A straightforward click-approach towards pretubulysin-analogues

Abstract

The [3+2]-cycloaddition of an azido tripeptide, corresponding to the left hand side of pretubulysin, with a range of alkynes, such as propiolic acid amides and propargyl ethers, allows the straightforward syntheses of libraries of tubulysin derivatives. Via this click approach, a chimera of pretubulysin and dolastatin 10, both highly potent antimitotic drug candidates, also becomes accessible.

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Introduction

The development of new selective anti-tumor drugs which can discriminate between normal cells and tumor cells is a challenging task in modern cancer therapy. The promising approach to bind anti-tumor compounds to monoclonal antibodies, to specifically target tumor cells, requires highly potent compounds.¹ Besides the well-established anti-tumor drugs, such as the taxanes² or the epothilones,³ especially the peptides dolastatin 10⁴ and the tubulysins⁵ (Fig. 1) are excellent candidates for this approach, based on their high biological activity. All these agents were found to interact with the tubulin skeleton, disturbing fundamental cellular processes.⁶

Fig. 1 Microtubuli-destabilising peptidic natural products.

Dolastatin 10 was isolated by Pettit from the sea hare Dolabella auricularia in 1987.⁷ It shows anti-tumor activity towards a wide range of tumor cells in the sub-nanomolar range,^4b,8 and found its way into clinical trials.⁹ At the time of its discovery, dolastatin 10 was the most powerful antimitotic natural product known. This antiproliferative agent binds to the so-called vinca domain of tubulin, inhibiting tubulin polymerization and inducing apoptosis.^8b,10 This mode of action is also found for the tubulysins, and differs from that of the taxanes and epothilones.^5c,11

The tubulysins are a group of peptidic natural products isolated from the myxobacteria Angiococcus Disciformis An d48 and Archangium Gephyra Ar 315 by Höfle and Reichenbach.¹² Very recently, Müller et al. discovered a set of new derivatives in Angiococcus Disciformis An d48 and Cystobacter SBCb 004.¹³ The different tubulysins vary mainly in the side chain of the N,O-acetal (Fig. 1). At the C-terminus an α-methylated aromatic γ-amino acid is found, generated biosynthetically either from phenylalanine [tubuphenylalanine (Tup)] or from tyrosine [tubutyrosine (Tut)]. The new discovered derivatives differ mainly in the oxidation and acylation state of the central unusual amino acid tubuvaline (Tuv), and are probably biosynthetic intermediates.¹⁴ Pretubulysin was identified as a central intermediate in the biosynthesis, showing only slightly lower anti-tumor activity compared to the tubulysins, although its structure is significantly less complex.¹⁵

From a structural point of view, the tubulysins and pretubulysins are closely related to dolastatin 10. Especially the left hand side of the molecule is rather conserved. At the N-terminus a tertiary amino acid is found, followed by an unpolar amino acid (Val or Ile), which is coupled to an N-alkylated unusual amino acid [dolaisoleucine (Dil) or tubuvaline (Tuv)]. SAR studies on tubulysin derivatives indicated, that the N,O-acetal is not required for high biological activity,¹⁶ an N-methyl group is sufficient.¹⁷ Only if the N-substituent is removed completely, the biological activity drops significantly.^5b,18 This also explains, why pretubulysin is still active in the nmol range.¹⁹ Therefore, pretubulysin should be compared to dolastatin 10. On both C-termini, unusual aromatic amino acid derivatives are found, while the aromatic rings probably are involved in tubulin binding. Further functionalities on the C-terminus seem to be less important. For example, dolastatin derivatives where the C-terminal dolaphenine (Doe) is replaced by a simple 2-phenylethylamine or 3-phenylpropane-1,2-diol unit show only a slightly reduced anti-tumor activity.²⁰ The major structural difference is found between the unpolar side chain of the central unusual amino acid and the C-terminal building block. The conformation of dolastatin 10 in solution was investigated in detail,²¹ and recent NMR studies by Carlomagno et al. indicate that the conformations of dolastatin 10 and tubulysin are very similar.²² The variable part in between the unpolar N-terminal and the aromatic C-terminal part probably acts as a spacer to bring the side chains interacting with the tubulin into the correct orientation for binding. On the other hand, this should allow scope for considerable variation of this spacer unit.²³

Results and discussion

Probably, one of the most attractive options is the replacement of the thiazole unit by a triazole (A). On one hand, the triazole unit is a common surrogate for an amide bond and therefore often used in the synthesis of peptidomimetics,²⁴ and on the other hand, it allows the use of click-chemistry to connect the left hand (B) and the right hand part (C) of the molecule (Scheme 1).²⁵

Scheme 1 Triazolpretubulysin analogues.

