Utilization of the 1, 2, 3, 5-Thiatriazolidin-3-one 1, 1-Dioxide Scaffold in the Design of Potential Inhibitors of Human Neutrophil Proteinase 3

Abstract

The S’ subsites of human neutrophil proteinase 3 (Pr 3) were probed by constructing diverse libraries of compounds based on the 1, 2, 3, 5-thiatriazolidin-3-one 1, 1-dioxide using combinational and click chemistry methods. The multiple points of diversity embodied in the heterocyclic scaffold render it well-suited to the exploration of the S’ subsites of Pr 3. Molecular modeling studies suggest that further exploration of the S’ subsites of Pr 3 using the aforementioned heterocyclic scaffold may lead to the identification of highly selective, reversible competitive inhibitors of Pr 3.

Introduction

Several lines of evidence suggest that proteases stored in the azurophilic granules of neutrophils, such as human neutrophil elastase (HNE) and proteinase 3 (Pr 3), play an important role in the pathophysiology of a range of inflammatory diseases, including chronic obstructive pulmonary disease (COPD),^1–5 cystic fibrosis,^6–8 Wegener’s granulomatosis,⁹ and others.^10–11 In addition to their ability to degrade elastin and other components of the extracellular matrix,^12–13 these endopeptidases play an important role in regulating chronic inflammation by modulating the activity of pro-inflammatory cytokines and chemokines.^14–15

COPD is characterized by an oxidant/antioxidant imbalance,^16–17 alveolar septal cell apoptosis,^18–19 chronic inflammation,^16,20 and a protease/antiprotease imbalance.^4,21 The molecular mechanisms which underlie the initiation and progression of the disorder are poorly understood. Furthermore, the precise role and actions of the proteases involved in COPD are not fully delineated, consequently there is a need for a better definition of which proteases and protease actions are of importance in COPD pathogenesis.²² Elucidation of the role these proteases play in COPD requires the availability of highly specific substrates and inhibitors.

Pr 3 and HNE share a high sequence homology (57%) and their primary specificity sites S1²³ are very similar, consequently, the design of covalent and non-covalent inhibitors that exhibit high specificity toward Pr 3 over HNE has been problematic.²⁴ We describe herein the results of exploratory studies related to the design and synthesis of potential non-covalent inhibitors of Pr 3 based on the 1, 2, 3, 5-thiatriazolidin 1, 1-dioxide scaffold that interact with and exploit key differences in the S’ subsites of the two enzymes.

Chemistry

The desired compounds were readily synthesized as shown in Scheme 1–Scheme 4. Heterocyclic template 1 was assembled in one step by condensing commercially available 1, 2-diethyl hydrazine dihydrochloride with N-chlorosulfonyl isocyanate in the presence of excess triethylamine (TEA) (Scheme 1). Treatment of the resulting 2,3-diethyl 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide intermediate 1 with TEA followed by the addition of t-butyl bromoacetate yielded the corresponding t-butyl ester which was readily deblocked and coupled to an array of structurally-diverse amines (Table 1) to yield compounds 4a–i (Scheme 2, Table 2). Mitsunobu reaction of intermediate 1 with (DL) 3-phenyl-2-hydroxy-propionic acid methyl ester²⁵ followed by hydrolysis afforded acid 6 which was coupled to a diverse set of amine inputs (Table 1) to give compounds 7a–e (Scheme 2, Table 2). Likewise, alkylation of 2,3-diethyl 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide (1) with N-Boc-m-aminobenzyl alcohol under Mitsunobu reaction conditions, followed by deprotection of the Boc group using TFA, yielded the corresponding aromatic amine which was further elaborated to yield compounds 10a–e (Scheme 2). Lastly, alkylation of 2, 3-diethyl 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide (1) with propargyl bromide followed by click chemistry²⁶ with a diverse set of azide inputs 12a–j (Scheme 3) gave compounds 13a–m (Scheme 2, compound 13d, 13k and 13m were generated from 13c, 13j and 13l, respectively). The desired azides 12a–h were readily prepared from the corresponding commercially-available halides²⁷ or halides prepared according to literature procedures²⁸ (Scheme 3, Scheme 4). The azide precursors of compounds 13k and 13m could not be prepared directly from the corresponding α-bromoacetyl compounds, consequently an alternative method was used. This involved α-bromination of an appropriate methyl ketone followed by reduction and treatment with base to form the corresponding epoxide (Scheme 4) which was sequentially subjected to ring opening (Scheme 3: 12i, 12j), click chemistry and oxidation with pyridinium chloro-chromate (PCC) (Scheme 2). The X-ray crystal structure of a representative member of this class of compounds (compound 13a) was determined (Figure 1).

Scheme 1.

Scheme 4.

Table 1.

Amine Inputs

Scheme 2.

Table 2.

Entry	R	R1
4a	H	phenyl
4b	H	benzyl
4c	H	phenethyl
4d	H	2,2–diphenylethyl
4e	H	2–(2–methoxyphenyl)ethyl
4f	H	4–morpholinophenyl
4g	H	benzo[d][1,3]dioxol–5–yl
4h	H	3–phenoxyphenyl
4i	H	2–morpholinoethyl
7a	bzl	2–(2–methoxyphenyl)ethyl
7b	bzl	4–morpholinophenyl
7c	bzl	benzo[d][1,3]dioxol–5–yl
7d	bzl	2–aminophenyl
7e	bzl	2-(isobutoxycarbonylamino)phenyl

^*Compounds 7a–e are DL isomers

Scheme 3.

Figure 1.

Mercury⁵³ drawing of compound 13a, showing the 50% thermal ellipsoids. Hydrogen atoms have been omitted for clarity.

Biochemical studies

Enzyme assays and inhibition studies using human proteinase 3 and neutrophil elastase were conducted as previously described.²⁹

Results and Discussion

Inhibitor Design Rationale

The biochemical rationale underlying the design of inhibitor (II) was based on the following considerations: (a) In previous studies we have demonstrated that the heterocyclic scaffold (I) (1,2,5-thiadiazolidin-3-one 1, 1-dioxide) (Figure 2) is a highly versatile peptidomimetic that embodies a structural motif that renders the platform capable of binding to the active site of HNE and related chymotrypsin-like serine proteases in a substrate-like fashion, orienting recognition elements R₁ and R₂ toward the S_n subsites and R₃ toward the S_n’ subsites.²⁴ Specifically, R₁ is accommodated at the primary substrate specificity subsite S₁ and its nature determines which subclass of serine proteases (neutral, basic, or acidic) will be inhibited, resulting in optimal enzyme selectivity;³⁰ (b) The encouraging results obtained with scaffold (I) suggested that it could serve as a prototype structure for the design of related scaffolds,^31–32 providing additional opportunities in terms of improving pharmacological and physicochemical properties. Thus, we reasoned that a functionalized surrogate scaffold (II) embellished with appropriate recognition elements could provide a structural framework for the rational design of non-covalent inhibitors of Pr 3 and related serine proteases. It was anticipated that the replacement of the α-carbon in scaffold (I) with nitrogen would decrease significantly the electrophilicity of the C=O carbon³³ and lead to a chemically robust ring system (II) that retained the substrate-like characteristics of (I) (Figure 2); (c) Pr 3 shows a strong preference for small aliphatic P₁ residues (ethyl, n-propyl)^34–36, while HNE prefers medium size P₁ alkyl groups (isopropyl/n-propyl or isobutyl/n-butyl). This is because the size of the S₁ subsite in Pr 3 is smaller due to the replacement of Val190 (HNE) by Ile (Pr 3).³⁷ Unlike HNE, S’-P’ interactions beyond S₁’ increase significantly the catalytic efficiency of Pr 3,³⁸ suggesting that S_n’ interactions play an important role in substrate specificity.^39–42 Furthermore, the substitution of Leu143 (HNE) by Arg143 (Pr 3) and the presence of Asp61 in Pr 3 make the S₁’-S₃’ subsites of Pr 3 more polar.³⁷

