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine. However, if the prodrug of the present invention is administered intravenously, it is predicted that the prodrug will release (S)-1-phenyl-2-(pyridin-2-yl)ethanamine at a slower rate than the intravenous administration of a corresponding dose of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine, thereby giving a lower Cmax. It is predicted that the use of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine prodrugs will improve the clinical safety profile of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine (e.g., in the event of drug overdose or drug abuse (e.g., tablet crushing)). Therefore, in summary, placing (S)-1-phenyl-2-(pyridin-2-yl)ethanamine directly into an oral formulation will lead to abuse. The compounds of the present invention are metabolized in vivo to give (S)-1-phenyl-2-(pyridin-2-yl)ethanamine at a slower rate than when (S)-1-phenyl-2-(pyridin-2-yl)ethanamine is administered intravenously, and therefore will not lead to the potential for abuse of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1a and 1bThe conversion of the prodrug (Example 5) of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine to (S)-1-phenyl-2-(pyridin-2-yl)ethylamine during incubation in human intestinal fluid is illustrated. Specifically, FIG. 1 illustrates the concentration of the prodrug (Example 5) of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine (initial concentrations of 30, 100, 300, and 600 μM) when converted to (S)-1-phenyl-2-(pyridin-2-yl)ethylamine during incubation in human intestinal fluid. Figure 1b The (S)-1-phenyl-2-(pyridin-2-yl)ethanamine concentrations when converted from the (S)-1-phenyl-2-(pyridin-2-yl)ethanamine prodrug during incubation in human intestinal fluid are illustrated (Example 5, initial concentrations of 30, 100, 300 and 600 μM). Summary of the invention

[0007] The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof

[0008]

[0009] Where R1 is C 1-6 Alkyl C(O)O(C 1-6 alkoxy), or

[0010]

[0011] AA is a natural amino acid linked by peptide bonds.

[0012] Alkyl is straight-chain or branched and contains 1 to 6, for example 1 to 4, carbon atoms. Alkyl is, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl.

[0013] Alkoxy is straight-chain or branched and contains 1 to 6, for example 1 to 4, carbon atoms.Alkoxy is, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy or tert-butoxy. DETAILED DESCRIPTION

[0014] In one aspect, the present invention provides a compound of formula (I), wherein R1 is C 1-6 Alkyl C(O)O(C 1-6 Alkoxy), for example, C 1-4 Alkyl C(O)O(C 1-4 Alkoxy). Examples include: (CH3)2CHC(O)OCH2O, (CH3)2CHC(O)OCH(CH(CH3)2)O, CH3C(O)OCH(CH3)O or (CH3)2CHC(O)OCH(CH3)O.

[0015] In another aspect, the present invention provides compounds of formula (I), wherein R1 is

[0016]

[0017] And AA is a natural amino acid linked by peptide bonds.

[0018] In another aspect, the present invention provides compounds of formula (I), wherein R1 is

[0019]

[0020] (The chiral center* therefore has the S absolute configuration).

[0021] In another aspect, the invention provides compounds of formula (I), wherein AA is, for example, glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, cystine, methionine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, hydroxylysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline or hydroxyproline. In another aspect, AA is selected from: tyrosine, tryptophan, phenylalanine, leucine, arginine, histidine, lysine and valine. In another aspect, AA is selected from: tyrosine, arginine, histidine, lysine and valine. In another aspect, AA is valine.

[0022] Suitable pharmaceutically acceptable salts are, for example, acid addition salts, such as hydrochloride, hydrobromide, sulfate, phosphate, acetate, fumarate, maleate, tartrate, lactate, citrate, pyruvate, succinate, oxalate, methanesulfonate, p-toluenesulfonate, hydrogensulfate, benzenesulfonate, ethanesulfonate, malonate, xinafoate, ascorbate, oleate, nicotinate, saccharinate, adipate, formate, glycolate, L-lactate, D-lactate, aspartate, malate, L- -tartrate, D-tartrate, stearate, 2-furoate, 3-furoate, naphthalene disulfonate (naphthalene-1,5-disulfonate or naphthalene-1-(sulfonic acid)-5-sulfonate), ethane disulfonate (ethane-1,2-disulfonate or ethane-1-(sulfonic acid)-2-sulfonate), isethionate (2-hydroxyethyl sulfonate), 2-mesitylene sulfonate, 2-naphthalene sulfonate, D-mandelate, L-mandelate, 2,5-dichlorobenzene sulfonate, cinnamate or benzoate.

[0023] A compound of formula (I) which is:

[0024] (S)-(1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)methyl isobutyrate;

[0025] 2-Methyl-1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)propyl isobutyrate;

[0026] 2-Methyl-1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)propyl isobutyrate diastereomer 1;

[0027] 2-Methyl-1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)propyl isobutyrate diastereomer 2;

[0028] 1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)ethyl acetate;

[0029] 1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)ethyl acetate diastereomer 1;

[0030] 1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)ethyl acetate diastereomer 2;

[0031] 1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)ethyl isobutyrate;

[0032] 1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)ethyl isobutyrate diastereomer 1;

[0033] 1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)ethyl isobutyrate diastereomer 2;

[0034] (S)-1-((S)-2-amino-3-methylbutanoyl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide;

[0035] (S)-1-((S)-2,6-diaminohexanoyl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide;

[0036] (S)-1-((S)-2-amino-3-(1H-imidazol-4-yl)propanoyl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide;

[0037] 2-((S)-2-((S)-1-((S)-2-amino-3-(4-hydroxyphenyl)propionyl)pyrrolidine-2-carboxamido)-2-phenylethyl)pyridine;

[0038] 2-((S)-2-((S)-1-((S)-2-amino-3-(1H-indol-3-yl)propanoyl)pyrrolidine-2-carboxamido)-2-phenylethyl)pyridine;

[0039] (S)-1-((S)-2-amino-3-phenylpropanoyl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide; or

[0040] (S)-1-((S)-2-amino-4-methylpentanoyl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide;

[0041] or a pharmaceutically acceptable salt of any one of the above.

[0042] The compounds of the present invention can be prepared by appropriate methods described in the art or by appropriate methods described in the examples. For example, compound (S)-1-phenyl-2-(pyridin-2-yl)ethylamine can be prepared by the methodology in EP-0633879, and the content of this document is incorporated by reference.

[0043] The compounds of the present invention, wherein R1 is

[0044]

[0045] It can be synthesized using a chemical approach via a 2-((S)-2-phenyl-2-((S)-pyrrolidine-2-carboxamido)ethyl)pyridine intermediate.

[0046] Therefore, in another aspect, the present invention provides the compound 2-((S)-2-phenyl-2-((S)-pyrrolidine-2-carboxamido)ethyl)pyridine or a salt thereof:

[0047]

[0048] The salts are, for example, hydrochlorides, hydrobromides, sulfates, phosphates, acetates, methanesulfonates, p-toluenesulfonates, formates or benzoates.

[0049] In another aspect, the present invention provides an intermediate compound 2-((S)-2-phenyl-2-((S)-pyrrolidin-2-iumcarboxamido)ethyl)pyridinium chloride

[0050]

[0051] The compound of formula (I) or a pharmaceutically acceptable salt thereof can be used to treat depression (such as major depression, for example, to treat refractory major depression).

[0052] The compound of formula (I) or a pharmaceutically acceptable salt thereof can be used to treat pain (such as neuropathic pain, chronic pain, phantom limb pain, nociceptive pain, psychogenic pain, incident pain or breakthrough pain).

[0053] The compound of formula (I) or a pharmaceutically acceptable salt thereof can be used to treat Rett syndrome, suicidal ideation, bipolar disorder, obsessive-compulsive disorder, sarin gas poisoning or status epilepticus.

[0054] Thus, the invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof as defined herein for use in therapy.Thus, the term "prodrug" as used herein may refer to a compound of formula (I) in either salt form or free base form.

[0055] In another aspect, the present invention provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as defined herein in the preparation of a medicament for use in therapy.

[0056] In another aspect, the present invention provides a method for administering (S)-1-phenyl-2-(pyridin-2-yl)ethanamine to a patient, the method comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof to a patient, wherein the compound of formula (I) is metabolized in the patient to produce (S)-1-phenyl-2-(pyridin-2-yl)ethanamine.

[0057] In the context of this specification, unless there is a specific indication to the contrary, the term "treatment" also includes "prevention". The terms "therapeutic" and "therapeutically" should also be interpreted accordingly.

[0058] Prevention is contemplated to be particularly relevant to the treatment of persons who have had a previous episode of the disease or condition in question or who are otherwise believed to be at increased risk for the disease or condition in question. Persons at risk for developing a particular disease or condition generally include those who have a family history of the disease or condition or who have been identified by genetic testing or screening as being particularly susceptible to developing the disease or condition.

[0059] The present invention further provides a method for treating depression, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof.

[0060] The present invention further provides a method for treating MDD, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof.

[0061] The present invention further provides a method for treating pain, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof.

[0062] The present invention further provides a method for treating neuropathic pain, chronic pain, phantom limb pain, nociceptive pain, psychiatric pain, incident pain or breakthrough pain, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof.

[0063] Use of the compound of formula (I) or a pharmaceutically acceptable salt thereof for treating depression.

[0064] Use of the compound of formula (I) or a pharmaceutically acceptable salt thereof for treating pain.

[0065] For the above-mentioned therapeutic uses, the dosage administered will necessarily vary with the compound, mode of administration, intended treatment, and specified condition employed. For example, if administered by inhalation, the daily dose of the compounds of the invention may be 0.05 micrograms (μg / kg) to 100 micrograms (μg / kg) per kilogram of body weight. Alternatively, if the compound is administered orally, the daily dose of the compounds of the invention may be 0.01 micrograms (μg / kg) to 100 micrograms (μg / kg) per kilogram of body weight.

[0066] The compound of formula (I) and its pharmaceutically acceptable salt can be used alone, but generally will be administered in the form of a pharmaceutical composition, wherein the compound / salt of formula (I) (active ingredient) is combined with a pharmaceutical excipient, diluent or carrier. Conventional methods for selecting and preparing appropriate pharmaceutical preparations are described, for example, in "Pharmaceuticals-The Science of Dosage Form Designs", ME Aulton, Churchill Livingstone, 1988.

