Biochemistry 1997, 36, 4739-4750
4739
Structure-Based Design of a General Class of Mechanism-Based Inhibitors of the Serine Proteinases Employing a Novel Amino Acid-Derived Heterocyclic Scaffold† William C. Groutas,*,‡ Rongze Kuang,‡ Radhika Venkataraman,‡ Jeffrey B. Epp,‡ Sumei Ruan,‡ and Om Prakash§ Department of Chemistry, Wichita State UniVersity, Wichita, Kansas 67260, and High-Field NMR Facility, Department of Biochemistry, Kansas State UniVersity, Manhattan, Kansas 66506-3702 ReceiVed NoVember 22, 1996; ReVised Manuscript ReceiVed February 10, 1997X
ABSTRACT:
We describe in this paper the structure-based design of a general class of heterocyclic mechanism-based inhibitors of the serine proteinases that embody in their structure a novel peptidomimetic scaffold (1,2,5-thiadiazolidin-3-one 1,1-dioxide). Sulfone derivatives of this class (I) were found to be time-dependent, potent, and highly efficient irreversible inhibitors of human leukocyte elastase, cathepsin G, and proteinase 3. The partition ratios for a select number of inhibitors were found to range between 0 and 1. We furthermore demonstrate that these inhibitors exhibit remarkable enzyme selectivity that is dictated by the nature of the P1 residue and is consistent with the known substrate specificity reported for these enzymes. Thus, inhibitors with small hydrophobic side chains were found to be effective inhibitors of elastase, those with aromatic side chains of cathepsin G, and those with a basic side chain of bovine trypsin. Taken together, the findings cited herein reveal the emergence of a general class of stable mechanism-based inhibitors of the serine proteinases which can be readily synthesized using amino acid precursors. Biochemical and high-field NMR studies show that the interaction of this class of inhibitors with a serine proteinase results in the formation of a stable acyl complex(es) and the release of benzenesulfinate, formaldehyde, and a low molecular weight heterocycle. The data are consistent with initial formation of a Michaelis-Menten complex, acylation of Ser195, and tandem loss of the leaving group. The initial HLE-inhibitor complex reacts with water generating formaldehyde and a stable HLEinhibitor complex. Whether the initial HLE-inhibitor complex also reacts with His57 to form a third complex is not known at this point. The desirable salient parameters associated with this class of inhibitors, including the expeditious generation of structurally diverse libraries of inhibitors based on I, suggest that this class of mechanism-based inhibitors is of general applicability and can be used in the development of inhibitors of human and viral serine proteinases of clinical relevance.
An array of inflammatory diseases are associated with a massive influx of neutrophils, the release of chemokines, the production of reactive oxygen species, and a compromised proteinase/antiproteinase screen (Weiss, 1989; Harada et al., 1994; Yoshimura et al., 1994; Piccioni et al., 1992). The inflammatory process also involves the extracellular release of the lysosomal serine endopeptidases elastase (HLE), cathepsin G (Cat G), and proteinase 3 (PR 3). Poor control of the activity of these enzymes due to depressed levels of their endogenous protein inhibitors (R-1-proteinase inhibitor, R-1-antichymotrypsin, secretory leukoproteinase inhibitor, elafin) is believed to result in the destruction of the major components of connective tissue, including elastin (Barrett, 1994; Janusz & Doherty, 1991; Birrer, 1993). The aberrant activity of these enzymes in disease states has provided the impetus behind efforts related to the design of agents capable of modulating the activity of these enzymes and redressing the proteinase/antiproteinase imbalance. Thus, there has been an intense and ongoing interest in the design of inhibitors, including mechanism-based inhibitors, of these †
We are grateful for the generous financial support provided by SPARTA Pharmaceuticals, Inc., Horsham, PA, and the National Institutes of Health (HL 38048). * Author to whom correspondence should be addressed: tel, (316) 978-3120; fax, (316) 978-3431; e-mail,
[email protected]. ‡ Wichita State University. § Kansas State University. X Abstract published in AdVance ACS Abstracts, April 1, 1997.