This approach should allow the syntheses of libraries of (pre)tubulysin analogues in a straightforward fashion. While the propiolic acid amides C can easily be obtained by standard peptide coupling protocols (see ESI†) and need no further explanation, the synthesis of the required azido tripeptide is shown in Scheme 2.

Scheme 2 Synthesis of triazoltubuvaline 6 and azido tripeptide 10.

Starting from N-Boc-protected (S)-β-leucine 1²⁶ the N-methyl derivative 2 was obtained in high yield. Interestingly, although a large excess of MeI was used in this methylation step no significant amount of the corresponding methylester was obtained. The Boc-protected amino acid was subsequently activated and reduced with NaBH₄ to give alcohol 3. Mesylation (4), followed by an S_N reaction with NaN₃ gave rise to the required azide 5. To evaluate and optimize the reaction conditions for the final click reaction, we first coupled 5 with methylpropiolate providing protected triazoltubuvaline 6. In a very clean reaction 6 was obtained in the presence of CuSO₄ and Na ascorbate (10 mol% each) in almost quantitative yield. With propiolic acid amide 7b the reaction was found to be much slower.²⁷ Even after a reaction time of 3 d only around 50% of triazole 8 was obtained, while 45% of starting material was recovered. But by using a 50% excess of the alkyne and by doubling the amount of catalyst used (20 mol%) the yield of 8 could also be increased to 90%.

Based on these encouraging results we proceeded with the synthesis of the azidotripeptide 10. Cleavage of the Boc-protecting group and subsequent coupling with Fmoc-Ile gave rise to dipeptide 9 in high yield. BEP (2-Bromo-1-ethyl-pyridinium-tetrafluoroborate), which was used here, is an especially suitable reagent for epimerization-free coupling of N-methyl amino acids.²⁸ In an initial attempt we coupled 5 with Cbz-Ile in comparable yield, but the Cbz-protecting group could not be removed (HBr in HOAc) without decomposition of the azide. In contrast, the Fmoc protecting group could easily be cleaved with tris(aminoethyl)amine according to Wipf et al.^16c This reagent can be removed under neutral conditions by extraction with water (no acidic workup necessary). The dipeptide obtained could be coupled with Cbz-protected pipecolic acid to tripeptide 10via a mixed anhydride.

With this protected azide 10 in hand we investigated several cycloadditions²⁹ with propiolic acid amides 7 (Scheme 3, Table 1). Under the previously optimized conditions the amide of tubuphenylalanine 7a gave rise to the protected tetrapeptide triazole 11a in almost quantitative yield (entry 1). Catalytic hydrogenation of the Cbz-protecting group and subsequent reductive methylation provided ester 12a. This could be saponified to triazolpretubulysin 13a, which was converted into the trifluoroacetate (TFA) for purification and storage. In an analogous manner, several other phenylalanine derivatives were coupled in overall high yields (entries 2 and 3).

Scheme 3 Synthesis of pretubulysin analogues 13.

Table 1 Synthesis of pretubulysin analogues 13

Entry	7	X–COOMe	11	Yield (%)	12	Yield (%)	13	Yield (%)
a Reaction was carried out in H2O/DMSO. b Product with hydrogenated double bond.
1	7a		11a	96	12a	78	13a	97
2	7b		11b	93	12b	79	13b	89
3	7c		11c ^a	81	12c ^b	77	13c ^b	99

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By coupling the azide 10 with the propiolic amide of dolaphenine (14),³⁰ the C-terminal building block of dolastatin 10, a chimera of pretubulysin and dolastatin was obtained (Scheme 4). The click reaction provided the triazole 15 in almost quantitative yield. Although dolaphenine derivatives are rather sensitive towards epimerization, no isomerization was observed in the cycloaddition step. In this case the Cbz-protecting group could not be removed via catalytic hydrogenation, instead HBr in acetic acid had to be used. After removal of the protecting group and N-methylation a slight epimerization at the stereogenic center of the C-terminal amino acid derivative was observed. But this was not a severe problem, diastereomerically pure 16 could be obtained by flash chromatography.