[0067] Depending on the mode of administration, the pharmaceutical composition preferably comprises the following percentage of active ingredient: 0.05-99%w (weight percentage), more preferably 0.05-80%w, still more preferably 0.10-70%w and even more preferably 0.10-50%w, all weight percentages based on the total composition. In some embodiments, the pharmaceutical composition comprises 0.5%w of active ingredient. In some embodiments, the pharmaceutical composition comprises 20%w of active ingredient.

[0068] The present invention also provides a pharmaceutical composition comprising a compound of formula (I) as defined herein, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable adjuvant, diluent or carrier.

[0069] The present invention further provides a process for preparing a pharmaceutical composition of the present invention, which comprises mixing a compound of formula (I) or a pharmaceutically acceptable salt thereof as defined herein with a pharmaceutically acceptable excipient, diluent or carrier.

[0070] For oral administration, the compounds of the present invention can be mixed with the following excipients or carriers: for example, lactose, sucrose, sorbitol, mannitol; starches such as potato starch, corn starch or pullulan; cellulose derivatives; binders such as gelatin or polyvinyl pyrrolidone; and / or lubricants such as magnesium stearate, calcium stearate, polyethylene glycol, wax, paraffin, etc., and then compressed into tablets. If coated tablets are required, the core part prepared as described above can be coated with a concentrated sugar solution containing, for example, gum arabic, gelatin, talc and titanium dioxide. Alternatively, the tablets can be coated with a suitable polymer dissolved in a volatile organic solvent.

[0071] For the preparation of soft gelatin capsules, the compounds of the present invention may be mixed with, for example, vegetable oils or polyethylene glycol. Hard gelatin capsules may contain particles of the compounds using the excipients described above for tablets. In addition, liquid or semisolid preparations of the compounds of the present invention may be filled in hard gelatin capsules.

[0072] The compounds of the present invention may also be administered in combination with other compounds useful in the treatment of the above-mentioned conditions.

[0073] The following examples illustrate the invention. In the examples, certain techniques were used and will now be described.

[0074] High performance liquid chromatography (HPLC) was performed on a reverse phase (RP) column. A linear gradient was used using, for example, mobile phases A (0.1% formic acid in MilliQ H2O or 0.1% NH3 in MilliQ H2O or 10 mM NH4OAc and 5% CH3CN in MilliQ H2O, or 0.05% trifluoroacetic acid in MilliQ H2O, or NH4HCO3 (10 mM) in MilliQ H2O) and B (CH3OH or CH3CN). Mass spectrometry (MS) analysis was performed using electrospray ionization (ESI+ / -), atmospheric pressure photoionization (APPI+ / -) and / or atmospheric pressure chemical ionization (APCI+ / -) in positive and / or negative ion mode.

[0075] Gas chromatography (GC) is performed on a GC equipped with a mass spectrometer (MS) or a flame ionization detector (FID). The MS ion source is electron impact (EI) or chemical ionization (CI, reaction gas: methane). For separation, a capillary column is used, such as DB-5MS (J&W Scientific). A linear temperature gradient is used.

[0076] Supercritical fluid chromatography (SFC) was performed on a normal phase column, using an isocratic flow with mobile phase A (CO2) and, for example, mobile phase B (MeOH, EtOH or IPA).

[0077] Alternatively, high performance liquid chromatography (HPLC) is performed on a normal phase column, using, for example, mobile phases A (heptane) and B (EtOH or IPA) with a linear gradient or isocratic flow.

[0078] NMR spectra were recorded on a 300 MHz (or higher field) NMR spectrometer equipped with an appropriately configured probe. Spectra were recorded at ambient temperature unless otherwise indicated. Chemical shifts are given in ppm low field and high field from TMS (0.00 ppm). The following reference signals were used: residual solvent signals for TMS δ 0.00 or less: DMSO-d6 δ 2.49, CD3OD δ 3.30, acetone-d6 2.04, CDCl3 δ 7.25, or D2O δ 4.79 (unless otherwise indicated). Resonance multiplicities are denoted as s, d, t, q, m, br, and app, respectively, for singlets, doublets, triplets, quartets, multiplets, broad peaks, and distinct peaks.

[0079] List of abbreviations

[0080] DBU 1,8-diazabicyclo[5.4.0]undec-7-ene

[0081] DCM Dichloromethane

[0082] DEA Diethylamine

[0083] DIPEA Diisopropylethylamine

[0084] DMF Dimethylformamide

[0085] DMSO Dimethyl sulfoxide

[0086] EtOAc Ethyl acetate

[0087] HATU 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

[0088] IPA Isopropyl Alcohol

[0089] MTBE Methyl tert-butyl ether

[0090] rt Room temperature or ambient temperature, about 20-25℃

[0091] sat saturated

[0092] T3P Propanephosphonic anhydride

[0093] Example 1

[0094] Isobutyric acid (S)-(1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)methyl ester

[0095]

[0096] Step A

[0097] Chloromethyl isobutyrate

[0098]

[0099] Sodium bicarbonate (2092 mg, 24.91 mmol), tetrabutylammonium hydrogen sulfate (220 mg, 0.65 mmol) and water (6 mL) were added to a solution of isobutyric acid (0.600 mL, 6.47 mmol) in DCM (6 mL). With rapid stirring, chloromethyl chlorosulfonate (0.767 mL, 7.44 mmol) was added at rt and the reaction mixture was subsequently stirred overnight at rt. The reaction mixture was then diluted with DCM (10 mL), washed with water (2 x 10 mL), dried over Na2SO4, filtered and concentrated to give chloromethyl isobutyrate (746 mg, 84%), which was used in the next step without further purification.

[0100] 1H NMR (500MHz, CDCl3) δppm 1.17 (m, 6H), 2.62 (m, 1H), 5.72 (s, 2H).

[0101] Step B

[0102] Isobutyric acid (S)-(1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)methyl ester

[0103]

[0104] Cesium carbonate (1543 mg, 4.74 mmol) and tetrabutylammonium iodide (1749 mg, 4.74 mmol) were added to a solution of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine (313 mg, 1.58 mmol) in anhydrous DMF (8 mL) at room temperature. Carbon dioxide gas was bubbled through the reaction mixture for 30 minutes, followed by a solution of chloromethyl isobutyrate (647 mg, 4.74 mmol) in DMF (2 mL). The reaction mixture was stirred overnight at rt by continuous CO2 gas bubbling and continued to stir over the weekend without further addition of CO2 gas. The reaction mixture was diluted with water and extracted with EtOAc (3x), the combined organic layers were washed with water (2x), brine, dried over Na2SO4 and concentrated. Purification was completed by column chromatography (using an EtOAc gradient (0-60%) in heptane) to give the title compound (239 mg, 44.2%).

[0105] 1H NMR (500 MHz, DMSO-d6) δ ppm 1.00 (m, 6H), 2.46 (m, 1H, partially hidden in DMSO-d6), 3.09 (m, 2H), 5.04 (m, 1H), 5.52 (m, 2H), 7.17-7.24 (m, 3H), 7.27-7.34 (m, 4H), 7.65 (td, 1H), 8.24 (d, 1H), 8.49 (m, 1H).

[0106] Example 2

[0107] 2-Methyl-1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)propyl isobutyrate

[0108] Since there are two possible configurations at the carbon atom indicated by *, Example 2 exists in two different diastereomers. They are referred to as Example 2 Diastereomer 1 and Example 2 Diastereomer 2. The absolute configuration has not been determined.

[0109]

[0110] Step A

[0111] 2-Methyl-1-((4-(methylsulfonyl)phenoxy)carbonyloxy)propyl isobutyrate.

[0112]

[0113] (i) 4-(Methylmercapto)phenol (8.46 g, 57.30 mmol) was taken up in DCM (60 mL), and the reaction flask was then cooled to 0 ° C. 1-chloro-2-methylpropyl chloroformate (4.27 mL, 28.65 mmol) was then added. A solution of 4-methylmorpholine (7.87 mL, 71.63 mmol) in DCM (40 mL) was added dropwise at 0 ° C for 50 minutes and the obtained mixture was stirred at this temperature for 5 minutes, and finally stirred at rt for 150 minutes. The reaction mixture was washed with water (2x), dried over Na2SO4, filtered and evaporated to give 1-chloro-2-methylpropyl carbonate 4-(methylthio)phenyl ester (13.84 g), which was used in the next step without further purification.

[0114] (ii) A mixture of 1-chloro-2-methylpropyl 4-(methylthio)phenyl carbonate (3.50 g, 12.74 mmol), silver(I) oxide (2.95 g, 12.74 mmol) and isobutyric acid (13.00 mL, 140.12 mmol) was heated to 95° C. under argon for 2 hours. The reaction mixture was cooled to rt and stirred overnight at rt, then diluted with MTBE, filtered through celite and washed with more MTBE. The combined filtrates were washed with water (4×25 mL), sat. aq. sodium bicarbonate (2×25 mL), dried (Na 2 SO 4 ), and evaporated to give 3.56 g of 2-methyl-1-((4-(methylthio)phenoxy)carbonyloxy)propyl isobutyrate, which was used in the next step without further purification.

[0115] (iii) 2-methyl-1-((4-(methylthio)phenoxy)carbonyloxy)propyl isobutyrate (3.56 g, 10.91 mmol) was taken up in a mixture of acetone (30 mL) and water (7.50 mL), followed by portionwise addition of oxone (13.41 g, 21.81 mmol) over 5 minutes and then stirred at rt for 2 hours. The reaction mixture was filtered and the filtrate was washed with MTBE (2x50 mL), the volume was reduced to 50 mL (acetone was distilled off) and the resulting mixture was then separated between MTBE and water. The aqueous layer was extracted with MTBE and the combined organics were dried over Na2SO4, filtered and evaporated to give 1.89 g of the title compound which was used in the next step without further purification.

