Cyclopenta[d]isoxazoline β-Turn Mimics: Synthetic Approach, Turn Driving Force, Scope, and Limitations

Abstract

Model β-turn inducers were prepared from constrained oxazanorbornene aminols. Taking advantage of the starting materials geometry, new diastereoisomeric compounds were synthesized, introducing different amino acidic residues. The products were spectroscopically characterized (VT and NMR titration). Temperature coefficients in dimethyl sulfoxide denote the existence of an intramolecular hydrogen bond. Chiroptical properties disclosed a β-turn arrangement of the synthesized compounds. The fused isoxazoline ring constraints the cyclopentane moiety, stabilizing a boatlike conformation that ensures the turn efficiency but limiting the accessibility to hindered amino acids.

Introduction

Turns are pivotal motifs that reverse the direction of peptide strands and helices.¹ Turns are crucial for protein structure and occur within protein-binding sites, at protein–protein interfaces and in bioactive peptides, playing a central role in recognition.² The growing interest in these structures pushes many research groups to intensify the efforts in this area³ by designing and developing novel structures for specific applications⁴ and using classical scaffolds for promising new synthetic targets.⁵

β-Turn motifs are the most common ones observed in small peptides and proteins,⁶ and their synthesis takes advantage of conformationally constrained molecules, to get information about the three-dimensional interplay between ligand and receptor. The key point is the correspondence among conformations in solution, their real physical meaning and conformations determined in silico.⁷ For these reasons, three-dimensional interactions are essential for the design of analogues in the drug discovery process. Spatial orientation determination deeply involves the conformational analysis on the basis of spectroscopic techniques and molecular modeling.⁶ Most β-turns contain intramolecular hydrogen bonds between the carbonyl oxygen of the first residue (i) and the amide NH proton of the fourth residue (i + 3), forming a pseudo-10-membered ring,^1,8 and must respect the critical distances between the Cα carbon atoms (d_crit < 7 Å) and the one between the carbonyl oxygen of the first amino acid and the amide hydrogen of the fourth one (<4 Å) (Figure 1). In general, the use of cage amino acids⁹⁻¹¹ or conformationally constrained β-amino acids¹² permits the induction of a β-turn by the complete replacement of the three amino acids, normally required.

Figure 1.

Criteria for the identification of β-turns.

Recently, we have presented our approach toward the preliminary design of original nonpeptidic turn inducers¹³ based on a new class of cyclopenta[d]isoxazoline aminols using the nitrosocarbonyl (RCONO, 3) chemistry (Scheme 1).¹⁴

Scheme 1. Synthetic Pathway of Cyclopenta[d]isoxazoline Aminols through Nitrosocarbonyl Intermediates Chemistry.

These fleeting intermediates, generated by the mild oxidation of nitrile oxides 1 with tertiary amine N-oxides or by periodate oxidation of hydroxamic acids 2, are efficiently trapped by cyclopentadiene (or other dienes), affording the hetero-Diels–Alder (HDA) cycloadducts 4, synthetically elaborated to give the conformationally restricted carbocyclic aminols 6 through amide hydrolysis and N–O bond cleavage of the cycloadducts 5.^9,15

In our “proof-of-concept” work,¹³ we promised to develop further experiments to verify the influence of the two side chain features on the type of turn, gaining a better understanding of the applicability/reliability/robustness of these scaffolds as β-turn inducers. We wish to present here the elongation studies from the hydroxy and amino groups with side chains incorporating amino acidic residues (alanine, glycine, and valine) and the characterizations of the corresponding diastereoisomeric turn structures through NMR and circular dichroism (CD) techniques.

Results and Discussion

The regioisomeric aminols 6a,6b were prepared according to the previously reported methodology,^14,15 using benzonitrile oxide as the 1,3-dipole in the cycloaddition step. The hydroxy group protection was performed under standard procedure affording the amines 7a,7b (85%). Compounds 7a,7b were coupled with the commercially available N-Boc-l-Alanine. Diastereoisomers 8a and 9a were isolated in good yields, and similarly, diastereoisomers 8b and 9b, respectively, were obtained from 7a and 7b. The hydroxy functionalities deprotection was secured by standard n-Bu₄NF treatment, and the alcohols of type 10a,10b/11a,11b were obtained in very good yields (Scheme 2). All of the compounds reported above were separated and found identical to authentic specimens previously synthesized.¹³

Scheme 2. Synthesis of the Turn Mimic Compounds.

