Design and synthesis of potent hydroxamate inhibitors with increased selectivity within the gelatinase family

Abstract

MMP-2 is a validated target for the development of anticancer agents. Herein we describe the synthesis of a new series of potent phenylalanine derived hydroxamates, with increased MMP-2/MMP-9 selectivity compared to analogous hydroxamates described previously. Docking and molecular dynamics experiments have been used to account for this selectivity, and to clarify the role of the triazole ring in the binding process.

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Introduction

Proteases are classified into serine proteases, cysteine proteases, aspartate proteases and metalloproteinases, based on differences in the hydrolysis mechanism of peptide bonds.¹ The human degradome (proteases produced by cells) consists of at least 569 proteases and homologues, with metalloproteinases representing the largest class (194 described in humans).²

The Metzincin superfamily of metalloproteinases can, in turn, be classified into the following four subfamilies, according to the slight differences in their catalytic site and the presence of additional domains: matrix metalloproteinases (MMPs), adamalysins (ADAM, ADAMTS and class III snake venom proteins), astacins (BMP1/TLL proteins and meprins) and bacterial serralysins.³ Metzincins are distinguished by a highly conserved HEXXHXXGXXH domain that bears three histidine residues that bind a zinc atom at the active site.⁴

The 26 MMPs described so far in the vertebrate family can be classified into several subfamilies according to their degree of sequence homology and substrate specificity: collagenases, gelatinases, stromelysins, matrilysines and membrane-type MMPs (MT-MMPs).⁵

Basically, all MMPs have been linked to disease development,^6,7 mainly in cardiovascular disorders,^8–10 arthritis,¹¹ acute lung injury,¹² and cancer.^13–15

Gelatinase A (MMP-2) and gelatinase B (MMP-9) play an important role in cancer. They are over-expressed or deregulated in a variety of malignant tumors.¹⁶ It has been shown, in an experiment in MMP-2 knockout mice, that angiogenesis and tumour progression can be controlled by inhibiting the activity of this enzyme, suggesting the use of MMP-2 inhibitors for chemotherapy of cancer and other diseases.¹⁷ However, while MMP-2 is a validated target in cancer therapy, MMP-9 has both pro- and anti-tumorigenic effects, and is an anti-target protein in advanced stages of the disease.¹⁸

A large number of MMP inhibitors (MMPIs) have been reported so far. MMPIs containing a Zinc Binding Group (ZBG) and a lipophilic chain (called the P1′-segment) which interacts with the hydrophobic S1′ pocket are very effective. The latter is considered the “selectivity pocket”, as it is the region where MMPs exhibit the largest differences.^19–22

In our research group we are interested in the design of MMP-2 inhibitors with selectivity over other metalloproteinases, especially over MMP-9. Because the active sites of both enzymes are very similar, it is necessary to carefully select the fragment that has to interact with the S1′ pocket. Our approach is based on the use of Cu(I)-catalyzed azide–alkyne cycloaddition, one of the most effective click reactions, to easily connect the selected ZBG with different non-polar P1′ substituents in order to explore the S1′ pocket. Following this strategy we have obtained inhibitors with high potency and MMP-2/MMP-9 selectivity (Fig. 1A).^23–25 The potent but not selective MMPI (2R)-[(4-biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide (BiPS)²⁶ (Fig. 1B) has been the starting point for the design of the new series of inhibitors described herein. With the aim of increasing the selectivity between both gelatinases we have followed the same approach based on click chemistry.

Fig. 1 Structure and inhibitory activity of (A) clicked MMP-2 selective inhibitors and (B) BiPS.

Results and discussion

Chemistry

Hydroxamates 1a–g were synthesized following the pathway outlined in Scheme 1. Compound 2 was obtained as described previously,²⁷ starting from phenylalanine and 4-nitrobenzenesulfonyl chloride under basic conditions. The nitro group was reduced by catalytic hydrogenation to give amino acid 3, which was transformed into azide 4 by reaction with tert-butyl nitrite and azidotrimethylsilane. Reaction of the carboxylic acid with O-THP protected hydroxylamine, using EDCI as an amide coupling agent, gave hydroxamate 5. Copper(I) catalyzed Huisgen cycloaddition (CuAAC) between 5 and different alkynes gave compounds 6a–g, which were deprotected to obtain the final hydroxamates 1a–g.

Scheme 1 Synthesis of triazoles 1a–g. (a) NaOH–H₂O; (b) H₂, Pd/C, MeOH; (c) t-BuONO, TMSiN₃, acetonitrile, 0 °C; (d) O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, EDCI, HOBt, NMM, DMF; (e) alkyne, CuSO₄, sodium ascorbate, DMF or t-BuOH–H₂O; (f) 4 M HCl–dioxane, MeOH.

(R) and (S) enantiomers of hydroxamates 1b, 1c and 1f were prepared separately following the same pathway starting from D or L phenylalanine.

In order to study the effect of the triazole ring on the activity, hydroxamate 7 was synthesized. This compound has a different substitution pattern in the triazole ring, with the biphenyl group attached to N1, instead of the C-4 position that is occupied in 1a–g. The synthetic pathway for 7 (Scheme 2) started from (±) phenylalanine methyl ester hydrochloride, which was transformed into sulphonamide 8 by reaction with pipsyl chloride. Sonogashira cross-coupling reaction with ethynyltrimethylsilane gave 9, which was deprotected under basic conditions to give 10, and was subsequently transformed into 11 by coupling with NH₂OTHP. The terminal alkyne was clicked with 4-azidobiphenyl²⁸ to give triazole 12, which was deprotected to give the desired compound 7.

Scheme 2 Synthesis of triazole 7: (a) pipsyl chloride, NMM, CH₂Cl₂; (b) TMSCCH, CuI, Et₃N, Ph₃P, Pd/C, reflux; (c) 1: KOH, dioxane, rt, 2: citric acid 20%; (d) NH₂OTHP, EDCI, HOBt, NMM, DMF; (e) biphenylazide, CuSO₄·5H₂O, sodium ascorbate, DMF, (f) 4 M HCl–dioxane, MeOH.

With the aim of improving the solubility of this class of compounds, a basic side chain was introduced into the sulphonamide nitrogen atom. We chose a morpholinoethyl moiety, as this fragment was used before to improve the solubility of valine-derived hydroxamates.²⁹

Thus, compounds 13 and 14 were synthesized following the synthetic pathway shown in Scheme 3. Carboxylic acid 4 was protected as tert-butyl ester 15, and after alkylation with 4-(2-chloroethyl)morpholine in the presence of K₂CO₃, 16 was obtained. A sequence of tert-butyl deprotection to give 17 and coupling of the carboxylic acid with O-THP hydroxylamine gave azide 18. The CuAAC reaction with two different alkynes gave the corresponding triazoles 19–20 which were converted into hydroxamates 13 and 14 by acidic O-THP deprotection.

Scheme 3 Synthesis of triazoles 13 and 14. (a) 2-Methylpropene, H₂SO₄, DCM, 86%; (b) 4-(2-chloroethyl)morpholine, K₂CO₃, DMF, 85%; (c) TFA, thioanisole, DCM, 80%; (d) NH₂OTHP, EDC, HOBt, DMAP, DMF, 91%; (e) alkyne, CuSO₄, sodium ascorbate, DMF; (f) HCl, dioxane, 60–75%.

Biological evaluation

The inhibitory activities of hydroxamates 1a–g, 7, 13 and 14 against MMP-2 and MMP-9 were determined by the colorimetric method using a thiopeptide as a chromogenic substrate (Enzo Life Science Inc.). The assay conditions were similar to those described before [Fabre, 2013 #34].²⁴ The results are collected as IC₅₀ in Table 1.

Table 1 MMP inhibition activities for compounds 1a–g, 7, 13 and 14

Compound	R^1	R^2	MMP-2 IC50 ^a (nM)	MMP-9 IC50 ^a (nM)	Selectivity for MMP-9/MMP-2
a IC50 values on the nM scale were determined using a colorimetric assay. Enzymatic data are the main values from three independent experiments. SD are within ±10%.
Rac.-1a	3-FC6H4	H	0.27	3.6	13
Rac.-1b	4-C6H5-C6H4	H	1.3	124	95
(R)-1b	4-C6H5-C6H4	H	0.75	58.5	78
(S)-1b	4-C6H5-C6H4	H	220	>1000	—
Rac.-1c	(CH3)2N-C6H4	H	0.88	2.11	2.4
(R)-1c	(CH3)2N-C6H4	H	0.32	1.06	3.4
(S)-1c	(CH3)2N-C6H4	H	49	>1000	—
Rac.-1d	PhSO2NHCONHCH2	H	>500	>1000	—
Rac.-1e	4-CH3O-C6H4	H	0.42	1.16	2.8
Rac.-1f	4-C6H5-O-C6H4	H	2.2	88	41
(R)-1f	4-C6H5-O-C6H4	H	1.08	36.8	34
(S)-1f	4-C6H5-O-C6H4	H	>500	>1000	—
Rac.-1g	4-C5H11-C6H4	H	8.18	372	46
Rac.-7			1.32	5.54	4
(R)-13	4-C6H5-C6H4	2-Morpholinoethyl	2.66	6.34	2.4
(R)-14	4-C6H5-O-C6H4	2-Morpholinoethyl	1.99	29.9	15

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All racemic compounds showed potent inhibitory activity against MMP-2, which was higher than the one reported for BiPS (IC₅₀ = 17 nM).²⁶

An exception was 1d, where the P1′ side chain is too polar to interact with the hydrophobic S1′ pocket of the enzyme. This result was expected, as we also found the lack of activity with this P1′ group in our previous studies.²³

The best result in potency and selectivity was found for 1b, with an IC₅₀ of 1.3 nM for MMP-2 and almost 100-fold less activity against MMP-9. This result shows that our click based strategy is a useful approach to improve the profile of this type of inhibitor, especially in the search for ligands capable of differentiating between the two gelatinases.

In the previously reported activity against MMP-3 of phenylalanine-derived hydroxamates, it was found that the preferred stereochemistry at the stereogenic centre corresponded to the R-isomer, which was more potent than the racemate.³⁰ A similar result was reported for BiPS, which was 28-fold more active in MMP-9 than the S-isomer.²⁶

In order to verify if the configuration of the stereogenic centre has an effect also on the inhibitory activity of 1b, 1c and 1f, both enantiomers of these compounds were synthesized and tested against both gelatinases. The results show that (R)-1b is 293-fold more potent against MMP-2 than (S)-1b, and 1.7-fold more potent than the racemic form, showing that the racemate activity is mainly due to the R-isomer. However, the MMP-2/MMP-9 selectivity of (R)-1b is slightly diminished with respect to the racemic mixture because the S-isomer was completely inactive in MMP-9 (IC₅₀ > 1000). A similar effect was observed for 1c and 1f.

Compounds 13 and 14, where a basic chain was introduced into the sulphonamide nitrogen atom, presented better solubility during sample preparation. However, although the inhibition against MMP-2 was kept at the same level, the inhibitory activities against MMP-9 were increased giving a worse selectivity profile.

Interestingly, the same effect was observed for compound 7. This compound was obtained through a click reaction between 4-azidobiphenyl and an alkyne bearing the hydroxamate ZBG. The structure is closely related to 1b, with the only difference being in the substitution pattern of the triazole ring: the biphenyl group in 7 is attached to the N1 of the triazole, instead of the C-4 position that is occupied in 1b. While the activity of 7 against MMP-2 was maintained (IC₅₀ = 1.32 nM), a high increase in the activity against MMP-9 was observed (IC₅₀ = 5.54 nM) compared to the activity of 1b (Rac.-1b: IC₅₀ = 124 nM; (R)-1b: 58.5 nM), with the corresponding loss of selectivity.

