Azapeptide compounds derived from melanocyte-stimulating hormone release-inhibiting factor-1 and a method to obtain the same

Abstract

The present application relates to azapeptide compounds of formula (I) derived from melanocyte-stimulating hormone release-inhibiting factor-1. A method to obtain compounds of formula (I) is also described herein. The compounds of formula (I) have been shown to be suitable for the treatment of dopamine-related disorders of the central nervous system. These compounds showed enhanced potency as positive allosteric modulators of the dopamine D2 receptors in comparison with the melanocyte-stimulating hormone release- inhibiting factor-1 and, in general, do not exhibit meaningful cytotoxicity.

Description

[0001] DESCRIPTION

[0002] " AZAPEPTIDE COMPOUNDS DERIVED FROM MELANOCYTE -STIMULATING HORMONE RELEASE -INHIBITING FACTOR- 1 AND A METHOD TO OBTAIN THE SAME"

[0003] Technical field

[0004] This application relates to azapeptide compounds of formula (I) derived from melanocyte-stimulating hormone releaseinhibiting factor-1 and a method to obtain said compounds.

[0005] Background art

[0006] Melanostatin, also known as melanocyte-stimulating hormone release-inhibiting factor-1 (MIF-1 ), is a short endogenous hypothalamic neuropeptide, which displays several biological functions within the central nervous system (CNS) [ 1-9]. In the brain, MIF-1 blocks the effects of opioid receptor activation [ 1-3 ], inhibits the release of other neuropeptides such as a-melanocyte-stimulating hormone

[0004] , and also potentiates melatonin activity [5]. Among the vast repertoire of biological activities associated with MIF-1, its interaction with dopamine receptors is one of the most extensively explored due to its potential applications in dopamine-related disorders. MIF-1 promotes the selective binding of agonists such as N-propylapomorphine and quinpirole to dopamine receptor subtypes D2L, D2S, and D4 in a dose-dependent manner to produce bell-shaped or "inverted U" concentration-response curves

[0010] . Interestingly, MIF-1 has no significant influence on the binding to Di, D3, and D5 receptors, or other G protein-coupled receptors, thus exhibiting D2-specif icity [ 11, 12 ]. This feature makes MIF-1 the only known endogenous positive allosteric modulator (PAM) of the dopamine D2 receptors (D2R)

[0013] . The main advantage of PAM molecules like MIF-1 over agonists is that they are more selective since allosteric binding sites are more conserved than orthosteric ones

[0013] . Moreover, PAM molecules are known to preserve the physiological signaling, i. e., the receptors are only active in the presence of the endogenous ligands or agonists and, therefore, exhibit fewer adverse effects

[0013] . Moreover, they can be used in combination therapy

[0013] . Due to its remarkable PAM activity and D2-selectivity, MIF-1 is deemed a lead candidate for the treatment of several CNS dopamine-related disorders in which the D2R are implicated, such as drug abuse, depression

[0006] , tardive dyskinesia

[0007] , restless legs syndrome

[0008] , and Parkinson' s disease [ 9, 14 ]. In fact, MIF-1 is a promising anti-Parkinson agent, as corroborated by the results obtained from animal models of Parkinson' s disease [ 15, 16 ] and clinical trials

[0017] . As a PAM, MIF-1 can activate the D2R at suboptimal concentrations of dopamine, making this neuropeptide clinically relevant in Parkinson' s therapy. In vitro binding assays conducted on transfected neuroblastoma cells show that the effect of MIF-1 is specific to the D2R, with high specificity towards the D2L receptor subtype, which is present in post-synaptic membranes

[0012] . Preliminary clinical studies showed that the administration of MIF-1 in small doses resulted in mood improvement and ameliorated motor symptoms of the patients (orally or intravenously) with no meaningful adverse ef f ects [ 17, 18-20 ]. Moreover, MIF-1 potentiated the effects of levodopa and reduced levodopa-induced dyskinesia [ 18, 20 ]. Despite significant alleviation of motor symptoms

[0017] , poor intestinal absorption associated with a short biologic half-life in CNS tissues were found to be the main drawbacks of MIF-1 neuropeptide when administrated orally, hindering the progression of the clinical trials

[0017] . Summary

[0007] The present invention relates to azapeptide compounds of formula (I) derived from melanocyte-stimulating hormone release-inhibiting factor-1, and salts thereof:

[0008]

[0009] Wherein,

[0010] n is 1, 2, or 3;

[0011] R1is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, including their salts, alkyl-aminocarbonyl, alkenylaminocarbonyl, alkynyl-aminocarbonyl, aryl-aminocarbonyl, aminocarbonyl, alkyl-oxycarbonyl, alkenyl-oxycarbonyl, alkynyl-oxycarbonyl, or aryl-oxycarbonyl groups;

[0012] R2is selected from hydrogen, alkyl, alkenyl, alkynyl, or aryl groups;

[0013] R3is selected from hydrogen, alkyl, alkenyl, alkynyl, or aryl groups;

[0014] *R2and R3are R or S configurations;

[0015] X is selected from OH, O-alkyl, O-alkenyl, O-alkynyl, d’aryl, NH2, NH- (Z), or N- (Z)2, in which Z is selected from an alkyl, alkenyl, alkynyl, or aryl groups.

[0016] In one embodiment, the compounds of formula (I) are selected from methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-leucylglycinate (8a), methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-leucyl-L-alaninate (8b), methyl N-( tert-butyloxycarbonyl) -2-aza-prolyl-L-isoleucylglycinate (8c), methyl N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-isoleucyl-L-alaninate (8d), methyl N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-valylglycinate (8e), methyl N-( tert-butyloxycarbonyl) -2-aza-prolyl-L-valyl-L-alaninate (8f), methyl 2-aza-prolyl-L-leucylglycinate trifluoroacetate salt (9a), methyl 2-aza-prolyl-L-leucyl-L-alaninate trifluoroacetate salt (9b), methyl 2-aza-prolyl-L-isoleucylglycinate trifluoroacetate salt (9c), methyl 2-aza-prolyl-L-isoleucyl-L-alaninate trifluoroacetate salt (9d), methyl 2-aza-prolyl-L-valylglycinate trifluoroacetate salt (9e), methyl 2-aza-prolyl-L-valyl-L-alaninate trifluoroacetate salt (9f), N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-leucylglycinamide (10a), N-( tert-butyloxycarbonyl) -2-aza-prolyl-L-leucyl-L-alaninamide (10b), N- ( tert -but yloxy carbonyl ) -2-aza-prolyl-L-isoleucylglycinamide (10c), N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-isoleucyl-L-alaninamide (10d), N-( tert-butyloxycarbonyl) -2-aza-prolyl-L-valylglycinamide (10e), N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-valyl-L-alaninamide (10f), 2-aza-prolyl-L-leucylglycinamide trifluoroacetate salt (11a), 2-aza-prolyl-L-leucyl-L-alaninamide trifluoroacetate salt (11b), 2 -aza-prolyl-L-isoleucylglycinamide trifluoroacetate salt (11c), 2-aza-prolyl-L-isoleucyl-L-alaninamide trifluoroacetate salt (11d), 2-aza-prolyl-L-valylglycinamide trifluoroacetate salt (11e), or 2-aza-prolyl-L-valyl-L-alaninamide trifluoroacetate salt (11f).

[0017] In one embodiment, the compounds of formula (I) are for use in the treatment of dopamine-related disorders of the central nervous system.

[0018] In one embodiment, the dopamine-related disorders of the central nervous system are selected from depression, drug abuse, tardive dyskinesia, restless legs syndrome, or Parkinson' s disease. In one embodiment, a pharmaceutical composition comprises at least one compound of formula (I).

[0019] In one embodiment, the pharmaceutical composition further comprises at least one other active ingredient for the treatment of dopamine-related disorders of the central nervous system.

[0020] In one embodiment, the pharmaceutical composition is for use in the treatment of dopamine-related disorders of the central nervous system.

[0021] The invention also relates to a method to obtain compounds of formula (I), comprising the steps of:

[0022] N-protection of hydrazine monohydrate (1) using between 200 and 300 mol% of di-tert-butyl dicarbonate in a polar aprotic organic solvent, in the presence of between 200 and 300 mol% of triethylamine, at a temperature between 20 and 25°C, to obtain the corresponding symmetrical protected 1, 2-di- ( tert -butyloxycarbonyl ) hydrazine (2);

[0023] alkylation of 1,2-di-(tert-butyloxycarbonyl)hydrazine (2) using between 100 and 200 mol% of 1,3-dibromopropane or 1, 4-dibromobutane under alkaline conditions of between 10 and 50% aqueous solution of NaOH (w / w) and toluene, at a temperature between 80 and 110 °C, in the presence of between 10 and 30 moll of tetraethylammonium bromide or tetrabutylammonium iodide as a phase-transfer agent, affording the corresponding protected cyclic 1, 2-di- ( tertbutyloxycarbonyl ) pyrazolidine (3a) or di- tert-butyl tetrahydropyridazine-1, 2-dicarboxylate (3b);

[0024] preparing 1- ( tert-butyloxycarbonyl ) pyrazolidine (5a) or tert-butyl tetrahydropyridazine-1 (2H) -carboxylate (5b) by acidolysis of compound (3a) or compound (3b), respectively, using between 3000 and 12000 mol% of trifluoroacetic acid in CH2Cl2, at a temperature between 20 and 25°C, followed by monoprotection of the hydrazinium intermediates pyrazolidine-1, 2-diium trifluoroacetate (4a) or hexahydropyridazine-1, 2-diium trifluoroacetate (4b), respectively, using between 90 and 110 mol% of Boc2O in the presence of between 300 and 500 mol% of Et3N;

[0025] acylation of cyclic 1-(tert-butyloxycarbonyl)pyrazolidine (5a) or tert-butyl tetrahydropyridazine-1(2H)-carboxylate (5b) with between 100 and 300 mol% of 4-nitrophenyl chloroformate in the presence of between 100 and 300 mol% of Et3N in CH2Cl2, at a temperature between 0 °C and 25°C, obtaining 4-nitrophenyl N-(tert-butyloxycarbonyl)-2-aza-prolinate (6a) or 4-nitrophenyl N-(tert-butyloxycarbonyl)-2-aza-pipecolate (6b), respectively;

[0026] dissolution of the 4-nitrophenyl N-(tert-butyloxycarbonyl)-2-aza-prolinate (6a) or 4-nitrophenyl N- ( tert-butyloxycarbonyl ) -2-aza-pipecolate (6b) and between 100 and 200 mol% of a dipeptide selected from methyl L-leucylglycinate trifluoroacetate salt (7a), methyl L-leucyl-L-alaninate trifluoroacetate salt (7b), methyl L-isoleucylglycinate trifluoroacetate salt (7c), methyl L-isoleucyl-L-alaninate trifluoroacetate salt (7d), methyl L-valylglycinate trifluoroacetate salt (7e), or methyl L-valyl-L-alaninate trifluoroacetate salt (7f), in N, N-dimethylformamide (DMF) comprising between 0 and 75% of CH2CI2; followed by the addition of between 20 and 40 mol% of 4- (dimethylamino) pyridine in the presence of between 10 and 20 mol% of p-TsOH and the mixture is heated at a temperature between 40 and 90 °C, for a time between 3 and 6 days, respectively, obtaining compounds methyl N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-leucylglycinate (8a), methyl N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-leucyl-L-alaninate (8b), methyl N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-isoleucylglycinate (8c), methyl N- ( tertbutyloxy carbonyl ) -2-aza-prolyl-L-isoleucyl-L-alaninate (8d), methyl N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-valylglycinate (8e), or methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-valyl-L-alaninate (8f).

[0027] In one embodiment, the method further comprises the step of:

[0028] acidolysis of a tripeptide selected from (8a) to (8f) using between 3000 and 12000 mol% of TFA in CH2Cl2, at a temperature between 20 and 25°C, respectively, obtaining tripeptides methyl 2-aza-prolyl-L-leucylglycinate trifluoroacetate salt (9a), methyl 2-aza-prolyl-L-leucyl-L-alaninate trifluoroacetate salt (9b), methyl 2-aza-prolyl-L-isoleucylglycinate trifluoroacetate salt (9c), methyl 2-aza-prolyl-L-isoleucyl-L-alaninate trifluoroacetate salt (9d), methyl 2-aza-prolyl-L-valylglycinate trifluoroacetate salt (9e), or methyl 2-aza-prolyl-L-valyl-L-alaninate trifluoroacetate salt (9f).

[0029] In one embodiment, the method further comprises the step of:

[0030] dissolution of a tripeptide selected from (8a) to (8f) in a methanolic solution of NH3 between 1 and 7 M, at a temperature between 20 and 25°C, respectively, obtaining tripeptides N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-leucylglycinamide (10a), N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-leucyl-L-alaninamide (10b), N- ( tertbutyloxy carbonyl ) -2-aza-prolyl-L-isoleucylglycinamide (10c), N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-isoleucyl-L-alaninamide (10d), N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-valylglycinamide (10e), or N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-valyl-L-alaninamide (10f). In one embodiment, the method further comprises the step of: acidolysis of a tripeptide selected from (10a) to (10f) using between 3000 and 12000 mol% of TFA in CH2Cl2, at a temperature between 20 and 25°C, respectively, obtaining tripeptides 2-aza-prolyl-L-leucylglycinamide trifluoroacetate salt (11a), 2-aza-prolyl-L-leucyl-L-alaninamide trifluoroacetate salt (11b), 2 -aza-prolyl-L-isoleucylglycinamide trifluoroacetate salt (11c), 2-aza-prolyl-L-isoleucyl-L-alaninamide trifluoroacetate salt (11d), 2 -aza-prolyl-L-valylglycinamide trifluoro acetate salt (He), or 2-aza-prolyl-L-valyl-L-alaninamide trifluoroacetate salt (11f).

[0031] General description

[0032] The invention herein disclosed relates to azapeptide compounds derived from MIF-1 with enhanced PAM potency at the D2R. The compounds are suitable for use in the treatment of Parkinson' s disease and other dopamine-related disorders of the CNS.

[0033] Additionally, the invention herein disclosed also relates to a more efficient method of obtaining these compounds including: a) a multigram, (tri ) phosgene-free, and high-yielding method to obtain two cyclic aza-amino acid derivatives in solution-phase, aza-proline and aza-pipecolic acid, from hydrazine monohydrate; and b) an unprecedented high-yielding method for the incorporation of these heterocyclic scaffolds in peptide matrixes using 4-toluenesulfonic acid monohydrate (p-TsOH) as an effective catalyst. The azapeptide compounds of formula (I) derived from MIF-1 (Figure 1 ) herein described are of pharmaceutical interest as potent PAM of the D2R and can be used as therapeutic agents.

[0034] Using functional and toxicological assays, it was demonstrated that these novel MIF-1-based azapeptides display interesting pharmacological profiles, denoting superior PAM potency in comparison with the parent neuropeptide at 1 nM concentration and, in general, do not exhibit meaningful cytotoxicity in human differentiated SH-SY5Y neuroblastoma cells (dopaminergic phenotype) up to 100 pM concentration (using the MTT reduction assay).

[0035] Brief description of drawings

[0036] For easier understanding of this application, figures are attached to represent the preferred forms of implementation which nevertheless are not intended to limit the invention disclosed herein.

[0037] Figure 1 shows the structure of compounds of formula (I). Figure 2 shows the synthesis of compounds (6a) and (6b). Reactants and conditions: i) Et3N, B0C2O, CH2CI2; ii ) 50% NaOH (aq), Et4NBr or BU4NI, 1, 3-dibromopropane (a) or 1, 4-dibromobutane (b), toluene ( 90 °C); iii) TEA, CH2CI2; iv) Et3N, NPCF, CH2CI2 ( 0 °C).

[0038] Figure 3 shows Oak Ridge thermal ellipsoid plot (ORTEP) drawings of the X-ray single crystal structures of (6a) (right) and (6b) (left). Ellipsoids are represented at 50% of probability.

[0039] Figure 4 shows the synthesis of compounds 8 (a) to 8 (f). Reactants and conditions: i) Et3N, 25% DMF in CH2CI2, DMAP, cat. p-TsOH. Key: a: R2= -CH2CH (CH3)2, R3= -H; b: R2= -CH2CH (CH3)2, R3= -CH3; C: R2= ( S) -CH (CH3) CH2CH3, R3= -H; d: R2= (S) -CH (CH3) CH2CH3, R3= -CH3; e: R2= -CH (CH3)2, R3= -H; f: R2= -CH (CH3)2, R3= -CH3.

[0040] Figure 5 shows the synthesis of compounds 8 (a) to 11 (f). Reactants and conditions: i) Et3N, 25% DMF in CH2CI2, DMAP, cat. p-TsOH; ii) 7 M NH3in methanol; iii) TFA, anhydrous CH2CI2; iv) TFA, anhydrous CH2CI2. Key: a: R2= -CH2CH (CH3) 2, R3= -H; b: R2= -CH2CH (CH3) 2, R3= -CH3; c: R2= (S) - CH (CH3) CH2CH3, R3= -H; d: R2= (S)-CH(CH3)CH2CH3, R3= -CH3; e: R2= -CH (CH3)2, R3= -H; f: R2= -CH (CH3)2, R3= -CH3.

