Pyrrole Carboxamidine Tryptase Inhibitors from Leptonychia pubescens

• Abstract
• Materials and Methods
• References

Abstract

The root and stem bark extracts of a Nigerian sample of Leptonychia pubescens Keay (Sterculiaceae) were found to inhibit the serine protease tryptase, a potential therapeutic target for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Bioassay-guided isolation led to the identification of 1-β-ribofuranosylbrunfelsamidine as the active component with a tryptase IC₅₀ of 3 μM. Brunfelsamidine was also isolated, but was only weakly active.

Asthma and chronic obstructive pulmonary disease (COPD) are chronic lung diseases which have experienced an alarming increase in recent years and, although the symptoms can be treated and the conditions managed in many cases, there are many treatment limitations and adverse reactions. Tryptase (EC 3.4. 21.59), a mast cell-specific trypsin-like serine protease, is a potential therapeutic target for the treatment of asthma and COPD, with several inhibitors currently being tested in preclinical and clinical studies [1]. Human tryptases appear to be quite resistant to inhibition by endogenous protease inhibitors like α₂-macroglobulin and serpins, and conventional protease inhibitors such as PMSF, aprotinin and classic Karzal-type inhibitors [1]. Serine protease inhibitors such as benzamidine (1), diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride, antipain, leupeptin [1], and cyclotheonamide E4 are more effective tryptase inhibitors [2].

Given the validation for tryptase as a novel target for asthma and COPD type of disease, and the relatively unique inhibitor profile known for this enzyme, a screening campaign was carried out against a library of natural product extracts. The root and stem bark extracts of Leptonychia pubescens collected in Nigeria were found to inhibit tryptase. Bioassay-guided isolation of the stem bark extract using a tryptase assay led to the isolation of 1-β-ribofuranosylbrunfelsamidine (2) as the active component (IC₅₀ = 3 μM) and the very weakly active brunfelsamidine (3). 1-β-Ribofuranosylbrunfelsamidine (2) and brunfelsamidine (3) were also present in the root extract. To the best of our knowledge, this represents the first published phytochemical report on the genus Leptonychia.[*]

Brunfelsamidine (3) was first isolated from Brunfelsia grandiflora D. Don (Solanaceae), a plant whose roots and bark water extracts have been used to treat rheumatism and arthritis in the upper Amazon region [3]. Brunfelsamidine (3) has also been isolated from Nierembergia hippomanica Miers (Solanaceae) as the active principle responsible for livestock toxicity [4]. The identity of brunfelsamidine (3) isolated in this study was confirmed by comparison with published data [3], [4].

The more active compound 2 was identified as the β-ribofuranosyl derivative of brunfelsamidine by MS and 2D NMR data [5], [6]. 1-β-Ribofuranosylbrunfelsamidine (2) is a new natural product but has been previously synthesised to probe the biological activity of a brunfelsamidine (3) analogue of the antiviral nucleoside ribavirin (4) [5]. However, biological evaluation indicated that 2 possessed no significant in vitro antiviral and antitumour activity [5]. As only the ¹H-NMR of 2 were available in the original synthesis paper [5], complete assignments of ¹H- and ¹³C-NMR data in D₂O were made and these are presented in the Materials and Methods section. In addition, ¹⁵N-HMBC NMR data of 2 obtained with J values at 3 and 8 Hz [7] allowed the assignment of N-1 at δ = 176. Similarly, a ¹⁵N-HMBC NMR experiment (J = 8 Hz) for brunfelsamidine (3) showed correlations from H-2, H-4 and H-5 to N-1 at δ = 166. The absolute stereochemistry of 2 remained unassigned as no optical rotation was reported in the original synthesis paper [5].

The guanidine, carboxamidine and sometimes amine functional groups are important for binding to the tryptase enzyme [1], [2]. For example, the simple carboxamidine, benzamidine (1), has been previously identified as a tryptase inhibitor and in our tryptase assay showed an IC₅₀ value of 14 μM (Fig. [1]). As a consequence, the identification of 1-β-ribofuranosylbrunfelsamidine (2) (IC₅₀ = 3 μM) and brunfelsamidine (3) (IC₅₀ = 265 μM) as tryptase inhibitors is not surprising given the presence of the carboxamidine functionality.