[0116] Step B:

[0117] 2-Methyl-1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)propyl isobutyrate

[0118]

[0119] A solution of 2-methyl-1-((4-(methylsulfonyl)phenoxy)carbonyloxy)propyl isobutyrate (0.389 g, 1.08 mmol) in acetonitrile (2 mL) was added to a stirred mixture of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine (0.215 g, 1.08 mmol) and sodium bicarbonate (0.182 g, 2.17 mmol) in acetonitrile (3 mL) and the reaction mixture was stirred at rt for 2 hours. EtOAc and sat aq NaHCO3 were separated, the organic layer was washed with sat aq NaHCO3 and evaporated to give 413 mg of material which was purified by column chromatography using a gradient of EtOAc (0-50%) in heptane to give 235 mg of a mixture of two diastereomers of 2-methyl-1-(S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)propyl isobutyrate.

[0120] The analysis and separation of diastereomers were completed on Chiralpak AD-H, 4.6*250mm; 5μm using 10% MeOH / 90% CO2 at a flow rate of 3mL / min, and on Chiralpak AD-H, 20*250mm; 5μm using 10% MeOH / 90% CO2 at a flow rate of 50mL / min, respectively.

[0121] Example 2, Diastereomer 1

[0122] 105 mg of diastereomer 1 was obtained by chiral separation as the first eluting diastereomer in 99% optical purity. Rotamer mixture:

[0123] 1 H NMR (500 MHz, DMSO-d6) δppm 0.66-1.07 (m), 1.86 (m), 2.39 (m), 2.98-3.21 (m), 5.01 (m), 6.31 (m), 7.10-7.26 (m), 7.26-7.37 (m), 7.56-7.73 (m), 8.10 (d), 8.43-8.55 (m). Complete absence of protons in the spectrum: 28. Major / minor rotamer ratio: 1 / 0.15.

[0124] MS (ES+APCI+) m / z 385 (M+H) +

[0125] Example 2, Diastereomer 2

[0126] 104 mg of diastereomer 2 was obtained as the second eluting diastereomer. Rotamer mixture:

[0127] 1H NMR (500 MHz, DMSO-d6) δppm 0.48-0.69 (m), 0.75-0.87 (m), 0.87-1.07 (m), 1.73 (m), 1.85 (m), 2.40 (m), 2.98 (m), 3.03-3.18 (m), 4.97 (m), 6.22-6.38 (m), 7.13-7.26 (m), 7.26-7.37 (m), 7.65 (m), 7.78 (d), 8.06 (d), 8.48 (m). The spectrum is completely free of protons: 28. Major / minor rotamer ratio: 1 / 0.17.

[0128] MS (ES+APCI+) m / z 385 (M+H) + .

[0129] Optical purity = 99%

[0130] Example 3

[0131] 1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)ethyl acetate

[0132] Since there are two possible configurations at the carbon atom indicated by *, there are two different diastereomers of Example 3. They are called Example 3 Diastereomer 1 and Example 3 Diastereomer 2. The absolute configuration has not been determined.

[0133]

[0134] Step A:

[0135] 1-((4-(methylsulfonyl)phenoxy)carbonyloxy)ethyl acetate

[0136]

[0137] (i) A mixture of 1-chloroethyl 4-(methylthio)phenyl carbonate (4.6 g, 18.65 mmol), silver(I) oxide (4.32 g, 18.65 mmol) and acetic acid (11.75 mL, 205.1 mmol) was heated to 95° C. under argon for 2 hours. The reaction mixture was cooled to rt and diluted with MTBE, filtered through celite and washed with more MTBE. The combined filtrates were washed with water (4 x 25 mL), sat. aq. sodium bicarbonate (2 x 25 mL), dried (Na2SO4), and evaporated to give 1.79 g of 1-((4-(methylthio)phenoxy)carbonyloxy)ethyl acetate which was used in the next step without further purification.

[0138] (ii) 1-((4-(methylthio)phenoxy)carbonyloxy)ethyl acetate (1.79 g, 6.62 mmol) was taken up in a mixture of acetone (16 mL) and water (4.00 mL). Potassium persulfate (8.14 g, 13.24 mmol) was added portionwise over 5 minutes and then stirred overnight at rt. The reaction mixture was filtered and washed with MTBE (2x50 mL), and the volume was reduced to 50 mL (acetone was distilled off). The product was separated between MTBE and water. The aqueous layer was extracted with MTBE and the combined organics (Na2SO4) were dried, filtered and evaporated to give 832 mg of the title compound, which was used in the next step without further purification.

[0139] Step B:

[0140] 1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)ethyl acetate

[0141]

[0142] A solution of 1-((4-(methylsulfonyl)phenoxy)carbonyloxy)ethyl acetate (302 mg, 1.00 mmol) in acetonitrile (1 mL) was added to a stirred mixture of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine (198 mg, 1 mmol) and sodium bicarbonate (168 mg, 2.00 mmol) in acetonitrile (4 mL) and the reaction mixture was stirred at rt for 4 hours. The reaction mixture was then partitioned between EtOAc and sat aq NaHCO3, the organic layer was washed with sat aq NaHCO3, brine, dried over Na2SO4 and evaporated. The mixture was pre-purified by column chromatography using a gradient of EtOAc (0-50%) in heptane to afford 160 mg of the title compound as a mixture of its two diastereomers.

[0143] The analysis and separation of the diastereomers were carried out on Phenomenex LuxC4, 4.6*250mm; 5μm using 30% MeOH+DEA / 70% CO2 at a flow rate of 3 mL / min, and on Phenomenex LuxC4, 20*250mm; 5μm using 25% MeOH+DEA / 75% CO2 at a flow rate of 50 mL / min.

[0144] Example 3, Diastereomer 1

[0145] 29 mg of diastereomer 1 was obtained by chiral separation as the first eluting diastereomer in 99% optical purity.

[0146] 1H NMR (500 MHz, DMSO-d6) δ ppm 1.31 (d, 3H), 1.92 (s, 3H), 3.00-3.16 (m, 2H), 5.00 (td, 1H), 6.52 (q, 1H), 7.14-7.25 (m, 3H), 7.30 (d, 4H), 7.65 (t, 1H), 8.13 (d, 1H), 8.50 (d, 1H). The signals in the spectrum were amplified and no DMSO-signal splitting was observed.

[0147] 13 C NMR (126MHz, DMSO-d6) δ19.6,20.7,44.6,54.9,88.7,121.6,123.8,126.4,126.9,128.3,136.2,143.0,149.0,153.2,158.0,168.6ppm. MS(ES+)m / z328(M+H) + .

[0148] Example 3, Diastereomer 2

[0149] 39 mg of diastereomer 2 was obtained as the second eluting diastereomer.

[0150] 1 H NMR (500 MHz, DMSO-d6) δ ppm 1.32 (d, 3H), 1.95 (s, 3H), 3.01-3.18 (m, 2H), 5.00 (td, 1H), 6.53 (q, 1H), 7.13-7.24 (m, 3H), 7.29 (d, 4H), 7.65 (t, 1H), 8.17 (d, 1H), 8.49 (d, 1H). The signals in the spectrum were amplified and no DMSO-signal splitting was observed.

[0151] 13 C NMR(126MHz,DMSO-d6)δ19.6,20.7,44.4,54.9,88.6,121.6,123.7,126.4,126.9,128.3,136.2,143.0,149.0,153.1,158.0,168.5.MS(ES+)m / z 329(M+H) + .

[0152] Optical purity 98%.

[0153] Example 4

[0154] 1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)ethyl isobutyrate

[0155] Since there are two possible configurations at the carbon atom indicated by *, Example 4 exists in two different diastereomers. They are referred to as Example 4 Diastereomer 1 and Example 4 Diastereomer 2. The absolute configuration has not been determined.

[0156]

[0157] Step A:

[0158] 1-((4-(methylsulfonyl)phenoxy)carbonyloxy)ethyl isobutyrate

[0159]

[0160] (i) 4-(Methylmercapto)phenol (4.91 g, 35.00 mmol) was taken up in DCM (100 mL) and cooled to 0°C. 1-Chloroethyl chloroformate (1.888 mL, 17.50 mmol) was added. A solution of 4-methylmorpholine (4.81 mL, 43.74 mmol) in DCM (20 mL) was added dropwise over 10 minutes at 0°C and the resulting mixture was stirred at this temperature for 5 minutes. The reaction mixture was stirred at rt for 180 minutes, then diluted with DCM, washed with water (2x), dried over Na2SO4, filtered and evaporated to give 1-chloroethyl carbonate 4-(methylthio)phenyl ester (6.94 g), which was used in the next step without further purification.

[0161] (ii) A mixture of 1-chloroethyl 4-(methylthio)phenyl carbonate (2.3 g, 9.32 mmol), silver(I) oxide (2.160 g, 9.32 mmol) and isobutyric acid (9.51 ml, 102.55 mmol) was heated to 95° C. under argon for 2 hours. The reaction mixture was cooled to rt and diluted with MTBE, filtered through celite, and washed with more MTBE. The combined filtrates were washed with water (4×25 mL), sat. aq. sodium bicarbonate (2×25 mL), dried (Na 2 SO 4 ) and evaporated to give 1.16 g of product which was used in the next step without further purification.

[0162] (iii) 1-((4-(methylthio)phenoxy)carbonyloxy)ethyl isobutyrate (1.16 g, 3.89 mmol) was taken up in a mixture of acetone (12 mL) and water (3.00 mL). Potassium oxone (4.78 g, 7.78 mmol) was added portionwise over 5 minutes and then stirred overnight at rt. The reaction mixture was filtered and the filtrate was washed with MTBE (2x50 mL), the volume was reduced to about 50 mL by distilling off acetone and then separated between MTBE and water. The aqueous layer was extracted with MTBE and the combined organics were dried over Na2SO4, filtered and evaporated to give 531 mg of the title compound, which was used in the next step without further purification.

[0163] Step B

[0164] 1-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyloxy)ethyl isobutyrate

[0165]

[0166] A solution of 1-((4-(methylsulfonyl)phenoxy)carbonyloxy)ethyl isobutyrate (0.500 g, 1.51 mmol) in acetonitrile (2 mL) was added to a stirred mixture of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine (0.300 g, 1.51 mmol) and sodium bicarbonate (0.254 g, 3.03 mmol) in acetonitrile (6 mL) and the reaction mixture was stirred overnight. The mixture was partitioned between EtOAc and sat aq NaHCO3, the organic layer was washed with sat aq NaHCO3, brine, dried over Na2SO4 and evaporated to afford 0.70 g., which was purified by column chromatography using a gradient of EtOAc (0-50%) in heptane to afford the title compound (0.309 g, 57.2%) as a mixture of two diastereomers.