Scheme 4 Synthesis of pretubulysin–dolastatin chimera 16.

The examples described so far are structurally closely related to the natural products, possessing an amide bond between the triazole unit and the C-terminal building block. Of course, these derivatives can also be obtained by incorporating triazoltubuvaline 6 into the N-terminal tripeptide and subsequent peptide coupling of the C-terminus. But the click approach is by far not limited to propiolic acid amides, but can also be applied to all kind of alkynes, allowing the introduction of non-hydrolysable C-termini (Scheme 5, Table 2). While the acetylenic amides required a reaction time of three days until complete conversion, the reaction with the propargylic ethers 17a and 17b were finished after one day. Cycloaddition with the coumarin-substituted alkyne 17c allows a straightforward synthesis of a fluorescent pretubulysin derivative 19, which might be an excellent candidate for binding studies.

Scheme 5 Synthesis of pretubulysin analogues 19.

Table 2 Synthesis of pretubulysin analogues 19

Entry	17	R	Time (d)	18	Yield (%)	19	Yield (%)
1	17a		1	18a	85	19a	87
2	17b		1	18b	85	19b	69
3	17c		3	18c	99	19c	72

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Conclusion

In conclusion, we have shown that the [3+2]-cycloaddition of azido tripeptides with a wide range of alkynes is a powerful tool for the straightforward synthesis of (pre)tubulysin derivatives, including a tubulysin–dolastatin chimera. Syntheses of small libraries of such pretubulysin analogues, and biological as well as activity studies are currently under investigation.

Experimental

General remarks

All reactions were carried out in oven-dried glassware (70 °C) under an atmosphere of nitrogen. Dried solvents were distilled before use: THF was distilled from LiAlH₄, DCM from CaH₂, dry DMF and MeOH was purchased from Sigma-Aldrich. The products were purified by flash chromatography on silica gel columns (Macherey-Nagel 60, 0.063–0.2 mm). Mixtures of ethyl acetate/petroleum ether and DCM/MeOH were generally used as eluents. Analytical TLC was performed on precoated silica gel plates (Macherey-Nagel, Polygram® SIL G/UV254). Visualization was accomplished with UV-light, KMnO₄ solution or ninhydrin solution. ¹H and ¹³C NMR spectra were recorded with a Bruker AC-400 [400 MHz (¹H) and 100 MHz (¹³C)] spectrometer in CDCl₃ or MeOH-d₄. Chemical shifts are reported in ppm (δ) with respect to TMS, CHCl₃ and MeOH-d₄ were used as the internal standards. Optical rotation measurements were performed on a Perkin-Elmer 341 polarimeter, with concentrations given in g per 100 mL. Melting points were determined with a MEL-TEMP II apparatus and are uncorrected. Mass spectra were recorded with a Finnigan MAT 95 spectrometer using the CI or EI technique. Elemental analyses were performed at Saarland University.

General procedure for click-reaction

The azide (1.0 mmol) and the alkyne (1.5 mmol) were dissolved in tert-butanol or DMSO (10 mL) and the clear solution was slowly diluted with water (10 mL). After addition of aqueous CuSO₄ solution (0.2 mmol, 1 M) and freshly prepared aqueous sodium ascorbate solution (0.2 mmol, 1 M), the reaction mixture was stirred for 1–3 days at room temperature (TLC monitoring). Then water was added and the reaction mixture was extracted thrice with ethyl acetate. The combined organic layers were washed with water (3×), brine and dried over Na₂SO₄. After evaporation of the solvent in vacuum and flash chromatography (SiO₂, petroleum ether/ethyl acetate) the pure product was obtained.