S0006-2960(96)02893-0 CCC: $14.00
and related serine endopeptidases (Edwards & Bernstein, 1994). Examples of inactivators of this type include haloenol and ynenol lactones (Katzenellenbogen et al., 1992; Copp et al., 1987), substituted isocoumarins (Hernandez et al., 1992), 3-alkyl-N-hydroxysuccinimide derivatives (Groutas et al., 1989, 1991, 1993a), substituted dihydrouracils (Groutas et al., 1994a), β-lactams (Underwood et al., 1995), and saccharin derivatives (Hlasta et al., 1995; Groutas et al., 1993b, 1996a). Herein we wish to describe the biochemical rationale underlying the design of a novel and general class of mechanism-based inhibitors of the serine proteinases (structure I (Groutas et al., 1994b), as well as the results of
pertinent biochemical and mechanistic studies related to the inhibition of HLE, Cat G, and PR 3 by sulfone derivatives of 1,2,5-thiadiazolidin-3-one 1,1-dioxide (I). We furthermore demonstrate that the heterocyclic ring in I is a highly effective peptidomimetic scaffold suitable for appending peptidyl and nonpeptidyl recognition elements that allow the optimization of binding interactions with both the Sn and © 1997 American Chemical Society
4740 Biochemistry, Vol. 36, No. 16, 1997
Groutas et al.
Scheme 1: Synthesis of Ia
a Reagents: a, NH SO Cl/TEA; b, NaH/THF; c, ArSCH Cl/TEA; 2 2 2 d, NaH/R1X/THF; e, m-chloroperbenzoic acid/CH2Cl2.
Table 1: Inhibitory Activity of Compounds 1-13 against Human Leukocyte Elastase, Cathepsin G, and Proteinase 3 kinact/KI (M-1 s-1) a
compound 1 2 3 4 5 6 7 8 9 10 11d 12d 13e
R1 DL-ethyl DL-ethyl
n-propyl n-propyl isopropyl isopropyl isobutyl isobutyl n-butyl n-butyl benzyl benzyl (CH2)4NH2
R2
HLE
PR 3
Cat G
methyl benzyl methyl benzyl methyl benzyl methyl benzyl methyl benzyl methyl benzyl methyl
190b 810b 780 7620 NA NA 9490 95190 1080 8060 NA NA NA
200b 80b 1830 4960 NA NA 2250 5200 NA NA NA NA NA
NAc NA NA 130 NA NA NA 110 NA 610 120 11210 NA
a All compounds are derived from the corresponding L-amino acid esters unless indicated otherwise, and L ) SO2Ph. b kobs/[I] (M-1 s-1) was determined by the incubation method. c NA: no activity. d Alternate substrate inhibitor of R-chymotrypsin. e Potent inhibitor of bovine trypsin (kinact/KI ) 16 820 M-1 s-1).
Sn′ subsites (Schecter & Berger, 1967) and yields efficient and highly specific inhibitors of serine proteinases. MATERIALS AND METHODS Materials. MeOSuc-AAPV-pNA,1 Suc-AAPF-pNA, BOCp-nitrophenyl ester, N-acetyl-L-tyrosine methyl ester, and NR-benzoyl-L-arginine p-nitroanilide were purchased from Sigma Chemical Co., St. Louis, MO. Aldrich 230400 mesh silica gel was used for flash chromatography. Human leukocyte elastase was purchased from Elastin Products Co., Owensville, MO. Human leukocyte cathepsin G and proteinase 3 were purchased from Athens Research and Technology Co., Athens, GA. Substrate and inhibitor stock solutions were prepared in DMSO. Chymotrypsin and trypsin were purchased from Sigma Chemical Co., St. Louis, MO. Active site titration of chymotrypsin (Schonbaum et al., 1961) yielded 80-90% active sites. The infrared and NMR spectra of the synthesized compounds were recorded on a Perkin-Elmer 1330 infrared spectrophotometer and a Varian XL-300 NMR spectrometer, respectively. The enzyme-inhibitor NMR studies were performed using a 500 MHz Varian Unity Plus NMR spectrometer. A Hewlett-Packard diode array UV/vis spectrophotometer was used in the enzyme assays and inhibition studies. Synthesis. Scheme 1 was used in the synthesis of inhibitors 1-12. These are listed in Table 1. A representative synthesis (compound 7 in Table 1) is described below. L-Ala
1 Abbreviations: MeOSuc-AAPV-pNA, methoxysuccinyl-Ala-AlaPro-Val p-nitroanilide; DMSO, dimethyl sulfoxide; MPCBA, mchloroperbenzoic acid; TEA, triethylamine; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; NCS, N-chlorosuccinimide.