(a) TBDMSiCl, imidazole, dichloromethane (DCM), rt, 18 h. (b) N-Boc-l-Ala, HBTU, DIEA, DCM, rt, 48 h. (c) n-Bu₄NF, tetrahydrofuran, rt, 3 h. (d) N-Boc-Gly, DIC, 4-dimethylaminopyridine (DMAP), DCM, rt, 48 h. (d′) N-Boc-Val, DIC, DMAP, DCM, rt, 48 h. Yields and [α]_D (MeOH) are reported (see Supporting Information (SI) for details).

Afterward, the alcoholic groups of compounds 10 and 11 were derivatized with two different N-Boc-protected amino acids: glycine and l-valine. Glycine was chosen to avoid, in a first advancement step, the presence of a further stereogenic center that was subsequently introduced with the l-valine residue. The esterifications were conducted with a typical DIC/DMAP coupling procedure in DCM at room temperature for 48 h. The ester derivatives 12/13 for glycine and 14/15 for the l-valine were obtained in very good yields. A full characterization of these final compounds, along with the detailed experimental procedures, is reported in the Supporting Information.

The conformational analysis of both the series of compounds¹⁶ relies on CD analyses coupled with NMR experiments.^17,18 The CD spectra were collected from MeOH 10^–4 M solutions of diastereoisomers 12/13 and 14/15 within a wavelength range of 200–300 nm (Figure 2). In the case of glycine derivatives, compound 12a CD profile has a positive absorption band at 213.9 nm and a second negative band centered at 227.3 nm, indicating a left β-turn in accordance with the data reported in the literature.¹⁹ On the other side, the CD spectrum of 13a shows a comparable and specular trend with a negative absorption band at 215.1 nm and a second positive band centered at 227.1 nm, consistent for a right β-turn.

Figure 2.

Experimental CD spectra of diastereoisomers 12/13 (top) and 14/15 (bottom).

In a similar way, compound 12b CD profile has a negative absorption band at 213.6 nm and a second positive band centered at 228.9 nm. This profile can be referred to a right β-turn.¹⁶

The CD spectrum of 13b shows a positive absorption band at 214.7 nm and a second negative band centered at 227.8 nm, consistent for a left β-turn. Noteworthy, these values are remarkably close to those observed for the model malonic derivatives.¹³ Regarding the l-valine derivatives, the CD profile of compound 14a, a left β-turn, has a positive absorption band at 213.5 nm and a second negative band centered at 227.1 nm.¹⁹ On the other side, the CD spectrum of 15a shows a comparable and specular trend with a negative absorption band at 211.3 nm and a second positive band centered at 225.9 nm, consistent for a right β-turn. Compound 14b CD profile has a negative absorption band at 215.4 nm and a second positive band centered at 226.3 nm. This profile can be addressed to a right β-turn.¹⁶ Finally, the CD spectrum of 15b shows a positive absorption band at 214.7 nm and a second negative band centered at 227.8 nm, consistent for a left β-turn. These values are again strictly close to those observed for the glycine derivatives and the model malonic ones.¹³

Previously reported simulations of the CD absorption profiles of the model malonic derivatives¹³ done by time-dependent density functional theory (DFT) calculations²⁰ at the B3LYP/6-31g(d,p) level in methanolic solution nicely fitted with the experimental profiles, indicating that the configuration inversions at the isoxazoline–norbornane moieties correspond to an inversion of the chiroptical properties of the products in hand. This fitting allowed assigning the product configurations beyond any reasonable doubt. These assignments are the same for all of the newly synthesized diastereoisomers because of the nice overlapping of CD spectra.¹³

H-bonding in diastereoisomers 12–15 in CDCl₃ and dimethyl sulfoxide (DMSO) was evaluated through temperature coefficients (tc) of the amide and NHBoc protons of the amino acidic residues at 298.15 and 318.15 K for CDCl₃ and up to 348.15 K for DMSO as well as DMSO titration experiments.¹⁹ Typically, lower tc values in CDCl₃ ≤ 2.4 ppb/K are related not only to shielded protons but also to accessible ones, and only values significantly larger than 2.4 ppb/K in CDCl₃ can be unambiguously assigned to NH protons initially shielded, which become exposed to the solvent upon increasing temperature.^2,21 Conversely, low tc in DMSO (<5 ppb/K) are related to inaccessible protons to the solvent.

Solutions (10^–3 M) of the compounds 12–15 in the deuterated solvent of choice were used to record the ¹H NMR spectra. Table 1 reports the tc values expressed in Δppb/ΔK highlighted in different colors for CDCl₃ and DMSO for the related diastereoisomers. The results indicate that in CDCl₃, the four glycine diastereoisomers 12 and 13 display somewhat a borderline situation since the values of the amide protons are in the range of 2.5–4.0 Δppb/ΔK similarly to that for the NHBoc protons.