(R)-Methyl 1-(3-(tert-butoxycarbonyl(methyl)amino)-4-methylpentyl)-1H-1,2,3-triazole-4-carboxylate (6). Following the general procedure for click-reaction 6 was obtained from azide 5 (386 mg, 1.51 mmol), methyl propiolate (0.2 mL, 2.27 mmol), CuSO₄ solution (151 μL, 151 μmol, 1 M) and sodium ascorbate solution (151 μL, 151 μmol, 1 M) in tert-butanol/water after 1 d. After purification by flash chromatography (SiO₂, hexanes/ethyl acetate = 1 : 1), triazole 6 (510 mg, 1.49 mmol, 99%) could be isolated as a colourless oil and a mixture of rotamers. R_f 0.19 (hexanes/EtOAc, 1 : 1). [α]²¹_D = +9.4 (c = 1.1, CHCl₃). Major rotamer: ¹H NMR (400 MHz, CDCl₃): δ 0.84 (d, ³J_7,6 = 6.6 Hz, 3 H, 7-H), 0.90 (d, ³J_7′,6 = 6.6 Hz, 3 H, 7′-H), 1.49 (s, 9 H, 1-H), 1.70 (m, 1 H, 6-H) , 2.01 (m, 1 H, 8-H_a), 2.24 (m, 1 H, 8-H_b), 2.62 (s, 3 H, 4-H), 3.78 (ddd, ³J_5,6 = ³J_5,8a = 11.1 Hz, ³J_5,8b = 2.9 Hz, 1 H, 5-H), 3.95 (s, 3 H, 13-H), 4.27 (m, 1 H, 9-H_a), 4.41 (m, 1 H, 9-H_b), 8.22 (s, 1 H, 10-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 19.4 (q, C-7), 20.0 (q, C-7′), 28.2 (q, C-4), 28.4 (q, C-1), 30.1 (t, C-8), 30.7 (d, C-6), 48.3 (t, C-9), 52.1 (q, C-13), 58.6 (d, C-5), 80.4 (s, C-2), 128.3 (d, C-10), 139.6 (s, C-11), 156.7 (s, C-3), 161.1 (s, C-12) ppm. Minor rotamer (selected signals): ¹H NMR (400 MHz, CDCl₃): δ 0.85 (d, ³J_7,6 = 6.6 Hz, 3 H, 7-H), 0.91 (d, ³J_7′,6 = 6.6 Hz, 3 H, 7′-H), 1.46 (s, 9 H, 1-H), 2.35 (m, 1 H, 8-H_a), 2.73 (s, 3 H, 4-H), 3.53 (bs, 1 H, 5-H), 3.96 (s, 3 H, 13-H), 8.09 (s, 1 H, 10-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 19.7 (q, C-7), 20.0 (q, C-7′), 30.2 (t, C-8), 47.9 (t, C-9), 52.1 (q, C-13), 79.8 (s, C-2), 127.9 (d, C-10), 139.7 (s, C-11), 156.2 (s, C-3), 161.1 (s, C-12) ppm. HRMS (CI) m/z calcd for C₁₆H₂₉N₄O₄ [M+H]⁺: 341.2189; found 341.2183. Anal. calcd for C₁₆H₂₈N₄O₄ (340.41): C 56.45, H 8.29, N 16.46; found C 56.33, H 8.10, N 15.84.