[0167] The analysis and separation of diastereomers were completed on Phenomenex LuxC4, 4.6*250mm; 5μm using 15% MeOH+DEA / 85% CO2 at a flow rate of 3mL / min, and on Phenomenex LuxC4, 20*250mm; 5μm using 15% MeOH+DEA / 85% CO2 at a flow rate of 50mL / min, respectively.

[0168] Example 4, Diastereomer 1

[0169] 25 mg of diastereomer 1 was obtained by chiral separation as the first eluting diastereomer in 99% optical purity. Rotamer mixture:

[0170] 1 H NMR (500 MHz, DMSO-d6) δ ppm 0.86-1.05 (m), 1.19 (d), 1.32 (d), 2.31-2.44 (m), 2.98-3.20 (m), 4.94-5.07 (m), 6.48 (d), 6.52 (q), 7.15-7.25 (m), 7.25-7.34 (m), 7.60-7.70 (m), 7.77 (d), 8.06-8.18 (m), 8.44-8.55 (m). Complete absence of protons in the spectrum: 24. Major / minor ratio: 1:0.07.

[0171] MS (ES+APCI+) m / z 357 (M+H) + .

[0172] UV purity = 100%.

[0173] Example 4, Diastereomer 2

[0174] 25 mg of diastereomer 2 was obtained as the second eluting isomer. Rotamer mixture:

[0175] 1 H NMR (500 MHz, DMSO-d6) d ppm 0.82-0.94 (m), 0.94-1.04 (m), 1.25 (d), 1.32 (d), 2.24-2.32 (m), 2.34-2.44 (m), 3.00-3.17 (m), 5.02 (td), 6.48 (d), 6.52 (q), 7.15-7.25 (m), 7.25-7.38 (m), 7.55-7.77 (m), 8.16 (d), 8.44-8.57 (m). Complete absence of protons in the spectrum: 24. Major / minor ratio: 1:0.08.

[0176] MS (ES+APCI+) m / z 357 (M+H) + .

[0177] UV purity = 100%.

[0178] Optical purity = 99%.

[0179] Examples 5-12 use common intermediates, and their preparation is now described.

[0180] Preparation of Common Intermediates for Examples 5-12

[0181] 2-((S)-2-phenyl-2-((S)-pyrrolidin-2-iumcarboxamido)ethyl)pyridinium dichloride (which may also be referred to as: (S)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide dihydrochloride)

[0182]

[0183] Step A

[0184] (S)-tert-Butyl 2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidine-1-carboxylate

[0185]

[0186] T3P (50% wt. in DMF, 0.86 mL, 1.47 mmol) was added dropwise to a solution of (1S)-1-phenyl-2-(pyridin-2-yl)ethan-1-amine (200 mg, 0.737 mmol), Boc-L-proline (159 mg, 0.737 mmol) and DIPEA (0.64 mL, 3.69 mmol) in CH2Cl2 (7.4 mL) at 0°C. The reaction mixture was gradually warmed to rt while stirring overnight. The organic layer was washed twice with 5% aq. NaHCO3 and once with brine, dried over Na2SO4, filtered and concentrated. The product was purified using a 50 g Snap column eluting with a gradient of MeOH and CH2Cl2 (0% MeOH / 100% CH2Cl2→10% MeOH / 90% CH2Cl2) to give the title compound 305 mg (>100%).

[0187] 1 H NMR (300MHz, CDCl3): ppm 1.35(s,9H),1.70–1.90(m,2H),2.05–2.19(m,2H),3.13(dd,1H),3.29–3.39(m,1H),3.46–3.62(m,2H),4.20– 4.29(m,2H),5.39(q,1H),6.91(d,1H),7.09–7.14(m,1H),7.16–7.23(m,5H),7.49(td,1H),8.45–8.59(m,1H).

[0188] Alternative methods:

[0189] Boc-L-Pro-OH (2.0 g, 9.29 mmol) was dissolved in anhydrous DMF (15 mL). HATU (3.7 g, 9.76 mmol) and Hunig's base (5.3 mL, 30.66 mmol) were added and the mixture was stirred at rt for 30 minutes. Subsequently, 2-[(2S)-2-amino-2-phenylethyl]pyridin-1-ium dichloride (2.5 g, 9.29 mmol) was added to the solution and the mixture was stirred at rt for 3 hours and 30 minutes. Water was added and the mixture was extracted with EtOAc 3x. The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The product was purified on a SiO2 column eluting with a gradient (0% MeOH / 50% EtOAc / 50% heptane→0% MeOH / 100% EtOAc / 0% heptane→10% MeOH / 90% EtOAc / 0% heptane) and then on a C-18 column eluting with a gradient of MeOH and water (0-100% MeOH) to give 2.9 g (78%) of the title compound.

[0190] 1 H NMR (300MHz, CDCl3): ppm 1.35(s,9H),1.70–1.90(m,2H),2.05–2.19(m,2H),3.13(dd,J=13.8,7.1Hz,1H),3.29–3.39(m,1H),3.46–3.62(m,2H),4.20–4.29(m,1 H),5.39(q,J=6.8Hz,1H),6.91(d,J=7.3Hz,1H),7.09–7.14(m,1H),7.16–7.23(m,5H),7.49(td,J=7.63,1.8Hz,1H),8.45–8.59(m,1H).

[0191] Step B

[0192] (S)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide dihydrochloride

[0193]

[0194] (S)-tert-Butyl 2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidine-1-carboxylate (305 mg, 0.771 mmol) was dissolved in 4 M HCl in 1,4-dioxane (7.7 mL, 30.8 mmol) at 0°C. The reaction mixture was gradually warmed to rt while stirring overnight. The volatiles were removed under vacuum and the product was ground in MTBE. The solid was recovered by filtration on a Buchner funnel, washed with MTBE and dried under vacuum to afford 263 mg (93%) of the title compound as the dihydrochloride salt.

[0195] 1 H NMR (300 MHz, DMSO-d6): ppm 1.61–1.84(m,3 H),2.30–2.36(m,1H),3.05–3.11(m,2 H),3.39–3.47(m,2 H),4.05–4.20(m,2 H),5.34(q,1 H),7.17–7.41(m,5 H),8.20–8.42(m,1 H),8.67–8.75(m,1 H),9.45–9.62(m,2H).

[0196] Alternative methods:

[0197] Tert-butyl (2S)-2-{[(1S)-1-phenyl-2-(pyridin-2-yl)ethyl]carbamoyl}pyrrolidine-1-carboxylate (2.9 g, 7.33 mmol) was dissolved in 4 M HCl in 1,4-dioxane (73 mL, 293.31 mmol). The reaction mixture was stirred at rt for 3 hours. The volatiles were removed under vacuum and the product was triturated in MTBE. The solid was recovered by filtration on a Buchner funnel, washed with MTBE and dried under vacuum to obtain 2.7 g (100%) of (2S)-2-{[(1S)-1-phenyl-2-(pyridin-1-ium-2-yl)ethyl]carbamoyl}pyrrolidine-1-ium.

[0198] 1H NMR (300 MHz, D2O): ppm 1.66–1.83 (m, 3 H), 2.20–2.26 (m, 1 H), 3.14 (t, J = 6.9 Hz, 2 H), 3.34 (dd, J = 14.1, 8.2 Hz, 1 H), 3.51 (dd, J = 14.4, 7.3 Hz, 1 H),4.18(dd,J=8.5,6.2 Hz,1 H),5.11(t,J=7.9 Hz,1 H),7.06–7.10(m,2 H),7.15–7.21(m,3 H),7.61–7.70(m,2 H),8.23(dt,J=7.9,1.5 Hz,1 H), 8.37 (d, J = 5.8 Hz, 1 H).

[0199] Example 5

[0200] (S)-1-((S)-2-amino-3-methylbutyryl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide

[0201]

[0202] The title compound was prepared as the dihydrochloride salt of (S)-1-((S)-2-amino-3-methylbutanoyl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide.

[0203] Step A

[0204] tert-Butyl (S)-3-methyl-1-oxo-1-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)butan-2-ylcarbamate

[0205]

[0206] T3P (50% wt. in DMF, 0.83 mL, 1.42 mmol) was added dropwise to a solution of (S)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide dihydrochloride (263 mg, 0.714 mmol), Boc-L-valine (155 mg, 0.714 mmol) and DIPEA (0.62 mL, 3.57 mmol) in DCM (7 mL) at 0°C. The reaction mixture was gradually warmed to rt overnight with stirring. The organic layer was washed twice with 5% aq. NaHCO3, once with brine, dried over Na2SO4, filtered and concentrated. The product was purified using a 50 g Snap column eluting with MeOH and CH2Cl2 gradient (5% MeOH / 95% CH2Cl2→10% MeOH / 90% CH2Cl2) to give the title compound 258 mg (73%).

[0207] 1 H NMR (300MHz, CDCl3): ppm 0.91(d,3H),1.00(d,3H),1.43(s,9H),1.85–1.99(m,2H),2.00–2.16(m ,1H),2.17–2.21(m,1H),3.12–3.27(m,2H),3.47–3.62(m,1H),3.63–3.7 8(m,1H),4.32(dd,1H),4.58(d,1H),5.23–5.35(m,2H),6.97(d,1H),7. 10(dd,1H),7.14–7.30(m,5H),7.50(td,1H),7.84(d,1H),8.48(dd,1H).

[0208] Step B

[0209] (S)-1-((S)-2-amino-3-methylbutyryl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide dihydrochloride

[0210]

[0211] (S)-3-methyl-1-oxo-1-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)butan-2-ylcarbamic acid tert-butyl ester (258 mg, 0.522 mmol) was dissolved in a solution of 4M HCl in 1,4-dioxane (5.2 mL, 30.8 mmol). The reaction mixture was stirred overnight. The volatiles were removed under vacuum and the product was ground in EtOAc. The solid was recovered by filtration on a Buchner funnel and then ground in Et2O. After filtration on a Buchner funnel, the solid was dried under vacuum to obtain 200 mg (82%) of the title compound as a dihydrochloride salt. The title compound as a dihydrochloride salt was then converted to a free base by techniques known to those skilled in the art. Alternatively, the title compound can be prepared as a fumarate salt by methods known to those skilled in the art.

