Exploring the Phe-Gly Dipeptide-derived Piperazinone Scaffold in the search for Antagonists of the Thrombin receptor PAR1

DOI: 10.3390/molecules19044814

Source and publish data:

Molecules p. 4814 - 4846 (2014)

Update date:2022-08-05

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Authors:

Valdivielso, Angel M.

Garcia-Lopez, M. Teresa

Gutierrez-Rodriguez, Marta

Herranz, Rosario

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Article abstract of DOI:10.3390/molecules19044814

A series of Phe-Gly dipeptide-derived piperazinones containing an aromatic urea moiety and a basic amino acid has been synthesized and evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents in human cancer cells. The synthetic strategy involves coupling of a protected basic amino acid benzyl amide to 1,2- and 1,2,4-substituted-piperazinone derivatives, through a carbonylmethyl group at the N1-position, followed by formation of an aromatic urea at the exocyclic moiety linked at the C2 position of the piperazine ring and removal of protecting groups. None of the compounds showed activity in the biological evaluation.

Full text of DOI:10.3390/molecules19044814

Molecules 2014, 19, 4814-4846; doi:10.3390/molecules19044814

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molecules

ISSN 1420-3049

www.mdpi.com/journal/molecules

Article

Exploring the Phe-Gly Dipeptide-Derived Piperazinone Scaffold

in the Search for Antagonists of the Thrombin Receptor PAR1

Ángel M. Valdivielso, M. Teresa García-López, Marta Gutiérrez-Rodríguez and

Rosario Herranz *

Instituto de Química Médica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain;

E-Mails: angel@iqm.csic.es (Á.M.V.); iqmgl37@iqm.csic.es (M.T.G.-L.);

mgutierrez@iqm.csic.es (M.G.-R.)

* Author to whom correspondence should be addressed; E-Mail: rosario@iqm.csic.es;

Tel.: +34-912-587-537; Fax: +34-915-644-853.

Received: 4 February 2014; in revised form: 8 April 2014 / Accepted: 13 April 2014 /

Published: 16 April 2014

Abstract: A series of Phe-Gly dipeptide-derived piperazinones containing an aromatic

urea moiety and a basic amino acid has been synthesized and evaluated as inhibitors of

human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents

in human cancer cells. The synthetic strategy involves coupling of a protected basic amino

acid benzyl amide to 1,2- and 1,2,4-substituted-piperazinone derivatives, through a

carbonylmethyl group at the N_1-position, followed by formation of an aromatic urea at the

exocyclic moiety linked at the C₂position of the piperazine ring and removal of protecting

groups. None of the compounds showed activity in the biological evaluation.

Keywords: peptidomimetics; regioselectivity; piperazinones; platelet antiaggregant activity;

PAR1 antagonists

1. Introduction

Most cellular effects of thrombin are mediated by activation of the protease-activated receptor 1

(PAR1) [1,2]. This receptor is mainly expressed in platelets, where its activation induces aggregation.

Therefore, PAR1 is considered a therapeutic target in cardiovascular diseases [1,3–5], whose

inactivation could inhibit platelet aggregation without affecting thrombin’s role in the coagulation

cascade [3,6,7]. In addition, numerous studies have shown that PAR1 is overexpressed in invasive and

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metastatic tumors and that its expression levels directly correlate with the degree of invasiveness of

the cancer [8–13]. Based on these facts, this receptor is starting to be also considered a promising

target for cancer therapy, particularly in the search of angiogenesis inhibitors [2].

Activation of PAR1 by thrombin involves the proteolytic cleavage of the N-terminal exodomain

between Arg⁴¹and Ser⁴². This cleavage unveils the recognition sequence SFLLRN that acts as

a tethered ligand, auto-activating the receptor [14]. The binding of this tethered ligand is followed by

the coupling of the receptor to heterotrimeric G proteins and activation of signal transduction. This

particular intramolecular activation mechanism makes PAR1 a target particularly difficult to address.

The first potent PAR1 antagonists were SFLLRN-based peptidomimetic ureas, represented by

the optimized antagonist RWJ-58259 (Figure 1) [7], which is considered a standard reference in

pharmacological studies on PAR1 receptor [15]. Later, a few series of antagonists have been

discovered by HTS of diverse libraries of non-peptide small molecules [7,16]. Up to now, only two of

these PAR1 antagonists are in advanced clinical development for the treatment of patients with acute

coronary syndrome, SCH-530348 (named vorapaxar, in phase III clinical trials) [17] and E-5555

(named atopaxar, in phase II clinical trials) [18] (Figure 1).

Figure 1. Reference PAR1 antagonists in pharmacological studies and/or advanced

clinical development.

Taking as reference the peptidomimetic antagonist RWJ-58259 we initiated a project directed to the

search of new PAR1 antagonists using a diversity oriented synthesis (DOS) strategy. To this aim, we

planned the synthesis of diverse small directed libraries of different scaffolds able to assemble, at least,

one or two aromatic groups and one or two basic groups at variable distances and orientations [19].

Among the scaffolds, we focused our attention on the piperazine ring, since this system is recognized

as a privileged scaffold, due to its recurrent presence in biological active compounds [20,21]. Firstly,

we synthesized the series of 1,2,4,6-tetrasusbtituted-piperazinone derivatives of general formula A

(Figure 2). Some of these derivatives showed moderate antagonist activity [22]. Trying to improve this

activity and to establish structure-activity relationships, we have synthesized and report herein the

analogues B, where the basic amino acid side chain has been moved from the piperazine C₆position to

the N₁. Now, the indazole moiety of the PAR1 antagonist RWJ-58259 has also been included among

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the selected arylureido groups at the piperazine C₂-substituent. The new piperazinone derivatives B

have been evaluated as human PAR1 antagonists in a platelet aggregation assay and as cytotoxic

agents in human cancer cell lines.

Figure 2. Piperazinone derivatives proposed as PAR1 antagonists.

2. Results and Discussion

Two alternative retrosynthetic routes were considered for the building of the desired piperazinones

derivatives B from the starting 1-(benzyloxycarbonyl)methyl-piperazinones 1 [23]. These routes differ

in the order of incorporation of the basic amino acid and the urea moieties. Firstly, we attempted the

formation of the urea at the exocyclic 1-amino-2-phenylethyl moiety, before coupling the basic amino

acid residue Xaa. However, as shown in Scheme 1, the Boc removal from the (3:1) epimeric mixture

of N₄-unsubstituted-piperazinones 1, by treatment with a 3 N solution of HCl in EtOAc, followed by

reaction with benzyl isocyanate in the presence of Et₃N, led to the corresponding epimeric mixture of

ureas 2 in 40% yield, along with 30% of the 1H-pyrazino[1,2-a]pyrazines 3. These bicyclic derivatives

resulted from the nucleophilic attack of the exocyclic amine, generated by the Boc removal, at the

(benzyloxycarbonyl)methyl group in the N₁-position. To minimize this cyclization, both the Boc

removal and the urea formation were carried out at 0 °C with an excess of benzyl isocyanate (2 equiv.)

to accelerate the urea formation. Nevertheless, in none of these attempts was the yield of the ureas 2

improved significantly.

Molecules 2014, 19

Scheme 1. Synthesis of the ureas 2 and the 1H-pyrazino[1,2-a]pyrazines 3.

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To avoid the cyclization, we decided to incorporate the basic amino acid prior to the

urea formation. As shown in Scheme 2, the Pd (C) catalyzed hydrogenolysis of the benzyl

ester of 1, followed by coupling with H-Orn(Z)-NHBn (6a) and H-Lys(Z)-NHBn (6b), using

diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazol (HOBt) as coupling agents, provided the

corresponding epimeric mixtures 7a,b in 60%–70% yield. The subsequent Boc removal, followed by

reaction with phenyl or benzyl isocyanate in the presence of Et₃N, gave the respective epimeric

mixtures of ureas 9a,b and 10a,b, which were chromatographically resolved into their respective

(R)- and (S)-epimers. Finally, the removal of the Z protecting group from the basic side chain, by

Pd(C)-catalysed hydrogenolysis, provided the corresponding deprotected pseudotripeptides 11a,b

and 12a,b. The (3:1) epimer ratio remained constant throughout the synthetic route.

Scheme 2. Synthesis of the 4-unsubstituted-piperazinone derivatives 11a,b and 12a,b.

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In view of the good results in the synthesis of the 4-unsubstituted-piperazinone derivatives 11a,b

and 12a,b, a parallel synthetic scheme was applied to the synthesis of the 4-benzyl-piperazinone

derivatives 19a–c and 20a,b from the (3:1) epimeric mixture of 4-benzyl-piperazinones 13 [23]

(Scheme 3). Based on the biological results of the previous library A, besides ornithine (a) and lysine

(b), arginine (c) was also included in this series. The Pbf protection was used for the guanidino group

of the side chain of this amino acid. This protection was removed in the last step of the synthesis by

treatment with a 90% solution of TFA in H₂O in the presence of triisopropylsilane (TIPS). The final

arylureido derivatives 19a–c and 20a,b were obtained in 39%–58% overall yields from 13, as (3:1)

epimeric mixtures that could not be separated in none of their synthetic steps.

Scheme 3. Synthesis of the N₄-benzyl-piperazinone derivatives 19a–c and 20a,b.

The 4-benzyl-piperazinones 16b,c were also used for the preparation of indazol-6-yl-ureido

derivatives analogues of the reference antagonist RWJ-58259. These analogues were prepared

according to our procedure developed for the synthesis of RWJ-58259 [24], which involves the in situ

formation of the isocyanate 22 (Scheme 4), by reaction of the corresponding 6-amino-indazole 21 with

triphosgene in the presence of propylene oxide as HCl acceptor, followed by reaction with the

epimeric mixture of the 4-benzyl-piperazinones 16b,c. The Z- or Pbf-removal, by hydrogenolysis and

TFA treatment, respectively, provided the proposed ureas 24b,c as (3:1) epimeric mixtures that, like

the analogues 19 and 20, could not be resolved at any of their synthetic steps.

To evaluate the PAR1 antagonist activity, all new compounds were screened as inhibitors of

human platelet aggregation induced by a 30 μM concentration of the PAR1 agonist SFLLRN [22].

The antagonist RWJ-58259 was used as a reference. At 10 µM concentration, this antagonist inhibited

98% the platelet aggregation. However, none of the new compounds displayed significant activity

at 0.1 mg/mL (≈150 μM). In the structural comparison of the inactive deprotected indazole-derived

ureas 24b,c with the potent peptidomimetic urea PAR1 antagonists, to which the reference antagonist

RWJ-58259 belongs [25], the main difference is localized at the linkage between the aromatic and the

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basic amino acids. Thus, the peptide bond of RWJ-58259 is replaced by the piperazinone ring and an

additional Gly residue in 24b,c. The results show that this replacement is completely detrimental for

PAR1 antagonist activity.

Scheme 4. Synthesis of the RWJ-58259 analogues 24b,c.

In a HTS of antitumor agents, none of the compound showed cytotoxicity on three representative

human cancer cell lines, such as breast (MDA-MB-231), lung (A549), and colon (HT-29).

3. Experimental

3.1. General

All reagents were of commercial quality. Solvents were dried and purified by standard methods.

Analytical TLC was performed on aluminum sheets coated with a 0.2 mm layer of silica gel 60 F₂₅₄.

Silica gel 60 (230–400 mesh) was used for flash chromatography. Analytical HPLC was performed on

a Sunfire C₁₈(4.6 × 150 mm, 3.5 μm) column, with a flow rate of 1 mL/min, and using a tunable UV

detector set at 214 nm. 10%–100% gradient of CH₃CN (solvent A) in 0.05% of TFA in H₂O (solvent

B) in 30 min was used as mobile phase. H-NMR spectra were recorded at 300 or 400 MHz, using

TMS as reference, and ¹³C-NMR spectra were recorded at 75 or 100 MHz. The NMR spectra

assignment was based on COSY, HSQC, and HMBC spectra. ESI-MS spectra were performed, in

positive mode, using MeOH as solvent. MW experiments were carried out in a EmrysTM Synthesizer

MW reactor (Biotage AB, surface IR sensor). Elemental analyses were obtained on a CH-O-RAOID

apparatus. Optical rotations were determined in a Perkin Elmer 141 polarimeter.

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3.2. Synthesis of Benzyl 2-[(2RS)-[(1S)-(3-benzylureido)-2-phenylethyl]-5-oxopiperazin-1-yl]acetate

(2) and (1S,9aRS)- 1-benzyl-3,7-dioxooctahydro-1H-pyrazino[1,2-a]pyrazine (3)

The epimeric mixture of piperazinones 1 [23] [(R:S) = (3:1)] (500 mg, 1.07 mmol) was dissolved in

a solution of HCl in EtOAc (3.4 N, 20 mL) and the mixture was stirred at room temperature for 30 min.

Afterwards, the solvent was evaporated to dryness, the residue was dissolved in CH₃CN/H₂O (1:3,

8 mL) and the solution was lyophilized. Benzyl isocyanate (199 μL, 1.61 mmol) and Et₃N (224 µL,

1.61 mmol) were added to a solution of the lyophilized powder in THF (40 mL) and the mixture was

stirred for 1 h. Afterwards, the solvent was removed under low pressure and the residue was dissolved

in CH₂Cl₂(60 mL). The solution was washed with H₂O (2 × 10 mL), brine (10 mL), dried over

Na₂SO₄and evaporated to dryness. The residue was purified by flash chromatography, with 0%–5%

MeOH gradient in EtOAc as mobile phase, to afford the epimeric mixture of ureas 2 [(R:S) = (3:1)]

as a foam (215 mg, 40%), along with the 1H-pyrazino[1,2-a]pyrazines 3 [23] (83 mg, 30%).

Benzyl 2-[(2RS)-[(1S)-(3-benzylureido)-2-phenylethyl]-5-oxopiperazin-1-yl]acetate (2). HPLC t_R:

20.02 min [(R)-2] and 21.24 min [(S)-2]; H-NMR (300 MHz, CDCl₃). (R)-2 δ (ppm): 2.56 (dd, 1H,

J = 10.5 and 13.5 Hz, CH₂-Ph), 2.82 (dd, 1H, J = 4 and 13.5 Hz, CH₂-Ph), 3.06 (dt, 1H, J = 4.5 and

9 Hz, 2-H), 3.24 (m, 1H, 3-H), 3.26 (d, J = 18 Hz, 6-H), 3.43 (s, 2H, CH₂CO₂Bn), 3.46 (d, 1H,

J = 18 Hz, 6-H), 3.60 (m, 1H, 3-H), 3.89 (m, 1H, 2-CH), 4.32 [d, 2H, J = 5.5 Hz, CH₂(NHBn)],

5.04 [m, 1H, NHBn], 5.10 [s, 2H, CH₂(CO₂Bn)], 5.45 (m, 1H, 4-H), 5.70 (m, 1H, 2-CHNH),

7.14–7.35 (m, 15H, Ar). (S)-2 δ (ppm): 2.56 (m, 1H, CH₂-Ph), 2.82 (m, 1H, CH₂-Ph), 3.06 (m, 1H,

2-H), 3.24 (m, 1H, 3-H), 3.50 (d, J = 17.5 Hz, 6-H), 3.43 (s, 2H, CH₂CO₂Bn), 3.58 (d, 1H, J = 17.5 Hz,

6-H), 3.60 (m, 1H, 3-H), 3.89 (m, 1H, 2-CH), 4.32 [m, 2H, CH₂(NHBn)], 5.04 (m, 1H, NHBn), 5.10

[s, 2H, CH₂(CO₂Bn)], 5.45 (m, 1H, 4-H), 5.70 (m, 1H, 2-CHNH), 7.14–7.35 (m, 15H, Ar); ¹³C-NMR

(75 MHz, CDCl₃). (R)-2 δ (ppm): 36.7 [C₃], 37.4 [CH₂-Ph], 44.5 [CH₂(NHBn)], 51.3 [C₆and

CH₂CO₂Bn], 53.0 [C₂-CH], 58.0 [C₂], 66.9 [CH₂(CO₂Bn)], 127.3, 127.4, 127.6, 128.4, 128.5, 128.6,

128.7, 129.0, 129.2 [15CH (Ar)], 135.2 [C (CO₂Bn)], 136.0 [C (Ph)], 139.3 [C (NHBn)], 158.2

[CO (Urea)], 169.2 [C₅], 171.0 [CO₂]. (S)-2 δ (ppm): 36.7 [C₃], 37.4 [CH₂-Ph], 44.5 [CH₂(NHBn)],

51.3 [CH₂CO₂Bn], 53.0 [C₂-CH], 55.6 [C₆], 58.0 [C₂], 66.9 [CH₂(CO₂Bn)], 127.3, 127.4, 127.6,

128.4, 128.5, 128.6, 128.7, 129.0, 129.2 [CH (Ar)], 135.2 [C (CO₂Bn)], 136.0 [C (Ph)], 139.3 [C

(NHBn)], 158.2 [CO (Urea)], 169.2 [C₅], 171.0 [CO₂]; ES-MS m/z 501.2 [M+1]⁺; C₂₉H₃₂N₄O₅(%): C:

69.58, H: 6.44, N: 11.19. Found (%): C: 69.73, H: 6.32, N: 11.45.

3.3. General Procedure for the Synthesis of the Piperazinone-Derived Acids 4 and 14

Pd(C) (10%) was added to a solution of the corresponding epimeric mixture of piperazinones 1 [23]

or 13 [23] [(R:S) = (3:1)] (1.00 mmol) in MeOH (50 mL) and the mixture was hydrogenated at 1 atm

of H₂at room temperature for 1 h. Afterwards, the reaction mixture was filtered and the solvent was

evaporated under reduced pressure to obtain the epimeric mixture of the corresponding acids 4 or 14

[(R:S) = (3:1)].

2-[(2RS)-[(1S)-((tert-Butoxycarbonyl)amino)-2-phenyl-ethyl]-5-oxopiperazin-1-yl] acetic acid (4).

Foam (377.4 mg, 100%); HPLC t_R: 13.99 min [(R)-4] and 13.39 min [(S)-4]; H-NMR (500 MHz,

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DMSO-d₆). (R)-4 δ (ppm): 1.24 (s, 9H, Boc), 2.56 (dd, 1H, J = 10 and 10.5 Hz, CH₂-Ph), 2.88 (m, 1H,

2-H), 2.97 (dd, 1H, J = 3.5 and 10.5 Hz, CH₂-Ph), 3.19 (m, 2H, 3-H), 3.30 (m, 1H, 6-H), 3.47 (d, 3H,

J = 17 Hz, 6-H and CH₂CO₂H), 3.80 (m, 1H, 2-CH), 6.80 (d, 1H, J = 9.5 Hz, NHBoc), 7.02–7.36

(m, 5H, Ph), 7.76 (s, 1H, 4-H). (S)-4 δ (ppm): 1.25 (s, 9H, Boc), 2.47 (m, 1H, CH₂-Ph), 2.82 (dd, 1H,

J = 2 and 13.5 Hz, CH₂-Ph), 2.92 (m, 1H, 2-H), 3.19 (m, 2H, 3-H), 3.31 (m, 1H, 6-H), 3.42 (m, 1H,

6-H), 3.47 (m, 2H, CH₂CO₂H), 3.80 (m, 1H, 2-CH), 6.89 (d, 1H, J = 9.5 Hz, NHBoc), 7.02–7.36

(m, 5H, Ph), 7.75 (s, 1H, 4-H); C-NMR (125 MHz, DMSO-d₆). (R)-4 δ (ppm): 28.6 [3CH₃(Boc)],

37.9 [CH₂-Ph], 38.9 [C₃], 51.8 [C₂-CH], 53.3 [CH₂CO₂H], 54.0 [C₆], 58.4 [C₂], 78.1 [C (Boc)], 126.2,

128.4, 129.7 [5CH (Ph)], 139.6 [C (Ph)], 155.8 [CO (Boc)], 168.3 [C₅], 172.6 [CO₂]. (S)-4 δ (ppm):

28.6 [3CH₃(Boc)], 35.7 [CH₂-Ph], 38.8 [C₃], 52.0 [C₂-CH], 53.0[C₆], 54.1 [CH₂CO₂H], 59.0 [C₂],

78.0 [C (Boc)], 126.3, 128.4, 129.4 [5CH (Ph)], 139.8 [C (Ph)], 155.5 [CO (Boc)], 169.2 [C₅], 172.4

[CO₂]; ES-MS m/z 378.0 [M+1]⁺; C₁₉H₂₇N₃O₅(%): C: 60.46, H: 7.21, N: 11.13. Found (%): C: 60.60,

H: 7.02, N: 11.25.

3.4. General Procedure for the Synthesis of the Piperazinone-Derived Pseudotripeptides 7a,b

HOBt (136 mg, 1.00 mmol), DIC (309 µL, 2.00 mmol) and a solution of the corresponding

benzylamides H-Orn(Boc)-NHBn (6a) [26] and H-Lys(Boc)-NHBn (6b) [27] (1.50 mmol) in dry DMF

(4 mL) were added to a solution of the epimeric mixture of the piperazinone-derived acid 4

(1.00 mmol) in dry CH₂Cl₂(16 mL) and stirred for 24 h. Afterwards, the solvent was removed under

reduced pressure and the residue was dissolved in EtOAc (100 mL). This solution was washed with a

solution of 10% citric acid (2 × 20 mL), a saturated solution of NaHCO₃(2 × 20 mL) and brine

(20 mL), dried over Na₂SO₄, and evaporated to dryness. The residue was purified by flash

chromatography, with 1%–10% MeOH gradient in CH₂Cl₂as mobile phase to afford the corresponding

epimeric mixture of piperazinone derivatives 7a,b [(R:S) = (3:1)].

