New transient receptor potential TRPV1, TRPM8 and TRPA1 channel antagonists from a single linear β,γ-diamino ester scaffold

Abstract

A high throughput screening campaign identified nine β,γ-diamino ester derivatives as TRP modulators. A discrete library of new derivatives (23 components) was prepared in a one-pot two step reductive amination reaction, and evaluated for their ability to block the agonist-induced calcium influx in cells expressing human TRPV1, TRPM8 and TRPA1 channels. Selective antagonists for each channel, as well as dual TRPV1/TRPM8 and TRPM8/TRPA1 ligands, were obtained after subtle modification of this linear scaffold. SAR studies revealed the preferred substituents for the selective blockade of the three TRP channels under study. The most potent TRPV1 antagonists displayed submicromolar IC₅₀ values.

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Introduction

Membrane transient receptor potential (TRP) proteins are voltage- and ligand-gated ion channels with a variety of biological functions and are apparently implicated in diverse pathological conditions. According to sequence similarities, mammalian TRP channels comprise six subfamilies with low sequence identity among them (as low as 20%), named TRPA (ankyrin), TRPC (canonical), TRPM (melastatin), TRPML (mucolipin), TRPP (polycystin), and TRPV (vanilloid).¹

A few members of the TRP family (TRPV1-4, TRPM8 and TRPA1), belonging to three different subfamilies, are the so-called thermoTRP channels, which participate in the detection of temperature changes and also integrate different noxious stimuli.² Thus, TRPV1 is a non-selective Ca²⁺ channel activated by noxious temperatures (>43 °C), acidic pH and vanilloid compounds. TRPV1 expression is overregulated under acute inflammatory states^3–5 and in chronic pain conditions,⁶ and its activity is potentiated by proalgesic mediators after inflammation and tissue injury.^7,8 TRPM8 channels have a physiological role in detecting low temperature (10–33 °C),⁹ and are also over-expressed in sensory neurons after nerve injury or inflammation,¹⁰ as well as involved in cold allodynia and hyperalgesia.¹¹ TRPA1 is also a non-selective Ca²⁺ channel activated by multiple stimuli, including harmful cold temperatures, acids, and numerous chemical pollutants.^12–15 TRPA1 receptors are coexpressed with TRPV1 channels in C-fiber sensory neurons,¹⁶ and seems to have a crucial role in neuronal and non-neuronal neuropathic pain.^17–19

Since patients with inflammatory or neuropathic pain suffer from hypersensitivity to mechanical, thermal and/or chemical stimuli, an approach to develop successful analgesic therapies may be to target TRPV1, TRPM8 and TRPA1 nociceptors.²⁰ However, despite the big number of compounds currently available in this field, their poor specificity and side effects justify the need for new compounds.²¹ A few representative modulators of these three types of TRP channels are depicted in Chart 1. Knowledge about the molecular requirements that makes a particular family of compounds to bind to one or more of these channels is still scarce and, in general, the described compounds for every TRP channel are structurally very different among them.²¹

Chart 1 Representative TRPA1, TRPM8 and TRPV1 antagonists.

Within our programs of innovative chemical libraries we have recently described a series of β,γ-diamino ester derivatives 1–4 as valuable building block intermediates to heterocyclic compounds (Chart 2).²² Following our interest in TRP modulators,^23–26 some of these derivatives were evaluated in our HTS screening platform for the search of new TRP channel ligands. Within them, we found either selective antagonists for TRPV1 and TRPM8 or dual TRPM8/TRPA1 blockers, although they are very closely related compounds. To explore further the interest of these β,γ-diamino ester derivatives as TRP ligands, and to look deeply into the structural particularities behind the TRP channel type preferences, in this paper we describe the results of the biological evaluation of compounds 1–4, along with the preparation and HTS screening of new derivatives within this series.

Chart 2 β,γ-Diamino ester derivatives previously synthesized by us.

Results and discussion

Chemistry

To understand the role of the benzyloxycarbonyl (Z) and ester groups in 4, we first aim to prepare the Boc analogue 8 and some ester variations, 9–11. Highly functionalized β,γ-diamino esters 8–11, derived from Phe and Ala, were prepared following our previously described method for analogues 4a–c.²² Briefly, the reaction of β-ketoesters 5–7 with the corresponding Ala-derived α-amino esters in the presence of AcOH led to imine intermediates, which were then reduced with NaBH₃CN. This two-step reductive amination procedure afforded compounds 4, 8–11 as mixtures of diastereoisomers that, in most cases, can be separated by column chromatography. As described, three isomers had been isolated for compound 4a–c, and their configurations had indirectly been determined through transformation to pyrrolidinone derivatives.²² Similarly, the formation of three major stereoisomers was observed in the case of compound 8–10, where only traces of a fourth diastereoisomer could be detected by HPLC-MS (not isolated). However, for the Ala-OBn derivative 11, all four possible diastereoisomers were formed, although isomers 11c and 11d could not be separated in pure form. In each case the configurational assignment was performed by comparison of NMR and HPLC data with those of 4a–c (Scheme 1).

Scheme 1

The loss of the stereochemical integrity of the initial amino acid-derived β-ketoester, leading to diastereoisomers c and d, could be explained through the existence of imine–enamine tautomerism, as previously determined by reduction with NaBD₃CN.²²

To set light on the importance of the starting amino acids side chains, we then prepared compounds 14–16, in which the initial Ala-O^tBu derivative was changed by Phe-O^tBu, and the Z-Phe-derived ketoester was substituted by those resulting from Z-Ala and Z-Ile (Scheme 2). Phe–Phe-derived compound 14 was obtained as a 23 : 44 : 23 : 10 mixture of a–d diastereoisomers, with 14c–14d isolated as an inseparable mix. However, three isomers were detected for the Ala–Phe analogue 15, although only isomers 15a and 15b were separated in pure form. Four main isomers were obtained in the case of compound 16, from which isomers a, b and d could be totally separated. As the Ile residue has an additional stereogenic center, other minor isomers were detected by HPLC and in certain NMR fractions (not isolated). As indicated above, the configuration assignment was tentatively done by comparison with isomers 4a–c.

Scheme 2

Finally, to explore the importance of the 3-NH group, we prepare the corresponding acetyl derivative 17a by treatment of compound 4a with acetyl chloride in the presence of propylene oxide as HCl scavenger (Scheme 3).

Scheme 3

Biological evaluation

All compounds were assayed on TRPV1, TRPM8 and TRPA1 channels, stably expressed in the appropriate cell lines (see Experimental for details). The agonist-induced intracellular Ca²⁺ signals were measured by microfluorography, using a fluorescence plate reader, in the absence and in the presence of test compounds. Capsaicin (TRPV1), menthol (TRPM8) and allylisothiocyanate (TRPA1) were used as the respective agonists. The obtained results were compared toward those of AMTB (TRPM8 antagonist) and ruthenium red (TRPV1, TRPA1 unspecific antagonist). The obtained results are recorded in Tables 1 and 2.

Table 1 Activities of compounds 1–4 against TRPV1, TRPM8 and TRPA1 (expressed as % blockade at the indicated concentrations)^d

Compd	Config. 3,4 or 3,4,1′	R	Concentration (μM)	TRPV1^a	TRPM8^b	TRPA1^c
a Blockade of Ca^2+ entry through TRPV1 channel by compounds (capsaicin, 10 μM, was used as the agonist).b Blockade of Ca^2+ entry through TRPM8 channel by compounds (menthol, 100 μM, was used as the agonist).c Blockade of Ca^2+ entry through TRPA1 channel by compounds (allyl isothiocyanate, 100 μM, was used as the agonist).d Compounds were assayed at 50 μM (up) and 5 μM (down) concentrations. NA: blockade lower than 25% at the higher concentration assayed.
1a	S,S	CH2Ph	50	31.50 ± 16.63	89.08 ± 5.40	NA
			5	29.48 ± 6.46	57.54 ± 1.67
1b	R,S	CH2Ph	50	65.05 ± 2.72	91.89 ± 4.07	NA
			5	14.60 ± 24.26	60.66 ± 5.34
2a	S,S	(CH2)3CH3	50	89.68 ± 11.88	74.93 ± 3.11	NA
			5	21.11 ± 8.00	52.98 ± 6.46
2b	R,S	(CH2)3CH3	50	54.95 ± 3.51	83.91 ± 4.42	NA
			5	15.14 ± 7.65	56.79 ± 6.11
3a	S,S	CH2CO2Me	50	57.38 ± 3.59	84.36 ± 5.41	80.14 ± 14.67
			5	22.00 ± 3.51	37.83 ± 11.03	70.34 ± 13.25
3b	R,S	CH2CO2Me	50	84.22 ± 9.08	79.51 ± 4.71	60.86 ± 10.71
			5	24.46 ± 15.73	36.96 ± 14.30	66.87 ± 17.04
4a	S,S,S	—	50	99.19 ± 12.10	NA	NA
			5	73.50 ± 9.62
4b	R,S,S	—	50	109.28 ± 6.54	62.29 ± 10.46	NA
			5	72.25 ± 11.10	−15.13 ± 19.57
4c	R,R,S	—	50	106.76 ± 9.82	57.45 ± 22.47	NA
			5	49.91 ± 22.57	−8.20 ± 17.97
Ruthenium red		—	10	100%		100%
AMTB		—	10		100%