Table 1. Temperature Coefficients (tc) of Amide and NHBoc Protons Δppb/ΔK in CDCl₃ (298.15–318.15 K) and in DMSO (298.15–348.15 K) for Glycine Derivatives 12a, 12b, 13a, and 13b^a.

	1-Ala		2		3-Gly or 3-Val
compounds	CDCl3	DMSO	CDCl3	DMSO	CDCl3	DMSO
12a	2.0	9.0	4.0	3.8	4.0	8.0
12b	2.0	9.4	2.5	3.2	1.0	8.2
13a	0.5	9.2	4.0	3.8	1.5	8.2
13b	4.0	8.8	3.5	2.4	0.5	7.6
14a	2.0	10.0	3.5	4.5	4.0	7.5
14b	2.0	10.0	4.5	3.0	3.0	7.0
15a	1.5	8.5	5.5	4.0	0.5	8.5
15b	3.5	10.5	4.5	5.0	1.0	8.0

^aTc of amide and NHBoc protons Δppb/ΔK in CDCl₃ (298.15–318.15 K) and in DMSO (298.15–348.15 K) for l-valine derivatives 14a, 14b, 15a, and 15b.

Some differences can be noted between compounds 12a,12b and 13a,13b: in these latter two compunds, tc values can reach remarkably low values as a mark of negligible temperature effect on proton accessibility. The tc values in DMSO indicate that the amide protons, ranging from 2.4 to 3.8 Δppb/ΔK for all of the four glycine substrates, are inaccessible to the solvent, while NHBoc protons have Δppb/ΔK values higher than the threshold (7.6–9.4 Δppb/ΔK), indicating the solvent accessibility of those protons. Analogous considerations can be done over the four l-valine diastereoisomers 14 and 15 that again display a borderline behavior for both the amide and NHBoc protons in CDCl₃, while the tc values in DMSO indicate that the amide protons, ranging from 4.0 to 5.0 Δppb/ΔK (a little higher with respect to the glycine ones) for all of the four l-valine substrates, are inaccessible to the solvent, while NHBoc protons have Δppb/ΔK higher values (7.0–10.5 Δppb/ΔK), indicating that those protons can be accessed by the solvent.

Furthermore, we performed DMSO titration experiments with gradual DMSO addition to CDCl₃ solutions (10^–3 M) of the eight diastereoisomers 12–15. Figure 3 shows the plots of the glycine (top) and l-valine (bottom) compounds. The first set of results concerning the glycine derivatives indicated clearly that amide protons chemical shifts remain almost unchanged over the addition of increasing amounts of DMSO to the CDCl₃ solutions; the range of variation ΔNH is 0.17–0.30 ppm, indicating a very low accessibility of the amide proton, engaged in H-bonding with the ester oxygen atom. A larger chemical shift variation is noted for the NHBoc protons with ΔNH values in the interval of 0.82–1.41 ppm, being these protons prone to be attached by the DMSO molecule through H-bond.

Figure 3.

DMSO titration of compounds 12–15. Top plots for glycine derivatives 12 and 13; bottom plots for l-valine derivatives 14 and 15.

The second set of results concerning the l-valine derivatives showed little variations with respect to previous results. Amide protons chemical shifts remain almost unchanged over the addition of increasing amounts of DMSO to the CDCl₃ solutions, but the range of variation ΔNH is shifted at slightly higher values (0.25–0.77 ppm), indicating a low accessibility of the amide proton, still engaged in a less tight H-bonding with the ester oxygen atom. A larger chemical shift variation is observed for the NHBoc protons with ΔNH values in the range of 1.27–1.85 ppm, indicating the accessibility through H-bonding.

The reported results enforce the indication that the oxazanorbornenes chemistry gives the possibility to synthesize stereo-ordinated constrained aminols holding the geometrical features to be used as turn inducers. The driving force that makes these products induce β-turns very efficiently is strictly related to the presence of a fused isoxazolinic ring to the cyclopentane moiety. This latter usually adopts an envelope conformation with the flap directed toward the isoxazoline ring, thus showing a boatlike appearance to the bicyclic system.