N-[2-[(2RS)-[(1S)-((tert-Butoxycarbonyl)amino)-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-Orn(Z)-

NHBn (7a). Foam (429 mg, 60%); HPLC t_R: 21.07 min; ¹H-NMR (400 MHz, CDCl₃) (R)-7a δ (ppm):

1.31 (s, 9H, Boc), 1.52 (m, 2H, γ-H), 1.67 (m, 1H, β-H), 1.84 (m, 1H, β-H), 2.82 (m, 1H, CH₂-Ph),

2.86 (m, 1H, 2-H), 3.02 (m, 1H, CH₂-Ph), 3.12 (m, 1H, δ-H), 3.30 (m, 1H, CH₂CO), 3.34 (m, 1H,

CH₂CO), 3.36 (m, 3H, 3-H and 6-H), 3.42 (m, 1H, δ-H), 3.44 (m, 1H, 6-H), 4.04 (m, 1H, 2-CH),

4.34 [dd, 1H, J = 5.5 and 15 Hz, CH₂(NHBn)], 4.42 [dd, 1H, J = 5.5 and 15 Hz, CH₂(NHBn)],

4.70 (m, 3H, α-H and NHBoc), 4.83 [d, 1H, J = 12 Hz, CH₂(Z)], 4.93 [d, 1H, J = 12 Hz, CH₂(Z)],

5.12 (t, 1H, J = 6 Hz, NHZ), 6.38 (m, 1H, 4-H), 7.11–7.39 (m, 16H, Ar and NHBn), 7.79 (d, 1H,

J = 8 Hz, α-NH). (S)-7a δ (ppm): 1.31 (s, 9H, Boc), 1.64(m, 1H, β-H), 1.86 (m, 1H, β-H), 3.26 (m, 1H,

CH₂CO), 3.32 (m, 1H, 6-H), 3.38 (m, 1H, CH₂CO), 3.46 (m, 1H, 6-H), 3.94 (m, 1H, 2-CH), 4.35,

4.47 [m, 2H, CH₂(NHBn)], 4.82 [m, 1H, CH₂(Z)], 4.95 [m, 1H, CH₂(Z)], 5.04 (m, 1H, NHZ), 6.62

(m, 1H, 4-H), 7.11–7.39 (m, 16H, Ar and NHBn), 7.79 (d, 1H, J = 8 Hz, α-NH); ¹³C-NMR (100 MHz,

CDCl₃) (R)-7a δ (ppm): 26.3 [C_γ], 28.2 [3CH₃(Boc)], 30.3 [C_β], 37.6 [CH₂-Ph], 39.4 [C₃], 39.7 [C_δ],

43.5 [CH₂(NHBn)], 51.3 [C₂-CH], 51.5 [C_α], 54.0 [C₆], 55.7 [CH₂CO], 58.8 [C₂], 66.6 [CH₂(Z)], 79.9

[C (Boc)], 126.7, 127.4, 127.7, 127.9, 128.1, 128.4, 128.6, 129.3 [15CH (Ar)], 136.4 [C (Ph)], 137.0

[C (Z)], 138.0 [C (NHBn)], 155.6 [CO (Boc)], 157.1 [CO (Z)], 168.9 [C₅], 169.7 [CO], 171.5

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[α-CONH]. (S)-7a δ (ppm): 28.2 [3CH₃(Boc)], 43.5 [CH₂(NHBn)], 66.6 [CH₂(Z)], 79.9 [C (Boc)],

126.6, 127.4, 127.6, 127.9, 128.0, 128.4, 128.6, 129.3 [15CH (Ar)], 136.4 [C (Ph)], 137.0 [C (Z)],

138.0 [C (NHBn)], 155.6 [CO (Boc)], 157.1 [CO (Z)], 171.5 [α-CONH]; ES-MS m/z 715.6 [M+1]⁺;

C₃₉H₅₀N₆O₇(%): C: 65.53, H: 7.05, N: 11.76. Found (%): C: 65.71, H: 6.98, N: 11.89.

N-[2-[(2RS)-[(1S)-((tert-Butoxycarbonyl)amino)-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-Lys(Z)-

NHBn (7b). Foam (481 mg, 66%); HPLC t_R: 21.56 min [(R)-7b] and 21.41 min [(S)-7b]; H-NMR

(400 MHz, CDCl₃) (R)-7b δ (ppm): 1.27 (s, 9H, Boc), 1.29 (m, 2H, γ-H), 1.44 (m, 2H, δ-H), 1.65

(m, 1H, β-H), 1.83 (m, 1H, β-H), 2.77 (m, 1H, CH₂-Ph), 2.78 (m, 1H, 2-H), 2.95 (d, 1H, J = 10 Hz,

CH₂-Ph), 3.05 (m, 2H, ε-H), 3.20 (m, 1H, 6-H), 3.22 (m, 2H, CH₂CO), 3.23 (m, 2H, 3-H), 3.35 (m,

1H, 6-H), 4.00 (m, 1H, 2-CH), 4.34 [dd, 1H, J = 8 and 15 Hz, CH₂(NHBn)], 4.40 [dd, 1H, J = 8 and

15 Hz, CH₂(NHBn)], 4.48 (m, 1H, α-H), 4.81 (d, 1H, J = 8 Hz, NHBoc), 5.03 [m, 2H, CH₂(Z)], 5.25

(m, 1H, NHZ), 6.85 (m, 1H, 4-H), 7.08–7.40 (m, 16H, Ar and NHBn), 7.79 (d, 1H, J = 8 Hz, α-NH).

(S)-7b δ (ppm): 1.27 (s, 9H, Boc), 1.29 (m, 2H, γ-H), 1.44 (m, 2H, δ-H), 1.65 (m, 1H, β-H), 1.83 (m,

1H, β-H), 2.78 (m, 1H, 2-H), 3.10 (m, 1H, 6-H), 3.15 (m, 2H, CH₂CO), 3.23 (m, 2H, 3-H), 3.36 (m,

1H, 6-H), 3.90 (m, 1H, 2-CH), 4.28, 4.42 [m, 2H, CH₂(NHBn)], 4.46 (m, 1H, α-H), 5.03 [m, 2H, CH₂

(Z)], 5.25 (m, 1H, NHZ), 6.77 (m, 1H, 4-H), 7.08–7.40 (m, 16H, Ar and NHBn), 7.73 (d, 1H, J = 8 Hz,

α-NH); C-NMR (100 MHz, CDCl₃) (R)-7b δ (ppm): 22.6 [C_γ], 28.1 [3CH₃(Boc)], 29.2 [C_δ], 32.1

[C_β], 37.6 [CH₂-Ph], 39.5 [C₃], 40.5 [C_ε], 43.4 [CH₂(NHBn)], 51.6 [C₂-CH], 52.7 [C_α], 53.9 [C₆], 55.7

[CH₂CO], 58.7 [C₂], 66.4 [CH₂(Z)], 79.6 [C (Boc)], 126.7, 127.3, 127.6, 128.0, 128.5, 128.6, 129.2

[15CH (Ar)], 136.6 [C (Ph)], 137.1 [C (Z)], 138.1 [C (NHBn)], 155.6 [CO (Boc)], 156.5 [CO (Z)],

169.4 [C₅], 169.8 [CO], 171.5 [α-CONH]. (S)-7b δ (ppm): 22.6 [C_γ], 28.1 [3CH₃(Boc)], 29.6 [C_δ],

31.8 [C_β], 39.4 [C₆], 43.4 [CH₂(NHBn)], 51.9 [C₂-CH], 52.7 [C_α], 53.9 [C₆], 55.7 [CH₂CO], 59.0 [C₂],

66.4 [CH₂(Z)], 79.7 [C (Boc)], 126.6, 127.3, 127.6, 128.0, 128.5, 128.6, 129.2 [15CH (Ar)], 136.6 [C

(Ph)], 137.1 [C (Z)], 138.1 [C (NHBn)], 155.8 [CO (Boc)], 156.5 [CO (Z)], 169.4 [C₅], 170.3 [CO],

171.6 [α-CONH]; ES-MS m/z 729.3 [M+1]⁺; C₄₀H₅₂N₆O₇(%): C: 65.91, H: 7.19, N: 11.53. Found

(%): C: 65.72, H: 7.40, N: 11.68.

3.5. General Procedure for the N-Boc Removal in 7a,b. Synthesis of the Hydrochlorides 8a,b

The epimeric corresponding epimeric mixture of piperazine derivatives 7a,b [(R:S) = (3:1)]

(0.60 mmol) was dissolved in 3.4 N HCl in EtOAc (15 mL) and the mixture was stirred at room

temperature for 30 min. Afterwards, the solvent was evaporated to dryness, the residue was dissolved

in CH₃CN/H₂O (1:3, 5 mL), and the solution was lyophilized. The desired epimeric mixture of

hydrochlorides [(R:S) = (3:1)] was obtained quantitatively.

N-[2-[(2RS)-[(1S)-Amino-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-Orn(Z)-NHBn

hydrochloride

(8a). Amorphous solid (391 mg, 100%); HPLC t_R: 14.86 min; ¹H-NMR (400 MHz, DMSO-d₆) (R)-8a

δ (ppm): 1.30 (m, 2H, γ-H), 1.57 (m, 1H, β-H), 1.72 (m, 1H, β-H), 2.59 (dd, 1H, J = 9 and 14 Hz,

CH₂-Ph), 2.86 (dd, 1H, J = 6.5 and 14 Hz, CH₂-Ph and 2-H), 2.95 (m, 4H, δ-H and 3-H), 3.02 (d, 1H,

J = 18 Hz, 6-H), 3.23 (d, 1H, J = 16.5 Hz, CH₂CO), 3.33 (d, 1H, J = 16.5 Hz, CH₂CO), 3.52 (d, 1H,

J = 18 Hz, 3-H), 4.16 (m, 1H, 2-CH), 4.28 (m, 1H, α-H), 4.40 [m, 2H, CH₂(NHBn)], 4.97 [m, 2H,

CH₂(Z)], 7.15–7.40 (m, 16H, Ar and NHZ), 7.63 (m, 1H, 4-H), 8.03 (m, 3H, NH₂·HCl), 8.14 (d, 1H,

Molecules 2014, 19

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J = 8.5 Hz, α-NH), 8.51 (t, 1H, J = 6 Hz, NHBn). (S)-8a δ (ppm): 1.30 (m, 2H, γ-H), 1.57 (m, 1H,

β-H), 1.72 (m, 1H, β-H), 4.32 (m, 1H, α-H), 4.40 [m, 2H, CH₂(NHBn)], 4.98 [m, 2H, CH₂(Z)], 7.15–7.40

(m, 16H, Ar and NHZ), 8.03 (m, 3H, NH₂·HCl), 8.20 (m, 1H, α-NH), 8.57 (m, 1H, NHBn); ¹³C-NMR

(100 MHz, DMSO-d₆) (R)-8a δ (ppm): 26.1 [C_γ], 28.8 [C_β], 34.9 [C₃], 36.0 [CH₂-Ph], 40.5 [C_δ], 42.0

[CH₂(NHBn)], 49.7 [C₆], 50.2 [C₂-CH], 52.4 [C_α], 55.9 [C₂], 58.5 [CH₂CO], 65.1 [CH₂(Z)], 126.7,

127.0, 127.7, 128.3, 128.4, 128.6, 128.8 [15CH (Ar)], 136.6 [C (Ph)], 137.2 [C (Z)], 139.4 [C

(NHBn)], 156.1 [CO (Z)], 168.2 [C₅], 169.7 [CO], 171.6 [α-CONH]. (S)-8a δ (ppm): 26.0 [C_γ], 28.8

[C_β], 42.0 [CH₂(NHBn)], 52.3 [C_α], 65.1 [CH₂(Z)], 126.6, 127.1, 127.8, 128.3, 128.4, 128.6, 128.8

[15CH (Ar)], 136.6 [C (Ph)], 137.2 [C (Z)], 139.4 [C (NHBn)], 156.1 [CO (Z)], 171.6 [α-CONH];

ES-MS m/z 615.8 [M−Cl]⁺; C₃₄H₄₂N₆O₅·HCl (%): C: 62.71, H: 6.66, N: 12.91. Found (%): C: 62.53,

H: 6.78, N: 12.98.

N-[2-[(2RS)-[(1S)-Amino-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-Lys(Z)-NHBn

hydrochloride

(8b). Amorphous solid (399 mg, 100%); HPLC t_R: 15.25 min; ¹H-NMR (400 MHz, DMSO-d₆) (R)-8b

δ (ppm): 1.22 (m, 2H, γ-H), 1.35 (m, 2H, δ-H), 1.66 (m, 2H, β-H), 2.59 (dd, 1H, J = 9 and 14 Hz,

CH₂-Ph), 2.84 (m, 1H, CH₂-Ph), 2.88 (m, 1H, 2-H), 3.94 (m, 2H, ε-H), 3.01 (m, 2H, 3-H), 3.02 (d, 1H,

J = 18 Hz, 6-H), 3.23 (d, 1H, J = 16.5 Hz, CH₂CO), 3.37 (d, 1H, J = 16.5 Hz, CH₂CO), 3.54 (d, 1H,

J = 18 Hz, 6-H), 4.18 (m, 1H, 2-CH), 4.24 [m, 2H, CH₂(NHBn)], 4.26 (m, 1H, α-H), 4.98 [m, 2H,

CH₂(Z)], 7.14–7.41 (m, 16H, Ar and NHZ), 7.63 (m, 1H, 4-H), 8.07 (m, 3H, NH₂·HCl), 8.15 (d, 1H,

J = 8.5 Hz, α-NH), 8.55 (t, 1H, J = 6 Hz, NHBn). (S)-8b δ (ppm): 1.22 (m, 2H, γ-H), 1.35 (m, 2H,

δ-H), 1.66 (m, 2H, β-H), 3.01 (m, 1H, 3-H), 3.37 (m, 1H, CH₂CO), 3.38 (m, 1H, CH₂CO), 3.55 (m,

1H, 6-H), 4.24 [m, 2H, CH₂(NHBn)], 4.26 (m, 1H, α-H), 4.98 [m, 2H, CH₂(Z)], 7.14–7.41 (m, 16H,

Ar and NHZ), 8.07 (m, 3H, NH₂·HCl), 8.23 (m, 1H, α-NH), 8.58 (m, 1H, NHBn); C-NMR (100

MHz, DMSO-d₆) (R)-8b δ (ppm): 22.9 [C_γ], 29.0 [C_δ], 31.2 [C_β], 34.8 [C₃], 36.0 [CH₂-Ph], 39.5 [C_ε],

42.0 [CH₂(NHBn)], 49.6 [C₆], 51.6 [C₂-CH], 52.8 [C_α], 55.9 [C₂], 58.5 [CH₂CO], 65.1 [CH₂(Z)],

126.6, 127.0, 127.7, 128.2, 128.3, 128.6, 128.8 [15CH (Ar)], 136.6 [C (Ph)], 137.3 [C (Z)], 139.4 [C

(NHBn)], 156.1 [CO (Z)], 168.2 [C₅], 169.6 [CO], 171.7 [α-CONH]. (S)-8b δ (ppm): 22.9 [C_γ], 29.0

[C_δ], 31.2 [C_β], 42.0 [CH₂(NHBn)], 49.6 [C₆], 58.5 [CH₂CO], 65.1 [CH₂(Z)], 126.6, 127.0, 127.7,

128.2, 128.3, 128.6, 128.8 [15CH (Ar)], 136.6 [C (Ph)], 137.3 [C (Z)], 139.4 [C (NHBn)], 156.1 [CO

(Z)], 171.7 [α-CONH]; ES-MS m/z 629.7 [M-Cl]⁺; C₃₅H₄₄N₆O₅·HCl (%): C: 63.19, H: 6.82, N: 12.63.

Found (%): C: 63.02, H: 6.94, N: 12.74.

3.6. General Procedure for the Synthesis of the Piperazinone-Derived Ureas 9a,b and 10a,b

Et₃N (168 µL, 1.20 mmol) and the corresponding isocyanate (phenyl or benzyl isocyanate)

(1.20 mmol) were added to a solution of the corresponding hydrochloride 8a,b (0.60 mmol) in dry

THF (30 mL). After stirring for 1 h at room temperature, the solvent was removed under reduced

pressure and the residue was dissolved in CH₂Cl₂(100 mL). The solution was washed with H₂O

(2 × 20 mL), brine (20 mL), dried over Na₂SO₄, and evaporated to dryness. The residue was purified

by flash chromatography using 1%–8% MeOH gradient in EtOAc as mobile phase. The respective

(R)- and (S)-epimers were resolved in this purification. The purified compounds were dissolved in

CH₃CN/H₂O (1:2, 2 mL) and the solution was lyophilized, to afford the desired ureas 9a,b and 10a,b.

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N-[2-[5-Oxo-(2R)-[2-phenyl-(1S)-(3-phenylureido)ethyl]piperazin-1-yl]acetyl]-Orn(Z)-NHBn [(R)-9a].

Amorphous solid (176 mg, 46%); α²_D⁰= −0.1 (c 1, MeOH); HPLC t_R: 20.30 min; ¹H-NMR (400 MHz,

CDCl₃) δ (ppm): 1.30 (m, 2H, γ-H), 1.50 (m, 1H, β-H), 1.70 (m, 1H, β-H), 2.82 (m, 1H, CH₂-Ph), 2.83

(m, 1H, δ-H), 2.84 (m, 1H, 2-H), 2.88 (m, 1H, CH₂-Ph), 3.03 (m, 1H, CH₂CO), 3.14 (m, 1H, 6-H),

3.35 (m, 1H, 6-H), 3.37 (m, 1H, 3-H), 3.24 (m, 1H, δ-H), 3.44 (m, 1H, CH₂CO), 4.20 (m, 1H, 3-H),

4.25 (m, 1H, 2-CH), 4.26 [m, 1H, CH₂(NHBn)], 4.36 [m, 1H, CH₂(NHBn)], 4.60 (m, 1H, α-H), 4.82

[d, 1H, J = 12.5 Hz, CH₂(Z)], 4.91 [d, 1H, J = 12.5 Hz, CH₂(Z)], 5.25 (m, 1H, NHZ), 5.97 (m, 1H, 4-H),

6.12 (m, 1H, 2-CHNH), 6.91–7.35 (m, 20H, Ar), 7.46 (m, 1H, NHBn), 7.65 [m, 1H, NHPh], 7.88 (m,

1H, α-NH); ¹³C-NMR (100 MHz, CDCl₃) δ (ppm): 26.7 [C_γ], 29.9 [C_β], 37.9 [CH₂-Ph], 39.7 [C_δ], 40.1

[C₃], 43.7 [CH₂(NHBn)], 51.6 [C₂-CH], 52.0 [C_α], 54.9 [C₆], 57.8 [CH₂CO], 59.6 [C₂], 67.0 [CH₂

(Z)], 116.7, 119.7, 123.0, 127.0, 127.9, 120.4, 128.8, 129.0, 129.3 [20CH (Ar)], 134.3 [C (Ph)], 136.1

[C (Z)], 137.9 [C (NHBn)], 139.5 [C (NHPh)], 157.2 [CO (Z) and CO (Urea)], 168.7 [C₅], 170.0 [CO],

171.1 [α-CONH]; ES-MS m/z 734.4 [M+1]⁺; C₄₁H₄₇N₇O₆(%): C: 67.10, H: 6.46, N: 13.36. Found

(%): C: 67.28, H: 6.59, N: 13.19.

N-[2-[5-Oxo-(2S)-[2-phenyl-(1S)-(3-phenylureido)ethyl]piperazin-1-yl]acetyl]-Orn(Z)-NHBn [(S)-9a].

Amorphous solid (79 mg, 18%); α²_D⁰= +9.2 (c 1.5, MeOH); t_R: 21.41 min; H-NMR (500 MHz,

CDCl₃) δ (ppm): 1.32 (m, 1H, γ-H), 1.40 (m, 1H, γ-H), 1.53 (m, 1H, β-H), 1.72 (m, 1H, β-H), 2.54

(dd, 1H, J = 11 and 13.5 Hz, CH₂-Ph), 2.90 (dd, 1H, J = 4 and 13.5 Hz, CH₂-Ph), 2.92 (m, 1H, δ-H),

3.08 (m, 1H, 5-H), 3.10 (m, 1H, 3-H), 3.14 (m, 1H, 6-H), 3.32 (m, 2H, CH₂CO), 3.35 (m, 1H, δ-H),

3.54 (d, 1H, J = 18 Hz, 6-H), 3.92 (m, 1H, 2-CH), 3.95 (m, 1H, 3-H), 4.28 [dd, 1H, J = 5 and 15 Hz,

CH₂(NHBn)], 4.44 [dd, 1H, J = 6 and 15 Hz, CH₂(NHBn)], 4.60 [d, 1H, J = 13 Hz, CH₂(Z)], 4.71

(m, 1H, α-H), 4.83 [d, 1H, J = 13 Hz, CH₂(Z)], 4.95 (m, 1H, NHZ), 5.67 (m, 1H, 4-H), 5.94 (d, 1H,

J = 6 Hz, 2-CHNH), 6.83–7.35 (m, 20H, Ar), 7.53 (m, 1H, NHBn), 7.93 [m, 1H, NHPh], 7.97 (d, 1H,

J = 8.5 Hz, α-NH); ¹³C-NMR (125 MHz, CDCl₃) δ (ppm): 26.4 [C_γ], 31.1 [C_β], 35.8 [C₃], 37.8

[CH₂-Ph], 38.9 [C_δ], 43.8 [CH₂(NHBn)], 50.8 [C_α], 51.9 [C₆], 52.3 [C₂-CH], 57.7 [CH₂CO], 58.6 [C₂],

66.7 [CH₂(Z)], 118.3, 122.2, 127.5, 127.8, 128.2, 128.5, 128.8, 128.9, 129.0, 129.3 [20CH (Ar)],

135.6 [C (Ph)], 136.2 [C (Z)], 137.3 [C (NHBn)], 139.6 [C (NHPh)], 155.6 [CO (Z)], 157.6

[CO (Urea)], 168.8 [C₅], 169.2 [CO], 172.9 [α-CONH]; ES-MS m/z 734.5 [M+1]⁺; C₄₁H₄₇N₇O₆(%):

C: 67.10, H: 6.46, N: 13.36. Found (%): C: 67.21, H: 6.30, N: 13.49.

N-[2-[5-Oxo-(2R)-[2-phenyl-(1S)-(3-phenylureido)ethyl]piperazin-1-yl]acetyl]-Lys(Z)-NHBn [(R)-9b].

Amorphous solid (206 mg, 46%); α_D²⁰= −3.7 (c 1.5, MeOH); HPLC t_R: 20.09 min; H-NMR

(400 MHz, CDCl₃) δ (ppm): 1.30 (m, 2H, γ-H), 1.40 (m, 2H, δ-H), 1.50 (m, 1H, β-H), 1.73 (m, 1H,

β-H), 2.80 (m, 1H, CH₂-Ph), 2.87 (m, 1H, CH₂-Ph), 2.85 (m, 1H, ε-H), 2.92 (m, 1H, 2-H), 3.03 (m,

1H, CH₂CO), 3.16 (m, 1H, 6-H), 3.20 (m, 2H, 3-H and ε-H), 3.38 (m, 1H, 6-H), 3.44 (m, 1H, CH₂CO),

4.25 (m, 1H, 3-H), 4.28 (m, 1H, 2-CH), 4.30 [m, 1H, CH₂(NHBn)], 4.38 [m, 1H, CH₂(NHBn)], 4.50

(m, 1H, α-H), 5.01 [m, 2H, CH₂(Z)], 5.27 (m, 1H, NHZ), 5.98 (m, 1H, 2-CHNH), 6.23 (m, 1H, 4-H),

6.78–7.59 (m, 21H, Ar and NHBn), 7.64 (m, 1H, NHPh), 7.90 (m, 1H, α-NH); C-NMR (100 MHz,

CDCl₃) δ (ppm): 23.1 [C_γ], 29.3 [C_δ], 31.9 [C_β], 38.3 [CH₂-Ph], 39.6 [C₃], 40.7 [C_ε], 43.6 [CH₂

(NHBn)], 51.7 [C₆], 53.9 [C₂-CH], 54.8 [C_α], 59.2 [CH₂CO], 59.8 [C₂], 66.8 [CH₂(Z)], 119.2, 119.9,

123.1, 127.1, 127.5, 127.6, 127.7, 128.1, 128.4, 128.8, 128.9, 129.0, 129.1 [20CH (Ar)], 136.8 [C

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(Ph)], 137.5 [C (Z)], 138.2 [C (NHBn)], 139.2 [C (NHPh)], 156.1 [CO (Z)], 156.9 [CO (Urea)], 170.5

[C₅and CO], 172.4 [α-CONH]; ES-MS m/z 748.6 [M+1]⁺; C₄₂H₄₉N₇O₆(%): C: 67.45, H: 6.60, N:

13.11. Found (%): C: 67.62, H: 6.74, N: 13.02.

N-[2-[5-Oxo-(2S)-[2-phenyl-(1S)-(3-phenylureido)ethyl]piperazin-1-yl]acetyl]-Lys(Z)-NHBn [(S)-9b].