(S)-Methyl 2-(1-((R)-3-(tert-butoxycarbonyl(methyl)amino)-4-methylpentyl)-1H-1,2,3-triazole-4-carboxamido)-3-phenylpropanoate (8). Following the general procedure for click-reaction 8 was obtained from azide 5 (53 mg, 0.21 mmol), alkyne 7b (71 mg, 0.31 mmol), CuSO₄ solution (21 μL, 21 μmol, 1 M) and sodium ascorbate solution (21 μL, 21 μmol, 1 M) in tert-butanol/water after 1 d. After purification by flash chromatography (SiO₂, petroleum ether/ethyl acetate = 1 : 1), triazole 8 (91 mg, 0.19 mmol, 90%) could be isolated as a colourless resin and a mixture of rotamers. R_f 0.17 (petroleum ether/EtOAc, 1 : 1). [α]²¹_D = +27.7 (c = 1.0, CHCl₃). Major rotamer: ¹H NMR (400 MHz, CDCl₃): δ 0.85 (d, ³J_16,15 = 6.6 Hz, 6 H, 16-H), 1.44 (s, 9 H, 20-H), 1.71 (m, 1 H, 15-H), 1.99 (m, 1 H, 13-H_a), 2.23 (m, 1 H, 13-H_b), 2.64 (s, 3 H, 17-H), 3.20 (dd, ²J_4a,4b = 14.2 Hz, ³J_4a,3 = 6.7 Hz, 1 H, 4-H_a), 3.25 (dd, ²J_4b,4a = 14.2 Hz, ³J_4b,3 = 5.9 Hz, 1 H, 4-H_b), 3.73 (s, 3 H, 1-H), 3.81 (m, 1 H, 14-H), 4.30 (m, 2 H, 12-H), 5.07 (m, 1 H, 3-H), 7.20 (m, 2 H, 7-H), 7.24–7.31 (m, 3 H, 6-H, 8-H), 7.54 (m, 1 H, NH), 8.03 (s, 1 H, 11-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 19.4 (q, C-16), 19.7 (q, C-16′), 28.3 (2 q, C-17, C-20), 30.2 (t, C-13), 30.3 (d, C-15), 38.2 (t, C-4), 47.9 (t, C-12), 52.3 (q, C-1), 53.0 (d, C-3), 58.7 (d, C-14), 79.7 (s, C-19), 125.7 (d, C-11), 127.1 (d, C-8), 128.6 (d, C-6), 129.2 (s, C-7), 135.8 (s, C-5), 142.5 (s, C-10), 156.5 (s, C-18), 159.6 (s, C-9), 171.4 (s, C-2) ppm. Minor rotamer (selected signals): ¹H NMR (400 MHz, CDCl₃): δ 0.92 (d, ³J_16,15 = 6.4 Hz, 3 H, 16-H), 0.93 (d, ³J_16′,15 = 6.4 Hz, 3 H, 16′-H), 1.48 (s, 9 H, 20-H), 2.74 (s, 3 H, 17-H), 3.54 (m, 1 H, 14-H), 8.12 (s, 11-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 20.0 (q, C-16), 30.7 (d, C-15), 48.4 (t, C-12), 52.3 (q, C-1), 53.0 (d, C-3), 80.3 (s, C-19), 126.0 (d, C-11), 142.6 (s, C-10), 156.2 (s, C-18), 159.5 (s, C-9), 171.4 (s, C-2) ppm. HRMS (CI) m/z calcd for C₂₅H₃₇N₅O₅ [M]⁺: 487.2795; found 487.2786.