Compounds 1b, 1f and 1g, showing a promising selectivity profile within gelatinases, were chosen for the analysis of their activity towards other metalloproteinases (Table 2). The obtained profile showed that these compounds are devoid of activity towards MMP-1, whose inhibition is hypothesized to be connected with the musculoskeletal syndrome,^31,32 and have lower activity towards MMP-8, an enzyme whose inhibition could enhance tumourigenesis and metastasis.³³ Moreover, 1b and 1f displayed good activity towards MMP-13, an enzyme whose inhibition is targeted in the treatment of osteoarthritis, while having much lower potency towards MMP-14, proposed recently as an anti-target in the treatment of this disease.³⁴

Table 2 Inhibitory activity (IC₅₀ values) ofRac.-1b, Rac.-1f and Rac.-1g towards a panel of metalloproteinases

	1b (nM)	1f (nM)	1g (nM)
a Enzymatic data are the mean values from two independent experiments. SD are within ±10%.
MMP-1	>10 000	>10 000	>1000
MMP-2	1.3	2.2	8.2
MMP-3	50	33.4	170
MMP-7	>500	>1000	>1000
MMP-8	>500	128.8	450
MMP-9	124	88	372.5
MMP-10	181	103.4	336
MMP-12	3.8	2	14.2
MMP-13	<5	1.5	36.8
MMP-14	555.7	256.6	>200

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In order to explain the difference in selectivity between compounds 1b and 7 we first studied both separately by means of ab initio quantum mechanics (QM) methods. Despite being structurally very similar, the difference in the substitution pattern of the triazole determines the relative orientation of the P1′-segment with the rest of the compound. The main difference arises from the energy profile of the dihedral angle that describes the torsion around C_sp2–C_sp2 and C_sp2–N_sp2. The first dihedral angle presents energy minima at ±15° and global maxima of 3.3 kcal mol⁻¹ at ±90°, whereas the dihedral angle between C_sp2–N_sp2 presents energy minima between ±30–45°, a local maximum of 0.7 kcal mol⁻¹ at 0°, and a global maximum of 1.6 kcal mol⁻¹ at ±90° (ESI† Fig. 1). This means that compound 1b will be almost flat from the triazole to the end of the P1′-segment, whereas compound 7 will form an angle of around 45° between the triazole and the P1′-segment.

The docking experiments for compound 1b were carried out with a deprotonated hydroxamic acid moiety on MMP-2 and MMP-9, and with a protonated side chain of Glu404. The obtained poses were overall similar to those obtained in our previous work,³⁵ so these complexes were used to build the complexes of 7 with MMP-2 and MMP-9 by changing the atom type in the triazole ring and allowing the geometries to relax in the molecular mechanics force field. During the 10 ns molecular dynamics (MD) simulations we monitored the per-residue protein–ligand interactions, especially those established between the P1′-segment and the S1′ pocket. Compounds 1b and 7 establish mainly van der Waals interactions in the complexes with both MMP-2 and MMP-9 (ESI† Fig. 2). Both compounds establish strong van der Waals interactions with the amino acids of the first segment of the Ω-loop within both MMPs, especially with Ile424(MMP-2)/Met422(MMP-9) that interacts with the triazole through the backbone; and Tyr425(MMP-2)/Tyr423(MMP-9) and Thr426(MMP-2)/Arg424(MMP-9) that stack their side chains with the triazole and biphenyl moieties. The slight difference in the interaction energy between both compounds and the side chain of Arg424 in the case of MMP-9 can be explained by the difference in flexibility at the end of the P1′-segment mentioned before. The C_sp2–C_sp2 bond present in the P1′ fragment of 1b is more rigid than the C_sp2–N_sp2 bond present in 7, which induces fewer fluctuations in the interaction with the highly flexible side chain of Arg424, resulting in a better interaction energy for 1b compared to 7. The interactions established between compounds 1b and 7 and the last segment of the Ω-loop can account for the lack of selectivity of compound 7. In our previous work we proposed that the selectivity of this type of molecules, especially the biphenyl derivatives, arises from the presence of the Phe431-Arg432 motif in MMP-2, which is mutated to Pro429-Pro430 in MMP-9. This difference makes the Ω-loop more rigid in MMP-9 than in MMP-2.²⁵ In the case of MMP-2, both compounds interact with the side chain of Phe431 (Fig. 2). Interestingly, the study of the MD trajectories and the minimized cooled structure obtained after the 10 ns simulations showed that compound 7, having a torsion of around 45° between the triazole and the P1′-segment, due to the C_sp2–N_sp2 bond, is able to adopt more easily the shape of the S1′ pocket, establishing a van der Waals interaction with Pro429 (Fig. 2). This interaction is absent in compound 1b which is flatter and has less flexibility. This difference could explain the different in vitro activity observed for both compounds. Although we have explained the difference in selectivity between compounds 1b and 7, we were not able to give the rationale of the difference in binding affinity between both enantiomers of compound 1b as they did not show any significant differences in the dynamic behaviour and interactions with the two proteins in the time span and conditions of our simulations.

Fig. 2 PyMOL cartoon representation of the complexes between MMP2 (green) and MMP9 (cyan) and compounds 1b (magenta) and 7 (yellow) respectively. For the sake of clarity, only polar hydrogens are displayed; and only the side chains of the amino acids that stabilize the ZBG-end and those that bring about the most different interactions in the Ω-loop are shown as sticks.

Experimental section

Molecular modeling

The proteins were prepared and the docking experiments were carried out as in the previous work.²⁵ The ligands (compounds 1b and 7) were built using the Maestro LigPrep module (http://www.Schrodinger.com). pK_a of titratable groups were calculated using the Sparc pK_a online calculator,³⁶ and the non-protonated form of the hydroxamate was used for the docking calculations making use of the Glide module.^37–39 The center of the box was positioned on the catalytic zinc ion present in the active site. The box size was set up to enclose the ligand-binding domain to ensure an adequate exploration of the binding poses. The docking procedure was performed with XP (extra precision) mode, and a van der Waals radii scale factor of 1.0/0.8 for the receptor and the ligand, respectively. The best-obtained result for each ligand in each complex was considered for analysis of the ligand–receptor interactions and subsequent molecular modeling simulations.

For the MD simulations the charge distribution for the ligands was obtained by fitting the quantum mechanically calculated (RHF/6-31G**//RHF/3-21G*) molecular electrostatic potential (MEP) of the geometry-optimized molecules to a point charge model, as implemented in Gaussian 03 (Gaussian, Inc., Wallingford, CT). Consistent bonded and non-bonded AMBER parameters for both molecules were assigned by analogy or through interpolation from those already present in the AMBER database (ff03). Each MMP molecular system was immersed in a truncated octahedron containing ∼10 000 TIP3P water molecules⁴⁰ and four Na⁺ ions⁴¹ to achieve system electroneutrality. The sander and pmemd modules of the AMBER12 suite (http://ambermd.org/) were used for the restrained and unrestrained MD simulations, respectively. Periodic boundary conditions were applied and electrostatic interactions were treated using the smooth particle mesh Ewald method⁴² with a grid spacing of 1 Å. The cutoff distance for the non-bonded interactions was 9 Å, the SHAKE⁴³ algorithm was applied to all bonds, and an integration step of 2.0 fs was used throughout. After an initial energy minimization of the water molecules and counterions, the system was heated to 300 K in 25 ps, after which the solvent was allowed to redistribute around the positionally restrained solute for 220 ps. After this time, the system was allowed to move freely so as to explore the mutual adaptation between the ligand and the protein. Snapshots from each 10 ns MD trajectory were collected every 20 ps for further analysis carried out with the ptraj module of AMBER to monitor the hydrogen bonding distances between the ligands and the protein. The per-residue energy decomposition of the 10 ns MD trajectory was carried out using the ISM program.^44,45

Chemistry

General procedures. Melting points (uncorrected) were determined on a Stuart Scientific SMP3 apparatus. Infrared (IR) spectra were recorded with a Perkin-Elmer Spectrum 100 Series infrared spectrophotometer. ¹H and ¹³C NMR data were recorded on a Bruker 300-AC or a Bruker 400-ultrashield instrument. Chemical shifts (δ) are expressed in parts per million relative to internal tetramethylsilane; coupling constants (J) are in hertz. Mass spectra were run on a Bruker Esquire 3000 spectrometer. Elemental analyses (C, H, N, S) were performed on a LECO CHNS-932 apparatus at the Microanalyses Service of the University Complutense of Madrid; unless otherwise stated, all reported values are within ±0.4% of the theoretical compositions. Thin-layer chromatography (TLC) was run on Merck silica gel 60 F-254 plates. Unless stated otherwise, the starting materials used were high-grade commercial products.

2-{[(4-Aminophenyl)sulfonyl]amino}-3-phenylpropanoic acid (3). A solution of 2²⁷ (2.2 g, 6.28 mmol) in EtOH (50 mL) and 10% Pd/C (220 mg) were introduced into a Parr shaker apparatus, and maintained under a hydrogen pressure of 60 p.s.i. for 18 h at room temperature. Palladium was filtered off and the solvent was removed. Then, it was evaporated to dryness, and the residue was purified by column chromatography on silica gel (hexane–EtOAc 1 : 4) to afford 3 (1.57 g, 78%) as a white solid, mp 205–206 °C (EtOH). (Found: C, 56.04; H, 5.00; N, 8.82; S, 9.97. C₁₅H₁₆N₂O₄S requires C, 56.24; H, 5.03; N, 8.74; S, 10.01%); ν_max (KBr)/cm⁻¹ 3372, 3306, 3284, 1719; δ_H (300 MHz, DMSO-d₆) 2.68 (1H, dd, J 13.4, 7.9, ½CH₂), 2.91 (1H, dd, J 13.4, 6.1, ½CH₂), 3.69–3.76 (1H, m, CH), 5.90 (2H, bs, NH₂), 6.48 (2H, d, J 8.5, ArH), 7.09–7.12 (2H, m, ArH), 7.17–7.27 (5H, m, ArH), 7.69 (1H, d, J 8.5, SO₂NH), 12.64 (1H, bs, COOH); δ_C (75.4 MHz, DMSO-d₆) 37.9, 57.1, 112.4, 125.8, 126.4, 128.1, 128.4, 129.2, 136.9, 152.4, 172.4. EM (ESI+) m/z 319.00 [M − H]⁺.

(2R)-2-{[(4-Aminophenyl)sulfonyl]amino}-3-phenylpropanoic acid ((R)-3). A solution of (R)-2²⁷ (2.42 g, 6.91 mmol) in EtOH (50 mL) and 10% Pd/C (200 mg) were introduced into a Parr shaker apparatus, and maintained under a hydrogen pressure of 60 p.s.i for 5 h at room temperature. Palladium was filtered off and the solvent was removed to afford after recrystallization from EtOH–H₂O (R)-3 (2.17 g, 98%) as a yellowish solid, mp 189–190 °C.

(2S)-2-{[(4-Aminophenyl)sulfonyl]amino}-3-phenylpropanoic acid ((S)-3). A solution of (S)-2²⁷ (2.75 g, 7.84 mmol) in EtOH (50 mL) and 10% Pd/C (225 mg) were introduced into a Parr shaker apparatus, and maintained under a hydrogen pressure of 60 p.s.i. for 5 h at room temperature. Palladium was filtered off and the solvent was removed to afford after recrystallization from EtOH–H₂O (S)-3 (2.49 g, 99%) as a yellowish solid, mp 189–190 °C.

2-{[(4-Azidophenyl)sulfonyl]amino}-3-phenylpropanoic acid (4). Compound 3 (1.45 g, 4.53 mmol) was suspended in anhydrous CH₃CN (30 mL) under argon and cooled to 0 °C in an ice bath. To this suspension was added t-BuONO (0.80 mL, 5.45 mmol), followed by TMSN₃ (0.72 mL, 5.42 mmol) dropwise. The reaction mixture was stirred at 0 °C for 30 min, and at room temperature for 6 h. Then, it was evaporated to dryness, and the residue was purified by column chromatography on silica gel (DCM–MeOH 5% MeOH) to afford 4 (1.4 g, 89%) as a yellowish solid, mp 197.0–198.4 °C. (Found: C, 52.11; H, 4.08; N, 15.87; S, 9.20. C₁₅H₁₄N₄O₄S requires C, 52.02; H, 4.07; N, 16.18; S, 9.26%); ν_max (KBr)/cm⁻¹ 3276, 2118, 2095, 1719; δ_H (300 MHz, DMSO-d₆) 2.71 (1H, dd, J 13.4, 9.2, ½CH₂Ar), 2.94 (1H, dd, J 13.4, 5.5, ½CH₂Ar), 3.82–3.89 (1H, m, CH), 7.11–7.21 (7H, m, ArH), 7.56 (2H, d, J 8.6, ArH), 8.34 (1H, d, J 9.2, SO₂NH), 12.78 (1H, bs, COOH); δ_C (75.4 MHz, DMSO-d₆) 37.8, 57.5, 119.4, 126.5, 128.2, 128.3, 129.2, 136.8, 137.3, 143.3, 172.4. EM (ESI+) m/z 328.06 [M − H₂O]⁺ 369.01 [M + Na]⁺.