Amorphous solid (67 mg, 15%); α²_D⁰= +6.7 (c 0.9, MeOH); HPLC t_R: 21.76 min; H-NMR

(500 MHz, CDCl₃) δ (ppm): 1.23 (m, 2H, γ-H), 1.34 (m, 2H, δ-H), 1.67 (m, 1H, β-H), 1.82 (m, 1H,

β-H), 2.58 (t, 1H, J = 12.5 Hz, CH₂-Ph), 2.87 (m, 1H, CH₂-Ph), 2.90 (m, 1H, ε-H), 2.98 (m, 1H, 2-H),

3.05 (m, 1H, ε-H), 3.10 (m, 1H, 3-H), 3.18 (m, 1H, 6-H), 3.30 (m, 1H, CH₂CO), 3.42 (m, 1H,

CH₂CO), 3.62 (m, 1H, 6-H), 3.95 (m, 1H, 2-CH), 4.05 (m, 1H, 3-H), 4.35 [m, 1H, CH₂(NHBn)], 4.50

[m, 2H, CH₂(NHBn) and α-H], 4.98 [m, 2H, CH₂(Z)], 5.02 (m, 1H, NHZ), 5.69 (m, 1H, 2-CHNH),

5.81 (m, 1H, 4-H), 6.94 (t, 1H, J = 7.5 Hz, NHBn), 6.98–7.14 (m, 20H, Ar), 7.98 (m, 1H, NHPh), 8.09

(m, 1H, α-NH); ¹³C-NMR (125 MHz, CDCl₃) δ (ppm): 22.2 [C_γ], 29.1 [C_δ], 32.6 [C_β], 35.7 [C₃], 37.7

[CH₂-Ph], 40.2 [C_ε], 43.8 [CH₂(NHBn)], 52.0 [C₆and C₂-CH], 52.8 [C_α], 57.8 [CH₂CO], 58.7 [C₂],

66.5 [CH₂(Z)], 118.4, 122.3, 127.7, 127.8, 128.1, 128.5, 128.9, 129.0, 129.3 [20CH (Ar)], 135.6 [C

(Ph)], 136.5 [C (Z)], 137.1 [C (NHBn)], 139.6 [C (NHPh)], 155.6 [CO (Z)], 156.7 [CO (Urea)], 168.6

[C₅], 169.9 [CO], 172.0 [α-CONH]; ES-MS m/z 748.7 [M+1]⁺; C₄₂H₄₉N₇O₆(%): C: 67.45, H: 6.60,

N: 13.11. Found (%): C: 67.31, H: 6.81, N: 13.25.

N-[2-[-(2R)-[(1S)-(3-Benzylureido)-2-phenylethyl]-5-oxo-piperazin-1-yl]acetyl]-Orn(Z)-NHBn [(R)-10a].

Amorphous solid (170 mg, 38%); α_D²⁰= −3.8 (c 1.2, MeOH); HPLC t_R: 20.79 min; H-NMR

(400 MHz, CDCl₃) δ (ppm): 1.40 (m, 2H, γ-H), 1.55 (m, 1H, β-H), 1.70 (m, 1H, β-H), 2.57 (dd, 1H,

J = 11 and 13.5, CH₂-Ph), 2.86 (dd, 1H, J = 3.5 and 13.5, CH₂-Ph), 3.00 (m, 1H, 2-H), 3.04 (m, 1H,

δ-H), 3.09 (m, 1H, 3-H), 3.15 (m, 1H, 3-H), 3.25 (m, 2H, CH₂CO), 3.32 (m, 1H, δ-H), 3.50 (d, 1H,

J = 18 Hz, 6-H), 3.85 (m, 1H, 3-H), 3.94 (m, 1H, 2-CH), 4.17 [dd, 1H, J = 5 and 15 Hz, CH₂(NHBn)],

4.23 [dd, 1H, J = 6 and 15 Hz, CH₂(NHBn)], 4.27 [dd, 1H, J = 5 and 15 Hz, CH₂(NHBn, Urea)], 4.36

[dd, 1H, J = 5.5 and 15 Hz, CH₂(NHBn, Urea)], 4.66 (m, 1H, α-H), 4.79 [d, 1H, J = 13 Hz, CH₂(Z)],

4.89 [d, 1H, J = 13 Hz, CH₂(Z)], 5.30 (t, 1H, J = 6 Hz, NHZ), 5.75 (m, 1H, 2-CHNH), 5.90 (m, 1H,

4-H), 6.07 [t, 1H, J = 5.5 Hz, NHBn (Urea)], 7.08–7.39 (m, 20H, Ar), 7.50 (t, 1H, J = 5.5 Hz, NHBn),

7.99 [d, 1H, J = 8.5, α-NH); C-NMR (100 MHz, CDCl₃) δ (ppm): 26.1 [C_γ], 30.6 [C_β], 35.8 [C₃],

37.5 [CH₂-Ph], 39.3 [C_δ], 43.5 [CH₂(NHBn)], 44.4 [CH₂(NHBn, Urea)], 51.0 [C_α], 51.8 [C₆], 52.5

[C₂-CH], 57.5 [CH₂CO], 58.3 [C₂], 66.5 [CH₂(Z)], 127.0, 127.4, 127.5, 127.8, 128.1, 128.4, 128.5,

128.7, 129.0, 129.2 [20CH (Ar)], 135.8 [C (Ph)], 136.5 [C (Z)], 137.5 [C (NHBn)], 139.6 [C (NHBn,

Urea)], 157.2 [CO (Z)], 158.5 [CO (Urea)], 168.9 [C₅], 170.0 [CO], 172.2 [α-CONH]; ES-MS m/z

748.6 [M+1]⁺; C₄₂H₄₉N₇O₆(%): C: 67.45, H: 6.60, N: 13.11. Found (%): C: 67.28, H: 6.82, N: 13.20.

N-[2-[-(2S)-[(1S)-(3-Benzylureido)-2-phenylethyl]-5-oxo-piperazin-1-yl]acetyl]-Orn(Z)-NHBn [(S)-10a].

Amorphous solid (76 mg, 17%); α²_D⁰= −8.2 (c 1.0, MeOH); HPLC t_R: 20.09 min; H-NMR

(500 MHz, (CD₃)₂CO) δ (ppm): 1.55 (m, 2H, γ-H), 1.71 (m, 1H, β-H), 1.87 (m, 1H, β-H), 2.80 (dd,

1H, J = 10 and 14, CH₂-Ph), 2.98 (dd, 1H, J = 6 and 13, 2-H), 3.06 (dd, 1H, J = 4 and 14, CH₂-Ph),

3.12 (m, 1H, δ-H), 3.16 (d, 1H, J = 16.5 Hz, 6-H), 3.35 (s, 2H, CH₂CO), 3.40 (d, 1H, J = 16.5 Hz,

6-H), 3.42 (m, 1H, 3-H), 3.51 (ddd, 1H, J = 4, 13 and 15 Hz, 3-H), 4.16 [dd, 1H, J = 6 and 15 Hz, CH₂

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(NHBn, Urea)], 4.27 [dd, 1H, J = 5.5 and 15 Hz, CH₂(NHBn, Urea)], 4.38 (m, 1H, 2-CH), 4.33 [d, 2H,

J = 6 Hz, CH₂(NHBn)], 4.56 (dt, 1H, J = 5 and 9 Hz, α-H), 5.00 [d, 1H, J = 4.5 Hz, CH₂(Z)], 4.89 [d,

1H, J = 13 Hz, CH₂(Z)], 6.00 [t, 1H, J = 6 Hz, NHBn (Urea)], 5.89 (d, 1H, J = 9 Hz, 2-CHNH), 5.90

(m, 1H, 4-H), 6.46 (t, 1H, J = 5.5 Hz, NHZ), 7.07–7.35 (m, 21H, Ar and 1-H), 8.03 (t, 1H, J = 6 Hz,

NHBn), 8.14 [d, 1H, J = 8.5, α-NH); C-NMR (125 MHz, (CD₃)₂CO) δ (ppm): 27.8 [C_γ], 31.5 [C_β],

39.4 [CH₂-Ph], 40.9 [C₃], 41.6 [C_δ], 44.1 [CH₂(NHBn)], 44.8 [CH₂(NHBn, Urea)], 53.5 [C₂-CH],

54.1 [C_α], 55.9 [C₆], 59.6 [CH₂CO], 62.5 [C₂], 67.1 [CH₂(Z)], 127.6, 128.0, 128.3, 128.4, 128.8,

129.2, 129.3, 129.7, 129.8, 130.9 [20CH (Ar)], 139.2 [C (Z)], 140.5 [C (Ph)], 140.9 [C (NHBn)], 142.3

[C (NHBn, Urea)], 158.1 [CO (Z)], 159.8 [CO (Urea)], 170.7 [C₅], 171.5 [CO], 173.5 [α-CONH];

ES-MS m/z 748.4 [M+1]⁺; C₄₂H₄₉N₇O₆(%): C: 67.45, H: 6.60, N: 13.11. Found (%): C: 67.60, H:

6.85, N: 13.01.

N-[2-[(2R)-[(1S)-(3-Benzylureido)-2-phenylethyl]-5-oxo-piperazin-1-yl]acetyl]-Lys(Z)-NHBn [(R)-10b].

Amorphous solid (165 mg, 36%); α_D²⁰= −5.6 (c 0.8, MeOH); HPLC t_R: 21.05 min; H-NMR

(400 MHz, CDCl₃) δ (ppm): 1.25 (m, 2H, γ-H), 1.42 (m, 2H, δ-H), 1.59 (dt, 1H, J = 7.5 and 14 Hz,

β-H), 1.75 (m, 1H, β-H), 2.54 (dd, 1H, J = 11 and 13 Hz, CH₂-Ph), 2.86 (dd, 1H, J = 2.5 and 13 Hz,

CH₂-Ph), 3.01 (m, 1H, 2-H), 3.04 (m, 1H, ε-H), 3.10 (m, 1H, ε-H), 3.12 (m, 1H, 6-H), 3.16 (m, 1H,

3-H), 3.22 (m, 1H, CH₂CO), 3.28 (m, 1H, CH₂CO), 3.53 (d, 1H, J = 18 Hz, 6-H), 3.86 (m, 1H, 3-H),

3.94 (m, 1H, 2-CH), 4.24 [d, 2H, J = 5.5 Hz, CH₂(NHBn)], 4.27 [d, 1H, J = 4.5 Hz, CH₂(NHBn,

Urea)], 4.37 [d, 1H, J = 4.5 Hz, CH₂(NHBn, Urea)], 4.43 (m, 1H, α-H), 5.00 [d, 2H, J = 7 Hz, CH₂

(Z)], 5.24 (t, 1H, J = 5 Hz, NHZ), 5.66 (m, 1H, 2-CHNH), 5.77 (m, 1H, 4-H), 6.04 [m, 1H, (NHBn,

Urea)], 7.09–7.41 (m, 21H, Ar and NHBn), 7.97 (d, 1H, J = 8 Hz, α-NH); C-NMR (100 MHz,

CDCl₃) δ (ppm): 22.4 [C_γ], 29.2 [C_δ], 32.5 [C_β], 35.9 [C₃], 37.5 [CH₂-Ph], 40.4 [C_ε], 43.6 [CH₂

(NHBn)], 44.2 [CH₂(NHBn, Urea)], 51.7 [C₆], 52.4 [C₂-CH], 52.6 [C_α], 57.7 [CH₂CO], 58.5 [C₂], 66.6

[CH₂(Z)], 127.1, 127.5, 127.6, 127.7, 127.9, 128.1, 128.5, 128.7, 129.0, 129.2 [20CH (Ar)], 135.8 [C

(Ph)], 136.5 [C (Z)], 137.4 [C (NHBn)], 139.6 [C (NHBn, Urea)], 156.7 [CO (Z)], 158.3 [CO (Urea)],

168.6 [C₅], 169.9 [CO], 172.0 [α-CONH]; ES-MS m/z 763.2 [M+1]⁺; C₄₃H₅₁N₇O₆(%): C: 67.79, H:

6.75, N: 12.87. Found (%): C: 67.60, H: 7.01, N: 12.69.

N-[2-[(2S)-[(1S)-(3-Benzylureido)-2-phenylethyl]-5-oxo-piperazin-1-yl]acetyl]-Lys(Z)-NHBn [(S)-10b].

Amorphous solid (59 mg, 13%); α²_D⁰= −11.2 (c 0.9, MeOH); HPLC t_R: 20.47 min; H-NMR

(500 MHz, (CD₃)₂CO) δ (ppm): 1.30 (m, 2H, δ-H), 1.56 (m, 2H, γ-H), 1.68 (m, 1H, β-H), 1.83 (m, 1H,

β-H), 2.82 (m, 1H, CH₂-Ph), 3.02 (m, 1H, 2-H), 3.05 (m, 1H, CH₂-Ph), 3.18 (m, 3H, ε-H and 6-H),

3.37 (m, 2H, CH₂CO), 3.16 (m, 1H, 3-H), 3.40 (m, 1H, 6-H), 3.55 (m, 2H, 3-H), 4.15 [m, 1H, CH₂

(NHBn, Urea)], 4.25 [m, 1H, CH₂(NHBn, Urea)], 4.28 [m, 2H, CH₂(NHBn)], 4.40 (m, 1H, 2-CH),

4.51 (m, 1H, α-H), 5.00 [m, 2H, CH₂(Z)], 5.98 (d, 1H, J = 7 Hz, 2-CHNH), 6.04 [m, 1H, (NHBn,

Urea)], 6.45 (m, 1H, NHZ), 7.03 (m, 1H, 4-H), 7.08–7.41 (m, 20H, Ar), 8.04 (m, 1H, NHBn), 8.15

(d, 1H, J = 8.5 Hz, α-NH); C-NMR (125 MHz, (CD₃)₂CO) δ (ppm): 26.2 [C_γ], 29.7 [C_δ], 29.9 [C_β],

37.9 [CH₂-Ph], 39.2 [C₃], 39.9 [C_ε], 42.5 [CH₂(NHBn)], 43.2 [CH₂(NHBn, Urea)], 52.0 [C₂-CH],

52.6 [C_α], 54.4 [C₆], 57.6 [CH₂CO], 61.0 [C₂], 65.5 [CH₂(Z)], 126.1, 126.5, 126.6, 126.8, 127.3,

127.7, 127.8, 128.3, 128.4, 129.3, 129.6 [20CH (Ar)], 137.5 [C (Z)], 138.9 [C (Ph)], 139.4 [C

(NHBn)], 140.7 [C (NHBn, Urea)], 156.5 [CO (Z)], 158.3 [CO (Urea)], 169.0 [C₅], 169.7 [CO], 171.9

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[α-CONH]; ES-MS m/z 763.3 [M+1]⁺; C₄₃H₅₁N₇O₆(%): C: 67.79, H: 6.75, N: 12.87. Found (%): C:

67.96, H: 6.93, N: 12.70.

3.7. General Procedure for the N-Z Removal in 9a,b and 10a,b. Synthesis of the Hydrochlorides

(R)-(11a,b and 12a,b) and (S)-(11a,b and 12a,b)

Pd(C) (10%) and a 3.4 N solution of HCl in EtOAc (134 µL, 0.40 mmol) were added to a solution

of (R)-(9a,b and -10a,b) and (S)-(9a,b and 10a,b) (0.20 mmol) in MeOH (5 mL), and the mixture was

hydrogenated at 1 atm of H₂and room temperature for 1 h. Afterwards, the reaction mixture was

filtered through celite and the solvent was evaporated under reduced pressure. The residue was

dissolved in CH₃CN/H₂O (1:3, 2 mL) and the solution was lyophilized. (R)-(11a,b and 12a,b) and

(S)-(11a,b and 12a,b) were obtained quantitatively.

N-[2-[5-Oxo-(2R)-[2-phenyl-(1S)-(3-phenylureido)ethyl]piperazin-1-yl]acetyl]-Orn-NHBn hydrochloride

[(R)-11a]. Amorphous solid (127 mg, 100%); α²_D⁰= +1.7 (c 0.7, MeOH); HPLC t_R: 14.65 min;

¹H-NMR (500 MHz, DMSO-d₆) δ (ppm): 1.58 (m, 2H, γ-H), 1.63 (m, 1H, β-H), 1.80 (m, 1H, β-H),

2.62 (m, 1H, CH₂-Ph), 2.63 (m, 1H, 2-H), 2.73 (m, 1H, CH₂-Ph), 2.75 (m, 2H, δ-H), 3.05 (m, 1H, 3-H),

3.38 (m, 1H, 6-H), 3.40 (m, 2H, CH₂CO and 3-H), 3.50 (m, 1H, CH₂CO), 3.55 (m, 1H, 6-H), 4.05

(m, 1H, 2-CH), 4.24 [dd, 1H, J = 6 and 15 Hz, CH₂(NHBn)], 4.31 [dd, 1H, J = 6 and 15 Hz, CH₂

(NHBn)], 4.39 (dd, 1H, J = 5 and 8 Hz, α-H), 6.58 (m, 1H, 2-CHNH), 6.80–7.35 (m, 15H, Ar), 7.86

(m, 4H, 4-H and NH₂·HCl), ], 8.20 (m, 1H, α-NH), 8.65 (m, 1H, NHBn), 8.74 [m, 1H, NHPh];

¹³C-NMR (125 MHz, DMSO-d₆) δ (ppm): 23.5 [C_γ], 29.2 [C_β], 38.2 [C_δ], 42.0 [CH₂(NHBn)], 51.5

[C_α], 117.6, 121.1, 126.1, 126.8, 127.1, 128.1, 128.3, 128.6, 129.3 [15CH (Ar)], 139.1 [C (NHBn)],

140.2 [C (NHPh)], 155.1 [CO (Urea)], 171.0 [α-CONH]; ES-MS m/z [M]⁺calculated for C₃₃H₄₁N₇O₄:

600.2; found: 600.5.

N-[2-[5-Oxo-(2S)-[2-phenyl-(1S)-(3-phenylureido)ethyl]piperazin-1-yl]acetyl]-Orn-NHBn hydrochloride

[(S)-11a]. Amorphous solid (127 mg, 100%); α²_D⁰= −1.6 (c 1.1, MeOH); HPLC t_R: 15.07 min;

¹H-NMR (500 MHz, DMSO-d₆) δ (ppm): 1.48 (m, 2H, γ-H), 1.58 (m, 1H, β-H), 1.74 (m, 1H, β-H),

2.62 (m, 2H, δ-H), 2.79 (m, 1H, CH₂-Ph), 2.83 (m, 1H, 2-H), 2.89 (m, 1H, CH₂-Ph), 3.00 (m, 1H, 3-H),

3.35 (m, 1H, CH₂CO), 3.40 (m, 1H, 6-H), 3.42 (m, 1H, 3-H), 3.50 (m, 1H, CH₂CO), 3.65 (m, 1H, 6-H),

4.04 (m, 1H, 2-CH), 4.25 [d, 2H, J = 6, CH₂(NHBn)], 4.31 (dd, 1H, J = 5 and 8 Hz, α-H), 6.70 (m,

1H, 2-CHNH), 6.79–7.49 (m, 15H, Ar), 7.81 (m, 4H, 4-H and NH₂·HCl), ], 8.21 (m, 1H, α-NH), 8.65

(t, 1H, J = 6 Hz, NHBn), 8.92 [m, 1H, NHPh]; C-NMR (125 MHz, DMSO-d₆) δ (ppm): 23.4 [C_γ],

28.9 [C_β], 38.1 [C_δ], 42.0 [CH₂(NHBn)], 51.5 [C_α], 117.6, 121.1, 126.4, 126.7, 127.0, 128.2, 128.4,

128.6, 129.0 [15CH (Ar)], 137.0 [C (Ph)], 139.1 [C (NHBn)], 140.3 [C (NHPh)], 155.3 [CO (Urea)],

170.9 [α-CONH]; ES-MS m/z [M]⁺calculated for C₃₃H₄₁N₇O₄: 600.2; found: 600.5.

N-[2-[5-Oxo-(2R)-[2-phenyl-(1S)-(3-phenylureido)ethyl]piperazin-1-yl]acetyl]-Lys-NHBn hydrochloride

[(R)-11b]. Amorphous solid (130 mg, 100%); α²_D⁰= −3.0 (c 1.7, MeOH); HPLC t_R: 14.97 min;

¹H-NMR (500 MHz, DMSO-d₆) δ (ppm): 1.27 (m, 2H, γ-H), 1.52 (m, 2H, δ-H), 1.58 (m, 1H, β-H),

1.70 (m, 1H, β-H), 2.61 (m, 1H, CH₂-Ph), 2.72 (m, 1H, ε-H), 2.84 (m, 1H, 2-H), 2.92 (m, 1H, ε-H),

2.95 (m, 1H, CH₂-Ph), 3.00 (m, 2H, 3-H), 3.38 (m, 1H, 3-H), 3.40 (m, 1H, CH₂CO), 3.42 (m, 1H,

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6-H), 3.50 (m, 1H, CH₂CO), 3.54 (m, 1H, 3-H), 4.22 [m, 1H, CH₂(NHBn)], 4.23 (m, 1H, 2-CH), 4.31

[m, 1H, CH₂(NHBn)], 4.32 (m, 1H, α-H), 6.80–7.37 (m, 16H, Ar and 2-CHNH), 7.88 (m, 4H,

NH₂·HCl and 4-H), 8.17 (m, 1H, α-NH), 8.61 (t, 1H, J = 6 Hz, NHBn), 8.90 (m, 1H, NHPh); ¹³C-NMR

(125 MHz, DMSO-d₆) δ (ppm): 22.2 [C_γ], 26.5 [C_δ], 30.7 [C_β], 38.4 [C_ε], 42.0 [CH₂(NHBn)], 52.5

[C_α], 117.7, 121.3, 126.3, 126.7, 127.0, 128.2, 128.3, 128.5, 129.2 [15CH (Ar)], 138.1 [C (Ph)], 139.2

[C (NHBn)], 140.0 [C (NHPh)], 155.3 [CO (Urea)], 171.3 [α-CONH]; ES-MS m/z [M]⁺calculated for

C₃₄H₄₃N₇O₄: 614.2; found: 614.5.

