Synthesis of N-urethane protected amino alkyl (S-methyl)-isothiouronium compounds and carbodiimide tethered peptidomimetics: an application for guanidino and substituted guanidino peptidomimetics synthesis

Abstract

The synthesis of N,N′-disubstituted and N,N′,N′′-trisubstituted guanidine linked peptidomimetic molecules suitably decorated in the peptide backbone has been delineated. N^α-Protected amino acid derived S-methyl isothiouronium derivatives are employed as the key intermediates for the synthesis of guanidinopeptide mimics. Synthesis of a new class of carbodiimide tethered dipeptidomimetics has also been outlined wherein a Staudinger-aza-Wittig type reaction between amino alkyl azide and isothiocyanato esters is employed. Thus obtained carbodiimides have been demonstrated as starting materials for the construction of guanidino peptide mimics as well as an array of trisubstituted guanidine mimetics bearing N-hydroxy, cyano and amino function as third substitutions at the guanidino unit in the backbone.

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Introduction

Peptides are not ideal therapeutical agents per se. Their in vivo action is constrained by factors such as proteolytic degradation by proteases and poor cell membrane permeability. One prominent approach to improve the pharmaco-kinetic properties is to substitute peptides with synthetic analogs called peptide mimics¹ and one such class is the backbone modified peptides.² In these compounds the peptide backbone contains one or more unnatural linkages. Our group has described different classes of backbone modified peptides by inserting ureido,³ thioureido⁴ and selenoureido⁵ subunits as unnatural tethers. We now delineate our studies on the synthesis of guanidino peptide mimics under simple and mild conditions employing commonly used N-protectors namely Fmoc, Boc and Cbz groups.

A major drawback of most of the non-native linkages is that they tend to decrease the solubility of the pseudo-peptides in aqueous solvents as well as cause aggregation in such solvents. The problem has been partly addressed by the incorporation into the peptide backbone of guanidino group/s, which remains protonated at physiological pH and hence renders hydrophilicity.⁶

This is due to the strong basicity of guanidine moiety which ensures protonation over a wide pH range. Also guanidinium functionality is useful as strong and selective anion-binding site in receptor designs for small oxoanionic target molecules or π-electron rich aromatic moieties of substrates.⁷

The oligomeric guanidines may be valuable in the design of protein ligands wherein the guanidine backbone may interact with target proteins by electrostatic interactions and/or hydrogen bonds.⁸ Also the replacement of one or more peptide bonds with unnatural linkages tend to improve the pharmacokinetic properties of the bioactive peptides.⁹

Though several amino acid derived guanidines (Fig. 1) exist in the literature, the insertion of guanidine into a peptide backbone are limited to two reports, wherein Pbf-activated thiourea and di-Boc-isothiouronium derivatives were utilized for guanidinylation. Fan et al.,⁸ reported the solid phase as well as solution phase synthesis of oligomeric guanidines by making use of Pbf-activated thiourea as monomer units (Fig. 2).

Fig. 1 Examples of amino acid derived guanidines.

Fig. 2 Reported oligomeric guanidines synthesized in N → C direction (B) and C → N direction (C).

The other one delineates the synthesis of dipeptide consisting of α- and γ-amino acid with guanidinium group as an amide bond replacement in the main chain employing 1,3-di-Boc-2-methyl-isothiourea as guanidinylating agent.⁷

Several methods have been developed for the preparation of substituted guanidines in solution.^10,11 The reaction of amines with S-alkylisothioureas (Rathke procedure) is an alternative to the use of guanidinylating agents (Fig. 3) for the synthesis of substituted guanidines.¹² Here, the reaction involves nucleophilic displacement of the alkyl thiol via an addition–elimination pathway. The S-alkylisothiouronium salt can be easily prepared by treatment of thiourea with an alkylating agent.

Fig. 3 Thiourea and non-thiourea based guanylating reagents.

In the present study, we describe the preparation of N-protected amino acid derived isothiouronium derivatives as guanylating agents and their utility in the preparation of guanidinopeptides under mild conditions. Isothiouronium compounds can serve as important intermediates for the synthesis of thiols.^13a Also they are reported to be useful in the condensation reactions with aldehyde and amine to yield triazine derivatives.^13b

Results and discussion

N-Protected amino acid derived S-methyl isothiouronium derivatives [Pg-Xaa-ψCH₂-N-C(SMe)-NH₂] 3

In the initial part of the study, the feasibility of thioureidopeptides to serve as the precursors for guanidine tethered peptide mimics was explored. The reaction of thioureidopeptides with ammonia or NH₄Cl in presence of HgCl₂ and Mukaiyama reagent was not successful. Consequently, the other route comprising of the S-methyl isothiouronium derivative of amino acids was conceived. Isothiouronium compounds are a class of amino acid derivatives that can be made starting from N^α-protected amino alkyl isothiocyanates and subsequently utilized as key intermediates for the desired target molecules.

Monosubstituted thiourea derivatives can be accessed through the reaction of primary amines with thiocyanates such as KSCN, NH₄SCN¹⁴ or coupling with benzoyl isothiocyanate followed by deprotection of benzoyl group.¹⁵ Stadlwieser et al.,¹⁶ reported the synthesis of resin bound amino ester derived thioureas, wherein amino acid ester was treated with allyloxycarbonyl (Alloc)-isothiocyanate followed by the removal of Alloc group.

Initially, N^α-protected amino alkyl monosubstituted thioureas 2 were obtained by the reaction of N^α-protected amino alkyl isothiocyanate⁴ with NH₄HCO₃ (ref. 17) in THF/H₂O at 0 °C (Scheme 1). The reaction is clean and complete conversion of isothiocyanate to thiourea was observed within 2–3 h and crude products were then purified through column chromatography (compounds 2a–h; Table 1). The products were characterized by ¹H and ¹³C NMR analyses. The chiral HPLC profile of Fmoc-L-Phg-ψ[CH₂-NH-CS-NH₂] 2e and Fmoc-D-Phg-ψ[CH₂-NH-CS-NH₂] 2e′ made from L-Phg and D-Phg (the amino acid with high tendency to lose optical integrity) showed only one peak R_t = 25.85 min and 29.81 min respectively, (80 : 20 n-hexane/isopropanol in isocratic mode in 40 min). Co-injection of 2e and 2e′ showed two well separated peaks (R_t = 25.31 and R_t = 30.21 min). Thus it can be concluded that the compounds 2 made via the present protocol were optically pure. Also, the synthesized compounds were found to be shelf stable to storage for several weeks at room temperature.

Scheme 1

Table 1 List of N-protected amino acid derived thioureas and S-methylisothiouronium derivatives

Entry	Thiourea	[α]^25D (c 1, CHCl3)	Yield (%)	Entry	Isothiouronium derivative	[α]^25D (c 1, CHCl3)	Yield (%)
2a	Fmoc-Val-ψCH2-NH-C(S)-NH2	30.16	81	3a	Fmoc-Val-ψCH2-N-C(SMe)-NH2	42.3	88
2b	Fmoc-Leu-ψCH2-NH-C(S)-NH2	26.08	84	3b	Fmoc-Leu-ψCH2-N-C(SMe)-NH2	62.4	90
2c	Fmoc-Gly-ψCH2-NH-C(S)-NH2	—	82	3c	Fmoc-Gly-ψCH2-N-C(SMe)-NH2	—	89
2d	Fmoc-Pro-ψCH2-NH-C(S)-NH2	41.04	79	3d	Fmoc-Pro-ψCH2-N-C(SMe)-NH2	16.3	86
2e	Fmoc-Phg-ψCH2-NH-C(S)-NH2	−29.2	72	3e	Fmoc-Phg-ψCH2-N-C(SMe)-NH2	−21.3	82
2f	Cbz-Ile-ψCH2-NH-C(S)-NH2	21.4	73	3f	Cbz-Ile-ψCH2-N-C(SMe)-NH2	4.6	88
2g	Boc-Phe-ψCH2-NH-C(S)-NH2	−5.04	88	3g	Boc-Phe-ψCH2-N-C(SMe)-NH2	−1.1	96
2h	Boc-Ala-ψCH2-NH-C(S)-NH2	38.12	94	3h	Boc-Ala-ψCH2-N-C(SMe)-NH2	18.5	98

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Compounds 2 and iodomethane were then refluxed in acetone to obtain corresponding S-methyl isothiouronium derivative 3 as the salts of HI (compounds 3a–h; Scheme 1 and Table 1). The reaction was complete within an hour affording the products in excellent yield. Evaporation of the solvent was sufficient to isolate the pure products.