For the sake of confirmation, we have located the relevant boat- and chair-like conformations of the turn structures 12a,12b–15a,15b obtained through DFT calculations at the B3LYP/6-31g(d) level.²⁰ The optimized structures of simplified compounds 12a,12b and 14a,14b are shown in Figure 4 (phenyl group of isoxazoline moiety was replaced with hydrogen). The other structures relative to compounds 13a,13b and 15a,15b are reported in the SI. The chairlike conformations of compounds 12a,12b and 14a,14b were found higher in energy than the boatlike ones by 4–6 kcal/mol. The boatlike conformations in fact allow for the relief of nonbonded interactions between the heterocyclic ring and the substituents on the adjacent cyclopentane carbons. Furthermore, the dihedral angles between the H4 and H5 isoxazoline protons and the adjacent trans cyclopentane protons are close to 90°.²² This geometry results in a flattening of the cyclopentane envelope pushing the amino and hydroxy groups close to each other at H-bond distances that, in the compounds in hand, range between 2.23 and 2.29 Å. These structures are maintained even when the amino and hydroxyl groups are the anchors for amino acid side chains that can emerge from other intramolecular H-bonding interactions. This determines of course a further stabilization, but it is not detrimental to be lost if polar substituents are needed to be located externally to the turn structures, leaving the amino acidic residues to float inside the turn arms or in closer positions. The cost of this conformation changes is modest, up to 1.4 kcal/mol.

Figure 4.

Boat- and chair-like conformations of simplified compounds 12a,12b and 14a,14b. Values in italics correspond to the intramolecular H-bonding (Å) and those close to the numbers to the relative energies (kcal/mol). Labels to the compound numbers: 2 H-bonds (b2), 1 H-bond (b1), and chairlike compounds (c).

These results can somewhat explain the failure of the insertion, in this type of turn structures, of more encumbered amino acids. A similar view can be found for compounds 13a,13b and 15a,15b (see Figure S3 in the SI).

Due to the specificity of the aminol structures, the elongation on both the arms with amino acids produces the situation illustrated in Figure 4. The boatlike conformation of the aminols guarantees a bonded arrangement for the turn structure. This happens precisely when the amino acids bear simple substituents, such as the cases of glycine and l-alanine. If larger groups are located on the amino acid chains, then the results depend on their steric demand. l-Valine could be easily inserted on one side of the aminol structure in the presence of the l-alanine on the other side. Again, spectroscopic investigations, including tc measurements and NMR titrations, as well as chiroptical properties, revealed a β-turn arrangement of all of the synthesized compounds, where a little divergence of the side chains is not detrimental. From the absolute configuration of the cyclopentane spacer, they can be labeled as left- or right-handed turns. Because of the constraints imposed by the fused isoxazoline ring, the nonbonded arrangement cannot be a preferred structure even when larger substituents have to be allocated. This constitutes a quite strong limitation for the use of aminols 6a,6b in the design of turn scaffolds, and it was experimentally verified by trying to link a l-tryptophan moiety instead of glycine/l-valine and in the presence of another amino acids in the opposite side. Every attempt failed reasonably because of the steric hindrance determined by the indole ring that cannot find space within the structure.

Conclusions

In conclusion, the synthesized diastereoisomeric compounds represent the reliable “proof” for the use of aminols, simple and easily prepared, for the synthesis of nonpeptidic turn inducers. We confirmed the crucial role of the cyclopenta[d]isoxazoline aminol structures in the formation of the β-turn arrangement. The performed experiments ascertained the influence of the two side chains features on the applicability/reliability/robustness of these scaffolds as β-turn inducers and the limitations in terms of amino acid choice determined by the strong conformational preference of the bidimensional heterocyclic aminols.

Experimental Section

Melting points (mp) are uncorrected and were determined by the capillary method. Elemental analyses were done on an elemental analyzer equipment available at the Department of Chemistry of the University of Pavia. ¹H and ¹³C NMR spectra were recorded on 300 and 400 MHz spectrometers (solvents are specified in the text). Chemical shifts (δ) are expressed in ppm from tetramethylsilane as internal reference and coupling constants (J) are in Hertz (Hz): b, broad; s, singlet; bs, broad singlet; bm, broad multiplet; d, doublet; t, triplet; and m, multiplet. IR spectra (nujol mulls) were recorded on a spectrophotometer available at the Department, and absorptions (ν) are measured in cm^–1. CD spectra were recorded on a spectropolarimeter at the Centro Grandi Strumenti (CGS) of the University of Pavia equipped with Spectra Analysis program. Column chromatography, thin-layer chromatography, and medium-pressure liquid chromatography: silica gel H60 and GF₂₅₄, respectively; eluants: from cyclohexane/ethyl acetate 9:1 to pure ethyl acetate.