N-[2-[5-Oxo-(2S)-[2-phenyl-(1S)-(3-phenylureido)ethyl]piperazin-1-yl]acetyl]-Lys-NHBn hydrochloride

[(S)-11b]. Amorphous solid (130 mg, 100%); α²_D⁰= −4.2 (c 0.4, MeOH); HPLC t_R: 15.21 min;

¹H-NMR (500 MHz, DMSO-d₆) δ (ppm): 1.29 (m, 2H, γ-H), 1.50 (m, 2H, δ-H), 1.55 (m, 1H, β-H),

1.73 (m, 1H, β-H), 2.72 (m, 1H, CH₂-Ph), 2.72 (m, 1H, ε-H), 2.74 (m, 2H, 3-H), 2.80 (m, 1H, ε-H),

2.89 (m, 1H, CH₂-Ph), 2.90 (m, 1H, 2-H), 3.13 (m, 1H, CH₂CO), 3.29 (m, 1H, 3-H), 3.33 (m, 1H,

6-H), 3.42 (m, 1H, CH₂CO), 3.63 (m, 1H, 6-H), 4.10 (m, 1H, 2-CH), 4.18 [m, 1H, CH₂(NHBn)], 4.20

(m, 1H, α-H), 4.35 [m, 1H, CH₂(NHBn)], 6.55 (m, 1H, 2-CHNH), 6.80–7.40 (m, 16H, Ar), 7.69 (m, 4H,

NH₂·HCl and 4-H), 7.92 (d, 1H, J = 9 Hz, α-NH), 8.56 (m, 1H, NHBn), 8.70 (m, 1H, NHPh); ¹³C-NMR

(125 MHz, DMSO-d₆) δ (ppm): 22.1 [C_γ], 26.8 [C_δ], 30.7 [C_β], 38.5 [C_ε], 42.0 [CH₂(NHBn)], 51.6

[C_α], 116.5, 118.0, 127.0, 127.2, 127.5, 128.7, 128.9, 129.1, 129.5 [15CH (Ar)], 138.2 [C (Ph)], 139.4

[C (NHBn)], 140.0 [C (NHPh)], 171.2 [α-CONH]; ES-MS m/z [M]⁺calculated for C₃₄H₄₃N₇O₄: 614.2;

found: 614.5.

N-[2-[(2R)-[(1S)-(3-Benzylureido)-2-phenylethyl]-5-oxo-piperazin-1-yl]acetyl]-Orn-NHBn hydrochloride

[(R)-12a]. Amorphous solid (130 mg, 100%); α²_D⁰= −7.9 (c 1.3, MeOH); HPLC t_R: 14.67 min;

¹H-NMR (500 MHz, DMSO-d₆) δ (ppm): 1.56 (m, 3H, γ-H and β-H), 1.78 (m, 1H, β-H), 2.60–3.16

(m, 6H, CH₂-Ph, 2-H, δ-H and 3-H), 3.20–3.86 (m, 5H, 3-H, 6-H and CH₂CO), 4.04 (m, 1H, 2-CH),

4.20 [m, 2H, CH₂(NHBn, Urea)], 4.26 [d, 2H, J = 6 Hz, CH₂(NHBn)], 4.34 (dd, 1H, J = 5 and 8 Hz,

α-H), 6.53 (m, 1H, 2-CHNH), 6.74 [m, 1H, NHBn (Urea)], 7.18–7.33 (m, 15H, Ar), 7.92 (m, 4H,

NH₂·HCl and 4-H), 8.34 [m, 1H, α-NH), 8.70 (t, 1H, J = 6 Hz, NHBn); ¹³C-NMR (125 MHz,

DMSO-d₆) δ (ppm): 23.8 [C_γ], 29.5 [C_β], 39.5 [C_δ], 42.5 [CH₂(NHBn)], 43.4 [CH₂(NHBn, Urea)],

52.0 [C_α], 126.9, 127.0, 127.2, 127.4, 127.5, 128.6, 128.7, 128.9, 129.3 [15CH (Ar)], 137.5 [C (Ph)],

139.6 [C (NHBn)], 141.0 [C (NHBn, Urea)], 158.7 [CO (Urea)], 171.4 [α-CONH]; ES-MS m/z [M]⁺

calculated for C₃₄H₄₃N₇O₄: 614.2; found: 614.5.

N-[2-[(2S)-[(1S)-(3-Benzylureido)-2-phenylethyl]-5-oxo-piperazin-1-yl]acetyl]-Orn-NHBn hydrochloride

[(S)-12a]. Amorphous solid (130 mg, 100%); α²_D⁰= −3.2 (c 1.2, MeOH); HPLC t_R: 15.01 min;

¹H-NMR (500 MHz, DMSO-d₆) δ (ppm): 1.58 (m, 3H, γ-H), 1.60 (m, 1H, β-H) 1.88 (m, 1H, β-H),

2.52–3.97 (m, 6H, CH₂-Ph, 2-H, δ-H and 3-H), 3.24–3.69 (m, 5H, 3-H, 6-H and CH₂CO), 4.03 (m, 1H,

2-CH), 4.05 [m, 1H, CH₂(NHBn, Urea)], 4.20 [m, 1H, CH₂(NHBn, Urea)], 4.25 [m, 2H, CH₂

(NHBn)], 4.39 (m, 1H, α-H), 6.48 (m, 1H, 2-CHNH), 6.96–7.36 [m, 16H, Ar and NHBn (Urea)], 7.90

(m, 4H, NH₂·HCl and 4-H), 8.30 [m, 1H, α-NH), 8.70 (m, 1H, NHBn); ¹³C-NMR (125 MHz,

DMSO-d₆) δ (ppm): 23.9 [C_γ], 29.6 [C_β], 38.7 [C_δ], 42.5 [CH₂(NHBn)], 43.1 [CH₂(NHBn, Urea)],

52.1 [C_α], 126.5, 126.8, 127.0, 127.1, 127.5, 128.5, 128.7, 129.7 [15CH (Ar)], 139.6 [C (NHBn)],

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141.0 [C (NHBn, Urea)], 158.5 [CO (Urea)], 171.3 [α-CONH]; ES-MS m/z [M]⁺calculated for

C₃₄H₄₃N₇O₄: 614.2; found: 614.5.

N-[2-[(2R)-[(1S)-(3-Benzylureido)-2-phenylethyl]-5-oxo-piperazin-1-yl]acetyl]-Lys-NHBn hydrochloride

[(R)-12b]. Amorphous solid (133 mg, 100%); α²_D⁰= −4.3 (c 0.6, MeOH); HPLC t_R: 14.80 min;

¹H-NMR (500 MHz, DMSO-d₆) δ (ppm): 1.21 (m, 2H, γ-H), 1.49 (m, 2H, δ-H), 1.57 (m, 1H, β-H),

1.69 (m, 1H, β-H), 2.68 (m, 2H, ε-H), 2.75 (m, 1H, CH₂-Ph), 2.80 (m, 1H, 2-H), 2.82 (m, 1H, CH₂-Ph),

3.02 (m, 1H, 3-H), 3.25 (m, 1H, CH₂CO), 3.37 (m, 1H, 6-H), 3.40 (m, 2H, CH₂CO and 3-H), 3.60

(m, 1H, 6-H), 3.98 (m, 1H, 2-CH), 4.20 [m, 2H, CH₂(NHBn, Urea)], 4.24 (m, 1H, α-H), 4.25 [m, 2H,

CH₂(NHBn)], 6.35 (m, 1H, 2-CHNH), 6.59 [m, 1H, (NHBn, Urea)], 7.08–7.35 (m, 15H, Ar), 7.82

(m, 4H, NH₂·HCl and 4-H), 8.08 (m, 1H, α-NH), 8.57 (t, 1H, J = 6 Hz, NHBn); ¹³C-NMR (125 MHz,

DMSO-d₆) δ (ppm): 22.1 [C_γ], 26.5 [C_δ], 31.4 [C_β], 38.5 [C_ε], 42.0 [CH₂(NHBn)], 42.9 [CH₂(NHBn,

Urea)], 52.0 [C_α], 126.4, 126.5, 126.7, 126.9, 127.0, 128.2, 128.4, 129.1 [15CH (Ar)], 137.3 [C (Ph)],

139.3 [C (NHBn)], 140.7 [C (NHBn, Urea)], 158.0 [CO (Urea)], 171.2 [α-CONH]; ES-MS m/z [M]⁺

calculated for C₃₅H₄₅N₇O₄: 628.2; found: 628.5.

N-[2-[(2S)-[(1S)-(3-Benzylureido)-2-phenylethyl]-5-oxo-piperazin-1-yl]acetyl]-Lys-NHBn hydrochloride

[(S)-12b]. Amorphous solid (133 mg, 100%); α²_D⁰= −1.6 (c 0.6, MeOH); HPLC t_R: 15.34 min;

¹H-NMR (500 MHz, DMSO-d₆) δ (ppm): 1.28 (m, 2H, γ-H), 1.47 (m, 1H, β-H), 1.50 (m, 2H, δ-H),

1.70 (m, 1H, β-H), 2.57–3.01 (m, 7H, CH₂-Ph, ε-H, 2-H and 3-H), 3.38 (m, 1H, 6-H), 3.42 (m, 1H,

CH₂CO), 3.53 (m, 1H, CH₂CO), 3.62 (m, 1H, 6-H), 4.00 [m, 1H, CH₂(NHBn, Urea)], 4.17 [m, 1H,

CH₂(NHBn, Urea)], 4.20 (m, 1H, 2-CH), 4.26 [m, 2H, CH₂(NHBn)], 4.30 (m, 1H, α-H), 6.45 (m, 1H,

2-CHNH), 6.96–7.34 [m, 16H, Ar and (NHBn, Urea)], 7.84 (m, 4H, NH₂·HCl and 4-H), 8.05 (m, 1H,

α-NH), 8.60 (m, 1H, NHBn); ¹³C-NMR (125 MHz, DMSO-d₆) δ (ppm): 22.2 [C_γ], 26.5 [C_δ], 31.4 [C_β],

38.5 [C_ε], 42.0 [CH₂(NHBn)], 42.6 [CH₂(NHBn, Urea)], 52.5 [C_α], 126.4, 126.6, 126.7, 127.0, 128.1,

128.2, 129.3 [15CH (Ar)], 137.4 [C (Ph)], 139.6 [C (NHBn)], 141.0 [C (NHBn, Urea)], 159.0

[CO (Urea)], 171.5 [α-CONH]; ES-MS m/z [M]⁺calculated for C₃₅H₄₅N₇O₄: 628.2; found: 628.5.

3.8. Synthesis of 2-[4-Benzyl-(2RS)-[(1S)-((tert-butoxycarbonyl)amino)-2-phenylethyl]-5-

oxopiperazin-1-yl]acetic Acid (14)

This compound was obtained from the benzyl ester 13 [23] by applying the general procedure of

benzyl ester hydrogenolysis above indicated for the synthesis of 4. Foam (467.6 mg, 100%); HPLC t_R:

19.72 min [(R)-14] and 19.00 min [(S)-14]; ¹H-NMR (500 MHz, CDCl₃). (R)-14 δ (ppm): 1.35 (s, 9H,

Boc), 2.73 (m, 2H, CH₂-Ph), 2.87 (m, 1H, 2-H), 3.23 (d, 1H, J = 7.5 and 13 Hz, 3-H), 3.33 (d, 1H,

J = 5 and 13 Hz, 3-H), 3.38 (d, 1H, J = 17 Hz, CH₂CO₂H), 3.49 (d, 2H, J = 17 Hz, 6-H and

CH₂CO₂H), 3.62 (d, 1H, J = 17 Hz, 6-H), 4.00 (m, 1H, 2-CH), 4.30 (d, 1H, J = 9 Hz, NHBoc), 4.52

[d, 1H, J = 14.5 Hz, 4-CH₂(Bn)], 4.68 [d, 1H, J = 14.5 Hz, 4-CH₂(Bn)], 6.93–7.40 (m, 10H, Ar).

(S)-14 δ (ppm): 1.35 (s, 9H, Boc), 2.73 (m, 2H, CH₂-Ph), 2.97 (m, 1H, 2-H), 3.17 (m, 1H, 3-H), 3.33

(m, 1H, 3-H), 3.23 (m, 1H, CH₂CO₂H), 3.38 (m, 1H, 3-H), 3.49 (m, 1H, CH₂CO₂H), 3.63 (d, 1H,

J = 17.5 Hz, 6-H), 3.82 (m, 1H, 2-CH), 4.30 (d, 1H, J = 9 Hz, NHBoc), 4.57 [m, 1H, 4-CH₂(Bn)], 4.82

[m, 1H, 4-CH₂(Bn)], 6.93–7.40 (m, 10H, Ar).¹³C-NMR (125 MHz, DMSO-d₆). (R)-14 δ (ppm): 28.2

[3CH₃(Boc)], 37.5 [CH₂-Ph], 44.1 [C₃], 49.7 [4-CH₂(Bn)], 51.6 [C₂-CH], 54.1 [C₆], 54.4 [CH₂CO₂H],

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58.7 [C₂], 80.2 [C (Boc)], 126.7, 127.9, 128.6, 128.9 [10CH (Ar)], 136.2 [C (Bn)], 136.8 [C (Ph)],

155.7 [CO (Boc)], 167.8 [C₅], 172.2 [CO₂]. (S)-14 δ (ppm): 28.2 [3CH₃(Boc)], 37.5 [CH₂-Ph], 44.1

[C₃], 49.6 [4-CH₂(Bn)], 51.6 [C₂-CH], 54.1 [C₆], 54.4 [CH₂CO₂H], 58.7 [C₂], 80.2 [C (Boc)], 128.0,

128.3, 128.4, 129.1 [10CH (Ar)], 136.2 [C (Bn)], 136.8 [C (Ph)], 155.7 [CO (Boc)], 167.8 [C₅], 172.2

[CO₂]; ES-MS m/z 468.2 [M+1]⁺; C₂₆H₃₃N₃O₅(%): C: 66.79, H: 7.11, N: 8.99. Found (%): C: 66.58,

H: 7.25, N: 9.14.

3.9. General Procedure for the Synthesis of the Piperazinone-Derived Pseudotripeptides 15a–c

These compounds were prepared by applying the general procedure described for the synthesis of

7a,b.

N-[2-[4-Benzyl-(2RS)-[(1S)-((tert-butoxycarbonyl)-amino)-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-

Orn(Z)-NHBn (15a). Foam (523 mg, 65%); HPLC t_R: 25.24 min; ¹H-NMR (500 MHz, CDCl₃) (R)-15a

δ (ppm): 1.34 (s, 9H, Boc), 1.43 (m, 2H, γ-H), 1.67 (m, 1H, β-H), 1.86 (m, 1H, β-H), 2.76 (m, 1H,

CH₂-Ph), 2.83 (m, 1H, CH₂-Ph), 2.86 (m, 1H, 2-H), 3.08 (m, 1H, 3-H), 3.23 (m, 2H, δ-H), 3.30 (m,

1H, 3-H), 3.35 (m, 1H, 6-H), 3.44 (m, 2H, CH₂CO), 3.55 (d, 1H, J = 18 Hz, 6-H), 4.00 (m, 1H, 2-CH),

4.34 [dd, 1H, J = 6 and 15 Hz, CH₂(NHBn)], 4.42 (m, 1H, α-H), 4.39 [m, 1H, CH₂(NBn)], 4.44

[m, 1H, CH₂(NHBn)], 4.70 (m, 1H, NHBoc), 4.75 [m, 1H, CH₂(NBn)], 4.86 [m, 2H, CH₂(Z)], 5.06

(m, 1H, NHZ), 6.70 (m, 1H, NHBn), 7.02–7.46 (m, 20H, Ar), 7.74 (m, 1H, α-NH). (S)-15a δ (ppm):

1.34 (s, 9H, Boc), 1.67 (m, 1H, β-H), 1.86 (m, 1H, β-H), 2.56 (m, 1H, CH₂-Ph), 2.74 (m, 1H, CH₂-Ph),

3.08 (m, 1H, 3-H), 3.23 (m, 2H, δ-H), 3.30 (m, 1H, 3-H), 3.85 (m, 1H, 2-CH), 4.34 [m, 1H, CH₂

(NHBn)], 4.42 (m, 1H, α-H), 4.44 [m, 1H, CH₂(NHBn)], 4.44 [m, 1H, CH₂(NHBn)], 4.50 [m, 1H,

CH₂(NBn)], 4.64 (m, 1H, NHBoc), 4.80 [m, 1H, CH₂(NBn)], 4.86 [m, 2H, CH₂(Z)], 5.06 (m, 1H,

NHZ), 6.70 (m, 1H, NHBn), 7.02–7.46 (m, 20H, Ar), 7.74 (m, 1H, α-NH); C-NMR (125 MHz,

CDCl₃) (R)-15a δ (ppm): 26.7 [C_γ], 28.4 [3CH₃(Boc)], 31.1 [C_β], 37.7 [CH₂-Ph], 39.5 [C_δ], 43.7 [C₃

and CH₂(NHBn)], 50.1 [CH₂(NBn)], 51.4 [C₂-CH], 51.7 [C_α], 54.6 [C₆], 56.3 [CH₂CO], 59.5 [C₂],

66.8 [CH₂(Z)], 80.1 [C (Boc)], 126.9, 127.9, 128.0, 128.3, 128.5, 128.8, 129.0, 129.3 [20CH (Ar)],

136.5 [C (Ph) and C (NBn)], 136.9 [C (Z)], 138.1 [C (NHBn)], 155.6 [CO (Boc)], 157.3 [CO (Z)],

166.9 [C₅], 169.6 [CO], 171.5 [α-CONH]. (S)-15a δ (ppm): 28.4 [3CH₃(Boc)], 30.5 [C_β], 37.7

[CH₂-Ph], 39.6 [C_δ], 43.7 [C₃and CH₂(NHBn)], 49.8 [CH₂(NBn)], 51.4 [C₂-CH], 51.7 [C_α], 66.8

[CH₂(Z)], 80.1 [C (Boc)], 126.8, 127.8, 128.0, 128.6, 128.8, 129.1, 129.3 [20CH (Ar)], 136.5 [C (Ph)

and C (NBn)], 136.9 [C (Z)], 138.1 [C (NHBn)], 155.6 [CO (Boc)], 157.3 [CO (Z)], 166.9 [C₂], 169.6

[CO], 171.5 [α-CONH]; ES-MS m/z 806.6 [M+1]⁺; C₄₆H₅₆N₆O₇(%): C: 68.63, H: 7.01, N: 10.44.

Found (%): C: 68.50, H: 7.19, N: 10.62.

N-[2-[4-Benzyl-(2RS)-[(1S)-((tert-butoxycarbonyl)-amino)-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-

Lys(Z)-NHBn (15b). Foam (639 mg, 78%); HPLC t_R: 25.42 min; ¹H-NMR (500 MHz, CDCl₃) (R)-15b

δ (ppm): 1.31 (m, 11H, Boc and γ-H), 1.47 (m, 2H, δ-H), 1.65 (m, 1H, β-H), 1.86 (m, 1H, β-H), 2.69

(dd, 1H, J = 8 and 13 Hz, CH₂-Ph), 2.78 (m, 1H, 2-H), 2.82 (m, 1H, CH₂-Ph), 3.12 (m, 2H, ε-H), 3.19

(m, 1H, 6-H), 3.20 (m, 1H, CH₂CO), 3.25 (m, 1H, 3-H), 3.35 (m, 1H, 6-H), 3.37 (m, 1H, CH₂CO),

3.50 (d, 1H, J = 17 Hz, 3-H), 3.95 (m, 1H, 2-CH), 4.29 (d, 1H, J = 9 Hz, NHBoc), 4.39 [m, 1H, CH₂

(NBn], 4.42 [m, 1H, CH₂(NHBn)], 4.44 (m, 1H, α-H), 4.45[m, 1H, CH₂(NHBn)], 4.75 [d, 1H,

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J = 14.5 Hz, CH₂(NBn)], 5.04 [m, 3H, CH₂(Z) and NHZ], 6.74 (m, 1H, NHBn), 7.01–7.38 (m, 20H,

Ar), 7.67 (d, 1H, J = 8 Hz, α-NH). (S)-15b δ (ppm): 1.31 (m, 9H, Boc), 1.65 (m, 1H, β-H), 1.86

(m, 1H, β-H), 3.12 (m, 1H, CH₂CO), 3.38 (m, 1H, 6-H), 3.39 (m, 1H, CH₂CO), 3.54 (m, 1H, 6-H),

3.82 (m, 1H, 2-CH), 4.33 (d, 1H, J = 9 Hz, NHBoc), 4.42 [m, 1H, CH₂(NHBn)], 4.43 (m, 1H, α-H),

4.45[m, 1H, CH₂(NHBn)], 4.72 [m, 1H, CH₂(NBn], 4.79 [m, 1H, CH₂(NBn], 5.04 [m, 3H, CH₂(Z)

and NHZ], 6.96 (m, 1H, NHBn), 7.01–7.38 (m, 20H, Ar), 7.55 (m, 1H, α-NH); C-NMR (125 MHz,

CDCl₃) (R)-15b δ (ppm): 22.7 [C_γ], 28.2 [3CH₃(Boc)], 29.4 [C_δ], 31.9 [C_β], 37.7 [CH₂-Ph], 40.5 [C_ε],

43.6 [C₃and CH₂(NHBn)], 49.8 [CH₂(NBn)], 51.7 [C₂-CH], 52.7 [C_α], 54.4 [C₆], 56.1 [CH₂CO], 59.4

[C₂], 66.5 [CH₂(Z)], 79.9 [C (Boc)], 127.0, 127.7, 127.8, 128.0, 128.2, 128.5, 128.6, 128.8, 128.9,

129.0, 129.3 [20CH (Ar)], 136.2 [C (NBn)], 136.6 [C (Ph)], 136.7 [C (Z)], 138.0 [C (NHBn)], 155.4

[CO (Boc)], 156.4 [CO (Z)], 166.9 [C₅], 169.6 [CO], 171.2 [α-CONH]. (S)-15b δ (ppm): 28.2 [3CH₃

(Boc)], 31.6 [C_β], 43.6 [C₃and CH₂(NHBn)], 49.6 [CH₂(NBn)], 51.7 [C₂-CH], 54.4 [C₆], 56.1

[CH₂CO], 66.5 [CH₂(Z)], 79.9 [C (Boc)], 126.8, 127.7, 127.8, 128.1, 128.2, 128.5, 128.7, 128.8,

128.9, 129.0, 129.2 [20CH (Ar)], 136.2 [C (NBn)], 136.6 [C (Ph)], 136.7 [C (Z)], 138.0 [C (NHBn)],

155.4 [CO (Boc)], 156.4 [CO (Z)], 169.6 [CO], 171.2 [α-CONH]; ES-MS m/z 819.7 [M+1]⁺;

C₄₇H₅₈N₆O₇(%): C: 68.93, H: 7.14, N: 10.26. Found (%): C: 68.67, H: 7.36, N: 10.20.