(2R)-2-{[(4-Azidophenyl)sulfonyl]amino}-3-phenylpropanoic acid ((R)-4). Compound (R)-3 (2.14 g, 6.68 mmol) was suspended into anhydrous CH₃CN (50 mL) under argon and cooled to 0 °C in an ice bath. To this suspension was added t-BuONO (1.19 mL, 10.02 mmol), followed by TMSN₃ (1.05 mL, 8.02 mmol) dropwise. The reaction mixture was stirred at 0 °C for 30 min, and at room temperature for 3 h. Then, it was evaporated to dryness, and the residue was purified by column chromatography on silica gel (DCM–MeOH 2.5% MeOH) to afford (R)-4 (2.16 g, 93%) as a yellowish solid, mp 127–128 °C.

(2S)-2-{[(4-Azidophenyl)sulfonyl]amino}-3-phenylpropanoic acid ((S)-4). Compound (S)-3 (2.48 g, 7.73 mmol) was suspended in anhydrous CH₃CN (50 mL) under argon and cooled to 0 °C in an ice bath. To this suspension was added t-BuONO (1.38 mL, 11.60 mmol), followed by TMSN₃ (1.22 mL, 9.28 mmol) dropwise. The reaction mixture was stirred at 0 °C for 30 min, and at room temperature for 3 h. Then, it was evaporated to dryness, and the residue was purified by column chromatography on silica gel (DCM–MeOH 2.5% MeOH) to afford (S)-4 (2.25 g, 84%) as a yellowish solid, mp 127–128 °C.

2-{[(4-Azidophenyl)sulfonyl]amino}-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide (5). To a solution of 4 (1.3 g, 3.75 mmol) in DMF (30 mL) were added HOBt (1.11 g, 8.25 mmol), O-tetrahydro-2H-pyran-2-yl-hydroxylamine (0.88 g, 7.50 mmol), EDCI (1.72 g, 9 mmol) and NMM (1.5 mL, 15 mmol). The reaction mixture was stirred overnight at room temperature and then diluted with EtOAc (50 mL) and washed successively with a saturated aqueous solution of NH₄Cl and brine. The organic layer was dried (MgSO₄), and evaporated to dryness, and the residue was purified by column chromatography on silica gel (hexane–AcOEt 7 : 3) to afford 5 (1.14 g, 68%) as a yellow solid. This compound is present as a diastereoisomeric mixture (55 : 45), mp 142–144 °C. (Found: C, 53.81; H, 5.14; N, 15.37; S, 7.18. C₂₀H₂₃N₅O₅S requires C, 53.92; H, 5.20; N, 15.72; S, 7.20%); ν_max (KBr)/cm⁻¹ 3213, 2130, 2097, 1673; δ_H (300 MHz, CDCl₃) 1.45–1.65 (4H, m, CH₂), 1.68–1.82 (2H, m, CH₂), 2.87–3.05 (2H, m, CH₂Ph), 3.54–3.58 (1H, m, CHN), 3.81–3.92 (2H, m, OCH₂), 4.72 (0.45H, m, O–CH–O, isomer b), 4.77 (0.55H, m, O–CH–O, isomer a), 5.47, (0.55H, d, J 7.8, ½SO₂NH, isomer a), 5.52 (0.45H, d, J 7.8, ½SO₂NH, isomer b), 6.96–7.02 (5H, m, ArH), 7.18–7.21 (2H, m, ArH), 7.59 (2H, d, J 8.4, ArH), 9.05 (1H, s, CONH, isomer a), 9.17 (1H, s, CONH, isomer b); δ_C (75.4 MHz, CDCl₃): 18.4, 18.6, 24.9, 27.9, 38.6, 39.0, 56.6, 62.5, 62.7, 102.5, 119.5, 127.4, 128.9, 129.0, 129.3, 134.9, 135.2, 145.0, 167.5. EM (ESI+) m/z 468.00 [M + Na]⁺.

(2R)-2-{[(4-Azidophenyl)sulfonyl]amino}-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide ((R)-5). To a solution of (R)-4 (0.5 g, 1.44 mmol) in DMF (5 mL) were added HOBt (234 mg, 1.73 mmol), O-tetrahydro-2H-pyran-2-yl-hydroxylamine (338 mg, 2.89 mmol), EDCI (387 mg, 2.02 mmol) and NMM (0.48 mL, 4.33 mmol). The reaction mixture was stirred overnight at room temperature and then diluted with EtOAc (50 mL) and washed successively with a saturated aqueous solution of NH₄Cl and brine. The organic layer was dried (MgSO₄), and evaporated to dryness, and the residue was purified by column chromatography on silica gel (hexane–AcOEt 3 : 2) to afford (R)-5 (594 mg, 92%) as a yellow solid. This compound is present as a diastereoisomeric mixture (55 : 45), mp 137–138 °C.

(2S)-2-{[(4-Azidophenyl)sulfonyl]amino}-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide ((S)-5). To a solution of (S)-4 (0.5 g, 1.44 mmol) in DMF (5 mL) were added HOBt (234 mg, 1.73 mmol), O-tetrahydro-2H-pyran-2-yl-hydroxylamine (338 mg, 2.89 mmol), EDCI (387 mg, 2.02 mmol) and NMM (0.48 mL, 4.33 mmol). The reaction mixture was stirred overnight at room temperature and then diluted with EtOAc (50 mL) and washed successively with a saturated aqueous solution of NH₄Cl and brine. The organic layer was dried (MgSO₄), evaporated to dryness, and the residue was purified by column chromatography on silica gel (hexane–AcOEt 3 : 2) to afford (S)-5 (589 mg, 92%) as a yellow solid. This compound is present as a diastereoisomeric mixture (55 : 45), mp 137–138 °C.

Preparation of triazoles: general procedure 1

To a suspension of azide 5 (1 equiv.) and the corresponding alkyne (1–1.5 equiv.) in t-BuOH–H₂O (1 : 1) or DMF were added sodium ascorbate (2 equiv. of freshly prepared 1 M solution in water) and copper(II) sulfate pentahydrate (0.5 equiv. of a 0.25 M solution in water) under argon. The mixture was stirred vigorously overnight, and then diluted with water (20 mL) and extracted with AcOEt. The organic layer was washed successively with saturated aqueous NH₄Cl and brine. The extract was dried (MgSO₄), filtered and evaporated to dryness, and the residue was chromatographed on silica gel.

2-[({4-{4-[(3-Fluorophenyl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonylamino]-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)-propanamide (6a). From 5 (100 mg, 0.224 mmol), 1-ethynyl-3-fluorobenzene (44 mg, 0.247 mmol), sodium ascorbate (448 μL, 0.448 mmol) and copper(II) sulfate pentahydrate (448 μL, 0.112 mmol) in DMF (3 mL), 6a (84.8 mg, 67%) was produced as a light yellow solid after chromatography purification (hexane–AcOEt 1 : 1 + 2% MeOH), mp 146–147 °C. (Found: C, 59.06; H, 5.08; N, 12.27; S 5.70. C₂₈H₂₈FN₅O₅S requires C, 59.46; H, 4.99; N, 12.38; S, 5.67%); ν_max (KBr)/cm⁻¹ 3203, 1665, 1620; δ_H (300 MHz, DMSO-d₆) 1.43–1.59 (6H, m, 3CH₂), 2.66–2.73 (1H, m, ½CH₂Ph), 2.79–2.89 (1H, m, ½CH₂Ph), 3.38–3.49 (1H, m, CHN), 3.76–3.85 (1H, m, OCH₂, isomer a), 3.93–3.98 (1H, m, OCH₂, isomer b), 4.39 (0.5H, s, O–CH–O, isomer a), 4.57 (0.5H, s, O–CH–O, isomer b), 7.11–7.29 (6H, m, ArH), 7.55–7.62 (1H, m, ArH), 7.76–7.84 (4H, m, ArH), 8.02 (2H, dd, J 8.7, 1.4, ArH), 8.54 (1H, bs, SO₂NH), 9.51 (1H, s, triazole), 11.25 (0.5H, bs, CONH, isomer a) 11.32 (0.5H, bs, CONH, isomer b); δ_C (75.4 MHz, DMSO-d₆): 18.2, 24.5, 27.7, 38.5, 55.2, 61.3 (61.4 b), 101.0, 112.0 (d, ²J_CF 22.5), 115.17 (d, ²J_CF 21.0), 119.9, 120.9 (d, ²J_CF 74.2), 126.4, 128.0, 128.1, 129.2 (129.3 b), 131.3 (d, ³J_CF 9.0), 132.31 (d, ³J_CF 9.0), 136.5, 136.8, 138.6, 140.9, 146.4, 162.6 (d, ¹J_CF 241.5), 166.6 (166.7 b). EM (ESI+): m/z 588.26 [M + Na]⁺.

2-{[(4-{4-[(Biphenyl-4-yl)]-1H-1,2,3-triazol-1-yl}phenyl)sulfonyl]amino}-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide (6b). From 5 (100 mg, 0.224 mmol), 4-ethynylbiphenyl (44 mg, 0.247 mmol), sodium ascorbate (448 μL, 0.448 mmol) and copper(II) sulfate pentahydrate (448 μL, 0.112 mmol) in DMF (3 mL), 6b (89 mg, 64%) was produced as a yellow solid after chromatography purification (DCM–MeOH 0.6% MeOH), mp 189–191 °C. (Found: C, 64.74; H, 5.45; N, 11.13; S, 5.13. C₃₄H₃₃N₅O₅S·0.5H₂O requires C, 64.54; H, 5.42; N, 11.07; S, 5.07%); ν_max (KBr)/cm⁻¹ 3284, 1668; δ_H (300 MHz, CDCl₃) 1.44–1.56 (6H, m, 3CH₂), 2.67–2.74 (1H, m, ½CH₂Ph), 2.80–2.86 (1H, m, ½CH₂Ph), 3.39–3.49 (1H, m, CHN), 3.75–3.85 (1H, m, OCH₂, isomer a), 3.86–3.97 (1H, m, OCH₂, isomer b), 4.38 (0.5H, s, O–CH–O, isomer a), 4.57 (0.5H, s, O–CH–O, isomer b), 7.11–7.22 (5H, m, ArH), 7.37–7.42 (1H, m, ArH), 7.48–7.53 (2H, m, ArH), 7.76 (2H, d, J 7.3, ArH), 7.80–7.86 (4H, m, ArH), 8.04–8.09 (4H, m, ArH), 8.53 (1H, m, SO₂NH), 9.5 (1H, s, triazole), 11.25 (0.5H, s, CONH, isomer b), 11.33 (0.5H, s, CONH, isomer a); δ_C (75.4 MHz, CDCl₃): 18.1, 24.4, 27.6, 38.6, 55.1, 61.3, 101.0, 119.6, 119.7, 125.8, 126.3, 126.5, 127.21, 127.6, 127.9, 128.1, 128.9, 129.1, 136.5, 136.7, 138.6, 139.3, 139.9, 140.8, 147.2, 166.5. EM (ESI+) m/z 646.26 [M + Na]⁺.

(2R)-2-{[(4-{4-[(Biphenyl-4-yl)]-1H-1,2,3-triazol-1-yl}phenyl)sulfonyl]amino}-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide ((R)-6b). From (R)-5 (150 mg, 0.337 mmol), 4-ethynylbiphenyl (90 mg, 0.505 mmol), sodium ascorbate (673 μL, 0.673 mmol) and copper(II) sulfate pentahydrate (673 μL, 0.168 mmol) in t-BuOH–H₂O (1 : 1, 5 mL), (R)-6b (68 mg, 32%) was produced as a white solid after chromatography purification (DCM–MeOH 0.6% MeOH), mp 199–200 °C (hexane–EtOAc).

(2S)-2-{[(4-{4-[(Biphenyl-4-yl)]-1H-1,2,3-triazol-1-yl}phenyl)sulfonyl]amino}-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide ((S)-6b). From (S)-5 (150 mg, 0.337 mmol), 4-ethynylbiphenyl (90 mg, 0.505 mmol), sodium ascorbate (673 μL, 0.673 mmol) and copper(II) sulfate pentahydrate (673 μL, 0.168 mmol) in t-BuOH–H₂O (1 : 1, 5 mL), (S)-6b (105 mg, 50%) was produced as a white solid after chromatography purification (DCM–MeOH 0.6% MeOH), mp 199–200 °C (hexane–EtOAc).