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Table 2 Activities of compounds 8–11 and 14–17 against TRPV1, TRPM8 and TRPA1 (expressed as % blockade at 50 and 5 μM concentrations)^d

Compd	Config. 3,4,1′	R^1	R^2	R^3	R^4	R^5	TRPV1^a	TRPM8^b	TRPA1^c
a Blockade of Ca^2+ entry through TRPV1 channel by compounds (capsaicin, 10 μM, was used as the agonist).b Blockade of Ca^2+ entry through TRPM8 channel by compounds (menthol, 100 μM, was used as the agonist).c Blockade of Ca^2+ entry through TRPA1 channel by compounds (allyl isothiocyanate, 100 μM, was used as the agonist).d Compounds were assayed at 50 μM (up) and 5 μM (down) concentrations. NA: blockade lower than 25% at the higher concentration assayed.
8a	S,S,S	Boc	Me	^tBu	Bn	Me	106.76 ± 9.82	88.55 ± 13.60	NA
							52.12 ± 12.39	14.64 ± 8.42
8b	R,S,S	Boc	Me	^tBu	Bn	Me	103.46 ± 8.27	33.14 ± 21.05	NA
							62.00 ± 10.03	9.56 ± 2.95
8c	R,R,S	Boc	Me	^tBu	Bn	Me	89.97 ± 2.62	38.96 ± 13.62	NA
							49.45 ± 4.86	8.34 ± 11.67
9a	S,S,S	Z	Et	^tBu	Bn	Me	88.30 ± 8.70	85.72 ± 5.01	NA
							74.58 ± 5.24	70.77 ± 5.49
9b	S,R,S	Z	Et	^tBu	Bn	Me	95.83 ± 5.81	87.90 ± 6.25	NA
							72.35 ± 17.12	39.78 ± 7.97
9c	R,R,S	Z	Et	^tBu	Bn	Me	92.95 ± 12.14	96.97 ± 6.91	NA
							50.49 ± 16.58	40.51 ± 8.85
10a	S,S,S	Z	Me	Me	Bn	Me	75.53 ± 1.24	57.83 ± 5.92	NA
							23.74 ± 15.03	13.48 ± 7.33
10b	R,S,S	Z	Me	Me	Bn	Me	28.41 ± 12.41	51.83 ± 8.30	NA
							9.65 ± 11.52	16.14 ± 16.27
10c	R,R,S	Z	Me	Me	Bn	Me	NA	46.54 ± 7.18	NA
								19.92 ± 7.52
11a	S,S,S	Z	Me	Bn	Bn	Me	NA	96.89 ± 2.67	93.46 ± 9.16
								69.79 ± 6.29	67.07 ± 7.47
11b	R,S,S	Z	Me	Bn	Bn	Me	42.49 ± 6.83	96.68 ± 3.01	90.61 ± 15.78
							17.92 ± 1.74	43.10 ± 8.24	67.84 ± 15.94
11c,d	R,R,S	Z	Me	Bn	Bn	Me	63.56 ± 5.18	95.91 ± 4.66	90.05 ± 18.55
	S,R,S						24.24 ± 7.06	65.65 ± 3.47	71.53 ± 4.30
14a	S,S,S	Z	Me	^tBu	Bn	Bn	67.05 ± 9.43	102.85 ± 3.00	57.07 ± 18.64
							15.16 ± 12.92	73.47 ± 7.41	44.20 ± 16.94
14b	R,S,S	Z	Me	^tBu	Bn	Bn	90.77 ± 2.17	86.91 ± 3.09	78.14 ± 9.53
							45.92 ± 14.74	47.09 ± 5.84	60.18 ± 11.21
14c,d	R,R,S	Z	Me	^tBu	Bn	Bn	85.37 ± 5.86	92.96 ± 4.22	81.99 ± 9.40
	S,R,S						42.17 ± 8.30	40.50 ± 13.24	54.25 ± 11.60
15a	S,S,S	Z	Me	^tBu	Me	Bn	54.46 ± 8.92	86.74 ± 5.26	NA
							17.90 ± 9.01	34.81 ± 5.85
15b	R,S,S	Z	Me	^tBu	Me	Bn	91.09 ± 3.78	86.35 ± 6.97	NA
							31.40 ± 4.84	29.10 ± 22.35
16b	R,S,S	Z	Me	^tBu	s-Bu	Me	61.88 ± 11.62	84.92 ± 13.64	NA
							6.46 ± 3.77	27.32 ± 8.42
17a	S,S,S	Z	Me	^tBu	Bn	Me	114.22 ± 9.16	72.98 ± 14.78	NA
							50.16 ± 11.85	−23.67 ± 10.83

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As shown in Table 1, N-benzyl and N-n-butyl derivatives 1a,b and 2a,b showed an interesting TRPM8 blockade activity (up to 91 and 60% inhibition of the menthol-induced channel activation at 50 and 5 μM, respectively). In addition, at the indicated concentrations, they are quite selective against TRPV1 (significant blockade was observed only at the high concentration) and especially against TRPA1. Interestingly, Gly analogues 3a,b at the lower concentration (5 μM) decreased TRPM8 activity but were able to nicely block TRPA1 channels. It seems that the incorporation of a polar, H-accepting ester moiety favors TRPA1 recognition, while more hydrophobic residues (Bn, Bu) are preferred at the TRPM8. Interestingly, Ala-derived compounds 4a–c behave as potent and selective TRPV1 antagonists. Thus, a minor modification of compound 3, by the incorporation of a small Me group in 4, gave to a shift in the selectivity, suggesting that this group could occupy a cavity in TRPV1 channels that is not present/accessible in TRPM8 and TRPA1. In general, each distereoisomer of the same compound displayed very similar activities and selectivities. It is interesting to note that the initial ketoester 1 (5 μM) did not show any significant antagonist activity on the channels under study (data not shown).

Modifications at the different parts of molecule 4 also provided us with valuable structural information. Thus, the benzyloxycarbonyl group in 4a,c might be interchanged by a tert-butoxycarbonyl moiety in derivatives 8a,c without apparent loss of TRPV1 antagonist activity, and similar selectivities. However, removal of the urethane moiety and cyclization to the corresponding pyrrolidinone derivatives led to inactive compounds in all studied TRP channels (data not shown), telling us on the importance of a hydrophobic substituent on the Phe nitrogen of 4 and 8.

When the R² substituent in 4 (Me) is changed by an ethyl group in 9, all diastereoisomers were able to maintain the potent TRPV1 antagonist properties, but an increase in the TRPM8 blocking activity was also detected, specially at the higher concentration. Therefore, Et-derivatives 9 can be considered as dual TRPV1/TRPM8 antagonists.

Concerning the R³ substituent, different results were obtained when the tert-butyl group was replaced by its methyl (10) or benzyl (11) counterparts. While Me derivatives 10a–c were mainly inactive in the three TRP channel assayed, Bn analogues 11a–c lost the TRPV1 antagonist activity, although they showed a significant ability to inhibit the Ca²⁺ entry through TRPM8 and TRPA1 channels, upon activation with their respective agonists. A similar dual TRPM8/TRPA1 antagonist activity was observed in the case of compounds 14a,c, in which a Phe-O^tBu residue was incorporated instead of the Ala-O^tBu in 4. The fact that Phe-O^tBu and Ala-OBn derived compounds 11 and 14 showed similar activity/selectivity profile could associate the presence of an aromatic ring by this part of the molecule with a preference for TRPM8 and TRPA1 channels.