N-[2-[4-Benzyl-(2RS)-[(1S)-((tert-butoxycarbonyl)-amino)-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-

Arg(Pbf)-NHBn (15c). Foam (705 mg, 73%); HPLC t_R: 26.88 min; H-NMR (500 MHz, CDCl₃)

(R)-15c δ (ppm): 1.28 (s, 9H, Boc), 1.45 [s, 6H, 2CH₃(Pbf)], 1.53 (m, 2H, γ-H), 1.67 (m, 1H, β-H),

1.90 (m, 1H, β-H), 2.07 [s, 3H, CH₃(Pbf)], 2.48 [s, 3H, CH₃(Pbf)], 2.55 [s, 3H, CH₃(Pbf)], 2.73

(d, 1H, J = 8.5 and 13.5 Hz, CH₂-Ph), 2.82 (m, 1H, 5-H), 2.84 (m, 1H, CH₂-Ph), 2.93 [m, 2H, CH₂

(Pbf)], 3.24 (m, 5H, 3-H, CH₂CO and δ-H), 3.30 (m, 1H, 3-H), 3.32 (m, 1H, 6-H), 3.49 (d, 1H,

J = 16.5 Hz, 6-H), 3.95 (m, 1H, 2-CH), 4.31 [dd, 1H, J = 6 and 15 Hz, CH₂(NHBn)], 4.38 [d, 1H,

J = 14.5 Hz, CH₂(NBn)], 4.41 [dd, 1H, J = 5.5 and 15 Hz, CH₂(NHBn)], 4.50 (d, 1H, J = 9 Hz,

NHBoc), 4.57 (dt, 1H, J = 4.5 and 9 Hz, α-H), 4.76 [d, 1H, J = 14.5 Hz, CH₂(NBn)], 6.41 [m, 3H,

NHC(NH₂) = N], 6.81–7.24 (m, 15H, Ar), 7.60 (m, 1H, NHBn), 7.74 (d, 1H, J = 8 Hz, α-NH). (S)-15c

δ (ppm): 1.28 (s, 9H, Boc), 1.45 [s, 6H, 2CH₃(Pbf)], 1.67 (m, 1H, β-H), 1.90 (m, 1H, β-H), 2.07 [s,

3H, CH₃(Pbf)], 2.48 [s, 3H, CH₃(Pbf)], 2.55 [s, 3H, CH₃(Pbf)], 2.73 (m, 1H, CH₂-Ph), 2.84 (m, 1H,

CH₂-Ph), 2.93 [m, 2H, CH₂(Pbf)], 3.20 (m, 1H, 3-H), 3.35 (m, 1H, 3-H), 3.83 (m, 1H, 2-CH), 4.31

[m, 1H, CH₂(NBn)], 4.41 [m, 1H, CH₂(NBn)], 4.50 (d, 1H, J = 9 Hz, NHBoc), 4.57 (m, 1H, α-H),

4.70 [m, 1H, CH₂(NBn)], 4.86 [m, 1H, CH₂(NBn)], 6.41 [m, 3H, NHC(NH₂) = N], 6.81–7.24 (m,

15H, Ar), 7.62 (m, 1H, NHBn), 7.83 (d, 1H, J = 8 Hz, α-NH); ¹³C-NMR (125 MHz, CDCl₃) (R)-15c δ

(ppm): 12.4, 18.0, 19.3 [3CH₃(Pbf)], 25.4 [C_γ], 28.2 [3CH₃(Boc)], 28.6 [2CH₃(Pbf)], 31.0 [C_β], 37.6

[CH₂-Ph], 40.4 [C_δ], 43.2 [CH₂(Pbf)], 43.4 [CH₂(NHBn)], 44.0 [C₃], 49.8 [CH₂(NBn)], 51.7 [C₂-CH],

52.2 [C_α], 54.4 [C₆], 56.5 [CH₂CO], 59.4 [C₂], 79.9 [C (Boc)], 86.4, 117.5, 124.6 [3C (Pbf)], 126.7,

127.2, 127.7, 127.9, 128.3, 128.5, 128.9, 129.1 [15CH (Ar)], 132.3 [2C (Pbf)], 136.1 [C (NBn)], 136.9

[C (Ph)], 138.2 [C (NHBn)], 138.4 [C (Pbf)], 155.5 [CO (Boc)], 156.3 [C (NHC(NH₂) = N)], 158.8 [C

(Pbf)], 167.3 [C₅], 169.9 [CO], 171.3 [α-CONH]. (S)-15c δ (ppm): 12.4, 18.0, 19.3 [3CH₃(Pbf)], 28.2

[3CH₃(Boc)], 28.6 [2CH₃(Pbf)], 30.9 [C_β], 37.6 [CH₂-Ph], 43.2 [CH₂(Pbf)], 43.3 [CH₂(NHBn)],

44.0 [C₃], 49.6 [CH₂(NBn)], 51.7 [C₂-CH], 52.2 [C_α], 79.9 [C (Boc)], 86.4, 117.5, 124.6 [3C (Pbf)],

126.7, 127.2, 127.6, 127.9, 128.3, 128.6, 128.9, 129.1 [15CH (Ar)], 132.3 [2C (Pbf)], 136.1 [C (NBn)],

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136.9 [C (Ph)], 138.2 [C (NHBn)], 138.4 [C (Pbf)], 155.5 [CO (Boc)], 156.3 [C (NHC(NH₂) = N)],

158.8 [C (Pbf)], 171.3 [α-CONH]; ES-MS m/z 966.8 [M+1]⁺; C₅₂H₆₈N₈O₈S (%): C: 64.71, H: 7.10, N:

11.61. Found (%): C: 64.58, H: 7.26, N: 11.81.

3.10. Synthesis of the Hydrochlorides 16a–c

These compounds were obtained by applying the above indicated method of N-Boc removal.

N-[2-[4-Benzyl-(2RS)-[(1S)-amino-2-phenylethyl]-5-oxo-piperazin-1-yl]acetyl]-Orn(Z)-NHBn

hydrochloride (16a). Amorphous solid (445 mg, 100%); HPLC t_R: 16.76 min; H-NMR (500 MHz,

DMSO-d₆) (R)-16a δ (ppm): 1.40 (m, 1H, γ-H), 1.47 (m, 1H, γ-H), 1.57 (m, 1H, β-H), 1.70 (m, 1H,

β-H), 2.85 (m, 1H, CH₂-Ph), 2.90 (m, 1H, CH₂-Ph), 2.98 (m, 1H, 2-H and δ-H), 3.21 (d, 1H, J = 17

Hz, 6-H), 3.30 (m, 2H, CH₂CO), 3.46 (m, 2H, 3-H), 3.56 (d, 1H, J = 17 Hz, 6-H), 3.70 (m, 1H, 2-CH),

4.28 [m, 2H, CH₂(NHBn)], 4.30 (m, 1H, α-H), 4.45 [d, 1H, J = 15 Hz, CH₂(NBn)], 4.62 [d, 1H,

J = 15 Hz, CH₂(NBn)], 4.97 [m, 2H, CH₂(Z)], 7.11–7.37 (m, 21H, Ar and NHZ), 8.11 (m, 3H,

NH₂·HCl), 8.19 (d, 1H, J = 8 Hz, α-NH), 8.54 (m, 1H, NHBn). (S)-16a δ (ppm): 1.40 (m, 1H, γ-H),

1.47 (m, 1H, γ-H), 1.57 (m, 1H, β-H), 1.70 (m, 1H, β-H), 3.15 (m, 1H, 2-H), 3.45 (m, 1H, 6-H), 3.46

(m, 2H, 3-H), 3.56 (m, 1H, 6-H), 3.60 (m, 1H, 2-CH), 4.28 [m, 3H, CH₂(NHBn) and CH₂(NBn)],

4.30 (m, 1H, α-H), 4.38 [d, 1H, J = 15 Hz, CH₂(NBn)], 4.97 [m, 2H, CH₂(Z)], 7.11–7.37 (m, 21H, Ar

and NHZ), 8.11 (m, 3H, NH₂·HCl), 8.43 (d, 1H, J = 8 Hz, α-NH), 8.52 (m, 1H, NHBn); C-NMR

(125 MHz, DMSO-d₆) (R)-16a δ (ppm): 26.0 [C_γ], 29.5 [C_β], 34.6 [CH₂-Ph], 40.5 [C_δ], 42.0 [CH₂

(NHBn)], 43.5 [C₃], 48.8 [CH₂(NBn)], 51.7 [C₂-CH], 52.3 [C_α], 54.7 [C₆], 56.1 [CH₂CO], 57.3 [C₂],

65.1 [CH₂(Z)], 126.8, 127.0, 127.7, 127.8, 128.3, 127.4, 128.6, 129.2 [20CH (Ar)], 135.8 [C (Ph)],

137.0 [C (NBn)], 137.2 [C (Z)], 139.2 [C (NHBn)], 156.1 [CO (Z)], 167.1 [C₅], 169.4 [CO], 171.4

[α-CONH]. (S)-16a δ (ppm): 26.2 [C_γ], 29.3 [C_β], 42.0 [CH₂(NHBn)], 43.4 [C₃], 48.9 [CH₂(NBn)],

51.3 [C₂-CH], 52.5 [C_α], 54.7 [C₆], 56.1 [CH₂CO], 58.6 [C₂], 65.1 [CH₂(Z)], 126.9, 127.4, 127.7,

127.8, 128.3, 127.4, 128.6, 129.3 [20CH (Ar)], 135.9 [C (Ph)], 136.9 [C (NBn)], 137.2 [C (Z)], 139.2

[C (NHBn)], 156.1 [CO (Z)], 167.1 [C₅], 169.4 [CO], 171.4 [α-CONH]; ES-MS m/z [M+1]⁺calculated

for C₄₁H₄₈N₆O₅: 706.3; found: 706.5.

N-[2-[4-Benzyl-(2RS)-[(1S)-amino-2-phenylethyl]-5-oxo-piperazin-1-yl]acetyl]-Lys(Z)-NHBn

hydrochloride (16b). Amorphous solid (453 mg, 100%); HPLC t_R: 16.93 min; H-NMR (500 MHz,

DMSO-d₆) (R)-16b δ (ppm): 1.22 (m, 1H, γ-H), 1.27 (m, 1H, γ-H), 1.41 (m, 2H, δ-H), 1.58 (m, 1H,

β-H), 1.70 (m, 1H, β-H), 2.83 (dd, 1H, J = 8 and 14 Hz, CH₂-Ph), 2.94 (m, 2H, ε-H), 2.96 (m, 2H, 2-H

and CH₂-Ph), 3.20 (d, 1H, J = 17 Hz, 6-H), 3.24 (d, 1H, J = 16.5 Hz, CH₂CO), 3.29 (d, 1H, J = 16.5

Hz, CH₂CO), 3.38 (m, 1H, 3-H), 3.46 (m, 1H, 3-H), 3.53 (d, 1H, J = 17 Hz, 6-H), 3.72 (m, 1H, 2-CH),

4.26 [m, 2H, CH₂(NHBn)], 4.28 (m, 1H, α-H), 4.45 [1d, 1H, J = 15 Hz, CH₂(NBn)], 4.63 [1d, 1H,

J = 15 Hz, CH₂(NBn)], 4.98 [m, 2H, CH₂(Z)], 7.16–7.37 (m, 21H, Ar and NHZ), 8.19 (m, 3H,

NH₂·HCl), 8.21 (m, 1H, α-NH), 8.57 (t, 1H, J = 6 Hz, NHBn). (S)-16b δ (ppm): 1.22 (m, 1H, γ-H),

1.27 (m, 1H, γ-H), 1.41 (m, 2H, δ-H), 1.58 (m, 1H, β-H), 1.70 (m, 1H, β-H), 3.15 (m, 1H, 2-H), 3.38

(m, 1H, 3-H), 3.46 (m, 1H, 3-H), 3.55 (m, 1H, 2-CH), 4.26 [m, 2H, CH₂(NHBn)], 4.28 [m, 2H, α-H

and CH₂(NBn)], 4.40 [d, 1H, J = 15 Hz, CH₂(NBn)], 4.98 [m, 2H, CH₂(Z)], 7.16–7.37 (m, 21H, Ar

and NHZ), 8.19 (m, 3H, NH₂·HCl), 8.43 (d, 1H, J = 8 Hz, α-NH), 8.57 (m, 1H, NHBn); C-NMR

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(125 MHz, DMSO-d₆) (R)-16b δ (ppm): 23.2 [C_γ], 29.5 [C_δ], 32.0 [C_β], 35.0 [CH₂-Ph], 41.1 [C_ε], 42.4

[CH₂(NHBn)], 44.0 [C₃], 49.2 [CH₂(NBn)], 52.1 [C₂-CH], 53.1 [C_α], 55.0 [C₆], 56.6 [CH₂CO], 57.7

[C₂], 65.5 [CH₂(Z)], 127.1, 127.5, 128.1, 128.2, 128.7, 128.8, 129.0, 129.7 [20CH (Ar)], 136.2

[C (Ph)], 137.4 [C (NBn)], 137.7 [C (Z)], 139.8 [C (NHBn)], 156.5 [CO (Z)], 167.5 [C₅], 169.8 [CO],

172.0 [α-CONH]. (S)-16b δ (ppm): 23.3 [C_γ], 31.9 [C_β], 42.4 [CH₂(NHBn)], 43.8 [C₃], 49.3 [CH₂

(NBn)], 51.7 [C₂-CH], 53.3 [C_α], 59.1 [C₂], 65.5 [CH₂(Z)], 127.3, 127.5, 127.8, 128.2, 128.7, 129.0,

129.1, 129.7 [20CH (Ar)], 136.3 [C (Ph)], 137.3 [C (NBn)], 137.7 [C (Z)], 139.2 [C (NHBn)], 162.3

[CO (Z)], 172.0 [α-CONH]; ES-MS m/z [M+1]⁺calculated for C₄₂H₅₀N₆O₅: 720.5; found: 720.8.

N-[2-[4-Benzyl-(2RS)-[(1S)-amino-2-phenylethyl]-5-oxo-piperazin-1-yl]acetyl]-Arg(Pbf)-NHBn

hydrochloride (16c). Amorphous solid (541 mg, 100%); HPLC t_R: 14.80 min [(R)-16c] and 19.68 min

[(S)-16c]; H-NMR (500 MHz, DMSO-d₆) (R)-16c δ (ppm): 1.38 [s, 6H, 2CH₃(Pbf)], 1.44 (m, 2H,

γ-H), 1.56 (m, 1H, β-H), 1.70 (m, 1H, β-H), 1.98 [s, 3H, CH₃(Pbf)], 2.40 [s, 3H, CH₃(Pbf)], 2.46

[s, 3H, CH₃(Pbf)], 2.83 (d, 1H, J = 6.5 and 14 Hz, CH₂-Ph), 2.94 (m, 1H, CH₂-Ph), 2.95 (m, 1H, 2-H),

2.96 [m, 2H, CH₂(Pbf)], 3.02 (dd, 2H, J = 6.5 and 12 Hz, δ-H), 3.19 (d, 1H, J = 16.5 Hz, 6-H), 3.29

(m, 2H, CH₂CO), 3.39 (m, 1H, 3-H), 3.44 (m, 1H, 3-H), 3.55 (d, 1H, J = 16.5 Hz, 6-H), 3.65 (m, 1H,

2-CH), 4.23 [m, 2H, CH₂(NHBn)], 4.30 (m, 1H, α-H), 4.45 [d, 1H, J = 15 Hz, CH₂(NBn)], 4.63 [d,

1H, J = 15 Hz, CH₂(NBn)], 6.45 [m, 3H, NHC(NH₂) = N], 6.91–7.37 (m, 15H, Ar), 8.18 (m, 3H,

NH₂·HCl), 8.23 (d, 1H, J = 8 Hz, α-NH), 8.59 (t, 1H, J = 6Hz, NHBn). (S)-16c δ (ppm): 1.38 [s, 6H,

2CH₃(Pbf)], 1.98 [s, 3H, CH₃(Pbf)], 2.40 [s, 3H, CH₃(Pbf)], 2.46 [s, 3H, CH₃(Pbf)], 2.96 [m, 2H,

CH₂(Pbf)], 3.15 (m, 1H, 2-H), 3.39 (m, 1H, 3-H), 3.44 (m, 1H, 3-H), 3.55 (m, 1H, 2-CH), 4.23 [m,

2H, CH₂(NHBn)], 4.30 (m, 2H, α-H and CH₂(NBn)), 4.40 [d, 2H, J = 15 Hz, CH₂(NBn)], 6.45 [m,

3H, NHC(NH₂) = N], 6.91–7.37 (m, 15H, Ar), 8.18 (m, 3H, NH₂·HCl), 8.46 (d, 1H, J = 8 Hz, α-NH),

8.56 (m, 1H, NHBn); ¹³C-NMR (125 MHz, DMSO-d₆) (R)-16c δ (ppm): 12.7, 18.1, 19.4 [3CH₃(Pbf)],

26.1 [C_γ], 28.2 [2CH₃(Pbf)], 29.9 [C_β], 35.0 [CH₂-Ph], 40.3 [C_δ], 42.4 [CH₂(NHBn)], 42.9 [CH₂

(Pbf)], 44.0 [C₃], 49.2 [CH₂(NBn)], 52.1 [C₂-CH], 52.8 [C_α], 55.0 [C₆], 56.7 [CH₂CO], 57.8 [C₂],

86.8, 116.8, 124.8 [4C (Pbf)], 127.1, 127.5, 127.8, 128.2, 128.7, 129.0, 129.7 [15CH (Ar)], 131.9,

134.5 [2C (Pbf)], 136.2 [C (Ph)], 137.7 [C (NBn)], 137.8 [C (Pbf)], 139.7 [C (NHBn)], 156.5 [C

(NHC(NH₂) = N)], 158.0 [C (Pbf)], 167.6 [C₅], 169.9 [CO], 171.8 [α-CONH]. (S)-16c δ (ppm): 12.7,

18.1, 19.4 [3CH₃(Pbf)], 28.2 [2CH₃(Pbf)], 42.4 [CH₂(NHBn)], 42.9 [CH₂(Pbf)], 43.9 [C₃], 49.1

[CH₂(NBn)], 51.8 [C₂-CH], 53.0 [C_α], 59.1 [C₂], 86.8, 116.8, 124.8 [4C (Pbf)], 127.3, 127.5, 127.8,

128.2, 128.7, 129.0, 129.1, 129.7 [15CH (Ar)], 131.9, 134.5 [2C (Pbf)], 136.3 [C (Ph)], 137.4 [C

(NBn)], 137.8 [C (Pbf)], 139.7 [C (NHBn)], 156.5 [C (NHC(NH₂) = N)], 158.0 [C (Pbf)], 167.5 [C₅],

171.8 [α-CONH]; ES-MS m/z [M+1]⁺calculated for C₄₇H₆₀N₈O₆S: 866.6; found: 866.0.

3.11. General Procedure for the Synthesis of the Piperazinone-Derived Ureas 17a–c and 18a,b

These compounds were obtained by applying the already indicated procedure for the synthesis of

the urea analogues 9a,b and 10a,b.