[0041] Figure 6 shows the ORTEP drawing of the X-ray single crystal structure of compound (10a). Ellipsoids are represented at 50% of probability. Hydrogen atoms are omitted for clarity. Figure 7 shows the cytotoxicological evaluation of compounds 8(a) to (11f) and MIF-1 (100 µM), and 6-OHDA ( 125 pM) by the MTT reduction assay in differentiated SH-SY5Y neuronal cells (dopaminergic phenotype). Data are expressed as a percentage of control (PBS or 0.5% DMSO in PBS) and are presented as mean ± standard deviation. The results were obtained from 12-72 wells and 3-9 independent experiments. Statistical analysis was performed using the one-way analysis of variance test followed by Tukey' s post hoc test (***p < 0.001, and ****p < 0.0001, versus control).

[0042] Figure 8 shows the chemical structures of the selected compounds of formula (I) for pharmacological characterization of PAM activity by functional assays at the human dopamine D2R.

[0043] Figure 9 shows the concentration-response curves of dopamine (DA) in the presence and absence of MIF-1 and the selected compounds of formula (I): (8c), (9c), (10a), (10b), (10c), (11a), and (11b) (1 nM). Data are expressed as a percentage of dopamine control and are presented as mean ± standard deviation. The results were obtained from 16 wells and 2 independent experiments.

[0044] Figure 10 shows the synthesis of 1, 2-di- ( tertbutyloxycarbonyl ) hydrazine (2).

[0045] Figure 11 shows the synthesis of 1, 2-di- ( tertbutyloxycarbonyl ) pyrazolidine (3a).

[0046] Figure 12 shows the synthesis of 1, 2-di- ( tertbutyloxycarbonyl ) pyrazolidine (3b).

[0047] Figure 13 shows the synthesis of 1- ( tertbutyloxycarbonyl ) pyrazolidine (5a).

[0048] Figure 14 shows the synthesis of tert-butyl tetrahydropyridazine-1 (2H) -carboxylate (5b).

[0049] Figure 15 shows the synthesis of 4-nitrophenyl - ( tertbutyloxycarbonyl ) -2-aza-prolinate (6a).

[0050] Figure 16 shows the synthesis of 4-nitrophenyl - ( tertbutyloxycarbonyl ) -2-aza-pipecolate (6b).

[0051] Figure 17 shows the synthesis of methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-leucylglycinate (8a).

[0052] Figure 18 shows the synthesis of methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-leucyl-L-alaninate (8b). Figure 19 shows the synthesis of methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-isoleucylglycinate (8c). Figure 20 shows the synthesis of methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-isoleucyl-L-alaninate (8d). Figure 21 shows the synthesis of methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-valylglycinate (8e).

[0053] Figure 22 shows the synthesis of methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-valyl-L-alaninate (8f). Figure 23 shows the synthesis of methyl 2-aza-prolyl-L-leucylglycinate trifluoroacetate salt (9a).

[0054] Figure 24 shows the synthesis of methyl 2-aza-prolyl-L-leucyl-L-alaninate trifluoroacetate salt (9b). Figure 25 shows the synthesis of methyl 2-aza-prolyl-L-isoleucylglycinate trifluoroacetate salt (9c).

[0055] Figure 26 shows the synthesis of methyl 2-aza-prolyl-L-isoleucyl-L-alaninate trifluoroacetate salt (9d).

[0056] Figure 27 shows the synthesis of methyl 2-aza-prolyl-L-valylglycinate trifluoroacetate salt (9e).

[0057] Figure 28 shows the synthesis of methyl 2-aza-prolyl-L-valyl-L-alaninate trifluoroacetate salt (9f).

[0058] Figure 29 shows the synthesis of N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-leucylglycinamide (10a).

[0059] Figure 30 shows the synthesis of N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-leucyl-L-alaninamide (10b).

[0060] Figure 31 shows the synthesis of N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-isoleucylglycinamide (10c).

[0061] Figure 32 shows the synthesis of N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-isoleucyl-L-alaninamide (lOd).

[0062] Figure 33 shows the synthesis of N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-valylglycinamide (lOe).

[0063] Figure 34 shows the synthesis of N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-valyl-L-alaninamide (lOf).

[0064] Figure 35 shows the synthesis of 2-aza-prolyl-L-leucylglycinamide trifluoroacetate salt (11a).

[0065] Figure 36 shows the synthesis of 2-aza-prolyl-L-leucyl-L-alaninamide trifluoroacetate salt (11b).

[0066] Figure 37 shows the synthesis of 2-aza-prolyl-L-isoleucylglycinamide trifluoroacetate salt (11c).

[0067] Figure 38 shows the synthesis of 2-aza-prolyl-L-isoleucyl-L-alaninamide trifluoroacetate salt (11d).

[0068] Figure 39 shows the synthesis of 2-aza-prolyl-L-valylglycinamide trifluoroacetate salt (11e).

[0069] Figure 40 shows the synthesis of 2-aza-prolyl-L-valyl-L-alaninamide trifluoroacetate salt (11f). Detailed description of embodiments

[0070] Now, preferred embodiments of the present application will be described in detail with reference to the annexed drawings. However, they are not intended to limit the scope of this application.

[0071] The present application relates to compounds of formula (I):

[0072] R1O R2

[0073] N

[0074] r O T R3 X

[0075]

[0076] Wherein,

[0077] n is 1, 2, or 3;

[0078] R1is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, including their salts, alkyl-aminocarbonyl, alkenylaminocarbonyl, alkynyl-aminocarbonyl, aryl-aminocarbonyl, aminocarbonyl, alkyl-oxycarbonyl, alkenyl-oxycarbonyl, alkynyl-oxycarbonyl, or aryl-oxycarbonyl groups;

[0079] R2is selected from hydrogen, alkyl, alkenyl, alkynyl, or aryl groups;

[0080] R3is selected from hydrogen, alkyl, alkenyl, alkynyl, or aryl groups;

[0081] *R2and R3are R or S configurations;

[0082] X is selected from OH, O-alkyl, O-alkenyl, O-alkynyl, d’aryl, NH2, NH- (Z ), or N- (Z)2, in which Z is selected from alkyl, alkenyl, alkynyl, or aryl groups.

[0083] Method to obtain compounds of formula (I)

[0084] a) Tri (phosgene) -free method for the preparation of stable aza-proline and aza-pipecolic acid carbazates on a multigram scale:

[0085] The current methods for the synthesis of aza-proline and azapipecolic acid intermediates rely on key advanced starting materials (ASM), and their application in peptide chemistry is restricted to solid-phase peptide synthesis protocols, being limited to milligram scale. Moreover, these methodologies rely on highly toxic reagents, such as phosgene or bis ( trichloromethyl ) carbonate (triphosgene)

[0021] as carbonylation agents to form semicarbazide bonds. While triphosgene is widely used as a less toxic alternative to phosgene, several studies show that both reagents pose similar health concerns

[0022] . Additionally, the high reactivity of the acyl intermediates obtained from ( tri ) phosgene make them highly unstable and therefore they should be used right after their preparation. Other methodologies require the use of hydride-based reagents for the cyclization of hydrazine derivatives, thus requiring anhydrous solvents and restricted conditions

[0023] .

[0086] In the present application, it is disclosed a cheap, straightforward, and high-yielding (tri) phosgene-free method to obtain stable aza-proline and aza-pipecolic acid carbazates that circumvents the limitations of previous methods and is compatible with multi-gram scale for obtaining azapeptides. Moreover, this method does not require anhydrous conditions, using aqueous NaOH as the base for cyclization in the presence of phase-transfer agents.

[0087] b) High-yielding methodology for the coupling of azaproline and aza-pipecolic acid carbazates for use in azapeptide synthesis:

[0088] Typically, in azapeptide synthesis, activated aryl esters either of the peptide N-terminus or the aza-residue (carbazic residue) are used to form semicarbazide bonds. However, coupling requires high temperature and long reaction times, and often results in low yields with numerous side products such as phenolic derivatives, hydantoins, and oxadiazolones

[0021] .

[0089] In this application, it is disclosed a more efficient and milder method that employs p-TsOH as the catalyst enabling not only to reduce the temperature from 90 to 60 °C, but also offering superior yields for including the aza-amino acids into peptide matrixes (up to 82%), as demonstrated for a series of six compounds of formula (I) obtained.

[0090] As shown in Figures 2 and 4, the method to obtain compounds of formula (I) comprises the steps of:

[0091] - -protection of hydrazine monohydrate (1) using between 200 and 300 mol% of di- tert-butyl dicarbonate (B0C2O) in a polar aprotic organic solvent, in the presence of between 200 and 300 mol% of triethylamine (Et₃N), at a temperature between 20 and 25°C, to obtain the corresponding symmetrical protected 1, 2-di- ( tertbutyloxycarbonyl ) hydrazine (2);

[0092] - alkylation of 1, 2-di- ( tert -butyloxycarbonyl ) hydrazine (2) using between 100 and 200 mol% of 1, 3-dibromopropane (a, n = 2 ) or 1, 4-dibromobutane (b, n = 3) under alkaline conditions of between 10 and 50% aqueous solution of NaOH (w / w) and toluene, at a temperature between 80 and 110 °C, in the presence of between 10 and 30 mol% of tetraethylammonium bromide (Et₄NBr) or tetrabutylammonium iodide (Bu₄NI) as a phase-transfer agent, affording the corresponding protected cyclic 1, 2-di- ( tert-butyloxycarbonyl ) pyrazolidine (3a) or di- tert-butyl tetrahydropyridazine-1, 2-dicarboxylate (3b);

[0093] - preparing 1- ( tert-butyloxycarbonyl ) pyrazolidine (5a) or tert-butyl tetrahydropyridazine-1 (2H) -carboxylate (5b) by acidolysis of compound (3a) or compound (3b), respectively, using between 3000 and 12000 mol% of trifluoroacetic acid (TFA) in CH₂Cl₂, at a temperature between 20 and 25°C, followed by monoprotection of the hydrazinium intermediates pyrazolidine-1,2-diium trifluoroacetate (4a) or hexahydropyridazine-1,2-diium trifluoroacetate (4b), respectively, using between 90 and 110 mol% of Boc₂O in the presence of between 300 and 500 mol% of Et₃N;

[0094] - acylation of cyclic 1- ( tertbutyloxycarbonyl ) pyrazolidine (5a) or tert-butyl tetrahydropyridazine-1 (2H) -carboxylate (5b) with between 100 and 300 mol% of 4-nitrophenyl chloroformate (NPCF) in the presence of between 100 and 300 mol% of Et₃N in CH₂Cl₂, at a temperature between 0 °C and 25°C, obtaining 4-nitrophenyl N- ( tert-butyloxycarbonyl ) -2- aza-prolinate (6a) or 4-nitrophenyl - ( tertbutyloxycarbonyl ) -2-aza-pipecolate (6b), respectively;

[0095] Obtaining compounds (8a) to (8f):

[0096] - dissolution of the 4-nitrophenyl N-(tert-butyloxycarbonyl)-2-aza-prolinate (6a) or 4- nitrophenyl N- ( tert-butyloxycarbonyl) -2-aza-pipecolate (6b) and between 100 and 200 mol% of a dipeptide selected from methyl L-leucylglycinate trifluoroacetate salt (7a), methyl L-leucyl-L-alaninate trifluoroacetate salt (7b), methyl L-isoleucylglycinate trifluoroacetate salt (7c), methyl L-isoleucyl-L-alaninate trifluoroacetate salt (7d), methyl L-valylglycinate trifluoroacetate salt (7e), or methyl L-valyl-L- alaninate trifluoroacetate salt (7f), in N, N- dimethylformamide (DMF) comprising between 0 and 75% of CH₂Cl₂; followed by the addition of between 20 and 40 mol% of 4- (dimethylamino) pyridine (DMAP) in the presence of between 10 and 20 moll of p-TsOH and the mixture is heated at a temperature between 40 and 90 °C, preferably 60-70 °C, for a time between 3 and 6 days, respectively obtaining compounds methyl N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-leucylglycinate (8a), methyl N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L- leucyl-L-alaninate (8b), methyl N- ( tertbutyloxy carbonyl ) -2-aza-prolyl-L-isoleucylglycinate (8c), methyl N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L- isoleucyl-L-alaninate (8d), methyl N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-valylglycinate (8e), or methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L- valyl-L-alaninate (8f);

[0097] Obtaining compounds (9a) to (9f):

[0098] - acidolysis of a tripeptide selected from (8a) to (8f) using between 3000 and 12000 mol% of TFA in CH2Cl2, at a temperature between 20 and 25°C, respectively obtaining tripeptides methyl 2-aza-prolyl-L- leucylglycinate trifluoroacetate salt (9a), methyl 2- aza-prolyl-L-leucyl-L-alaninate trifluoroacetate salt (9b), methyl 2-aza-prolyl-L-isoleucylglycinate trifluoroacetate salt (9c), methyl 2-aza-prolyl-L- isoleucyl-L-alaninate trifluoroacetate salt (9d), methyl 2-aza-prolyl-L-valylglycinate trifluoroacetate salt (9e), or methyl 2-aza-prolyl-L-valyl-L-alaninate trifluoroacetate salt (9f).

[0099] Obtaining compounds (10a) to (10f):

[0100] - dissolution of a tripeptide selected from (8a) to (8f) in a methanolic solution of NH3 between 1 and 7 M, at a temperature between 20 and 25°C, respectively obtaining tripeptides N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-leucylglycinamide (10a), N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-leucyl-L-alaninamide (10b), N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-isoleucylglycinamide (10c), N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-isoleucyl-L-alaninamide (10d), N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-valylglycinamide (10e), or N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-valyl-L-alaninamide (10f);

[0101] Obtaining compounds (11a) to (11f):

[0102] - acidolysis of a tripeptide selected from (10a) to (10f) using between 3000 and 12000 mol% of TFA in CH2Cl2, at a temperature between 20 and 25°C, respectively obtaining tripeptides 2-aza-prolyl-L-leucylglycinamide trifluoroacetate salt (11a), 2 -aza-prolyl-L-leucyl-L- alaninamide trifluoroacetate salt (11b), 2 -aza-prolyl- L-isoleucylglycinamide trifluoroacetate salt (11c), 2- aza-prolyl-L-isoleucyl-L-alaninamide trifluoro acetate salt (Hd), 2-aza-prolyl-L-valylglycinamide trifluoroacetate salt (He), or 2-aza-prolyl-L-valyl-L- alaninamide trifluoroacetate salt (11f).

[0103] As shown above, the presently disclosed method is suitable to obtain compounds (8a) to (8f), and / or respectively obtaining compounds (9a) to (9f) from compounds (8a) to (8f), and / or respectively obtaining compounds (10a) to (lOf) from compounds (9a) to (9f), and / or respectively obtaining compounds (11a) to (11f) from compounds (10a) to (10f).

[0104] As shown in Figure 5, after obtaining compounds (8a) to (8f) from 4-nitrophenyl N- ( tert-butyloxycarbonyl ) -2-aza-prolinate (6a) with dipeptides selected from (7a) to (7f) catalyzed by p-TsOH (i), the methyl ester functional group can be converted to the primary amide by ammonolysis with NH₃ in methanol (ii), respectively affording compounds (10a) to (lOf) with high yields (77-91%). The tert-butyl carbamate from compounds (8a) to (8f) or (10a) to (lOf) can be cleaved by acidolysis using TFA to afford the corresponding trifluoroacetate salts (9a) to (9f) (iii) or (11a) to (11f) (iv), with good yields ( 66-86%).

[0105] Carbazate compounds (6a) and (6b) were obtained with 79 and 67% global yield, respectively.

[0106] The crystals of carbazate compounds (6a) and (6b) obtained enabled to study their structures in the solid-state by X-ray crystallography for the first time. The ORTEP diagram of carbazate compounds (6a) and (6b) is presented in Figure 3. The data obtained by X-ray crystallography corroborate the structures of carbazate compounds (6a) and (6b). Carbazate compounds (6a) and (6b) were stable for at least 12 months in the presence of moisture without noticeable decomposition.

[0107] The use of p-TsOH is unprecedented. In the absence of p-TsOH, the maximum yield obtained for the compounds of formula (I) herein disclosed was 42% using the conditions described in the literature

[0024] . The addition of catalytic amounts of p-TsOH greatly improved the yield between 68 and 82%. Besides the target compounds of formula (I) and p-nitrophenol no other side products were detected. Additionally, unreacted carbazate compounds can be recovered and reused without noticeable decomposition.