N-protected amino acid derived guanidinopeptide mimics [Pg-Xaa-ψCH₂-NH-C(NH)-NH-Xbb-COOY] 4a–c, 4e, 4g and h

The S-methylisothiouronium salts 3 were deprotonated with 1.0 equiv of NMM and then treated with amino acid ester in acetonitrile. The reaction mixture was heated at 60 °C to obtain the crude (Scheme 1). Thus obtained products were column chromatographed using neutral alumina to obtain pure products (compounds 4a–c, 4e, 4g and h; Table 2). The compounds were then characterized by mass spectrometry, ¹H and ¹³C NMR analyses.

Table 2 List of N-protected guanidine tethered dipeptidomimetics

Entry	Pg	R^1	R^2	X	Yield (%)
4a	Fmoc	–CH(CH3)2	–CH3	Me	59
4b	Fmoc	–CH2CH(CH3)2	–CH2CH2	Me	66
4c	Fmoc	–H	–CH2C6H5	Me	63
4e	Fmoc	–C6H5	–CH3	Me	61
4g	Boc	–CH2C6H5	–CH(CH3)2	Me	71
4h	Boc	–CH3	–CH(CH3)2	Et	68

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Test for epimerisation

Fan et al., have not emphasized on establishing the optical purity of the monomers as well as the oligomeric guanidines. The optical purity of the N-protected guanidinopeptide mimic was evaluated by the ¹H NMR study of the model compounds 4e and 4e′ prepared via the present protocol is illustrated in Fig. 4. The diastereomeric guanidinopeptide mimics 4e and 4e′ were synthesized by coupling Fmoc-Phg-ψ[CH₂NC(SMe)NH₂] 3e with L- and D-Ala-OMe respectively in separate experiments. ¹H NMR of 4e and 4e′ showed a single distinct methyl group doublet. Observed δ values for the –CH₃ group of the compounds are, 4e: 1.17, 1.18 and 4e′: 1.24, 1.25 ppm. Further, the mixture prepared by coupling 3e with racemic L,D-Ala-OMe showed methyl groups as two doublets with δ values 1.15, 1.16 and 1.23, 1.24 indicating the presence of two isomers and confirming that the protocol is free from epimerisation. The optical purity of compounds 4e and 4e′ was demonstrated to be high through chiral HPLC studies.¹⁸

Fig. 4 Diastereomeric guanidinodipeptide mimics 4e and 4e′ synthesized for epimerisation studies.

Synthesis of N-protected carbodiimide tethered dipeptidomimetics [Pg-Xaa-ψCH₂-(N=C=N)-Xbb-COOY] 5

For the synthesis of N,N′,N′′-trisubstituted guanidino peptide mimics such as, N-hydroxyguanidines,¹⁹ aminoguanidines²⁰ and cyanoguanidines,²¹ an alternative route need to be envisaged. Thus, we extended our study to develop a common protocol for the synthesis of diverse classes of trisubstituted guanidines. To access these molecules one of the best routes is the reaction of a suitable substituted amine with a carbodiimide.²²

The envisaged backbone carbodiimide mimics are itself a novel class of peptide mimetics. Previously peptidic carbodiimides were obtained in situ by the coupling of peptidyl amine with aryl isothiocyanate followed by desulfurization.^23,24 Carbodiimide linked amino esters illustrated in Fig. 5 were prepared through the reaction of azido ester with aryl isocyanates or by the dehydration of corresponding urea, which were N-terminal derived ones.^25–27

Fig. 5 Reported amino acid derived carbodiimides and peptidic carbodiimides.

Fan et al.,⁸ carried out the oligomeric guanidine synthesis employing Pbf-activated thiourea as monomer units and performed the guanidinylation in presence of EDC, wherein carbodiimides were key intermediates.

As we aimed to introduce carbodiimide at C-terminus of peptide backbone, we sought to employ Staudinger-aza-Wittig type reaction using N-protected amino alkyl azides and isothiocyanato esters. The intermolecular aza-Wittig type reaction of iminophosphoranes with heterocumulenes such as isocyanates/isothiocyanates can be carried out under neutral conditions²⁸ and thus amenable to the preparation of carbodiimide tethered peptidomimetics. Isothiocyanato esters undergo desired transformation with the extrusion of triphenylphosphinethioxide leading to hitherto unreported class of N-protected carbodiimide tethered dipeptidomimetics, which can be satisfactorily converted into guanidinopeptide mimics with diverse substitutions as demonstrated below.

In a typical experiment, Cbz-Phe-CH₂OH derived amino alkyl azide⁴ was treated with triphenylphosphine in dry CH₂Cl₂ at 0 °C and then allowed to reach room temperature. Upon disappearance of the azide (TLC), alaninyl isothiocyanato ester²⁹ was added and the mixture was stirred for 2–3 h (Scheme 2). The IR analysis of the crude reaction mixture showed a strong absorption band at 2118 cm⁻¹ corresponding to –N=C=N– stretch of the carbodiimide³⁰ (see characterisation data for compounds 5a–f). Carbodiimides could not be purified through silica gel column chromatography due to their conversion to the corresponding ureas (∼75%). However careful purification through neutral alumina afforded the desired carbodiimide 5d. Employing the same protocol, a series of carbodiimides, 5a–c as illustrated in Table 3 were then prepared, isolated and characterized.

Scheme 2

Table 3 List of carbodiimide tethered peptidomimetics

Entry	Pg	R^1	R^2	X	Yield (%)
5a	Fmoc	–CH3	–CH(CH3)2	Me	71
5b	Cbz	–CH2COOBzl	–H	Bzl	70
5c	Cbz	–(CH2)2COOBzl	–CH2C6H5	Et	68
5d	Cbz	–CH2C6H5	–CH3	Me	75

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Carbodiimides were confirmed to be optically pure by carrying out ¹H NMR analysis of the model compounds 5e and 5f. These were prepared starting from Cbz-Phe-ψ[CH₂-N₃] with isothiocyanates derived from (R)- and (S)-1-phenethylamine respectively. ¹H NMR of the crude 5e and 5f showed a single distinct methyl group doublet. Observed δ values for the –CH₃ group are as follows: 5e: 1.48, 1.49 and 5f: 1.54, 1.55. The equimolar mixture of 5e and 5f showed two distinct doublets at δ 1.46, 1.48, 1.52, 1.54, suggesting the presence of two isomers (see pages S47–48 of ESI†). This study infers the absence of epimerisation during their synthesis from corresponding azides.

N,N′-Disubstituted and N,N′,N′′-trisubstituted guanidine tethered dipeptidomimetics [Pg-Xaa-ψCH₂-NH-C(NY)-NH-Xbb-COOX] 6a–e, 7–10

Carbodiimide prepared as in the above protocol was treated with NH₄Cl in acetonitrile at 50–60 °C to afford the guanidinopeptides 6a–e (Scheme 2 and Table 4). Further, the synthetic utility of the carbodiimide as useful intermediate was also demonstrated by its reaction with hydroxylamine to yield hydroxyguanidines (compounds 7a and b, Table 4), sodium cyanamide to yield cyanoguanidine (compounds 8a and b, Table 4), hydrazine to yield aminoguanidine (compounds 9a and b, Table 4), and aryl amines to yield N,N′,N′′-trisubstituted guanidine (compounds 10a–c, Table 4) as illustrated in Scheme 2. Acetonitrile serve as a good solvent among the solvents screened such as DMF, THF and 1,4-dioxane and the reaction requires higher temperature to provide desired products in moderate to good yields. The products were purified on neutral alumina column chromatography and the compounds were confirmed by mass spectrometry, ¹H and ¹³C NMR analyses.