The role of the R⁴ benzyl group is not negligible, since compounds with the reverse sequence 15a–b were only able to maintain certain TRPV1 and TRPM8 antagonist activity at high concentrations, and completely loss it for TRPA1 channels. The importance of the phenyl group of this benzyl moiety was corroborated by the lack of activity of the Ile analogue 16b at 5 μM concentration, compared to 4b.

Finally, the decreased TRPV1 antagonist activity of the acetyl derivative 17a, compared to its free NH analogue 4a, suggests a possible direct participation of the NH group either in a saline bridge or in an H-bond formation.

As mentioned previously, no big differences in activity were found among diastereoisomers of the same compound, suggesting that the pocket within the studied TRP channels responsible for the interaction with this family of compounds is quite big, allowing different spatial arrangements to accommodate.

To further validate the TRP-blocking activity for the apparently most potent and selective TRP antagonists, the corresponding dose–response curves were obtained for selected compounds. Table 3 summarizes the IC₅₀ values of N-benzyl derivatives 1a–b for TRPM8 receptors, Gly-analogue 3a for TRPA1, and Ala-derived compounds 4a–c and 8a–c against TRPV1 channels. TRPM8 selective antagonists 1a and 1b show similar micromolar IC₅₀ values, regardless of the configuration at C3. A related value was obtained for the TRPA1 antagonists 3a. Diastereoisomeric Phe–Ala diamino esters 4 and 8 display micromolar or submicromolar IC₅₀ values for TRPV1, quite similar among them, with compound 4b showing the highest potency. Although the configuration does not play a crucial role in the antagonist activity, confirming previous results, the 3R,4S,1′S diastereoisomers seems to give to slightly higher potencies.

Table 3 Potency of selected compounds for TRP channels

Compd	Configuration 3,4 or 3,4,1′	Channel	IC50 (μM)
1a	S,S	TRPM8	3.64 ± 0.34
1b	R,S	TRPM8	2.75 ± 0.18
3a	S,S	TRPA1	1.40 ± 0.70
4a	S,S,S	TRPV1	0.62 ± 0.32
4b	R,S,S	TRPV1	0.30 ± 0.33
4c	S,S,R	TRPV1	1.75 ± 0.50
8a	S,S,S	TRPV1	1.13 ± 1.80
8b	R,S,S	TRPV1	0.47 ± 0.80
8c	S,S,R	TRPV1	3.17 ± 0.76

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In in vivo experiments, compound 4b produced some elevations of PWL in the plantar test, at a 2 mg kg⁻¹ dose ip (see ESI, Fig S1†). Although not statistically significant, it seems that this compound could decrease the thermal hyperalgesia induced in mice by CFA injection. However, no positive signs of activity were observed in the mechanical von Frey test (mechanical hypersensitivity) at this dose. Unfortunately, we could not evaluate the effect at higher doses due to low solubility issues.

Conclusions

In summary, medicinal chemistry efforts initiated from a HTS of synthetic intermediates resulted in the discovery of new selective hits for TRPV1, TRPM8 and TRPA1 blockade, just by incorporating minor changes in the structure of a single β,γ-diaminoester linear scaffold. Compounds were prepared by a two-steps reductive amination process that afforded separable mixtures of diastereoisomers. Starting with Phe-derived β-ketoesters, the SAR demonstrated that the addition of simple 3-NHBn or 3-NHBu substituents resulted in TRPM8 antagonists. Compounds including a second amino acid residue indicated that 3-Gly-OMe-containing derivatives are selective TRPA1 antagonists (3), while the corresponding 3-Ala-O^tBu analogues are potent and selective TRPV1 blockers (4). An increase in the hydrophobicity around the R² substituent enhanced the ability to block the TRPM8 activation, while keeping the TRPV1 antagonists activity, thus resulting in dual TRPV1/TRPM8 blockers. At R³, a tert-butyl ester is clearly preferred for the vanilloid channel while Bn analogues are better for the inhibition of the agonist-activation of both TRPM8 and TRPA1 channels. Dual TRPM8/TRPA1 blockers were also found when the 3-Ala-O^tBu moiety was substituted for the more hydrophobic 3-Phe-OBn residue. Concerning the stereochemistry, no strict requirements were observed, with all SSS, RSS and SSR configurations allowed (slightly better results for the RSS diastereoisomers). Although further pharmacological characterization of compounds within this series is limited by their low solubility, and probably poor pharmacokinetics, due in part to the high number of rotable bonds, this work allowed the identification of substituents and amino acid residues that led to selective modulators of the indicated three types of TRP channels. Attaching these particular combinations of substituent on more rigid scaffolds that allowed different 3D-arrangements could serve to discover new potent and selective TRP blockers with improved PK profile. Efforts in this direction are in progress in our labs.

Experimental

General methods

All reagents were of commercial quality. Solvents were dried and purified by standard methods. ¹H NMR spectra were recorded at 300 or 400 MHz in CDCl₃. ¹³C NMR spectra were registered at 75 MHz. Electrospray mass spectra (positive mode) were also recorded. Analytical TLC was performed on aluminium sheets with a 0.2 mm layer of silica gel F254. Silica gel 60 (230–400 mesh) was used for column chromatography. Silica gel SPE cartridges (Supelco) were also used for compound purification. Analytical HPLC was performed on a Novapak C₁₈ (3.9 × 150 mm, 0.004 mm), Deltapak C₁₈ (3.9 × 150 mm, 0.004 mm) or on a Sunfire C₁₈ (3.9 × 150 mm, 4 μM) column, with a flow rate of 1 mL min⁻¹, using a tuneable UV detector set at 214 nm. Mixtures of MeCN (solvent A) and 0.05% TFA in H₂O (solvent B) were used in the mobile phase. The solvent mixtures are specified in each case. Electrospray mass spectra (positive mode) were also recorded. Compounds 1a,b–4a–c were prepared as described.²¹ β-Ketoesters 5–7, 12 and 13 were prepared by standard procedures, and their analytical and spectroscopic data are coincident with those described.^27–30

Methyl (4S)-tert-butoxycarbonylamino-(3S)-[(1′S-tert-butoxy-carbonyl)eth-1′-yl]amino-5-phenylpentanoate (8a). (EtOAc : hexane, 1 : 8). Yield: 19% (syrup). t_R = 13.04 min (5 to 100% A in 15 min). ¹H NMR (CDCl₃) δ: 7.29–7.18 (m, 5H, Ar), 4.81 (d, 1H, J = 9.2, 4-NH), 3.88 (m, 1H, H-4), 3.63 (s, 3H, OCH₃), 3.28 (q, 1H, J = 6.9, H-1′), 2.95 (m, 1H, H-3), 2.80 (d, 2H, J = 7.4, H-5), 2.49 (dd, 1H, J = 15.5, 6.2, H-2), 2.42 (dd, 1H, J = 15.5, 7.2, H-2), 1.68 (bs, 1H, 3-NH), 1.44 (s, 9H, CH₃ ^tBu, Boc), 1.34 (s, 9H, CH₃ ^tBu), 1.24 (d, 3H, J = 6.9, H-2′). ¹³C NMR (75 MHz, CDCl₃) δ: 175.4, 172.2 (CO), 155.5 (NCO), 138.3 (C Ar), 129.2, 128.3, 126.2 (CH Ar), 81.1, 79.9 (C ^tBu), 57.0 (C1′), 54.9 (C4, C3), 51.6 (OCH₃), 39.2 (C5), 38.1 (C2), 28.3, 27.9 (CH₃ ^tBu), 20.20 (C2′). MS: 451.6 [M + 1]⁺. Anal. calc. for C₂₄H₃₈N₂O₆: C, 63.98; H, 8.50; N, 6.22. Found: C, 63.75; H, 8.44; N, 6.04.