(R)-Benzyl 2-((2S,3S)-1-(((R)-1-(4-((2R,4S)-5-methoxy-4-methyl-5-oxo-1-phenylpentan-2-ylcarbamoyl)-1H-1,2,3-triazol-1-yl)-4-methylpentan-3-yl)(methyl)amino)-3-methyl-1-oxopentan-2-ylcarbamoyl)piperidine-1-carboxylate (11a). Following the general procedure for click-reaction 11a was obtained from azide 10 (213 mg, 0.41 mmol), alkyne 7a (165 mg, 0.60 mmol), CuSO₄ solution (120 μL, 120 μmol, 1 M) and sodium ascorbate solution (120 μL, 120 μmol, 1 M) in tert-butanol/water after 3 d. After purification by flash chromatography (SiO₂, hexanes/ethyl acetate = 3 : 7), triazole 11a (310 mg, 0.39 mmol, 96%) could be isolated as a white solid and a mixture of rotamers; m. p. 92–94 °C. R_f 0.19 (hexanes/EtOAc, 3 : 7). [α]²²_D = +14.5 (c = 1.0, CHCl₃). Major rotamer: ¹H NMR (400 MHz, CDCl₃): δ 0.78 (d, ³J_18,17 = 6.6 Hz, 3 H, 18-H), 0.87 (m, 3 H, 24-H), 0.94–0.97 (sh, 6 H, 18′-H, 25-H), 1.08 (m, 1 H, 23-H_a), 1.16 (d, ³J_3,2 = 7.1 Hz, 3 H, 3-H), 1.38–1.49 (sh, 2 H, 29-H_ax, 30-H_ax), 1.49–1.70 (sh, 5 H, 4-H_a, 23-H_b, 28-H_ax, 29-H_eq, 30-H_eq), 1.70–1.87 (sh, 2 H, 4-H_b, 15-H_a), 2.23–2.34 (sh, 2 H, 15-H_b, 28-H_eq), 2.62 (m, 1 H, 2-H), 2.86–2.96 (sh, 3 H, 29-H, 31-H_ax), 3.04 (s, 3 H, 19-H), 3.06 (s, 3 H, 38-H), 4.09 (m, 1 H, 31-H_eq), 4.18–4.31 (sh, 3 H, 14-H, 16-H), 4.43 (m, 1 H, 5-H), 4.79 (dd, ³J_21,22 = ³J_21,NH = 8.3 Hz, 1 H, 21-H), 4.87 (m, 1 H, 27-H), 5.21 (d, ²J_33a,33b = 12.3 Hz, 1 H, 33-H_a), 5.16 (d, ²J_33b,33a = 12.5 Hz, 1 H, 33-H_b), 6.66 (bs, 1 H, NH), 6.96 (d, ³J_NH,5 = 9.2 Hz, 1 H, NH), 7.19–7.24 (sh, 3 H, 8-H, 10-H), 7.27–7.40 (sh, 7 H, 9-H, 35-H, 36-H, 37-H), 8.11 (s, 1 H, 13-H). ppm. ¹³C NMR (100 MHz, CDCl₃): δ 10.9 (q, C-24), 15.9 (q, C-25), 17.7 (q, C-3), 19.3 (q, C-18), 19.7 (q, C-18′), 29.0 (t, C-29), 24.4 (t, C-30), 24.5 (t, C-23), 26.1 (t, C-28), 29.6 (q, C-19), 29.8 (d, C-17), 30.4 (t, C-15), 36.5 (2 d, C-2, C-22), 37.0 (t, C-4), 41.3 (t, C-6), 42.3 (t, C-31), 48.1 (t, C-14), 51.9 (q, C-38), 54.0 (d, C-21), 54.7 (d, C-27), 57.8 (d, C-16), 67.9 (t, C-33), 125.8 (d, C-13), 126.5 (d, C-10), 127.9, 128.1, 128.4, 128.5, 129.4 (5 d, C-8, C-9, C-35, C-36, C-37), 136.1 (s, C-34), 137.3 (s, C-7), 143.0 (s, C-12), 159.7 (s, C-11), 171.1 (s, C-20), 174.3 (s, C-26), 176.7 (s, C-1) ppm. Minor rotamer (selected signals): ¹H NMR (400 MHz, CDCl₃): δ 1.05 (d, ³J_18,17 = 6.5 Hz, 3 H, 18-H), 3.65 (s, 3 H, 38-H), 3.73 (m, 1 H, 31-H_ax), 6.51 (bs, 1 H, NH) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 11.2 (q, C-24), 14.1 (q, C-25), 16.1 (q, C-3), 60.3 (d, C-16), 67.5 (t, C-33) ppm. HRMS (CI) m/z calcd for C₄₃H₆₂N₇O₇ [M+H]⁺: 788.4710; found 788.4730.