N-[2-[4-Benzyl-5-oxo-(2RS)-[2-phenyl-(1S)-(3-phenyl-ureido)ethyl]-piperazin-1-yl]acetyl]-Orn(Z)-

NHBn (17a). Amorphous solid (R:S) = (3:1)] (346 mg, 70%); HPLC t_R: 23.73 min [(R)-17a] and

24.44 min [(S)-17a]; H-NMR (500 MHz, CDCl₃) (R)-17a δ (ppm): 1.50 (m, 2H, γ-H), 1.70 (m, 1H,

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β-H), 1.80 (m, 1H, β-H), 2.69 (dd, 1H, J = 6 and 14 Hz, CH₂-Ph), 2.88 (m, 1H, CH₂-Ph), 2.94 (m, 1H,

2-H), 3.10 (m, 1H, δ-H), 3.20 (m, 2H, 3-H and 6-H), 3.35 (m, 1H, CH₂CO), 3.38 (m, 1H, 3-H),

3.40 (m, 1H, CH₂CO), 3.42 (m, 1H, δ-H), 3.49 (d, 1H, J = 17 Hz, 6-H), 4.07 (m, 1H, 2-CH), 4.18

[m, 1H, CH₂(NHBn)], 4.32 [m, 1H, CH₂(NBn)], 4.40 [m, 1H, CH₂(NHBn)], 4.78 [m, 2H, α-H and

CH₂(Z)], 4.82 [m, 1H, CH₂(NBn)], 4.87 [m, 1H, CH₂(Z)], 5.09 (t, 1H, J = 6 Hz, NHZ), 5.57 (m, 1H,

2-CHNH), 6.84–7.30 (m, 26H, Ar and NHPh), 7.34 (m, 1H, NHBn), 7.79 (m, 1H, α-NH). (S)-17a

δ (ppm): 1.40 (m, 2H, γ-H), 1.70 (m, 1H, β-H), 1.80 (m, 1H, β-H), 2.81 (m, 1H, CH₂-Ph), 2.94 (m, 1H,

CH₂-Ph), 3.05 (m, 1H, δ-H), 3.08 (m, 1H, 3-H), 3.35 (m, 1H, CH₂CO), 3.39 (m, 1H, 3-H), 3.40 (m,

1H, CH₂CO), 3.43 (m, 1H, δ-H), 4.07 (m, 1H, 2-CH), 4.16 [dd, 1H, J = 5 and 15 Hz, CH₂(NHBn)],

4.38 [m, 1H, CH₂(NHBn)], 4.50 [m, 2H, CH₂(NBn)], 4.72 (m, 1H, α-H), 4.76 [m, 1H, CH₂(Z)], 4.85

[m, 1H, CH₂(Z)], 4.96 (m, 1H, NHZ), 5.39 (d, 1H, J = 6.5 Hz, 5-CHNH), 6.84–7.30 (m, 26H, Ar and

NHPh), 7.41 (m, 1H, NHBn), 7.73 (m, 1H, α-NH); C-NMR (125 MHz, CDCl₃) (R)-17a δ (ppm):

26.6 [C_γ], 30.1 [C_β], 36.9 [CH₂-Ph], 39.1 [C_δ], 42.0 [CH₂(NHBn)], 44.8 [C₃], 49.5 [CH₂(NBn)], 51.2

[C_α], 52.3 [C₂-CH], 55.8 [C₆], 59.0 [CH₂CO], 61.2 [C₂], 66.7 [CH₂(Z)], 120.0, 123.1, 126.7, 127.5,

127.8, 127.9, 128.1, 128.3, 128.5, 128.8 [25CH (Ar)], 136.1 [C (NBn)], 136.2 [C (Z)], 137.2 [C (Ph)],

137.6 [C (NHBn)], 138.7 [C (NHPh)], 155.1 [CO (Z)], 157.5 [CO (Urea)], 167.9 [C₅], 170.4 [CO],

172.7 [α-CONH]. (S)-17a δ (ppm): 26.5 [C_γ], 30.6 [C_β], 37.6 [CH₂-Ph], 38.8 [C_δ], 42.1 [CH₂(NHBn)],

44.8 [C₃], 49.9 [CH₂(NBn)], 50.6 [C_α], 51.9 [C₂-CH], 66.7 [CH₂(Z)], 118.9, 122.3, 126.7, 127.5,

127.8, 127.9, 128.2, 128.5, 128.6, 128.7, 128.9, 129.4 [25CH (Ar)], 136.1 [C (NBn)], 136.2 [C (Z)],

137.2 [C (Ph)], 137.4 [C (NHBn)], 139.5 [C (NHPh)], 155.0 [CO (Z)], 157.6 [CO (Urea)], 166.9 [C₅],

169.7 [CO], 172.9 [α-CONH]; ES-MS m/z 825.7 [M+1]⁺; C₄₈H₅₃N₇O₆(%): C: 69.97, H: 6.48, N:

11.90. Found (%): C: 69.75, H: 6.65, N: 12.02.

N-[2-[4-Benzyl-5-oxo-(2RS)-[2-phenyl-(1S)-(3-phenyl-ureido)ethyl]-piperazin-1-yl]acetyl]-Lys(Z)-

NHBn (17b). Amorphous solid [(R:S) = (3:1)] (327 mg, 65%); HPLC t_R: 24.06 min; H-NMR

(500 MHz, CDCl₃) (R)-17b δ (ppm): 1.33 (m, 2H, γ-H), 1.46 (m, 2H, δ-H), 1.67 (m, 1H, β-H), 1.85

(m, 1H, β-H), 2.68 (m, 1H, CH₂-Ph), 2.82 (m, 1H, CH₂-Ph), 2.89 (m, 1H, 2-H), 3.05 (m, 2H, ε-H),

3.15 (m, 1H, 6-H), 3.25 (m, 2H, 3-H), 3.31 (m, 2H, CH₂CO), 3.53 (d, 1H, J = 16.5 Hz, 6-H), 4.20

(m, 1H, 2-CH), 4.25 [m, 3H, CH₂(NHBn and NBn)], 4.44 (m, 1H, α-H), 4.77 [d, 1H, J = 14.5 Hz, CH₂

(NBn], 5.00 [s, 2H, CH₂(Z)], 5.23 (m, 1H, NHZ), 5.45 (m, 1H, 2-CHNH), 6.84–7.52 (m, 27H, Ar,

NHBn and NHPh), 7.79 (m, 1H, α-NH). (S)-17b δ (ppm): 2.78 (m, 1H, CH₂-Ph), 2.89 (m, 1H, CH₂-Ph),

2.94 (m, 1H, 2-H), 4.20 (m, 1H, 2-CH), 4.25 [m, 2H, CH₂(NHBn)], 4.52 [d, 1H, J = 14.5 Hz, CH₂

(NBn], 4.58 [d, 1H, J = 14.5 Hz, CH₂(NBn], 5.03 [m, 2H, CH₂(Z)], 5.09 (m, 1H, NHZ), 5.55 (m, 1H,

2-CHNH), 6.84–7.52 (m, 27H, Ar, NHBn and NHPh), 7.89 (m, 1H, α-NH); C-NMR (125 MHz,

CDCl₃) (R)-17b δ (ppm): 22.7 [C_γ], 29.1 [C_δ], 31.9 [C_β], 37.2 [CH₂-Ph], 40.3 [C_ε], 43.6 [CH₂

(NHBn)], 44.6 [C₃], 49.5 [CH₂(NBn)], 52.0 [C₂-CH], 53.0 [C_α], 55.6 [C₆], 58.8 [CH₂CO], 60.9 [C₂],

66.0 [CH₂(Z)], 120.1, 123.2, 126.8, 127.5, 128.0, 128.1, 128.4, 128.5, 128.7, 128.8, 128.9 [25CH

(Ar)], 136.2 [C (NBn)], 136.5 [C (Z)], 137.0 [C (Ph)], 137.7 [C (NHBn)], 138.6 [C (NHPh)], 155.3

[CO (Z)], 156.6 [CO (Urea)], 167.6 [C₅], 170.1 [CO], 172.3 [α-CONH]. (S)-17b δ (ppm): 43.6 [CH₂

(NHBn)], 44.6 [C₃], 52.1 [C₂-CH], 60.8 [C₂], 66.0 [CH₂(Z)], 120.1, 123.2, 126.8, 127.5, 128.0, 128.1,

128.4, 128.5, 128.7, 128.8, 129.0 [25CH (Ar)], 136.5 [C (Z)], 156.4 [CO (Z)], 172.3 [α-CONH];

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ES-MS m/z 839.7 [M+1]⁺; C₄₉H₅₅N₇O₆(%): C: 70.23, H: 6.62, N: 11.70. Found (%): C: 70.46, H:

6.75, N: 11.54.

N-[2-[4-Benzyl-5-oxo-(2RS)-[2-phenyl-(1S)-(3-phenyl-ureido)ethyl]-piperazin-1-yl]acetyl]-Arg(Pbf)-

NHBn (17c). Amorphous solid [(R:S) = (3:1)] (443 mg, 75%); HPLC t_R: 25.80 min [(R)-17c] and

23.82 min [(S)-17c]; H-NMR (500 MHz, CDCl₃) (R)-17c δ (ppm): 1.45 [s, 3H, CH₃(Pbf)], 1.46

[s, 3H, CH₃(Pbf)], 1.40 (m, 2H, γ-H), 1.52 (m, 1H, β-H), 1.68 (m, 1H, β-H), 2.10 [s, 3H, CH₃(Pbf)],

2.50 [s, 3H, CH₃(Pbf)], 2.58 [s, 3H, CH₃(Pbf)], 2.64 (m, 1H, 2-H), 2.68 (m, 1H, CH₂-Ph), 2.77 (m,

1H, CH₂-Ph), 2.94 [s, 2H, CH₂(Pbf)], 2.98 (m, 1H, CH₂CO), 3.06 (m, 1H, 6-H), 3.25 (m, 1H, δ-H),

3.28 (dd, 1H, J = 5 and 13 Hz, 3-H), 3.29 (m, 1H, δ-H), 3.50 (d, 1H, J = 15.5 Hz, CH₂CO), 3.58 (m,

1H, 3-H), 3.64 (d, 1H, J = 16.5 Hz, 6-H), 4.13 [d, 1H, J = 14.5 Hz, CH₂(NBn)], 4.30 (m, 1H, 2-CH),

4.32 (m, 1H, α-H), 4.36 [m, 2H, CH₂(NHBn)], 5.00 [d, 1H, J = 14.5 Hz, CH₂(NBn)], 5.98 (m, 1H,

2-CHNH), 6.18 [m, 2H, NHC(NH₂) = N], 6.36 [m, 1H, NHC(NH₂) = N], 6.86–7.37 (m, 21H, Ar and

NHPh), 7.64 (m, 1H, NHBn), 7.88 (d, 1H, J = 8 Hz, α-NH). (S)-17c δ (ppm): 1.45 [s, 3H, CH₃(Pbf)],

1.46 [s, 3H, CH₃(Pbf)], 2.10 [s, 3H, CH₃(Pbf)], 2.50 [s, 3H, CH₃(Pbf)], 2.58 [s, 3H, CH₃(Pbf)], 2.68

(m, 1H, CH₂-Ph), 2.77 (m, 1H, CH₂-Ph), 2.94 [s, 2H, CH₂(Pbf)], 3.23 (m, 1H, δ-H), 3.29 (m, 1H, δ-H),

3.28 (m, 1H, 3-H), 3.58 (m, 1H, 3-H), 4.05 [m, 1H, CH₂(NHBn)], 4.29 (m, 1H, α-H), 4.30 [m, 1H,

CH₂(NHBn)], 4.46 [d, 1H, J = 14 Hz, CH₂(NBn)], 4.59 [d, 1H, J = 14 Hz, CH₂(NBn)], 5.98 (m, 1H,

2-CHNH), 6.18 [m, 2H, NHC(NH₂) = N], 6.36 [m, 1H, NHC(NH₂) = N], 6.86–7.37 (m, 21H, Ar and

NHPh), 7.64 (m, 1H, NHBn), 7.78 (d, 1H, J = 8 Hz, α-NH); C-NMR (125 MHz, CDCl₃) (R)-17c

δ (ppm): 12.5, 18.0, 19.4 [3CH₃(Pbf)], 25.4 [C_γ], 28.6 [2CH₃(Pbf)], 29.3 [C_β], 38.1 [CH₂-Ph], 40.3

[C_δ], 43.2 [2CH₂(Pbf and NHBn)], 44.3 [C₃], 49.2 [CH₂(NBn)], 51.2 [C₂-CH], 53.1 [C_α], 55.4 [C₆],

59.5 [CH₂CO], 60.2 [C₂], 86.6, 117.8, 124.9 [3C (Pbf)], 119.5, 122.7, 126.7, 127.1, 127.3, 127.9,

128.1, 128.5, 128.6, 129.0, 129.3 [20CH (Ar)], 132.2 [2C (Pbf)], 136.2 [C (NBn)], 137.2 [C (Ph)],

138.1 [C (NHBn)], 138.3 [C (Pbf)], 139.0 [C (NHPh)], 156.2 [CO (Urea)], 156.4 [C (NHC(NH₂) = N)],

159.0 [C (Pbf)], 168.3 [C₅], 171.0 [CO], 172.0 [α-CONH]. (S)-17c δ (ppm): 12.5, 18.0, 19.4 [3CH₃

(Pbf)], 28.6 [2CH₃(Pbf)], 38.4 [CH₂-Ph], 40.3 [C_δ], 43.2 [2CH₂(Pbf and NHBn)], 44.2 [C₃], 49.2

[CH₂(NBn)], 53.1 [C_α], 86.6, 117.8, 124.9 [3C (Pbf)], 118.9, 122.2, 126.6, 127.1, 127.2, 127.9, 128.1,

128.4, 128.7, 129.0, 129.3 [20CH (Ar)], 132.2 [2C (Pbf)], 136.1 [C (NBn)], 137.1 [C (Ph)], 138.0

[C (NHBn)], 138.3 [C (Pbf)], 139.4 [C (NHPh)], 156.4 [C (NHC(NH₂) = N)], 159.0 [C (Pbf)], 172.0

[α-CONH]; ES-MS m/z 985.1 [M+1]⁺; C₅₄H₆₅N₉O₇S (%): C: 65.90, H: 6.66, N: 12.81. Found (%): C:

65.72, H: 6.90, N: 12.63.

N-[2-[4-Benzyl-(2RS)-[(1S)-(3-benzylureido)-2-phenyl-ethyl]-5-oxopiperazin-1-yl]acetyl]-Orn(Z)-NHBn

(18a). Amorphous solid [(R:S) = (3:1)] (375 mg, 65%); HPLC t_R: 23.30 min [(R)-18a] and 23.82 min

[(S)-18a]; H-NMR (500 MHz, CDCl₃) (R)-18a δ (ppm): 1.52 (m, 2H, γ-H), 1.65 (m, 1H,

β-H), 1.82 (m, 1H, β-H), 2.69 (m, 1H, CH₂-Ph), 2.88 (m, 1H, CH₂-Ph), 2.90 (m, 1H, 2-H), 3.11 (m,

1H, δ-H), 3.20 (m, 1H, CH₂CO), 3.23 (m, 1H, 3-H), 3.32 (m, 2H, CH₂CO and 6-H), 3.36 (m, 1H, 3-H),

3.42 (m, 1H, δ-H), 3.55 (m, 1H, 6-H), 4.05 [m, 1H, CH₂(NHBn)], 4.08 [m, 1H, CH₂(NHBn, Urea)],

4.15 [m, 1H, CH₂(NHBn, Urea)], 4.18 (m, 1H, 2-CH), 4.25 [m, 1H, CH₂(NHBn)], 4.32 [m, 1H, CH₂

(NBn)], 4.68 (m, 1H, α-H), 4.75 [m, 1H, CH₂(Z)], 4.79 [m, 1H, CH₂(NBn)], 4.88 [d, 1H, J = 12.5 Hz,

CH₂(Z)], 5.08 (m, 1H, 2-CHNH), 5.20 (m, 1H, NHZ), 5.95 [m, 1H, NHBn (Urea)], 6.95–7.40 (m,

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25H, Ar), 7.40 (m, 1H, NHBn), 7.86 (d, 1H, J = 9 Hz, α-NH). (S)-18a δ (ppm): 1.46 (m, 2H, γ-H), 1.63

(m, 1H, β-H), 1.80 (m, 1H, β-H), 2.57 (m, 1H, CH₂-Ph), 2.85 (m, 1H, CH₂-Ph), 3.04 (m, 1H, 3-H),

3.07 (m, 1H, δ-H), 3.34 (m, 1H, 3-H), 3.38 (m, 1H, 6-H), 3.44 (m, 1H, δ-H), 3.57 (m, 1H, 6-H), 3.96

[dd, 1H, J = 5 and 15 Hz, CH₂(NHBn)], 4.02 (m, 1H, 2-CH), 4.08 [m, 1H, CH₂(NHBn, Urea)], 4.15

[m, 1H, CH₂(NHBn, Urea)], 4.20 [m, 1H, CH₂(NHBn)], 4.50 [m, 1H, CH₂(NBn)], 4.66 [m, 1H, CH₂

(Z)], 4.68 (m, 1H, α-H), 4.79 [m, 1H, CH₂(NBn)], 4.82 [m, 1H, CH₂(Z)], 5.02 (t, 1H, J = 6 Hz, NHZ),

5.08 (m, 1H, 5-CHNH), 5.95 [m, 1H, NHBn (Urea)], 6.95–7.40 (m, 25H, Ar), 7.40 (m, 1H, NHBn),

7.78 (d, 1H, J = 8.5 Hz, α-NH); C-NMR (125 MHz, CDCl₃) (R)-18a δ (ppm): 26.6 [C_γ], 30.2 [C_β],

37.3 [CH₂-Ph], 39.0 [C_δ], 43.5 [CH₂(NHBn)], 44.0 [CH₂(NHBn, Urea)], 44.6 [C₃], 49.5 [CH₂(NBn)],

51.1 [C_α], 52.2 [C₂-CH], 55.6 [C₆and CH₂CO], 60.8 [C₂], 66.7 [CH₂(Z)], 126.6, 127.0, 127.4, 127.6,

127.9, 128.1, 128.4, 128.5, 128.7, 128.9 [25CH (Ar)], 136.3 [C (NBn) and C (Z)], 137.2 [C (Ph)],

137.7 [C (NHBn)], 139.3 [C (NHBn, Urea)], 155.4 [CO (Z)], 157.7 [CO (Urea)], 167.6 [C₅], 170.0

[CO], 172.4 [α-CONH]. (S)-18a δ (ppm): 26.6 [C_γ], 30.7 [C_β], 38.2 [CH₂-Ph], 38.7 [C_δ], 43.5 [CH₂

(NHBn)], 43.8 [CH₂(NHBn, Urea)], 45.0 [C₃], 49.9 [CH₂(NBn)], 51.4 [C_α], 52.2 [C₂-CH], 55.6

[CH₂CO], 66.7 [CH₂(Z)], 126.9, 127.1, 127.4, 127.5, 127.8, 127.9, 128.2, 128.5, 128.6, 128.9, 129.6

[25CH (Ar)], 136.1 [C (NBn) and C (Z)], 137.1 [C (Ph)], 137.7 [C (NHBn)], 139.8 [C (NHBn, Urea)],

155.2 [CO (Z)], 157.9 [CO (Urea)], 167.6 [C₅], 170.0 [CO], 172.6 [α-CONH]; ES-MS m/z 839.6

[M+1]⁺; C₄₉H₅₅N₇O₆(%): C: 70.23, H: 6.22, N: 11.70. Found (%): C: 70.01, H: 6.46, N: 11.59.

N-[2-[4-Benzyl-(2RS)-[(1S)-(3-benzylureido)-2-phenyl-ethyl]-5-oxopiperazin-1-yl]acetyl]-Lys(Z)-NHBn

(18b). Amorphous solid [(R:S) = (3:1)] (317 mg, 62%); HPLC t_R: 23.69 min [(R)-18b] and 24.16 min

[(S)-19b]; H-NMR (500 MHz, CDCl₃) (R)-18b δ (ppm): 1.27 (m, 2H, γ-H), 1.40 (m, 2H,

δ-H), 1.60 (m, 1H, β-H), 1.78(m, 1H, β-H), 2.64 (m, 2H, CH₂-Ph), 2.76 (m, 1H, 2-H), 3.08 (m, 1H,

ε-H), 3.12 (m, 2H, CH₂CO and 6-H), 3.14 (m, 1H, 3-H), 3.15 (m, 1H, ε-H), 3.24 (m, 1H, CH₂CO),

3.25 (m, 1H, 3-H), 3.41 (d, 1H, J = 16.5 Hz, 6-H), 4.10 (m, 1H, 2-CH), 3.95 [dd, 1H, J = 5.5 and 15,

CH₂(NHBn, Urea)], 4.04 [m, 1H, CH₂(NHBn, Urea)], 4.18 [m, 2H, CH₂(NHBn)], 4.32 [m, 1H, CH₂

(NBn], 4.38 (m, 1H, α-H), 4.62 [d, 1H, J = 14.5 Hz, CH₂(NBn], 4.92 (m, 1H, 2-CHNH), 5.00 [s, 2H,

CH₂(Z)], 5.20 (m, 1H, NHZ), 5.70 [m, 1H, NHBn (Urea)], 6.81–7.33 (m, 26H, Ar and NHBn), 7.71

(d, 1H, J = 8 Hz, α-NH). (S)-18b δ (ppm): 1.27 (m, 2H, γ-H), 1.40 (m, 2H, δ-H), 1.60 (m, 1H, β-H),

1.78(m, 1H, β-H), 2.68 (m, 2H, CH₂-Ph), 2.76 (m, 1H, 2-H), 2.98 (m, 1H, 3-H), 3.10 (m, 1H, CH₂CO),

3.18 (m, 1H, 3-H), 3.22 (m, 1H, CH₂CO), 3.76 (m, 1H, 2-CH), 3.95 [m, 1H, CH₂(NHBn, Urea)], 4.04

[m, 1H, CH₂(NHBn, Urea)], 4.10 [m, 1H, CH₂(NHBn)], 4.18 [m, 1H, CH₂(NHBn)], 4.34 (m, 1H,

α-H), 4.43 [d, 1H, J = 14.5 Hz, CH₂(NBn], 4.52 [d, 1H, J = 14.5 Hz, CH₂(NBn], 4.92 (m, 1H,

2-CHNH), 4.96 [s, 2H, CH₂(Z)], 5.10 (m, 1H, NHZ), 5.70 [m, 1H, NHBn (Urea)], 6.81–7.33 (m, 26H,

Ar and NHBn), 7.64 (d, 1H, J = 7.5 Hz, α-NH); C-NMR (125 MHz, CDCl₃) (R)-18b δ (ppm): 22.7

[C_γ], 29.2 [C_δ], 31.9 [C_β], 37.5 [CH₂-Ph], 40.4 [C_ε], 43.4 [CH₂(NHBn)], 44.0 [CH₂(NHBn, Urea)],

44.3 [C₃], 49.5 [CH₂(NBn)], 52.0 [C₂-CH], 53.0 [C_α], 55.3 [C₆], 58.6 [CH₂CO], 60.1 [C₂], 66.6

[CH₂(Z)], 126.7, 127.0, 127.4, 127.5, 128.0, 128.2, 128.4, 128.5, 128.6, 128.7, 128.9 [25CH (Ar)],

136.4 [C (NBn)], 136.6 [C (Z)], 137.1 [C (Ph)], 137.8 [C (NHBn)], 139.1 [C (NHBn, Urea)], 156.6

[CO (Z)], 157.7 [CO (Urea)], 167.8 [C₅], 170.1 [CO], 172.1 [α-CONH]. (S)-18b δ (ppm): 22.4 [C_γ],

29.7 [C_δ], 30.9 [C_β], 38.2 [CH₂-Ph], 43.5 [CH₂(NHBn)], 43.9 [CH₂(NHBn, Urea)], 44.3 [C₃], 49.7

[CH₂(NBn)], 51.2 [C₂-CH], 52.8 [C_α], 58.6 [CH₂CO], 60.0 [C₂], 66.7 [CH₂(Z)], 126.7, 127.2, 127.4,

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127.5, 128.0, 128.1, 128.4, 128.5, 128.6, 128.7, 128.9, 129.5 [25CH (Ar)], 136.1 [C (NBn)], 136.6

[C (Z)], 137.1 [C (Ph)], 137.8 [C (NHBn)], 139.6 [C (NHBn, Urea)], 156.7 [CO (Z)], 158.0

[CO (Urea)], 170.1 [CO], 172.1 [α-CONH]; ES-MS m/z 853.7 [M+1]⁺; C₅₀H₅₇N₇O₆(%): C: 70.48,

H: 6.74, N: 11.51. Found (%): C: 70.31, H: 6.95, N: 11.69.

3.12. General Procedure for the Synthesis of the Hydrochlorides 19a,b and 20a,b

These compounds were prepared following the general procedure for the removal of the N-Z

protecting group, already indicated for the synthesis of 11a,b and 12a,b.