Figure 2.

Inhibitor (II) design rationale.

Based on the aforementioned considerations, as well as modeling studies using the X-ray crystal structure of Pr 3,³⁷ it was envisaged that an entity based on scaffold (II) with attached recognition elements that can potentially interact with the S_n’ subsites of the enzyme may function as non-covalent inhibitors of Pr 3. Thus, the S_n’ subsites of Pr 3 were initially explored via the construction of focused libraries based on (II). Based on the initial assumption that inhibitor (II) would bind to the active site of Pr 3 in the same orientation as inhibitor (I), a ring nitrogen substitution (ethyl) was chosen that is congruent with the primary substrate specificity (S1) of Pr 3 and a diversity of amides and polar substituted triazoles were initially utilized to probe the S' subsites. We speculated that a derivative of 4 having a lysine side chain on the α carbon could potentially provide a favorable ion-ion interaction with Asp 51 (see Figure 3 for Pr 3 active site), however, Mitsunobu reaction of 1 with the α-hydroxyester of Cbz-L-lysine failed to give the expected product. Fortuitously, the Mitsunobu reaction with the α-hydroxyester of (DL) Phe was successful and made possible the synthesis of a wide range of derivatives of 7 and their subsequent use in the exploration of the S2'-S3' subsites along with compound 4. The resulting compounds were either inactive or marginally active (selected examples are shown in Figure 4). Then the nature, polarity, and geometry of the inhibitor component projecting toward the S’ subsites were changed by utilizing meta-substituted phenyl derivatives having a carboxyl group that could potentially interact with the Arg 241 side chain located in the vicinity of the S2’ subsite of the enzyme. The inhibitory activity of compounds 10a–e was also disappointingly low.

Figure 3.

Compound 13g bound to Pr3. The structure was generated from molecular simulation. Ligand rendered as CPK-colored sticks. Receptor surface colors correspond to: yellow = nonpolar, white = polar alkyls, blue = polar N, cyan = polar H, red = O.

Figure 4.

Inhibitory activity of selected compounds against human neutrophil elastase and proteinase 3.

We then turned our attention to the use of click chemistry to generate a focused library of structurally-diverse electron-rich compounds having multiple sites capable of interacting with the S’ subsites of Pr 3. Molecular modeling studies using compound 13g suggested that it fits into the Pr 3 active site well and engages in multiple interactions with the enzyme, including the following: a) the phenyl ring binds to a hydrophobic pocket defined by Ile190, Phe192; b) the triazole ring appears to accept H-bonds from both the backbone Val216 NH and from the Lys99 side chain; c) the heterocyclic carbonyl O is well positioned to H-bond with the Lys99 side chain; d) one of the sulfamide O's is capable of H-bonding with the backbone NH of Ile217; e) the two ethyl groups interact with the second hydrophobic pocket, defined by side chains of Phe215, Ile217 and Trp218 (Figure 3; the docked structure is derived from computational studies). Quite unexpectedly, inhibitor 13g is predicted to bind to the active site in a reverse mode, namely, with the phenyl ring occupying the S1 pocket and the rest of the molecule projecting toward the S’ subsites. The molecular modeling studies showed that the ketone carbonyl of 13g is hydrogen bonded to the side chain hydroxyl of the catalytic Ser195 residue. We reasoned that the introduction of strongly electron-withdrawing substituents into the phenyl ring or replacement of the phenyl ring by a heteroaromatic 5-membered ring would enhance the electrophilicity of the carbonyl carbon, potentially transforming 13g into a transition state inhibitor of Pr 3 (Figure 5). Thus, structural variants 13h and 13k having a 2-fluorophenyl or a 2, 6-difluorophenyl group respectively, were synthesized. The phenyl ring was also replaced by a thiazole ring (compound 13m) for the same purpose⁴³. The few compounds generated by these modifications were also devoid of any inhibitory activity. Future studies involving the generation of a focused library of potential inhibitors of Pr 3 incorporating in their structure a triazole scaffold are currently in progress and may provide a fruitful avenue of investigation for the development of potent and selective inhibitors of Pr 3.

Figure 5.

Mechanism of transition state inhibitor.

Experimental

General

The ¹H spectra were recorded on a Varian XL-300 or XL-400 NMR spectrometer. A Hewlett–Packard diode array UV/VIS spectrophotometer was used in the in vitro evaluation of the inhibitors. Human neutrophil elastase, proteinase 3 and Boc-Ala-Ala-Nva thiobenzyl ester were purchased from Elastin Products Company, Owensville, MO. Methoxysuccinyl Ala-Ala-Pro-Val p-nitroanilide and 5,5’-dithio-bis(2-nitrobenzoic acid) were purchased from Sigma Chemicals, St. Louis, MO. Melting points were determined on a Mel-Temp apparatus and are uncorrected. Reagents and solvents were purchased from various chemical suppliers (Aldrich, Acros Organics, TCI America, and Bachem). Silica gel (230– 450 mesh) used for flash chromatography was purchased from Sorbent Technologies (Atlanta, GA). Thin layer chromatography was performed using Analtech silica gel plates. The TLC plates were visualized using iodine and/or UV light. The high resolution mass spectra were performed by the Mass Spectrometry Lab at the University of Kansas, Lawrence, KS.

Representative synthesis

2,3-Diethyl 1,2,3,5-thiatriazolidin-3-one 1, 1-dioxide 1

1, 2-Diethylhydrazine dihydrochloride (33.0 g; 187 mmol) suspended in dry methylene chloride (300 mL) was cooled in an ice-bath under N₂ and treated with triethylamine (76.0 g; 747 mmol). After stirring for 20 minutes, a solution of N-chlorosulfonyl isocyanate (27.0 g; 187 mmol) in dry methylene chloride (100 mL) was added dropwise. The reaction mixture was stirred overnight at room temperature. The solvent was removed and 6M HCl (150 mL) was added. The aqueous solution was extracted with ethyl acetate (3 × 200 mL) and the combined organic extracts were dried over anhydrous sodium sulfate. The drying agent was filtered and the solvent was removed, and then the crude product was purified by flash chromatography (silica gel/ ethyl acetate/ hexanes) to give compound 1 as a white solid (12.0 g; 33% yield), mp 64–66 °C. ¹H NMR (CDCl₃): δ 1.25–1.31 (m, 6H), 3.39–3.45 (q, 2H), 3.58–3.64 (q, 2H).