General procedure for Cbz-deprotection by hydrogenation

A solution of the tetrapeptide (1.0 mmol) in MeOH (5 mL) was stirred under a hydrogen atmosphere in the presence of Pd/C (10 mass%, 10% Pd) until the deprotection was complete (1–4 h, TLC monitoring). After filtration through celite, the solvent was removed and the free amine was dried in high vacuum. The amine was used in the reductive methylation without any further purification.

General procedure for reductive methylation

The crude amine (1.0 mmol) was dissolved in dry MeOH (10 mL), and paraformaldehyde (1.0 mmol) was added. After the system had been stirred for 3 h at room temperature, sodium cyanoborohydride (1.05 mmol) was added and the reaction mixture was stirred overnight at room temperature. The methanol was removed, the residue was diluted with saturated aqueous NaHCO₃ and extracted with DCM (3×). The combined organic layers were washed with brine and dried over Na₂SO₄. After evaporation of the solvent in vacuum and flash chromatography (SiO₂, DCM/MeOH) the pure product was obtained.

(2S,4R)-Methyl 4-(1-((R)-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-methylpentyl)-1H-1,2,3-triazole-4-carboxamido)-2-methyl-5-phenylpentanoate (12a). According to the general procedure for Cbz-deprotection 11a (193 mg, 0.25 mmol) was hydrogenated. Following the general procedure for reductive methylation 12a was obtained from the crude amine, paraformaldehyde (22 mg, 0.25 mmol) and sodium cyanoborohydride (17 mg, 0.26 mmol). After purification by flash chromatography (SiO₂, DCM/MeOH = 95 : 5), 12a (168 mg, 0.25 mmol, 78% over 2 steps) could be isolated as a white solid and a mixture of rotamers; m. p. 56–58 °C. R_f 0.20 (DCM/MeOH, 95 : 5). [α]²²_D = +19.8 (c = 1.0, CHCl₃). Major rotamer: ¹H NMR (400 MHz, CDCl₃): δ = 0.78 (d, ³J_18,17 = 6.6 Hz, 3 H, 18-H), 0.89–0.96 (sh, 6 H, 18′-H, 24-H), 1.00 (d, ³J_25,22 = 6.7 Hz, 3 H, 25-H), 1.16 (d, ³J_3,2 = 7.1 Hz, 3 H, 3-H), 1.19–1.26 (sh, 2 H, 23-H_a, 29-H_ax), 1.35 (m, 1 H, 28-H_ax), 1.48–1.84 (sh, 7 H, 4-H_a, 17-H, 23-H_b, 28-H_eq, 29-H_eq, 30-H), 1.91 (m, 1 H, 22-H), 1.96–2.08 (sh, 3 H, 4-H_b, 15-H_a, 31-H_ax), 2.20–2.32 (sh, 4 H, 15-H_b, 32-H), 2.49 (m, 1 H, 27-H), 2.62 (m, 1 H, 2-H), 2.86–2.96 (sh, 3 H, 6-H, 31-H_eq), 3.05 (s, 3 H, 19-H), 3.65 (s, 3 H, 33-H), 4.19–4.30 (sh, 3 H, 14-H, 16-H), 4.43 (m, 1 H, 5-H), 4.77 (dd, ³J_21,22 = ³J_21,NH = 8.8 Hz, 1 H, 21-H), 6.96 (d, ³J_NH,5 = 9.2 Hz, 1 H, NH), 7.04 (d, ³J_NH,21 = 7.7 Hz, 1 H, NH), 7.19–7.23 (sh, 3 H, 8-H, 10-H), 7.28 (m, 2 H, 9-H), 8.13 (s, 1 H, 13-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 10.9 (q, C-24), 16.0 (q, C-25), 17.7 (q, C-3), 19.4 (q, C-18), 19.8 (q, C-18′), 23.2 (t, C-29), 24.6 (t, C-23), 25.0 (t, C-30), 29.7 (d, C-16), 29.9 (q, C-19), 30.2 (t, C-15), 30.3 (t, C-28), 36.3 (d, C-2), 36.8 (d, C-22), 37.9 (t, C-4), 41.4 (t, C-6), 44.8 (q, C-32), 48.1 (t, C-14), 48.2 (d, C-5), 51.7 (d, C-33), 53.0 (d, C-21), 55.3 (t, C-31), 57.4 (d, C-16), 69.5 (d, C-27), 125.6 (d, C-13), 126.4 (d, C-10), 128.4 (d, C-8), 129.4 (d, C-9), 137.4 (s, C-7), 143.2 (s, C-12), 159.5 (s, C-11), 173.6 (s, C-20), 174.3 (s, C-26), 176.4 (s, C-1) ppm. Minor rotamer (selected signals): ¹H NMR (400 MHz, CDCl₃): δ 1.05 (d, ³J_18,17 = 6.6 Hz, 3 H, 18-H), 2.83 (s, 3 H, 19-H), 3.75 (m, 1 H, 16-H), 4.09 (m, 3 H, 14-H), 4.86 (m, 1 H, 21-H), 8.06 (s, 1 H, 13-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ 11.3 (q, C-24), 16.5 (q, C-25), 20.1 (q, C-18), 20.4 (q, C-18′), 27.3 (q, C-19) ppm. HRMS (CI) m/z calcd for C₃₆H₅₈N₇O₅ [M+H]⁺: 668.4499; found 668.4489.