Starting and Reference Materials

Aminols 6a,6b were prepared according to the established procedures.^14,15

TBDMSiCl, imidazole, N-Boc-l-Ala, HBTU, DIEA, n-Bu₄NF, N-Boc-Gly, DIC, DMAP, and N-Boc-Val were purchased from chemical suppliers. Solvents and other reagents were also purchased and used as they were, without any further purification.

The protected amines 7a,7b, the alanine diastereomeric compounds 8a,8b and 9a,9b, and the deprotected alanine ones 10a,10b and 11a,11b were prepared according to the well-established procedure reported in the literature¹³ and were found identical to authentic samples available in our laboratories.

General Procedure for the Coupling of the Alcohol Derivatives 10a,10b and 11a,11b with N-Boc-Glycine and N-Boc-Valine

To a solution of the deprotected alanine compounds 10a,10b and 11a,11b (typically 1.0 g scale) in 50 mL CH₂Cl₂, 2 equiv. of DIC were added along with a catalytic amount of DMAP. N-Boc-glycine or N-Boc-valine (1.30 equiv.) were then added to the reaction mixtures, which were left under stirring at room temperature for 48 h. The diluted solutions (50 mL of DCM was added) were washed with brine (50 mL) and water (2 × 50 mL), and the collected organic phases were dried over anhydrous Na₂SO₄. The residues, obtained from evaporation of the solvent, were submitted to chromatographic separation to yield desired compounds 12a,12b–15a,15b. The products were fully characterized.

Synthesis of Compound 12a

Colorless crystals, mp 110–114 °C from ethyl acetate. IR: ν_max 3365, 3322, 1740, 1687, 1656 cm^–1. [α]_D = +7 (c = 1, MeOH). ¹H NMR (DMSO) δ: 1.22 (d, 3H, J = 6 Hz, CH₃); 1.40 (s, 18H t-Bu); 1.83 and 2.00 (m, 1H + 1H, CH₂); 3.82 (AB syst., 2H, CH₂ gly); 4.01 (bm, 1H, CH); 4.28 (bs, 1H CH-N); 4.30 (d, 1H, J = 9.5 Hz, H_4ISOX); 5.11 (d, 1H, J = 9.5 Hz, H_5ISOX); 5.15 (bs, 1H, CH-O); 7.12 (d, 1H, J = 7.5 Hz, NH ala), 7.33 (t, 1H, J = 6 Hz, NH gly); 7.45 (m, 3H, Ph); 7.80 (bs, 1H, NH_AMID); 7.84 (m, 2H, Ph). ¹³C NMR (DMSO, 25 °C, 75 MHz) δ: 17.8; 28.1; 30.7; 35.2; 42.0; 49.8; 53.6; 58.1; 78.4; 79.6; 89.0; 127.0; 128.1; 128.9; 130.2; 155.2; 155.9; 156.9; 169.7; 172.0. ¹H NMR (CDCl₃, 25 °C, 300 MHz) δ: 1.17 (d, 3H, J = 7 Hz, CH₃); 1.46 (s, 18H, Boc); 1.99 e 2.06 (m, 1H + 1H, CH₂); 3.85 (m, 1H, CH Ala); 4.09 (bm, AB syst., 2H, CH₂, Gly); 4.22 (d, 1H, J = 9 Hz, H_4ISOX); 4.67 (bs, 1H, CH-N); 5.15 (d, 1H, J = 9 Hz, H_5ISOX); 5.34 (d, 1H, J = 5.5 Hz, NH Ala); 5.43 (bs, 1H, CH-O); 5.82 (m, 1H, NH Gly); 7.15 (d, 1H, J = 7.5 Hz, NH_AMID); 7.46 (m, 3H, Ph); 8.10 (m, 2H, Ph). Anal. Calcd. for C₂₇H₃₈N₄O₈ (546.62): C, 59.33; H, 7.01; N, 10.25. Found: C, 59.35; H, 7.01; N, 10.26.