2-{[(4-{4-[(4-Dimethylamino)phenyl]-1H-[1,2,3]-triazol-1-yl}phenyl)sulfonyl]amino}-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide (6c). From 5 (100 mg, 0.224 mmol), 4-ethynyl-N,N-dimethylaniline (36 mg, 0.248 mmol), sodium ascorbate (448 μL, 0.448 mmol) and copper(II) sulfate pentahydrate (448 μL, 0.112 mmol) in DMF (3 mL), 6c (71.6 mg, 54%) was produced as a yellow solid after chromatography purification (DCM–MeOH 0.75% MeOH), mp 192–194 °C. (Found: C, 59.92; H, 5.91; N, 13.96; S, 5.27. C₃₀H₃₄N₆O₅S·0.5H₂O requires: C, 60.08; H, 5.88; N, 14.01; S, 5.35%); ν_max (KBr)/cm⁻¹ 3276, 1668, 1616. δ_H (300 MHz, DMSO-d₆) 1.43–1.58 (6H, m, 3CH₂), 2.65–2.73 (1H, m, ½CH₂Ph), 2.79–2.85 (1H, m, ½CH₂Ph), 2.96 (6H, s, 2CH₃), 3.38–3.49 (1H, m, CH), 3.75–3.85 (1H, m, OCH₂, isomer a), 3.85–3.95 (1H, m, OCH₂, isomer b), 4.36 (0.5H, s, O–CH–O, isomer a), 4.56 (0.5H, s, O–CH–O, isomer b), 6.84 (2H, d, J 8.9, ArH), 7.1–7.2 (5H, m, Ar), 7.76–7.81 (4H, m, Ar), 8.02 (2H, d, J 8.6, ArH), 8.53 (1H, bs, SO₂NH), 9.2 (1H, s, triazole), 11.23 (0.5H, s, CONH, isomer b), 11.32 (0.5H, s, CONH, isomer a); δ_C (75.4 MHz, DMSO-d₆): 18.1, 24.4, 27.6, 37.2, 39.8, 55.1, 61.3, 100.9, 112.2, 117.3, 117.4, 119.4, 126.2, 127.9, 128.0, 129.1, 136.5, 136.7, 138.7, 140.3, 148.2, 150.2, 166.5. EM (ESI+) m/z 613.30 [M + Na]⁺.

(2R)-2-{[(4-{4-[(4-Dimethylamino)phenyl]-1H-[1,2,3]-triazol-1-yl}phenyl)sulfonyl]amino}-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide ((R)-6c). From (R)-5 (150 mg, 0.337 mmol), 4-ethynyl-N,N-dimethylaniline (73 mg, 0.505 mmol), sodium ascorbate (673 μL, 0.673 mmol) and copper(II) sulfate pentahydrate (673 μL, 0.168 mmol) in t-BuOH–H₂O (1 : 1, 5 mL), (R)-6c (130 mg, 65%) was produced as a yellow solid after chromatography purification (DCM–MeOH 0.75% MeOH), mp 203–204 °C (hexane–EtOAc).

(2S)-2-{[(4-{4-[(4-Dimethylamino)phenyl]-1H-[1,2,3]-triazol-1-yl}phenyl)sulfonyl]amino}-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide ((S)-6c). From (S)-5 (150 mg, 0.337 mmol), 4-ethynyl-N,N-dimethylaniline (73 mg, 0.505 mmol), sodium ascorbate (673 μL, 0.673 mmol) and copper(II) sulfate pentahydrate (673 μL, 0.168 mmol) in t-BuOH–H₂O (1 : 1, 5 mL), (S)-6c (130 mg, 65%) was produced as a yellow solid after chromatography purification (DCM–MeOH 0.75% MeOH), mp 203–204 °C (hexane–EtOAc).

3-Phenyl-2-[({4-[4-({[(phenylsulfonyl)amino]carbonyl}aminomethyl)-1H-1,2,3-triazol-1-yl]-phenyl}sulfonyl)amino]-N-(tetrahydro-2H-pyran-2-yloxy)propanamide (6d). From 5 (100 mg, 0.224 mmol), N-[(prop-2-yn-1-yl-amino)carbonyl)]benzenesulfonamide²³ (58.5 mg, 0.247 mmol), sodium ascorbate (448 μL, 0.448 mmol) and copper(II) sulfate pentahydrate (448 μL, 0.112 mmol) in DMF (3 mL), 6d (87.6 mg, 60%) was produced as a beige solid after chromatography purification (DCM–MeOH, 5% MeOH), mp 165–167 °C. (Found: C, 52.64; H, 4.96; N, 14.10; S, 9.15. C₃₀H₃₃N₇O₈S₂ requires: C, 52.70; H, 4.86; N, 14.34; S, 9.38) ν_max (KBr)/cm⁻¹ 3343, 3284, 1694, 1675 cm⁻¹. δ_H (300 MHz, DMSO-d₆) 1.35–1.58 (6H, m, 3CH₂), 2.64–2.73 (1H, m, ½CH₂Ph), 2.78–2.84 (1H, m, ½CH₂Ph), 3.37–3.45 (2H, m, OCH₂), 3.75–3.80 (0.5H, m, CHN, isomer a), 3.89–3.94 (0.5H, m, CHN, isomer b), 4.31 (2H, s, NHCH₂), 4.33 (0.5H, s, O–CH–O, isomer a), 4.55 (0.5H, s, O–CH–O, isomer b), 7.09–7.2 (7H, m, ArH + NHCONH), 7.54–7.63 (3H, m, Ar), 7.73–7.76 (2H, m, ArH), 7.89–7.93 (4H, m, ArH), 8.47–8.54 (1H, m, SO₂NH), 8.61 (1H, s, triazole), 11.23 (0.5H, s, CONH, isomer b), 11.31 (0.5H, s, CONH, isomer a); δ_C (75.4 MHz, DMSO-d₆) 18.0, 24.4, 27.5, 34.6, 37.2, 55.1 (55.2 b), 61.3, 100.9 (101.1 b), 119.8, 120.9, 126.2, 127.08, 127.9 (127.9 b), 128.8, 129.1 (129.1 b), 133.0, 136.4, 136.7, 138.5, 140.2, 140.7, 145.9, 151.4, 166.6. MS (ESI−) m/z 682.30 [M − H]⁻.

2-[({4-[4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)amino]-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide (6e). From 5 (100 mg, 0.224 mmol), 4-ethynylanisole (33 mg, 0.25 mmol), sodium ascorbate (448 μL, 0.448 mmol) and copper(II) sulfate pentahydrate (448 μL, 0.112 mmol) in DMF (3 mL), 6e (98.8 mg, 76%) was produced as a yellow solid after chromatography purification (DCM–MeOH, 5% MeOH), mp 151–153 °C. (Found: C, 59.94; H, 5.46; N, 12.04; S, 5.54; C₂₉H₃₁N₅O₆S requires: C, 60.3; H, 5.41; N, 12.12; S, 5.55%); ν_max (KBr)/cm⁻¹ 3202, 1668, 1620. δ_H (300 MHz, DMSO-d₆) 1.43–1.58 (6H, m, 3CH₂), 2.66–2.73 (1H, m, ½CH₂Ph), 2.79–2.85 (1H, m, ½CH₂Ph), 3.35–3.48 (1H, m, CH), 3.76–3.85 (1H, m, OCH₂, isomer a), 3.82 (3H, s, OCH₃), 3.87–3.95 (1H, m, OCH₂, isomer b), 4.37 (0.5H, s, O–CH–O, isomer a), 4.56 (0.5H, s, O–CH–O, isomer b), 6.84 (2H, d, J 8.9, ArH), 7.1–7.2 (7H, m, ArH), 7.78–7.81 (2H, dd, J 8.7, 2.3, ArH), 7.89 (2H, d, J 8.7, ArH), 8.00–8.04 (2H, dd, J 8.7, 1.5, ArH), 8.44 (1H, bs, SO₂NH), 9.32 (1H, s, triazole), 11.24 (0.5H, s, CONH, isomer b), 11.32 (0.5H, s, CONH, isomer a); δ_C (75.4 MHz, DMSO-d₆): 18.1, 24.4, 27.5, 38.5, 55.1, 55.2, 61.3, 100.9 (101.1 b), 114.4, 119.6, 122.3, 126.3, 126.7, 127.9, 128.0, 129.1 (129.2 b), 136.5, 136.7, 138.7, 140.6, 147.5, 159.3, 166.5 (166.6 b). MS (ESI+): m/z 600.27 [M + Na]⁺.

2-[({4-[4-(4-Phenoxyphenyl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)amino]-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide (6f). From 5 (100 mg, 0.224 mmol), 1-ethynyl-4-phenoxybenzene (48 mg, 0.247 mmol), sodium ascorbate (448 μL, 0.448 mmol) and copper(II) sulfate pentahydrate (448 μL, 0.112 mmol) in DMF (3 mL), 6f (115 mg, 80%) was produced as a pale yellow solid after chromatography purification (hexane–AcOEt 1 : 1 + 2% MeOH), mp 189–190 °C. (Found: C, 63.70; H, 5.26; N, 10.87; S, 4.95; C₃₄H₃₃N₅O₆S requires: C, 63.83; H, 5.20; N, 10.95; S, 5.01%); ν_max (KBr)/cm⁻¹ 3166, 1657. δ_H (300 MHz, DMSO-d₆): 1.43–1.58 (6H, m, 3CH₂), 2.66–2.73 (1H, m, ½CH₂Ph), 2.79–2.86 (1H, m, ½CH₂Ph), 3.42–3.49 (1H, m, CH), 3.76–3.85 (1H, m, OCH₂, isomer a), 3.93–3.98 (1H, m, OCH₂, isomer b), 4.38 (0.5H, s, O–CH–O, isomer a), 4.57 (0.5H, s, O–CH–O, isomer b), 7.08–7.21 (10H, m, ArH), 7.41–7.46 (2H, m, ArH), 7.78–7.82 (2H, dd, J 8.6, 2,1, ArH), 7.89 (2H, d, J 8.6, ArH), 8.03 (2H, d, J 7.4, ArH), 8.5 (1H, bs, SO₂NH), 9.39 (1H, s, triazole), 11.32 (1H, bs, CONH); δ_C (75.4 MHz, DMSO-d₆) 18.2, 24.5, 27.6, 38.5, 55.2, 61.4, 101.1, 118.93, (118.98 b), 119.2, 119.7, 123.7, 125.1, 126.4, 127.2, 128.0, 128.15, 129.2 (129.3 b), 130.1, 136.5, 136.8, 138.7, 140.7, 147.1, 156.3, 156.9, 166.5 (166.7 b). MS (ESI+): m/z 662.26 [M + Na]⁺.

(2R)-2-[({4-[4-(4-Phenoxyphenyl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)amino]-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide ((R)-6f). From (R)-5 (150 mg, 0.337 mmol), 1-ethynyl-4-phenoxybenzene (98 mg, 0.505 mmol), sodium ascorbate (673 μL, 0.673 mmol) and copper(II) sulfate pentahydrate (673 μL, 0.168 mmol) in t-BuOH–H₂O (1 : 1, 5 mL), (R)-6f (170 mg, 79%) was produced as a white solid after chromatography purification (DCM–MeOH 0.6% MeOH), mp 169–170 °C (hexane–EtOAc).

(2S)-2-[({4-[4-(4-Phenoxyphenyl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)amino]-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide ((S)-6f). From (S)-5 (150 mg, 0.337 mmol), 1-ethynyl-4-phenoxybenzene (98 mg, 0.505 mmol), sodium ascorbate (673 μL, 0.673 mmol) and copper(II) sulfate pentahydrate (673 μL, 0.168 mmol) in t-BuOH–H₂O (1 : 1, 5 mL), (S)-6f (175 mg, 81%) was produced as a white solid after chromatography purification (DCM–MeOH 0.6% MeOH), mp 169–170 °C (hexane–EtOAc).