[0212] 1 H NMR(300MHz,CD3OD):ppm 0.97(d,3H),1.04(d,3H),1.68-1.79(m,1H),1.86-2.05(m,2H),2.13-2.27(m,2H),3.50-3.61(m,3H),3.68-3.75(m,1 H),4.02(d,1H),4.45(dd,1H),5.40(dd,1H),7.31-7.43(m,5H),7.90(t,1H),8.02(d,1H),8.51(td,1H),8.75(d,1H).

[0213] [M+H]+=395.27

[0214] Example 6

[0215] (S)-1-((S)-2,6-Diaminohexanoyl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide

[0216]

[0217] Preparation of the title compound, 2-[(2S)-2-{[(2S)-1-[(2S)-2,5-diamidinohexanoyl]pyrrolidin-2-yl]formamido}-2-phenylethyl]pyridin-1-ium trichloride

[0218] Step A

[0219] (S)-tert-Butyl 6-oxo-6-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)hexane-1,5-diyldicarbamate

[0220]

[0221] N-α, N-ε-di(tert-butoxycarbonyl)-L-lysine dicyclohexyl ammonium salt (287mg, 0.54mmol) is dissolved in anhydrous DMF (4mL). HATU (217mg, 0.57mmol) and DIPEA (0.21mL, 1.19mmol) are added and the mixture is stirred at rt for 30 minutes. Subsequently, (2S)-2-{[(1S)-1-phenyl-2-(pyridin-1-ium-2-yl)ethyl]carbamoyl}pyrrolidine-1-ium dichloride (200mg, 0.54mmol) is added to the solution and the mixture is stirred at rt for 18 hours. Water is added and the mixture is extracted with EtOAc 3x. The organic phase is washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The product was purified on a SiO2 column eluting with a gradient (0% MeOH / 50% EtOAc / 50% heptane→0% MeOH / 100% EtOAc / 0% heptane→10% MeOH / 90% EtOAc / 0% heptane) followed by purification on a C-18 column eluting with a gradient of MeOH and water (0-100% MeOH) to give 80 mg (24%) of the title compound.

[0222] 1 H NMR (300MHz, CDCl3): ppm 1.34–1.39(m,2H),1.41(s,9H),1.43(s,9H),1.52–1.64(m,3H),1.68–2.02(m,4H ),2.08–2.22(m,2H),3.02–3.16(m,3H),3.20–3.28(m,1H),3.48–3.57(m,1H),3.6 0–3.70(m,1H),4.40–4.51(m,1H),4.54–4.57(m,1H),4.97–5.08(m,1H),5,29-5.3 9(m,2H),7.00(d,1H),7.10–7.32(m,6H),7.52(td,1H),7.86(d,1H),8.49(d,1H).

[0223] Step B

[0224] Synthesis of 2-[(2S)-2-{[(2S)-1-[(2S)-2,5-diamidinohexanoyl]pyrrolidin-2-yl]formamido}-2-phenylethyl]pyridin-1-ium trichloride

[0225]

[0226] Tert-butyl (S)-6-oxo-6-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)hexan-1,5-diyldicarbamate (80 mg, 0.13 mmol) was dissolved in 4M HCl in 1,4-dioxane (1.6 mL, 6.4 mmol). The reaction mixture was stirred at rt for 18 hours. The volatiles were removed under vacuum and the product was triturated in MTBE. The solid was recovered by filtration on a Buchner funnel, washed with MTBE and dried under vacuum. The solid was dissolved in water and freeze-dried to give 60 mg (88%) of the title compound.

[0227] 1 H NMR(300MHz,D2O):ppm 1.23-1.35(m,2H),1.46-1.59(m,3H),1.65-1.80(m,4H),2.04-2.13(m,1H),2.80(t,2H),3.31-3.43(m,2H),3.47-3.57(m,2H) ,4.16(t,1H),4.28(t,1H),5.11(t,1H),7.10-7.13(m,2H),7.16-7.26(m,3H),7.68-7.73(m,2H),8.31(td,1H),8.42(dd,1H);

[0228] [M+H] + =424.2.

[0229] Example 7

[0230] (S)-1-((S)-2-amino-3-(1H-imidazol-4-yl)propanoyl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide

[0231]

[0232] Preparation of the title compound, 2-[(2S)-2-{[(2S)-1-[(2S)-2-amino-3-(1H-imidazol-1-4-yl)propionyl]pyrrolidin-2-yl]formamido}-2-phenylethyl]pyridin-1-ium trichloride

[0233] Step A

[0234] tert-Butyl (S)-3-(1H-imidazol-4-yl)-1-oxo-1-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)propan-2-ylcarbamate

[0235]

[0236] Boc-His-OH (166 mg, 0.65 mmol) was dissolved in anhydrous DMF (4 mL). HATU (260 mg, 0.68 mmol) and DIPEA (0.38 mL, 2.85 mmol) were added and the mixture was stirred at rt for 30 mol. Subsequently, 2-((S)-2-phenyl-2-((S)-pyrrolidine-2-ium formamido)ethyl)pyridinium chloride (240 mg, 0.65 mmol) was added and the mixture was stirred at rt for 66 hours. Water was added and the mixture was extracted with EtOAc 3x. The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The product was purified on a SiO2 column eluting with a gradient of MeOH and CH2Cl2 (0%-13% MeOH) followed by purification on a C-18 column eluting with a gradient of MeOH and water (0-100% MeOH) to give 100 mg (29%) of the title compound.

[0237] 1 H NMR (300MHz, CDCl3): ppm 1.44(s,9H),1.77–2.00(m,3H),2.05–2.19(m,1H),3.03–3.14(m,2H),3.23(dd,1H),3.40(dd,1H),3.52–3.58(m,1H),4.50 –4.62(m,2H),5,43-5.50(m,2H),6.91–6.97(m,2H),7.13–7.27(m,5H),7.53(t,1H),7.65(s,1H),8.53(d,1H),8.67(d,1H).

[0238] Step B

[0239] 2-((S)-2-((S)-1-((S)-2-Amino-3-(1H-imidazol-1-4-yl)propionyl)pyrrolidine-2-carboxamido)-2-phenylethyl)pyridinium trichloride

[0240]

[0241] Tert-butyl (S)-3-(1H-imidazole-4-yl)-1-oxo-1-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)propan-2-ylcarbamate (100 mg, 0.19 mmol) was dissolved in 4M HCl in 1,4-dioxane (2.3 mL, 9.39 mmol). The reaction mixture was stirred at rt for 18 hours. The volatiles were removed under vacuum and the product was ground in MTBE. The solid was recovered by filtration on a Buchner funnel, washed with MTBE and dried under vacuum. The solid was dissolved in water and freeze-dried to give 85 mg (69%) of the title compound.

[0242] 1 H NMR(300MHz,D2O):ppm 1.50-1.58(m,1H),1.69-1.76(m,2H),2.07-2.14(m,1H),3.12-3.21(m,3H),3.38(dd,7.3Hz,1H),3.48-3.56(m,2H),4.32(t,1H),4.4 3(t,1H),5.14(t,1H),7.08-7.12(m,2H),7.15-7.23(m,3H),7.26(s,1H),7.65-7.70(m,2H),8.26(td,1H),8.39(d,1H),8.49(d,1H);

[0243] [M+H] + =433.2;

[0244] [M+Na] + =455.1.

[0245] Example 8

[0246] 2-((S)-2-((S)-1-((S)-2-amino-3-(4-hydroxyphenyl)propionyl)pyrrolidine-2-carboxamido)-2-phenylethyl)pyridine dihydrochloride

[0247]

[0248] The title compound may also be referred to as: 2-[(2S)-2-{[(2S)-1-[(2S)-2-amino-3-(4-hydroxyphenyl)propionyl]pyrrolidin-2-yl]formamido}-2-phenylethyl]pyridin-1-ium dichloride

[0249] Step A

[0250] tert-Butyl (S)-3-(4-hydroxyphenyl)-1-oxo-1-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)propan-2-ylcarbamate

[0251]

[0252] Boc-Tyr-OH (229 mg, 0.81 mmol) was dissolved in anhydrous DMF (5 mL). HATU (325 mg, 0.86 mmol) and DIPEA (0.47 mL, 2.69 mmol) were added and the mixture was stirred at rt for 30 minutes. Subsequently, 2-((S)-2-phenyl-2-((S)-pyrrolidine-2-ium formamido)ethyl)pyridinium chloride (300 mg, 0.81 mmol) was added to the solution and the mixture was stirred at rt for 66 hours. Water was added and the mixture was extracted with EtOAc 3x. The organic phase was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The product was purified on a SiO2 column eluting with (0% MeOH / 50% EtOAc / 50% heptane→0% MeOH / 100% EtOAc / 0% heptane→15% MeOH / 85% EtOAc / 0% heptane) followed by a C-18 column eluting with a gradient of MeOH and water (0-100% MeOH) to give 180 mg (40%) of the title compound.

[0253] 1 H NMR (300MHz,CDCl3):ppm 1.44(s,9H),1.73–2.02(m,3H),2.16–2.22(m,1H),2.92–3.09(m,4H),3.35 –3.42(m,1H),3.48–3.51(m,1H),3.53–3.68(m,1H),4.48–4.51(m,1H),4.65 –4.73(m,1H),5.06–5.14(m,1H),5.43(d,1H),6.61(d,1H),6.86(d,,2H),6. 94–6.99(m,2H),7.08–7.26(m,6H),7.42(dd,1H),8.49(d,1H),8.63(s,1H).