2,2-Diethyl-5-carboxymethyl-1,2,3,5-thiatriazolidin-3-one 1, 1-dioxide t-butyl ester 2

To a solution of compound 1 (14 g; 72.5 mmol) in dry N, N-dimethyl-formamide (70 mL) kept in an ice bath was added sodium hydride (60%, w/w; 4.5 g; 112 mmol) and the reaction mixture was stirred for 15 minutes before adding t-butyl bromoacetate (18.5 g; 92.7 mmol). The reaction mixture was refluxed overnight with stirring. The solvent was removed in vacuo and the crude product was purified by flash chromatography (silica gel/ ethyl acetate/ hexanes) to give compound 2 as a colorless oil (17.7g; 79% yield). ¹H NMR (CDCl3): δ 1.22–1.28 (t, 3H), 1.28–1.34 (t, 3H), 1.47 (s, 9H), 3.39–3.44 (q, 2H), 3.60–3.65 (q, 2H), 4.10 (s, 2H).

2,2-Diethyl-5-carboxymethyl-1,2,3,5-thiatriazolidin-3-one 1, 1-dioxide 3

To a solution of compound 2 (3.34 g; 10.9 mmol) in dry methylene chloride (3 mL) was added trifluoroacetic acid (15 mL) and the reaction was stirred at room temperature for 1 h. The solvent and trifluoroacetic acid were removed and the residue was re-dissolved in ethyl acetate (200 mL). The organic solution was washed with saturated sodium bicarbonate (3 × 40 mL) and then brine (40 mL). The organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered and the solvent was removed to give compound 3 as colorless oil (2.74 g; 100% yield). ¹H NMR (CDCl₃): δ 1.22–1.32 (m, 6H), 3.38–3.43 (q, 2H), 3.60–3.65 (q, 2H), 4.25 (s, 2H).

General coupling procedure for preparation of compounds 4a–i

A solution of acid 3 (2.4 mmol) in dry N, N-dimethylform-amide (5 mL) was treated with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.51g; 2.64 mmol), followed by the addition of amine (2.4 mmol) from Table 1. The reaction mixture was stirred at room temperature overnight. The solvent was removed in vacuo and the residue was taken up with ethyl acetate (30 mL). The organic layer was washed sequentially with 5% HCl (3×10 mL), saturated NaHCO₃ (3×0 mL), and then brine (10 mL). The organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered and the solvent removed on the rotary evaporator. The crude product was purified by flash chromatography (silica gel/ ethyl acetate/ hexanes) to give pure amide product (4a–i).

Compound 4a

White solid (52% yield), mp 108–110 °C. ¹H NMR (CDCl₃): δ 1.22–1.28 (t, 3H), 1.29–1.35 (t, 3H), 3.42–3.48 (q, 2H), 3.62–3.68 (q, 2H), 4.25 (s, 2H), 7.10–7.50 (m, 5H), 8.08 (s, 1H); HRMS (ESI) calculated for C₁₃H₁₈N₄O₄SNa [M+Na]⁺ 349.0946, found 349.0936.

Compound 4b

White solid (53% yield), mp 62–63 °C. ¹H NMR (CDCl₃): δ 1.18–1.25 (m, 6H), 3.34–3.42 (q, 2H), 3.58–3.66 (q, 2H), 4.20 (s, 2H), 4.46–4.50 (d, 2H), 6.28 (s, 1H), 7.22–7.36 (m, 5H); HRMS (ESI) calculated for C₁₄H₂₁N₄O₄S [M+H]⁺ 341.1284, found 341.1295; C₁₄H₂₀N₄O₄SNa [M+Na]⁺ 363.1103, found 363.1090.

Compound 4c

White solid (20% yield), mp 91–92 °C. ¹H NMR (CDCl₃): δ 1.18–1.25 (m, 6H), 2.80–2.85 (t, 2H), 3.22–3.28 (q, 2H), 3.56–3.63 (m, 4H), 4.10 (s, 2H), 5.98 (s, 1H), 7.18–7.32 (m, 5H); HRMS (ESI) calculated for C₁₅H₂₃N₄O₄S [M+H]⁺ 355.1440, found 355.1451; C₁₅H₂₂N₄O₄SNa [M+Na]⁺ 377.1259, found 377.1255.

Compound 4d

Oil (51% yield). ¹H NMR (CDCl₃): δ 1.10–1.18 (m, 6H), 3.02–3.08 (q, 2H), 3.52–3.58 (q, 2H), 3.92–3.96 (m, 2H), 4.08 (s, 2H), 4.10–4.18 (m, 1H), 5.96 (s, 1H), 7.20–7.32 (m, 10H); HRMS (ESI) calculated for C₂₁H₂₆N₄O₄SNa [M+Na]⁺ 453.1572, found 453.1575.

Compound 4e

Oil (39% yield). ¹H NMR (CDCl₃): δ 1.20–1.26 (m, 6H), 2.80–2.85 (t, 2H), 3.28–3.33 (q, 2H), 3.50–3.58 (q, 2H), 3.58–3.66 (q, 2H), 3.80 (s, 3H), 4.06 (s, 2H), 6.20 (s, 1H), 6.82–6.88 (m, 2H), 7.08–7.11 (m, 1H), 7.17–7.21(m, 1H); HRMS (ESI) calculated for C₁₆H₂₄N₄O₅SNa [M+Na]⁺ 407.1365, found 407.1355.

Compound 4f

Yellow solid (32% yield), mp 135–137 °C. ¹H NMR (CDCl₃): δ 1.22–1.36 (m, 6H), 3.08–3.12 (t, 4H), 3.40–3.48 (q, 2H), 3.52–3.60 (q, 2H), 3.82–3.86 (t, 4H), 4.24 (s, 2H), 6.82–6.86 (d, 2H), 7.34–7.38 (d, 2H), 7.72 (s, 1H); HRMS (ESI) calculated for C₁₇H₂₆N₅O₅S [M+H]⁺ 412.1655, found 412.1643.

Compound 4g

Brown solid (35% yield), mp 83–85 °C. ¹H NMR (CDCl₃): δ 1.22–1.36 (m, 6H), 3.40–3.46 (q, 2H), 3.52–3.58 (q, 2H), 4.10 (s, 2H), 5.90 (s, 2H), 6.65–6.78 (dd, 2H), 7.10 (s, 1H), 8.22 (s, 1H); HRMS (ESI) calculated for C₁₄H₁₉N₄O₆S [M+H]⁺ 371.1025, found 371.0984; C₁₄H₁₈N₄O₆SNa [M+Na]⁺ 393.0845, found 393.0847.