Table 4 List of N^α-protected guanidine tethered peptidomimetics and trisubstituted guanidinopeptide mimics

Entry	Pg	R^1	R^2	Y	X	Yield (%)
6a	Cbz	–CH2C6H5	–CH3	H	Me	69
6b	Cbz	–CH(CH3)2	–CH2CH(CH3)2	H	Me	67
6c	Cbz	–CH2S(Bzl)	–CH3	H	Me	59
6d	Fmoc	–CH3	–CH2CH2	H	Me	63
6e	Fmoc	–CH2C6H5	–CH2CH2	H	Me	58
7a	Fmoc	–CH3	–CH(CH3)2	OH	Me	56
7b	Fmoc	–CH2C6H5	–CH2C6H5	OH	Me	53
8a	Fmoc	–CH2C6H5	–H	CN	Et	64
8b	Fmoc	–CH2C6H5	–CH2CH(CH3)2	CN	Bzl	67
9a	Cbz	–CH2CH(CH3)2	–CH2C6H5	NH2	Me	71
9b	Fmoc	–CH3	–CH(CH3)2	NH2	Me	51
10a	Fmoc	–CH3	–CH(CH3)CH2CH3	C6H5	Me	65
10b	Fmoc	–H	–CH2CH(CH3)2	CH2C6H5	Me	58
10c	Fmoc	–CH3	–CH(CH3)2	4-Cl–C6H4	Me	62

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Conclusion

We have demonstrated the synthesis of N^α-protected amino alkyl S-methyl isothiouronium derivatives using a variation of Rathke protocol and accomplished the synthesis of novel class of carbodiimide tethered dipeptidomimetics through a Staudinger-aza-wittig type reaction. These compounds were then demonstrated as intermediates for the synthesis of guanidine tethered backbone modified peptidomimetics. A range of guanidinium derivatives such as free guanidine, N-hydroxy, cyano, amino and N,N′,N′′-trisubstituted guanidinium units were accomplished under mild experimental conditions so as to obtain them in good yields. The access to guanidinium and its derivatives serves to expand the repertoire of these unique entities in drug and medicinal fields as they mimic the biological roles played by Arg and its variants.

Experimental section

General procedure for the synthesis of N^α-protected amino alkyl thioureas 2a–h

To a stirred solution of N^α-protected amino alkyl isothiocyanate (1.0 mmol) in THF (5.0 mL) was added 3.0 eq of ammonium bicarbonate in 3.0 mL of water. The reaction mixture was cooled to 0 °C and stirred further for 3 h. After completion of the reaction as monitored through TLC, the reaction mixture was concentrated and diluted with EtOAc. The organic layer was washed with water (10 mL) and brine (5 mL) and dried over anhydrous Na₂SO₄. The organic layer was evaporated in vacuo to afford the product 2 in quantitative yield.

General procedure for the synthesis of N^α-protected amino alkyl isothiouronium derivatives 3a–h

To a solution of N^α-protected amino alkyl thiourea 2 (1.0 mmol) in acetone (8.0 mL) was added methyl iodide (1.5 mmol) and the reaction mixture was heated at reflux. The reaction was completed in 1–2 h as judged by TLC. Then the solvent was evaporated in vacuo and the resultant product 3 was obtained in excellent yield.

General procedure for the synthesis of guanidine tethered peptidomimetics 4a–c, 4e, 4g and h

To a neutralized solution of isothiouronium derivative 3 (1.0 mmol) in acetonitrile (6.0 mL) was added amino acid ester (1.3 mmol). The resultant mixture was heated at 60 °C till the completion of the reaction as monitored through TLC. After completion of the reaction, solvent was evaporated in vacuo and the obtained crude product 4 was then purified through column chromatography. [Caution: reaction has to be carried out in a fume hood as it involves the expulsion of toxic methyl mercaptan (CH₃SH) as by-product].

General procedure for the synthesis of carbodiimide 5a–f

To a solution of N-protected amino alkyl azide in dry CH₂Cl₂ at 0 °C was added triphenylphosphine (1.2 mmol). The reaction mixture was stirred for 10–15 min. then requisite isothiocyanate (1.3 mmol) was added and the mixture was stirred for about 2–3 h at same temperature till the completion of the reaction. The progress of the reaction was monitored through IR, where it showed a strong absorption band in the region 2116–2130 cm⁻¹ characteristic of carbodiimide 5. The solvent was evaporated under reduced pressure and the product was purified through neutral alumna to afford the desired carbodiimide 5.

General procedure for the synthesis of guanidine tethered peptidomimetics, 6a–e

To a solution of N-protected amino alkyl azide in dry CH₂Cl₂ at 0 °C was added triphenylphosphine (1.2 mmol). The reaction mixture was stirred for 10–15 min. then isothiocyanato ester (1.3 mmol) was added and the mixture was stirred for about 2–3 h at same temperature till the completion of the reaction. The progress of the reaction was monitored through IR, where it showed a strong absorption band in the region 2116–2130 cm⁻¹ characteristic of carbodiimide. The solvent was evaporated under reduced pressure and then the product without isolation was treated with NH₄Cl (2.0 mmol) in acetonitrile. The reaction mixture was heated at 60 °C till the completion of the reaction as monitored through TLC. The organic layer was then evaporated in vacuo to afford the crude product 6 in quantitative yield. The product was then purified through column chromatography.

General procedure for the synthesis of N-hydroxyguanidine (7a and b), aminoguanidine (8a and b) and cyanoguanidines (9a and b) and trisubstituted guanidine mimetics (10a–c)

To a solution of N-protected amino alkyl azide in dry CH₂Cl₂ at 0 °C was added triphenylphosphine (1.2 mmol). The reaction mixture was stirred for 10–15 min. then isothiocyanato ester (1.3 mmol) was added and the mixture was stirred for about 2–3 h at same temperature till the completion of the reaction. The progress of the reaction was monitored through IR, where it showed a strong absorption band in the region 2116–2130 cm⁻¹ characteristic of carbodiimide. The solvent was evaporated under reduced pressure and then the product without isolation was treated with a 1.5 mmol solution of hydroxylamine in acetonitrile to yield hydroxyguanidines (compounds 7a and b), sodium cyanamide in acetonitrile to yield cyanoguanidine (compounds 8a and b), hydrazine in acetonitrile to yield aminoguanidines (compounds 9a and b), and aryl amines in acetonitrile to yield N,N′,N′′-trisubstituted guanidines (compounds 10a–c). The resulting mixture was heated at 60 °C till the completion of the reaction as monitored by TLC. Then the organic layer was evaporated in vacuo to afford the crude products in quantitative yield. The products were then purified through column chromatography.

(9H-Fluoren-9-yl)methyl-3-methyl-1-thioureidobutan-2-ylcarbamate, Fmoc-Val-ψCH₂-NH-C(S)-NH₂ (2a)

Yield 81%; ¹H NMR (400 MHz, DMSO-d₆) δ 0.89 (d, J = 6.7 Hz, 3H), 0.98 (d, J = 6.7 Hz, 3H), 2.11 (m, 1H), 3.45 (m, 2H), 4.14–4.23 (m, 2H), 4.44 (m, 2H), 5.34 (br, s, 1H), 6.14 (s, br, 1H), 7.33 (t, J = 7.2 Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.61 (m, 2H), 7.78 (d, J = 7.2 Hz, 2H), 8.02 (s, br, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 18.05, 19.16, 30.39, 45.23,47.01, 55.84, 65.27, 120.12, 120.15, 127.09, 127.12, 127.61, 127.64, 140.72, 140.75, 143.81, 143.97, 156.24, 183.40; HRMS calcd for C₂₁H₂₅N₃O₂S m/z 406.1554 (M⁺ + Na), found 406.1556.

(9H-Fluoren-9-yl)methyl-4-methyl-1-thioureidobutan-2-ylcarbamate, Fmoc-Leu-ψCH₂-NH-C(S)-NH₂ (2b)

Yield 84%; ¹H NMR (400 MHz, DMSO-d₆) δ 0.91 (d, J = 6.2 Hz, 6H), 1.32–1.78 (m, 3H), 3.38 (d, J = 6.8 Hz, 2H), 4.12–4.28 (m, 2H), 4.43 (m, 2H), 5.31 (br s, 1H), 6.10 (s, br, 1H), 7.28 (t, J = 7.4 Hz, 2H), 7.37 (t, J = 7.4 Hz, 2H), 7.57 (t, J = 7.4 Hz, 2H), 7.73 (d, J = 7.4 Hz, 2H), 8.01 (br s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 23.22, 23.31, 24.21, 41.42, 46.80, 48.13, 49.02, 65.10, 120.10, 120.13, 125.20, 127.04, 127.07, 127.59, 140.73, 140.75, 143.77, 143.99, 155.87, 183.44; HRMS calcd for C₂₂H₂₇N₃O₂S m/z 420.1836 (M⁺ + Na), found 420.1844.

(9H-Fluoren-9-yl)methyl-2-thioureidoethylcarbamate, Fmoc-Gly-ψCH₂-NH-C(S)-NH₂ (2c)

Yield 82%; ¹H NMR (400 MHz, DMSO-d₆) δ 3.16 (m, 2H), 3.65 (m, 2H), 4.22 (t, J = 6.6 Hz, 1H), 4.45 (d, J = 6.6 Hz, 2H), 5.27 (s, br, 1H), 6.12 (s, br, 1H), 7.28–7.43 (m, 4H), 7.60 (m, 2H), 7.77 (d, J = 7.5 Hz, 2H), 8.05 (s, br, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 40.11, 43.68, 46.90, 65.41, 120.14, 125.18, 127.10, 127.63, 140.73, 143.88, 156.24, 183.40; HRMS calcd for C₁₈H₁₉N₃O₂S m/z 364.4170 (M⁺ + Na), found 364.4177.