2-[({4-[4-(4-Pentylphenyl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)amino]-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide (6g). From 5 (100 mg, 0.224 mmol), 1-ethynyl-4-pentylbenzene (42.5 mg, 0.247 mmol), sodium ascorbate (448 μL, 0.448 mmol) and copper(II) sulfate pentahydrate (448 μL, 0.112 mmol) in DMF (3 mL), 6g (107 mg, 77%) was produced as a pale yellow solid after chromatography purification (hexane–AcOEt 1 : 1 + 2% MeOH), mp 189–190 °C. (Found: C, 63.72; H, 6.43; N, 11.32; S, 5.17; C₃₃H₃₉N₅O₅S requires: C, 64.16; H, 6.36; N, 11.34; S, 5.19; ν_max (KBr)/cm⁻¹ 3291, 1690. δ_H (300 MHz, DMSO-d₆) 0.87 (3H, t, J 6.8, CH₃), 1.30–1.63 (12H, m, 6CH₂), 2.62–2.73 (3H, t, J 7.6, CH₂Ar + m, ½CH₂Ph), 2.79–2.89 (1H, m, ½CH₂Ph), 3.42–3.49 (1H, m, CH), 3.78–3.85 (1H, m, OCH₂, isomer a), 3.92–3.99 (1H, m, OCH₂, isomer b), 4.37 (0.5H, s, O–CH–O, isomer a), 4.57 (0.5H, s, O–CH–O, isomer b), 7.01–7.21 (5H, m, ArH), 7.34 (2H, d, J 8.1, ArH), 7.80 (2H, dd, J 8.7, 2.1, ArH), 7.87 (2H, d, J 8.0, ArH), 8.03 (2H, dd, J 8.6, 1.6, ArH), 8.52 (1H, bs, SO₂NH), 9.38 (1H, s, triazole), 11.24 (0.5H, bs, CONH isomer a), 11.32 (0.5H, bs, CONH, isomer b); δ_C (75.4 MHz, DMSO-d₆) 13.9, 18.2, 21.9, 24.5, 27.7, 30.5, 30.8, 34.8, 38.5, 55.3, 61.4, 101.0, 119.1 (119.7 b), 125.3, 126.4, 127.4, 128.04, 128.1, 128.9, 129.2 (129.3 b), 136.5, 136.8, 138.7, 140.79, 142.7, 147.7, 166.5 (166.7 b). MS (ESI+): m/z 640.33 [M + Na]⁺.

Cleavage of the tetrahydropyrane protecting group: general procedure 2

To a suspension of the corresponding tetrahydro-2H-pyranyl derivative in DCM (5 mL) were added 4 M HCl in dioxane (4 equivalents) and MeOH (0.1 mL). After stirring at room temperature for 1 h the reaction mixture was concentrated under vacuum and the obtained solid was washed with DCM–hexane 1 : 1.

2-[({4-[4-(3-Fluorophenyl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)amino]-N-hydroxy-3-phenylpropanamide (1a). From 6a (111 mg, 0.178 mmol), MeOH (0.18 mL) and 4 M HCl–dioxane (0.18 mL, 0.72 mmol), 1a was produced as a light beige solid (73.2 mg, 76%), mp 210 °C (dec.). (Found: C, 56.67; H, 4.22; N, 14.35; S, 6.54. C₂₃H₂₀FN₅O₄S requires: C, 57.37; H, 4.19; N, 14.54; S, 6.66; ν_max (KBr)/cm⁻¹ 3313, 1675, 1620; δ_H (300 MHz, DMSO-d₆) 2.68 (1H, dd, J 13.5, 9.1, ½CH₂Ph), 2.83 (1H, dd, J 13.5, 5.8, ½CH₂Ph), 3.84–3.92 (1H, m, CH), 7.09–7.16 (5H, m, ArH), 7.25 (1H, dt, J 8.5, 2.1, ArH), 7.54–7.61 (1H, m, ArH), 7.77 (2H, d, J 8.7, ArH), 7.83 (1H, d, J 7.7, ArH), 7.99 (2H, d, J 8.7, ArH), 8.43 (1H, d, J 8.9, SO₂NH), 8.87 (1H, bs, CONHOH), 9.47 (1H, s, CH, triazole), 10.67 (1H, s, CONHOH); δ_C (75.4 MHz, DMSO-d₆) 38.4, 55.5, 111.8–112.1 (d, ²J_CF 22.5), 114.9–115.2 (d, ²J_CF 21.0), 119.85, 120.8 (d, ²J_CF 74.2), 126.2, 127.9, 129.1, 131.15 (d, ³J_CF 9.0), 132.26 (d, ³J_CF 9.0), 136.8, 138.4, 141.0, 146.4, 162.5 (d, J 241.5, Ar–F), 166.6. MS (ESI): m/z 504.20 [M + Na]⁺.

2-[({4-[4-(Biphenyl-4-yl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)amino]-N-hydroxy-3-phenyl-propanamide (1b). From 6b (83.8 mg, 0.147 mmol), MeOH (0.14 mL) and 4 M HCl–dioxane (0.14 mL, 0.56 mmol), 1b was produced as a light yellow solid (54.0 mg, 75%), mp 182–183 °C (Found: C, 62.81; H, 4.72; N, 12.63; S, 5.57. C₂₉H₂₅N₅O₄S·H₂O requires: C, 62.46; H, 4.88; N, 12.56; S, 5.75%). ν_max (KBr)/cm⁻¹ 3557, 3284, 1683, 1668. δ_H (300 MHz, DMSO-d₆): 2.69 (1H, dd, J 13.5, 9.1, ½CH₂Ar), 2.84 (1H, dd, J 13.5, 5.8, ½CH₂Ar), 3.84–3.93 (1H, m, CH), 7.10–7.16 (5H, m, ArH), 7.39 (1H, t, J 7.3, ArH), 7.48–7.52 (2H, m, ArH), 7.75–7.86 (6H, m, ArH), 8.02 (2H, d, J 8.6, ArH), 8.07 (2H, d, J 8.1, ArH), 8.43 (1H, d, J 8.9, SO₂NH), 8.86 (1H, bs, OH), 9.47 (s, 1H, CH, triazole), 10.68 (s, 1H, CONH); δ_C (75 MHz, DMSO-d₆) 38.4, 55.5, 119.6, 119.7, 125.8, 126.2, 126.5, 127.2, 127.5, 127.9, 127.9, 128.9, 128.9, 129.1, 136.8, 138.5, 139.4, 139.9, 140.8, 147.2, 166.6. MS (ESI+): m/z 562.27 [M + Na]⁺.

(2R)-2-[({4-[4-(Biphenyl-4-yl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)amino]-N-hydroxy-3-phenyl-propanamide ((R)-1b). From (R)-6b (48 mg, 0.077 mmol), MeOH (1 mL) and 4 M HCl–dioxane (1 mL, 4 mmol), (R)-1b was produced as a white solid (34.1 mg, 82%), mp 240 °C (dec.) (EtOH) (Found: C, 63.91; H, 4.70; N, 12.93; S, 5.99. C₂₉H₂₅N₅O₄S·H₂O requires: C, 64.01; H, 4.72; N, 12.87; S, 5.89%). [α]²⁵_D − 8.3 (c 0.0012 g cm⁻³ in DMSO).

(2S)-2-[({4-[4-(Biphenyl-4-yl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)amino]-N-hydroxy-3-phenyl-propanamide ((S)-1b). From (S)-6b (78 mg, 0.125 mmol), MeOH (1 mL) and 4 M HCl–dioxane (3 mL, 12 mmol), (S)-1b was produced as a white solid (58 mg, 86%), mp 240 °C (dec.) (EtOH) (Found: C, 64.32; H, 4.68; N, 12.89; S, 5.92. C₂₉H₂₅N₅O₄S requires: C, 64.55; H, 4.67; N, 12.98; S, 5.94%). [α]²⁵_D + 7.9 (c 0.007 g cm⁻³ in DMSO).

2-{[(4-{[4-(4-Dimethylamino)phenyl]-1H-1,2,3-triazol-1-yl}phenyl)sulfonyl]amino}-N-hydroxy-3-phenylpropanamide hydrochloride (1c). From 6c (79.8 mg, 0.135 mmol), MeOH (0.14 mL) and 4 M HCl–dioxane (0.14 mL, 0.56 mmol), 1c was produced as a light orange solid (62.9 mg, 92%), mp 223 °C (dec.) (Found: C, 54.71; H, 4.97; N, 15.40; S, 5.83; C₂₅H₂₆N₆O₄S·HCl·1/3H₂O requires: C, 54.69; H, 5.08; N, 15.31; S, 5.84%); ν_max (KBr)/cm⁻¹ 3431, 3188, 2428, 1679. δ_H (300 MHz, DMSO-d₆) 2.68 (1H, dd, J 13.5, 9.1, ½CH₂Ph), 2.83 (1H, dd, J 13.5, 5.8, ½CH₂Ph), 3.07 (6H, s, 2CH₃) 3.84–3.92 (1H, m, CH), 7.09–7.16 (5H, m, ArH), 7.42 (1H, bs, ArH), 7.75 (2H, d, J 8.6, ArH), 7.95–8.01 (4H, m, ArH), 8.42 (1H, d, J 8.9, SO₂NH), 9.38 (1H, s, CH, triazole), 10.72 (1H, s, CONH); δ_C (75.4 MHz, DMSO-d₆) 38.4, 42.6, 55.5, 116.9, 118.9, 119.6, 126.2, 126.4, 127.9, 127.9, 129.1, 136.8, 138.5, 140.7, 147.1, 166.6. MS (ESI−): m/z 505.19 [M − H]⁻.

(2R)-2-{[(4-{[4-(4-Dimethylamino)phenyl]-1H-1,2,3-triazol-1-yl}phenyl)sulfonyl]amino}-N-hydroxy-3-phenylpropanamide hydrochloride ((R)-1c). From (R)-6c (74 mg, 0.125 mmol), MeOH (1 mL) and 4 M HCl–dioxane (3 mL, 12 mmol), (R)-1c was produced as a yellow solid (53.6 mg, 79%), mp 210 °C (dec.) (EtOH) (Found: C, 58.66; H, 5.20; N, 16.39; S, 6.26; C₂₅H₂₆N₆O₄S·1/3H₂O requires: C, 58.58; H, 5.24; N, 16.40; S, 6.26%). [α]²⁵_D – 0.81 (c 0.005 g cm⁻³ in DMSO).

(2S)-2-{[(4-{[4-(4-Dimethylamino)phenyl]-1H-1,2,3-triazol-1-yl}phenyl)sulfonyl]amino}-N-hydroxy-3-phenylpropanamide hydrochloride ((S)-1c). From (S)-6c (80 mg, 0.135 mmol), MeOH (1 mL) and 4 M HCl–dioxane (3 mL, 12 mmol), (S)-1c was produced as a yellow solid (61.4 mg, 83%), mp 210 °C (dec.) (EtOH) (Found: C, 58.11; H, 5.11; N, 16.24; S, 6.18; C₂₅H₂₆N₆O₄S·1/2H₂O requires: C, 58.24; H, 5.28; N, 16.30; S, 6.22%). [α]²⁵_D + 0.99 (c 0.006 g cm⁻³ in DMSO).

N-Hydroxy-3-phenyl-2-{[(4-{4-[({[(phenylsulfonyl)amino]carbonyl}amino)methyl]-1H-1,2,3-triazol-1-yl}phenyl)sulfonyl]amino}propanamide (1d). From 6d (117 mg, 0.17 mmol), MeOH (0.17 mL) and 4 M HCl–dioxane (0.17 mL, 0.68 mmol), 1d was produced as a white solid (93 mg, 90%), mp 179–180 °C (Found: C, 49.53; H, 4.25; N, 16.08; S, 10.52. C₂₅H₂₅N₇O₇S₂·1/3H₂O requires: C, 49.58; H, 4.27; N, 16.19; S, 10.59%); ν_max (KBr)/cm⁻¹ 3579, 1690, 1642. δ_H (300 MHz, DMSO-d₆) 2.66 (1H, dd, J 13.6, 9.1, ½CH₂Ph), 2.81 (1H, dd, J 13.6, 5,9, ½CH₂Ph), 3.81–3.89 (1H, m, CH), 4.33 (2H, d, J 5.5, CH₂NHCO), 7.04–7.16 (7H, m, NHCONH + ArH), 7.56–7.72 (5H, m, ArH), 7.88–7.93 (4H, m, ArH), 8.41 (1H, d, J 8.9, SO₂NH), 8.59 (1H, s, CH, triazole), 10.65 (1H, s, CONHOH), 10.79 (1H, s, CONHOH); δ_C (75.4 MHz, DMSO-d₆) 34.6, 38.3, 55.5, 119.8, 121.0, 126.20, 127.1, 127.8, 127.9, 128.9, 129.0, 133.1, 136.7, 138.4, 140.0, 140.7, 145.8, 151.3, 166.6. MS (ESI−) m/z 598.09 [M − H]⁻.