Compound 4h

Oil (62% yield). ¹H NMR (CDCl₃): δ 1.22–1.36 (m, 6H), 3.42–3.48 (q, 2H), 3.62–3.68 (q, 2H), 4.25 (s, 2H), 6.74–7.37 (m, 9H), 7.95 (s, 1H); HRMS (ESI) calculated for C₁₉H₂₂N₄O₅SNa [M+Na]⁺ 441.1209, found 441.1206.

Compound 4i

Oil (45% yield). ¹H NMR (CDCl₃): δ 1.23–1.30 (t, 3H), 1.30–1.37 (t, 3H), 2.42–2.52 (m, 6H), 3.37–3.46 (m, 4H), 3.63–3.74 (m, 6H), 4.18 (s, 2H), 6.61 (s, 1H); HRMS (ESI) calculated for C₁₃H₂₆N₅O₅S [M+H]⁺ 364.1655, found 364.1635; C₁₃H₂₅N₅O₅SNa [M+Na]⁺ 386.1474, found 386.1490.

(DL) Methyl 2-(2,3-diethyl-1,1-dioxido-4-oxo-1,2,3,5-thiatriazolidin-5-yl)-3-phenylpropanoate 5

To a stirred solution of compound 1 (9.95 g; 55.2 mmol) and (DL) 3-phenyl-2-hydroxy propanoic acid methyl ester (10.6 g; 55.2 mmol) in dry tetrahydrofuran was added triphenyl phosphine (28.96 g; 110.4 mmol), followed by the dropwise addition of a solution of diethyl azodicarboxylate (97%; 19.9 g; 110.4 mmol) in dry tetrahydrofuran (30 mL). The reaction was stirred at room temperature overnight and the resulting precipitate was filtered off. The filtrate was evaporated to give the crude product which was purified by flash chromatography (silica gel/ ethyl acetate/ hexanes) to give compound 5 as a colorless oil (3.2 g; 16% yield). ¹H NMR (CDCl₃): δ 1.00–1.06 (t, 3H), 1.16–1.22 (t, 3H), 2.82–3.08 (m, 2H), 3.40–3.75 (m, 4H), 3.80 (s, 3H), 4.63–4.70 (m, 1H), 7.20–7.32 (m, 5H).

(DL) 2-(2,3-diethyl-1,1-dioxido-4-oxo-1,2,3,5-thiatriazolidin-5-yl)-3-phenyl-propanoic acid 6

A solution of compound 5 (3.74 g; 10.5 mmol) in 1,4-dioxane (40 mL) was treated with 17.5 mL 6N potassium hydroxide solution at room temperature for 0.5 hr. The pH was adjusted to 7 by the addition of 5% hydrochloride solution and then the solvent was removed. The residue was acidified to pH 2 and extracted with ethyl acetate (3 × 50 mL). The combined organic extracts were dried over anhydrous sodium sulfate. The drying agent was filtered and the solvent was removed. The crude product was purified by flash chromatography (silica gel/ ethyl acetate/ hexanes) to give compound 6 as a colorless oil (2.4 g; 59% yield). ¹H NMR (CDCl₃): δ 1.00–1.06 (t, 3H), 1.16–1.22 (t, 3H), 2.76–2.86 (m, 1H), 2.94–3.04 (m, 1H), 3.39–3.75 (m, 4H), 4.65–4.73 (m, 1H), 7.20–7.32 (m, 5H).

Synthesis of amides 7a–e

The same coupling procedures were used as described above.

Compound 7a

Oil (15% yield). ¹H NMR (CDCl₃): δ 0.92–0.98 (t, 3H), 1.12–1.18 (t, 3H), 2.50–2.61 (m, 1H), 2.72–2.84 (m, 3H), 3.36–3.75 (m, 6H), 3.81 (s, 3H), 4.47–4.55 (m,1H), 6.50 (s, 1H), 6.80–7.30 (m, 9H); HRMS (ESI) calculated for C₂₃H₃₀N₄O₅SNa [M+Na]⁺ 497.1835, found 497.1835.

Compound 7b

White solid (23% yield), mp 155–156 °C. ¹H NMR (CDCl₃): δ 0.92–0.98 (t, 3H), 1.12–1.18 (t, 3H), 2.60–2.68 (m, 1H), 2.85–2.94 (m, 1H), 3.08–3.12 (m, 4H),3.38–3.78 (m, 4H), 3.91–3.95 (m, 4H), 4.62–4.68 (m,1H), 6.81–6.85 (d, 2H), 7.20–7.35 (m, 5H), 7.36–7.40 (d, 2H), 8.25 (s, 1H); HRMS (ESI) calculated for C₂₄H₃₂N₅O₅S [M+H]⁺ 502.2124, found 502.2101; C₂₄H₃₁N₅O₅SNa [M+Na]⁺ 524.1944, found 524.1939.

Compound 7c

Brown solid (14% yield), mp 100–101 °C. ¹H NMR (CDCl₃): δ 0.94–1.00 (t, 3H), 1.16–1.20 (t, 3H), 2.60–2.68 (m, 1H), 2.86–2.94 (m, 1H), 3.38–3.77 (m, 4H), 4.62–4.68 (m,1H), 5.93 (s, 2H), 6.68–6.78 (m, 2H), 7.18–7.30 (m, 6H), 8.32 (s, 1H); HRMS (ESI) calculated for C₂₁H₂₄N₄O₆SNa [M+Na]⁺ 483.1314, found 483.1306.

Compound 7d

White solid (33% yield), mp 128–130 °C. ¹H NMR (CDCl₃): δ 0.94–1.00 (t, 3H), 1.16–1.20 (t, 3H), 2.60–2.75 (m, 1H), 2.85–3.00 (m, 1H), 3.37–3.80 (m, 4H), 3.78 (bs, 2H), 4.69–4.77 (m, 1H), 6.68–7.34 (m, 9H), 7.90 (s, 1H); HRMS (ESI) calculated for C₂₀H₂₆N₅O₄S [M+H]⁺ 432.1706, found 432.1698; C₂₀H₂₅N₅O₄SNa [M+Na]⁺ 454.1525, found 454.1506.

Compound 7e

White solid (8% yield), mp 113–115 °C. ¹H NMR (CDCl₃): δ 0.94–1.02 (m, 9H), 1.16–1.20 (t, 3H), 1.90–2.01 (m, 1H), 2.69–2.80 (m, 1H), 2.89–3.00 (m, 1H), 3.40–3.80 (m, 4H), 3.92–3.96 (d, 2H), 4.68–4.74 (m, 1H), 7.00 (s, 1H), 7.04–7.32 (m, 9H), 8.20 (s, 1H); HRMS (ESI) calculated for C₂₅H₃₃N₅O₆SNa [M+Na]⁺ 554.2049, found 554.2053.