[0108] In one embodiment, the compounds of formula (I) are selected from methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L- leucylglycinate (8a), methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-leucyl-L-alaninate (8b), methyl N-( tert-butyloxycarbonyl) -2-aza-prolyl-L-isoleucylglycinate (8c), methyl N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-isoleucyl-L-alaninate (8d), methyl N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-valylglycinate (8e), methyl N-( tert-butyloxycarbonyl) -2-aza-prolyl-L-valyl-L-alaninate (8f), methyl 2-aza-prolyl-L-leucylglycinate trifluoroacetate salt (9a), methyl 2-aza-prolyl-L-leucyl-L-alaninate trifluoroacetate salt (9b), methyl 2-aza-prolyl-L-isoleucylglycinate trifluoroacetate salt (9c), methyl 2-aza-prolyl-L-isoleucyl-L-alaninate trifluoroacetate salt (9d), methyl 2-aza-prolyl-L-valylglycinate trifluoroacetate salt (9e), methyl 2-aza-prolyl-L-valyl-L-alaninate trifluoroacetate salt (9f), N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-leucylglycinamide ( 10a), N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-leucyl-L-alaninamide (10b), N- ( tert -but yloxy carbonyl ) -2-aza-prolyl-L-isoleucylglycinamide (10c), N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-isoleucyl-L-alaninamide (10d), N-( tert-butyloxycarbonyl) -2-aza-prolyl-L-valylglycinamide (10e), N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-valyl-L-alaninamide (10f), 2-aza-prolyl-L-leucylglycinamide trifluoroacetate salt (11a), 2-aza-prolyl-L-leucyl-L-alaninamide trifluoroacetate salt (11b), 2 -aza-prolyl-L-isoleucylglycinamide trifluoroacetate salt (11c), 2-aza-prolyl-L-isoleucyl-L-alaninamide trifluoroacetate salt (11d), 2-aza-prolyl-L-valylglycinamide trifluoroacetate salt (11e), or 2-aza-prolyl-L-valyl-L-alaninamide trifluoroacetate salt (11f). In one embodiment, the compounds of formula (I) are for use in the treatment of dopamine-related disorders of the central nervous system.

[0109] In one embodiment, a pharmaceutical composition comprises at least one compound of formula (I).

[0110] In one embodiment, a pharmaceutical composition comprises at least one compound of formula (I) and further comprising at least one other active ingredient for the treatment of dopamine-related disorders of the central nervous system.

[0111] In one embodiment, the dopamine-related disorders of the central nervous system are selected from, but not limited to, depression, drug abuse, tardive dyskinesia, restless legs syndrome, or Parkinson' s disease.

[0112] Compounds of formula (I) as nontoxic PAM of the D2R with improved potency and resistance to peptidase activity.

[0113] MIF-1 is a potent and D2-selective PAM that has intrinsic therapeutical potential for the treatment of Parkinson' s disease and other dopamine-related disorders, as corroborated in several clinical assays. Moreover, it can be used in coadministration with the levodopa regimen, allowing the reduction of the therapeutical doses of levodopa and minimizing its adverse effects. However, despite its pharmacological PAM activity and therapeutical potential in Parkinson' s disease, its clinical translation is hampered due to pharmacokinetic issues, such as low gastrointestinal absorption and lability towards peptidases from CNS tissues, as a consequence of its intrinsic peptide nature. Pharmacokinetic studies demonstrate that the L-prolyl-L-leucyl bond present at MIF-1 is highly susceptible to peptidase activity, resulting in the formation of L-proline along with L-leucylglycinamide dipeptide.

[0114] In this application, a series of 24 compounds of formula (I) were obtained using aza-proline as a proline surrogate, which is less prone to peptidase activity due to limited biochemical recognition.

[0115] The results showed that the compounds of formula (I) display, in general, a toxicological profile similar to that of MIF-1 neuropeptide with no significative toxicity using differentiated human SH-SY5Y neuroblastoma cells (dopaminergic phenotype) up to 100 pM concentration (MTT reduction assay). Functional assays at human D2R were performed for the nontoxic compounds (20 azapeptides) of formula (I) with different chemical diversity. Among these, 7 of the tested compounds of formula (I) were able to enhance the dopamine potency at 1 nM concentration, with the identification and characterization of two compounds of formula (I) with superior PAM activity than MIF-1 neuropeptide (up to a 2.19-fold increase in potency).

[0116] The nontoxic and potent PAM activity of these compounds at the D2R with expected improved pharmacokinetic profiles offers the possibility to explore the clinical translation of MIF-1-based azapeptide derivatives to be used in the early stages of Parkinson' s disease, when dopamine is still present in the CNS, thus delaying the introduction of levodopa therapy and extending its clinical use. On the other hand, co-administration of levodopa with PAM molecules of the D2R allows to reduce the therapeutic doses of levodopa and, therefore, its adverse effects. Examples

[0117] In the context of the present application, room temperature is considered to be within the range of 20 and 25°C. The yields reported were determined after purification of the compounds and drying with a high vacuum pump. The melting points (MP) were determined on a Stuart Scientific MEL-TEMP SMP10 electrothermal apparatus (United Kingdom) and are not corrected. The specific rotation ( [a]) values were determined on a JASCO P-2000 fully automatic polarimeter (United Kingdom) using a JASCO CG3-100 cylindrical glass cell (United Kingdom) and the data is reported as follows: [a]p expressed in ° dm-1g-1, whereas 0 represents temperature in °C and D indicates the wavelength of the radiation used (589 nm, sodium vapor lamp), and the concentration ( c) is expressed in g / 100 mL. Electrospray ionization (ESI ) high-resolution mass spectrometry (HRMS) exact mass measurements were performed with a Bruker Impact II spectrometer. Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker Advance III 400 spectrometer at 400.15 MHz for1H and 100.62 MHz for13C{1H} and distortionless enhancement by polarization transfer 135 (DEPT-135). The sample volume was approximately 0.5 mL in NMR economy (class B) quartz tubes. The chemical shifts ( 5) are reported in ppm and referenced to the residual solvent signals (CDCl3: δH= 7.26, δC= 77.16; DMSO-d6: δH= 2.50, δC= 39.52; CD3OD: δH= 4.87, δC= 49.00). NMR data is specified as follows: (1)1H NMR (solvent, frequency, temperature): δH– multiplicity (s: singlet, br s: broad singlet, d: doublet, dd: double of doublets, t: triplet, dt: double of triplets, appt: apparent triplet, q: quartet, dq: double of quartets, ABq: AB quartet, p: quintet, dp: double of quintets, m: multiplet), coupling constant (J), area under the signal (XH, where X represents the relative number of protons), and location of the proton in the molecule (indication of the functional group, or as HResidue-Y, where Y represents the number of the carbon attached to the proton, or as ArH for aromatic hydrogens). (2 )13C{1H} & DEPT-135 NMR (solvent, frequency, temperature): 5c - carbon type (CH3: primary, CH2: secondary, CH: tertiary, C: quaternary) and location of the carbon in the molecule (indication of the functional group, or as CResidue-Y, where Y represents the number of the carbon, or as ArC for aromatic carbons).

[0118] For all effects, the nomenclature used to identify the amino acid residues in this work was based on the three-letter code of the amino acids (Ala: L-alanine, Gly: glycine, Ile: L-isoleucine, Leu: L-leucine, Vai: L-valine). The numeration of the carbon atoms within a given amino acid residue starts at the carbonyl carbon.

[0119] 1. Synthesis of compounds:

[0120] a) 1, 2-Di- ( tert-butyloxycarbonyl ) hydrazine (2) (Figure 10) Hydrazine monohydrate (1) ( 1.94 mL, 39.9 mmol) was transferred to a round-bottom flask and then dissolved in CH2CI2 (40 mL). To the resulting solution, EtsN (11.50 mL, 82.51 mmol) was added, followed by B0C2O (21.8536 g, 100.132 mmol). The mixture was stirred overnight at room temperature. Workup: After the reaction, the solvent was eliminated using a rotary evaporator. To the resulting residue, hexane ( 100 mL) was added, obtaining a white precipitate that was filtered and washed with hexane. Hydrazine (2) (white solid) was obtained in 90% yield ( 8.3022 g).

[0121] Rr = 0.53 in hexane / AcOEt (3: 1 ). MP = [ 105; 107 ] °C.

[0122] 1H NMR (CDCl3, 400 MHz, 25 °C) δ: 6.33 (br s, 2H, 2NH), 1.45 (s, 18H, 2 xtBu).

[0123] 13C{1H} and DEPT-135 NMR (CDCl3, 101 MHz, 25 °C) δ: 155.9 (2C, 2 x OCON), 81.6 (2C, 2 xtBu), 28.3 (6CH3, 2 xtBu). b) 1, 2-Di- ( tert-butyloxycarbonyl ) pyrazolidine (3a) (Figure 11 )

[0124] Hydrazine (2) (5.3194 g, 22.901 mmol) was transferred to a round-bottom flask and then dissolved in toluene (75 mL). To the resulting solution, an aqueous solution of NaOH (25 mL) at 50% g / mL was added, followed by Et4NBr ( 1.0080 g, 4.7963 mmol) and 1, 3-dibromopropane (3.50 mL, 34.5 mmol). The mixture originated a gel that became a clear solution at 120 °C. Then, the mixture was stirred with reflux for 90 minutes at 90 °C. Workup: After the reaction, the solvent was eliminated using a rotary evaporator. Then, EtOAc (50 mL) was added, and the mixture was washed with aqueous NaHCOs (3 x 75 mL). Afterwards, the organic layer was collected and dried over with anhydrous Na2SO4, and the suspension was filtered. The filtrate was collected, and the solvent was removed using a rotary evaporator. The resulting residue was purified by column chromatography using hexane / EtOAc (3: 1 ) as the eluent. Pyrazolidine (3a) (clear crystalline solid) was obtained in 91% yield (5. 6757 g).

[0125] Rr = 0.42 in hexane / EtOAc (3: 1 ). MP = [32; 34 ] °C.

[0126] 1H NMR (CDCl3, 400 MHz, 25 °C) δ: 3.85 (p, J = 6.6 Hz, 2H, CHantiHsynCH2CHantiHsyn), 3.19 (q, J = 9.9, 8.8 Hz, 2H, CHantiHsynCH2CHantiHsyn), 1.98 (p, J = 7.2 Hz, 2H, CH2CH2CH2), 1.45 (s, 18H, 2 xtBu).

[0127] 13C{1H} and DEPT-135 NMR (CDCl3, 101 MHz, 25 °C) δ: 156.2 (2C, 2 x OCON), 81.1 (2C, 2 xtBu), 46.4 (2CH2, CH2CH2CH2), 28.3 (6CH3, 2 xtBu), 25.7 (CH2, CH2CH2CH2).

[0128] c) 1, 2-Di- ( tert -butyloxycarbonyl ) pyrazolidine (3b)

[0129]

[0130] (Figure 12 )

[0131] Hydrazine (2) (3.9524 g, 17.016 mmol) was transferred to a round-bottom flask and then dissolved in toluene (75 mL). To the resulting solution, an aqueous solution of NaOH (25 mL) at 50% g / mL was added, followed by Et4NBr ( 1.2570 g, 3.4031 mmol) and 1, -dibromobutane (3.06 mL, 25.5 mmol). The mixture originated a gel that became a clear solution at 120 °C. Then, the mixture was stirred with reflux for 90 minutes at 90 °C. Workup: After the reaction, the solvent was eliminated using a rotary evaporator. Then, EtOAc (50 mL) was added, and the mixture was washed with aqueous NaHCO3(3 x 75 mL). Afterwards, the organic layer was collected and dried over with anhydrous Na2SO4, and the suspension was filtered. The filtrate was collected, and the solvent was removed using a rotary evaporator. The resulting residue was purified by column chromatography using hexane / EtOAc (4: 1 ) as the eluent. Pyrazolidine (3b) (clear crystalline solid) was obtained in 89% yield (4.337 g).

[0132] Rr = 0. 65 in hexane / EtOAc ( 1: 1 ). MP = [45; 47] °C.

[0133] ! H NMR (DMSO-d6, 400 MHz, 100 °C) 5: 3.96 (d, J = 12.0 Hz, 2H, H-2 + H-2' ), 2.91 - 2.74 (m, 2H, H-2 + H-2' ), 1. 68 -1.47 (m, 4H, H-3 + H-3' ), 1.43 (s, 18H, 2 x tBu).

[0134] 13C{1H} and DEPT-135 NMR (DMSO-d6, 101 MHz, 100 °C) δ: 153.1 (2C, 2 x C=O), 79.5 (2C, 2 xtBu), 44.3 (2CH2, C-2 + C-2'), 27.5 (6CH3, 2 xtBu), 22.6 (2CH2, C-3 + C-3').

[0135] d) 1- ( tert-Butyloxycarbonyl ) pyrazolidine (5a) (Figure 13) Cyclic hydrazine (3a) (3.3226 g, 12.200 mmol) was transferred to a round-bottom flask and then dissolved in anhydrous CH2C12(40 mL). To the resulting solution, TFA (40.0 mL, 522 mmol) was added. The mixture was stirred for 5 hours at room temperature. Workup: After the reaction, the volatiles were eliminated using a rotary evaporator. The resulting residue (yellow oil) was dried (high vacuum pump). Then, the residue obtained was dissolved in anhydrous CH2C12(40 mL). To the resulting solution, Et3N ( 6.80 mL, 48.8 mmol) was added, followed by Boc20 (2.9289 g, 13.420 mmol). The mixture was stirred overnight at room temperature. Workup: After the reaction, the solvent was removed using a rotary evaporator. The resulting residue was purified by column chromatography using EtOAc as the eluent. Compound (5a) (clear oil) was obtained in 97% yield (2.0382 g).

[0136] Rr = 0.09 in EtOAc.

[0137] 1H NMR (CDCl3, 400 MHz, 25 °C) δ: 3.79 (br s, 1H, NH), [3.56 – 3.37 (m, 2H), 3.00 (t, J = 6.6 Hz, 2H), CH2CH2CH2], 2.06 – 1.94 (m, 2H, CH2CH2CH2), 1.46 (s, 9H,tBu).

[0138] 13C{1H} and DEPT-135 NMR (CDCl3, 101 MHz, 25 °C) δ: 155.1 (C, OCON), 80.2 (C,tBu), [47.9 (CH2), 45.6 (CH2), CH2CH2CH2], 28.6 (3CH3,tBu), 28.2 (CH2, CH2CH2CH2).

[0139] e ) tert-Butyl tetrahydropyridazine-1 (2A) -carboxylate (5b) (Figure 14 )

[0140] Cyclic hydrazine (3b) (4.3171 g, 15.076 mmol) was transferred to a round-bottom flask and then dissolved in anhydrous CH2CI2 (40 mL). To the resulting solution, TFA (57.8 mL, 754 mmol) was added. The mixture was stirred for 5 hours at room temperature. Workup: After the reaction, the volatiles were eliminated using a rotary evaporator. The resulting residue (yellow oil) was dried (high vacuum pump). Then, the residue obtained was dissolved in anhydrous CH2CI2 (40 mL). To the resulting solution, Et3N (8.41 mL, 60.3 mmol) was added, followed by B0C2O (3. 6194 g, 16.584 mmol). The mixture was stirred overnight at room temperature. Workup: After the reaction, the solvent was removed using a rotary evaporator. The resulting residue was purified by column chromatography using Et2O as the eluent. Compound (5b) (white solid) was obtained in 84% yield (2.3588 g).

[0141] Rr = 0.09 in Et2O. MP = [24; 26] °C.1H NMR (CDCl3, 400 MHz, 25 °C) δ: 3.56 – 3.42 (m, 2H, H-2), 2.90 – 2.74 (m, 2H, H-5), 1.68 – 1.47 (m, 4H, H-3 + H-4), 1.43 (s, 18H, 2 xtBu).

[0142] 13C{1H} and DEPT-135 NMR (CDCl3, 101 MHz, 25 °C) δ: 154.9 (C, OCON), 80.6 (C,tBu), [47.9 (CH2), 45.7 (CH2), C-2 + C-5], 28.6 (3CH3,tBu), [24.9 (CH2), 24.5 (CH2), C-3 + C-4).

[0143] f ) 4 -Nitrophenyl _ N- ( tert -butyloxycarbonyl ) -2-aza-

[0144]

[0145] prolinate (6a) ( Figure 15 )

[0146] Compound (5a) ( 1. 7364 g, 10. 082 mmol ) was trans ferred to a round-bottom flas k and then dissolved in anhydrous CH2C12( 40 mL ). The solution was placed on ice. Then, Et3N (2.10 mL, 15.1 mmol) was added, followed by NPCF ( 4. 0642 g, 20. 164 mmol ). The mixture was stirred for 3 hours at 0 ° C under an atmosphere of argon ( 1 atm). Workup: After the reaction, the solvent was removed using a rotary evaporator. Then, EtOAc ( 50 mL ) was added, and the mixture was washed with aqueous NaHCOs ( 3 x 50 mL ). Afterwards, the organic layer was collected and dried over with anhydrous Na2SO4, and the suspension was filtered. The filtrate was collected, and the solvent was removed using a rotary evaporator. The resulting residue was puri fied by column chromatography using hexane / EtOAc ( 2: 1 ) as the eluent. Compound (6a) (pale yellow solid) was obtained in 86% yield ( 2. 9367 g).