N-Hydroxy-2-[({4-[4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)amino]-3-phenylpropanamide (1e). From 6e (112 mg, 0.194 mmol), MeOH (0.20 mL) and 4 M HCl–dioxane (0.20 mL, 0.80 mmol), 1e was produced as a yellow solid (93 mg, 90%), mp 160 °C (dec.) (Found: C, 57.49; H, 4.70; N, 13.96; S, 6.35. C₂₄H₂₃N₅O₅S·1/3H₂O requires: C, 57.70; H, 4.78; N, 14.02; S, 6.42%); ν_max (KBr)/cm⁻¹ 3571, 3298, 1668, 1635. δ_H (300 MHz, DMSO-d₆) 2.67(1H, dd, J 13.5, 9.0, ½CH₂Ph), 2.83 (1H, dd, J 13.5, 5,6 ½CH₂Ph), 3.82 (3H, s, OCH₃), 3.85–3.91 (1H, m, CH), 7.07–7.16 (8H, m, ArH), 7.75 (2H, d, J 8.7, ArH), 7.89 (2H, d, J 8.7, ArH), 7.98 (2H, d, J 8.7, ArH), 8.42 (1H, d, J 8.9, SO₂NH), 9.28 (s, 1H, CH, triazole), 10.67 (s, 1H, CONHOH); δ_C (75.4 MHz, DMSO-d₆) 38.4, 55.2, 55.6, 114.4, 118.6, 119.7, 122.5, 126.3, 126.8, 127.9, 128.0, 129.1, 136.8, 138.7, 140.8, 147.5, 159.4, 166.7. MS (ESI+) m/z 516.22 [M + Na]⁺.

N-Hydroxy-2-{[(4-[4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl]phenyl)sulfonyl]amino}-3-phenylpropanamide (1f). From 6f (94 mg, 0.147 mmol), MeOH (0.15 mL) and 4 M HCl–dioxane (0.15 mL, 0.60 mmol), 1f was produced as a pale yellow solid (64 mg, 78%), mp 229 °C (dec.) (Found: C, 61.59; H, 4.54; N, 12.32; S, 5.66; C₂₉H₂₅N₅O₅S·1/2H₂O requires: C, 61.69; H, 4.64; N, 12.40; S, 5.68%); ν_max (KBr)/cm⁻¹ 3564, 3306, 1675. δ_H (300 MHz, DMSO-d₆) 2.67 (1H, dd, J 13.6, 9.0, ½CH₂Ph), 2.82 (1H, dd, J 13.6, 5.8, ½CH₂Ph), 3.83–3.91 (1H, m, CH), 7.08–7.21 (10H, m, ArH), 7.41–7.46 (2H, m, ArH), 7.76 (2H, d, J 8.7, ArH), 7.96–8.01 (4H, m, ArH), 8.42 (1H, d, J 8.9, SO₂NH), 8.85 (1H, s, CONHOH), 9.35 (s, 1H, CH, triazole), 10.66 (s, 1H, CONHOH); δ_C (75.4 MHz, DMSO-d₆) 38.4, 55.5, 118.8, 118.9, 119.1, 119.7, 123.6, 125.1, 126.2, 127.1, 127.9, 127.9, 129.1, 130.0, 136.8, 138.5, 140.8, 147.0, 156.2, 156.8, 166.6. MS (ESI−) m/z 554.27 [M − H]⁻.

(2R)-N-Hydroxy-2-{[(4-[4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl]phenyl)sulfonyl]amino}-3-phenylpropanamide ((R)-1f). From (R)-6f (130 mg, 0.203 mmol), MeOH (1 mL) and 4 M HCl–dioxane (3 mL, 12 mmol), (R)-1f was produced as a white solid (87.5 mg, 78%), mp 199–200 °C (EtOH). (Found: C, 62.59; H, 4.52; N, 12.52; S, 5.68; C₂₉H₂₅N₅O₅S requires: C, 62.69; H, 4.54; N, 12.60; S, 5.77%). [α]²⁵_D – 2.06 (c 0.004 g cm⁻³ in DMSO).

(2S)-N-Hydroxy-2-{[(4-[4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl]phenyl)sulfonyl]amino}-3-phenylpropanamide ((S)-1f). From (S)-6f (125 mg, 0.195 mmol), MeOH (1 mL) and 4 M HCl–dioxane (3 mL, 12 mmol), (S)-1f was produced as a white solid (100.9 mg, 93%), mp 199–200 °C (EtOH). (Found: C, 62.56; H, 4.51; N, 12.63; S, 5.75; C₂₉H₂₅N₅O₅S requires: C, 62.69; H, 4.54; N, 12.60; S, 5.77%). [α]²⁵_D + 1.97 (c 0.010 g cm⁻³ in DMSO).

N-Hydroxy-2-[({4-[4-(4-pentylphenyl)-1H-1,2,3-triazol-1-yl]-phenyl}sulfonyl)amino]-3-phenylpropanamide (1g). From 6g (83.8 mg, 0.147 mmol), MeOH (0.15 mL) and 4 M HCl–dioxane (0.15 mL, 0.60 mmol), 1g was produced as a yellow solid (54 mg, 75%), mp 196 °C (dec.) (Found: C, 68.11; H, 6.53; N, 14.12; S, 6.47; C₂₈H₃₁N₅O₄S requires: C, 63.02; H, 5.86; N, 13.12; S, 6.01%); ν_max (KBr)/cm⁻¹ 3549, 3291, 2450, 1664, 1635. δ_H (300 MHz, DMSO-d₆) 0.87 (3H, t, J 6.8, CH₃), 1.29–1.32 (4H, m, 2CH₂), 1.59–1.63 (2H, m, CH₂), 2.60–2.71 (3H, t, J 7.6, ArCH₂CH₂ + m, ½CH₂ Ph), 2.79–2.86 (1H, dd, J 13.66, 5.8, ½CH₂Ar), 3.84–3.92 (1H, m, CH), 7.09–7.16 (5H, m, ArH), 7.33 (2H, d, J 8.1, ArH), 7.76 (2H, d, J 8.6, ArH), 7.87 (2H, d, J 8.0, ArH), 8.00 (2H, d, J 8.7, ArH), 8.42 (1H, d, J 8.9, SO₂NH), 9.35 (s, 1H, CH, triazole), 10.68 (s, 1H, CONHOH); δ_C (75.4 MHz, DMSO-d₆) 13.8, 21.9, 30.4, 30.8, 34.8, 38.4, 55.6, 119.2, 119.7, 125.4, 126.3, 127.4, 127.9, 128.0, 128.9, 129.1, 136.8, 138.6, 140.8, 142.7, 147.7, 166.7. MS (ESI+) m/z 556.31 [M + Na]⁺.

Methyl 2-{[(4-iodophenyl)sulfonyl]amino}-3-phenylpropanoate (8). To a stirred mixture of the L-alanine methyl ester (1 g, 4.63 mmol) and NMM (1.3 mL, 11.6 mmol) in CH₂Cl₂ (5 mL) was slowly added a solution of 4-iodobenzenesulfonyl chloride (1.54 g, 5.1 mmol) in CH₂Cl₂ (5 mL) under a N₂ atmosphere, and the reaction mixture was stirred for 30 min at 0 °C and 24 h at RT. The solvent was removed under reduced pressure, the residue was dissolved in ethyl acetate, and the solution was washed with HCl aqueous solution and brine. The extracts were dried over anhydrous MgSO₄, filtered, and evaporated. The residue was crystallized from EtOH to obtain 8 as colorless crystals (1.89 g, 92%), mp 117–118 °C. (Found C, 43.15; H, 3.67; N, 3.38; S, 7.19; C₁₆H₁₆INO₄S requires: C, 43.16; H, 3.62; N, 3.15; S, 7.20%). ν_max (KBr)/cm⁻¹ 3291, 1731, 1569. δ_H (300 MHz, CDCl₃) 2.95–3.11 (2H, m, CH₂Ar), 3.56 (3H, s, CH₃), 4.15–4.23 (1H, m, CH), 5.18 (1H, d, J 9.2, SO₂NH), 7.04–7.07 (2H, m, ArH), 7.24–7.25 (3H, m, ArH), 7.41 (2H, d, J 8.7, ArH), 7.77 (2H, d, J 8.7, ArH); δ_C (75.4 MHz, CDCl₃) 39.2, 52.5, 56.7, 100.1, 127.3, 128.4, 128.6, 129.2, 134.7, 138.1, 139.1, 171.1. MS (ESI+) m/z 467.88 [M + Na]⁺.

Methyl 3-phenyl-2-{[(4-trimethylsilanylethynyl)phenylsulfonyl]amino}propanoate (9). To a mixture of 8 (1 g, 2.24 mmol), triphenylphosphine (94 mg, 359 μmol), bis(triphenylphosphine)palladium(II) dichloride (120 mg, 170 μmol), and copper(I) iodide (20 mg, 110 μmol) in degassed acetonitrile–NEt₃ (5 mL/3.5 mL) was added (trimethylsilyl)acetylene (0.38 mL, 2.70 mmol) under a nitrogen atmosphere. The reaction mixture was refluxed for 20 h. After cooling to room temperature, methanol was added and the mixture was filtered over Celite and the solvent was evaporated under reduced pressure. The residue was dissolved in dichloromethane, and the solution was washed with an HCl aqueous solution. The extracts were dried using anhydrous MgSO₄, filtered, and evaporated. The residue was purified by column chromatography on silica gel with ethyl hexane–EtOAc (4 : 1) to give 9 (0.69 g, 74%) as a white solid, mp. 118–119 °C (DCM–hexane). (C, 60.74; H, 5.96; N, 3.50; S, 7.66; C₂₁H₂₅NO₄SSi requires: C, 60.69; H, 6.06; N, 3.37; S, 7.72%). ]⁺. ν_max (KBr)/cm^-1 3239, 2154, 1745, 1587. δ_H (300 MHz, CDCl₃) 0.27 (9H, s, (CH₃)₃Si), 2.97–3.09 (2H, m, CH₂Ar), 3.52 (3H, s, OCH₃), 4.17–4.24 (1H, m, CH), 5.29 (1H, d, J 9.2, SO₂NH), 7.05–7.23 (2H, m, ArH), 7.24–7.26 (3H, m, ArH), 7.49 (2H, d, J 8.3 Hz, ArH), 7.66 (2H, d, J 8.3, ArH); δ_C (75.4 MHz, CDCl₃) −0.3, 39.1, 52.4, 56.6, 98.4, 102.9, 126.8, 127.2, 127.7, 129.3, 132.2, 134.7, 138.9, 171.0. MS (ESI+) m/z 438.10 [M + Na]⁺.

2-{[(4-Ethynylphenyl)sulfonyl]amino}-3-phenylpropanoic acid (10). To a solution of 9 (0.25 g, 0.6 mmol) in dioxane (3 mL) was added KOH (1 M in water, 3 mL), and the mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure. The residue was diluted in water (50 mL), acidified to pH 3 with 20% solution of citric acid, and extracted with EtOAc (2 × 50 mL). The collected organic phase was washed with saturated solution of NH₄Cl (2 × 50 mL) and brine (50 mL), dried over MgSO₄ and evaporated. The residue was purified by column chromatography on silica gel (hexane–AcOEt 2 : 8, 1 : 9) to give 10 as a white solid (0.165 g, 83%), mp 136–137 °C. ν_max (KBr)/cm⁻¹ 3335, 3261, 2108, 1705, 1590. δ_H (300 MHz, CDCl₃) 2.98 (1H, dd, J 13.9, 7.3, ½CH₂Ar), 3.15 (1H, dd, J 13.9, 4.8, ½CH₂Ar), 3.26 (1H, s, ArCCH), 4.19–4.27 (1H, m, CH), 5.28 (1H, d, J 8.3, SO₂NH), 7.08–7.09 (2H, m, ArH), 7.24–7.27 (3H, m, ArH), 7.48 (2H, d, J 8.3, ArH), 7.67 (2H, d, J 8.3, ArH); δ_C (75.4 MHz, CDCl₃) 38.7, 56.5, 80.8, 81.9, 126.8, 126.9, 127.4, 128.7, 129.3, 132.5, 134.5, 139.5, 176.0. MS (ESI+) m/z 352.04 [M + Na]⁺.