Synthesis of compound 8

To a stirred solution of compound 1a (0.77 g; 4 mmol) and 3-N-Boc amino benzyl alcohol (0.90 g; 4 mmol) in dry tetrahydrofuran (10 mL) was added triphenyl phosphine (2.12 g; 8 mmol) followed by the dropwise addition of a solution of diethyl azodicarboxylate (1.44 g; 8 mmol) in dry tetrahydrofuran (5 mL). The reaction was stirred at room temperature overnight. The solvent was removed under vacuum and the residue was taken up with ethyl acetate (40 mL), and then washed with water (20 mL). The organic layer was dried over anhydrous sodium sulfate. The solvent was evaporated and the crude product was purified by flash chromatography (silica gel/ ethyl acetate/ hexanes) to give compound 8 as colorless oil (0.49 g; 30% yield). ¹H NMR (CDCl₃): δ 1.12–1.30 (m, 6H), 1.50 (s, 9H), 3.25–3.40 (q, 2H), 3.50–3.65 (q, 2H), 4.60 (s, 2H), 6.72 (s, 1H), 6.98–7.40 (m, 4H).

Synthesis of compound 9

To a solution of compound 8 (0.47 g; 1.18 mmol) was added trifluoroacetic acid (5 mL) and the reaction was stirred at room temperature for 1 h. Trifluoroacetic acid was removed under vacuum and the residue was redissolved in 20 mL methylene chloride. The solvent was removed and the crude product was purified by flash chromatography (silica gel/ ethyl acetate/ hexanes) to give compound 9 as a colorless oil (0.30 g; 85% yield). ¹H NMR (CDCl₃): δ 1.16–1.24 (m, 6H), 3.26–3.36 (q, 2H), 3.54–3.64 (q, 2H), 4.58 (s, 2H), 5.25 (bs, 2H), 6.98–7.30 (m, 4H).

Synthesis of compounds 10a–b

A solution of free amine 12 (0.4 mmol) in dry methylene chloride (3 mL) kept in an ice-bath was treated with phthalic or succinic anhydride (0.4 mmol) and then the reaction mixture was refluxed for 1 h. The solvent was removed and the crude product was purified by flash chromatography (silica gel/ ethyl acetate/ hexanes) to give the corresponding product (10a–b).

Compound 10a

White solid (70% yield), mp 134–136 °C. ¹H NMR (DMSO-D₆): δ 1.10–1.20 (m, 6H), 3.32–3.40 (q, 2H), 3.55–3.63 (q, 2H), 4.64 (s, 2H), 7.02–7.70 (m, 7H), 7.72 (s, 1H), 7.88–7.90 (m, 1H), 10.40 (s, 1H); HRMS (ESI) calculated for C₂₀H₂₂N₄O₆SNa [M+Na]⁺ 469.1158, found 469.1161.

Compound 10b

White solid (85% yield), mp 109–111 °C. ¹H NMR (DMSO-D₆): δ 1.18–1.26 (m, 6H), 2.64–2.72 (t, 2H), 2.72–2.80 (t, 2H), 3.31–3.37 (q, 2H), 3.58–3.64 (q, 2H), 4.60 (s, 2H), 7.08–7.62 (m, 4H), 7.95 (s, 1H); HRMS (ESI) calculated for C₁₆H₂₂N₄O₆SNa [M+Na]⁺ 421.1158, found 421.1139.

Compound 10c

A solution of compound 9 (0.37 g; 0.81 mmol) and phenethyl isocyanate (0.18 g; 1.21 mmol) in dry methylene chloride (3 mL) was refluxed for 1 h. The solvent was removed under vacuum and ethyl acetate (80 mL) was added. The organic layer was washed with 5% HCl (20 mL) and dried over anhydrous sodium sulfate. The drying agent was filtered and the solvent was removed. The crude product was purified by flash chromatography (silica gel/ ethyl acetate/ hexanes) to give compound 10c as colorless oil (0.15 g; 42% yield). ¹H NMR (CDCl₃): δ 1.16–1.24 (m, 6H), 2.75–2.80 (t, 2H), 3.26–3.34 (q, 2H), 3.42–3.48 (q, 2H), 3.56–3.62 (q, 2H), 4.55 (s, 2H), 5.23 (s, 1H), 7.00–7.28 (m, 10H); HRMS (ESI) calculated for C₂₁H₂₇N₅O₄SNa [M+Na]⁺ 468.1681, found 468.1679.

Compounds 10d–e

Amides 13d–e were prepared from 12 and appropriate acids using a same coupling procedure described above.

Compound 10d

Oil (76% yield). ¹H NMR (CDCl₃): δ 1.20–1.30 (m, 6H), 3.30–3.38 (q, 2H), 3.57–3.65 (q, 2H), 4.80 (s, 2H), 7.12–7.85 (m, 9H), 8.00 (s, 1H); HRMS (ESI) calculated for C₁₉H₂₂N₄O₄SNa [M+Na]⁺ 425.1259, found 425.1272.

Compound 10e

Oil (10% yield). ¹H NMR (CDCl₃): δ 1.20–1.28 (m, 6H), 3.30–3.38 (q, 2H), 3.57–3.65 (q, 2H), 4.60 (s, 2H), 7.00–7.64 (m, 13H), 7.86(s, 1H); HRMS (ESI) calculated for C₂₅H₂₆N₄O₅SNa [M+Na]⁺ 517.1522, found 517.1527.

Compound 11

Compound 1 (1.93 g; 10 mmol) was dissolved in 20 mL dry acetonitrile and triethylamine (1.01g; 10 mmol) was added, followed by propargyl bromide (1.19 g; 10 mmol). The resulting mixture was refluxed overnight. The solvent was removed, and the residue was dissolved in 40 mL ethyl acetate. The organic solution was washed with 5% HCl (3 × 20 mL), 5% saturated NaHCO₃ (3 × 20 mL) and then brine (20 mL). The organic layer was dried over anhydrous sodium sulfate. The drying agent was filtered off and the filtrate was evaporated under vacuum to give compound 11 as a yellow oil (1.26 g; 54% yield). ¹H NMR (CDCl₃): δ 1.20–1.32 (m, 6H), 2.38–2.40 (t, 1H), 3.36–3.42 (q, 2H), 3.60–3.66(q, 2H), 4.26–4.29 (d, 2H).

General procedure for preparation of azide 12a–h

To a solution of NaN₃ (1.44 g; 22 mmol) in 20 mL DMSO was added appropriate commercial or self-prepared halide (20 mmol), and the reaction was stirred at room temperature for overnight. The reaction mixture was added 70 mL H₂O under a water bath, and then extracted with 3×50 mL of diethyl ether. The combined organic layers were washed with 2×50 mL brine and dried over anhydrous sodium sulfate. Removal of solvent gave a pure product.

Compound 12a

Oil (100% yield). ¹H NMR (CDCl₃): 4.35 (s, 2H), 7.20–7.38 (m, 4H).