Synthesis of Compound 12b

Dark yellow oil. IR: ν_max 3345, 3333, 1748, 1704, 1652 cm^–1. [α]_D = −26 (c = 1, MeOH). ¹H NMR (DMSO, 25 °C, 75 MHz) δ: 1.21 (d, 3H, J = 6 Hz, CH₃); 1.40 (s, 18H t-Bu); 1.85 e 2.01 (m, 1H + 1H, CH₂); 3.79 (AB syst., 2H, CH₂ gly); 4.04 (bm, 1H, CH); 4.30 (bs, 1H CH-N); 4.39 (d, 1H, J = 9.5 Hz, H_4ISOX); 5.06 (d, 1H, J = 9.5 Hz, H_5ISOX); 5.19 (bs, 1H, CH-O); 7.11 (d, 1H, J = 7.5 Hz, NH ala); 7.29 (t, 1H, J = 6 Hz, NH gly); 7.48 (m, 3H, Ph); 7.63 (d, 1H, NH_AMID); 7.83 (m, 2H, Ph). ¹³C NMR (DMSO, 25 °C, 75 MHz) δ: 17.9; 28.1; 30.7; 35.2; 42.2; 49.8; 56.3; 58.2; 78.3; 78.4; 90.1; 126.9; 128.2; 128.9; 130.2; 155.1; 155.2; 155.9; 169.9; 171.9. ¹H NMR (CDCl₃, 25 °C, 300 MHz) δ: 1.21 (d, 3H, J = 7 Hz, CH₃); 1.46 (s, 18H, Boc); 1.97 e 2.11 (m, 1H + 1H, CH₂); 3.85 (m, 1H, CH Ala); 4.01 (bm, AB syst., 2H, CH₂, Gly); 4.26 (d, 1H, J = 9 Hz, H_4ISOX); 4.75 (bs, 1H, CH-N); 5.03 (d, 1H, J = 9 Hz, H_5ISOX); 5.33 (d, 1H, J = 5.5 Hz, NH Ala); 5.40 (bs, 1H, CH-O); 6.10 (m, 1H, NH Gly); 7.07 (d, 1H, J = 7.5 Hz, NH_AMID); 7.43 (m, 3H, Ph); 7.97 (m, 2H, Ph). Anal. Calcd. for C₂₇H₃₈N₄O₈ (546.62): C, 59.33; H, 7.01; N, 10.25. Found: C, 59.32; H, 7.02; N, 10.24.

Synthesis of Compound 13a

Pale yellow oil. IR: ν_max 3365, 3353, 1773, 1741, 1634 cm^–1. [α]_D = −64 (c = 1, MeOH). ¹H NMR (DMSO, 25 °C, 300 MHz) δ: 1.22 (d, 3H, J = 6 Hz, CH₃); 1.40 (s, 18H t-Bu); 1.85 e 2.01 (m, 1H + 1H, CH₂); 3.82 (AB syst., 2H, CH₂ gly); 4.03 (bm, 1H, CH); 4.26 (bs, 1H CH-N); 4.32 (d, 1H, J = 9.5 Hz, H_4ISOX); 5.11 (d, 1H, J = 9.5 Hz, H_5ISOX); 5.15 (bs, 1H, CH-O); 7.12 (d, 1H, J = 7.5 Hz, NH ala); 7.31 (t, 1H, J = 6 Hz, NH gly); 7.46 (m, 3H, Ph); 7.75 (d, 1H, NH_AMID); 7.84 (m, 2H, Ph). ¹³C NMR (DMSO, 25 °C, 75 MHz) δ: 14.1; 17.9; 28.1; 35.1; 42.1; 49.9; 53.8; 58.1; 78.4; 79.5; 89.0; 127.1; 128.1; 128.8; 130.2; 155.2; 155.9; 156.8; 169.6; 172.3. ¹H NMR (CDCl₃, 25 °C, 300 MHz) δ: 1.17 (d, 3H, J = 7 Hz, CH₃); 1.46 (s, 18H, Boc); 1.95 e 2.06 (m, 1H + 1H, CH₂); 3.75 (m, 1H, CH Ala); 4.16 (bm, AB syst., 2H, CH₂, Gly); 4.25 (d, 1H, J = 9 Hz, H_4ISOX); 4.68 (bs, 1H, CH-N); 5.18 (d, 1H, J = 9 Hz, H_5ISOX); 5.34 (d, 1H, J = 5.5 Hz, NH Ala); 5.41 (bs, 1H, CH-O); 5.70 (m, 1H, NH Gly); 7.21 (d, 1H, J = 7.5 Hz, NH_AMID); 7.43 (m, 3H, Ph); 8.05 (m, 2H, Ph). Anal. Calcd. for C₂₇H₃₈N₄O₈ (546.62): C, 59.33; H, 7.01; N, 10.25. Found: C, 59.36; H, 7.02; N, 10.27.