(9H-fluoren-9-yl)methyl-2-(thioureidomethyl)pyrrolidine-1-carboxylate, Fmoc-Pro-ψCH₂-NH-C(S)-NH₂ (2d)

Yield 79%; ¹H NMR (400 MHz, DMSO-d₆) δ 1.72–2.02 (br, 4H), 3.18–3.54 (m, 4H), 3.99–4.44 (m, 4H), 6.18 (s, br, 1H), 7.26–7.75 (m, 8H), 8.11 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 23.00, 28.32, 45.68, 46.90, 56.27, 57.30, 66.32, 120.13, 125.13, 125.28, 127.15, 127.67, 140.66, 140.77, 143.78, 154.20, 183.69; HRMS calcd for C₂₁H₂₃N₃O₂S m/z 404.1412 (M⁺ + Na), found 404.4804.

(9H-Fluoren-9-yl)methyl-1-phenyl-2-thioureidoethyl-carbamate, Fmoc-Phg-ψCH₂-NH-C(S)-NH₂ (2e)

Yield 72%; ¹H NMR (400 MHz, DMSO-d₆) δ 3.21 (m, 2H), 4.01 (m, 1H), 4.15 (t, J = 6.8 Hz, 1H), 4.37 (d, J = 8.0 Hz, 2H), 5.52 (s, br, 1H), 6.17 (s, br, 1H), 7.20–7.80 (m, 13H), 8.19 (s, br, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 47.67, 50.04, 55.40, 67.54, 120.55, 125.53, 127.02, 127.64, 128.27, 129.18, 129.67, 134.00, 137.96, 141.83, 144.15, 155.76, 182.86; HRMS calcd for C₂₄H₂₃N₃O₂S m/z 440.1435 (M⁺ + Na), found 404.1439.

Benzyl-3-methyl-1-thioureidopentan-2-ylcarbamate, Cbz-Ile-ψCH₂-NH-C(S)-NH₂ (2f)

Yield 73%; ¹H NMR (400 MHz, CDCl₃) δ 0.97 (m, 6H), 1.18 (m, 1H), 1.49 (m, 1H), 1.80 (m, 1H), 3.15 (m, 2H), 4.14 (m, 1H), 5.38 (s, br, 1H), 6.08 (s, br, 1H), 7.30–7.49 (m, 5H), 7.89 (s, br, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 11.35, 15.34, 25.21, 37.58, 47.17, 56.13, 66.84, 127.55, 128.08, 128.55, 136.26, 157.56, 183.95; HRMS calcd for C₁₅H₂₃N₃O₂S m/z 332.1390 (M⁺ + Na), found 332.1392.

Tert-Butyl-3-phenyl-1-thioureidopropan-2-ylcarbamate, Boc-Phe-ψCH₂-NH-C(S)-NH₂ (2g)

Yield 88%; ¹H NMR (400 MHz, CDCl₃) δ 1.49 (s, 9H), 3.05 (2 dd, J = 13.7 and 6.9 Hz, 2H), 3.18 (m, 2H), 4.43 (m, 1H), 5.18 (br, s, 1H), 6.14 (s, br, 1H), 7.21–7.36 (m, 5H), 7.98 (s, br, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 28.22, 39.28, 51.03, 53.40, 80.04, 128.47, 128.75, 128.83, 129.08, 156.54, 183.92; HRMS calcd for C₁₅H₂₃N₃O₂S m/z 332.1246 (M⁺ + Na), found 332.1250.

Tert-Butyl-1-thioureidopropan-2-ylcarbamate, Boc-Ala-ψCH₂-NH-C(S)-NH₂ (2h)

Yield 94%; ¹H NMR (400 MHz, CDCl₃) δ 1.41 (s, 9H), 1.58 (d, J = 7.1 Hz, 3H), 3.01 (m, 2H), 4.23 (m, 1H), 5.31 (s, br, 1H), 6.07 (s, br, 1H), 7.82 (s, br, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 18.41, 30.18, 41.17, 50.54, 80.93, 155.53, 181.19; HRMS calcd for C₉H₁₉N₃O₂S m/z 256.1076 (M⁺ + Na), found 256.1080.

(S,Z)-(9H-Fluoren-9-yl)methyl-1-(amino(methylthio)methyl-eneamino)3-methylbutan-2-ylcarbamate, Fmoc-Val-ψCH₂-NH-C(SMe)-NH₂ (3a)

Yield 88%; ¹H NMR (400 MHz, CDCl₃) δ 0.98 (d, J = 7.81 Hz, 6H), 1.91 (m, 1H), 2.23 (s, 2H), 3.08 (m, 2H), 3.52 (m, 1H), 4.20 (t, J = 13.28 Hz, 1H), 4.44 (d, J = 6.72 Hz, 2H), 5.25 (s, br, 1H), 6.13 (s, br, 1H), 7.30 (t, J = 14.78 Hz, 2H), 7.40 (t, J = 14.7 Hz, 2H), 7.58 (d, J = 7.32 Hz, 2H), 7.76 (d, J = 7.44 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 19.64, 29.64, 41.08, 47.06, 56.05, 67.03, 119.86, 119.97, 125.33, 127.10, 127.63, 141.15, 143.54, 143.70, 143.89, 14.78, 156.36, 168.77; HRMS calcd for C₂₂H₂₇N₃O₂S m/z 398.1904 (M⁺ + H), found 398.1906.

(S,Z)-(9H-Fluoren-9-yl)methyl-1-(amino(methylthio)methyl-eneeamino)4-methylpentan-2-ylcarbamate, Fmoc-Leu-ψCH₂-NH-C(SMe)-NH₂ (3b)

Yield 90%; ¹H NMR (400 MHz, CDCl₃) δ 0.92 (dd, J = 2.28 Hz, J = 6.2 Hz, 6H), 1.32 (m,1H), 1.63 (m, 2H), 2.19 (s, 3H), 3.51 (m, 1H), 3.68 (m, 1H), 3.76 (m, 1H), 4.21 (t, J = 6.88 Hz, 1H), 4.45 (d, J = 6.88 Hz, 2H), 5.24 (s, br, 1H), 6.19 (s, br, 1H), 7.32 (t, J = 7.32 Hz, 2H), 7.40 (t, J = 7.32 Hz, 2H), 7.59 (d, J = 7.32 Hz, 2H), 7.77 (d, J = 7.76 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 14.64, 22.96, 24.72, 40.44, 47.03, 48.47, 51.60, 66.93, 119.85, 119.96, 125.36, 127.10, 127.63, 127.82, 141.15, 143.35, 143.69, 143.84, 156.06, 168.78; HRMS calcd for C₃₀H₂₉N₃O₂S m/z 412.2070 (M⁺ + H), found 412.2071.

(Z)-(9H-Fluoren-9-yl)methyl-2-(amino(methylthio)methylene-amino)ethylcarbamate, Fmoc-Gly-ψCH₂-NH-C(SMe)-NH₂ (3c)

Yield 89%; ¹H NMR (400 MHz, CDCl₃) δ 2.10 (s, 3H), 3.42 (m, 2H), 3.52 (m, 2H), 4.19 (t, J = 12.66 Hz, 1H), 4.42 (d, J = 6.57 Hz, 2H), 5.28 (s, br, 1H), 6.12 (s, br, 1H), 7.29 (t, J = 14.45 Hz, 2H), 7.38 (t, J = 14.59 Hz, 2H), 7.57 (d, J = 7.08 Hz, 2H), 7.75 (d, J = 7.38 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 14.92, 38.76, 44.37, 46.86, 67.46, 119.89, 125.12, 125.25, 127.09, 127.65, 127.73, 141.02, 143.41, 143.62, 158.32, 168.85; HRMS calcd for C₁₉H₂₁N₃O₂S m/z 356.1442 (M⁺ + H), found 356.1440.

(S,Z)-(9H-Fluoren-9-yl)methyl-2-(amino(methylthio)methyl-eneamino)methyl)pyrrolidines-1-carboxylate, Fmoc-Pro-ψCH₂-NH-C(SMe)-NH₂ (3d)

Yield 82%; ¹H NMR (400 MHz, CDCl₃) δ 1.29 (m, 4H), 2.10 (s, 3H), 3.23 (m, 2H), 3.52 (m, 1H), 3.66 (m, 2H), 4.23 (t, J = 7.0 Hz, 1H), 4.44 (m, 2H), 6.10 (s, br, 1H), 7.31–7.43 (m, 4H), 7.60 (d, J = 7.2 Hz, 2H), 7.77 (d, J = 7.6 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 14.07, 23.66, 29.36, 47.19, 50.77, 54.89, 56.18, 68.10, 119.93, 120.06, 124.87, 127.07, 127.68, 127.89, 141.25, 143.26, 143.53, 143.79, 157.82, 168.56; HRMS calcd for C₂₁H₂₆N₃O₂S m/z 396.1750 (M⁺ + H), found 396.1748.