Methyl (4S)-tert-butoxycarbonylamino-(3R)-[(1′S-tert-butoxy-carbonyl)eth-1′-yl]amino-5-phenylpentanoate (8b). (EtOAc : hexane, 1 : 8). Yield: 29% (syrup). t_R = 12.42 min (5 to 100% A in 15 min). ¹H NMR (CDCl₃) δ: 7.31–7.19 (m, 5H, Ar), 4.98 (d, 1H, J = 8.5, 4-NH), 3.89 (m, 1H, H-4), 3.65 (s, 3H, OCH₃), 3.29 (q, 1H, J = 6.8, H-1′), 3.11 (dt, 1H, J = 6.2, 2.8, H-3), 2.89 (dd, 1H, J = 13.6, 6.1, H-5), 2.78 (dd, 1H, J = 13.6, 7.7, H-5), 2.49 (dd, 1H, J = 15.6, 6.3, H-2), 2.39 (dd, 1H, J = 15.6, 6.5, H-2), 1.69 (bs, 1H, 3-NH), 1.47 (s, 9H, CH₃ ^tBu), 1.37 (s, 9H, CH₃ ^tBu), 1.25 (d, 3H, J = 6.8, H-2′). ¹³C NMR (75 MHz, CDCl₃) δ: 174.9, 172.2 (CO), 155.6 (NCO), 138.1 (C Ar), 129.3, 128.3, 126.2 (CH Ar), 81.1, 79.0 (C ^tBu), 55.1 (C1′), 54.9 (C4), 54.5 (C3), 51.6 (OCH₃), 38.5 (C5), 37.5 (C2), 28.3, 27.9 (CH₃ ^tBu), 19.8 (C2′). MS: 451.6 [M + 1]⁺. Anal. calc. for C₂₄H₃₈N₂O₆: C, 63.98; H, 8.50; N, 6.22. Found: C, 63.59; H, 8.61; N, 6.07.

Methyl (4R)-tert-butoxycarbonylamino-(3R)-[(1′S-tert-butoxycarbonyl)eth-1′-yl]amino-5-phenylpentanoate (8c). (EtOAc : hexane, 1 : 8). Yield: 20% (syrup). t_R = 12.21 min (5 to 100% A in 15 min). ¹H NMR (CDCl₃) δ: 7.22–7.09 (m, 5H, Ar), 4.81 (m, 1H, 4-NH), 3.85 (m, 1H, H-4), 3.61 (s, 3H, OCH₃), 3.25 (q, 1H, J = 6.9, H-1′), 3.04 (dt, 1H, J = 7.2, 5.7, H-3), 2.86 (dd, 1H, J = 13.6, 5.2, H-5), 2.66 (m, 1H, H-5), 2.50 (dd, 1H, J = 15.2, 5.7, H-2), 2.40 (dd, 1H, J = 15.2, 7.2, H-2), 1.69 (bs, 1H, 3-NH), 1.38 (s, 9H, CH₃ ^tBu), 1.24 (s, 9H, CH₃ ^tBu), 1.14 (d, 3H, J = 6.8, H-2′). ¹³C NMR (75 MHz, CDCl₃) δ: 174.9, 172.6 (CO), 155.5 (NCO), 138.2 (C Ar), 129.2, 128.3, 126.3 (CH Ar), 80.9, 79.0 (C ^tBu), 56.7 (C3), 55.2 (C1′), 54.5 (C4), 51.7 (OCH₃), 37.4 (C5), 37.0 (C2), 28.2, 27.9 (CH₃ ^tBu), 19.6 (C2′). MS: 451.6 [M + 1]⁺. Anal. calc. for C₂₄H₃₈N₂O₆: C, 63.98; H, 8.50; N, 6.22. Found: C, 63.62; H, 8.70; N, 5.98.

Ethyl (4S)-benzyloxycarbonylamino-(3S)-[(1′S-tert-butoxy-carbonyl)eth-1′-yl]amino-5-phenylpentanoate (9a). (EtOAc : hexane, 1 : 8). Yield: 16% (syrup). t_R = 14.03 min (5 to 100% A in 15 min). ¹H NMR (CDCl₃) δ: 7.32–7.19 (m, 10H, Ar), 5.17 (d, 1H, J = 9.3, 4-NH), 5.05, 4.98 (d, 1H, J = 12.1, OCH₂ Z), 4.07 (m, 2H, OCH₂), 3.97 (m, 1H, H-4), 3.31 (q, 1H, J = 6.8, H-1′), 2.98 (t, 1H, J = 6.1, H-3), 2.84 (d, 2H, J = 7.4, H-5), 2.46 (dd, 1H, J = 15.1, 7.0, H-2), 2.44 (dd, 1H, J = 15.1, 5.5, H-2), 1.69 (bs, 1H, 3-NH), 1.44 (s, 9H, CH₃ ^tBu), 1.25 (d, 3H, J = 7.0, H-2′), 1.20 (t, 3H, J = 7.1, CH₃ OEt). ¹³C NMR (75 MHz, CDCl₃) δ: 175.2, 171.5 (CO), 156.1 (NCO), 138.1, 136.0 (C Ar), 129.1, 128.4, 128.3, 127.9, 126.3 (CH Ar), 81.1 (C ^tBu), 66.4 (OCH₂ Z), 60.5 (OCH₂), 57.1 (C1′), 55.5 (C4), 54.7 (C3), 39.1 (C5), 38.3 (C2), 27.9 (CH₃ ^tBu), 19.9 (C2′), 14.0 (CH₃ OEt). MS: 499.6 [M + 1]⁺.

Anal. calc. for C₂₈H₃₈N₂O₆: C, 67.45; H, 7.68; N, 5.62. Found: C, 67.21; H, 7.98; N, 5.49.

Ethyl (4S)-benzyloxycarbonylamino-(3R)-[(1′S-tert-butoxy-carbonyl)eth-1′-yl]amino-5-phenylpentanoate (9b). (EtOAc : hexane, 1 : 8). Yield: 31% (syrup). t_R = 13.22 min (5 to 100% A in 15 min). ¹H NMR (CDCl₃) δ: 7.32–7.17 (m, 10H, Ar), 5.36 (d, 1H, J = 8.6, 4-NH), 5.07, 5.00 (d, 1H, J = 12.3, OCH₂ Z), 4.01 (m, 2H, OCH₂ OEt), 3.95 (m, 1H, H-4), 3.28 (q, 1H, J = 6.9, H-1′), 3.12 (dt, 1H, J = 6.2, 2.9, H-3), 2.93 (dd, 1H, J = 14.0, 5.7, H-5), 2.81 (dd, 1H, J = 14.0, 7.5, H-5), 2.96 (dd, 1H, J = 15.6, 6.0, H-2), 2.81 (dd, 1H, J = 15.6, 6.3, H-2), 1.71 (bs, 1H, 3-NH), 1.45 (s, 9H, CH₃ ^tBu), 1.23 (d, 3H, J = 6.9, H-2′), 1.20 (t, 3H, J = 6.9, CH₃ OEt). ¹³C NMR (75 MHz, CDCl₃) δ: 174.9, 171.6 (CO), 156.1 (NCO), 137.8, 136.6 (C Ar), 129.3, 129.2, 128.4, 128.3, 127.9, 127.9, 126.3 (CH Ar), 81.2 (C ^tBu), 66.4 (OCH₂ Z), 60.5 (OCH₂), 55.5 (C4), 55.0 (C1′), 54.5 (C3), 39.3 (C5), 37.6 (C2), 27.9 (CH₃ ^tBu), 19.7 (C2′), 14.1 (CH₃ OEt). MS: 499.6 [M + 1]⁺. Anal. calc. for C₂₈H₃₈N₂O₆: C, 67.45; H, 7.68; N, 5.62. Found: C, 67.33; H, 7.96; N, 5.23.