Materials and Methods

The roots and stem bark of Leptonychia pubescens were collected in Egba Obafemi, Ogun State, Nigeria on 29 May 1993, under contract as part of a collaborative programme between GlaxoWellcome (now GlaxoSmithKline) and the University of Illinois at Chicago. The plant was identified by D.D.S. and a voucher specimen (No. 10 492) has been deposited at the John. G. Searle Herbarium of the Field Museum, Chicago.

The stem bark of L. pubescens (50 g, dry and ground) was extracted with CH₂Cl₂/MeOH (1 : 1; 2 × 500 mL) and dried using rotary evaporation. The resulting extract (2.5 g) was partitioned using 200 mL of H₂O/MeOH/CHCl₃ (9 : 1:1) and the aqueous MeOH fraction (1.7 g) was separated using a C18 open column (8 × 3.5 cm I.D., 40 g Waters Preparative C18, 125 Å, 55 - 105 μm, eluted with a 160 mL MeOH/H₂O, 0 : 10, 1 : 9, 2 : 8, 3 : 7 and 10 : 0). The active 100 % H₂O fraction (450 mg) was separated by repeated HPLC using a YMC-Pack ODS-AQ HPLC column (150 × 10 mm I.D., 3 mL/min, isocratic H₂O) to give brunfelsamidine (3) (tR = 4.5 min) and a mixture of 2 and 3 (tR = 6 min,10 mg). The mixture of 2 and 3 was separated using Sephadex LH-20 (10 × 2 cm I.D., 18 g gel, eluted with MeOH) to give brunfelsamidine (3) (4 mg, overall 50 mg, 0.1 % dry weight) and 1-β-ribofuranosylbrunfelsamidine (2) (5 mg, 0.01 % dry weight) in order of elution.

Human lung tryptase was purchased from Elastin Products Company Inc, fluorescent tryptase substrate peptide (Tos-Gly-Pro-Lys-AMC) from Bachem and heparin from Sigma. Tryptase activity was measured by following the cleavage of the Tos-Gly-Pro-Lys-AMC peptide to release free AMC using a Tecan Ultra plate-reader at 360 nm excitation/465 nm emission wavelength. The assay contained 50 μM substrate in 0.1 M Tris-HCl, pH 8.0, 10 % glycerol, 10 μM heparin, 0.01 % CHAPS, 0.05 μg/mL of tryptase and test samples at varying concentrations. This homogeneous, continuous assay was carried out in a 50-μL final volume in 384-well black microtitre plates and read using a Tecan Ultra or SpectraFluor Plus using 360/465 excitation/emission filters. Under these conditions the assay was linear over at least 2 hours. Leupeptin was used as a standard (average IC₅₀ = 0.4 μM).

1-β-Ribofuranosylbrunfelsamidine (2): white solid, [α]_D ³⁰: -1° (c 0.5, H₂O); UV (H₂O): λ_max (log ε) = 199 (4.05), 236 nm (3.18); (+)-HR-ESI-MS: m/z = 242.1145 [M + H]⁺ (calcd. 242.1135); ¹H-NMR (500 MHz, D₂O): δ = 7.61 (1H, t, J = 2 Hz, H-2), 6.56 (1H, dd, J = 3, 2 Hz, H-4), 7.01 (1H, dd, J = 3, 2 Hz, H-5), 5.54 (1H, d, J = 5.5 Hz, H-1′), 4.27 (1H, t, J = 5.5 Hz, H-2′), 4.15 (1H, dd, J = 5.5, 3.8 Hz, H-3′), 4.01 (1H, m, H-4′), 3.68 (1H, dd, J = 12.6, 3.4 Hz, H-5′a), 3.62 (1H, dd, J = 12.6, 4.7 Hz, H-5′b); ¹³C-NMR (125 MHz, D₂O): δ = 125.2 (C-2), 114.0 (C-3), 109.9 (C-4), 123.0 (C-5), 162.0 (C-6), 92.7 (C-1′), 76.3 (C-2′), 71.5 (C-3′), 86.3 (C-4′), 62.9 (C-5′); ¹⁵N-NMR (50 MHz, D₂O): δ = 176 (N-1) from ¹⁵N-HMBC (J = 8 Hz, correlation H-2, H-4, H-2′ to N-1 and J = 3 Hz, correlation H-5, H-1′, H-2’ to N-1).