(S,Z)-(9H-Fluoren-9-yl)methyl-2-(amino(methylthio)methyleneamino)1-phenylethyl-carbamate, Fmoc-Phg-ψCH₂-NH-C(SMe)-NH₂ (3e)

Yield 88%; ¹H NMR (400 MHz, CDCl₃) δ 2.14 (s, 3H), 3.47 (m, 2H), 4.10–4.40 (m, 4H), 5.16 (s, br, 1H), 6.18 (s, br, 1H), 7.20–7.78 (m, 13H); ¹³C NMR (100 MHz, CDCl₃) δ 14.75, 39.05, 47.15, 55.23, 66.92, 124.86, 126.45, 127.06, 127.70, 128.47, 128.74, 129.17, 138.12, 141.28, 143.62, 158.06, 168.28; HRMS calcd for C₂₅H₂₅N₃O₂S m/z 432.2030 (M⁺ + H), found 432.2031.

Benzyl(2S,3R)-1-(Z-amino(methylthio)methyleneamino)3-methylpentan-2-ylcarbamate, Cbz-Ile-ψCH₂-NH-C(SMe)-NH₂ (3f)

Yield 96%; ¹H NMR (400 MHz, CDCl₃): δ 0.75–1.26 (m, 9H), 3.13–3.39 (m, 2H), 4.10 (br, 1H), 4.30 (br, 2H), 4.63 (br, 1H), 6.20 (br, 1H), 7.31–7.77 (m, 8H), 8.14 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 12.93, 14.15, 15.40, 25.21, 41.37, 49.72, 49.83, 67.54, 128.77, 128.95, 129.08, 132.80, 133.78, 136.58, 157.94, 168.14; HRMS calcd for C₁₆H₂₅N₃O₂S m/z 324.1736 (M⁺ + H), found 324.1738.

(S,Z)-tert-Butyl-1-(amino(methylthio)methyleneamino)3-phenylpropan-2-ylcarbamate, Boc-Phe-ψCH₂-NH-C(SMe)-NH₂ (3g)

Yield 98%; ¹H NMR (400 MHz, CDCl₃) δ 1.42 (s, 9H), 2.96 (m, 2H), 3.67 (d, J = 3.6 Hz, 2H), 4.02 (m, 1H), 5.10 (s, br, 1H), 6.19 (s, br, 1H), 7.12–7.37 (m, 5H); ¹³C NMR (100 MHz, CDCl₃) δ 15.13, 28.32, 38.04, 46.76, 51.22, 80.10, 128.71, 128.77, 129.18, 129.37, 136.65, 155.32, 168.88; HRMS calcd for C₁₆H₂₅N₃O₂S m/z 324.1751 (M⁺ + H), found 324.1754.

(S,Z)-tert-Butyl-1-(amino(methylthio)methyleneamino) propan-2-ylcarbamate, Boc-Ala-ψCH₂-NH-C(SMe)-NH₂ (3h)

Yield 97%; ¹H NMR (300 MHz, CDCl₃) δ 1.18 (d, J = 6.8 Hz, 3H), 1.39 (s, 9H), 2.08 (s, 3H), 3.45 (m, 2H), 3.83 (m, 1H), 5.14 (s, br, 1H), 5.86 (s, br, 1H); ¹³C NMR (750 MHz, CDCl₃) δ 15.13, 18.17, 28.13, 45.45, 50.98, 80.34, 156.99, 168.54; HRMS calcd for C₁₀H₂₁N₃O₂S m/z 248.1431 (M⁺ + H), found 248.1434.

(S)-Methyl-2-(3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)-3-methylbutyl)guanidino) propanoate, Fmoc-Val-ψ[CH₂NHC(NH)NH]-Ala-OMe, (4a)

Yield 59%; ¹H NMR (400 MHz, DMSO-d₆) δ 0.97 (dd, J = 7.1 Hz, 6H), 1.40 (d, J = 7.2 Hz, 3H), 2.05–2.20 (m, 1H), 3.12 (m, 2H), 3.73 (s, 3H), 4.00–4.10 (m, 1H), 4.21 (t, J = 7.0 Hz, 1H), 4.25–4.50 (m, 2H), 4.50–4.65 (m, 1H), 5.56 (d, J = 8.8 Hz, 1H), 6.56 (s, br, 2H), 7.30 (m, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.59 (d, J = 7.1 Hz, 2H), 7.76 (d, J = 7.5 Hz, 2H); HRMS calcd for C₂₅H₃₄N₄O₄ m/z 453.0953 (M⁺ + H), found 453.0954.

(S)-Methyl-3-(3-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-methylpentyl)guanidino) propanoate, Fmoc-Leu-ψ[CH₂NHC(NH)NH]-β-Ala-OMe, (4b)

Yield 66%; ¹H NMR (400 MHz, CDCl₃) δ 0.92 (d, J = 6.8 Hz, 6H), 1.37 (m, 2H), 1.66 (m, 1H), 2.58 (m, 2H), 3.58 (s, 3H), 3.65–3.80 (m, 4H), 4.02 (m, 1H), 4.19 (t, J = 6.8 Hz, 1H), 4.35 (m, 2H), 5.21 (br, 1H), 6.96 (br, 2H), 7.22–7.76 (m, 8H); ¹³C NMR (100 MHz, CDCl₃) δ 22.44, 23.55, 25.29, 34.00, 42.35, 47.64, 52.31, 54.09, 64.32, 67.37, 120.48, 125.60, 127.61, 128.25, 141.75, 144.17, 157.77, 158.75, 173.44; HRMS calcd for C₂₆H₃₄N₄O₄ m/z 467.1827 (M⁺ + H), found 467.1894.

(S)-Methyl-2-(3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)ethyl)guanidino)-3-phenylpropanoate, Fmoc-Gly-ψ[CH₂NHC(NH)NH]-Phe-OMe, (4c)

Yield 63%; ¹H NMR (400 MHz, CDCl₃) δ 3.12–3.24 (m, 4H), 3.67 (s, 3H), 3.77–3.82 (m, 1H), 4.12–4.28 (m, 3H), 4.66 (d, J = 5.6 Hz, 2H), 5.17 (s, br, 1H), 6.45 (s, br, 2H), 7.15–7.41 (m, 9H), 7.53 (d, J = 7.2 Hz, 2H), 7.68 (d, J = 7.6 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 37.64, 39.81, 41.29, 47.69, 56.96, 60.71, 67.30, 120.44, 125.10, 125.65, 127.54, 128.15, 129.44, 129.83, 134.90, 141.78, 144.38, 156.16, 159.90, 173.89; HRMS calcd for C₂₈H₃₀N₄O₄ m/z 487.0804 (M⁺ + H), found 487.0806.

(S)-Methyl-2-(3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)-2-phenylethyl)guanidino) propanoate, Fmoc-Phg-ψ[CH₂-NHC(NH)NH]-(R)-Ala-OMe, (4e)

Yield 61%; ¹H NMR (400 MHz, CDCl₃) δ 1.17–1.18 (d, J = 4.0 Hz, 3H), 3.21–3.57 (m, 2H), 3.72 (s, 3H), 4.13–4.20 (m, 3H), 4.32 (d, J = 8.0 Hz, 2H), 5.24 (br, 1H), 6.10 (br, 1H), 7.28–7.48 (m, 8H), 7.56 (d, J = 4.0 Hz, 2H), 7.74 (d, J = 8.0 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 17.12, 43.91, 46.31, 53.99, 58.26, 67.18, 120.03, 125.03, 127.09, 127.77, 128.33, 128.40, 128.63, 135.54, 141.30, 143.75, 155.14, 156.29, 171.17.

(S)-Methyl-2-(3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)-2-phenylethyl)guanidino)propanoate, Fmoc-(R-)Phg-ψ[CH₂-NHC(NH)NH]-(S)-Ala-OMe, (4e′)

Yield 60%; ¹H NMR (400 MHz, CDCl₃) δ 1.24–1.25 (d, J = 4.0 Hz, 3H), 3.20–3.51 (m, 2H), 3.74 (s, 3H), 4.10–4.19 (m, 3H), 4.32 (d, J = 8.0 Hz, 2H), 5.23 (br, 1H), 6.07 (br, 1H), 7.25–7.49 (m, 8H), 7.54 (d, J = 4.0 Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H).