(S)-Methyl 2-Sulfamido-4-methylpentanoate. To a mixture of L-Leu-OCH3 hydrochloride (7.23 g; 0.04 mol) and sulfamoyl chloride (4.62 g; 0.04 mol) in 50 mL of methylene chloride was added dropwise a solution of triethylamine (8.1 g; 0.08 mol) in 30 mL of methylene chloride at 0 °C. After the mixture was stirred at room temperature for 2 h, the triethylamine hydrochloride salt was filtered off, and the filtrate was washed with water, 5% HCl, and brine and dried over anhydrous sodium sulfate. Removal of the solvent yielded 6.4 g (71% yield) of an oily product. 1H NMR (CDCl3): δ 0.99 (dd, 6H), 1.57 (t, 2H), 1.85 (m, 1H), 3.78 (s, 3H), 4.10 (m, 1H), 5.12 (s, 2H), 5.73 (d, 1H). 13C NMR (CDCl3): δ 21.41, 22.70, 24.44, 41.53, 52.65, 54.89, 174.55. [R]D -26.9 (c 1, CH2Cl2) (Dewynter et al., 1996). (S)-4-Isobutyl-1,2,5-thiadiazolidin-3-one 1,1-Dioxide. A solution of (S)-methyl 2-sulfamido-4-methylpentanoate (6.4 g; 0.285 mol) in 60 mL of dry THF was treated portionwise with 60% sodium hydride (1.5 g; 0.037 mol) at 0 °C. The reaction mixture was stirred at room temperature overnight. The solvent was removed in Vacuo, and the residue was dissolved in 30 mL of cold water. The pH was adjusted to 6-7 with concentrated HCl and extracted with ethyl acetate (30 mL). The aqueous layer was separated and acidified with concentrated HCl to pH 1. The product was extracted with ethyl acetate (3 × 30 mL), and the combined organic extracts were dried over anhydrous sodium sulfate and evaporated. The crude product (4.2 g; 77% yield) was recrystallized from methanol, mp 196-197 °C. 1H NMR (DMSO-d6): δ 0.96 (dd, 6H), 1.72 (m, 2H), 1.84 (m, 1H), 6.95 (s, 1H),7.36 (s, 1H). 13C NMR (CDCl3): δ 21.04, 22.83, 24.60, 39.87, 59.71, 172.48 (Dewynter et al., 1996). (S)-4-Isobutyl-2-[(phenylthio)methyl]-1,2,5-thiadiazolidin3-one 1,1-Dioxide. A solution of (S)-4-isobutyl-1,2,5thiadiazolidin-3-one 1,1-dioxide (1.92 g; 10 mmol), chloromethyl phenyl sulfide (1.80 g; 12 mmol), and triethylamine (1.01 g; 10 mmol) in 15 mL of dry acetonitrile was refluxed for 20 h. The solvent was removed in Vacuo, and the residue was taken up in 30 mL of ethyl acetate, washed with water, 5% aqueous HCl (10 mL), and 5% aqueous sodium bicarbonate, and dried over anhydrous sodium sulfate. Evaporation of the solvent left a crude product which was purified by flash chromatography, using a hexane/methylene chloride gradient, mp 48-49 °C (1.64 g; 52% yield). 1H NMR (CDCl3): δ 0.94 (dd, 6H), 1.60 (m, 1H), 1.75 (m, 2H), 4.08 (m, 1H), 4.94 (d, 2H), 5.05 (d, 1H), 7.33 (m, 3H), 7.56 (m, 2H). 13C NMR (CDCl3): δ 21.17, 22.73, 25.00, 39.88,45.64, 59.15, 128.44, 129.14, 132.33, 132.89, 168.63. Anal. Calcd for C13H18N2O3S2: C, 49.66; H, 5.77; N, 8.91. Found: C, 49.61; H, 5.81; N, 8.87. (S)-4-Isobutyl-5-methyl-2-[(phenylthio)methyl]-1,2,5-thiadiazolidin-3-one 1,1-Dioxide. Methyl iodide (3.92 g; 27.6 mmol) was added to a solution of (S)-4-isobutyl-2-[(phenylthio)methyl]-1,2,5-thiadiazolidin-3-one 1,1-dioxide (1.74 g; 5.52 mmol) in dry acetonitrile (100 mL). The solution was cooled in an ice bath, and 60% NaH (2.21 g; 5.52 mmol) was added in small portions over a period of 5 min. The reaction mixture was stirred at room temperature overnight, and the solvent and excess methyl iodide were removed using a rotary evaporator. The residue was dissolved in methylene chloride (25 mL) and washed with water (10 mL). The organic layer was separated and dried over anhydrous sodium sulfate. Removal of the solvent left a crude product which was purified by flash chromatography (hexane/methylene
Mechanism-Based Serine Proteinase Inhibitors Scheme 2: Synthesis of Compound 13a
a Reagents: a, NH2SO2Cl/TEA/CH2Cl2 (51%); b, NaH/THF, then HCl (88%); c, PhSCH2Cl/TEA/CH3CN (55%); d, NaH, then CH3I/ CH3CN (52%); e, m-chloroperbenzoic acid (100%); f, (CH3)3SiI/CH3CN (90%).