2-{[(4-Ethynyl)phenyl]sulfonyl}amino-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide (11). To a solution of 10 (0.15 g, 0.45 mmol) in DMF (5 mL) were added HOBt (0.135 g, 1 mmol), O-tetrahydro-2H-pyran-2-yl-hydroxylamine (0.107 g, 0.9 mmol), EDCI (0.2 g, 1.09 mmol) and NMM (0.15 mL, 1.37 mmol). The reaction mixture was stirred overnight at room temperature and then diluted with EtOAc (50 mL) and washed successively with a saturated aqueous solution of NH₄Cl and brine. The organic layer was dried (MgSO₄), evaporated to dryness, and the residue was purified by column chromatography on silica gel (hexane–AcOEt 7 : 3) to afford 11 (0.135 g, 69%) as a white solid. This compound is present as a diastereoisomeric mixture (55 : 45), mp 148–149 °C. (C, 61.86; H, 5.69; N, 6.61; S, 7.34; C₂₂H₂₄N₂O₅S requires: C, 61.67; H, 5.65; N, 6.54; S, 7.48%). ν_max (KBr)/cm⁻¹ 3276, 3202, 2110, 1660. δ_H (300 MHz, CDCl₃) 1.57–1.77 (6H, m, 3 × CH₂), 2.88–3.07 (2H, m, CH₂Ar), 3.27 (1H, s, ArCCH), 3.55–3.59 (1H, m, CH), 3.80–3.89 (2H, m, CH₂O), 4.7 (0.55H, s, O–CH–O, isomer a), 4.79 (0.45H, s, O–CH–O, isomer b), 5.43 (1H, bs, SO₂NH), 6.99 (2H, d, J 7.32, ArH), 7.18–7.22 (3H, m, ArH), 7.48 (2H dd, J 8.3, 2.8, ArH) 7.58 (2H dd, J 8.3, 2.8, ArH), 8.79 (0.55H, s, CONH, isomer a), 9.09 (0.45H, s, CONH, isomer b); δ_C (75.4 MHz, CDCl₃) 18.4, 24.8, 27.8, 38.9, 56.4, 62.4, 80.9, 82.2, 102.5, 126.8, 126.9, 127.3, 128.8, 128.9, 129.1, 132.7, 134.9, 162.7. MS (ESI+) m/z = 451.14 [M + Na]⁺.

2-{[4-(1-Biphenyl-4-yl-1H-1,2,3-triazol-4-yl)phenyl]sulfonyl}amino-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide (12). General procedure 1: from 11 (50 mg, 0.117 mmol), 4-azidobiphenyl²⁸ (23 mg, 0.117 mmol), sodium ascorbate (234 μL, 0.234 mmol) and copper(II) sulfate pentahydrate (234 μL, 0.06 mmol) in DMF (3 mL), 12 (84.8 mg, 67%) was produced. The reaction mixture was diluted in EtOAc and washed with 30% aqueous NH₃, 6 M HCl and brine. The organic layer was dried (MgSO₄), evaporated to dryness, and the residue was suspended in CHCl₃ and filtered off to obtain a white solid (47 mg, 64%), mp 203–204 °C. (C, 65.26; H, 5.27; N, 11.26; S, 5.13; C₃₄H₃₃N₅O₅S requires: C, 65.47; H, 5.33; N, 11.23; S, 5.14%); found: ν_max (KBr)/cm⁻¹ 3210, 1664, 1602. δ_H (300 MHz, DMSO-d₆) 1.36–1.60 (6H, m, 3 × CH₂), 2.64–2.72 (1H, m, ½CH₂Ar), 2.79–2.85 (1H, m, ½CH₂Ar), 3.39–3.51 (1H, m, CH), 3.81–3.85 (1H, m, OCH₂, isomer a), 3.91–3.96 (1H, m, OCH₂, isomer b), 4.38 (0.55H, s, O–CH–O, isomer a), 4.56 (0.45H, s, O–CH–O, isomer b), 7.10–7.13 (1H, m, ArH), 7.16–7.23 (4H, m, ArH), 7.43–7.45 (1H, m, ArH), 7.50–7.55 (2H, m, ArH), 7.70–7.74 (2H, m, ArH), 7.79 (2H, d, J 8.6, ArH), 7.95–8.09 (6H, m, ArH), 8.34–8.42 (1H, m, SO₂NH), 9.54 (1H, s, triazole), 11.23 (0.55H, s, CONH, isomer b), 11.33 (0.45H, s, CONH, isomer a); δ_C (75.4 MHz, DMSO-d₆) 18.6, 24.9, 28.0, 38.8, 55.5 (55.6 b), 61.3, 101.4, (101.6 b), 120.8, 121.1, 125.8, 126.8, 127.1, 127.4, 128.4 (128.4 b), 129.4, 129.5, 129.6, 133.9, 136.0, 136.9, 137.2, 139.0, 140.8 (140.8 b), 140.9, 146.4, 167.0 (167.1 b). MS (ESI+) m/z 646.24 [M + Na]⁺.

2-{[4-(1-Biphenyl-4-yl-1H-1,2,3-triazol-4-yl)phenyl]sulfonyl}amino-N-hydroxy-3-phenylpropanamide (7). General procedure 2: from 12 (54.2 mg, 0.073 mmol), MeOH (0.1 mL) and 4 M HCl–dioxane (0.1 mL, 0.4 mmol), 7 was produced as a white solid (30.0 mg, 77%), mp 223 °C (dec.); (C, 63.66; H, 4.77; N, 12.71; S, 5.85, C₂₉H₂₅N₅O₄S·1/3H₂O requires: C, 63.84; H, 4.74; N, 12.84; S, 5.88%) ν_max (KBr)/cm⁻¹ 3254, 1649, 1605. δ_H (300 MHz, DMSO-d₆) 2.65 (1H, dd, J 13.5, 8.7, ½CH₂Ar), 2.83 (1H, dd, J 13.5, 6.1, ½CH₂), 3.82–3.90 (1H, m, CH), 7.09–7.18 (6H, m, ArH), 7.40–7.45 (1H, m, ArH), 7.50–7.55 (2H, m, ArH), 7.69 (2H, d, J 8.4, ArH), 7.78 (2H, d, J 7.4, ArH), 7.94–8.01 (4H, m, ArH), 8.08 (2H, d, J 8.6, ArH), 8.28 (1H, d, J 8.8, SO₂NH), 8.86 (1H, s, CONHOH), 9.50 (1H, s, triazole), 10.65 (1H, s, CONHOH); δ_C (75.4 MHz, DMSO-d₆) 38.4, 55.4, 120.4, 120.6, 125.3, 126.2, 126.6, 126.8, 127.9, 128.0, 128.9, 129.1, 133.4, 135.6, 136.8, 138.6, 140.4, 140.5, 146.0, 166.0. MS (ESI+): m/z = 562.25 [M + Na]⁺.

tert-Butyl (2R)-2-{[(4-azidophenyl)sulfonyl]amino}-3-phenylpropanoate (15). To a solution of (R)-4 (4.08 g, 11.77 mmol) in DCM (100 mL) was added concentrated H₂SO₄ (0.2 mL) and the solvent was saturated with 2-methylpropene. After 24 h, water was added, neutralized with NaHCO₃ and extracted with DCM. The organic layer was dried (MgSO₄), filtered and evaporated to dryness, and the residue was chromatographed on silica gel (hexane–AcOEt 4 : 1) to give 15 (4.05 g, 86%) as a yellowish solid, mp 75.5–77.4 °C. ν_max (KBr)/cm⁻¹ 3271, 2135, 2106, 1730, 1589. δ_H (400 MHz, DMSO-d₆) 1.15 (9H, s, (CH₃)₃), 2.91 (1H, dd, J 6.4, 1.3, CH₂Ar), 3.94–4.04 (1H, m, CH), 5.43 (1H, d, J 9.3 NH), 6.93 (2H, d, J 8.7 ArH), 7.01–7.08 (2H, m, ArH), 7.09–7.20 (3H, m, ArH), 7.64 (2H, d, J 8.7, ArH); δ_C (100.6 MHz, DMSO-d₆) 27.7, 39.5, 57.1, 82.7, 119.3, 127.1, 128.4, 129.2, 129.6, 135.4, 136.2, 144.6, 170.0. MS (ESI+): m/z = 401.2 [M − H]⁺.

tert-Butyl (2R)-2-{[(4-azidophenyl)sulfonyl][2-(morpholin-4-yl)ethyl]amino}-3-phenylpropanoate (16). To a solution of 15 (0.8 g, 1.99 mmol) in DMF (10 mL) were added K₂CO₃ (0.69 g, 4.97 mmol) and 4-(2-chloroethyl)morpholine hydrochloride (0.44 g, 2.39 mmol). The reaction was heated for 24 h at 80 °C and, after cooling, was diluted with ethyl acetate and washed successively with saturated aqueous NH₄Cl and brine. The extract was dried (MgSO₄), filtered off and evaporated to dryness, and the residue was chromatographed on silica gel (hexane–AcOEt 3 : 2) to give 16 (872 mg, 85%) as a yellow oil. ν_max (KBr)/cm⁻¹ 2971, 2853, 2806, 2129, 2106, 2106, 1730, 1589. δ_H (400 MHz, DMSO-d₆) 1.25 (9H, s), 2.38–2.56 (5H, m), 2.57–2.65 (1H, m), 2.97 (1H, dd, J = 14.0, 7.2), 2.97 (1H, dd, J = 14.0, 8.2), 3.34–3.53 (2H, m), 3.68 (4H, t, J 4.6), 4.67 (1H, t, J 7.7), 7.04 (2H, d, J 8.7 ArH), 7.18–7.31 (5H, m, ArH), 7.73 (2H, d, J 8.7, ArH); δ_C (100.6 MHz, DMSO-d₆) 27.7, 37.3, 42.7, 53.9, 58.8, 61.7, 66.9, 82.2, 119.2, 126.9, 128.5, 129.2, 129.4, 136.3, 136.6, 144.6, 169.4. MS (ESI+): m/z = 516.1 [M + H]⁺.

(2R)-2-{[(4-Azidophenyl)sulfonyl][2-(morpholin-4-yl)ethyl]amino}-3-phenylpropanoic acid (17). To a mixture of 16 (0.82 g, 1.58 mmol) and thioanisole (3.34 mL, 28.45 mmol) in DCM (4 mL) at 0 °C was added TFA (3.65 mL, 47.4 mmol). The reaction was stirred at room temperature overnight and evaporated to dryness. The residue was purified by column chromatography on silica gel (DCM–MeOH 9 : 1) to afford 17 (580 mg, 80%) as a white foam, mp 160.2 °C (dec.). ν_max (KBr)/cm⁻¹ 3430, 2926, 2131, 2101, 1617, 1591. δ_H (400 MHz, DMSO-d₆) 2.70–3.05 (7H, m), 3.45–3.60 (3H, m), 3.60–3.80 (4H, m), 4.79 (1H, dd, J = 9.7, 3.9), 6.94 (2H, d, J 8.4 ArH), 7.12–7.23 (5H, m, ArH), 7.60 (2H, d, J 8.4, ArH); δ_C (100.6 MHz, DMSO-d₆) 36.9, 41.0, 52.4, 56.9, 62.9, 64.5, 119.1, 126.6, 128.5, 128.9, 129.3, 135.6, 137.8, 144.5, 174.7. MS (ESI+): m/z = 460.1 [M + H]⁺.

(2R)-2-{[(4-Azidophenyl)sulfonyl][2-(morpholin-4-yl)ethyl]amino}-N-(tetrahydro-2H-pyran-2-yl)-3-phenylpropanamide (18). To a solution of 17 (543 mg, 1.18 mmol) in DMF (5 mL) were added HOBt (192 mg, 1.42 mmol), NMM (0.39 mL, 3.55 mmol), O-tetrahydro-2H-pyran-2-yl-hydroxylamine (277 mg, 2.36 mmol) and EDCI (317 mg, 1.65 mmol). The reaction mixture was stirred overnight at room temperature and then diluted with AcOEt and washed successively with a saturated aqueous solution of NH₄Cl and brine. The organic layer was dried (MgSO₄), evaporated to dryness and the solid obtained was purified by column chromatography on silica gel (hexane–AcOEt 2 : 3) to afford 18 (600 mg, 91%). This compound is present as a diastereoisomeric a : b mixture (60 : 40), mp 59.8–61.5 °C. ν_max (KBr)/cm⁻¹ 3206, 2126, 2101, 1693, 1592; δ_H (400 MHz, CDCl₃) 1.50–1.65 (3H, m), 1.68–1.85 (3H, m), 2.35–2.47 (2H, m), 2.50–2.65 (4H, m), 2.65–2.85 (1H, m), 3.25–3.52 (2H, m), 3.54–3.82 (6H, m), 3.94–4.02 (0.6H, m, isomer a), 4.05–4.13 (0.4H, m, isomer b), 4.42–4.49 (0.4H, m, isomer b), 4.50–4.55 (0.6H, m, isomer a), 4.84 (0.6H, d, J 2.6, isomer a), 4.97 (0.4H, s, isomer b), 6.90–7.03 (4H, m, ArH), 7.08–7.18 (3H, m, ArH), 7.58 (1.2H, d, J 8.6, ArH, isomer a), 7.64 (0.8H, d, J 8.5, ArH, isomer b), 11.01 (0.4H, bs, CONH, isomer b), 11.13 (0.6H, bs, CONH, isomer a); δ_C (100.6 MHz, CDCl₃): 18.9 (isomer b), 19.5 (isomer a), 25.0, 28.1 (isomer b), 28.3 (isomer a), 34.4 (isomer a), 34.7 (isomer b), 41.6 (isomer a), 42.0 (isomer b), 53.5, 57.0 (isomer b), 57.2 (isomer a), 59.9 (isomer a), 60.0 (isomer b), 62.9 (isomer b), 63.4 (isomer a), 66.4 (isomer a), 66.5 (isomer b), 102.5 (isomer b), 103.3 (isomer a), 119.4, 126.7, 128.6, 128.8, 128.9, 135.4 (isomer a), 135.6 (isomer b), 136.7 (isomer a), 137.0 (isomer b), 144.7, 167.9 (isomer a), 168.1 (isomer b). MS (ESI) m/z 559.1 [M + H]⁺.