(S)-Methyl-2-(3-((S)-2-(tert-butoxycarbonyl)-3-phenylpropyl)guanidino)-3-ethylbutanoate, Boc-Phe-ψ[CH₂NHC(NH)NH]-Val-OMe, (4g)

Yield 71%; ¹H NMR (400 MHz, DMSO-d₆) 0.87 (d, J = 6.4 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H), 1.45 (s, 9H), 2.00–2.20 (m, 1H), 3.05–3.20 (m, 4H), 3.71 (s, 3H), 3.85–4.00 (m, 1H), 4.80–4.95 (m, 1H), 5.04 (d, J = 7.7 Hz, 1H), 6.37 (s, br, 2H), 7.05–7.15 (m, 2H), 7.25–7.35 (m, 3H); HRMS calcd for C₂₁H₃₄N₄O₄ m/z 407.2275 (M⁺ + H), found 407.2275.

(S)-Ethyl-2-(3-((S)-2-(tert-butoxycarbonyl)propyl)guanidino)-3-methylbutanoate, Boc-Ala-ψ[CH₂NHC(NH)NH]-Val-OEt, (4h)

Yield 68%; ¹H NMR (400 MHz, CDCl₃) δ 1.04 (m, 6H), 1.21 (d, J = 5.8 Hz, 3H), 1.41 (s, 9H), 1.96 (t, J = 6.9 Hz, 3H), 2.95 (m, 2H), 3.61 (m, 1H), 4.21 (m, 1H), 4.73 (m, 2H), 5.14 (s, br, 1H), 6.36 (br, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 16.92, 19.29, 21.47, 29.69, 30.22, 43.66, 48.24, 53.44, 65.23, 82.35, 158.07, 160.93, 172.93; HRMS calcd for C₁₆H₃₂N₄O₄ m/z 345.1477 (M⁺ + H), found 345.1472.

(S)-Methyl-2-(((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)propylimino)methyleneamino)-3-methylbutanoate, Fmoc-Ala-ψ[CH₂-N=C=N]-Val-OMe, (5a)

Yield 71%; IR (neat) ν_max 2116 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 0.94 (dd, J = 6.8 Hz and J = 14.6 Hz, 6H), 1.34 (d, J = 7.2 Hz, 3H), 2.00–2.20 (m, 1H), 3.09 (m, 2H), 3.73 (s, 3H), 3.85–4.00 (m, 1H), 4.50–4.65 (m, 3H), 5.11 (d, J = 7.9 Hz, 1H), 7.03–7.60 (m, 8H); ¹³C NMR (100 MHz, CDCl₃) δ 19.59, 19.91, 20.60, 31.18, 44.59, 47.42, 52.47, 56.48, 62.60, 67.92, 120.54, 125.44, 125.52, 127.60, 128.33, 136.48, 141.75, 143.97, 157.24, 168.04; HRMS calcd for C₂₅H₂₉N₃O₄ m/z 436.5222 (M⁺ + H), found 436.5218.

(3S)-Benzyl-4-(2-benzyloxy)-2-oxoethylimino)methylene-amino)-3-benzyloxycarbonyl)butanoate, Cbz-Asp(OBzl)-ψ[CH₂-N=C=N]-Gly-OBzl, (5b)

Yield 70%; IR (neat) ν_max 2120 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 3.06–3.11 (m, 2H), 4.30 (m, 2H), 4.98 (s, 2H), 5.03 (s, 2H), 5.08 (s, 2H), 5.29 (s, 2H), 5.36 (m, 1H), 5.87 (d, J = 8.0 Hz, 1H), 7.18–7.28 (m, 15H); ¹³C NMR (100 MHz, CDCl₃) δ 44.39, 48.84, 55.14, 62.14, 67.66, 68.08, 68.84, 128.60, 128.86, 128.93, 129.04, 129.10, 129.13, 129.23, 129.35, 131.46, 132.68, 134.75, 135.56, 136.02, 156.28, 168.16, 170.53; HRMS calcd for C₂₉H₂₉N₃O₆ m/z 516.0697 (M⁺ + H), found 516.0665.

(4S)-Benzyl-4-(benzyloxycarbonyl)-5-((1-ethoxy-1-oxo-3-phenylpropan-2-ylimino)methyleneamino)pentanoate, Cbz-Glu(OBzl)-ψ[CH₂-N=C=N]-Phe-OEt, (5c)

Yield 68%; IR (neat) ν_max 2116 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 1.20 (t, J = 5.6 Hz, 3H), 1.82–1.94 (m, 2H), 2.13 (t, J = 4.5 Hz, 2H), 2.94–3.18 (m, 4H), 3.49 (m, 1H), 3.77 (m, 1H), 4.09–4.15 (m, 2H), 5.11 (s, 4H), 5.61 (s, br, 1H), 7.03–7.51 (m, 15H); ¹³C NMR (100 MHz, CDCl₃) δ 13.98, 28.01, 30.22, 37.79, 44.19, 50.01, 53.91, 61.47, 64.11, 67.18, 127.03, 127.95, 128.10, 128.17, 128.47, 129.21, 135.68, 136.13, 138.50, 155.27, 170.69, 173.01; HRMS calcd for C₃₂H₃₅N₃O₆ m/z 558.1796 (M⁺ + H), found 558.1702.

(S)-Methyl-2-(((S)-2-(benzyloxycarbonyl)-3-phenylpropylimino)methyleneamino)propanoate, Cbz-Phe-ψ[CH₂-N=C=N]-Ala-OMe, (5d)

Yield 75%; IR (neat) ν_max 2118 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 1.17 (d, J = 6.8 Hz, 3H), 3.11 (d, J = 4.8 Hz, 2H), 3.51 (m, 2H), 3.69 (s, 3H), 4.50 (t, J = 8 Hz, 1H), 5.04 (s, 2H), 5.22 (br, 1H), 5.66 (q, J = 8.4 Hz, 1H), 6.93–7.33 (m, 10H); ¹³C NMR (100 MHz, CDCl₃) δ 17.01, 34.05, 42.56, 50.78, 53.95, 57.61, 67.02, 126.52, 128.12, 128.26, 128.69, 128.83, 129.07, 136.85, 137.79, 139.94, 155.73, 173.56; HRMS calcd for C₂₂H₂₅N₃O₄ m/z 396.0901 (M⁺ + H), found 396.0908.

Benzyl(S)-phenyl-1-(((R)-1-phenylethylimino)methyleneamino)propan-2-ylcarbamate, Cbz-Phe-ψ[CH₂-N=C=N]-R-phenethylamine, (5e)

Yield 74%; IR (neat) ν_max 2119 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 1.48–1.49 (d, J = 6.0 Hz, 3H), 2.75 (m, 2H), 3.68 (m, 1H), 3.87 (m, 1H), 4.24 (m, 2H), 5.02 (s, 2H), 5.20 (br, 1H), 7.07–7.49 (m, 15H); ¹³C NMR (75 MHz, CDCl₃) δ 22.8, 38.5, 45.8, 52.3, 53.9, 66.8, 125.9, 126.8, 127.2, 127.7, 128.1, 128.5, 128.6, 128.9, 129.0, 136.2, 136.6, 139.8, 156.9, 179.0.

Benzyl(S)-phenyl-1-(((S)-1-phenylethylimino) methyleneamino)propan-2-ylcarbamate, Cbz-Phe-ψ[CH₂-N=C=N]-S-phenethylamine, (5f)

Yield 73%; IR (neat) ν_max 2120 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 1.54–1.55 (d, J = 6.0 Hz, 3H), 2.78 (m, 2H), 3.72 (m, 1H), 3.90 (m, 1H), 4.30 (m, 2H), 5.00 (s, 2H), 5.61 (br, 1H), 6.97–7.48 (m, 15H).

(S)-Methyl-2-(3-((S)-2-(benzyloxycarbonyl)-3-phenylpropyl) guanidino)propanoate, Cbz-Phe-ψ[CH₂NHC(NH)NH]-Ala-OMe, (6a)

Yield 69%; ¹H NMR (400 MHz, CDCl₃) δ 1.36 (d, J = 7.1 Hz, 3H), 3.08 (m, 4H), 3.72 (s, 3H), 4.30–4.45 (m, 1H), 4.45–4.60 (m, 1H), 5.00 (s, 2H), 5.45 (s, br, 1H), 6.81 (br, 2H), 7.15–7.35 (m, 10H); ¹³C NMR (100 MHz, CDCl₃) δ 16.97, 33.65, 38.68, 47.32, 53.35, 60.73, 66.62, 126.60, 127.62, 127.94, 128.35, 128.50, 129.04, 136.14, 136.97, 156.79, 158.60, 172.54. HRMS calcd for C₂₂H₂₈N₄O₄ m/z 413.1994 (M⁺ + H), found 413.1974.