Compound 12b

Oil (98% yield). ¹H NMR (CDCl₃): 4.56 (s, 2H), 7.29–7.56 (m, 5H).

Compound 12c

Oil (99% yield). ¹H NMR (CDCl₃): 1.51 (s, 9H), 3.77 (s, 2H).

Compound 12d

Oil (90% yield). ¹H NMR (CDCl₃): 4.40 (s, 2H), 7.33–7.39 (m, 1H), 7.67–7.82 (m, 1H), 8.53–8.63 (m, 2H).

Compound 12e

Oil (98% yield). ¹H NMR (CDCl₃): 3.81 (s, 3H), 4.25 (s, 2H), 6.88–6.94 (d, 2H), 7.23–7.29 (d, 2H).

Compound 12f

Oil (95% yield). ¹H NMR (CDCl₃): 4.58 (s, 2H), 7.45–7.68 (m, 3H), 7.92–7.96 (m, 2H).

Compound 12g

Oil (92% yield). ¹H NMR (CDCl₃): 4.52 (s, 2H), 7.10–7.30 (m, 2H), 7.57–7.64 (m, 1H), 7.97–8.02 (m, 1H).

Compound 12h

Oil (76% yield). ¹H NMR (CDCl₃): 0.90–1.00 (t, 3H), 1.62–1.70 (q, 2H), 2.39–2.47 (t, 2H), 3.93 (s, 2H).

Azides 12i–j were prepared using the following procedure⁴⁴: Epoxide 15 or 17 (2.63 mmol) was dissolved in 15 mL methanol and then sodium azide (0.60 g; 9 mmol) and ammonium chloride (0.36 g; 6.6 mmol) were added. The resulting mixture was refluxed for 15 h. The solvent was removed and 15 mL of water was added. The aqueous solution was extracted with 2 × 15 mL ethyl acetate, and the combined organic layers were dried over anhydrous sodium sulfate. The drying agent was filtered and the filtrate was pumped to dryness, yielding a pure product.

Compound 12i

Oil (96% yield). ¹H NMR (CDCl₃): 3.49–3.55 (m, 1H), 3.77–3.85 (m, 3H), 5.09–5.15 (m, 1H), 6.89–7.00 (m, 2H), 7.24–7.40 (m, 1H).

Compound 12j

Oil (64% yield). ¹H NMR (CDCl₃): 3.63–3.80 (m, 2H), 4.72 (bs, 1H), 5.17–5.21 (m, 1H), 7.37–7.39 (d, 1H), 7.76–7.78 (d, 1H).

General alkyne-azide cycloaddition of compounds 13a–c, 13e–j and 13l

To a solution of 11 (1.8 mmol) and appropriate azide (1.8 mmol) in 5 mL of 1:1 t-BuOH and H₂O solution were added sodium ascorbate (0.04 g; 0.18 mmol) and copper sulfate pentahydrate (0.005 g; 0.018 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was removed and the residue was taken up with ethyl acetate (30 mL) (any insoluble impurities were filtered off). The organic solution was washed with water (2 × 20 mL) and then dried over anhydrous sodium sulfate. The drying agent was filtered and the solvent was removed. The residue was purified by flash chromatography (silica gel/ ethyl acetate/ hexanes) to give pure compounds 13a–c, 13e–j and 13l.

Compound 13a

White solid (75% yield), mp 83–84 °C. ¹H NMR (CDCl₃): 1.18–1.22 (t, 6H), 3.28–3.36 (q, 2H), 3.55–3.63 (q, 2H), 4.80 (s, 2H), 5.52 (s, 2H), 7.09–7.14 (t, 1H), 7.21 (s, 1H), 7.28–7.30 (d, 2H), 7.70 (s, 1H); HRMS (ESI) calculated for C₁₅H₂₀ClN₆O₃S [M+H]⁺ 399.1006, found 399.1016; C₁₅H₁₉ClN₆O₃SNa [M+Na]⁺ 421.0826, found 421.0824.

Compound 13b

Oil (63% yield). ¹H NMR (CDCl₃): 1.20–1.25 (t, 6H), 3.30–3.38 (q, 2H), 3.58–3.66 (q, 2H), 4.80 (s, 2H), 5.60 (s, 2H), 7.28 (s, 5H), 7.64 (s, 1H); HRMS (ESI) calculated for C₁₅H₂₁N₆O₃S₂ [M+H]⁺ 397.1117, found 397.1137; C₁₅H₂₀N₆O₃S₂Na [M+Na]⁺ 419.0936, found 419.0932.

Compound 13c

White solid (52% yield), mp 96–97 °C. ¹H NMR (CDCl₃): 1.20–1.24 (t, 6H), 1.46 (s, 9H), 3.30–3.38 (q, 2H), 3.58–3.66 (q, 2H), 4.82 (s, 2H), 5.06 (s, 2H), 7.78 (s, 1H); HRMS (ESI) calculated for C₁₄H₂₄N₆O₅SNa [M+Na]⁺ 411.1427, found 411.1440.

Compound 13e

White solid (63% yield), mp 85–86 °C. ¹H NMR (CDCl₃): 1.20–1.24 (m, 6H), 3.30–3.40 (q, 2H), 3.55–3.65 (q, 2H), 4.80 (s, 2H), 5.59 (s, 2H), 7.29–7.34 (m, 1H), 7.55–7.59 (m, 1H), 7.63 (s, 1H), 8.59–8.62 (m, 2H); HRMS (ESI) calculated for C₁₄H₂₀N₇O₃S [M+H]⁺ 366.1348, found 366.1354.

Compound 13f

White solid (66% yield), mp 71–72 °C. ¹H NMR (CDCl₃): 1.18–1.22 (t, 6H), 3.28–3.36 (q, 2H), 3.55–3.63 (q, 2H), 3.80 (s, 3H), 4.78 (s, 2H), 5.43 (s, 2H), 6.85–6.89 (d, 2H), 7.18–7.22 (d, 2H), 7.51 (s, 1H); HRMS (ESI) calculated for C₁₆H₂₂N₆O₄SNa [M+Na]⁺ 417.1321, found 417.1321.

Compound 13g

White solid (86% yield), mp 107–109 °C. ¹H NMR (CDCl₃): 1.20–1.26 (t, 6H), 3.34–3.40 (q, 2H), 3.60–3.66 (q, 2H), 4.86 (s, 2H), 5.85 (s, 2H), 7.52–7.60 (t, 2H), 7.64–7.72 (t, 1H), 7.82 (s, 1H), 7.98–8.02 (d, 2H); HRMS (ESI) calculated for C₁₆H₂₀N₆O₄SNa [M+Na]⁺ 415.1164, found 415.1164.