[0147] Rr = 0. 51 in hexane / EtOAc ( 2: 1 ). MP = [ 68; 72 ] ° C.

[0148] 1H NMR (CDCl3, 400 MHz, 25 °C) δ: 8.24 (d, J = 9.2 Hz, 2H, ArH), 7.34 (d, J = 9.2 Hz, 2H, ArH), [4.06 (br s, 1H), 3.95 (br s, 1H), 3.49 (br s, 1H), 3.30 (br s, 1H), CH2CH2CH2], 2.28 – 2.04 (m, 2H, CH2CH2CH2), 1.48 (s, 9H,tBu).

[0149] 13C{1H} and DEPT-135 NMR (CDCl3, 101 MHz, 25 °C) δ: [156.3 (2C), 156.2 (C), 2OCON + ArC-O], 145.1 (C, ArC-NO2), 125.3 (2CH, 2ArC), 122.1 (2CH, 2ArC), 82.5 (C,tBu), [47.5 (CH2), 46.2 (CH2), CH2CH2CH2], 28.2 (3CH3,tBu), 25.6 (CH2, CH2CH2CH2). g) 4 -Nitrophenyl N- ( tert -butyloxycarbonyl ) -2-aza-pipecolate (6b) ( Figure 16 )

[0150] Compound (5b) ( 2. 6623 g, 14. 2945 mmol ) was transferred to a round-bottom flas k and then dissolved in anhydrous CH2CI2 ( 40 mL ). The solution was placed on ice. Then, Et3N ( 3. 00 mL, 21. 5 mmol ) was added, followed by NPCF ( 5. 7620 g, 28. 587 mmol ). The mixture was stirred for 3 hours at 0 ° C under an atmosphere of argon ( 1 atm). Workup: After the reaction, the solvent was removed using a rotary evaporator. Then, EtOAc ( 50 mL ) was added, and the mixture was washed with aqueous NaHCO3( 3 x 50 mL ). Afterwards, the organic layer was collected and dried over with anhydrous Na2SO4, and the suspension was filtered. The filtrate was collected, and the solvent was removed using a rotary evaporator. The resulting residue was puri fied by column chromatography using hexane / EtOAc ( 3: 1 ) as the eluent. Compound (6b) (pale yellow solid) was obtained in 79% yield ( 3. 9678 g).

[0151] Rr = 0. 56 in hexane / EtOAc ( 3: 1 ). MP = [ 103; 106 ] ° C.

[0152] 1H NMR (DMSO-d6, 400 MHz, 25 °C) δ: 8.43 – 8.18 (m, 2H, ArH), 7.58 – 7.26 (m, 2H, ArH), [4.34 – 3.85 (m, 2H), 3.26 – 2.82 (m, 2H), H-2 + H-5], 1.83 – 1.49 (m, 4H, H-3 + H-4), 1.43 (s, 9H,tBu).

[0153] 13C{1H} and DEPT-135 NMR (DMSO-d6, 101 MHz, 25 °C) δ: [ 155. 8 ( C ), 155. 6 ( C ), 153. 9 ( C ), 153. 6 ( C ), 153. 3 ( C ), 151. 7 ( C ), 151. 5 ( C ), 2 x CO + ArC ], 144. 7 ( C, ArC ), 125. 3 ( 2CH, ArC ), 122 ( 2CH, ArC ), [81.6 (C), 81.1 (C),tBu], [ 47. 2 ( CH2), 46. 7 ( CH2), 44. 9 ( CH2), 44. 5 ( CH2), 44. 2 ( CH2), 43. 9 ( CH2), C-2 + C-5 ], 27.8 (3CH3,tBu), [ 23. 4 ( CH2), 22. 9 ( CH2), 22. 6 ( CH2), 2CH2, C-3 + C-4 ].

[0154] h) Methyl N- ( tert -butyloxy carbonyl ) -2-aza-prolyl-L-leucylglycinate (8a) ( Figure 17 ) To a solution of compound (6a) ( 0. 5636 g, 1. 6708 mmol ) in DMF ( 5 mL ), was added a solution of dipeptide (7a) tri fluoroacetate salt ( 0. 6325 g, 1. 9998 mmol ) in anhydrous CH2CI2 ( 15 mL ) and Et3N ( 1. 30 mL, 9. 33 mmol ) followed by DMAP ( 0. 0718 g, 0. 588 mmol ) and catalytic amounts of p-TsOH monohydrate. The mixture was stirred for 3 days at 60-70 ° C. Workup: After the reaction, the volatiles were removed using a rotary evaporator. Then, EtOAc ( 50 mL ) was added, and the mixture was washed with aqueous NaHCOs ( 3 x 50 mL ). Then, the aqueous phase was washed with EtOAc ( 1 x 50 mL ). Afterwards, the organic layers were collected and dried over with anhydrous Na2SO4, the suspension was filtered. The filtrate was collected, and the solvent was removed using a rotary evaporator. The resulting residue was puri fied by column chromatography using EtOAc as the eluent. Tripeptide Compound (8a) (pale yellow oil ) was obtained in 79% yield ( 0. 5293 g).

[0155] Rr = 0. 44 in EtOAc. [a]^0= -0. 19 ± 0. 13 ( cl. 08, CHCI3 ).

[0156] HRMS-ESI+m / z: 401.2400 calculated for C18H33N4O6+, 401.2392 found.

[0157] ! H NMR ( CDCI3, 400 MHz, 25 ° C ) 5: 7. 01 (br s, 1H, CONH), 5. 84 ( d, J = 8. 3 Hz, 1H, NCONH), 4. 39 - 4. 27 (m, 1H, HLeu-2 ), 4. 05 - 3. 91 (m, 2H, Hciy-2 ), 3.88 – 2.96 [3.70 (s), 7H, COOCH3+ CH2CH2CH2], 1. 98 (p, J = 7. 1 Hz, 2H, CH2CH2CH2 ), 1. 76 - 1. 59 (m, 2H, HLeu- 3 ), 1. 58 - 1. 48 (m, 1H, HLeu-4 ), 1. 45 ( s, 9H,fcBu), 0. 94 - 0. 86 (m, 3H, HLeu-5 ).

[0158] 13C{1H} and DEPT-135 NMR (CDCl3, 101 MHz, 25 °C) δ: 173.0 (C, COOCH3) 170.3 (C, CONH), 159.7 (C, OCON), 157.6 (C, NCONH), 82.7 (C,tBu), 52.6, (CH, CLeu-2), 52.3 (CH3, COOCH3), [47.0 (CH2), 45.7 (CH2), CH2CH2CH2], 41.2 (CH2, CGly-2), 40.9 (CH2, CLeu-3), 28.1 (3CH3,tBu), 25.7 (CH2, CH2CH2CH2), 24.8 (CH, CLeu-4), [23.1 (CH3), 21.9 (CH3), CLeu-5]. i ) Methyl N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-leucyl-L-alaninate (8b) ( Figure 18 )

[0159] Following the method described for the synthesis of compound (8a), compound (8b) was prepared using dipeptide (7b) tri fluoroacetate salt ( 1. 2175 g, 3. 6860 mmol ), anhydrous CH2CI2 ( 30 mL ), EtsN ( 2. 00 mL, 14. 3 mmol ), a solution of compound ( 6a) ( 1. 0325 g, 3. 0608 mmol ) in DMF ( 10 mL ), DMAP ( 0. 0935 g, 0. 765 mmol ), and catalytic amounts of p-TsOH monohydrate. After the typical workup, the resulting residue was puri fied by column chromatography using EtOAc as the eluent. Tripeptide (8b) (pale yellow oil ) was obtained in 55% yield ( 0. 6922 g).

[0160] Rr = 0. 44 in EtOAc. [a] >9= -15. 22 ± 0. 13 ( cl. 04, CHCI3 ).

[0161] HRMS-ES I+ m / z: 415. 2557 calculated for C19H35N4O6, 415. 2552 found.

[0162] iH NMR ( CDCI3, 400 MHz, 25 ° C ) 5:: 6. 89 (br s, 1H, CONH), 5. 84 ( d, J = 8. 3 Hz, 1H, NCONH), 4. 48 (p, J = 7. 2 Hz, 1H, HAla-2 ), 4. 31 ( dt, J = 9. 5, 4. 8 Hz, 1H,, HLeu-2 ), 4. 10 - 2. 86 [ 3. 69 ( s ), 7H, COOCH3 + CH2CH2CH2 ], 1. 97 (p, J = 7. 2 Hz, 2H, CH2CH2CH2 ), 1. 74 - 1. 57 (m, 2H, HLeu-3 ), 1. 52 - 1. 47 (m, 1H, HLeu- 4 ), 1. 44 ( s, 9H,fcBu), 1. 35 ( d, J = 7. 2 Hz, 3H, HAia-3 ), [ 0. 90 ( d, J = 6. 4 Hz ), 0. 89 ( d, J = 6. 1 Hz ), 6H, HLeu-5 ].13C {1H } and DEPT- 135 NMR ( CDCI3, 101 MHz, 25 ° C ) 5: 173. 3 ( C, COOCH3 ) 172. 2 ( C, CONH), 159. 6 ( C, OCON), 157. 6 ( C, NCONH), 82. 7 ( C,fcBu), 52. 7 ( CH, CLeu-2 ), 52. 4 ( CH3, COOCH3 ), 48. 1 ( CH, CAIS- 2 ), [ 46. 9 ( CH2), 45. 8 ( CH2), CH2CH2CH2 ], 41. 3 ( CH2, CLeu- 3 ), 28. 1 ( 3CH3,fcBu), 25. 7 ( CH2, CH2CH2CH2 ), 24. 8 ( CH, CLeu-4 ), [ 23. 1 ( CH3), 21. 9 ( CH3), CLeu- 51, 18. 2 ( CH3, CAia-3 ).

[0163] j ) Methyl _ N- ( tert -butyloxy carbonyl ) -2-aza-prolyl-L-isoleucylglycinate (8c) ( Figure 19 )

[0164] Following the method described for the synthesis of compound (8a), compound (8c) was prepared using dipeptide (7c) tri fluoroacetate salt ( 1. 2474 g, 3. 9440 mmol ), anhydrous CH2CI2 ( 30 mL ), EtsN ( 2. 00 mL, 14. 3 mmol ), a solution of compound (6a) ( 1. 0145 g, 3. 0074 mmol ) in DMF ( 10 mL ), DMAP ( 0. 0929 g, 0. 760 mmol ), and catalytic amounts of p-TsOH monohydrate. After the typical workup, the resulting residue was puri fied by column chromatography using EtOAc as the eluent. Tripeptide (8c) (white wax ) was obtained in 68 % yield ( 0. 8227 g).

[0165] Rr = 0. 59 in EtOAc. MP = [ 91; 93 ] ° C. [a]^>7= -23. 59 ± 0. 17 ( cl. 01, CHCI3 ).

[0166] HRMS-ES I+ m / z: 401. 2400 calculated for CI8H33N4O6+, 401. 2410 found.

[0167] iH NMR ( CDCI3, 400 MHz, 25 ° C ) 5: 7. 02 (br s, 1H, CONH), 5. 98 ( d, J = 8. 1 Hz, 1H, NCONH), 4. 18 ( dd, J = 8. 9, 6. 3 Hz, 1H, Hue-2 ), 4. 11 - 2. 89 [ 3. 97 ( d, J = 5. 4 Hz ), 3. 68 ( s ), 9H, Hciy-2 + COOCH3 + CH2CH2CH2 ], 2. 06 - 1. 86 (m, 3H, CH2CH2CH2 + Hile-3 ), 1. 56 - 1. 35 [ 1. 44 ( s ), 10H, Hne-4a + tBu], 1. 13 -0. 99 (m, 1H, Hne- 4b ), 0. 92 (d, J = 6. 8 Hz, 3H, Hne- 6 ), 0. 86 ( t, J = 7. 4 Hz, 3H, Hue-5 ).

[0168] 13C {1H } and DEPT- 135 NMR ( CDCI3, 101 MHz, 25 ° C ) 5: 172. 1 ( C, COOCH3 ) 170. 2 ( C, CONH), 159. 6 ( C, OCON), 157. 5 ( C, NCONH), 82. 6 ( C,fcBu), 58. 9, ( CH, Cne-2 ), 52. 2 ( CH3, COOCH3 ), [ 46. 9 ( CH2), 45. 7 ( CH2), CH2CH2CH2 ], 41. 1 ( CH2, CGiy-2 ), 37. 0 ( CH, Cue-3 ), 28. 1 ( 3CH3,fcBu), 25. 7 ( CH2, CH2CH2CH2 ), 24. 5 ( CH2, Cue- 4 ), 15. 7 ( CH3, Cue- 6 ), 11. 4 ( CH3, Cne-5 ).

[0169] k) Methyl N- ( tert -butyloxy carbonyl ) -2-aza-prolyl-L-isoleucyl-L-alaninate (8d) ( Figure 20 )

[0170] Following the method described for the synthesis of compound (8a), compound (8d) was prepared using dipeptide (7d) tri fluoroacetate salt ( 1. 2158 g, 3. 6809 mmol ), anhydrous CH2CI2 ( 30 mL ), EtsN ( 2. 00 mL, 14. 3 mmol ), a solution of compound (6a) ( 1. 0125 g, 3. 0015 mmol ) in DMF ( 10 mL ), DMAP ( 0. 0920 g, 0. 753 mmol ), and catalytic amounts of p-TsOH monohydrate. After the typical workup, the resulting residue was puri fied by column chromatography using EtOAc as the eluent. Tripeptide (8d) (pale yellow oil ) was obtained in 70% yield ( 0. 8660 g).

[0171] Rr = 0. 61 in EtOAc. [a] >9= -15. 22 ± 0. 13 ( cl. 04, CHC13).

[0172] HRMS-ES I+m / z: 415. 2557 calculated for CigHss^Oe, 415. 2553 found.

[0173] ! H NMR ( CDCI3, 400 MHz, 25 ° C ) 5: 6. 80 (br s, 1H, CONH), 6. 02 ( d, J = 8. 7 Hz, 1H, NCONH), 4. 52 (p, J = 7. 2 Hz, 1H, HAia-2 ), 4. 17 ( dd, J = 8. 9, 6. 3 Hz, 1H, Hne-2 ), 4. 08 - 2. 88 [ 3. 70 ( s ), 7H, COOCH3 + CH2CH2CH2 ], 2. 06 - 1. 84 (m, 3H, CH2CH2CH2 + Hue-3 ), 1. 52 - 1. 41 [ 1. 46 ( s ), 10H, Hne-4a +fcBu), 1. 37 ( d, J = 7. 2 Hz, 3H, HAia- 3 ), 1. 14 - 1. 03 (m, 1H, Hne-4b ), 0. 93 ( d, J = 6. 8 Hz, 3H, Hue- 6 ), 0. 88 ( t, J = 7. 4 Hz, 3H, Hne-5 ).13C {1H } and DEPT- 135 NMR ( CDCI3, 101 MHz, 25 ° C ) 5: 173. 2 ( C, COOCH3 ) 171. 3 ( C, CONH), 159. 7 ( C, OCON), 157. 6 ( C, NCONH), 82. 5 ( C,fcBu), 58. 8, ( CH, Cne-2 ), 52. 4 ( CH3, COOCH3 ), 48. 1 ( CH, CAia- 2 ), [ 46. 9 ( CH2), 45. 8 ( CH2), CH2CH2CH2], 37. 4 ( CH, Cue-3 ), 28. 1 ( 3CH3,fcBu), 25. 8 ( CH2, CH2CH2CH2 ), 24. 7 ( CH2, Cue- 4 ), 18. 2 ( CH3, CAia- 3 ), 15. 6 ( CH3, Cne-6 ), 11. 5 ( CH3, Cne-5 ).

[0174] 1 ) Methyl N- ( tert -butyloxy carbonyl ) -2-aza-prolyl-L-valylglycinate (8e) ( Figure 21 )

[0175] Following the method described for the synthesis of compound (8a), compound (8e) was prepared using dipeptide (7e) tri fluoroacetate salt ( 1. 2856 g, 4. 2534 mmol ), anhydrous CH2CI2 ( 30 mL ), EtsN ( 2. 00 mL, 14. 3 mmol ), a solution of compound (6a) ( 1. 1992 g, 3. 5550 mmol ) in DMF ( 10 mL ), DMAP ( 0. 1083 g, 0. 8865 mmol ), and catalytic amounts of p-TsOH monohydrate. After the typical workup, the resulting residue was puri fied by column chromatography using EtOAc as the eluent. Tripeptide (8e) (pale yellow oil ) was obtained in 78 % yield ( 1. 0654 g).

[0176] Rr = 0. 86 in EtOAc. [a]p0= + 15. 88 ± 0. 08 ( cl. 06, CHC13).

[0177] HRMS-ES I+ m / z: 387. 2244 calculated for Ci7H3iN4O6+, 387. 2236 found.