Ethyl (4R)-benzyloxycarbonylamino-(3R)-[(1′S-tert-butoxy-carbonyl)eth-1′-yl]amino-5-phenylpentanoate (9c). (EtOAc : hexane, 1 : 8). Yield: 16% (syrup). t_R = 13.36 min (5 to 100% A in 15 min). ¹H NMR (300 MHz, CDCl₃) δ: 7.32–7.19 (m, 10H, Ar), 5.40 (bd, 1H, J = 8.2, 4-NH), 5.03, 4.96 (d, 1H, J = 12.6, OCH₂ Z), 4.13 (q, 2H, J = 7.1, OCH₂ OEt), 4.00 (m, 1H, H-4), 3.33 (q, 1H, J = 6.9, H-1′), 3.13 (q, 1H, J = 6.5, H-3), 2.93 (dd, 1H, J = 13.8, 5.3, H-5), 2.73 (dd, 1H, J = 13.8, 8.6, H-5), 2.50 (dd, 1H, J = 15.2, 5.9, H-2), 2.42 (dd, 1H, J = 15.2, 7.1 H-2), 1.67 (bs, 1H, 3-NH), 1.42 (s, 9H, CH₃ ^tBu), 1.24 (d, 3H, J = 6.9, H-2′), 1.21 (t, 3H, J = 6.9, CH₃ OEt). ¹³C NMR (75 MHz, CDCl₃) δ: 174.9, 172.1 (CO), 156.1 (NCO), 138.0, 136.7 (C Ar), 129.2, 128.4, 128.3, 127.8, 127.7, 126.4 (CH Ar), 81.0 (C ^tBu), 66.3 (OCH₂ Z), 60.6 (OCH₂ OEt), 56.8 (C3), 55.4 (C4, C1′), 37.3 (C2, C5), 27.9 (CH₃ ^tBu), 19.6 (C2′), 14.1 (CH₃ OEt). MS: 499.6 [M + 1]⁺. Anal. calc. for C₂₈H₃₈N₂O₆: C, 67.45; H, 7.68; N, 5.62. Found: C, 67.36; H, 7.77; N, 5.35.

Methyl (4S)-benzyloxycarbonylamino-(3S)-[(1′S-methoxy-carbonyl)eth-1′-yl]amino-5-phenylpentanoate (10a). (CH₂Cl₂ : ether : hexane, 1 : 1 : 2). Yield: 16% (syrup). t_R = 8.89 min (20 to 100% A in 20 min). ¹H NMR (400 MHz, CDCl₃) δ: 7.26–7.13 (m, 10H, Ar), 5.12 (m, 1H, 4-NH), 4.98, 4.89 (d, 1H, J = 12.3, OCH₂), 3.92 (m, 1H, H-4), 3.62 (s, 3H, OCH₃), 3.56 (s, 3H, OCH₃), 3.39 (m, 1H, H-1′), 2.99 (m, 1H, H-3), 2.78 (m, 2H, H-5), 2.45 (m, 2H, H-2), 1.56 (bs, 1H, 3-NH), 1.25 (d, 3H, J = 6.9, H-2′). ¹³C NMR (75 MHz, CDCl₃) δ: 175.8, 172.0 (CO), 156.1 (NCO), 137.8, 136.5 (C Ar), 129.1, 128.4, 127.9, 127.8, 126.4 (CH Ar), 66.5 (OCH₂), 56.6 (C4), 55.6 (C3), 55.4 (C1′), 52.0, 51.7 (OCH₃), 38.9 (C5), 37.8 (C2), 19.7 (C2′). MS: 443.6 [M + 1]⁺. Anal. calc. for C₂₄H₃₀N₂O₆: C, 65.14; H, 6.83; N, 6.33. Found: C, 65.01; H, 6.50; N, 6.39.

Methyl (4S)-benzyloxycarbonylamino-(3R)-[(1′S-methoxy-carbonyl)eth-1′-yl]amino-5-phenylpentanoate (10b). (CH₂Cl₂ : ether : hexane, 1 : 1 : 2). Yield: 22% (syrup). t_R = 7.85 min (20 to 100% A in 20 min). ¹H NMR (400 MHz, CDCl₃) δ: 7.28–7.13 (m, 10H, Ar), 5.24 (m, 1H, 4-NH), 4.98, 4.91 (d, 1H, J = 12.6, OCH₂), 3.91 (m, 1H, H-4), 3.63 (s, 3H, OCH₃), 3.55 (s, 3H, OCH₃), 3.40 (m, 1H, H-1′), 3.10 (m, 1H, H-3), 2.80 (m, 2H, H-5), 2.41 (m, 2H, H-2), 1.38 (bs, 1H, 3-NH), 1.22 (d, 3H, J = 6.9, H-2′). ¹³C NMR (75 MHz, CDCl₃) δ: 175.4, 172.1 (CO), 156.2 (NCO), 137.7, 136.5 (C Ar), 129.2, 128.4, 128.3, 127.9, 127.8, 126.4 (CH Ar), 66.5 (OCH₂), 55.5 (C4), 54.8 (C3), 54.6 (C1′), 52.0, 51.7 (OCH₃), 38.2 (C5), 37.2 (C2), 19.4 (C2′). MS: 443.6 [M + 1]⁺. Calc. for C₂₄H₃₀N₂O₆: C, 65.14; H, 6.83; N, 6.33. Found: C, 64.88; H, 7.16; N, 6.00.

Methyl (4R)-benzyloxycarbonylamino-(3R)-[(1′S-methoxy-carbonyl)eth-1′-yl]amino-5-phenylpentanoate (10c). (CH₂Cl₂ : ether : hexane, 1 : 1 : 2). Yield: 23% (syrup). t_R = 7.05 min (20 to 100% A in 20 min). ¹H NMR (400 MHz, CDCl₃) δ: 7.32–7.18 (m, 10H, Ar), 5.25 (m, 1H, 4-NH), 5.00 (m, 2H, OCH₂), 3.99 (m, 1H, H-4), 3.66 (s, 3H, OCH₃), 3.65 (s, 3H, OCH₃), 3.46 (m, 1H, H-1′), 3.14 (m, 1H, H-3), 2.93 (dd, 1H, J = 14.0, 5.2, H-5), 2.75 (m, 1H, H-5), 2.46 (m, 2H, H-2), 1.65 (bs, 1H, 3-NH), 1.25 (d, 3H, J = 6.9, H-2′). ¹³C NMR (75 MHz, CDCl₃) δ: 175.6, 172.4 (CO), 156.1 (NCO), 137.8, 136.5 (C Ar), 129.1, 128.4, 128.3, 127.9, 127.8, 126.5 (CH Ar), 66.5 (OCH₂), 56.7 (C3), 55.3 (C4), 54.8 (C1′), 51.9, 51.8 (OCH₃), 37.1 (C5), 36.9 (C2), 19.5 (C2′). MS: 443.6 [M + 1]⁺. Calc. for C₂₄H₃₀N₂O₆: C, 65.14; H, 6.83; N, 6.33. Found: C, 64.95; H, 6.71; N, 6.05.

Methyl (4S)-benzyloxycarbonylamino-(3S)-[(1′S-benzyloxy-carbonyl)eth-1′-yl]amino-5-phenylpentanoate (11a). (CH₂Cl₂ : ether : hexane, 1 : 1 : 3). Yield: 12% (syrup). t_R = 12.05 min (20 to 100% A in 20 min). ¹H NMR (400 MHz, CDCl₃) δ: 7.39–7.19 (m, 15H, Ar), 5.19 (m, 1H, 4-NH), 5.17, 5.11 (d, 1H, J = 12.3, OCH₂), 5.06, 4.95 (d, 1H, J = 12.4, OCH₂), 3.99 (m, 1H, H-4), 3.59 (s, 3H, OCH₃), 3.51 (m, 1H, H-1′), 3.08 (m, 1H, H-3), 2.85 (m, 2H, H-5), 2.50 (m, 2H, H-2), 2.05 (bs, 1H, 3-NH), 1.33 (d, 3H, J = 6.8, H-2′). ¹³C NMR (75 MHz, CDCl₃) δ: 175.3, 171.9 (CO), 156.1 (NCO), 137.8, 136.5, 135.5 (C Ar), 129.1, 128.6, 128.4, 128.3, 128.2, 127.9, 126.4 (CH Ar), 66.8, 66.5 (OCH₂), 56.8 (C1′), 55.5 (C4), 55.4 (C3), 51.7 (OCH₃), 38.9 (C5), 37.8 (C2), 19.7 (C2′). MS: 519.6 [M + 1]⁺. Anal. calc. for C₃₀H₃₄N₂O₆: C, 69.48; H, 6.61; N, 5.40. Found: C, 69.22; H, 6.62; N, 4.97.