N-[2-[4-Benzyl-5-oxo-(2RS)-[2-phenyl-(1S)-(3-phenylureido)ethyl]-piperazin-1-yl]acetyl]-Orn-NHBn

hydrochloride (19a). Amorphous solid [(R:S) = (3:1)] (145 mg, 100%); HPLC t_R: 16.27 min [(R)-19a]

and 16.52 min [(S)-19a]; H-NMR (500 MHz, DMSO-d₆) (R)-19a δ (ppm): 1.62 (m, 3H, γ-H and

β-H), 1.82 (m, 1H, β-H), 2.70 (m, 1H, CH₂-Ph), 2.75 (m, 3H, δ-H and 2-H), 2.91 (d, 1H, J = 11 Hz,

CH₂-Ph), 3.33–4.11 (m, 6H, 3-H, 6-H and CH₂CO), 4.22 [m, 1H, CH₂(NHBn)], 4.34 [m, 1H, CH₂

(NHBn)], 4.38 (m, 1H, 2-CH), 4.39 (m, 1H, α-H), 4.48 [m, 1H, CH₂(NBn)], 4.62 [m, 1H, CH₂(NBn)],

6.80 (m, 1H, 2-CHNH), 6.76–6.95 (m, 2H, Ar), 7.08–7.38 (m, 18H, Ar), 7.96 (m, 3H, NH₂·HCl), 8.60

(m, 1H, α-NH), 8.72 (m, 1H, NHBn), 8.85 (m, 1H, NHPh). (S)-19a δ (ppm): 1.58 (m, 3H, γ-H and

β-H), 1.78 (m, 1H, β-H), 2.55 (dd, 1H, J = 6 and 14 Hz, CH₂-Ph), 2.75 (m, 3H, δ-H and 2-H), 2.85

(m, 1H, CH₂-Ph), 3.33–4.11 (m, 7H, 3-H, 6-H, CH₂CO and 2-CH), 4.22 [m, 1H, CH₂(NHBn)], 4.34

[m, 1H, CH₂(NHBn)], 4.51 [m, 1H, CH₂(NBn)], 4.64 [m, 1H, CH₂(NBn)], 6.80 (m, 1H, 2-CHNH),

6.76–6.95 (m, 2H, Ar), 7.08–7.38 (m, 18H, Ar), 7.96 (m, 3H, NH₂·HCl), 8.60 (m, 1H, α-NH), 8.70

(m, 1H, NHBn), 8.81 (m, 1H, NHPh); C-NMR (125 MHz, DMSO-d₆) (R)-19a δ (ppm): 23.4 [C_γ],

28.9 [C_β], 37.8 [CH₂-Ph], 38.1 [C_δ], 42.0 [CH₂(NHBn)], 43.7 [C₃], 49.2 [CH₂(NBn)], 49.8 [C₂-CH],

51.9 [C_α], 53.6 [C₆], 53.9 [CH₂CO], 60.5 [C₂], 117.7, 121.2, 126.2, 126.6, 127.0, 127.2, 127.5, 128.2,

128.4, 128.5, 129.1 [20CH (Ar)], 136.3 [C (NBn)], 137.8 [C (Ph)], 139.1 [C (NHBn)], 139.9

[C (NHPh)], 155.2 [CO (Urea)], 170.7 [α-CONH]. (S)-19a δ (ppm): 23.3 [C_γ], 29.0 [C_β], 37.8

[CH₂-Ph], 38.2 [C_δ], 42.0 [CH₂(NHBn)], 43.7 [C₃], 49.2 [CH₂(NBn)], 49.8 [C₂-CH], 53.5 [C₆], 53.9

[CH₂CO], 60.2 [C₂], 117.5, 121.0, 126.1, 126.6, 127.0, 127.3, 127.7, 127.9, 128.1, 128.4, 128.5, 129.1

[20CH (Ar)], 136.3 [C (NBn)], 138.0 [C (Ph)], 139.1 [C (NHBn)], 140.1 [C (NHPh)], 155.0

[CO (Urea)], 170.8 [α-CONH]; ES-MS m/z [M+2]⁺calculated for C₄₀H₄₇N₇O₄: 690.3; found: 690.6.

N-[2-[4-Benzyl-5-oxo-(2RS)-[2-phenyl-(1S)-(3-phenylureido)ethyl]-piperazin-1-yl}acetyl}-Lys-NHBn

hydrochloride (19b). Amorphous solid [(R:S) = (3:1)] (148 mg, 100%); HPLC t_R: 16.44 min; ¹H-NMR

(500 MHz, DMSO-d₆) (R)-19b δ (ppm): 1.30 (m, 2H, γ-H), 1.50 (m, 2H, δ-H), 1.60 (m, 1H, β-H), 1.72

(m, 1H, β-H), 2.70 (m, 3H, ε-H and 2-H), 2.72 (m, 1H, CH₂-Ph), 2.92 (m, 1H, CH₂-Ph), 3.26–4.20

(m, 6H, 3-H, 6-H and CH₂CO), 4.32 (m, 1H, 2-CH), 4.24 [dd, 1H, J = 6 and 15 Hz, CH₂(NHBn)],

4.30 [m, 2H, α-H and CH₂(NHBn)], 4.50 [m, 1H, CH₂(NBn)], 4.70 [m, 1H, CH₂(NBn)], 6.55 (m, 1H,

2-CHNH), 6.86 (t, 1H, J = 7 Hz, Ar), 6.97–7.41 (m, 19H, Ar), 7.84 (m, 3H, NH₂·HCl), 8.51 (m, 1H,

α-NH), 8.62 (m, 1H, NHBn), 8.80 (m, 1H, NHPh). (S)-19b δ (ppm): 1.60 (m, 1H, β-H), 1.72 (m, 1H,

β-H), 2.55 (m, 1H, CH₂-Ph), 2.88 (m, 1H, CH₂-Ph), 4.24 [m, 1H, CH₂(NHBn)], 4.30 [m, 1H, CH₂

(NHBn)], 6.55 (m, 1H, 2-CHNH), 6.86 (t, 1H, J = 7 Hz, Ar), 6.97–7.41 (m, 19H, Ar), 7.84 (m, 3H,

NH₂·HCl), 8.58 (m, 1H, NHBn), 8.83 (m, 1H, NHPh); ¹³C-NMR (125 MHz, DMSO-d₆) (R)-19b

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δ (ppm): 22.2 [C_γ], 26.5 [C_δ], 31.3 [C_β], 37.8 [CH₂-Ph], 38.4 [C_ε], 42.0 [CH₂(NHBn)], 43.8 [C₃], 49.2

[CH₂(NBn) and C₂-CH], 52.5 [C_α], 53.7 [C₆], 60.4 [C₂], 117.8, 121.3, 126.7, 127.0, 127.3, 127.6,

128.2, 128.5, 128.6, 129.2 [20CH (Ar)], 136.5 [C (NBn)], 137.9 [C (Ph)], 139.2 [C (NHBn)], 139.9

[C (NHPh)], 155.3 [CO (Urea)], 171.1 [α-CONH]. (S)-19b δ (ppm): 31.5 [C_β], 37.8 [CH₂-Ph], 42.0

[CH₂(NHBn)], 117.8, 121.3, 126.3, 127.0, 127.3, 127.6, 128.2, 128.5, 128.6, 129.2 [20CH (Ar)],

136.5 [C (NBn)], 137.9 [C (Ph)], 139.2 [C (NHBn)], 139.9 [C (NHPh)], 155.5 [CO (Urea)], 171.1

[α-CONH]; ES-MS m/z [M+2]⁺calculated for C₄₁H₄₉N₇O₄: 705.3; found: 705.6.

N-[2-[4-Benzyl-(2RS)-[(1S)-(3-benzylureido)-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-Orn-NHBn

hydrochloride (20a). Amorphous solid [(R:S) = (3:1)] (148 mg, 100%); HPLC t_R: 16.32 min [(R)-20a]

and 16.78 min [(S)-20a]; H-NMR (500 MHz, DMSO-d₆) (R)-20a δ (ppm): 1.60 (m, 2H, γ-H), 1.65

(m, 1H, β-H), 1.75 (m, 1H, β-H), 2.65 (dd, 1H, J = 10 and 14 Hz, CH₂-Ph), 2.74 (m, 1H, 2-H), 2.78

(m, 1H, δ-H), 2.93 (m, 1H, CH₂-Ph), 3.35–3.82 (m, 6H, 3-H, 6-H and CH₂CO), 4.03 [d, 1H, J = 15 Hz,

CH₂(NHBn, Urea)], 4.15 [d, 1H, J = 15 Hz, CH₂(NHBn, Urea)], 4.25 [m, 1H, CH₂(NHBn)], 4.30

[m, 1H, CH₂(NHBn)], 4.36 (m, 1H, 2-CH), 4.38 (m, 1H, α-H), 4.47 [d, 1H, J = 15 Hz, CH₂(NBn)],

4.62 [d, 1H, J = 15 Hz, CH₂(NBn)], 6.49 (m, 1H, 2-CHNH), 6.90–7.40 [m, 21H, Ar and NHBn

(Urea)], 7.92 (m, 3H, NH₂·HCl), 8.56 (m, 1H, α-NH), 8.72 (t, 1H, J = 6 Hz, NHBn). (S)-20a δ (ppm):

1.58 (m, 1H, β-H), 1.72 (m, 1H, β-H), 2.50 (m, 1H, CH₂-Ph), 2.78 (m, 1H, δ-H), 2.79 (m, 1H, CH₂-Ph),

3.97 [d, 1H, J = 15 Hz, CH₂(NHBn, Urea) and 5-CH], 4.14 [m, 1H, CH₂(NHBn, Urea)], 4.25 [m, 1H,

CH₂(NHBn)], 4.30 [m, 1H, CH₂(NHBn)], 4.44 [m, 1H, CH₂(NBn)], 4.58 [m, 1H, CH₂(NBn)], 6.42

(m, 1H, 2-CHNH), 6.90–7.40 [m, 21H, Ar and NHBn (Urea)], 7.92 (m, 3H, NH₂·HCl), 8.74 (t, 1H,

J = 6 Hz, NHBn); ¹³C-NMR (125 MHz, DMSO-d₆) (R)-20a δ (ppm): 23.4 [C_γ], 29.2 [C_β], 37.8

[CH₂-Ph], 38.1 [C_δ], 42.0 [CH₂(NHBn)], 42.7 [CH₂(NHBn, Urea)], 43.7 [C₃], 49.1 [CH₂(NBn)], 51.9

[C_α], 49.7 [C₂-CH], 53.7 [C₆], 53.9 [CH₂CO], 60.8 [C₂], 126.4, 126.6, 126.7, 127.1, 127.6, 128.1,

128.3, 128.5, 129.2 [20CH (Ar)], 136.4 [C (NBn)], 137.1 [C (Ph)], 139.0 [C (NHBn)], 140.4

[C (NHBn, Urea)], 158.1 [CO (Urea)], 170.9 [α-CONH]. (S)-20a δ (ppm): 29.4 [C_β], 37.8 [CH₂-Ph],

38.2 [C_δ], 42.0 [CH₂(NHBn)], 42.7 [CH₂(NHBn, Urea)], 49.1 [CH₂(NBn)], 49.7 [C₂-CH], 126.4,

126.5, 126.7, 127.3, 127.8, 128.0, 128.3, 128.6, 129.2 [20CH (Ar)], 136.4 [C (NBn)], 137.1 [C (Ph)],

139.0 [C (NHBn)], 140.4 [C (NHBn, Urea)], 157.8 [CO (Urea)], 170.9 [α-CONH]; ES-MS m/z [M+2]⁺

calculated for C₄₁H₄₉N₇O₄: 705.3; found: 705.6.

N-[2-[4-Benzyl-(2RS)-[(1S)-(3-benzylureido)-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-Lys-NHBn

hydrochloride (20b). Amorphous solid [(R:S) = (3:1)] (151 mg, 100%); HPLC t_R: 15.83 min [(R)-20b]

and 16.25 min [(S)-20b]; H-NMR (500 MHz, DMSO-d₆) (R)-20b δ (ppm): 1.29 (m, 2H, γ-H), 1.52

(m, 2H, δ-H), 1.54 (m, 1H, β-H), 1.72 (m, 1H, β-H), 2.67 (m, 1H, 2-H), 2.68 (m, 2H, ε-H), 2.69 (m,

1H, CH₂-Ph), 2.87 (m, 1H, CH₂-Ph), 3.34–3.87 (m, 6H, 3-H, 6-H and CH₂CO), 4.03 [d, 1H, J = 15 Hz,

CH₂(NHBn, Urea)], 4.14 [d, 1H, J = 15 Hz, CH₂(NHBn, Urea)], 4.20 [m, 1H, CH₂(NHBn)], 4.29

[m, 1H, CH₂(NHBn)], 4.30 (m, 1H, α-H), 4.33 (m, 1H, 2-CH), 4.50 [m, 1H, CH₂(NBn], 4.64 [m, 1H,

CH₂(NBn], 6.48 (m, 1H, 2-CHNH), 6.94–7.41 [m, 21H, Ar and NHBn (Urea)], 7.87 (m, 3H,

NH₂·HCl), 8.50 (m, 1H, α-NH), 8.60 (m, 1H, NHBn). (S)-20b δ (ppm): 1.25 (m, 1H, γ-H), 1.35 (m,

1H, γ-H), 1.54 (m, 1H, β-H), 1.72 (m, 1H, β-H), 2.52 (m, 1H, CH₂-Ph), 2.80 (m, 1H, CH₂-Ph), 3.98 [d,

1H, J = 15 Hz, CH₂(NHBn, Urea)], 4.14 [m, 1H, CH₂(NHBn, Urea)], 4.20 [m, 1H, CH₂(NHBn)],

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4.29 [m, 1H, CH₂(NHBn)], 6.40 (m, 1H, 2-CHNH), 6.94–7.41 [m, 21H, Ar and NHBn (Urea)], 7.87

(m, 3H, NH₂·HCl), 8.62 (m, 1H, NHBn); ¹³C-NMR (125 MHz, DMSO-d₆) (R)-20b δ (ppm): 22.7 [C_γ],

26.9 [C_δ], 31.7 [C_β], 38.2 [CH₂-Ph], 38.9 [C_ε], 42.5 [CH₂(NHBn)], 43.0 [CH₂(NHBn, Urea)], 44.2

[C₃], 49.5 [C₂-CH], 49.7 [CH₂(NBn)], 53.0 [C_α], 54.0 [C₆], 60.1 [C₂], 126.7, 126.8, 127.0, 127.2,

127.5, 128.0, 128.5, 128.6, 129.0, 129.6 [20CH (Ar)], 136.9 [C (NBn)], 138.3 [C (Ph)], 139.7 [C

(NHBn)], 140.8 [C (NHBn, Urea)], 158.6 [CO (Urea)], 171.5 [α-CONH]. (S)-20b δ (ppm): 22.7 [C_γ],

31.5 [C_β], 38.0 [CH₂-Ph], 42.5 [CH₂(NHBn)], 43.0 [CH₂(NHBn, Urea)], 126.7, 126.8, 127.0, 127.2,

127.7, 128.2, 128.5, 128.6, 129.0, 129.6 [20CH (Ar)], 136.9 [C (NBn)], 138.3 [C (Ph)], 139.7 [C

(NHBn)], 141.0 [C (NHBn, Urea)], 158.2 [CO (Urea)], 171.5 [α-CONH]; ES-MS m/z [M+2]⁺

calculated for C₄₂H₅₁N₇O₄: 719.4; found: 719.9.

3.13. General Procedure for Removal of the N-Pbf Protecting Group. Synthesis of N-[2-[4-benzyl-5-

oxo-(2RS)-[2-phenyl-(1S)-(3-phenyl-ureido)ethyl]piperazin-1-yl]acetyl]-Arg-NHBn

Trifluoroacetate (19c)

The epimeric mixture of the Arg(Pbf) -derived phehylureido-piperazine 17c [(R:S) = (3:1)]

(295 mg, 0.30 mmol) was dissolved in TFA/H₂O/TIS mixture (90:5:5; 5 mL) and the mixture was

stirred at room temperature for 5 h. Afterwards, the TFA was evaporated under stream of argon and the

residue was centrifuged three times in diethyl ether (10 mL) at 5000 rpm and −15 °C for 15 min. The

residue was dissolved in CH₃CN/H₂O (1:3, 2 mL) and the solution was lyophilized. The epimeric

mixture of trifluoroacetate salts 19c [(R:S) = (3:1)] was obtained quantitatively (254 mg, 100%). HPLC

t_R: 16.60 min [(R)-19c] and 16.87 min [(S)-19c]; H-NMR (500 MHz, DMSO-d₆) (R)-19c δ (ppm):

1.44 (m, 2H, γ-H), 1.63 (m, 1H, β-H), 1.72 (m, 1H, β-H), 2.71 (dd, 1H, J = 9 and 13.5 Hz, CH₂-Ph),

2.95 (m, 1H, 2-H), 2.96 (m, 1H, CH₂-Ph), 3.03 (m, 2H, δ-H), 3.20 (m, 1H, 3-H), 3.28 (m, 1H, 6-H),

3.30 (m, 1H, 3-H), 3.31 (m, 2H, CH₂CO), 3.48 (d, 1H, J = 17 Hz, 6-H), 4.10 (m, 1H, 2-CH), 4.23

[m, 2H, CH₂(NHBn)], 4.30 [m, 1H, CH₂(NBn)], 4.32 (m, 1H, α-H), 4.72 [d, 1H, J = 15 Hz, CH₂

(NBn)], 6.49 (m, 1H, 2-CHNH), 6.73–7.36 (m, 20H, Ar), 7.76 [m, 1H, NHC(NH₂·CF₃CO₂H) = NH],

8.23 (d, 1H, J = 7 Hz, α-NH), 8.57 (t, 1H, J = 6 Hz, NHBn), 9.00 (m, 1H, NHPh). (S)-19c δ (ppm):

2.97 (m, 1H, δ-H), 3.07 (m, 1H, δ-H), 3.12 (m, 1H, CH₂CO), 3.14 (m, 1H, 3-H), 3.23 (m, 1H, 6-H),

3.30 (m, 1H, 3-H), 3.42 (m, 1H, CH₂CO), 3.77 (d, 1H, J = 16.5 Hz, 6-H), 3.91 (m, 1H, 2-CH), 4.04

[m, 1H, CH₂(NHBn)], 4.30 [m, 1H, CH₂(NHBn)], 4.43 [d, 1H, J = 15 Hz, CH₂(NBn)], 4.54 [d, 1H,

J = 15 Hz, CH₂(NBn)], 6.73–7.36 (m, 20H, Ar), 7.76 [m, 1H, NHC(NH₂·CF₃CO₂H) = NH], 8.59

(m, 1H, NHBn), 9.00 (m, 1H, NHPh); C-NMR (125 MHz, DMSO-d₆) (R)-19c δ (ppm): 25.4 [C_γ],

29.7 [C_β], 37.6 [CH₂-Ph], 40.8 [C_δ], 42.5 [CH₂(NHBn)], 45.3 [C₃], 49.5 [CH₂(NBn)], 50.8 [C₂-CH],

52.6 [C_α], 54.9 [C₆], 55.4 [CH₂CO], 59.9 [C₂], 118.2, 121.5, 126.4, 127.2, 217.3, 127.5, 128.1, 128.6,

128.7, 129.0, 129.7 [20CH (Ar)], 137.6 [C (NBn)], 139.2 [C (Ph)], 139.7 [C (NHBn)], 140.9

[C (NHPh)], 157.1 [CO (Urea)], 155.4 [C (NHC(NH₂) = N)], 167.1 [C₅], 170.0 [CO], 171.8

[α-CONH]. (S)-19c δ (ppm): 40.8 [C_δ], 42.3 [CH₂(NHBn)], 46.1 [C₃], 48.9 [CH₂(NBn)], 51.6

[C₂-CH], 54.9 [C₆], 55.4 [CH₂CO], 118.0, 121.5, 126.4, 127.2, 217.3, 127.6, 128.2, 128.4, 128.6,

129.0, 129.7 [20CH (Ar)], 138.0 [C (NBn)], 139.2 [C (Ph)], 139.7 [C (NHBn)], 140.9 [C (NHPh)],

157.5 [CO (Urea)], 155.8 [C (NHC(NH₂) = N)], 170.6 [CO], 173.0 [α-CONH]; ES-MS m/z [M+1]⁺

calculated for C₄₁H₄₉N₉O₄: 732.4; found: 732.7.

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3.14. General Procedure for the Synthesis of the Indazole-Derived Ureas 23b,c

Propylene oxide (19 µL, 0.27 mmol) was added to a 0 °C cooled solution of 1-(2,6-dichlorobenzyl)-

6-amino-3-(pyrrolidin-1-ylmethyl)-1H-indazol [24] (83 mg, 0.22 mmol) in dry THF (4 mL). Then,

a solution of bis(trichloromethyl)carbonate (24 mg, 0.082 mmol) in dry THF (1 mL) was added

dropwise and stirring was maintened at 0 °C for 15 min. Afterwards, the mixture was added dropwise

to a 0 °C cooled solution of the corresponding epimeric mixture of hydrochlorides 16b,c (0.22 mmol)

and Et₃N (17 µL, 0.48 mmol) in dry THF (5 mL) and stirred for 2h. Then, the solvent was removed

under reduced pressure and the residue was dissolved in CH₂Cl₂(50 mL). The solution was washed

with H₂O (2 × 10 mL), brine (10 mL), dried over Na₂SO₄, and evaporated to dryness. The residue was

purified by reverse phase chromatography, using 10%–100% CH₃CN gradient in 0.05% TFA solution

in H₂O as mobile phase, to afford the desired compounds 23b,c.