[0254] Step B:

[0255] 2-((S)-2-((S)-1-((S)-2-Amino-3-(4-hydroxyphenyl)propionyl)pyrrolidine-2-carboxamido)-2-phenylethyl)pyridinium dichloride

[0256]

[0257] Tert-butyl (S)-3-(4-hydroxyphenyl)-1-oxo-1-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)propan-2-ylcarbamate (180 mg, 0.32 mmol) was dissolved in 4M HCl in 1,4-dioxane (3.2 mL, 12.89 mmol). The reaction mixture was stirred at rt for 18 hours. The volatiles were removed under vacuum and the product was ground in MTBE. The solid was recovered by filtration on a Buchner funnel, washed with MTBE and dried under vacuum. The solid was dissolved in water and freeze-dried to give 150 mg (88%) of the title compound.

[0258] 1 H NMR(300MHz,D2O):ppm 1.48-1.57(m,1H),1.68-1.73(m,2H),1.98-2.05(m,1H),2.68-2.76(m,1H),2.93-3.00(m,1H),3.10-3.19(m,1H),3.40-3.63(m,3H ),4.20-4.28(m,2H),5.14(t,1H),6.65(d,2H),6.96(d,2H),7.13-7.22(m,5H),7.63-7.66(m,2H),8.19-8.22(m,1H),8.40(d,1H);

[0259] [M+H] + =459.2.

[0260] Example 9

[0261] 2-((S)-2-((S)-1-((S)-2-amino-3-(1H-indol-3-yl)propionyl)pyrrolidine-2-carboxamido)-2-phenylethyl)pyridine dihydrochloride

[0262]

[0263] The title compound may also be referred to as: 2-[(2S)-2-{[(2S)-1-[(2S)-2-amino-3-(1H-indol-3-yl)propionyl]pyrrolidin-2-yl]formamido}-2-phenylethyl]pyridin-1-ium dichloride

[0264] Step A

[0265] tert-Butyl (S)-3-(1H-indol-3-yl)-1-oxo-1-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)propan-2-ylcarbamate

[0266]

[0267] Boc-Trp-OH (207mg, 0.68mmol) is dissolved in anhydrous DMF (4mL). HATU (271mg, 0.71mmol) and DIPEA (0.39mL, 2.24mmol) are added and the mixture is stirred at room temperature for 30 minutes. Subsequently, (2S)-2-{[(1S)-1-phenyl-2-(pyridin-1-ium-2-yl)ethyl]carbamoyl}pyrrolidine-1-ium dichloride (250mg, 0.68mmol) is added and the mixture is stirred at rt for 18 hours. Water is added and the mixture is extracted with EtOAc 3x. The organic phase is washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The product was purified on a SiO2 column eluting with a gradient (0% MeOH / 50% EtOAc / 50% heptane→0% MeOH / 100% EtOAc / 0% heptane→15% MeOH / 85% EtOAc / 0% heptane) to give 410 mg (quantified) of the title compound.

[0268] 1 H NMR (300MHz, CDCl3): ppm 1.44(s,9H),1.77–1.89(m,3H),2.14–2.24(m,1H),3.03–3.24(m,4H), 3.28–3.36(m,2H),3.54–3.65(m,1H),4.52–4.58(m,1H),4.79–4.88(m ,1H),5,12-5.27(m,1H),5.34–5.41(m,1H),6.63(d,1H),6.94–7.31(m ,8H),7.38–7.53(m,3H),7.62–7.67(m,1H),8.59(d,1H),9.87(s,1H).

[0269] Step B

[0270] 2-((S)-2-((S)-1-((S)-2-Amino-3-(1H-indol-3-yl)propionyl)pyrrolidine-2-carboxamido)-2-phenylethyl)pyridinium dichloride

[0271]

[0272] Tert-butyl (S)-3-(1H-indol-3-yl)-1-oxo-1-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)propan-2-ylcarbamate (395 mg, 0.68 mmol) was dissolved in 1M HCl in Et2O (27.0 mL, 27.20 mmol). The reaction mixture was stirred at rt for 4 hours. The product was triturated in Et2O and the solid was recovered by filtration on a Buchner funnel, washed with Et2O and dried under vacuum. The solid was dissolved in water and cooled to dryness to give 175 mg (44%) of the title compound.

[0273] 1 H NMR(300MHz,D2O):ppm 1.41-1.53(m,1H),1.63-1.72(m,2H),1.93-2.04(m,1H),2.85(dd,1H),2.94-3.55(m,5H),4.19-4.3 1(m,2H),5.08(t,1H),6.90-7.31(m,9H),7.38(d,1H),7.55-7.58(m,2H),8.15(t,1H),8.31(d,1H);

[0274] [M+H] + =482.2,

[0275] [M+Na] + =504.1.

[0276] Example 10

[0277] (S)-1-((S)-2-amino-3-phenylpropanoyl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide

[0278]

[0279] Preparation of the title compound, 2-[(2S)-2-{[(2S)-1-[(2S)-2-amino-3-phenylpropionyl]pyrrolidin-2-yl]formamido}-2-phenylethyl]pyridin-1-ium dichloride

[0280] Step A

[0281] tert-Butyl (S)-1-oxo-3-phenyl-1-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)propan-2-ylcarbamate

[0282]

[0283] Boc-Phe-OH (180mg, 0.68mmol) is dissolved in anhydrous DMF (4mL). HATU (271mg, 0.71mmol) and DIPEA (0.39mL, 2.24mmol) are added and the mixture is stirred at rt for 30 minutes. Subsequently, (2S)-2-{[(1S)-1-phenyl-2-(pyridin-1-ium-2-yl)ethyl]carbamoyl}pyrrolidine-1-ium dichloride (250mg, 0.68mmol) is added to the solution and the mixture is stirred at rt for 18 hours. Water is added and the mixture is extracted with EtOAc 3x. The organic phase is washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The product was purified on a SiO2 column eluting with a gradient (0% MeOH / 50% EtOAc / 50% heptane→0% MeOH / 100% EtOAc / 0% heptane→15% MeOH / 85% EtOAc / 0% heptane) followed by purification on a C-18 column eluting with a gradient of MeOH and water (0-100% MeOH) to give 170 mg (46%) of the title compound.

[0284] 1 H NMR (300MHz, CDCl3): ppm 1.44(s,9H),1.73–1.91(m,3H),2.10–2.19(m,1H),3.10–3.40(m,3H),3.45–3.61(m,2H),4.27–4.35(m,1H),4.50–4.56(m,1 H),4.60–4.69(m,1H),5,17-5.41(m,3H),6.93–7.03(m,2H),7.06–7.32(m,9H),7.47–7.56(m,1H),7.82(d,1H),8.48(d,1H).

[0285] Step B

[0286] 2-((S)-2-((S)-1-((S)-2-Amino-3-phenylpropionyl)pyrrolidine-2-carboxamido)-2-phenylethyl)pyridinium dichloride

[0287]

[0288] Tert-butyl (S)-1-oxo-3-phenyl-1-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)propan-2-ylcarbamate (170 mg, 0.31 mmol) was dissolved in 1M HCl in Et2O (12.5 mL, 12.53 mmol). The reaction mixture was stirred at rt for 18 hours. The product was triturated in Et2O. The solid was recovered by filtration on a Buchner funnel, washed with Et2O and dried under vacuum. The solid was dissolved in water and freeze-dried to give 150 mg (93%) of the title compound.

[0289] 1 H NMR(300MHz,D2O):ppm 1.39-1.48(m,1H),1.58-1.65(m,2H),1.92-1.99(m,1H),2.66-2.74(m,1H),2.95-3.10(m,2H),3.29-3.45 (m,3H),4.15-4.27(m,2H),5.09(t,1H),6.97-7.17(m,10H),7.59-7.64(m,2H),8.18(t,1H),8.35(d,1H);

[0290] [M+H] + =443.3,

[0291] [M+Na] + =465.2.

[0292] Embodiment 11

[0293] (S)-1-((S)-2-amino-4-methylpentanoyl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide

[0294]

[0295] The title compound was prepared as 2-[(2S)-2-{[(2S)-1-[(2S)-2-aminoglycanyl-4-methylpentanoyl]pyrrolidin-2-yl]formamido}-2-phenylethyl]pyridin-1-ium dichloride.

[0296] Step A

[0297] tert-Butyl (S)-4-methyl-1-oxo-1-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)pentan-2-ylcarbamate

[0298]

[0299] N-(tert-butoxycarbonyl)-L-leucine (157mg, 0.68mmol) is dissolved in anhydrous DMF (4mL). HATU (271mg, 0.71mmol) and DIPEA (0.39mL, 2.24mmol) are added and the mixture is stirred at rt for 30 minutes. Subsequently, (2S)-2-{[(1S)-1-phenyl-2-(pyridin-1-ium-2-yl)ethyl]carbamoyl}pyrrolidine-1-ium dichloride (250mg, 0.68mmol) is added to the solution and the mixture is stirred at rt for 18 hours. Water is added and the mixture is extracted with EtOAc 3x. The organic phase is washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The product was purified on a SiO2 column eluting with a gradient (0% MeOH / 50% EtOAc / 50% heptane→0% MeOH / 100% EtOAc / 0% heptane→15% MeOH / 85% EtOAc / 0% heptane) followed by a C-18 column eluting with a gradient of MeOH and water (0-100% MeOH) to give 200 mg (58%) of the title compound.

[0300] 1 H NMR (300MHz,CDCl3):ppm 0.91(d,3H),0.98(d,3H),1.34–1.52(m,1H),1.41(s,9H),1.70–1.84(m,2H), 1.85–1.96(m,3H),2.11–2.18(m,1H),3.07–3.27(m,2H),3.47–3.56(m,1H),3 .61–3.71(m,1H),4.43–4.56(m,2H),5.14–5.18(m,1H),5,26-5.34(m,1H),6. 98(d,J=7.7Hz,1H),7.08–7.25(m,5H),7.52(t,1H),7.83(d,1H),8.48(d,1H).

[0301] Step B

[0302] 2-((S)-2-((S)-1-((S)-2-Amino-4-methylpentanoyl)pyrrolidine-2-carboxamido)-2-phenylethyl)pyridinium dichloride

[0303]

[0304] Tert-butyl (S)-4-methyl-1-oxo-1-((S)-2-((S)-1-phenyl-2-(pyridin-2-yl)ethylcarbamoyl)pyrrolidin-1-yl)pentan-2-ylcarbamate (200 mg, 0.39 mmol) was dissolved in 1M HCl in Et2O (15.7 mL, 15.7 mmol). The reaction mixture was stirred at rt for 18 hours. The product was triturated in Et2O. The solid was recovered by filtration on a Buchner funnel, washed with Et2O and dried under vacuum. The solid was dissolved in water and freeze-dried to give 130 mg (69%) of the title compound.