References

• 1 Cairns J A. Inhibitors of mast cell tryptase beta as therapeutics for the treatment of asthma and inflammatory disorders.  Pulm Pharmacol Ther. 2005;  18 55-66
  PubMedSuche in Google Scholar
• 2 Murakami Y, Takei M, Shindo K, Kitazume C, Tanaka J, Higa T. et al . Cyclotheonamide E4 and E5, new potent tryptase inhibitors from an Ircinia species of sponge.  J Nat Prod. 2002;  65 259-61
  CrossrefPubMedSuche in Google Scholar
• 3 Lloyd H A, Fales H M, Goldman M E, Jerina D M, Plowman T, Schultes R E. Brunfelsamidine: a novel convulsant from the medicinal plant Brunfelsia grandiflora .  Tetrahedron Lett. 1985;  26 2623-4
  CrossrefPubMedSuche in Google Scholar
• 4 Buschi C A, Pomilio A B. Pyrrole-3-carbamidine: a lethal principle from Nierembergia hippomanica .  Phytochemistry. 1987;  26 863-5
  CrossrefPubMedSuche in Google Scholar
• 5 Ramasamy K, Robins R K, Revankar G R. Synthesis of brunfelsamidine ribonucleoside and certain related compounds by the stereospecific sodium salt glycosylation procedure.  Nucleosides Nucleotides. 1988;  7 385-92
  PubMedSuche in Google Scholar
• 6 Bock K, Pedersen C. ¹³C N.M.R. spectroscopy of monosaccharides.  Adv Carbohydr Chem Biochem. 1983;  41 44-66
  PubMedSuche in Google Scholar
• 7 Martin G E, Hadden C E. Long-range ¹H-¹⁵N heteronuclear shift correlation at natural abundance.  J Nat Prod. 2000;  63 543-85
  CrossrefPubMedSuche in Google Scholar

Mark S. Butler

MerLion Pharmaceuticals

1 Science Park Road

The Capricorn #05-01

Singapore Science Park II

Singapore 117528

Telefon: +65-6829-5611

Fax: +65-6829-5601

eMail: mark@merlionpharma.com

References

• 1 Cairns J A. Inhibitors of mast cell tryptase beta as therapeutics for the treatment of asthma and inflammatory disorders.  Pulm Pharmacol Ther. 2005;  18 55-66
  PubMedSuche in Google Scholar
• 2 Murakami Y, Takei M, Shindo K, Kitazume C, Tanaka J, Higa T. et al . Cyclotheonamide E4 and E5, new potent tryptase inhibitors from an Ircinia species of sponge.  J Nat Prod. 2002;  65 259-61
  CrossrefPubMedSuche in Google Scholar
• 3 Lloyd H A, Fales H M, Goldman M E, Jerina D M, Plowman T, Schultes R E. Brunfelsamidine: a novel convulsant from the medicinal plant Brunfelsia grandiflora .  Tetrahedron Lett. 1985;  26 2623-4
  CrossrefPubMedSuche in Google Scholar
• 4 Buschi C A, Pomilio A B. Pyrrole-3-carbamidine: a lethal principle from Nierembergia hippomanica .  Phytochemistry. 1987;  26 863-5
  CrossrefPubMedSuche in Google Scholar
• 5 Ramasamy K, Robins R K, Revankar G R. Synthesis of brunfelsamidine ribonucleoside and certain related compounds by the stereospecific sodium salt glycosylation procedure.  Nucleosides Nucleotides. 1988;  7 385-92
  PubMedSuche in Google Scholar
• 6 Bock K, Pedersen C. ¹³C N.M.R. spectroscopy of monosaccharides.  Adv Carbohydr Chem Biochem. 1983;  41 44-66
  PubMedSuche in Google Scholar
• 7 Martin G E, Hadden C E. Long-range ¹H-¹⁵N heteronuclear shift correlation at natural abundance.  J Nat Prod. 2000;  63 543-85
  CrossrefPubMedSuche in Google Scholar

Mark S. Butler

MerLion Pharmaceuticals

1 Science Park Road

The Capricorn #05-01

Singapore Science Park II

Singapore 117528

Telefon: +65-6829-5611

Fax: +65-6829-5601

eMail: mark@merlionpharma.com