Compound 13h

Oil (34% yield). ¹H NMR (CDCl₃): 1.20–1.28 (t, 6H), 3.32–3.39 (q, 2H), 3.58–3.65 (q, 2H), 4.85 (s, 2H), 5.79–5.83 (d, 2H), 7.20–7.37 (m, 2H), 7.62–7.70 (m, 1H), 7.80 (s, 1H), 7.95–8.02 (m, 1H); HRMS (ESI) calculated for C₁₆H₁₉FN₆O₄SNa [M+Na]⁺ 433.1070, found 433.1049.

Compound 13i

Oil (76% yield). ¹H NMR (CDCl₃): 0.92–0.98 (t, 3H), 1.20–1.25 (t, 6H), 1.60–1.74 (m, 2H), 2.43–2.49 (t, 2H), 3.32–3.40 (q, 2H), 3.59–3.67 (q, 2H), 4.93 (s, 2H), 5.20 (s, 2H), 7.73 (s, 1H); HRMS (ESI) calculated for C₁₃H₂₂N₆O₄SNa [M+Na]⁺ 381.1321, found 381.1327.

Compound 13j

White solid (77% yield), mp 127–129 °C. ¹H NMR (CDCl₃): 1.16–1.22 (t, 6H), 3.28–3.35 (q, 2H), 3.55–3.62 (q, 2H), 4.16–4.22 (m, 1H), 4.68–4.74 (m, 1H), 4.77 (s, 2H), 6.06–6.13 (m, 1H), 6.89–6.97 (t, 2H), 7.28–7.40 (m, 1H), 7.73 (s, 1H); HRMS (ESI) calculated for C₁₆H₂₀F₂N₆O₄SNa [M+Na]⁺ 435.1133, found 453.1134.

Compound 13l

Oil (77% yield). ¹H NMR (CDCl₃): 1.19–1.25 (t, 6H), 3.28–3.36 (q, 2H), 3.55–3.63 (q, 2H), 4.60–4.71 (m, 1H), 4.75 (s, 2H), 4.96–5.04 (m, 1H), 5.43–5.51 (m, 1H), 5.56–5.61 (d, 1H), 7.34–7.36 (d, 1H), 7.73–7.76 (d, 2H); HRMS (ESI) calculated for C₁₃H₁₉N₇O₄S₂Na [M+Na]⁺ 424.0838, found 424.0827.

Compound 13k, 13m

To a suspension of pyridinium chlorochromate (PCC, 0.53g; 2.5 mmol) in 5 mL methylene chloride was added compound 13j or 13l (1mmol) and the reaction was stirred at room temperature for 3 days. The supernatant was decanted and the gummy residue was washed with 10 mL of methylene chloride. The combined organic solutions were concentrated and the residue was purified using flash chromatography (silica gel/ ethyl acetate/ hexanes) to give pure compound 13k or 13m.

Compound 13k

Oil (28% yield). ¹H NMR (CDCl₃): 1.20–1.26 (t, 6H), 3.32–3.40 (q, 2H), 3.59–3.67 (q, 2H), 4.86 (s, 2H), 5.70 (s, 2H), 7.01–7.08 (t, 2H), 7.51–7.60 (m, 1H), 7.81 (s, 1H); HRMS (ESI) calculated for C₁₆H₁₈F₂N₆O₄SNa [M+Na]⁺ 451.0976, found 451.0964.

Compound 13m

Oil (8% yield). ¹H NMR (CDCl₃): 1.20–1.26 (t, 6H), 3.32–3.40 (q, 2H), 3.59–3.67 (q, 2H), 4.87 (s, 2H), 6.03 (s, 2H), 7.82–7.84 (m, 2H), 8.15–8.15 (d, 1H); HRMS (ESI) calculated for C₁₃H₁₇N₇O₄S₂Na [M+Na]⁺ 422.0681, found 422.0686.

Compound 13d

Compound 13c (0.11g; 0.28 mmol) was added 3 mL 4M HCl in dry 1,4-dioxane, and the reaction was stirred at room temperature for 2 h. The solvent was removed and the residue was washed with 1 mL chloroform, leaving the pure product 13d as a white solid (0.09 g; 97% yield), mp 145–147 °C. ¹H NMR (CD₃OD): 1.18–1.22 (m, 6H), 3.30–3.40 (q, 2H), 3.60–3.70 (q, 2H), 4.80 (s, 2H), 5.26 (s, 2H), 8.03 (s, 1H); HRMS (ESI) calculated for C₁₀H₁₆N₆O₅SNa [M+Na]⁺ 355.0801, found 355.0802.

Synthesis of compound 14

Copper (II) bromide (4.46 g; 20 mmol) was ground and placed into a RB flask, ethyl acetate (10 mL) was added and the mixture was brought to boiling. 2’, 6’-difluoroacetylphenone (1.72 g; 11 mmol) in 10 mL chloroform was added through the top of the condenser. The reaction started immediately and the mixture was refluxed for 30 min whereupon a large amount of amber precipitate formed and gas release ceased. The reaction mixture was cooled down, 10 mL H₂O was added and stirred for 1 min. The precipitate was filtered and the filtrate was extracted with 30 mL ethyl acetate. The organic layer was washed with brine (2 × 20 mL) and dried over anhydrous sodium sulfate. The drying agent was filtered off and the filtrate was concentrated to dryness to give compound 14 as a colorless oil (2.35g; 100% yield). ¹H NMR (CDCl₃): 4.39 (s, 2H), 6.95–7.05 (m, 2H), 7.42–7.55 (m, 1H).

Synthesis of compound 15

Compound 14 (2.35 g; 10 mmol) was dissolved in 30 mL methanol and sodium borohydride (0.40 g; 10 mmol) was added at 0 °C in small portions. The reaction was then stirred for 1 h at 0 °C. Two milliliters of 12 N sulfuric acid and 10 mL water were added and the reaction was stirred for 2 min before the solvent was removed. The residue was dissolved in 40 mL ethyl acetate, and the organic solution was washed with 30 mL water. The organic layer was separated and dried over anhydrous sodium sulfate. The drying agent was filtered and the filtrate was pumped to dryness. The residue was dissolved in 10 mL DMF, and solid potassium carbonate (3.45 g; 25 mmol) was added. The resulting mixture was stirred at room temperature overnight. Water (15 mL) was added and the solution was extracted ethyl acetate (2 × 40 mL). The combined organic layers were washed with brine (2 × 30 mL) and dried over anhydrous sodium sulfate. The drying agent was filtered off and the filtrate was concentrated to give pure compound 15 (0.82 g; 53% yield) as a colorless oil. ¹H NMR (CDCl₃): 3.14–3.18 (m, 1H), 3.32–3.36 (m, 1H), 4.01–4.04 (m, 1H), 6.82–6.95 (m, 2H), 7.21–7.33 (m, 1H).