[0178] ! H NMR ( CDCI3, 400 MHz, 25 ° C ) 5: 7. 00 (br s, 1H, CONH), 6. 00 ( d, J = 8. 1 Hz, 1H, NCONH), 4. 18 ( dd, J = 8. 9, 5. 8 Hz, 1H, Hvai-2 ), 4. 13 - 3. 15 [ 3. 99 ( d, J = 5. 3 Hz ), 3. 70 ( s ), 9H, HGiy-2 + COOCH3 + CH2CH2CH2 ], 2. 22 ( dq, J = 13. 2, 6. 7 Hz, 1H, HVai- 3 ), 1. 99 (p, J = 6. 5 Hz, 2H, CH2CH2CH2 ), 1. 46 ( s, 9H,fcBu), [ 0. 96 ( d, J = 6. 8 Hz, 3H), 0. 90 ( d, J = 6. 8 Hz, 3H), HVai-4 ].

[0179] 13C {1H } and DEPT- 135 NMR ( CDCI3, 101 MHz, 25 ° C ) 5: 172. 1 ( C, COOCH3 ) 170. 2 ( C, CONH), 159. 7 ( C, OCON), 157. 6 ( C, NCONH), 82. 7 ( C,fcBu), 59. 4, ( CH, CVai-2 ), 52. 3 ( CH3, COOCH3 ), [ 47. 0 ( CH2), 45. 7 ( CH2), CH2CH2CH2 ], 41. 1 ( CH2, CGiy-2 ), 30. 6 ( CH, Cvai-3 ), 28. 1 ( 3CH3,fcBu), 25. 8 ( CH2, CH2CH2CH2 ), [ 19. 4 ( CH3), 17. 5 ( CH3), Cvai-4 ].

[0180] m) Methyl N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-valyl-L-alaninate (8f) ( Figure 22 )

[0181] Following the protocol described for the synthesis of compound (8a), compound (8f) was prepared using dipeptide (7f) tri fluoroacetate salt ( 1. 2091 g, 3. 8229 mmol ), anhydrous CH2CI2 ( 30 mL ), Et3N ( 2. 00 mL, 14. 3 mmol ), a solution of compound (6a) ( 1. 0016 g, 2. 9692 mmol ) in DMF ( 10 mL ), DMAP ( 0. 0904 g, 0. 740 mmol ), and catalytic amounts of TsOH monohydrate. After the typical workup, the resulting residue was puri fied by column chromatography using EtOAc as the eluent. Tripeptide (8f) (pale yellow oil ) was obtained in 82 % yield ( 0. 9729 g).

[0182] Rr = 0. 56 in EtOAc. [a]^3= +26. 40 ± 0. 18 ( cl. 00, CHCI3 ).

[0183] HRMS-ES I+ m / z: 401. 2400 calculated for CI8H33N4O6+, 401. 2400 found. ! H NMR ( CDCI3, 400 MHz, 25 ° C ) 5: 6. 79 (br s, 1H, CONH), 6. 06 ( d, J = 8. 4 Hz, 1H, NCONH), 4. 53 (p, J = 7. 2 Hz, 1H, HAia-2 ), 4. 16 ( dd, J = 8. 9, 6. 0 Hz, 1H, HVai-2 ), 4. 08 - 2. 90 [ 3. 72 ( s ), 7H, COOCH3 + CH2CH2CH2 ], 2. 18 ( dq, J = 13. 5, 6. 7 Hz, HVai-3 ), 1. 99 (p, J = 7. 2 Hz, 2H, CH2CH2CH2 ), 1. 47 ( s, 9H,fcBu), 1. 38 ( d, J = 7. 1 Hz, 3H, HAIS- 3 ), [ 0. 97 ( d, J = 6. 8 Hz, 3H), 0. 90 ( d, J = 6. 8 Hz, 3H), HVai-4 ].

[0184] 13C {1H } and DEPT- 135 NMR ( CDCI3, 101 MHz, 25 ° C ) 5: 173. 3 ( C, COOCH3 ) 171. 2 ( C, CONH), 159. 8 ( C, OCON), 157. 6 ( C, NCONH), 82. 6 ( C,fcBu), 59. 4, ( CH, CVai-2 ), 52. 5 ( CH3, COOCH3 ), 48. 1 ( CH, CAia-2 ), [ 46. 9 ( CH2), 45. 8 ( CH2), CH2CH2CH2], 31. 1 ( CH, CVai-3 ), 28. 2 ( 3CH3,fcBu), 25. 8 ( CH2, CH2CH2CH2 ), [ 19. 4 ( CH3), 17. 7 ( CH3), CVai-4 ], 18. 2 (CH3, CAia-3 ).

[0185] n) Methyl 2-aza-prolyl-L-leucylglycinate tri fluoroacetate salt (9a) ( Figure 23 )

[0186] Tripeptide (8a) ( 0. 2894 g, 0. 7226 mmol ) was trans ferred to a round-bottom flask and then dissolved in anhydrous CH2CI2 ( 5 mL ). To the resulting solution, TFA ( 1. 80 mL, 23. 5 mmol ) was added. The mixture was stirred for 5 hours at room temperature. Workup: After the reaction, the volati les were eliminated using a rotary evaporator. The resulting was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (9a) (pale yellow oil ) was obtained in 66% yield ( 0. 1972 g).

[0187] Rr = 0. 04 in EtOAc. [a]^1= -0. 73 ± 0. 13 ( cl. 02, CH3OH).

[0188] HRMS-ES I+ m / z: 301. 1870 calculated for CI3H25N4O4+, 301. 1785 found.

[0189] iH NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 32 ( dd, J = 9. 1, 5. 8 Hz, 1H, HLeu-2 ), [ 4. 00 ( d, ABq, J = 17. 5 Hz, 1H), 3. 93 ( d, ABq, J = 17. 6 Hz, 1H), Hciy-2 ], 3. 74 ( s, 3H, COOCH3 ), [ 3. 59 - 3. 37 (m, 2H), 3. 09 - 2. 82 (m, 2H), CH2CH2CH2 ], 2. 10 (p, J = 7. 0 Hz, 2H, CH2CH2CH2 ), 1. 83 - 1. 55 (m, 3H, HLeu-3 + HLeu-4 ), [ 0. 99 ( d, J = 6. 5 Hz, 3H), 0. 96 ( d, J = 6. 5 Hz, 3H), HLeu-5 ].

[0190] 13C {1H } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: 176. 3 ( C, COOCH3 ), 171. 7 ( C, CONH), 161. 5 ( C, NCONH), 53. 9 ( CH, CLeu-2 ), 52. 6 ( CH3, COOCH3 ), [ 47. 9 ( CH2), 46. 5 ( CH2), CH2CH2CH2], 42. 5 ( CH2, Cay-2 ), 41. 8 ( CH2, CLeu-3 ), 28. 7 ( CH2, CH2CH2CH2), 25. 9 ( CH, CLeu- 4 ), [ 23. 4 ( CH3), 22. 1 ( CH3), CLeu-5 ].

[0191] o ) Methyl 2-aza-prolyl-L-leucyl-L-alaninate tri fluoroacetate salt (9b) ( Figure 24 )

[0192] Following the method described for the synthesis of compound (9a), compound (9b) was prepared using tripeptide (8b) ( 0. 1928 g, 0. 4651 mmol ), anhydrous CH2CI2 ( 5 mL ), and TFA ( 1. 80 mL, 23. 5 mmol ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (9b) (pale yellow oil ) was obtained in 72 % yield ( 0. 1429 g).

[0193] Rr = 0. 05 in EtOAc. [a]^3= -33. 46 ± 0. 23 ( cl. 01, CH3OH).

[0194] HRMS-ES I+ m / z: 315. 2027 calculated for Ci4H27N4O4+, 315. 1910 found.

[0195] ! H NMR ( DMSO-cte, 400 MHz, 25 ° C ) 5: 8. 39 ( d, J = 6. 9 Hz, 1H, CONH), 6. 77 ( d, J = 9. 2 Hz, 1H, NCONH), 4. 33 - 4. 14 (m, 2H, HAIS- 2 + HLeu- 2 ), 3. 60 ( s, 3H, COOCH3 ), [ 3. 28 ( t, J = 7. 5 Hz, 2H), 2. 71 ( t, J = 6. 7 Hz, 2H), CH2CH2CH2 ], 1. 88 ( dp, J = 6. 9, 2. 5 Hz, 2H, CH2CH2CH2 ), 1. 67 - 1. 55 (m, 1H, HLeu-4 ), 1. 49 -1. 33 (m, 2H, HLeu-3 ), 1. 27 ( d, J = 7. 3 Hz, 3H, HAia-3 ), 0. 96 - 0. 79 (m, 6H, HLeu-5 ).

[0196] 13C {1H } and DEPT- 135 NMR ( DMSO-cte, 101 MHz, 25 ° C ) 5: [ 172. 9 ( C ), 172. 6 ( C ), COOCH3 + CONH], 159. 5 ( C, NCONH), 51. 8 ( CH, CLeu-2 ), 51. 3 ( CH3, COOCH3 ), 47. 4 ( CH, CAia-2 ), [ 46. 5 ( CH2), 45. 1 ( CH2), CH2CH2CH2], 42. 2 ( CH2, CLeu-3 ), 27. 5 ( CH2, CH2CH2CH2), 24. 2 ( CH, CLeu-4 ), [ 23. 1 ( CH3), 22. 0 ( CH3), CLeu-5 ], 16. 8 ( CH3, CAia-3 ). p ) Methyl 2-aza-prolyl-L-isoleucylglycinate tri fluoroacetate salt (9c) ( Figure 25 )

[0197] Following the method described for the synthesis of compound (9a), compound (9c) was prepared using tripeptide (8c) ( 0. 2815 g, 0. 7029 mmol ), anhydrous CH2CI2 ( 5 mL ), and TFA ( 1. 80 mL, 23. 5 mmol ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (9c) (pale yellow oil ) was obtained in 71 % yield ( 0. 2072 g).

[0198] Rr = 0. 04 in EtOAc. [a]^2= -23. 64 ± 0. 12 ( cl. 06, CH3OH).

[0199] HRMS-ES I+ m / z: 301. 1870 calculated for CI3H25N4O4+, 301. 1760 found.

[0200] iH NMR ( CD3OD, 400 MHz, 25 ° C ) 5: [ 4. 28 ( d, J = 6. 7 Hz ), 4. 18 ( d, J = 6. 6 Hz ), 1H, Hue-2 ], [ 4. 03 ( d, ABq, J = 17. 5 Hz, 1H), 3. 93 ( d, ABq, J = 17. 5 Hz, 1H), HGiy-2 ], 3. 75 ( s, 3H, COOCH3 ), [ 3. 48 ( dt, J = 7. 2, 3. 5 Hz, 2H), 3. 05 - 2. 86 (m, 2H), CH2CH2CH2 ], 2. 10 (p, J = 7. 0 Hz, 2H, CH2CH2CH2 ), 1. 99 - 1. 83 (m, 1H, Hue-3 ), 1. 69 - 1. 53 (m, 1H, Hne-4a ), 1. 26 -1. 13 (m, 1H, Hne-4b ), 1. 08 - 0. 90 [ 1. 01 ( d, J = 6. 8 Hz ), 0. 97 ( t, J = 7. 4 Hz ), 6H, Hne-5 + Hne- 6 ].

[0201] 13C {1H } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: 175. 1 ( C, COOCH3 ), 171. 5 ( C, CONH), 162. 0 ( C, NCONH), 59. 8 ( CH, Cne-2 ), 52. 5 ( CH3, COOCH3 ), [ 48. 0 ( CH2), 46. 4 ( CH2), CH2CH2CH2], 41. 8 ( CH2, Cciy-2 ), 38. 9 ( CH, Cne-3 ), 28. 9 ( CH2, CH2CH2CH2 ), 25. 7 ( CH2, Cue- 4 ), 16. 0 ( CH3, Cne-6 ), 11. 7 ( CH3, Cne-5).

[0202] q) Methyl 2-aza-prolyl-L-isoleucyl-L-alaninate tri fluoroacetate salt (9d) ( Figure 26 )

[0203] Following the method described for the synthesis of compound (9a), compound (9d) was prepared using tripeptide (8d) ( 0. 2262 g, 0. 5457 mmol ), anhydrous CH2CI2 ( 5 mL ), and TFA ( 1. 80 mL, 23. 5 mmol ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (9d) (pale yellow oil ) was obtained in 72 % yield ( 0. 1679 g).

[0204] Rr = 0. 06 in EtOAc. [a]^3= -41. 49 ± 0. 17 ( cl. 08, CH3OH).

[0205] HRMS-ES I+ m / z: 315. 2027 calculated for Ci4H27N4O4+, 315. 1929 found.

[0206] ! H NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 50 - 4. 37 (m, 1H, HAia-2 ), [ 4. 25 ( d, J = 7. 1 Hz ), 4. 15 ( d, J = 7. 1 Hz ), 1H, Hne-2 ], 3. 73 ( s, 3H, COOCH3 ), [ 3. 48 ( t, J = 7. 4 Hz, 2H), 3. 02 - 2. 89 (m, 2H), CH2CH2CH2 ], 2. 10 (p, J = 6. 9 Hz, 2H, CH2CH2CH2 ), 1. 91 - 1. 79 (m, 1H, Hue-3 ), 1. 64 - 1. 52 (m, 1H, Hne-4a ), 1. 42 ( d, J = 7. 3 Hz, 3H, HAia- 3 ), 1. 23 - 1. 12 (m, 1H, Hne-4b ), 1. 02 -0. 92 [ 1. 00 ( d, J = 6. 8 Hz ), 0. 95 ( t, J = 7. 4 Hz ), 6H, Hne-5 + HIle- 6 ].

[0207] ^C pH } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: [ 174. 4 ( C ), 174. 4 ( C ), COOCH3 + CONH], 161. 9 ( C, NCONH), 59. 5 ( CH, Cue- 2 ), 52. 6 ( CH3, COOCH3 ), 49. 4 ( CH, CAia-2 ), [ 47. 9 ( CH2), 46. 5 ( CH2), CH2CH2CH2 ], 39. 0 ( CH, Cne-3 ), 28. 8 ( CH2, CH2CH2CH2 ), 25. 8 ( CH2, Cue- 4 ), 17. 3 ( CH3, CAia-3 ), 15. 9 ( CH3, Cue- 6 ), 11. 5 ( CH3, Cue-5 ).

[0208] r ) Methyl 2-aza-prolyl-L-valylglycinate tri fluoroacetate salt (9e) ( Figure 27 )

[0209] Following the protocol described for the synthesis of compound (9a), compound (9e) was prepared using tripeptide (8e) ( 0. 2658 g, 0. 6878 mmol ), anhydrous CH2CI2 ( 5 mL ), and TFA ( 1. 80 mL, 23. 5 mmol ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10 % ) in EtOAc as the eluent. Tripeptide (9e) (pale yellow oil ) was obtained in 72 % yield ( 0. 1973 g). Rr = 0. 04 in EtOAc. [a]^1= + 18. 12 ± 0. 12 ( cl. 07, CH3OH).

[0210] HRMS-ES I+ m / z: 287. 1714 calculated for Ci2H23N4O4+, 287. 1629 found. ! H NMR ( DMSO-cte, 400 MHz, 25 ° C ) 5: 8. 51 ( t, J = 5. 8 Hz, 1H, CONH), 6. 91 ( d, J = 9. 3 Hz, 1H, NCONH), 4. 04 ( dd, J = 9. 3, 6. 1 Hz, 1H, Hvai-2 ), [ 3. 88 ( dd, ABX, JAB = 17. 3 Hz, JAX = 5. 9 Hz, 1H), 3. 79 ( dd, ABX, JAB = 17. 3 Hz, JAX = 5. 8 Hz, 1H), HGiy-2 ], 3. 61 ( s, 3H, COOCH3), [ 3. 45 - 3. 17 (m, 2H), 2. 79 (br s, 2H), CH2CH2CH2 ], 2. 02 - 1. 86 (m, 3H, CH2CH2CH2 + HVai-3 ), [ 0. 86 ( d, J = 6. 7 Hz ), 0. 81 ( d, J = 6. 7 Hz ), 6H, HVai-4 ].

[0211] 13C {1H } and DEPT- 135 NMR ( DMSO-cte, 101 MHz, 25 ° C ) 5: [ 172. 1 ( C ), 170. 3 ( C ), COOCH3 + CONH], 159. 6 ( C, NCONH), 58. 1 ( CH, Cvai-2 ), 51. 7 ( CH3, COOCH3 ), [ 46. 4 ( CH2), 45. 5 ( CH2), CH2CH2CH2 ], 40. 5 ( CH2, Cciy-2 ), 31. 3 ( CH, CVai-3 ), 27. 3 ( CH2, CH2CH2CH2 ), [ 19. 2 ( CH3), 17. 8 ( CH3), Cvai-4 ].

[0212] s ) Methyl 2-aza-prolyl-L-valyl-L-alaninate tri fluoroacetate salt ( 9f) ( Figure 28 )

[0213] Following the method described for the synthesis of compound (9a), compound (9f) was prepared using tripeptide (8f) ( 0. 2415 g, 0. 6030 mmol ), anhydrous CH2CI2 ( 5 mL ), and TFA ( 1. 80 mL, 23. 5 mmol ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (9f) (pale yellow oil ) was obtained in 69% yield ( 0. 1732 g).