(S)-Methyl-2-(3-((S)-2-(benzyloxycarbonyl)-3-methylbutyl)guanidino)-4-methylpentanoate, Cbz-Val-ψ[CH₂NHC(NH)NH]-Leu-OMe, (6b)

Yield 67%; ¹H NMR (400 MHz, CDCl₃) 0.91 (d, J = 5.9 Hz, 6H), 0.93 (dd, J = 6.8 Hz and J = 12.6 Hz, 6H), 1.45–1.75 (m, 3H), 2.00–2.20 (m, 1H), 3.71 (s, 3H), 3.89 (m, 1H), 4.55–4.65 (m, 1H), 5.02 (s, 2H), 5.51 (s, br, 1H), 6.62 (br, 2H), 7.05–7.32 (m, 5H); HRMS calcd for C₂₁H₃₄N₄O₄ m/z 407.0785 (M⁺ + H), found 407.0781.

(S)-Methyl-2-(((3-((S)-2-(benzyloxycarbonyl)-3-benzylthio)propyl)guanidino)propanoate, Cbz-Cys(Bzl)-ψ[CH₂NHC(NH)NH]-Ala-OMe, (6c)

Yield 59%; ¹H NMR (400 MHz, CDCl₃) δ 1.32 (d, J = 7.2 Hz, 3H), 2.58 (m, 2H), 3.65 (s, 2H), 3.77 (s, 3H), 3.92–3.99 (m, 3H), 4.35 (m, 1H), 5.06 (s, 2H), 5.28 (d, J = 7.6 Hz, 1H), 6.84 (br, 2H), 7.23–7.32 (m, 10H); ¹³C NMR (100 MHz, CDCl₃) δ 16.76, 34.14, 36.91, 44.62, 49.61, 55.41, 55.56, 67.26, 127.69, 128.43, 128.63, 129.11, 129.56, 136.95, 138.07, 156.70, 158.80, 172.72; HRMS calcd for C₂₃H₃₀N₄O₄S m/z 459.1534 (M⁺ + H), found 459.1532.

(S)-Methyl-3-(3-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)propyl)guanidino)propanoate, Fmoc-Ala-ψ[CH₂NHC(NH)NH]-β-Ala-OMe, (6d)

Yield 63%; ¹H NMR (400 MHz, CDCl₃) δ 1.12 (d, J = 6.8 Hz, 3H), 2.52 (br, 2H), 3.51 (s, 3H), 3.69 (br, 4H), 4.10 (m, 1H), 4.26 (t, J = 6.8 Hz, 2H), 4.45 (d, J = 6.2 Hz, 1H), 5.36 (br, 1H), 6.92 (br, 2H), 7.18–7.32 (m, 4H), 7.50 (d, J = 7.2 Hz, 2H), 7.67 (d, J = 7.6 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 19.15, 34.02, 40.26, 47.59, 47.95, 51.15, 52.34, 67.44, 120.48, 125.57, 127.59, 128.25, 141.75, 144.14, 157.46, 159.44, 173.46; HRMS calcd for C₂₃H₂₈N₄O₄ m/z 425.0537 (M⁺ + H), found 425.0530.

(S)-Methyl-3-(3-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)-3-phenylpropyl)guanidino)propanoate, Fmoc-Phe-ψ[CH₂NHC(NH)NH]-β-Ala-OMe, (6e)

Yield 58%; ¹H NMR (400 MHz, DMSO-d₆) δ 2.56 (d, J = 6.4 Hz, 2H), 2.85 (m, 2H), 3.56 (s, 3H), 3.73 (m, 4H), 3.99 (br, 1H), 4.13 (t, J = 6.8 Hz, 1H), 4.22–4.34 (m, 2H), 5.45 (br, 1H), 6.78 (br, 2H), 7.18–7.51 (m, 13H); ¹³C NMR (100 MHz, DMSO-d₆) δ 23.60, 30.19, 32.57, 33.99, 39.41, 47.54, 52.37, 67.45, 120.48, 125.58, 127.37, 127.60, 128.25, 129.23, 129.65, 137.50, 141.74, 144.21, 157.51, 159.53, 173.61; HRMS calcd for C₂₉H₃₂N₄O₄ m/z 501.1528 (M⁺ + H), found 501.1522.

(S)-Methyl-2-((E)-3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)propyl)-2-hydroxyguanidino)-3-methylbutanoate, Fmoc-Ala-ψ[CH₂NHC(NOH)NH]-Val-OMe, (7a)

Yield 56%; ¹H NMR (300 MHz, CDCl₃) δ 0.98 (d, J = 4.8 Hz, 6H), 1.14 (d, J = 6.4 Hz, 3H), 2.64 (m, 3H), 3.56 (d, J = 7.1 Hz, 1H), 3.69 (s, 3H), 3.94 (m, 1H), 4.41 (t, J = 4.2 Hz, 1H), 4.67 (d, J = 5.6 Hz, 2H), 5.78 (br, 1H), 6.41 (br, 1H), 7.24–7.71 (m, 8H), 8.50 (br, 1H); ¹³C NMR (75 MHz, CDCl₃) δ 19.5, 19.9, 20.6, 30.1, 31.1, 40.5, 42.4, 47.4, 53.4, 66.6, 67.9, 120.5, 125.4, 125.5, 127.6, 128.3, 141.7, 143.9, 156.0, 159.2, 168.0; HRMS calcd for C₂₅H₃₂N₄O₅ m/z 491.1249 (M⁺ + Na), found 491.1240.

(S)-Methyl-2-((E)-3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)-3-phenylpropyl)-2-hydroxyguanidino)-3-phenyl propanoate, Fmoc-Phe-ψ[CH₂NHC(NOH)NH]-Phe-OMe, (7b)

Yield 53%; ¹H NMR (300 MHz, CDCl₃) δ 2.72 (m, 1H), 2.96 (m, 4H), 3.14 (m, 1H), 3.69 (s, 3H), 3.78 (t, J = 7.0 Hz, 1H), 3.89 (t, J = 8.8 Hz, 1H), 4.46 (t, J = 6.2 Hz, 1H), 4.68 (d, J = 5.9 Hz, 2H), 5.48 (br, 1H), 6.68 (br, 1H), 7.17–7.81 (m, 18H), 8.46 (br, 1H); ¹³C NMR (75 MHz, CDCl₃) δ 35.8, 39.6, 42.0, 46.9, 53.4, 56.4, 66.7, 68.3, 126.3, 126.4, 126.6, 127.7, 127.8, 127.9, 128.0, 128.6, 128.7, 128.9, 138.4, 138.6, 141.0, 143.1, 156.0, 160.3, 170.8; HRMS calcd for C₃₅H₃₆N₄O₅ m/z 615.1477 (M⁺ + Na), found 615.1472.

(S,E)-Ethyl-2-(3-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)-3-phenylpropyl)-2-cyanoguanidino)acetate, Fmoc-Phe-ψ[CH₂NHC(NCN)NH]-Gly-OEt, (8a)

Yield 64%; ¹H NMR (300 MHz, CDCl₃) δ 1.21 (t, J = 4.7 Hz, 3H), 2.59 (d, J = 3.2 Hz, 1H), 2.67 (d, J = 5.6 Hz, 1H), 2.75 (d, J = 5.7 Hz, 2H), 3.49 (s, 2H), 3.85 (m, 1H), 4.05 (m, 2H), 4.32 (t, J = 5.9 Hz, 1H), 4.65 (d, J = 3.1 Hz, 2H), 5.48 (s, br, 1H), 6.10 (s, br, 1H), 6.82 (s, br, 1H), 7.15–7.79 (m, 13H); ¹³C NMR (75 MHz, CDCl₃) δ 14.1, 38.4, 41.8, 47.6, 50.0, 60.2, 67.5, 116.5, 125.4, 127.0, 127.6, 128.2, 129.1, 129.6, 130.5, 130.9, 137.9, 141.8, 144.1, 153.9, 156.0, 170.1; HRMS calcd for C₃₀H₃₁N₅O₄ m/z 548.0785 (M⁺ + Na), found 548.0781.