N-[2-[4-Benzyl-(2RS)-[(1S)-[3-(1-(2,6-dichlorobenzyl)-3-(pyrrolidin-1-ylmethyl)-1H-indazol-6-yl)ureido]

-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-Lys(Z)-NHBn (23b). Amorphous solid [(R:S) = (3:1)]

(74 mg, 30%); HPLC t_R: 19.99 min; ¹H-NMR (500 MHz, CDCl₃) (R)-23b δ (ppm): 1.26 (m, 2H, γ-H),

1.33 (m, 2H, δ-H), 1.70 (m, 1H, β-H), 1.72 (m, 2H, pyrrolidine), 1.82 (m, 1H, β-H), 1.88 (m, 2H,

pyrrolidine), 2.68 (dd, 1H, J = 6.5 and 14 Hz, CH₂-Ph), 2.90 (m, 1H, J = 8 and 14 Hz, CH₂-Ph), 2.96

(m, 1H, 2-H), 2.98 (m, 2H, ε-H), 3.00 (m, 2H, pyrrolidine), 3.27 (m, 1H, 6-H), 3.42 (m, 1H, 3-H), 3.45

(m, 4H, pyrrolidine and CH₂CO), 3.47 (m, 1H, 3-H), 3.53 (d, 1H, J = 16 Hz, 6-H), 4.18 [m, 1H,

J = 14.5 Hz, CH₂(NBn)], 4.28 [m, 1H, CH₂(NHBn), 4.30 (m, 1H, 2-CH), 4.32 [m, 1H, CH₂(NHBn),

4.35 (s, 2H, CH₂-pyrrolidine), 4.40 (m, 1H, α-H), 4.95 [m, 1H, CH₂(NBn], 4.98 [s, 2H, CH₂(Z)], 5.36

(m, 1H, NHZ), 5.50 (s, 2H, CH₂-diClPh), 6.83 (d, 2H, J = 8 Hz, Ar), 7.02 (d, 2H, J = 7 Hz, Ar),

7.12–7.35 (m, 22H, Ar and NHBn), 7.91 (s, 1H, Ar), 8.06 (m, 1H, α-NH), 8.58 (m, 1H, 2-CHNH),

11.67 [m, 1H, Indz-NH (Urea)]. (S)-23b δ (ppm): 1.35 (m, 2H, δ-H), 1.72 (m, 2H, pyrrolidine), 1.88

(m, 2H, pyrrolidine), 3.00 (m, 2H, pyrrolidine), 3.24 (m, 1H, 3-H), 3.43 (m, 2H, pyrrolidine), 3.45

(m, 1H, 3-H), 4.35 (s, 2H, CH₂-Pyrrolidine), 4.38 (m, 1H, α-H), 4.94 [s, 2H, CH₂(Z)], 5.36 (m, 1H,

NHZ), 5.50 (s, 2H, CH₂-diClPh), 6.76 (m, 2H, Ar), 7.02 (m, 2H, Ar), 7.12–7.35 (m, 22H, Ar and

NHBn), 7.91 (s, 1H, Ar), 8.10 (m, 1H, α-NH), 8.70 (m, 1H, 2-CHNH), 11.67 [m, 1H, Indz-NH (Urea)];

¹³C-NMR (125 MHz, CDCl₃) (R)-23b δ (ppm): 22.8 [C_γ], 23.4 [2CH₂(pyrrolidine)], 29.1 [C_δ], 31.5

[C_β], 37.7 [CH₂-Ph], 40.3 [C_ε], 43.5 [CH₂(NHBn)], 44.2 [C₃], 47.6 [CH₂-diClPh], 48.2 [CH₂-pyrrolidine],

49.5 [CH₂(NBn)], 51.6 [C₂-CH], 52.4 [2CH₂(pyrrolidine)], 53.4 [C_α], 55.2 [C₆], 57.5 [CH₂CO], 60.7

[C₂], 66.6 [CH₂(Z)], 98.0, 116.0, 118.6 [3CH (Ar)], 119.2 [C (Ar)], 126.8, 127.3, 127.9, 128.0, 128.2,

128.4, 128.5, 128.6, 128.7, 128.9, 130.1 [23CH (Ar)], 131.3, 133.7 [2C (Ar)], 135.9 [C (NBn)], 136.5

[C (Z)], 136.8 [3C (Ph and Ar)], 137.7 [C (NHBn)], 139.3, 137.7 [2C (Ar)], 155.8 [CO (Z)], 156.8

[CO (Urea)], 166.9 [C₅], 169.6 [CO], 172.5 [α-CONH]. (S)-23b δ (ppm): 23.4 [2CH₂(pyrrolidine)],

29.1 [C_δ], 44.2 [C₃], 47.6 [CH₂-diClPh], 48.2 [CH₂-pyrrolidine], 52.4 [2CH₂(pyrrolidine)], 53.4 [C_α],

66.6 [CH₂(Z)], 98.0, 115.2, 117.5 [3CH (Ar)], 119.2 [C (Ar)], 126.8, 127.4, 127.9, 128.0, 128.2,

128.4, 128.5, 128.6, 128.7, 128.9, 130.1 [23CH (Ar)], 131.3, 133.7 [2C (Ar)], 135.9 [C (NBn)], 136.5

[C (Z)], 136.8 [3C (Ph and Ar)], 137.7 [C (NHBn)], 139.3, 137.7 [2C (Ar)], 155.8 [CO (Z)], 156.8 [CO

(urea)], 172.5 [α-CONH]; ES-MS m/z 1120.9 [M+1]⁺; C₆₂H₆₈Cl₂N₁₀O₆(%): C: 66.48, H: 6.12, N:

12.50. Found (%): C: 66.79, H: 6.38, N: 12.34.

Molecules 2014, 19

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N-[2-[4-Benzyl-(2RS)-[(1S)-[3-(1-(2,6-dichlorobenzyl)-3-(pyrrolidin-1-ylmethyl)-1H-indazol-6-yl)ureido]

-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-Arg(Pbf)-NHBn (23c). Amorphous solid [(R:S) = (3:1)]

(106 mg, 38%); HPLC t_R: 20.38 min [(R)-23c] and 21.40 min [(S)-23c]; H-NMR (500 MHz,

(CD₃)₂CO) (R)-23c δ (ppm): 1.30 [s, 6H, 2CH₃(Pbf)], 1.40 (m, 2H, γ-H), 1.60 (m, 1H, β-H), 1.73

(m, 1H, β-H), 1.86 [s, 3H, CH₃(Pbf)], 1.88 (m, 2H, pyrrolidine), 1.97 (m, 2H, pyrrolidine), 2.36 [s,

3H, CH₃(Pbf)], 2.44 [s, 3H, CH₃(Pbf)], 2.72 (m, 2H, CH₂-Ph), 2.86 [s, 2H, CH₂(Pbf)], 3.00 (m, 1H,

δ-H), 3.08 (m, 1H, 2-H), 3.10 (m, 1H, δ-H), 3.26 (m, 1H, CH₂CO), 3.32 (m, 1H, 6-H), 3.34 (m, 2H,

pyrrolidine), 3.52 (m, 1H, CH₂CO), 3.57 (m, 1H, 6-H), 3.58 (m, 1H, 3-H), 3.62 (m, 1H, 3-H), 3.68 (m,

2H, pyrrolidine), 4.20 [m, 1H, CH₂(NBn)], 4.34 [m, 1H, CH₂(NHBn)], 4.38 [m, 2H, 2-CH and CH₂

(NHBn)], 4.39 (m, 1H, α-H), 4.66 (s, 2H, CH₂-pyrrolidine), 4.88 [d, 1H, J = 15 Hz, CH₂(NBn)], 5.55

(d, 2H, J = 6 Hz, CH₂-diClPh), 6.39 [m, 3H, NHC(NH₂) = N], 6.95–7.34 (m, 21H, Ar), 7.86 (m, 1H,

NHBn), 8.26 (d, 1H, J = 6 Hz, α-NH), 9.26 (m, 1H, 2-CHNH), 10.32 [m, 1H, Indz-NH (Urea)]. (S)-23c

δ (ppm): 1.30 [s, 6H, 2CH₃(Pbf)], 1.86 [s, 3H, CH₃(Pbf)], 1.88 (m, 2H, pyrrolidine), 1.97 (m, 2H,

pyrrolidine), 2.36 [s, 3H, CH₃(Pbf)], 2.44 [s, 3H, CH₃(Pbf)], 2.68 (m, 2H, CH₂-Ph), 2.80 (m, 2H,

CH₂-Ph), 2.86 [s, 2H, CH₂(Pbf)], 3.34 (m, 2H, pyrrolidine), 3.68 (m, 2H, pyrrolidine), 4.10 [dd, 1H,

J = 6 and 15 Hz, CH₂(NHBn)], 4.37 (m, 1H, α-H), 4.40 [m, 1H, CH₂(NHBn)], 4.43 [d, 1H, J = 14 Hz,

CH₂(NBn)], 4.58 [m, 1H, CH₂(NBn)], 4.66 (m, 2H, CH₂-pyrrolidine), 5.55 (m, 2H, J = 6 Hz,

CH₂-diClPh), 6.50 [m, 3H, NHC(NH₂) = N], 6.95–7.34 (m, 21H, Ar), 7.91 (m, 1H, NHBn), 8.26 (m,

1H, α-NH), 9.26 (m, 1H, 2-CHNH), 10.32 [m, 1H, Indz-NH (urea)]; ¹³C-NMR (125 MHz, (CD₃)₂CO)

(R)-23c δ (ppm): 13.2, 19.0, 20.2 [3CH₃(Pbf)], 24.5 [2CH₂(pyrrolidine)], 27.5 [C_γ], 28.6 [2CH₃

(Pbf)], 31.3 [C_β], 39.2 [CH₂-Ph], 41.4 [C_δ], 44.0 [CH₂(NHBn)], 44.3 [CH₂(Pbf)], 45.7 [C₃], 49.0

[CH₂-diClPh], 50.5 [CH₂(NBn)], 50.7 [CH₂-pyrrolidine], 54.1 [C₂-CH and C_α], 55.1 [2CH₂

(pyrrolidine)], 56.0 [C₆], 59.5 [CH₂CO], 63.2 [C₂], 87.5 [C (Pbf)], 98.9, 117.2 [2CH (Ar)], 118.4

[C (Pbf)], 118.6 [CH (Ar)], 119.7 [C (Ar)], 121.4 [CH (Ar)], 126.2 [C (Pbf)], 127.7, 128.2, 128.7,

129.6, 129.8, 130.1, 130.2, 130.8, 132.1 [17CH (Ar)], 133.5, 135.7 [2C (Pbf)], 136.7, 138.3 [3C (Ar)],

139.0 [C (NBn)], 139.5 [C (Pbf)], 140.3 [C (Ph)], 140.9 [C (NHBn)], 141.9, 143.5 [3C (Ar)], 157.6

[CO (Urea)], 160.9 [C (NHC(NH₂) = N)], 159.7 [C (Pbf)], 168.3 [C₅], 171.1 [CO], 173.2 [α-CONH].

(S)-23c δ (ppm): 13.2, 19.0, 20.2 [3CH₃(Pbf)], 24.5 [2CH₂(pyrrolidine)], 28.6 [2CH₃(Pbf)], 39.2

[CH₂-Ph], 44.0 [CH₂(NHBn)], 44.3 [CH₂(Pbf)], 49.0 [CH₂-diClPh], 50.4 [CH₂(NBn)], 50.7

[CH₂-pyrrolidine], 54.1 [C_α], 55.1 [2CH₂(pyrrolidine)], 87.5 [C (Pbf)], 98.9, 117.2 [2CH (Ar)], 118.4

[C (Pbf)], 118.6 [CH (Ar)], 119.1 [C (Ar)], 121.4 [CH (Ar)], 126.2 [C (Pbf)], 127.7, 128.2, 128.8,

129.6, 129.8, 130.1, 130.2, 131.0, 132.1 [17CH (Ar)], 133.5, 135.7 [2C (Pbf)], 136.7, 138.3 [3C (Ar)],

139.1 [C (NBn)], 139.5 [C (Pbf)], 140.3 [C (Ph)], 140.9 [C (NHBn)], 141.9, 143.5 [3C (Ar)], 158.1

[CO (Urea)], 160.9 [C (NHC(NH₂) = N)], 159.7 [C (Pbf)]; ES-MS m/z 1267.7 [M+1]⁺;

C₆₇H₇₈Cl₂N₁₂O₇S (%): C: 63.54, H: 6.21, N: 13.27. Found (%): C: 63.78, H: 6.02, N: 13.39.

3.15. N-Z Removal in 23b. Synthesis of N-[2-[4-Benzyl-(2RS)-[(1S)-[3-(1-(2,6-dichlorobenzyl)-3-

(pyrrolidin-1-ylmethyl)-1H-indazol-6-yl)ureido]-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-Lys-

NHBn hydrochloride (24b)

It was carried out by applying the general methodology for N-Z removal above described for

the synthesis of (11–12)a,b. Amorphous solid [(R:S) = (3:1)] (70 mg, 100%); HPLC t_R: 14.99 min;

Molecules 2014, 19

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¹H-NMR (500 MHz, DMSO-d₆) (R)-24b δ (ppm): 1.30 (m, 2H, γ-H), 1.50 (m, 2H, δ-H), 1.62 (m, 1H,

β-H), 1.72 (m, 1H, β-H), 1.79 (m, 2H, pyrrolidine), 1.85 (m, 2H, pyrrolidine), 2.74 (dd, 1H, J = 10 and

14 Hz, CH₂-Ph), 2.96 (m, 1H, CH₂-Ph), 2.65 (m, 3H, 5-H and ε-H), 3.05 (m, 2H, pyrrolidine), 3.35

(m, 2H, pyrrolidine), 3.44–3.98 (m, 6H, 3-H, 6-H and CH₂CO), 4.25 [d, 1H, J = 6 Hz, CH₂(NHBn),

4.27 [d, 1H, J = 6 Hz, CH₂(NHBn), 4.33 (m, 2H, 2-CH and α-H), 4.46 [m, 1H, CH₂(NBn], 4.57

(d, 2H, J = 5 Hz, CH₂-pyrrolidine), 4.72 [d, 1H, J = 15 Hz, CH₂(NBn], 5.59 (s, 2H, CH₂-diClPh), 7.01

(d, 1H, J = 8 Hz, Ar), 6.96–7.46 (m, 16H, Ar), 7.52 (d, 2H, J = 8 Hz, Ar), 7.83 (d, 1H, J = 9 Hz, Ar),

7.86 (s, 1H, Ar), 7.95 (m, 3H, NH₂·HCl), 8.45 (m, 1H, α-NH), 8.63 (t, 1H, J = 6 Hz, NHBn), 9.35

(s, 1H, 2-CHNH), 11.68 [m, 2H, Indz-NH (urea) and N·HCl (pyrrolidine)]. (S)-24b δ (ppm): 1.79

(m, 2H, pyrrolidine), 1.85 (m, 2H, pyrrolidine), 2.74 (m, 1H, CH₂-Ph), 2.96 (m, 1H, CH₂-Ph), 3.05

(m, 2H, pyrrolidine), 3.35 (m, 2H, pyrrolidine), 4.22 [m, 1H, CH₂(NHBn), 4.28 [m, 1H, CH₂(NHBn),

4.57 (d, 2H, J = 5 Hz, CH₂-pyrrolidine), 4.72 [d, 1H, J = 15 Hz, CH₂(NBn], 5.59 (s, 2H, CH₂-diClPh),

7.01 (d, 1H, J = 8 Hz, Ar), 6.96–7.46 (m, 16H, Ar), 7.52 (d, 2H, J = 8 Hz, Ar), 7.83 (d, 1H, J = 9 Hz,

Ar), 7.86 (s, 1H, Ar), 7.95 (m, 3H, NH₂·HCl), 8.45 (m, 1H, α-NH), 8.63 (m, 1H, NHBn), 9.35 (s, 1H,

2-CHNH), 11.68 [m, 2H, Indz-NH (Urea) and N·HCl (pyrrolidine)]; ¹³C-NMR (125 MHz, DMSO-d₆)

(R)-24b δ (ppm): 22.3 [C_γ], 22.6 [2CH₂(pyrrolidine)], 26.4 [C_δ], 31.4 [C_β], 37.9 [CH₂-Ph], 38.4 [C_ε],

42.0 [CH₂(NHBn)], 44.1 [C₃], 47.2 [CH₂-diClPh], 47.7 [CH₂-pyrrolidine], 49.2 [CH₂(NBn)], 49.6

[C₂-CH], 52.5 [2CH₂(pyrrolidine), C_αand CH₂CO], 53.9 [C₆], 60.3 [C₂], 96.1, 114.5 [2CH (Ar)],

117.7 [C (Ar)], 120.6, 126.7, 127.0, 127.2, 127.6, 128.2, 128.5, 128.7, 129.2 [19CH (Ar)], 130.8, 131.4

[4C (Ar)], 135.4 [C (NBn)], 136.0 [C (Ph)], 139.2 [C (NHBn)], 141.2 [2C (Ar)], 155.2 [CO (urea)],

171.1 [α-CONH]. (S)-24b δ (ppm): 22.6 [2CH₂(pyrrolidine)], 37.8 [CH₂-Ph], 42.0 [CH₂(NHBn)],

47.2 [CH₂-diClPh], 47.7 [CH₂-pyrrolidine], 52.5 [2CH₂(pyrrolidine)], 96.1, 114.5 [2CH (Ar)], 117.7

[C (Ar)], 120.6, 126.7, 127.0, 127.2, 127.6, 128.1, 128.5, 128.7, 129.2 [19CH (Ar)], 130.8, 131.4 [4C

(Ar)], 135.4 [C (NBn)], 136.0 [C (Ph)], 139.6 [C (NHBn)], 141.2 [2C (Ar)], 155.2 [CO (urea)], 171.1

[α-CONH]; ES-MS m/z [(M+2)/2]⁺calculated for C₅₄H₆₂Cl₂N₁₀O₄: 493.2; found: 493.6.

3.16. N-Pbf Removal in 23c. Synthesis of N-[2-[4-Benzyl-(2RS)-[(1S)-[3-(1-(2,6-dichlorobenzyl)-3-

(pyrrolidin-1-ylmethyl)-1H-indazol-6-yl)ureido]-2-phenylethyl]-5-oxopiperazin-1-yl]acetyl]-Arg-

NHBn Trifluoroacetate (24c)

It was carried out by applying the above described methodology for N-Pbf removal in the Arg

derivative 19c. Amorphous solid [(R:S) = (3:1)] (104 mg, 100%); HPLC t_R: 15.14 min [(R)-24c] and

15.72 min [(S)-24c]; H-NMR (500 MHz, (CD₃)₂CO) (R)-24c δ (ppm): 1.44 (m, 2H, γ-H), 1.60 (m,

1H, β-H), 1.84 (m, 1H, β-H), 1.83 (m, 4H, pyrrolidine), 2.75 (dd, 1H, J = 9.5 and 14 Hz, CH₂-Ph), 3.00

(m, 1H, 2-H), 3.04 (m, 2H, δ-H), 3.06 (m, 1H, CH₂-Ph), 3.20 (m, 1H, CH₂CO), 3.26 (m, 1H, 3-H),

3.32 (m, 1H, 6-H), 3.35 (m, 4H, pyrrolidine), 3.39 (m, 1H, 3-H), 3.42 (m, 1H, CH₂CO), 3.47 (m, 1H,

6-H), 4.21 (m, 1H, 2-CH), 4.27 [d, 2H, J = 6 Hz, CH₂(NHBn)], 4.33 [d, 1H, J = 15 Hz, CH₂(NBn)],

4.40 (td, 1H, J = 5.5 and 8 Hz, α-H), 4.58 (s, 2H, CH₂-pyrrolidine), 4.74 [d, 1H, J = 15 Hz, CH₂

(NBn)], 5.60 (s, 2H, CH₂-diClPh), 6.37 (m, 1H, 2-CHNH), 6.90–7.44 (m, 18H, Ar), 7.52 [m, 2H,

NHC(NH₂·CF₃CO₂H) = NH and Ar], 7.72 (d, 1H, J = 9 Hz, Ar), 7.92 (s, 1H, Ar), 8.09 (d, 1H,

J = 8 Hz, α-NH), 8.58 (t, 1H, J = 6 Hz, NHBn), 8.77 [m, 1H, Indz-NH (Urea)], 9.95 [m, 1H,

N·CF₃CO₂H (Pyrrolidine)]. (S)-24c δ (ppm): 1.83 (m, 4H, pyrrolidine), 2.63 (m, 1H, CH₂-Ph), 2.82

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(m, 1H, CH₂-Ph), 3.30 (m, 1H, 6-H), 3.35 (m, 4H, pyrrolidine), 3.50 (m, 1H, 6-H), 4.04 (m, 1H,

2-CH), 4.14 [m, 1H, CH₂(NHBn)], 4.28 [m, 1H, CH₂(NHBn)], 4.58 (s, 2H, CH₂-pyrrolidine), 5.57

(s, 2H, CH₂-diClPh), 6.90–7.44 (m, 18H, Ar), 7.52 [m, 2H, NHC(NH₂·CF₃CO₂H) = NH and Ar], 7.70

(d, 1H, J = 9 Hz, Ar), 7.95 (s, 1H, Ar), 8.57 (m, 1H, NHBn), 9.95 [m, 1H, N·CF₃CO₂H (pyrrolidine)];

¹³C-NMR (125 MHz, (CD₃)₂CO) (R)-24c δ (ppm): 23.0 [2CH₂(pyrrolidine)], 25.5 [C_γ], 30.0 [C_β],

37.8 [CH₂-Ph], 40.4 [C_δ], 42.5 [CH₂(NHBn)], 44.9 [C₃], 47.7 [CH₂-diClPh], 48.6 [CH₂-pyrrolidine],

49.6 [CH₂(NBn)], 50.5 [C₂-CH], 52.2 [C_α], 53.5 [2CH₂(pyrrolidine)], 54.4 [C₆], 55.2 [CH₂CO], 59.7

[C₂], 96.8, 115.1 [2CH (Ar)], 118.0 [C (Ar)], 120.7 [CH (Ar)], 126.5, 127.2, 127.5, 128.0, 128.5,

128.7, 129.0, 129.2, 129.7 [18CH (Ar)], 131.4, 131.9, 136.5 [4C (Ar)], 137.4 [C (NBn)], 139.0 [C

(Ph)], 139.6 [C (NHBn)], 140.2, 141.9 [2C (Ar)], 157.0 [CO (urea)], 155.3 [C (NHC(NH₂) = N)],

167.0 [CO], 169.8 [C₅], 171.6 [α-CONH]. (S)-24c δ (ppm): 23.0 [2CH₂(pyrrolidine)], 37.8 [CH₂-Ph],

42.5 [CH₂(NHBn)], 47.7 [CH₂-diClPh], 48.6 [CH₂-pyrrolidine], 50.4 [C₂-CH], 53.5 [2CH₂

(pyrrolidine)], 54.4 [C₆], 96.8, 115.1 [2CH (Ar)], 118.0 [C (Ar)], 120.7 [CH (Ar)], 126.5, 127.4, 127.6,

128.3, 128.5, 128.7, 129.0, 129.2, 129.6 [18CH (Ar)], 131.4, 131.9, 136.5 [4C (Ar)], 137.4 [C (NBn)],

139.0 [C (Ph)], 139.6 [C (NHBn)], 140.2, 141.9 [2C (Ar)], 155.3 [C (NHC(NH₂) = N)]; ES-MS m/z

[(M+2)/2]⁺calculated for C₅₄H₆₂Cl₂N₁₂O₄: 507.2; found: 507.

4. Conclusions

In summary, a series of highly functionalized Phe-Gly dipeptide-derived piperazinones containing

an aromatic urea moiety and a basic amino acid has been prepared and evaluated as human PAR1

antagonists in a platelet aggregation assay. The synthetic strategy involves coupling of a protected

basic amino acid benzyl amide to 1,2- and 1,2,4-substituted-piperazinone derivatives, through a

carbonylmethyl group at the N_1-position, followed by formation of an aromatic urea at the exocyclic

moiety linked at the C₂position of the piperazine ring and removal of protecting groups. In comparison

with the 1,2,4,6-tetrasusbtituted-piperazinone analogues A, the change of position of the basic amino

acid side chain from C₆to N₁in B has led to the complete loss of PAR1 antagonist activity and tumor

cell cytotoxicity.

Supplementary Materials

Copies of H and ¹³C-NMR spectra for all new compounds. Supplementary materials can be

accessed at: http://www.mdpi.com/1420-3049/19/4/4814/s1.

Acknowledgments

This work was supported by the Spanish Ministerio de Ciencia e Innovación grant SAF2009-09323

and the Ministerio de Economía y competitividad grant SAF2012-32209. A. M. Valdivielso held

a postgraduate FPU fellowship from the Spanish Ministerio de Educación. A. M. Valdivielso thanks

the SEQT (Spanish Society of Therapeutic Chemistry) for the Jassen-Cilag Award for young

researches (XV Edition, 2011).

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DOI: 10.3390/molecules19044814

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Article abstract of DOI:10.3390/molecules19044814

Full text of DOI:10.3390/molecules19044814

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