(2S,4R)-4-(1-((R)-3-((2S,3S)-N,3-Dimethyl-2-((R)-1-methyl-piperidine-2-carboxamido)pentanamido)-4-methylpentyl)-1H-1,2,3-triazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid (13a). The free carboxylic acid 13a was obtained from 12a (57 mg, 85 μmol) by saponification with aqueous NaOH solution (200 μL, 200 μmol, 1 M) after 4 h at 80 °C. The solvent was removed in vacuum, and the residue was dissolved in water, acidified to pH 1 with TFA, and extracted with ethyl acetate (3×). The combined organic layers were dried over Na₂SO₄. After evaporation of the solvent in vacuum and flash chromatography (SiO₂, DCM/MeOH) the pure product 13a (63 mg, 82 μmol, 97%) could be isolated as a white solid and a mixture of rotamers; m. p. 100–102 °C. R_f 0.16 (DCM/MeOH, 9 : 1). [α]²⁰_D −19.2 (c 1.1, MeOH). Major rotamer: ¹H NMR (400 MHz, MeOH-d₄): δ = 0.79 (d, ³J_18,17 = 6.5 Hz, 3 H, 18-H), 0.92–0.97 (sh, 6 H, 18′-H, 24-H), 1.04 (d, ³J_25,23 = 6.7 Hz, 3 H, 25-H), 1.17 (d, ³J_3,2 = 7.0 Hz, 3 H, 3-H), 1.25 (m, 1 H, 23-H_a), 1.52–1.70 (sh, 3 H, 4-H_a, 23-H_b, 29-H_ax), 1.72–1.87 (sh, 3 H, 17-H, 28-H_ax, 30-H_ax), 1.88–2.04 (sh, 4 H, 4-H_b, 22-H, 29-H_eq, 30-H_eq), 2.08–2.20 (sh, 2 H, 15-H_a, 28-H_eq), 2.33 (m, 1 H, 15-H_b), 2.56 (m, 1 H, 2-H), 2.73 (s, 3 H, 32-H), 2.88 (d, ³J_6,5 = 6.7 Hz, 2 H, 6-H), 3.02–3.14 (sh, 4 H, 19-H, 31-H_ax), 3.49 (m, 1 H, 31-H_eq), 3.77 (dd, ³J_27,28eq = 11.8 Hz, ³J_27,28ax = 2.4 Hz, 1 H, 27-H), 4.11 (m, 1 H, 16-H), 4.26–4.47 (sh, 3 H, 5-H, 14-H), 4.69 (d, ³J_21,22 = 7.8 Hz, 1 H, 21-H), 7.15 (m, 1 H, 10-H), 7.18–7.28 (sh, 3 H, 8-H, 9-H), 8.32 (s, 1 H, 13-H) ppm. ¹³C NMR (100 MHz, MeOH-d₄): δ 11.3 (q, C-24), 16.2 (q, C-25), 18.6 (q, C-3), 20.2 (q, C-18), 20.4 (q, C-18′), 22.3 (t, C-29), 24.0 (t, C-30), 25.5 (t, C-23), 30.2 (d, C-17), 30.9 (t, C-28), 31.0 (t, q, C-15, C-19), 37.4 (d, C-22), 37.8 (d, C-2), 39.2 (t, C-3), 42.4 (q, C-32), 42.9 (t, C-6), 49.2 (t, C-14), 50.5 (d, C-5), 56.1 (t, C-31), 56.2 (d, C-21), 59.7 (d, C-16), 68.0 (d, C-27), 118.3 (q, ²J_C,F = 292 Hz, C̲F₃COOH), 127.3, 127.4 (2 d, C-10, C-13), 129.3 (d, C-8), 130.5 (d, C-9), 139.6 (s, C-7), 144.0 (s, C-12), 162.1 (s, C-11), 163.0 (q, ³J_C,F = 34.3 Hz, CF₃C̲OOH), 169.3 (s, C-20), 174.7 (s, C-27), 179.9 (s, C-1) ppm. Minor rotamer (selected signals): ¹H NMR (400 MHz, MeOH-d₄): δ 0.90 (m, 3 H, 24-H), 0.99 (m, 3 H, 25-H), 1.06 (m, 3 H, 18-H), 1.13 (m, 3 H, 3-H), 2.78 (s, 3 H, 32-H), 3.85 (dd, ³J_27,28eq = 11.9 Hz, ³J_27,28ax = 2.4 Hz, 1 H, 27-H), 4.79 (d, ³J_21,22 = 4.6 Hz, 1 H, 21-H), 8.30 (s, 1 H, 13-H) ppm. ¹³C NMR (100 MHz, MeOH-d₄): δ 11.6 (q, C-24), 16.3 (q, C-25), 20.6 (q, C-18), 28.4 (q, C-19), 37.5 (d, C-22), 39.3 (t, C-3), 43.2 (q, C-32), 52.3 (d, C-5), 54.9 (t, C-31), 56.9 (d, C-21), 62.3 (d, C-16), 139.7 (s, C-7), 143.9 (s, C-12), 170.0 (s, C-20), 173.4 (s, C-27), 178.3 (s, C-1) ppm. HRMS (CI) m/z calcd for C₃₅H₅₆N₇O₅ [M+H]⁺: 654.4343; found 654.4368.

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (FOR 1406, Ka 880/10-1).

References

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Footnote

† Electronic supplementary information (ESI) available: Experimental procedures and spectral data for all new compounds, including copies of ¹H and ¹³C NMR spectra. See DOI: 10.1039/c2ra20191g

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