[0305] 1 H NMR(300MHz,D2O):ppm 0.72(d,6H),1.31-1.49(m,4H),1.65-1.74(m,2H),1.93-2.02(m,1H),3.23-3.49(m,4H),3.99-4.04(m, 1H),4.16-4.21(m,1H),5.07(t,1H),7.08-7.20(m,5H),7.63-7.67(m,2H),8.22(td,1H),8.37(dd,1H);

[0306] [M+H] + =409.2,

[0307] [M+Na] + =431.2.

[0308] Example 12

[0309] 2-[(2S)-2-{[(2S)-1-[(2S)-2-Aminonia-5-{[amino(imino)methyl]amino}pentanoyl]-pyrrolidin-2-yl]formamido}-2-phenylethyl]pyridin-1-ium tetrachloride

[0310]

[0311] Step A

[0312] tert-Butyl N-[(1Z)-{[(4S)-4-{[(tert-butoxy)carbonyl]amino}-5-oxo-5-[(2S)-2-{[(1S)-1-phenyl-2-(pyridin-2-yl)ethyl]carbamoyl}pyrrolidin-1-yl]pentyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]carbamate

[0313]

[0314] Boc-Arg (Boc) 2-OH (644mg, 1.36mmol) is dissolved in anhydrous DMF (7mL). HATU (542mg, 1.43mmol) and DIPEA (0.78mL, 4.48mmol) are added and the mixture is stirred at rt for 30 minutes. Subsequently, (2S) -2- { [(1S) -1- phenyl -2- (pyridin-1-ium -2-yl) ethyl] carbamoyl} pyrrolidine -1-ium dichloride (500mg, 1.36mmol) is added to the solution and stirred at rt for 18 hours. Water is added and the mixture is extracted with EtOAc 3x. The organic phase is washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. Purification on a SiO2 column eluting with a gradient (0% MeOH / 50% EtOAc / 50% heptanes→0% MeOH / 100% EtOAc / 0% heptanes→10% MeOH / 90% EtOAc / 0% heptanes) followed by purification on a C-18 column eluting with a gradient of MeOH and water (0-100% MeOH) afforded 200 mg (20%) of the title compound.

[0315] 1 H NMR (300MHz, CDCl3): ppm 1.43(s,9H),1.46(s,9H),1.51(s,9H),1.57–1.76(m,4H),1.86–1.99(m,3H),2.12–2.25(m,1H),3.11(dd,1H),3.23–3.30(m,1H),3.5 8–3.95(m,4H),4.42(t,,1H),4.56(d,1H),5,31-5.38(m,2H),6.98(d,1H),7.17–7.33(m,5H),7.54(t,1H),7.93(d,1H),8.56(d,1H).

[0316] Step B

[0317] 2-[(2S)-2-{[(2S)-1-[(2S)-2-Aminonia-5-{[amino(imino)methyl]amino}pentanoyl]pyrrolidin-2-yl]formamido}-2-phenylethyl]pyridin-1-ium tetrachloride

[0318]

[0319] Tert-butyl N-[(1Z)-{[(4S)-4-{[(tert-butoxy)carbonyl]amino}-5-oxo-5-[(2S)-2-{[(1S)-1-phenyl-2-(pyridin-2-yl)ethyl]carbamoyl}pyrrolidin-1-yl]pentyl]amino}({[(tert-butoxy)carbonyl]imino})methyl]carbamate (200 mg, 0.27 mmol) was dissolved in 1M HCl in Et2O (5.3 mL, 10.64 mmol). The reaction mixture was stirred at rt for 18 hours. The product was triturated in Et2O. The solid was recovered by filtration on a Buchner funnel, washed with Et2O and dried under vacuum. The solid was dissolved in water and freeze-dried to give 140 mg (88%) of the title compound.

[0320] 1 H NMR(300MHz,D2O):ppm 1.31-1.57(m,3H),1.66-1.78(m,4H),2.00-2.10(m,1H),2.99(t,2H),3.28-3.40(m,4H),4.16(t,1H),4.2 6(t,1H),5.09(t,1H),7.10-7.13(m,2H),7.16-7.21(m,3H),7.67-7.71(m,2H),8.27(t,1H),8.40(d,1H);

[0321] [M+H] + =452.2.

[0322] Biological Activity

[0323] The prodrugs described herein are contemplated for oral administration to a subject suffering from depression or pain.

[0324] Embodiment 13

[0325] This example illustrates various prodrugs of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine, wherein R1 is C 1-6 Alkyl C(O)O(C 1-6 alkoxy), which can be used to achieve different rates (i.e., slow or fast) of conversion to (S)-1-phenyl-2-(pyridin-2-yl)ethanamine to explore the appropriate PK profile. Thus, varying R1 to produce different prodrugs results in different pharmacokinetic profiles of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine. In addition, this example shows that there is no expected loss in oral exposure when the prodrug is converted to the free base. Where R1 is C 1-6 Alkyl C(O)O(C 1-6The conversion of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrugs to (S)-1-phenyl-2-(pyridin-2-yl)ethylamine is expected to occur via an initial enzymatic hydrolysis of a functional group such as an ester, followed by a spontaneous conversion to (S)-1-phenyl-2-(pyridin-2-yl)ethylamine. The enzymes involved are expected to be several non-specific, high-capacity esterases, which can be shown by inhibiting the conversion using non-selective esterase inhibitors, while not inhibiting the conversion using selective inhibitors. Such esterases are expected to be distributed throughout the human body. Human intestinal fluid (HIF), human liver S9 fractions, and human whole blood have been used to examine the pharmacokinetic properties of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrugs and the rate and extent of formation of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine in different body compartments. The rates of formation of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine from different prodrugs of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine varied significantly in all assays tested, as shown in Table 1.

[0326] Table 1

[0327]

[0328] The in vitro results shown in Table 1 indicate that different pharmacokinetic plasma profiles of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine can be achieved by using different (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrugs. Thus, the selection of a particular prodrug will allow for different pharmacokinetic profiles of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine, that is, different prodrugs can be used to achieve a slow or fast conversion to (S)-1-phenyl-2-(pyridin-2-yl)ethylamine. The human intestinal fluid (HIF), human liver, and human blood assays used to generate the results shown in Table 1 have been described in Malmborg J & Ploeger BA,. J Pharmacol Toxicol Methods (2013) May-Jun 67 (3) 203-13, which is incorporated herein by reference.

[0329] Embodiment 14

[0330] This example shows that the use of Different prodrugs of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine are used to achieve different rates (i.e., slow or fast) of conversion to (S)-1-phenyl-2-(pyridin-2-yl)ethylamine to explore appropriate PK profiles. Therefore, changing R1 to produce different prodrugs leads to different pharmacokinetic profiles of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine. In addition, this example shows that there is no expected loss in oral exposure when the prodrug is converted to the free base.

[0331] Where R1 is The conversion of the prodrug of the compound of formula (I) to (S)-1-phenyl-2-(pyridin-2-yl)ethylamine is expected to occur via enzymatic hydrolysis at the C-terminus of proline, thereby releasing a dipeptide and (S)-1-phenyl-2-(pyridin-2-yl)ethylamine. The enzyme involved is expected to be dipeptidyl peptidase IV (DPPIV), which can be shown by inhibiting the conversion with a selective inhibitor of the enzyme. DPPIV is expected to be distributed throughout the human body. Human intestinal fluid (HIF), human liver S9 fraction and human whole blood have been used to examine the pharmacokinetic properties of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrugs and the rate and extent of formation of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine in different body compartments. Caco-2 cells have been used to evaluate the permeability and absorption potential of prodrugs of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine. The rates of formation of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine from different (S)-1-phenyl-2-(pyridin-2-yl)ethanamine prodrugs varied in the assays tested, particularly in human blood, as shown in Table 2. The different permeabilities exhibited by the different prodrugs of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine suggest that the systemic exposure of the prodrugs of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine will vary depending on the prodrug selected.

[0332] Table 2

[0333]

[0334] In vitro assays have shown that different pharmacokinetic plasma profiles of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine can be achieved by using different prodrugs. Therefore, different pharmacokinetic profiles of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine can be achieved by using different prodrugs.

[0335] Embodiment 15

[0336] This example demonstrates that oral administration of a prodrug of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine has a similar pharmacokinetic profile to an IV 1 hour infusion of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine.

[0337] Based on in vitro data, (S)-1-phenyl-2-(pyridin-2-yl)ethanamine (lanicemine) has been shown to have good oral bioavailability in humans (>75%), but has a higher than acceptable Cmax (relative to its safety profile for a 1-hour infusion) when given as an intravenous bolus. One selection criterion for evaluating the utility and usability of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine prodrugs is whether the prodrug retains the oral / 1-hour iv infusion characteristics of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine when the prodrug is administered directly into the bloodstream. max , C max , AUC) while reducing the C of the active part max The potential for abuse (e.g., abuse tendency) and / or Cmax-driven safety hazards are reduced. As a way to identify prodrugs with appropriate pharmacokinetic distribution in humans, a multi-compartment, physiologically based pharmacokinetic model (PBPK) was constructed for (S)-1-phenyl-2-(pyridin-2-yl)ethylamine and each of the prodrugs considered. The PBPK model was verified using a series of known prodrugs for which human PK data can be obtained (see Malmborg J & Ploeger BA,. J Pharmacol Toxicol Methods (2013) May-Jun 67 (3) 203-13, which is incorporated into the present application by introduction). The rate constant used in the model is based on an in vitro stability study (i.e., human intestinal fluid, human blood, etc.) conducted with prodrugs and (S)-1-phenyl-2-(pyridin-2-yl)ethylamine (example values ​​are shown in Tables 1 and 2).