Methyl (4S)-benzyloxycarbonylamino-(3R)-[(1′S-benzyloxy-carbonyl)eth-1′-yl]amino-5-phenylpentanoate (11b). (CH₂Cl₂ : ether : hexane, 1 : 1 : 3). Yield: 19% (syrup). t_R = 11.60 min (20 to 100% A in 20 min). ¹H NMR (400 MHz, CDCl₃) δ: 7.34–7.19 (m, 15H, Ar), 5.20 (m, 1H, 4-NH), 5.15 (m, 2H, OCH₂), 5.06, 4.99 (d, 1H, J = 12.3, OCH₂), 3.96 (m, 1H, H-4), 3.60 (s, 3H, OCH₃), 3.47 (m, 1H, H-1′), 3.15 (m, 1H, H-3), 2.76 (m, 2H, H-5), 2.45 (m, 2H, H-2), 1.67 (bs, 1H, 3-NH), 1.29 (d, 3H, J = 6.9, H-2′). ¹³C NMR (75 MHz, CDCl₃) δ: 174.9, 172.0 (CO), 156.1 (NCO), 137.7, 136.5, 135.5 (C Ar), 129.2, 128.6, 128.4, 128.3, 128.2, 127.9, 126.4 (CH Ar), 66.7, 66.5 (OCH₂Ph), 55.5 (C4), 54.8 (C3), 54.6 (C1′), 51.6 (OCH₃), 38.3 (C5), 37.3 (C2), 19.5 (C2′). MS: 519.6 [M + 1]⁺. Anal. calc. for C₃₀H₃₄N₂O₆: C, 69.48; H, 6.61; N, 5.40. Found: C, 69.31; H, 6.38; N, 5.48.

Methyl (4R)-benzyloxycarbonylamino-(3S,R)-[(1′S-benzyloxy-carbonyl)eth-1′-yl]amino-5-phenylpentanoate (11c,d). (CH₂Cl₂ : ether : hexane, 1 : 1 : 3). Yield: 29% (syrup). t_R = 10.19 min (20 to 100% A in 20 min). Diastereosiomeric mixture c : d = 1 : 1. ¹H NMR (300 MHz, CDCl₃) δ: 7.35–7.15 (m, 15H, Ar), 5.24 (d, 1H, J = 8.0, 4-NH), 5.15, 5.09 (d, 1H, J = 12.3, OCH₂), 4.98 (m, 2H, OCH₂), 3.98 (m, 1H, H-4), 3.64, 3.62 (s, 3H, OCH₃), 3.51 (m, 1H, H-3′), 3.16, 3.10 (q, 1H, J = 6.0, H-1′), 2.92 (m, 1H, H-5), 2.72 (m, 1H, H-5), 2.48 (m, 2H, H-2), 1.60 (bs, 1H, 3-NH), 1.28, 1.27 (d, 3H, J = 7.0, H-2′). ¹³C NMR (75 MHz, CDCl₃) δ: 175.7, 175.4 (COO), 172.6, 172.4 (COO), 156.3, 156.1 (NCO), 138.1, 138.0, 136.8, 136.7, 135.85, 135.8 (C Ar), 128.7, 128.6, 128.4, 128.3, 128.1, 127.9, 126.6 (CH Ar), 66.8, 66.6 (OCH₂), 57.0, 56.7 (C3), 55.0 (C4), 55.1, 55.0 (C1′), 51.1, 51.0 (OCH₃), 37.4, 37.2, 37.1 (C5, C2), 19.9, 19.6 (C2′). MS: 519.5 [M + 1]⁺.

Methyl (4S)-benzyloxycarbonylamino-(3S)-[(1′S-tert-butoxy-carbonyl-2′-phenyl)eth-1′-yl]amino-5-phenylpentanoate (14a). (CH₂Cl₂ : ether : hexane, 1 : 1 : 4). Yield: 15% (syrup). t_R = 15.43 min (20 to 100% A in 20 min). ¹H NMR (CDCl₃) δ: 7.33–6.96 (m, 15H, Ar), 5.03, 4.95 (d, 1H, J = 12.4 Hz, OCH₂), 4.90 (bd, 1H, J = 10.0 Hz, 4-NH), 3.81 (m, 1H, H-4), 3.61 (s, 3H, OCH₃), 3.42 (m, 1H, H-1′), 2.95 (m, 2H, H-3, H-5), 2.71 (dd, 1H, J = 8.6, 13.3 Hz, H-5), 2.46 (m, 4H, H-2, H-2′), 1.38 (s, 9H, CH₃ ^tBu). ¹³C NMR (75 MHz, CDCl₃) δ: 174.5, 171.7 (COO), 156.0 (NCO), 138.0, 136.6 (C Ar), 129.6, 129.1, 128.4, 128.3, 128.2, 127.9, 127.8, 126.6, 126.2 (CH Ar), 81.5 (C ^tBu), 66.4 (OCH₂), 64.0 (C1′), 55.7 (C4), 55.3 (C3), 51.6 (OCH₃), 40.7 (C5), 38.9 (C2′), 38.3 (C2), 27.9 (CH₃ ^tBu). MS: 561.3 [M + 1]⁺, 583.3 [M + 23]⁺. Anal. calc. for C₃₃H₄₀N₂O₆: C, 70.69; H, 7.19; N, 5.00. Found: C, 70.34; H, 7.02; N, 5.21.

Methyl (4S)-benzyloxycarbonylamino-(3R)-[(1′S-tert-butoxy-carbonyl-2′-phenyl)eth-1′-yl]amino-5-phenylpentanoate (14b). (CH₂Cl₂ : ether : hexane, 1 : 1 : 4). Yield: 28% (syrup). t_R = 13.48 min (20 to 100% A in 20 min). ¹H NMR (CDCl₃) δ: 7.33–7.15 (m, 15H, Ar), 5.23 (bd, 1H, J = 7.2 Hz, 4-NH), 5.06, 5.00 (d, 1H, J = 12.3 Hz, OCH₂), 3.91 (m, 1H, H-4), 3.56 (s, 3H, OCH₃), 3.44 (m, 1H, H-1′), 3.07 (m, 1H, H-3), 2.85 (m, 4H, H-5, H-2′), 2.31 (m, 2H, H-2), 1.33 (s, 9H, CH₃ ^tBu). ¹³C NMR (75 MHz, CDCl₃) δ: 173.6, 171.9 (COO), 156.0 (NCO), 137.7, 137.1, 136.6 (C Ar), 129.4, 129.3, 128.5, 128.4, 128.3, 128.0, 127.9, 126.7, 126.4 (CH Ar), 81.6 (C ^tBu), 66.5 (OCH₂), 61.1 (C1′), 55.3 (C4), 54.5 (C3), 51.6 (OCH₃), 40.3, 38.4 (C5, C2′), 36.9 (C2), 27.9 (CH₃ ^tBu). MS: 561.3 [M + 1]⁺, 583.3 [M + 23]⁺. Anal. calc. for C₃₃H₄₀N₂O₆: C, 70.69; H, 7.19; N, 5.00. Found: C, 70.85; H, 7.15; N, 4.86.

Methyl (4R)-benzyloxycarbonylamino-(3S,R)-[(1′S-tert-butoxy-carbonyl-2′-phenyl)eth-1′-yl]amino-5-phenylpentanoate (14c,d). (CH₂Cl₂ : ether : hexane, 1 : 1 : 4). Yield: 21% (syrup). t_R = 13.48 min (20 to 100% A in 20 min). Diastereoisomeric mixture c : d = 2.5 : 1. ¹H NMR (300 MHz, CDCl₃) δ: 7.34–7.05 (m, 15H, Ar), 5.33 (bd, 1H, J = 10.6 Hz, 4-NH isomer d), 5.00 (s, 2H, OCH₂ d), 4.93 (s, 2H, OCH₂ isomer c), 4.59 (bd, 1H, J = 8.2 Hz, 4-NH isomer c), 3.97 (m, 1H, H-4 d), 3.83 (m, 1H, H-4 c), 3.63 (s, 3H, OCH₃ c), 3.56 (s, 3H, OCH₃ d), 3.47 (dd, 1H, J = 8.0, 6.0, H-2′ d), 3.47 (dd, 1H, J = 8.5, 5.7, H-2′ c), 3.08 (m, 1H, H-3 d), 2.97 (m, 1H, H-3 d), 2.96–2.64 (m, 2H, H-5, H-3′), 2.46 (m, 4H, H-2 c), 2.25 (m, 4H, H-2 d), 1.37 (s, 9H, CH₃ ^tBu c), 1.36 (s, 9H, CH₃ ^tBu d). MS: 561.2 [M + 1]⁺.