Synthesis of Compound 13b

Pale yellow oil. IR: ν_max 3345, 3338, 1752, 1701, 1650 cm^–1. [α]_D = +4 (c = 1, MeOH). ¹H NMR (DMSO, 25 °C, 300 MHz) δ: 1.21 (d, 3H, J = 6 Hz, CH₃); 1.39 (s, 18H t-Bu); 1.85 e 2.01 (m, 1H + 1H, CH₂); 3.80 (AB syst., 2H, CH₂ gly); 4.03 (bm, 1H, CH); 4.26 (bs, 1H CH-N); 4.26 (d, 1H, J = 9.5 Hz, H_4ISOX); 5.06 (d, 1H, J = 9.5 Hz, H_5ISOX); 5.22 (bs, 1H, CH-O); 7.09 (d, 1H, J = 7.5 Hz, NH ala); 7.31 (t, 1H, J = 6 Hz, NH gly); 7.48 (m, 3H, Ph); 7.56 (d, 1H, NH_AMID); 7.78 (m, 2H, Ph). ¹³C NMR (DMSO, 25 °C, 75 MHz) δ: 18.0; 28.1; 34.9; 42.2; 49.7; 55.8; 58.0; 78.1; 78.4; 90.2; 126.8; 128.2; 128.9; 130.3; 155.2; 155.8; 155.9; 169.8; 172.2. ¹H NMR (CDCl₃, 25 °C, 300 MHz) δ: 1.21 (d, 3H, J = 7 Hz, CH₃); 1.44 (s, 18H, Boc); 1.94 e 2.09 (m, 1H + 1H, CH₂); 4.00 (m, 1H, CH Ala); 4.19 (bm, AB syst., 2H, CH₂, Gly); 4.52 (d, 1H, J = 9 Hz, H_4ISOX); 4.78 (bs, 1H, CH-N); 5.13 (d, 1H, J = 9 Hz, H_5ISOX); 5.32 (bs, 1H, CH-O); 5.46 (d, 1H, J = 5.5 Hz, NH Ala); 6.02 (m, 1H, NH Gly); 7.02 (d, 1H, J = 7.5 Hz, NH_AMID); 7.45 (m, 3H, Ph); 7.83 (m, 2H, Ph). Anal. Calcd. for C₂₇H₃₈N₄O₈ (546.62): C, 59.33; H, 7.01; N, 10.25. Found: C, 59.35; H, 7.03; N, 10.24.

Synthesis of Compound 14a

Colorless crystals, mp 62–64 °C from ethyl acetate. IR: ν_max 3365, 3322, 1740, 1687, 1656 cm^–1. [α]_D = +9 (c = 0.11, MeOH). ¹H NMR (CDCl₃, 25 °C, 300 MHz) δ: 1.04 (s, 9H, Boc); 1.06 (s, 9H, Boc); 1.09 (d, 6H, J = 7 Hz, CH₃); 1.43 (d, 3H, CH₃ Ala); 1.48 e 2.02 (m, 1H + 1H, CH₂); 2.03 (m, 1H, CH iPr); 3.91 (m, 1H, CH Ala); 4.00 (d, 1H, J = 9 Hz, H_4ISOX); 4.21 (m, 1H, CH, Val); 4.45 (bs, 1H, CH-N); 5.08 (d, 1H, J = 9 Hz, H_5ISOX); 5.40 (bs, 1H, NH Ala); 5.40 (bs, 1H, CH-O); 5.65 (bs, 1H, NH Val); 7.27 (d, 1H, J = 6 Hz, NH_AMID); 7.47 (m, 3H, Ph); 8.23 (m, 2H, Ph). ¹³C NMR (CDCl₃, 25 °C, 75 MHz) δ: 18.4; 18.5; 18.6; 26.4; 27.8; 27.9; 29.4; 34.1; 54.8; 59.5; 59.9; 80.4; 80.9; 88.3; 88.7; 127.1; 128.3; 128.4; 129.8; 154.7; 155.9; 156.3; 171.6; 172.7; 173.0. Anal. Calcd. for C₃₀H₄₄N₄O₈ (588.70): C, 61.21; H, 7.53; N, 9.52. Found: C, 61.23; H, 7.51; N, 9.56.