(S)-Benzyl-2-((E)-3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)-3-phenylpropyl)-2-cyanoguanidino)-4-methyl pentanoate, Fmoc-Phe-ψ[CH₂NHC(NCN)NH]-Leu-OBzl, (8b)

Yield 67%; ¹H NMR (300 MHz, CDCl₃) δ 0.82 (d, J = 5.4 Hz, 6H), 1.42 (t, J = 7.1 Hz, 2H), 1.49–1.63 (m, 1H), 2.51 (d, J = 3.9 Hz, 1H), 2.59 (d, J = 6.5 Hz, 1H), 2.65 (d, J = 6.2 Hz, 2H), 3.29 (t, J = 11.1 Hz, 1H), 3.89 (m, 1H), 4.36 (t, J = 4.9 Hz, 1H), 4.74 (d, J = 3.9 Hz, 2H), 5.22 (s, 2H), 6.01 (s, br, 1H), 6.39 (s, br, 1H), 6.92–7.75 (m, 18H and NH); ¹³C NMR (75 MHz, CDCl₃) δ 22.8., 23.0, 34.3, 40.4, 42.2, 47.4, 48.0, 60.03, 65.3, 67.7, 117.5, 125.7, 126.4, 127.6, 128.2, 128.6, 128.9, 129.2, 129.8, 136.6, 139.7, 141.7, 143.1, 154.4, 156.2, 171.1; HRMS calcd for C₃₅H₃₃N₅O₄ m/z 610.1067 (M⁺ + Na), found 610.1061.

(S)-Methyl-2-((E)-3-((S)-2-(benzyloxycarbonyl)-4-methylpentyl)-2-aminoguanidino)-3-phenylpropanoate, Z-Leu-ψ[CH₂NHC(NNH₂)NH]-Phe-OMe, (9a)

Yield 71%; ¹H NMR (300 MHz, CDCl₃) δ 0.98 (d, J = 6.8 Hz, 6H), 1.42 (m, 2H), 1.91 (m, 1H), 2.79 (m, 2H), 3.12 (m, 1H), 3.26 (m, 1H), 3.71 (s, 3H), 4.38 (m, 1H), 4.92 (m, 1H), 5.04 (s, 2H), 6.08 (br, 1H), 6.72 (br, 1H), 7.12–7.22 (m, 10H), 7.84 (br, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 18.6, 21.2, 36.3, 43.2, 45.1, 52.3, 56.7, 64.8, 67.9, 127.1, 127.8, 128.2, 128.6, 129.4, 129.7, 137.2, 138.2, 155.4, 156.1, 170.1; HRMS calcd for C₂₅H₃₅N₅O₄ m/z 470.0275 (M⁺ + H), found 470.0275.

(S)-Methyl-2-((E)-3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)propyl)-2-aminoguanidino)-3-methylbutanoate, Fmoc-Ala-ψ[CH₂NHC(NNH₂)NH]-Val-OMe, (9b)

Yield 51%; ¹H NMR (300 MHz, CDCl₃) δ 0.84 (d, J = 6.4 Hz, 3H), 0.99 (d, J = 6.8 Hz, 3H), 1.65 (d, J = 6.8 Hz, 3H), 2.81 (m, 3H), 3.52 (s, 3H), 4.23 (m, 1H), 4.36 (m, 1H), 4.99 (d, J = 8.4 Hz, 2H), 5.16 (m, 1H), 5.49 (br, 1H), 6.76 (br, 1H), 7.21 (m, 2H), 7.32 (m, 2H), 7.45 (d, 2H, J = 6.8 Hz), 7.67 (d, 2H, J = 7.6 Hz), 8.07 (br, 2H); ¹³C NMR (75 MHz, CDCl₃) δ 16.80, 21.6, 29.8, 44.2, 46.8, 52.4, 59.6, 63.2, 65.8, 126.7, 127.2, 127.9, 128.4, 141.1, 142.8, 154.1, 156.0, 170.4; HRMS calcd for C₂₅H₃₃N₅O₄ m/z 468.0642 (M⁺ + H), found 468.0649.

(2S,3R)-methyl-2-((E)-3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)propyl)-2-phenylguanidino)-3-methylpentanoate, Fmoc-Ala-ψ[CH₂NHC(NC₆H₅)NH]-Ile-OMe, (10a)

Yield 65%; ¹H NMR (300 MHz, CDCl₃) δ 0.94 (t, J = 4.2 Hz, 3H), 0.99 (d, J = 4.6 Hz, 3H), 1.24 (m, 2H), 1.29 (d, J = 6.8 Hz, 3H), 2.04 (m, 1H), 2.94 (m, 2H), 3.71 (s, 3H), 4.02 (m, 1H), 4.42 (t, J = 6.1 Hz, 1H), 4.62 (d, J = 7.1 Hz, 2H), 4.89 (m, 1H), 5.41 (br, 1H), 6.12 (br, 1H), 6.73 (br, 1H), 7.10–7.75 (m, 13H); ¹³C NMR (75 MHz, CDCl₃) δ 10.4, 16.3, 18.9, 28.6, 35.0, 42.4, 46.4, 48.2, 50.6, 52.3, 66.4, 120.6, 125.4, 125.8, 127.4, 127.6, 128.4, 128.8, 139.1, 141.0, 143.2, 155.2, 155.9, 172.1; HRMS calcd for C₃₂H₃₈N₄O₄ m/z 565.1796 (M⁺ + Na), found 565.1702.

(S,E)-methyl-2-(3-(2-(((9H-fluoren-9-yl)methoxy)carbonyl)ethyl)-2-benzylguanidino)-4-methylpentanoate, Fmoc-Gly-ψ[CH₂NHC(NBn)NH]-Leu-OMe, (10b)

Yield 58%; ¹H NMR (300 MHz, CDCl₃) δ 0.92 (d, J = 5.4 Hz, 6H), 1.45–1.71 (m, 3H), 2.52–2.63 (m, 2H), 2.79 (m, 2H), 2.97 (s, 2H), 3.31 (t, J = 4.2 Hz, 1H), 3.69 (s, 3H), 4.32 (t, J = 2.9 Hz, 1H), 4.49 (d, J = 7.2 Hz, 2H), 5.69 (s, br, 1H), 6.24 (s, br, 1H), 6.75 (s, br, 1H), 7.09–7.81 (m, 13H), ¹³C NMR (75 MHz, CDCl₃) δ 21.8, 22.5, 38.4, 41.1, 44.2, 46.9, 50.8, 52.0, 54.3, 67.2, 119.8, 125.0, 126.8, 126.9, 127.6, 128.3, 129.2, 136.2, 141.1, 143.7, 154.3, 156.6, 170.7; HRMS calcd for C₃₂H₃₈N₄O₄ m/z 565.1615 (M⁺ + Na), found 565.1603.

(S)-Methyl-2-((E)-3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)propyl)-2-(3-chlorophenyl)guanidino)-3-methylbutanoate, Fmoc-Ala-ψ[CH2NHC(NC₆H₄Cl)NH]-Val-OMe, (10c)

Yield 62%; ¹H NMR (300 MHz, CDCl₃) δ 0.89 (d, J = 3.7 Hz, 6H), 1.17 (d, J = 6.4 Hz, 3H), 2.12 (m, 1H), 2.41 (d, J = 4.8 Hz, 1H), 2.82 (d, J = 7.1 Hz, 1H), 2.89 (d, J = 11.2 Hz, 1H), 3.52 (s, 3H), 3.85 (m, 1H), 4.51 (t, J = 6.6 Hz, 1H), 4.81 (d, J = 5.6 Hz, 2H), 6.12 (s, br, 1H), 6.93 (s, br, 1H), 7.12–7.75 (m, 12H); ¹³C NMR (75 MHz, CDCl₃) δ 18.0, 18.9, 30.9, 42.7, 46.9, 49.4, 55.4, 57.9, 66.6, 119.7, 124.9, 126.8, 127.5, 128.0, 128.3, 135.4, 141.0, 143.6, 143.7, 153.9, 156.6, 170.1,; ES-MS calcd for C₃₁H₃₅ClN₄O₄ m/z 586.09 (M⁺ + Na), found 586.07.

Acknowledgements

We thank Professor K. N. Ganesh, IISER Pune, for useful discussions at the beginning of this study. Also we thank DST, and BRNS for their generous financial assistance. Authors also thank Dr H. N. Gopi, IISER, Pune for Mass spectrometry and SAIF, IPC, IISc, Bangalore for ¹H and ¹³C NMR spectra. One of the authors Basavaprabhu thanks CSIR for SRF fellowship.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Details of mass spectrometry, ¹H and ¹³C NMR spectra of compounds prepared were given in ESI. See DOI: 10.1039/c4ra07252a

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