Methyl (4S)-benzyloxycarbonylamino-(3S)-[(1′S-tert-butoxy-carbonyl-2′-phenyl)eth-1′-yl]aminopentanoate (15a). (CH₂Cl₂ : ether : hexane, 1 : 1 : 3). Yield: 6% (syrup). t_R = 13.97 min (20 to 100% A in 30 min). ¹H NMR (CDCl₃) δ: 7.28–7.10 (m, 10H, Ar), 4.99 (m, 2H, OCH₂), 4.84 (m, 1H, 4-NH), 3.57 (m, 4H, H-1′, OCH₃), 3.32 (m, 1H, H-4), 2.80 (m, 2H, H-3, H-2′), 2.67 (dd, 1H, J = 13.4, 8.0, H-2′), 2.34 (m, 2H, H-2), 1.52 (bs, 1H, 3-NH), 1.30 (s, 9H, CH₃ ^tBu), 0.85 (d, 3H, J = 6.7, H-5). ¹³C NMR (75 MHz, CDCl₃) δ: 174.3, 172.1 (CO), 155.9 (NCO), 137.7, 136.6 (C Ar), 129.5, 128.5, 128.2, 128.0, 126.5 (CH Ar), 81.5 (C ^tBu), 66.5 (OCH₂), 63.7 (C4), 57.6 (C3), 51.7 (OCH₃), 49.9 (C1′), 40.5 (C2′), 37.9 (C2), 27.9 (CH₃ ^tBu), 18.5 (C5). MS: 485.6 [M + 1]⁺. Anal. calc. for C₂₇H₃₆N₂O₆: C, 66.92; H, 7.49; N, 5.78. Found: C, 66.73; H, 7.12; N, 5.32.

Methyl (4S)-benzyloxycarbonylamino-(3R)-[(1′S-tert-butoxy-carbonyl-2′-phenyl)eth-1′-yl]aminopentanoate (15b). (CH₂Cl₂ : ether : hexane, 1 : 1 : 3). Yield: 12% (syrup). t_R = 13.37 min (20 to 100% A in 30 min). ¹H NMR (CDCl₃) δ: 7.37–7.18 (m, 10H, Ar), 5.29 (bd, 1H, J = 7.9, 4-NH), 5.11 (m, 2H, OCH₂), 3.73 (m, 1H, H-4), 3.60 (s, 3H, OCH₃), 3.43 (m, 1H, H-1′), 2.98 (m, 1H, H-3), 2.85 (m, 2H, H-2′), 2.36 (dd, 1H, J = 15.6, 6.3, H-2), 2.27 (dd, 1H, J = 15.6, 6.1, H-2), 1.45 (s, 9H, CH₃ ^tBu), 1.17 (d, 3H, J = 6.7, H-5). ¹³C NMR (75 MHz, CDCl₃) δ: 173.8, 172.5 (COO), 156.2 (NCO), 137.4, 136.8 (C Ar), 129.5, 128.6, 128.4, 128.1, 126.7 (CH Ar), 81.7 (C ^tBu), 66.7 (OCH₂), 61.0 (C1′), 57.1 (C3), 51.8 (OCH₃), 49.9 (C4), 40.3 (C2′), 36.7 (C2), 28.1 (CH₃ ^tBu), 18.3 (C5). MS: 485.2 [M + 1]⁺. Anal. calc. for C₂₇H₃₆N₂O₆: C, 66.92; H, 7.49; N, 5.78. Found: C, 66.58; H, 7.83; N, 5.40.

Methyl (4R)-benzyloxycarbonylamino-(3R)-[(1′S-tert-butoxy-carbonyl-2′-phenyl)eth-1′-yl]aminopentanoate (15c). Data extracted from a 2 : 1 mixture of 15b,c. (CH₂Cl₂ : ether : hexane, 1 : 1 : 3). Yield: 42% (syrup). t_R = 13.24 min (20 to 100% A in 30 min). ¹H NMR (CDCl₃) δ: 7.37–7.18 (m, 10H, Ar), 5.77 (bd, 1H, J = 8.1, 4-NH), 5.11 (m, 2H, OCH₂), 3.66 (m, 1H, H-4), 3.60 (s, 3H, OCH₃), 3.48 (m, 1H, H-1′), 2.98 (m, 2H, H-3, H-2′), 2.75 (dd, 1H, J = 13.5, 8.3, H-2′), 2.19 (m, 2H, H-2), 1.46 (s, 9H, CH₃ ^tBu), 1.05 (d, 3H, J = 6.7, H-5). ¹³C NMR (75 MHz, CDCl₃) δ: 174.1, 172.2 (COO), 156.0 (NCO), 137.4, 137.0 (C Ar), 129.4, 128.6, 128.5, 128.1, 126.9 (CH Ar), 81.7 (C ^tBu), 66.5 (OCH₂), 61.6 (C1′), 57.5 (C3), 51.9 (OCH₃), 49.6 (C4), 40.0 (C2′), 37.3 (C2), 28.1 (CH₃ ^tBu), 15.5 (C5). MS: 485.4 [M + 1]⁺.

Methyl (4S)-benzyloxycarbonylamino-(3S)-[(1′S-tert-butoxy-carbonyl)eth-1′-yl]amino-(5S)-methylheptanoate (16a). (EtOAc : hexane, 1 : 7). Yield: 13% (syrup). t_R = 4.78 min (15 to 95% A in 5 min). ¹H NMR (CDCl₃) δ: 7.36 (m, 5H, Ar), 5.20 (bd, 1H, J = 7.2 Hz, 4-NH), 5.12, 5.06 (d, 1H, J = 12.1 Hz, OCH₂), 3.66 (s, 3H, OCH₃), 3.25 (m, 1H, H-3, H-4, H-1′), 2.45 (m, 2H, H-2), 1.51 (m, 2H, H-5, H-6), 1.45 (s, 9H, CH₃ ^tBu), 1.19 (d, 3H, J = 6.9, H-2′), 1.08 (m, 1H, H-6), 0.96 (d, 3H, J = 6.7, 5-CH₃), 0.86 (t, 3H, J = 7.3, H-7). ¹³C NMR (CDCl₃) δ: 175.5, 172.3 (COO), 156.8 (NCO), 136.9 (C Ar), 128.6, 128.2, 127.9 (CH Ar), 81.3 (C ^tBu), 66.8 (OCH₂), 59.3 (C4), 57.2 (C-1′), 53.3 (C3), 51.8 (OCH₃), 38.8 (C2), 37.1 (C5), 28.1 (CH₃ ^tBu), 26.7 (C6), 20.0 (C2′), 15.9 (5-CH₃), 11.2 (C7). MS: 451.4 [M + 1]⁺. Anal. calc. for C₂₄H₃₈N₂O₆: C, 63.98; H, 8.50; N, 6.22. Found: C, 63.91; H, 8.58; N, 5.89.

Methyl (4S)-benzyloxycarbonylamino-(3R)-[(1′S-tert-butoxy-carbonyl)eth-1′-yl]amino-(5S)-methylheptanoate (16b). (EtOAc : hexane, 1 : 7). Yield: 21% (syrup). t_R = 4.36 min (15 to 95% A in 5 min). ¹H NMR (CDCl₃) δ: 7.34 (m, 5H, Ar), 5.27 (bs, 1H, 4-NH), 5.11 (s, 2H, OCH₂), 3.66 (s, 3H, OCH₃), 3.53 (m, 1H, H-4), 3.24 (m, 2H, H-3, H-1′), 2.43 (m, 2H, H-2), 1.56 (m, 1H, H-5), 1.45 (s, 9H, CH₃ ^tBu), 1.24 (m, 2H, H-6), 1.20 (d, 3H, J = 6.9, H-2′), 0.91 (t, 3H, J = 7.2, H-7), 0.88 (d, 3H, J = 6.5, 5-CH₃). ¹³C NMR (CDCl₃) δ: 175.2, 172.4 (COO), 157.1 (NCO), 136.9 (C Ar), 128.6, 128.1, 128.0 (CH Ar), 81.3 (C ^tBu), 66.7 (OCH₂), 58.0 (C4), 54.9 (C-1′), 53.2 (C3), 51.8 (OCH₃), 37.7 (C2), 36.4 (C5), 28.0 (CH₃ ^tBu), 26.3 (C6), 20.0 (C2′), 15.1 (5-CH₃), 11.0 (C7). MS: 451.1 [M + 1]. Anal. calc. for C₂₄H₃₈N₂O₆: C, 63.98; H, 8.50; N, 6.22. Found: C, 63.71; H, 8.20; N, 5.95.