[0214] Rr = 0. 06 in EtOAc. [a]p0= -35. 03 ± 0. 13 ( cl. 03, CH3OH).

[0215] HRMS-ES I+ m / z: 301. 1870 calculated for CI3H25N4O4+, 301. 1772 found.

[0216] ! H NMR ( DMSO-cte, 400 MHz, 25 ° C ) 5: 8. 47 ( d, J = 6. 6 Hz, 1H, CONH), 6. 86 ( d, J = 9. 4 Hz, 1H, NCONH), 4. 24 (p, J = 7. 2 Hz, 1H, HAia- 2 ), 4. 06 ( dd, J = 9. 4, 6. 1 Hz, 1H, HVai-2 ), 3. 60 ( s, 3H, COOCH3 ), [ 3. 30 ( dt, J = 7. 4, 4. 3 Hz, 2H), 2. 75 ( t, J = 6. 8 Hz, 2H), CH2CH2CH2 ], 2. 04 - 1. 81 (m, 3H, CH2CH2CH2 + HVai- 3 ), 1. 27 ( d, J = 7. 3 Hz, 3H, HAia- 3 ), [ 0. 86 ( d, J = 6. 8 Hz ), 0. 80 ( d, J" = 6. 8 Hz ), 6H, HVai-4 ].13C {1H } and DEPT- 135 NMR ( DMSO-cte, 101 MHz, 25 ° C ) 5: [ 173. 0 ( C ), 171. 5 ( C ), COOCH3+ CONH], 159. 7 ( C, NCONH), 57. 6 ( CH, Cvai-2 ), 51. 8 ( CH3, COOCH3 ), 47. 6 ( CH, CAia-2 ), [ 46. 4 ( CH2), 45. 4 ( CH2), CH2CH2CH2], 31. 5 ( CH, Cvai-3 ), 27. 4 ( CH2, CH2CH2CH2), [ 19. 1 ( CH3), 17. 7 ( CH3), Cvai-4 ], 16. 8 ( CH3, CAia-3 ).

[0217] t ) N- ( tert -Butyloxycarbonyl ) -2-aza-prolyl-L-leucylglycinamide (10a) ( Figure 29 )

[0218] Tripeptide (8a) ( 0. 3251 g, 0. 8118 mmol ) was trans ferred to a round-bottom flask and then dissolved in a solution of ammonia 7 M in CH3OH ( 25 mL ). The mixture was stirred for 48 hours at room temperature. Workup: After the reaction, the volatiles were eliminated using a rotary evaporator. The resulting was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (10a) (white solid) was obtained in 83% yield ( 0. 2603 g).

[0219] Rr = 0. 06 in EtOAc.

[0220] MP = [ 144; 146 ] ° C. [a]^2= + 9. 74 ± 0. 11 ( cl. 12, CH3OH).

[0221] HRMS-ES I+ m / z: 386. 2403 calculated for Ci7H32N5O5+, 386. 2401 found.

[0222] ! H NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 31 ( dd, J = 9. 4, 5. 6 Hz, 1H, HLeu-2 ), 4. 09 - 3. 20 [ 3. 95 ( d, ABq, J = 17. 0 Hz ), 3. 82 ( d, ABq, J = 17. 0 Hz ), 6H, HGiy-2 + CH2CH2CH2], 2. 15 - 2. 00 (m, 2H, CH2CH2CH2), 1. 85 - 1. 64 (m, 3H, HLeu-3 + HLeu-4 ), 1. 54 ( s, 9H,fcBu), [ 1. 01 ( d, J = 6. 5 Hz ), 0. 99 ( d, J = 6. 4 Hz ), 6H, HLeu-5 ].

[0223] 13C {1H } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: 175. 9 ( C, CONH2), 174. 3 ( C, CONH), 161. 8 ( C, NCON), 159.2 ( C, OCONH), 83. 6 ( C,fcBu), 54. 5 ( CH, CLeu-2 ), [ 48. 1 ( CH2), 47. 1 ( CH2), CH2CH2CH2], 43. 1 ( CH2, CGIY- 2 ), 41. 5 ( CH2, CLeu-3 ), 28. 4 ( 3CH3,fcBu), 26. 6 ( CH2, CH2CH2CH2), 25. 8 ( CH, CLeu-4 ), [ 23. 6 ( CH3), 21. 7 ( CH3), CLeu-5 ]. u) N- ( tert -Butyloxycarbonyl ) -2-aza-prolyl-L-leucyl-L-alaninamide ( 10b) ( Figure 30 )

[0224] Following the method described for the synthesis of compound ( 10a), compound ( 10b) was prepared using tripeptide (8b) ( 0. 1951 g, 0. 4707 mmol ) and a solution of ammonia 7 M in CH3OH ( 20 mL ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide ( 10b) (white solid) was obtained in 77 % yield ( 0. 1449 g).

[0225] Rf= 0 in EtOAc. MP = [ 65; 67 ] ° C. [a]^0= +3. 13 ± 0. 16 ( cl. 07, CH3OH).

[0226] HRMS-ESI+m / z: 400. 2560 calculated for C18H34N5O5+, 400. 2550 found.

[0227] ! H NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 47 - 4. 25 (m, 2H, HLeu-2 + HAia- 2 ), 4. 16 - 3. 08 ( 4H, CH2CH2CH2), 2. 18 - 1. 96 (m, 2H, CH2CH2CH2), 1. 86 - 1. 71 (m, 1H, HLeu-4 ), 1. 71 - 1. 61 (m, 2H, HLeu-3 ), 1. 55 ( s, 9H,fcBu), 1. 42 ( d, J = 7. 2 Hz, 3H, HAia-3 ), 1. 00 ( appt, J = 6. 9 Hz, 6H, HLeu-5 ).

[0228] ^C pH } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: 177. 4 ( C, CONH2), 175. 2 ( C, CONH), 161. 6 ( C, NCONH), 159. 1 ( C, OCON), 83. 6 ( C,fcBu), [ 54. 5 ( CH), 54. 4 ( CH), CLeu-2 ], 50. 0 ( CH, CAia-2 ), [ 48. 1 ( CH2), 47. 0 ( CH2), CH2CH2CH2], 41. 8 ( CH2, CLeu-3 ), 28. 4 ( 3CH3,fcBu), 26. 6 ( CH2, CH2CH2CH2), 25. 9 ( CH, CLeu-4 ), [ 23. 6 ( CH3), 21. 7 ( CH3), CLeu-5 ], 18. 4 ( CH3, CAia-3 ).

[0229] v) N- ( tert -Butyloxycarbonyl ) -2-aza-prolyl-L-isoleucylglycinamide ( 10c) ( Figure 31 )

[0230] Following the method described for the synthesis of compound ( 10a), compound ( 10c) was prepared using tripeptide (8c) ( 0. 4007 g, 1. 001 mmol ) and a solution of ammonia 7 M in CH3OH ( 25 mL ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (10c) (white solid) was obtained in 82 % yield ( 0. 3181 g).

[0231] Rf= 0. 07 in EtOAc. MP = [ 45; 48 ] ° C. [α]D19= + 19. 86 ± 0. 23 ( c0. 99, CH3OH).

[0232] HRMS-ESI+m / z: 386. 2403 calculated for C17H32N5O5+, 386. 2402 found.

[0233] ! H NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 12 ( d, J = 7. 3 Hz, 1H, Hue-2 ), 4. 06 - 3. 11 [ 3. 96 ( d, ABq, J = 16. 9 Hz ), 3. 83 ( d, ABq, J = 17. 0 Hz ), 6H, HGiy-2 + CH2CH2CH2 ], 2. 06 (p, J = 6. 9 Hz, 2H, CH2CH2CH2 ), 1. 98 - 1. 86 (m, 1H, Hne-3 ), 1. 69 - 1. 48 [ 1. 55 ( s ), 10H,fcBu + Hne-4a ], 1. 29 - 1. 16 (m, 1H, Hne-4b ), 1. 09 - 0. 89 [ 1. 01 ( d, J = 6. 8 Hz ), 0. 97 ( t, J = 7. 4 Hz ), 6H, Hile_5 + Hile-6 ].

[0234] 13C {1H } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: 174. 8 ( C, CONH2 ), 174. 1 ( C, CONH), [ 161. 9 ( C ), 161. 8 ( C ), NOON], 159. 0 ( C, OCONH), 83. 6 ( C,fcBu), [ 60. 7 ( CH), 60. 6 ( CH), Cne-2 ], [ 48. 1 ( CH2), 47. 1 ( CH2), CH2CH2CH2 ], 43. 0 ( CH2, CGiy-2 ), 38. 1 ( CH, Cue-3 ), 28. 3 ( 3CH3,fcBu), 26. 6 ( CH2, CH2CH2CH2 ), 26. 0 ( CH2, Cue- 4 ), 16. 0 ( CH3, Cue- 6 ), 11. 4 ( CH3, Cne-5 ).

[0235] w) N- ( tert -Butyloxycarbonyl ) -2-aza-prolyl-L-isoleucyl-L-alaninamide (lOd) ( Figure 32 )

[0236] Following the method described for the synthesis of compound (10a), compound (lOd) was prepared using tripeptide (8d) ( 0. 4097 g, 0. 9884 mmol ) and a solution of ammonia 7 M in CH3OH ( 25 mL ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (lOd) (white solid) was obtained in 78 % yield ( 0. 3076 g).

[0237] Rf= 0. 13 in EtOAc. MP = [ 72; 74 ] ° C. [α]D21= + 17. 51 ± 0. 07 ( c1. 03, CH3OH).

[0238] HRMS-ES I+ m / z: 400. 2560 calculated for C18H34N5O5+, 400. 2559 found. ! H NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 41 ( q, J = 7. 1 Hz, 1H, HAIS- 2 ), 4. 22 - 4. 12 (m, 1H, Hne-2 ), 4. 06 - 3. 12 ( 4H, CH2CH2CH2 ), 2. 07 (p, J = 7. 1 Hz, 2H, CH2CH2CH2 ), 1. 95 - 1. 83 (m, 1H, Hue-3 ), 1. 69 - 1. 47 [ 1. 55 ( s ), 10H, tBu + Hne-4a ], 1. 41 ( d, J = 7. 2 Hz, 3H, HAia-3 ), 1. 27 - 1. 15 (m, 1H, Hne-4b ), 1. 07 - 0. 90 [ 1. 01 ( d, J = 6. 8 Hz ), 0. 96 ( t, J = 7. Hz ), 6H, Hue-5 + Hue- 6 ].

[0239] ^C pH } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: 177. 2 ( C, CONH2 ), 173. 8 ( C, CONH), 161. 5 ( C, NCONH), 159. 0 ( C, OCON), 83. 7 ( C,fcBu), [ 60.2 ( CH), 60. 2 ( CH), Cne-2 ], 49. 9 ( CH, CAia-2 ), [ 48. 1 ( CH2), 47. 0 ( CH2), CH2CH2CH2], 38. 7 ( CH, Cne-3 ), 28. 3 ( 3CH3,fcBu), 26. 6 ( CH2, CH2CH2CH2 ), 25. 8 ( CH2, Cne-4 ), 18. 3 ( CH3, CAia- 3 ), 16. 0 ( CH3, Cne-6 ), 11. 5 ( CH3, Cne-5 ).

[0240] x ) N- ( tert -Butyloxycarbonyl ) -2-aza-prolyl-L-valylglycinamide (lOe) ( Figure 33 )

[0241] Following the method described for the synthesis of compound (10a), compound (lOe) was prepared using tripeptide (8e) ( 0. 6303 g, 1. 631 mmol ) and a solution of ammonia 7 M in CH3OH ( 25 mL ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (lOe) (pale yellow solid) was obtained in 79% yield ( 0. 4811 g).

[0242] Rf= 0. 07 in EtOAc. MP = [ 41; 43 ] ° C. [α]D21= +5. 24 ± 0. 11 ( c1. 05, CH3OH).

[0243] HRMS-ESI+m / z: 372. 2247 calculated for C16H30N5O5+, 372. 2240 found.

[0244] iH NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 11 - 4. 05 (m, 1H, HVai-2 ), 4. 00 - 3. 00 [ 3. 95 ( d, ABq, J = 16. 9 Hz ), 3. 82 ( d, ABq, J = 17. 0 Hz ), 6H, Hciy-2 + CH2CH2CH2 ], 2. 21 - 1. 99 (m, 3H, CH2CH2CH2 + Hvai-3 ), 1. 53 ( s, 9H,fcBu), [ 1. 01 ( d, J = 6. 8 Hz ), 0. 98 ( d, J = 6. 8 Hz ), 6H, Hvai-4 ].13C {1H } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: 174. 7 ( C, CONH2 ), 174. 1 ( C, CONH), [ 161. 9 ( C ), 161. 8 ( C ), NCON], 159. 1 ( C, OCONH), 83. 7 ( C,fcBu), [ 61. 5 ( CH), 61. 4 ( CH), Cvai-2 ], [ 48. 1 ( CH2), 47. 1 (CH2), CH2CH2CH2], 43. 0 ( CH2, CGiy-2 ), [ 32. 0 ( CH), 31. 9 ( CH), Cvai-3 ], 28. 4 ( 3CH3,fcBu), 26. 6 ( CH2, CH2CH2CH2), [ 19. 8 ( CH3), 18. 6 ( CH3), Cvai-4 ].

[0245] y ) N- ( tert -Butyloxycarbonyl ) -2-aza-prolyl-L-valyl-L-alaninamide (lOf) ( Figure 34 )

[0246] Following the method described for the synthesis of compound (10a), compound (lOf) was prepared using tripeptide (8f) ( 0. 4897 g, 1. 223 mmol ) and a solution of ammonia 7 M in CH3OH ( 25 mL ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (lOf) (white solid) was obtained in 91 % yield ( 0. 4279 g).

[0247] Rf= 0. 07 in EtOAc. MP = [ 42; 45 ] ° C. [α]D20= - 18. 21 ± 0. 38 ( c1. 13, CH3OH).

[0248] HRMS-ESI+m / z: 386. 2403 calculated for C12H32N5O5+, 386. 2401 found.

[0249] ! H NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 39 ( q, J = 7. 2 Hz, 1H, HAia- 2 ), 4. 13 ( d, J = 6. 5 Hz, 1H, HVai-2 ), 4. 06 - 2. 86 ( 4H, CH2CH2CH2), 2. 21 - 1. 95 (m, 3H, CH2CH2CH2+ HVai-3 ), 1. 53 ( s, 9H,fcBu), 1. 40 ( d, J = 7. 2 Hz, 3H, HAia-3 ), [ 1. 01 ( d, J = 6. 8 Hz, 3H), 0. 95 ( d, J = 6. 8 Hz, 3H), HVai-4 ].

[0250] 13C {1H } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: 177. 3 ( C, CONH2), 173. 7 ( C, CONH), 161. 5 ( C, NCONH), 159. 0 ( C, OCON), 83. 7 ( C,fcBu), 60. 8 ( CH, Cvai-2 ), 49. 9 ( CH, CAia-2 ), [ 48. 1 ( CH2), 47. 0 ( CH2), CH2CH2CH2], 32. 5 ( CH, Cvai-3 ), 28. 3 ( 3CH3,fcBu), 26. 6 ( CH2, CH2CH2CH2), 19. 8 ( CH3, Cvai-4 ), 18. 4 ( CH3, CVai- 4 ), 18. 3 ( CH3, CAia-3 ). z ) 2-Aza-prolyl-L-leucylglycinamide tri fluoroacetate salt (11a) ( Figure 35 )

[0251] Tripeptide (10a) ( 0. 2472 g, 0. 6413 mmol ) was trans ferred to a round-bottom flask and then dissolved in anhydrous CH2CI2 ( 5 mL ). To the resulting solution, TFA ( 1. 80 mL, 23. 5 mmol ) was added. The mixture was stirred for 5 hours at room temperature. Workup: After the reaction, the volati les were eliminated using a rotary evaporator. The resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (11a) (white solid) was obtained in 79% yield ( 0. 2017 g).

[0252] Rf= 0 in EtOAc. MP = [ 44; 46 ] ° C. [α]D21= -17. 88 ± 0. 10 ( c1. 04, CH3OH).

[0253] HRMS-ESI+m / z: 286. 1874 calculated for C12H24N5O3+, 286. 1862 found.

[0254] iH NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 19 ( dd, J = 9. 1, 5. 8 Hz, 1H, HLeu- 2 ), 3. 87 - 3. 52 [ 3. 83 ( d, ABq, J = 17. 0 Hz ), 3. 70 ( d, ABq, J = 17. 0 Hz ), 4H, HGiy-2 + CH2CH2CH2], 3. 35 - 3. 26 (m, 2H, CH2CH2CH2 ), 2. 24 (p, J = 7. 1 Hz, 2H, CH2CH2CH2 ), 1. 85 - 1. 40 (m, 3H, HLeu-3 + HLeu-4 ), [ 0. 90 ( d, J = 6. 3 Hz ), 0. 87 ( d, J = 6. 4 Hz ), 6H, HLeu-5 ].