(2R)-2-[({4-[4-(Biphenyl-4-yl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)(2-[morpholin-4-yl]ethyl)amino]-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide (19). The product was prepared by General procedure 1 (preparation of triazoles), using 18 (150 mg, 0.269 mmol), 4-ethynylbiphenyl (57.4 mg, 0.322 mmol), sodium ascorbate (106 mg, 0.537 mmol) and copper(II) sulfate pentahydrate (33.5 mg, 0.134 mmol) in DMF (5 mL); 19 (145 mg, 73%) was produced as a white solid after chromatography purification (DCM–MeOH 1% MeOH). This compound is present as a diastereoisomeric a : b mixture (60 : 40), mp 196.1 °C (dec.). ν_max (KBr)/cm⁻¹ 3374, 2951, 2854, 2814, 1704, 1597; δ_H (400 MHz, CDCl₃) 1.50–1.65 (3H, m), 1.70–1.88 (3H, m), 2.40–2.54 (2H, m), 2.56–2.70 (4H, m), 2.75–2.85 (1H, m), 3.35–3.85 (8H, m), 3.94–4.02 (0.6H, m, isomer a), 4.05–4.13 (0.4H, m, isomer b), 4.50–4.59 (1H, m), 4.89 (0.6H, d, J 2.7, isomer a), 5.01 (0.4H, s, isomer b), 6.92–7.00 (2H, m, ArH), 7.04–7.08 (3H, m, ArH), 7.36 (1H, t, J 7.3, ArH), 7.45 (2H, t, J 7.5, ArH), 7.63 (2H, d, J 7.5, ArH), 7.62–7.78 (6H, m, ArH), 7.99 (2H, d, J 8.2, ArH), 8.34 (1H, s, triazole), 11.12 (0.4H, bs, CONH, isomer b), 11.24 (0.6H, bs, CONH, isomer a); δ_C (100.6 MHz, CDCl₃): 19.0 (isomer b), 19.6 (isomer a), 25.0, 28.2 (isomer b), 28.4 (isomer a), 34.4, 41.8 (isomer a), 42.2 (isomer b), 53.4, 57.0 (isomer b), 57.2 (isomer a), 60.4 (isomer a), 60.5 (isomer b), 63.0 (isomer b), 63.6 (isomer a), 66.5 (isomer a), 66.6 (isomer b), 102.8 (isomer b), 103.5 (isomer a), 114.1, 117.3 (isomer a), 117.4 (isomer b), 120.3, 123.6, 126.4, 126.9, 127.0, 127.7, 128.6, 128.7, 128.8, 128.9, 128.9, 129.0, 136.6 (isomer a), 136.9 (isomer b), 139.2, 139.3, 139.3, 139.7, 140.3, 140.5, 141.5, 148.7, 168.0 (isomer a), 168.2 (isomer b). MS (ESI) m/z 737.1 [M + H]⁺.

(2R)-2-[(2-[Morpholin-4-yl]ethyl)({4-[4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)amino]-3-phenyl-N-(tetrahydro-2H-pyran-2-yloxy)propanamide (20). The product was prepared by General procedure 1 (preparation of triazoles), using 18 (150 mg, 0.269 mmol), 4-ethynylbiphenyl (57.4 mg, 0.322 mmol), sodium ascorbate (106 mg, 0.537 mmol) and copper(II) sulfate pentahydrate (33.5 mg, 0.134 mmol) in DMF (5 mL); 20 (150 mg, 74%) was produced as a gray solid after chromatography purification (DCM–MeOH 1% MeOH). This compound is present as a diastereoisomeric a : b mixture (60 : 40), mp 81.5–82.9 °C. ν_max (KBr)/cm⁻¹ 2956, 1693, 1597; δ_H (400 MHz, CDCl₃) 1.50–1.65 (3H, m), 1.70–1.85 (3H, m), 2.44–2.52 (2H, m), 2.56–2.72 (4H, m), 2.75–2.86 (1H, m), 3.38–3.85 (8H, m), 3.97–4.03 (0.6H, m, isomer a), 4.10–4.15 (0.4H, m, isomer b), 4.47–4.59 (1H, m), 4.89 (0.6H, d, J 2.7, isomer a), 5.01 (0.4H, s, isomer b), 6.94–7.00 (2H, m, ArH), 7.04–7.11 (7H, m, ArH), 7.15 (1H, t, J 7.4, ArH), 7.33–7.40 (2H, m, ArH), 7.71–7.80 (4H, m, ArH), 7.89 (2H, d, J 8.5, ArH), 8.27 (1H, s, triazole), 11.12 (0.4H, bs, CONH, isomer b), 11.25 (0.6H, bs, CONH, isomer a); δ_C (100.6 MHz, CDCl₃): 19.0 (isomer b), 19.6 (isomer a), 25.0, 28.2 (isomer b), 28.4 (isomer a), 34.4 (isomer a), 34.6 (isomer b), 41.7 (isomer a), 42.2 (isomer b), 53.4, 57.0 (isomer b), 57.1 (isomer a), 60.4 (isomer a), 60.5 (isomer b), 63.1 (isomer b), 63.6 (isomer a), 66.5 (isomer a), 66.6 (isomer b), 102.8 (isomer b), 103.5 (isomer a), 114.1, 116.8 (isomer a), 116.9 (isomer b), 119.0, 119.3, 120.3, 123.8, 124.6, 126.9, 127.5, 128.7, 128.8, 128.8, 128.9, 128.9, 129.0, 136.5 (isomer a), 136.8 (isomer b), 139.1, 139.3, 139.7, 148.6, 156.6, 158.0 168.0 (isomer a), 168.2 (isomer b). MS (ESI) m/z 753.1 [M + H]⁺.

(2R)-2-[({4-[4-(Biphenyl-4-yl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)(2-[morpholin-4-yl]ethyl)amino]-N-hydroxy-3-phenylpropanamide (13). The product was prepared by General procedure 2 (cleavage of tetrahydropyrane protecting group) using 19 (125 mg, 0.17 mmol), MeOH (0.5 mL) and 4 M HCl–dioxane (2 mL). The crude was purified by chromatography (DCM–MeOH 4% MeOH) to obtain 13 (66.0 mg, 60%) as a white solid, mp 208 °C (dec.). (Found: C, 64.31; H, 5.68; N, 12.51; S, 4.67. C₂₉H₂₅N₅O₄S·H₂O requires: C, 64.40; H, 5.56; N, 12.87; S, 4.91%). ν_max (KBr)/cm⁻¹ 3470, 3129, 1668, 1622, 1597. δ_H (400 MHz, DMSO-d₆): 2.38–2.59 (6H, m, 3 × NCH₂), 2.80 (1H, dd, J = 13.9, 7.1, ½CH₂Ph), 2.97 (1H, dd, J = 14.0, 8.1, ½CH₂Ph), 3.40–3.46 (1H, m, ½SO₂NCH₂), 3.58 (4H, t, J 4.5, 2 × OCH₂), 3.63–3.74 (1H, m, ½SO₂NCH₂), 4.48 (1H, t, J 7.5, CH), 7.14–7.24 (5H, m, ArH), 7.41 (1H, t, J 7.3, ArH), 7.51 (2H, t, J 7.6, ArH), 7.76 (2H, d, J 7.5, ArH), 7.85 (2H, d, J 8.3, ArH), 8.03 (2H, d, J 8.8, ArH), 8.08 (2H, d, J 8.3, ArH), 8.14 (2H, d, J 8.7, ArH), 8.93 (1H, bs, OH), 9.54 (s, 1H, CH triazole), 10.96 (s, 1H, CONH); δ_C (100.6 MHz, DMSO-d₆) 36.0, 41.7, 53.33, 58.0, 58.3, 66.1, 119.8, 120.1, 125.9, 126.5, 126.6, 127.2, 127.7, 128.3, 129.0, 136.6, 139.0, 139.3, 139.4, 140.0, 147.3, 165.5. MS (ESI+): m/z 653.1 [M + H]⁺. [α]²⁵_D + 12.2 (c 0.0018 g cm⁻³ in DMSO).

(2R)-N-Hydroxy-2-[(2-[morpholin-4-yl]ethyl)({4-[4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl]phenyl}sulfonyl)amino]-3-phenylpropanamide (14). The product was prepared by General procedure 2 (cleavage of tetrahydropyrane protecting group) using 20 (130 mg, 0.17 mmol), MeOH (0.5 mL) and 4 M HCl–dioxane (2 mL). The crude was purified by chromatography (DCM–MeOH 4% MeOH) to obtain 14 (64.0 mg, 56%) as a gray solid, mp 131–133 °C. (Found: C, 64.31; H, 5.68; N, 12.51; S, 4.67. C₂₉H₂₅N₅O₄S·H₂O requires: C, 64.40; H, 5.56; N, 12.87; S, 4.91%). ν_max (KBr)/cm⁻¹ 3368, 3140, 1673, 1597. δ_H (400 MHz, DMSO-d₆): 2.33–2.60 (6H, m, 3 × NCH₂), 2.79 (1H, dd, J = 13.8, 7.1, ½CH₂Ph), 3.18 (1H, dd, J = 13.8, 7.8, ½CH₂Ph), 3.37–3.48 (1H, m, ½SO₂NCH₂), 3.58 (4H, t, J 4.5, 2 × OCH₂), 3.63–3.74 (1H, m, ½SO₂NCH₂), 4.47 (1H, t, J 7.5, CH), 7.09–7.24 (10H, m, ArH), 7.44 (1H, t, J 8.0, ArH), 7.97–8.02 (4H, m, ArH), 8.12 (2H, d, J 8.7, ArH), 8.93 (1H, bs, OH), 9.43 (s, 1H, CH triazole), 10.96 (s, 1H, CONH); δ_C (100.6 MHz, DMSO-d₆) 36.0, 41.7, 53.3, 58.0, 58.3, 66.1, 118.9, 119.0, 119.3, 120.1, 123.8, 125.1, 126.5, 127.2, 128.3, 128.9, 129.0, 130.1, 136.6, 139.0, 139.3, 147.2, 156.3, 156.9, 165.5. MS (ESI+): m/z 669.1 [M + H]⁺. [α]²⁵_D + 4.0 (c 0.0005 g cm⁻³ in DMSO).

Conclusions

We have used a click chemistry approach to synthesize a series of phenylalanine derived hydroxamates. The substitution pattern in the resulting triazole seems to be crucial to obtain a high degree of selectivity between both gelatinases. The best compound 1b displayed subnanomolar IC₅₀ against MMP-2 and was 95-fold less potent against MMP-9, while the closely structurally related compound 7 was devoid of selectivity. We have used docking and molecular dynamics tools to shed light on the reason for this difference in selectivity between 1b and 7. The difference in the energetic profile of the dihedral angle that describes the torsion around C_sp2–C_sp2 and C_sp2–N_sp2 induces a change in the dynamic behaviour of the P1′-segment that allows 7 to establish good interactions with both MMP-2 and MMP-9.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Torsional energies of the C_sp2–C_sp2 and C_sp2–N_sp2 scan; and per-residue energy decomposition of the four systems. ¹H NMR and ¹³C NMR spectra of compounds 1a–g, 7, 13 and 14. See DOI: 10.1039/c4ob01516a

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