Methyl (4R)-benzyloxycarbonylamino-(3R)-[(1′S-tert-butoxy-carbonyl)eth-1′-yl]amino-(5S)-methylheptanoate (16c). Data extracted from a diastereoisomeric mixture of 16b,c 1 : 2 (EtOAc : hexane, 1 : 7). Yield: 16% (syrup), mixture of D2 and D3 in 1 : 2 ratio. t_R = 3.82 min (15 to 95% A in 5 min). ¹H NMR (CDCl₃) δ D3: 7.34 (m, 5H, Ar), 5.09, 5.05 (d, 1H, J = 12.3 Hz, OCH₂), 4.67 (d, 1H, J = 10.5, 4-NH), 3.64 (m, 1H, H-4), 3.58 (s, 3H, OCH₃), 3.20 (m, 1H, H-1′), 3.05 (m, 2H, H-3), 2.41 (m, 2H, H-2), 1.82 (m, 1H, H-5), 1.54 (m, 1H, H-6), 1.44 (s, 9H, CH₃ ^tBu), 1.37 (m, 1H, H-6), 1.18 (d, 3H, J = 6.9, H-2′), 0.90 (t, 3H, J = 7.2, H-7), 0.84 (d, 3H, J = 6.8, 5-CH₃). ¹³C NMR (CDCl₃) δ: 175.1, 173.7 (COO), 157.0 (NCO), 136.9 (C Ar), 128.6, 128.3, 128.1 (CH Ar), 82.5 (C ^tBu), 66.8 (OCH₂), 57.3 (C4), 55.2 (C-1′), 53.9 (C3), 51.7 (OCH₃), 37.1 (C2), 34.9 (C5), 28.0 (CH₃ ^tBu), 27.1 (C6), 19.7 (C2′), 15.7 (5-CH₃), 11.5 (C7). MS: 451.5 [M + 1]⁺.

Methyl (4R)-benzyloxycarbonylamino-(3S)-[(1′S-tert-butoxy-carbonyl)eth-1′-yl]amino-(5S)-methylheptanoate (16d). (EtOAc : hexane, 1 : 7). Yield: 6% (syrup). t_R = 3.64 min (15 to 95% A in 5 min). ¹H NMR (CDCl₃) δ: 7.35 (m, 5H, Ar), 5.08 (s, 2H, OCH₂), 5.20 (d, 1H, J = 10.3 Hz, 4-NH), 3.61 (m, 4H, H-4, OCH₃), 3.30 (m, 1H, H-1′), 3.19 (m, 1H, H-3), 2.42 (dd, 1H, J = 15.3, 5.5, H-2), 2.32 (dd, 1H, J = 15.3, 6.3, H-2), 1.56 (m, 2H, H-5, H-6), 1.45 (s, 9H, CH₃ ^tBu), 1.21 (d, 3H, J = 6.9, H-2′), 1.00 (m, 1H, H-6), 0.93 (d, 3H, J = 6.5, 5-CH₃), 0.90 (t, 3H, J = 7.2, H-7). ¹³C NMR (CDCl₃) δ: 174.9, 173.3 (COO), 157.0 (NCO), 136.8 (C Ar), 128.6, 128.2, 128.1 (CH Ar), 80.9 (C ^tBu), 67.0 (OCH₂), 59.1 (C4), 54.8 (C-1′), 53.3 (C3), 51.7 (OCH₃), 36.4 (C2), 35.9 (C5), 28.1 (CH₃ ^tBu), 24.4 (C6), 19.7 (C2′), 16.5 (5-CH₃), 11.5 (C7). MS: 451.1 [M + 1]⁺. Anal. calc. for C₂₄H₃₈N₂O₆: C, 63.98; H, 8.50; N, 6.22. Found: C, 63.86; H, 8.09; N, 6.05.

Methyl (4S)-benzyloxycarbonylamino-(3S)-[[N-(1′S-tert-butoxy-carbonyl)eth-1′-yl]-N-acetyl]amino-5-phenylpentanoate (17a). A solution of compound 4a (52 mg, 0.107 mmol) in THF (2 mL) was cooled to 0 °C and treated with propylene oxide (113 μL, 1.61 mmol) and acetyl chloride (29 μL, 0.322 mmol). Stirring at room temperature was continued for three days. After evaporation of the solvent, the resulting residue was purified on a column using EtOAc–hexane (3 : 1) as eluent. The title compound, 48 mg (86%) was obtained as a syrup. t_R = 8.52 min (15 to 95% A in 10 min). ¹H NMR (300 MHz, CDCl₃) δ: 7.30–7.00 (m, 10H, Ar), 5.81 (d, 1H, J = 8.5, 4-NH), 4.90 (m, 2H, OCH₂), 4.59 (m, 1H, H-4), 4.00 (m, 1H, H-2′), 3.65 (m, 1H, H-3), 3.59 (s, 3H, OCH₃), 3.12 (m, 1H, H-5), 2.91 (m, 1H, H-5), 2.68 (m, 2H, H-2), 2.15 (s, 3H, CH₃ Ac), 1.45 (s, 9H, CH₃ ^tBu), 1.26 (d, 3H, J = 7.1, H-3′). MS: 527.6 [M + 1]⁺. Anal. calc. for C₂₉H₃₈N₂O₇: C, 66.14; H, 7.27; N, 5.32. Found: C, 65.87; H, 6.95; N, 5.01.

Calcium microfluorography

For fluorescence assays, cells expressing TRP channels (TRPV1-SH-SY5Y, TRPM8-HEK and TRPA1-IMR90) were seeded in 96-well plates (Corning Incorporated, Corning, NY) at a cell density of 40 000 cells 2 days before treatment. The day of treatment the medium was replaced with 100 μL of the dye loading solution Fluo-4 NW supplemented with probenecid 2.5 mM. Then the compounds dissolved in DMSO were added at the desired concentrations and the plate(s) were incubated at 37 °C in a humidified atmosphere of 5% CO₂ for 60 minutes.

The fluorescence was measured using instrument settings appropriate for excitation at 485 nm and emission at 535 nm (POLARstar Omega BMG LAB tech). A baseline recording of 7 cycles was recorded prior to stimulation with the agonist (10 μM capsaicin for TRPV1, 100 μM menthol for TRPM8, and 100 μM AITC for TRPA1). The corresponding antagonist (10 μM ruthenium red for TRPV1 and TRPA1, 100 μM AMTB for TRPM8) was added for the blockade. The changes in fluorescence intensity were recorded during 15 cycles more. DMSO, at the higher concentration used in the experiment, was added to the control wells.

The degree of blockage (%) of TRP channel activity was calculated by:

where F₀ is the fluorescence after the addition of agonist in the presence of the compound, F₁ is the fluorescence before the addition of agonist in the presence of the compound, F_c0 is the fluorescence after the addition of agonist in the absence of the compound, F_c1 is the fluorescence before the addition of agonist in the absence of the compound.

The Z factor was calculated using the following equation:

where mean_max is the mean of the maximum fluorescence in the presence of agonist, mean_min is the mean of the maximum fluorescence in the presence of agonist and antagonist.

Acknowledgements

This work was supported by the Spanish MINECO: CSD2008-00005, The Spanish Ion Channel Initiative-CONSOLIDER INGENIO 2010, and BFU2012-39092-C02.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Protocol for the evaluation of compound 4b in the CFA-induced paw inflammation model and obtained results. See DOI: 10.1039/c5ra25709c

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