[0255] 13C {1H } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: 175. 7 ( C, CONH2 ), 174. 3 ( C, CONH), 159. 6 ( C, NCONH), 54. 9 ( CH, CLeu-2 ), [ 48. 2 ( CH2), 46. 8 ( CH2), CH2CH2CH2], 43. 1 ( CH2, CGiy-2 ), 41. 5 ( CH2, CLeu- 3 ), 26. 9 ( CH2, CH2CH2CH2 ), 26. 0 ( CH, CLeu- 4 ), [ 23. 4 ( CH3), 21. 9 ( CH3), CLeu-5 ].

[0256] aa ) 2-Aza-prolyl-L-leucyl-L-alaninamide tri fluoro ace fate salt (lib) ( Figure 36 )

[0257] Following the method described for the synthesis of compound (11a), compound (lib) was prepared using tripeptide (10b) ( 0. 1547 g, 0. 3872 mmol ), anhydrous CH2CI2 ( 5 mL ), and TFA ( 1. 80 mL, 23. 5 mmol ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (lib) (white solid) was obtained in 81 % yield ( 0. 1298 g).

[0258] Rf= 0 in EtOAc. MP = [ 129; 131 ] ° C. [α]D22= -28. 72 ± 0. 21 ( c1. 02, CH3OH).

[0259] HRMS-ESI+m / z: 300. 2030 calculated for C13H26N5O3+, 300. 1926 found.

[0260] ! H NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 38 ( q, J = 7. 2 Hz, 1H, HAia- 2 ), 4. 26 ( dd, J = 9. 0, 5. 8 Hz, 1H, HLeu-2 ), [ 3. 47 ( t, J = 7. 4 Hz, 2H), 3. 08 - 2. 79 (m, 2H), CH2CH2CH2 ], 2. 09 (p, J = 6. 9 Hz, 2H, CH2CH2CH2 ), 1. 81 - 1. 55 (m, 3H, HLeu-3 + HLeu-4 ), 1. 40 ( d, J = 7. 2 Hz, 3H, HAia-3 ), [ 1. 01 ( d, J = 6. 5 Hz ), 0. 98 ( d, J = 6. 4 Hz ), HLeu-5 ].

[0261] ^C pH } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: 177. 5 ( C, CONH2 ), 175. 6 ( C, CONH), 162. 0 ( C, NCONH), 54.2 ( CH, Cne-2 ), 49. 9 ( CH, CAia- 2 ), [ 48. 1 ( CH2), 46. 3 ( CH2), CH2CH2CH2], 42. 4 ( CH2, CLeu-3 ), 29. 0 ( CH2, CH2CH2CH2 ), [ 26. 1 ( CH2), 26. 0 ( CH2), CLeu-4 ], [ 23. 5 ( CH3), 22. 0 ( CH3), CLeu-5 ], 18. 2 ( CH3, CAia-3 ).

[0262] bb ) 2-Aza-prolyl-L-isoleucylglycinamide tri fluoro ace fate salt (11c) ( Figure 37 )

[0263] Following the method described for the synthesis of compound (11a), compound (11c) was prepared using tripeptide (10c) ( 0. 2681 g, 0. 6955 mmol ), anhydrous CH2CI2 ( 5 mL ), and TEA ( 1. 80 mL, 23. 5 mmol ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (11c) (white solid) was obtained in 86% yield ( 0. 2397 g).

[0264] Rf= 0 in EtOAc. MP = [ 39; 41 ] ° C. [α]D22= + 9. 25 ± 0. 15 ( c1. 12, CH3OH).

[0265] HRMS-ESI+m / z: 286. 1874 calculated for C12H24N5O3+, 286. 1872 found. ! H NMR ( CD3OD, 400 MHz, 25 ° C ) 5: [ 4. 17 ( d, J = 6. 8 Hz ), 4. 06 ( d, J = 6. 8 Hz ), 1H, Hue-2 ], [ 3. 93 ( d, ABq, J = 17. 0 Hz, 1H), 3. 85 ( d, ABq, J = 17. 0 Hz, 1H), HGiy-2 ], [ 3. 47 ( t, J = 7. 3 Hz, 2H), 3. 08 - 2. 80 (m, 2H), CH2CH2CH2 ], 2. 09 (p, J = 6. 8 Hz, 2H, CH2CH2CH2 ), 1. 99 - 1. 83 (m, 1H, Hne-3 ), [ 1. 68 -1. 52 (m, 1H), 1. 30 - 1. 13 (m, 1H), Hne-4 ], 1. 07 - 0. 87 (m, 6H, Hile-5 + Hile-6 ).

[0266] ^C pH } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: 175. 3 ( C, CONH2 ), 174. 3 ( C, CONH), 162. 4 ( C, NCONH), 60. 5 ( CH, Cne-2 ), [ 48. 0 ( CH2), 46. 3 ( CH2), CH2CH2CH2], 43. 1 ( CH2, CGiy-2 ), 38. 2 ( CH, Cue- 3 ), 29. 0 ( CH2, CH2CH2CH2 ), 26. 0 ( CH2, Cne-4 ), 16. 1 ( CH3, Cue- 6 ), 11. 5 ( CH3, Cue-5 ).

[0267] cc ) 2-Aza-prolyl-L-isoleucyl-L-alaninamide tri fluoro acetate salt (lid) ( Figure 38 )

[0268] Following the method described for the synthesis of compound (11a), compound (lid) was prepared using tripeptide (lOd) ( 0. 2544 g, 0. 6368 mmol ), anhydrous CH2CI2 ( 5 mL ), and TFA ( 1. 80 mL, 23. 5 mmol ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (lid) (white solid) was obtained in 76% yield ( 0. 2008 g).

[0269] Rf= 0 in EtOAc. MP = [ 43; 45 ] ° C. [α]D21= -10. 83 ± 0. 33 ( c1. 00, CH3OH).

[0270] HRMS-ESI+m / z: 300. 2030 calculated for C13H26N5O3+, 300. 1933 found.

[0271] iH NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 41 ( q, J = 7. 2 Hz, 1H, HAIS- 2 ), [ 4. 21 ( d, J = 6. 8 Hz ), 4. 10 ( d, J = 6. 7 Hz ), 1H, Hue-2 ], [ 3. 79 - 3. 40 (m, 2H), 3. 06 - 2. 81 (m, 2H), CH2CH2CH2 ], 2. 09 (p, J = 7. 0 Hz, 2H, CH2CH2CH2 ), 1. 95 - 1. 82 (m, 1H, Hne-3 ), 1. 63 - 1. 50 (m, 1H, Hne-4a ), 1. 40 ( d, J = 7. 2 Hz, 3H, HAIS- 3 ), 1. 27 - 1. 12 (m, 1H, Hne-4b ), [ 0. 99 ( d, J = 6. 8 Hz ), 0. 96 ( t, J = 7. 4 Hz ), 6H, Hne-5 + Hne- 6 ].13C {1H } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: 177. 3 ( C, CONH2 ), 174. 3 ( C, CONH), 162. 2 ( C, NCONH), 60. 0 ( CH, Cne-2 ), 49. 9 ( CH, CAIS- 2 ), [ 48. 0 ( CH2), 46. 3 ( CH2), CH2CH2CH2], 38. 7 ( CH, Cue- 3 ), 29. 0 ( CH2, CH2CH2CH2), 25. 9 ( CH2, Cne-4 ), 18. 2 ( CH3, CAia-3 ), 16. 1 ( CH3, Cue- 6 ), 11. 6 ( CH3, Cne-5 ).

[0272] dd) 2-Aza-prolyl-L-valylglycinamide tri fluoroacetate salt (lie) ( Figure 39 )

[0273] Following the method described for the synthesis of compound (11a), compound (lie) was prepared using tripeptide (lOe) ( 0. 4151 g, 1. 118 mmol ), anhydrous CH2C12( 5 mL ), and TFA ( 1. 80 mL, 23. 5 mmol ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide (lie) ( clear vitreous solid) was obtained in 83% yield ( 0. 3557 g).

[0274] Rf= 0 in EtOAc. MP = [ 38; 40 ] ° C. [α]D20= +41. 26 ± 0. 22 ( c1. 12, CH3OH).

[0275] HRMS-ESI+m / z: 272. 1717 calculated for C11H22N5O3+, 272. 1721 found.

[0276] ! H NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 02 ( d, J = 6. 5 Hz, 1H, Hvai-2 ), [ 3. 94 ( d, ABq, J = 17. 0 Hz, 1H), 3. 85 ( d, ABq, J = 17. 0 Hz, 1H), HGiy-2 ], [ 3. 48 ( t, J = 7. 0 Hz, 2H), 3. 04 - 2. 88 (m, 2H), CH2CH2CH2], 2. 21 - 2. 03 (m, 3H, CH2CH2CH2+ HVai-3 ), 1. 02 ( appt, J = 6. 4 Hz, 1H, HVai-4 ).

[0277] 13C {1H } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: [ 175. 3 ( C ), 175. 2 ( C ), CONH2], 174. 3 ( C, CONH), 162. 4 ( C, NCONH), 61. 2 ( CH, Cvai-2 ), [ 48. 0 ( CH2), 46. 3 ( CH2), CH2CH2CH2], 43. 1 ( CH2, CGIY- 2 ), 31. 8 ( CH, Cvai-3 ), 29. 0 ( CH2, CH2CH2CH2), [ 19. 8 ( CH3), 18. 4 ( CH3), Cvai-4 ].

[0278] ee ) 2-Aza-prolyl-L-valyl-L-alaninamide tri fluoro acetate

[0279]

[0280] Following the method described for the synthesis of compound ( 11a), compound ( Ilf) was prepared using tripeptide ( lOf) ( 0. 3548 g, 0. 9204 mmol ), anhydrous CH2CI2 ( 5 mL ), and TFA ( 1. 80 mL, 23. 5 mmol ). After the typical workup, the resulting residue was puri fied by column chromatography using CH3OH ( 0- 10% ) in EtOAc as the eluent. Tripeptide ( Ilf) (pale yellow solid) was obtained in 85% yield ( 0. 3129 g).

[0281] Rf= 0. 08 in EtOAc. MP = [ 43; 45 ] ° C. [α]D20= + 18. 97 ± 0. 35 ( c1. 15, CH3OH).

[0282] HRMS-ESI+m / z: 286. 1874 calculated for C12H24N5O3+, 286. 1955 found.

[0283] ! H NMR ( CD3OD, 400 MHz, 25 ° C ) 5: 4. 48 - 4. 32 (m, 1H, HAia-2 ), 4. 06 ( d, J = 6. 2 Hz, 1H, HVai-2 ), [ 3. 46 ( dt, J = 7. 3, 3. 9 Hz, 2H), 3. 02 - 2. 83 (m, 2H), CH2CH2CH2 ], 2. 20 - 2. 01 (m, 3H, CH2CH2CH2 + Hvai-3 ), 1. 40 ( d, J = 7. 2 Hz, 3H, HAia-3 ), [ 1. 01 ( d, J = 6. 8 Hz ), 0. 96 ( d, J = 6. 8 Hz ), 6H, HVai-4 ].

[0284] ^C pH } and DEPT- 135 NMR ( CD3OD, 101 MHz, 25 ° C ) 5: [ 177. 4 ( C ), 177. 4 ( C ), CONH2 ], [ 174. 4 ( C ), 174. 3 ( C ), CONH], 162. 3 ( C, NCONH), 60. 6 ( CH, Cvai-2 ), [ 50. 0 ( CH), 49. 9 ( CH), CAia-2 ], [ 48. 1 ( CH2), 46. 3 ( CH2), CH2CH2CH2], 32. 3 ( CH, Cvai-3 ), 29. 0 ( CH2, CH2CH2CH2 ), 19. 8 ( CH3, Cvai-4 ), 18. 2 ( CH3, CAia-3 ).

[0285] 2. Compounds of formula ( I ) ( Figure 5 ):

[0286]

[0287] A group of 24 compounds of formula ( I ) ( 8a-f; 9a-f; lOa-f; lla-f ) were success fully synthesi zed demonstrating the feasibility of the synthesis route.

[0288] After obtaining the compounds (8a) to (8f) from compound ( 6a) with dipeptides (7a) to (7f) catalyzed by p-TsOH ( 1 ), the methyl ester functional group was converted to the primary amide by ammonolysis with 7 M NH3 in methanol ( if ), af fording compounds ( 10a) to ( lOf) with high yields ( 77-91 % ). The tert -butyl carbamate from compounds (8a) to (8f) and (10a) to (lOf) can be cleaved by acidolysis using TFA to afford the corresponding trifluoroacetate salts (9a) to (9f) (iff ) and (11a) to (Ilf) (iv), with good yields ( 66-86%).

[0289] Crystals of compound (10a) were obtained, enabling to study its structure in the solid-state by X-ray crystallography. The ORTEP diagram of (10a) is given in Figure 6. The data obtained by X-ray crystallography unequivocally corroborates the structure of (10a) and advocates the success of the protocol for obtaining compounds of formula (I).

[0290] 3. Cytotoxicity assays in dopaminergic-dif f erentiated human SH-SH5Y neuroblastoma cells

[0291] 3. 1 Toxicological evaluation

[0292] Neuroblastoma SH-SY5Y cells provided by the European Collection of Authenticated Cell Cultures were purchased from Sigma-Aldrich (Germany). The cells were incubated at 37 °C in a CO2 atmosphere (5%) throughout all procedures using a BINDER CB 150 incubator (Germany). The culture medium, Dulbecco' s Modified Eagle Medium ( lx) high glucose, containing sodium pyruvate, GlutaMAX, and phenol red, was obtained from Thermo Fisher Scientific (United States of America) and supplemented with 10% fetal bovine serum from Thermo Fisher Scientific (United States of America) and 1% antibiotics ( 100 units / mL of penicillin and 100 pg / mL of streptomycin) from Biochrom (Germany). DMSO (CAS 67-68-5), RA (R2625), TPA (R79346), 0.4% trypan blue solution (T8154 ), 0.04% trypsin / 0. 03% ethylenediamine tetraacetic acid (EDTA) solution, and 6-OHDA hydrobromide (CAS 636-00-0) were supplied by Sigma-Aldrich (Germany). PBS was obtained from Biochrom (Germany). MTT (CAS 298-93-1 ) was obtained from Thermo Fisher Scientific (United States of America). MIF-1 (CAS 2002-44-0) was obtained from Bachem Holding (Switzerland). Sterile culture flasks, test plates, and centrifuge tubes were obtained from TPP Techno Plastic Products (Switzerland). Sterile serological pipettes were obtained from Nerbe Plus & Company (Germany). Cells were counted using a Nikon Eclipse TS100 inverted optic microscope (Japan) with a hemocytometer (Neubauer improved bright line blood counting chamber) from BOECO (Germany). The MTT test plates were analysed with a Synergy HT microplate reader from BioTek Instruments (United States of America) for absorbance measurement.

[0293] 3. 2 Cell seeding and dopaminergic differentiation

[0294] SH-SY5Y cells were grown in the incubator until confluence in 25 cm2culture flasks with supplemented culture medium. When the cells reached confluence, the medium was discarded, and the cells were washed with PBS (5 mL, without calcium and magnesium). Following, the PBS was discarded, and a 0.04% trypsin / 0. 03% EDTA solution ( 1.5 mL) was added. The cells were incubated for 3 min. Then, 8 mL of warm supplemented culture medium were added, and the cell suspension was stirred gently and transferred to a 50 mL centrifuge tube. A sample of the suspension (20 pL) was collected and dyed with a 0.4% solution of trypan blue ( 180 pL). The sample was placed in an assembled Neubauer chamber, and the cells were counted under an inverted optic microscope. After counting, the cell suspension was diluted with warm supplemented culture medium to a final concentration of 100 cells / pL and a solution of RA ( 10 mM) was added ( final concentration of 10 pM). Afterwards, the cells were seeded in 48-well test plates. For this, 250 pL (25000 cells) of suspension were placed in each well to achieve a density of 25000 cells / cm2. The cells were incubated for 72 h at 37 °C with a 5% CO2 flow. Then, 50 pL of a TPA solution were added to each well ( final concentration of 80 nM) and the cells were incubated for another 72 h. After the differentiation, the culture medium was discarded and 250 pL of warm supplemented culture medium were added to each well. Next, 10 pL of solution of the test compounds (2. 6 mM) or 6-OHDA (3.25 mM) were added to each well and the cells were incubated for 48 h. The solutions of the test compounds are as follows: ( 1 ) PBS as control; (2 ) solution of 125 pM for 6-OHDA ( freshly prepared in PBS) as a positive control for cytotoxicity; (3) solution of 100 pM for all compounds (in PBS at a final well concentration except (9b) ) and MIF-1; (4 ) solution of 13% DMSO / PBS at a final well concentration of 0.5% for DMSO as vehicle control; (5) a condition with a final well concentration of 0.5% for DMSO as a vehicle control ( 13% DMSO / PBS stock solution); and ( 6) solution with a final well concentration of 100 pM for compound (9b) that was solubilized in 13% DMSO / PBS at ( final well concentration of 0.5% for DMSO in those conditions).