chloride), yielding an oily product (1.41 g; 78% yield). 1H NMR (CDCl3): δ 0.92 (dd, 6H), 1.60-1.88 (m, 3H), 2.84 (s, 3H), 3.72 (t, 1H), 4.97 (s, 2H), 7.30-7.60 (m, 5H). 13C NMR (CDCl3): δ 22.37, 22.48, 24.58, 33.36, 38.53, 45.38, 65.18, 128.44, 129.07, 132.32, 133.14, 166.34. Anal. Calcd for C14H20N2S2O3: C, 51.19; H, 6.14; N, 8.53. Found: C, 51.34; H, 6.40; N, 8.44. (S)-4-Isobutyl-5-methyl-2-[(phenylsulfonyl)methyl]-1,2,5thiadiazolidine 1,1-Dioxide (7). A solution of (S)-4-isobutyl5-methyl-2-[(phenylthio)methyl]-1,2,5-thiadiazolidin-3-one 1,1-dioxide (0.3 g; 0.74 mmol) and m-chloroperbenzoic acid (0.58 g; 1.85 mmol) in 10 mL of methylene chloride was stirred at room temperature overnight. The mixture was diluted with 10 mL of methylene chloride, washed with 5% sodium bicarbonate (10 mL) and water (10 mL), and dried over anhydrous sodium sulfate. Removal of the solvent left a crude product, which was purified by flash chromatography (hexane/methylene chloride) to yield 0.31 g (99% yield) of pure product, mp 133-134 °C. 1H NMR (CDCl3): δ 0.85 (dd, 6H), 1.55-1.80 (m, 3H), 2.81 (s, 3H), 4.75 (t, 1H), 4.82 (s, 2H), 7.50-7.68 (m, 3H), 7.93 (d, 2H). 13C NMR (CDCl3): δ 22.20, 22.43, 24.65, 34.41, 38.64, 60.08, 65.62, 129.20, 129.26, 134.67, 136.88, 166.57. Anal. Calcd for C14H20N2O5S2: C, 46.65; H, 5.59; N, 7.77. Found: C, 46.49; H, 5.77; N, 7.58. The synthesis of compound 13 was accomplished using Scheme 2. (S)-Methyl 6-[(benzyloxycarbonyl)amino]-2-sulfamidohexanoate (14). N-Cbz-L-lysine methyl ester hydrochloride (25.00 g; 75.6 mmol) was added to a stirred solution of sulfamoyl chloride, prepared by adding dropwise 95% formic acid (3.14 g; 68.3 mmol) to chlorosulfonyl isocyanate (11.77 g; 83 mmol) in methylene chloride (Kristinsson et al., 1994), at 0 °C under nitrogen. A solution of triethylamine (15.27 g; 151.2 mmol) in 40 mL of methylene chloride was then added dropwise, and the resulting mixture was stirred at room temperature for 1 h. The solvent was removed in Vacuo, and the residue was taken up in ethyl acetate (100 mL). The organic layer was then washed with 5% HCl (50 mL), 5% NaHCO3 (50 mL), and brine (50 mL) and dried over anhydrous sodium sulfate. Evaporation of the solvent left a crude product which was purified by flash chromatography (hexane/methylene chloride) to give 14.5 g (51% yield) of pure product, mp 90-91 °C. 1H NMR (CDCl3): δ 1.38-
Biochemistry, Vol. 36, No. 16, 1997 4741 1.58 (m, 4H), 1.69 (m, 1H), 3.18 (q, 2H), 3.75 (s, 3H), 4.05 (m, 1H), 4.98 (br t, 1H), 5.04 (br s, 2H), 5.09 (s, 2H), 5.62 (d, 1H), 7.34 (m, 5H). 13C NMR (CDCl3): δ 22.02, 29.10, 31.85, 40.36, 52.75, 56.02, 66.70, 128.07, 128.49, 136.52, 156.64, 173.52. (S)-4-[4-[(Benzyloxycarbonyl)amino]butyl]-1,2,5-thiadiazolidin-3-one 1,1-Dioxide (15). A solution of compound 14 (12.35 g; 33.1 mmol) in 50 mL of dry THF kept at 0 °C was treated portionwise with 60% sodium hydride (1.7 g; 1.3 equiv) with stirring. The solution was stirred at room temperature overnight, and the solvent was removed on the rotary evaporator. The residue was dissolved in 40 mL of cold water and the solution extracted with ethyl acetate (40 mL). The aqueous layer was cooled in ice and carefully acidified with concentrated HCl to pH