Synthesis of compound 16

2-Acetylthiazole (1.27 g; 10 mmol) was dissolved in 8 mL glacial acetic acid, 33% HBr in acetic acid (2.5 mL; ~10 mmol) was added and the resulting mixture was placed in an ice-bath. Bromine (0.57 mL; 11 mmol) was added and then the reaction was allowed to warm to 70–75 °C for 2 h with stirring. The disappearance of reddish color indicated the reaction has completed. The reaction mixture was cooled to room temperature, and the precipitate was filtered and washed with 15 mL ethyl acetate. The filter cake was transferred into a round-bottom flask, 20 mL of water was added and pH was adjusted to ~ 10 using 6 N NaOH. The oily product was extracted with ethyl acetate (2 × 30 mL) and the combined organic layers were dried over anhydrous sodium sulfate. The drying agent was filtered and the filtrate was concentrated to dryness, yielding a pure compound 16 (1.63 g; 79% yield) as a reddish oil. ¹H NMR (CDCl₃): 4.73 (s, 2H), 7.78–7.80 (d, 1H), 8.05–8.07 (d, 1H). Compound 17 was prepared using the same procedure as compound 15.

Compound 17

oil (63% yield). ¹H NMR (CDCl₃): 3.01–3.05 (m, 1H), 3.24–3.28 (m, 1H), 4.28–4.32 (m, 1H), 7.32–7.34 (d, 1H), 7.77–7.79 (d, 1H).

Enzyme assays and inhibition studies

Human neutrophil proteinase 3

Twenty microliters of 32.0 mM 5, 5’-dithio-bis(2-nitrobenzoic acid) in dimethyl sulfoxide and 10 µL of 3.45 µM human proteinase 3 in 0.1 M phosphate buffer /pH 6.50 (final enzyme concentration: 34.5 nM) were added to a cuvette containing a solution of 940 µL 0.1 M HEPES buffer/0.5 M NaCl/pH 7.25, 10 µL 862.5 µM inhibitor in dimethyl sulfoxide (final inhibitor concentration: 8.62 µM) and 20 µL 12.98 mM Boc-Ala-Ala-NVa-SBzl (final substrate concentration: 259.6 µM) at 25 °C. The change in absorbance was monitored at 410 nM for 2 minutes. A control (hydrolysis run) was also run under the same conditions by adding 5, 5’-dithio-bis(2-nitrobenzoic acid) in dimethyl sulfoxide and 10 µL of 3.45 µM solution of human proteinase 3 in 0.1 M phosphate buffer/pH 6.50 (final enzyme concentration: 34.5 nM) to a cuvette containing a solution of 940 µL 0.1 M HEPES buffer/0.5 M NaCl/pH 7.25, 10 µL dimethyl sulfoxide and 20 µL 12.98 mM Boc-Ala-Ala-NVa-SBzl (final substrate concentration: 259.6 µM). The change in absorbance was monitored at 410 nM for 2 minutes. The percent inhibition of Pr 3 was determined using % inhibition = (1-v/v_o) × 100 and is the average of duplicate or triplicate determinations.

Human neutrophil elastase

Ten microliters of 7.0 µM human elastase in 0.05 mM sodium acetate buffer/0.5 M NaCl/pH 5.50 (final enzyme concentration: 70 nM) was added to a cuvette containing a solution of 970 µL 0.1 M HEPES buffer /0.5 M NaCl/pH 7.25, 10 µL of 3.5 mM solution of inhibitor in dimethyl sulfoxide (final inhibitor concentration: 35 µM) and 10 µL of 70 mM methoxysuccinyl-Ala-Ala-Pro-Val p-nitroaniline (final substrate concentration: 700 µM) at 25 °C. The change in absorbance was monitored at 410 nM for 2 minutes. A control (hydrolysis run) was also run under the same conditions by adding 10 µL of a 7.0 µM solution of human elastase (final enzyme concentration: 70 nM) to a cuvette containing 970 µL 0.1 M HEPES buffer/0.5 M NaCl/pH 7.25, 10 µL DMSO and 10 µL of 70 mM methoxysuccinyl-Ala-Ala-Pro-Val p-nitroaniline (final substrate concentration: 700 µM). The change in absorbance was monitored at 410 nM for 2 minutes. The percent inhibition of HNE was determined using % inhibition = (1-v/v_o) × 100 and is the average of duplicate or triplicate determinations.

Computational method

Molecular docking simulation was performed using the SURFLEX program.⁴⁵ The structure of compound 16f was constructed in SYBYL⁴⁶ and was structurally optimized to default convergence thresholds using the Tripos Force Field⁴⁷ and Gasteiger–Marsili partial atomic charges.⁴⁸ A receptor model was prepared for Pr 3 using the 1FUJ³⁷ crystal structure. This structure was protonated in SYBYL according to pH = 7.0 protonation states, stripped of all water molecules and bound ligands, and electrostatically represented with Gasteiger–Marsili charges.

X-ray Crystallography

A crystal was affixed to a nylon cryoloop using oil (Paratone-n, Exxon) and mounted in the cold stream of a Bruker Kappa-Apex-II area-detector diffractometer. The temperature at the crystal was maintained at 150 K using a Cryostream 700EX low-temperature apparatus (Oxford Cryosystems). The unit cell was determined from the setting angles of the reflections collected in 36 frames of data. Data were measured using graphite mono-chromated molybdenum K_α radiation (λ= 0.71073 Å) collimated to a 0.6 mm diameter and a CCD detector at a distance of 50 mm from the crystal with a combination of phi and omega scans. A scan width of 0.5 degrees and scan time of 10 seconds were employed. Data collection, reduction, structure solution, and refinement were performed using the Bruker Apex2 suite (v2.0–2).⁴⁹ All available reflections to 2θ_max = 52° were harvested and corrected for Lorentz and polarization factors with Bruker SAINT (v6.45).⁵⁰ Reflections were then corrected for absorption, interframe scaling, and other systematic errors with SADABS 2004/1.⁵¹ The structure was solved (direct methods) and refined (full-matrix least-squares against F²) with the Bruker SHELXTL package (v6.14–1).⁵² All non-hydrogen atoms were refined using anisotropic thermal parameters. Hydrogen atoms were included at idealized positions and were not refined. Pertinent details are given in Table 3.

Table 3.

X-ray Data Collection and Structure Solution Parameters

molecular formula	C15H19ClN3O6S
fw	398.87
diffractometer	Bruker Kappa Apex II
radiation/λ, Ǻ	Mo Kα/0.71073
temp, K	150
color, habit	colorless, needle
crystal system	Monoclinic
space group	P21/n
crystal size(mm3)	0.17 × 0.25 × 0.69
a, Ǻ	10.2729(12)
b, Ǻ	8.4972(9)
c, Ǻ	41.227(5)
β, deg	94.384(6)
V, Ǻ3	3588.2(7)
Z	8
calcd density(g cm−1)	1.477
octants collected	±h,±k,±l
Max. h, k, l	12, 10, 50
⊝̣ range (°)	2.45–26.00
µ, mm−1	0.359
rflns/unique (Rint)	80192/7069 (0.0270)
obs,[>2σ]/params	7069/473
Robs, Rall	0.0326/0.0383
GOF	1.083
ρmax/ρmin, e Ǻ−3	0.333, –0.466