[0295] 3. 3 MTT reduction assay

[0296] After incubation with the test compounds for 48 h, the culture medium was discarded and 200 pL of warm supplemented culture medium were added in each well followed by 20 pL of a 5 mg / mL solution of MTT in PBS ( final MTT well concentration of 0.5 mg / mL). The cells were incubated for 90 minutes protected from light. After that, the culture medium was discarded and 200 pL of DMSO were added to each well to solubilize the formazan crystals meanwhile formed. The plates were gently stirred for 15 minutes protected from light. The absorbance was measured at 570 nm and 690 nm (reference wavelength). MTT reduction was determined by the difference between the absorbance at 570 nm and 690 nm and expressed as a percentage of control. 3. 4 Statistical analysis

[0297] Data are presented as mean ± standard deviation and expressed as a percentage of control. Statistical analysis was performed using GraphPad Prism 8.3.0 software (United States of America) by one-way analysis of variance test followed by Tukey' s post hoc test. Statistical significance is regarded at a probability value (p) less than or equal to 5%.

[0298] The cytotoxicological profile of compounds (8a) to (8f) and (11a) to (Ilf) was evaluated at 100 pM. The neurotoxin 6-OHDA ( 125 pM) was used as a positive control for cytotoxicity, as it induces the phenotype features of Parkinson' s disease. Moreover, commercial MIF-1 was also tested for comparison. Compound (9b) was dissolved in a solution of 13% DMSO ( final well concentration 0.5% DMSO) in phosphate-buff ered saline solution (PBS), while the remaining compounds were dissolved solely in PBS. The results obtained in the MTT reduction assay are presented in Figure 7.

[0299] In this assay ( Figure 7 ), 6-OHDA caused a statistically significant decrease (p < 0.0001 ) in the reduction of MTT to 50.37 ± 6.39% when compared to PBS control ( 100.00 ± 3.42%). Compounds (9b) ( 86.34 ± 4. 63%, p < 0.0001 ), 8d ( 91.03 ± 5.18%, p < 0.001 ), 9d ( 89.79 ± 7.84%, p < 0.0001 ), and compound (9f) ( 83.40 ± 6.35%, p < 0.0001 ) exhibited a mild statistically significant cytotoxicity (Figure 7 ). MIF-1 and the remaining compounds showed no significant cytotoxicity when compared to control (Figure 7 ).

[0300] Functional assays at human dopamine D2R Functional assays were performed on human dopamine D2R expressed in CHO cells using a Cisbio cAMP kit for the determination of the cAMP mobilization by means of HTRF measurements as previously reported [25-27 ]. Briefly, in the presence of 500 pM of IBMX (a phosphodiesterase Paninhibitor), 5000 cells / well were seeded in a 96-well black plate in stimB buffer (provided in the kit), guaranteeing a high level of cAMP accumulation in the cells. Then, test compounds and dopamine were added and incubated for 10 min at 37 °C followed by a 5 min period of incubation with 10 pM forskolin. After the addition of the kit reagents and an incubation period of 1 h at room temperature, the HTRF signal was measured using a Tecan M1000 Pro multilabel reader. Data were converted to cAMP using a standard cAMP curve, and the activity of the test compounds was determined as the percentage of increase in the activity of 0.1 pM DA. A dopamine curve was included as the positive control in all assays.

[0301] All the nontoxic compounds of formula (I) were subj ect to preliminary pharmacological evaluation of the activity of the compounds over 0.1 pM of dopamine at 1 nM concentration. The compounds that showed an increase in dopamine effect higher than 20% were then selected to undergo in vitro cell-based functional assays at human dopamine D2R by means of cAMP mobilization in the presence of dopamine and preactivation of adenyl cyclase with forskolin to characterize their PAM activity

[0025] . In this series, the most promising compounds that elicited more than a 20% increase in 0.1 pM dopamine effect at 1 nM concentration were compounds (8c), (9c), (10a), (10b), (10c), (11a), and (11b) (Figure 8). For that, CHO cells transfected with human dopamine D2R were used as reported in the literature

[0025] . The half-maximal effective concentration (EC50) and the maximum effect (Emax) of dopamine were determined in the absence and the presence of the tested compounds at 1 nM concentration. The concentration-response curves of dopamine in the presence of MIF-1 and compounds (8c), (9c), (10a), (10b), (10c), (11a), and (11b) (1 nM) are shown in Figure 9. The curve of dopamine alone was included in all graphics for comparison purposes. As observed, MIF-1 and all the selected compounds of formula (I) induce a left-shift of the dopamine concentrationresponse curve, demonstrating to increase the potency of dopamine (Figure 9).

[0302] Table 1 summarizes the EC50 and the Emaxparameters obtained in the functional assays at the human dopamine D2R.

[0303] In these experiments, the EC50obtained for dopamine alone was 172.71 nM. When co-incubated with dopamine, all the tested compounds of formula (I) ( 1 nM) were able to effectively reduce the EC50of dopamine between 1.62 and 8.82 times, while the parent neuropeptide led to a 4.02-fold reduction of the EC50of dopamine. In this assay, compounds (9c) and (lib) outperformed MIF-1 by reducing the EC50of dopamine by 8.82 and 5.41 times (EC50= 19.58 and 31.94 nM), being 2.19 and 1.34 times more potent than the parent neuropeptide (EC50= 42.91 nM), respectively (Table 1). Except for compound (10a) (Emax= 83.71%), all compounds of formula (I) ( 87.27 < Emax< 102.8%) exhibited a superior Emaxin comparison to MIF-1 (Emax= 84.43%), as shown in Table 1.

[0304] Table 1. Potency (EC50) and efficacy (Emax) of dopamine (DA) in the absence and the presence of 1 nM of the studied compounds. Compound / 1 nM EC50of DA Emaxof DA (%) DA alone 172.71 100.53

[0305] 8c 89. 41 87. 41

[0306] 9c 19. 58 88. 18

[0307] 10a 72. 39 83. 71

[0308] 10b 92. 98 95. 81

[0309] 10c 106. 93 94. 28

[0310] 11a 78. 29 102. 80

[0311] 11b 31.94 87.27

[0312] DA + MI F- 1 42. 91 84. 43

[0313]

[0314] Compounds of formula ( I ): a ) display absent or negligible cytotoxicity to human di f ferentiated SH-SY5Y neuroblastoma cells comparable to MI F- 1 at 100 pM; b ) retain the PAM activity at the D2R; c) exhibit up to 2.19-fold increase in PAM potency in comparison to MI F- 1 at 1 nM.

[0315] Bibliography

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[0338] 2023, 14 ( 12 ), 1656-1663.

[0339] This description is of course not in any way restricted to the forms of implementation presented herein and any person with an average knowledge of the area can provide many possibilities for modification thereof without departing from the general idea as defined by the claims. The preferred forms of implementation described above can obviously be combined with each other. The following claims further define the preferred forms of implementation.

Claims

CLAIMS1. Azapeptide compounds of formula (I) derived from melanocyte-stimulating hormone release-inhibiting factor-1, and salts thereof:(I)Wherein,n is 1, 2, or 3;R1is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, including their salts, alkyl-aminocarbonyl, alkenylaminocarbonyl, alkynyl-aminocarbonyl, aryl-aminocarbonyl, aminocarbonyl, alkyl-oxycarbonyl, alkenyl-oxycarbonyl, alkynyl-oxycarbonyl, or aryl-oxycarbonyl groups;R2is selected from hydrogen, alkyl, alkenyl, alkynyl, or aryl groups;R3is selected from hydrogen, alkyl, alkenyl, alkynyl, or aryl groups;*R2and R3are R or S configurations;X is selected from OH, O-alkyl, O-alkenyl, O-alkynyl, d’aryl, NH2, NH- (Z), or N- (Z)2, in which Z is selected from an alkyl, alkenyl, alkynyl, or aryl groups.

2. Compounds of formula (I) according to the previous claim, wherein the compounds are selected from methyl N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-leucylglycinate (8a), methyl N- ( tert -but yloxy carbonyl ) -2-aza-prolyl-L-leucyl-L-alaninate (8b), methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-isoleucylglycinate (8c), methyl - ( tertbutyloxycarbonyl) -2-aza-prolyl-L-isoleucyl-L-alaninate (8d), methyl N- ( tert -but yloxy carbonyl ) -2-aza-prolyl-L-valylglycinate (8e), methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-valyl-L-alaninate (8f), methyl 2-aza-prolyl-L-leucylglycinate trifluoroacetate salt (9a), methyl 2-aza-prolyl-L-leucyl-L-alaninate trifluoroacetate salt (9b), methyl 2-aza-prolyl-L-isoleucylglycinate trifluoroacetate salt (9c), methyl 2-aza-prolyl-L-isoleucyl-L-alaninate trifluoroacetate salt (9d), methyl 2-aza-prolyl-L-valylglycinate trifluoroacetate salt (9e), methyl 2-aza-prolyl-L-valyl-L-alaninate trifluoroacetate salt (9f), N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-leucylglycinamide (10a), N- ( tert -butyloxycarbonyl ) -2-aza-prolyl-L-leucyl-L-alaninamide (10b), N- ( tert -but yloxy carbonyl ) -2-aza-prolyl-L-isoleucylglycinamide (10c), N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-isoleucyl-L-alaninamide (10d), N-( tert-butyloxycarbonyl) -2-aza-prolyl-L-valylglycinamide (10e), N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-valyl-L-alaninamide (10f), 2-aza-prolyl-L-leucylglycinamide trifluoroacetate salt (11a), 2-aza-prolyl-L-leucyl-L-alaninamide trifluoroacetate salt (11b), 2 -aza-prolyl-L-isoleucylglycinamide trifluoroacetate salt (11c), 2-aza-prolyl-L-isoleucyl-L-alaninamide trifluoroacetate salt (11d), 2-aza-prolyl-L-valylglycinamide trifluoroacetate salt (11e), or 2-aza-prolyl-L-valyl-L-alaninamide trifluoroacetate salt (11f).

3. Compounds of formula (I) according to any of the previous claims for use in the treatment of dopamine-related disorders of the central nervous system.

4. Compounds of formula (I) according to the previous claim, wherein the dopamine-related disorders of the central nervous system are selected from depression, drug abuse,tardive dyskinesia, restless legs syndrome, or Parkinson' s disease.

5. A pharmaceutical composition comprising at least one compound of formula (I) described in any of the claims 1 to 2.

6. The pharmaceutical composition according to the previous claim, further comprising at least one other active ingredient for the treatment of dopamine-related disorders of the central nervous system.

7. The pharmaceutical composition according to any of the claims 5 or 6, for use in the treatment of dopamine-related disorders of the central nervous system.

8. The pharmaceutical composition according to the claims 5 to 7, wherein the dopamine-related disorders of the central nervous system are selected from depression, drug abuse, tardive dyskinesia, restless legs syndrome, or Parkinson' s disease.

9. A method to obtain compounds of formula ( I ) described in any of the claims 1 to 2, comprising the steps of:N-protection of hydrazine monohydrate (1) using between 200 and 300 mol% of di-tert-butyl dicarbonate in a polar aprotic organic solvent, in the presence of between 200 and 300 mol% of triethylamine, at a temperature between 20 and 25°C, to obtain the corresponding symmetrical protected 1, 2-di- ( tert -butyloxycarbonyl ) hydrazine (2);alkylation of 1,2-di-(tert-butyloxycarbonyl)hydrazine (2) using between 100 and 200 mol% of 1,3-dibromopropane or 1, 4-dibromobutane under alkaline conditions of between 10and 50% aqueous solution of NaOH (w / w) and toluene, at a temperature between 80 and 110 °C, in the presence of between 10 and 30 mol% of tetraethylammonium bromide or tetrabutylammonium iodide as a phase-transfer agent, affording the corresponding protected cyclic 1, 2-di- ( tertbutyloxycarbonyl ) pyrazolidine (3a) or di- tert-butyl tetrahydropyridazine-1, 2-dicarboxylate (3b);preparing 1- ( tert-butyloxycarbonyl ) pyrazolidine (5a) or tert-butyl tetrahydropyridazine-1 (2H) -carboxylate (5b) by acidolysis of compound (3a) or compound (3b) respectively, using between 3000 and 12000 mol% of trifluoroacetic acid in CH2Cl2, at a temperature between 20 and 25°C, followed by monoprotection of the hydrazinium intermediates pyrazolidine-1, 2-diium trifluoroacetate (4a) or hexahydropyridazine-1, 2-diium trifluoroacetate (4b) respectively, using between 90 and 110 mol% of Boc2O in the presence of between 300 and 500 mol% of Et3N;acylation of cyclic 1-(tert-butyloxycarbonyl)pyrazolidine (5a) or tert-butyl tetrahydropyridazine-1(2H)-carboxylate (5b) with between 100 and 300 mol% of 4-nitrophenyl chloroformate in the presence of between 100 and 300 mol% of Et3N in CH2Cl2, at a temperature between 0 °C and 25°C, obtaining 4-nitrophenyl - ( tertbutyloxycarbonyl ) -2-aza-prolinate (6a) or 4-nitrophenyl N- ( tert-butyloxycarbonyl ) -2-aza-pipecolate (6b) respectively;dissolution of the 4-nitrophenyl N-(tert-butyloxycarbonyl)-2-aza-prolinate (6a) or 4-nitrophenyl N- ( tert-butyloxycarbonyl ) -2-aza-pipecolate (6b) and between 100 and 200 mol% of a dipeptide selected from methyl L-leucylglycinate trifluoroacetate salt (7a), methyl L-leucyl-L-alaninate trifluoroacetate salt (7b), methyl L-isoleucylglycinate trifluoroacetate salt (7c), methyl L-isoleucyl-L-alaninate trifluoroacetate salt (7d), methyl L-valylglycinate trifluoroacetate salt (7e), or methyl L-valyl-L-alaninate trifluoroacetate salt (7f), in N,N-dimethylformamide (DMF) comprising between 0 and 75% of CH₂Cl₂; followed by the addition of between 20 and 40 mol% of 4-(dimethylamino)pyridine in the presence of between 10 and 20 mol% of p-TsOH and the mixture is heated at a temperature between 40 and 90 °C, for a time between 3 and 6 days, respectively obtaining compounds methyl N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-leucylglycinate (8a), methyl N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-leucyl-L-alaninate (8b), methyl N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-isoleucylglycinate (8c), methyl N- ( tertbutyloxy carbonyl ) -2-aza-prolyl-L-isoleucyl-L-alaninate (8d), methyl N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-valylglycinate (8e), or methyl N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-valyl-L-alaninate (8f).

10. Method according to the previous claim, wherein it further comprises the step of:acidolysis of a tripeptide selected from (8a) to (8f) using between 3000 and 12000 mol% of TFA in CH2CI2, at a temperature between 20 and 25°C, respectively obtaining tripeptides methyl 2-aza-prolyl-L-leucylglycinate trifluoroacetate salt (9a), methyl 2-aza-prolyl-L-leucyl-L-alaninate trifluoroacetate salt (9b), methyl 2-aza-prolyl-L-isoleucylglycinate trifluoroacetate salt (9c), methyl 2-aza-prolyl-L-isoleucyl-L-alaninate trifluoroacetate salt (9d), methyl 2-aza-prolyl-L-valylglycinate trifluoroacetate salt (9e), or methyl 2-aza-prolyl-L-valyl-L-alaninate trifluoroacetate salt (9f).

11. Method according to claim 9, wherein it further comprises the step of:dissolution of a tripeptide selected from (8a) to (8f) in a methanolic solution of NH3 between 1 and 7 M, at a temperature between 20 and 25°C, respectively obtaining tripeptides N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-leucylglycinamide (10a), N- ( tert-butyloxycarbonyl ) -2-aza-prolyl-L-leucyl-L-alaninamide (10b), N- ( tertbutyloxy carbonyl ) -2-aza-prolyl-L-isoleucylglycinamide (10c), N- ( tert-butyloxycarbonyl) -2-aza-prolyl-L-isoleucyl-L-alaninamide (10d), N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-valylglycinamide (10e), or N-(tert-butyloxycarbonyl)-2-aza-prolyl-L-valyl-L-alaninamide (10f).

12. Method according to the previous claim, wherein it further comprises the step of:acidolysis of a tripeptide selected from (10a) to (10f) using between 3000 and 12000 mol% of TFA in CH2Cl2, at a temperature between 20 and 25°C, respectively obtaining tripeptides 2-aza-prolyl-L-leucylglycinamide trifluoroacetate salt (11a), 2-aza-prolyl-L-leucyl-L-alaninamide trifluoroacetate salt (11b), 2 -aza-prolyl-L-isoleucylglycinamide trifluoroacetate salt (11c), 2-aza-prolyl-L-isoleucyl-L-alaninamide trifluoroacetate salt (11d), 2 -aza-prolyl-L-valylglycinamide trifluoro acetate salt (He), or 2-aza-prolyl-L-valyl-L-alaninamide trifluoroacetate salt (11f).

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