[0338] Table 3 shows the key pharmacokinetic predictions for Example 5 - a DPPIV-fragmented prodrug. As exemplified in Table 3, when a given oral dosage form is crushed and not properly delivered as a bolus iv, the Cmax exposure of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine is predicted to increase by >2x. In contrast, (S)-1-phenyl-2-(pyridin-2-yl)ethylamine released from the orally administered prodrug has a PK profile similar to that of a 1-hour infusion of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine, and when administered as a bolus iv, there is no Cmax doubling. In addition, since the conversion to (S)-1-phenyl-2-(pyridin-2-yl)ethylamine is selectively driven by DPPIV, DPPIV inhibitors can be used to stop the conversion in intentional or unintentional abuse, which allows the added safety features to be built into the DPPIV prodrug design.

[0339] In Table 3, Compound 1 is (S)-1-phenyl-2-(pyridin-2-yl)ethylamine. In Table 3, all doses are selected to contain an equal amount of Compound 1 [(S)-1-phenyl-2-(pyridin-2-yl)ethylamine free base], i.e., each dose contains the same amount of Compound 1 therein and potentially releases (S)-1-phenyl-2-(pyridin-2-yl)ethylamine in free base form. Therefore, as shown in Table 3, the modeled Cmax concentration of free base Compound 1 can be directly compared with different administrations of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine and (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrug.

[0340] Table 3

[0341]

[0342] Example 16

[0343] This example demonstrates that when the prodrug of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine dihydrochloride (Example 5) and (S)-1-phenyl-2-(pyridin-2-yl)ethanamine dihydrochloride are administered to dogs, there is less risk of seizures with the prodrug as shown by the NOEL and LOEL.

[0344] (S)-1-phenyl-2-(pyridin-2-yl)ethylamine dihydrochloride and vehicle were administered via oral gavage (water) once daily. Formulations were prepared twice a week, once. Dosage formulations were stored and refrigerated (2-8°C) in brown glass containers until administration was required. A range of concentrations of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine dihydrochloride was prepared in the vehicle to maintain the dose volume (1 mL / kg) for all doses. Individual doses were based on the most recent body weight of the dog used in the study.

[0345] (S)-1-phenyl-2-(pyridin-2-yl) ethylamine was administered once daily for 14 days (vehicle was 0.3M gluconic acid pH 3.0) as the prodrug (embodiment 5) of fumarate and vehicle via oral gavage at approximately the same time (± 1 hour) every day. (S)-1-phenyl-2-(pyridin-2-yl) ethylamine of certain concentration range was prepared in vehicle as the prodrug of fumarate (for example (S)-1-phenyl-2-(pyridin-2-yl) ethylamine prodrug of 3,10 and 30mg / mL), and then the dosage volume (2mL / kg) was maintained for all dosages. Before administration, the administration preparation was warmed to room temperature by stirring at least 30 minutes, and continued to stir throughout the administration operation. Independent dosage was based on the latest body weight of the dog used in this research.

[0346] Table 4 Compound 1 is (S)-1-phenyl-2-(pyridin-2-yl)ethylamine. Table 4 illustrates the highest blood levels where seizures did not occur (no observed action limit, NOEL) and the lowest blood levels where seizures occurred (lowest observed action limit, LOEL) after administration of Compound 1 dihydrochloride and after administration of Compound 1 fumarate prodrug. In each case, the highest blood concentration (Cmax) of the free base (S)-1-phenyl-2-(pyridin-2-yl)ethylamine was measured. In the first case, the free base (S)-1-phenyl-2-(pyridin-2-yl)ethylamine was generated directly from Compound 1 administered as the dihydrochloride. In the second case, the free base (S)-1-phenyl-2-(pyridin-2-yl)ethylamine was generated via in vivo conversion of the prodrug of Compound 1 administered as the fumarate. The NOEL for epilepsy was higher for the (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrug administered as the fumarate salt compared to the (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrug administered as the dihydrochloride salt.

[0347] Table 4

[0348]

[0349] *All doses administered (in parentheses) reflect free base concentrations.

[0350] Embodiment 17

[0351] This example illustrates that increasing doses of the (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrug of Example 5 produce less than proportional increases in the concentration of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine in human intestinal fluid, as shown in Figure 1a and 1b As shown. As a result, it makes it difficult to obtain (S)-1-phenyl-2-(pyridin-2-yl) ethylamine prodrug administration relative to directly administering (S)-1-phenyl-2-(pyridin-2-yl) ethylamine at a high concentration of (S)-1-phenyl-2-(pyridin-2-yl) ethylamine, which is required for drug abuse. Without wishing to be limited by theory, the prodrug of (S)-1-phenyl-2-(pyridin-2-yl) ethylamine where R1 is first broken by DPPIV. There is endogenous and / or exogenous regulation of potential DPPIV to affect the conversion of prodrug to (S)-1-phenyl-2-(pyridin-2-yl) ethylamine.

[0352] Stock solutions of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrug (0.33, 1, 3, and 6 mmol / L) were prepared by adding a certain volume (8.25, 25, 75, and 150 μL) of a 20 mmol / L solution of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrug in DMSO to FaSSIF-v2 (491.75, 475, 425, and 350 μL). Subsequently, 10 μL of the stock solution of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrug was added to the supernatant (90 μL HIF) in a glass vial, mixed for 1 min, and injected on the LC–UV system. The initial concentrations of the incubations were 33, 100, 300, and 600 μmol / L and samples were taken at 5, 20, 35, 65, and 125 min from the start of the incubation. An incubation experiment with (S)-1-phenyl-2-(pyridin-2-yl)ethylamine (μmol / L) was used as a standard sample (single point calibration) to determine the formation rate and concentration of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine. The analysis was performed on a photodiode array detector coupled to a WatersAcquity UPLC (the analytical wavelength used was 261 nm). The column used was a BEH C-18, 1.7 μm, 2.1×50 mm ID maintained at 40° C. and using a 0.2 μL inline prefilter. The mobile phase used consisted of A: 0.03% TFA in H2O (v / v) and B: 0.03% TFA in acetonitrile (v / v). Example gradient: Initial 1% B was increased to 95% B in 7 minutes at a flow rate of 0.6 mL / min and then maintained at 95% B for 0.4 minutes. The conversion rate of the prodrug to (S)-1-phenyl-2-(pyridin-2-yl)ethanamine was determined by fitting the measured prodrug and (S)-1-phenyl-2-(pyridin-2-yl)ethanamine concentrations versus time.

[0353] Embodiment 18

[0354] Consistent with the in vitro observations from Example 17, in vivo studies in rats (Example 18 below) demonstrated that the Cmax exposure of (S)-1-phenyl-2-(pyridin-2-yl)ethanamine as described above increased at less than proportional levels following the transition process between therapeutic and supratherapeutic doses of the (S)-1-phenyl-2-(pyridin-2-yl)ethanamine prodrug. The results of these in vivo studies are listed in Table 5.

[0355] 52 male and 52 female rats were given (a) (S)-1-phenyl-2-(pyridin-2-yl) ethylamine prodrug as fumarate in vehicle (0.3M gluconic acid, pH 3.0) or (b) vehicle (0.3M gluconic acid, pH 3.0), once daily at approximately the same time (09:26+ / -124 minutes) for 14 days. The dose (30, 100 and 300mg / kg of (S)-1-phenyl-2-(pyridin-2-yl) ethylamine prodrug) was given in a dose volume of 10mL / kg. After 14 days, blood samples were collected from the lateral tail vein of the restricted animals 24 hours after administration. Blood samples were collected using 50ul neutral Sarstedt MinivettePOCT capillaries. The blood was then transferred to a pre-cooled tube containing frozen sodium citrate, shaken 5-10 times by hand and frozen in dry ice within 10 seconds of collection. Whole blood samples were transported frozen on dry ice to Covance Laboratories Inc. for analysis. The concentrations of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrug and (S)-1-phenyl-2-(pyridin-2-yl)ethylamine of the analyzed samples were measured. All analytical work was performed by Covance Laboratories, Inc., Madison, Wisconsin using the LC / MS / MS analytical method developed and verified by the laboratory.

[0356] Table 5 illustrates the toxicokinetic parameters following administration of therapeutic and supratherapeutic doses of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine prodrug to rats.

[0357] Table 5

[0358] Mean toxicokinetic parameters of (S)-1-phenyl-2-(pyridin-2-yl)ethylamine in rats 14 days after oral administration of (S)-1-((S)-2-amino-3-methylbutanoyl)-N-((S)-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide fumarate (Example 5)

[0359]

Claims

1. A pharmaceutical composition comprising 0.10 to 50% by weight of a compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the compound is ( S)-1 -(( S )-2-amino-3-methylbutyryl)-N-(( S )-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable salt is: ( S)-1 -(( S )-2-amino-3-methylbutyryl)-N-(( S )-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide dihydrochloride.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable salt is ( S )-1-(( S )-2-amino-3-methylbutyryl)-N-(( S )-1-phenyl-2-(pyridin-2-yl)ethyl)pyrrolidine-2-carboxamide fumarate.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the carrier is selected from lactose, sucrose, sorbitol, mannitol, starch and cellulose derivatives.

5. The pharmaceutical composition according to any one of claims 1 to 3, further comprising a binder.

6. The pharmaceutical composition according to claim 5, wherein the binder is selected from gelatin and polyvinylpyrrolidone.

7. The pharmaceutical composition according to any one of claims 1 to 3, further comprising a lubricant.

8. The pharmaceutical composition according to claim 7, wherein the lubricant is selected from the group consisting of magnesium stearate, calcium stearate, polyethylene glycol and wax.

9. The pharmaceutical composition according to any one of claims 1 to 3, which is in the form of a tablet.

10. The pharmaceutical composition according to claim 9, wherein the tablet has a coating.

11. The pharmaceutical composition of claim 10, wherein the coating comprises gum arabic, gelatin, talc or titanium dioxide.

12. The pharmaceutical composition according to any one of claims 1 to 3, in the form of a soft gelatin capsule.

13. The pharmaceutical composition of claim 12, wherein the soft gelatin capsule comprises vegetable oil or polyethylene glycol.

14. The pharmaceutical composition of any one of claims 1 to 3, which is in the form of a hard gelatin capsule.

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