Synthesis of Compound 14b

Pale yellow oil. IR: ν_max 3271, 3320, 1744, 1706, 1684 cm^–1. [α]_D = +5 (c = 0.12, MeOH). ¹H NMR (CDCl₃, 25 °C, 300 MHz) δ: 1.03 (d, 6H, J = 7 Hz, CH₃); 1.08 (s, 9H, Boc); 1.11 (s, 9H, Boc); 1.41(d, 3H, CH₃ Ala); 1.85 e 2.06 (m, 1H + 1H, CH₂); 2.16 (m, 1H, CH iPr); 3.92 (m, 1H, CH Ala); 4.18 (d, 1H, J = 9 Hz, H_4ISOX); 4.22 (m, 1H, CH, Val); 4.27 (bs, 1H, CH-N); 4.93 (d, 1H, J = 9 Hz, H_5ISOX); 5.06 (bs, 1H, NH Ala); 5.38 (bs, 1H, NH Val); 5.39 (bs, 1H, CH-O); 7.08 (d, 1H, J = 6 Hz, NH_AMID); 7.44 (m, 3H, Ph); 8.01 (m, 2H, Ph). ¹³C NMR (CDCl₃, 25 °C, 75 MHz): 18.7; 18.9; 19.2; 28.3; 28.4; 29.5; 29.8; 30.0; 34.6; 34.9; 51.7; 56.2; 57.0; 59.4; 61.8; 79.8; 80.8; 91.7; 127.2; 128.8; 130.2; 130.3; 154.4; 154.7; 171.2; 173.0. Anal. Calcd. for C₃₀H₄₄N₄O₈ (588.70): C, 61.21; H, 7.53; N, 9.52. Found: C, 61.20; H, 7.55; N, 9.51.

Synthesis of Compound 15a

Yellowish oil. IR: ν_max 3306, 3322, 1744, 1706, 1684 cm^–1. [α]_D = −3 (c = 0.26, MeOH). ¹H NMR (CDCl₃, 25 °C, 300 MHz) δ: 1.05 (s, 9H, Boc); 1.07 (s, 9H, Boc); 1.11 (d, 6H, J = 7 Hz, CH₃); 1.39 (d, 3H, CH₃ Ala); 1.71 e 2.14 (m, 1H + 1H, CH₂); 2.21 (m, 1H, CH iPr); 3.98 (m, 1H, CH Ala); 4.07 (d, 1H, J = 9 Hz, H_4ISOX); 4.34 (m, 1H, CH, Val); 4.76 (bs, 1H, CH-N); 5.02 (d, 1H, J = 9 Hz, H_5ISOX); 5.12 (bs, 1H, NH Ala); 5.74 (bs, 1H, NH Val); 5.77 (bs, 1H, CH-O); 7.06 (d, 1H, J = 6 Hz, NH_AMID); 7.40 (m, 3H, Ph); 8.06 (m, 2H, Ph). ¹³C NMR (CDCl₃, 25 °C, 75 MHz): 18.5; 18.9; 19.1; 19.2; 21.2; 21.4; 22.1; 28.1; 28.2; 28.4; 31.2; 43.2; 46.9; 58.9; 80.2; 80.7; 88.8; 89.4; 127.3; 128.3; 128.8; 130.5; 153.4; 155.6; 169.8; 171.5; 172.5; 173.8. Anal. Calcd. for C₃₀H₄₄N₄O₈ (588.70): C, 61.21; H, 7.53; N, 9.52. Found: C, 61.22; H, 7.50; N, 9.53.

Synthesis of Compound 15b

Pale yellow oil. IR: ν_max 3251, 3308, 1743, 1706, 1684 cm^–1. [α]_D = +28 (c = 0.23, MeOH). ¹H NMR (CDCl₃, 25 °C, 300 MHz) δ: 1.04 (d, 3H, CH₃ Ala); 1.13 (d, 6H, J = 7 Hz, CH₃); 1.46 (s, 18H, Boc); 2.02 (m, 2H, CH₂); 2.17 (m, 1H, CH iPr); 4.03 (d, 1H, J = 9 Hz, H_4ISOX); 4.22 (m, 1H, CH Ala); 4.80 (bs, 1H, CH-N); 4.89 (m, 1H, CH, Val); 5.07 (d, 1H, J = 9 Hz, H_5ISOX); 5.28 (bs, 1H, CH-O); 5.32 (bs, 1H, NH Ala); 5.70 (bs, 1H, NH Val); 7.13 (d, 1H, J = 6 Hz, NH_AMID); 7.44 (m, 3H, Ph); 7.96 (m, 2H, Ph). ¹³C NMR (CDCl₃, 25 °C, 75 MHz): 18.2; 18.4; 19.1; 28.2; 29.8; 30.1; 35.1; 35.4; 55.9; 59.5; 59.9; 61.3; 77.1; 79.7; 80.8; 91.6; 126.8; 127.1; 128.8; 130.2; 130.4; 154.8; 155.4; 155.8; 170.6; 171.9; 173.2. Anal. Calcd. for C₃₀H₄₄N₄O₈ (588.70): C, 61.21; H, 7.53; N, 9.52. Found: C, 61.22; H, 7.51; N, 9.54.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01670.

• ¹H and ¹³C NMR spectra of all new compounds, CD spectra, and NMR double-resonance experiments (PDF)

The authors declare no competing financial interest.