Synthesis and biological evaluation of nigranoic acid esters as novel human neutrophil elastase inhibitors

Article abstract of DOI:10.1080/14786419.2014.996149

Human neutrophil elastase (HNE) has been implicated as a major contributor in the pathogenesis of diseases, such as lung disorders and other inflammatory diseases. A series of 12 new nigranoic acid esters were regioselectively synthesised in good yields and evaluated for HNE inhibitory activity. Nigranoic acid exhibited significant inhibitory activity against HNE with the IC₅₀ value of 3.77 M, and six esters displayed considerable inhibitory effects on HNE with IC₅₀ values in the range of 2.61-8.95 M. The nigranoic acid esters having phenyls substituted with bromine and trimethoxyls (3h and 3b) showed stronger inhibitory activity on HNE than nigranoic acid.

Full text of DOI:10.1080/14786419.2014.996149

This article was downloaded by: [New York University]
On: 13 January 2015, At: 05:46
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Natural Product Research: Formerly
Natural Product Letters
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/gnpl20
Synthesis and biological evaluation of
nigranoic acid esters as novel human
neutrophil elastase inhibitors
Guoli Huanga, Li Fengb, Bo Liua, Yi Heb, Yiming Lib & Yegao Chena
a School of Chemistry and Chemical Engineering, Yunnan Normal
University, Kunming 650050, P.R. China
b School of Pharmacy, Shanghai University of Traditional Chinese
Medicine, Shanghai 201203, P.R. China
Published online: 06 Jan 2015.
Click for updates
To cite this article: Guoli Huang, Li Feng, Bo Liu, Yi He, Yiming Li & Yegao Chen (2015): Synthesis
and biological evaluation of nigranoic acid esters as novel human neutrophil elastase inhibitors,
Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.996149
To link to this article: http://dx.doi.org/10.1080/14786419.2014.996149
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Natural Product Research, 2015
http://dx.doi.org/10.1080/14786419.2014.996149
Synthesis and biological evaluation of nigranoic acid esters as novel
human neutrophil elastase inhibitors
Guoli Huanga1, Li Fengb1, Bo Liua, Yi Heb, Yiming Lib and Yegao Chena*
aSchool of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650050,
P.R. China; bSchool of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai
201203, P.R. China
(Received 25 August 2014; final version received 30 November 2014)
Ar
N
HO
O
O
O
O
OH
O
O
NH
DCC/DMAP
THF
+
OH
Ar
1a–l
2
3a–l
Ar =4–BrC6H4 (3h) IC50 = 2.61 µM
Ar =3,4,5–(OCH3)3C6H2 (3b) IC50 = 3.25 µM
Human neutrophil elastase (HNE) has been implicated as a major contributor in the
pathogenesis of diseases, such as lung disorders and other inflammatory diseases.
A series of 12 new nigranoic acid esters were regioselectively synthesised in good
yields and evaluated for HNE inhibitory activity. Nigranoic acid exhibited significant
inhibitory activity against HNE with the IC50 value of 3.77 mM, and six esters
displayed considerable inhibitory effects on HNE with IC50 values in the range of
2.61–8.95 mM. The nigranoic acid esters having phenyls substituted with bromine
and trimethoxyls (3h and 3b) showed stronger inhibitory activity on HNE than
nigranoic acid.
Keywords: nigranoic acid esters; human neutrophil elastase; inhibition
1. Introduction
Human neutrophil elastase (HNE, E.C. 3.4.21.37) is a 30 kDa serine protease stored in the
azurophilic granules of neutrophils. The intracellular HNE at high concentration (5 mM) breaks
down foreign proteins (e.g. those from invading bacteria), whereas extracellular HNE, released
by neutrophils and mostly bound to the neutrophil plasma membrane, assists neutrophil
migration to inflammation sites by degrading various host proteins, such as extracellular matrix
proteins (Siedle et al. 2007). Under normal physiological conditions, HNE is controlled by
endogenous inhibitors, including a1-trypsin (a1-AT), secretory leucocyte proteinase inhibitor,
a2-macroglobulin and elafin (Rubin 1996; Fitch et al. 2006; Pham 2006; Williams et al. 2006).
However, an imbalance between HNE and its endogenous inhibitors can stimulate inflammatory
lung disorders through HNE’s role in the inflammatory process, mucus overproduction and lung
tissue damage (Wright et al. 2002; Caldwell et al. 2005; Bergin et al. 2008). HNE plays an
important role in a variety of lung inflammatory diseases, including acute lung injury, acute
*Corresponding author. Email: ygchen48@gmail.com
q 2015 Taylor & Francis

2
G. Huang et al.
respiratory distress syndrome, bronchiectasis, pulmonary emphysema and chronic obstructive
pulmonary disease (Stevens et al. 2011). Sivelestat sodium hydrate (ONO-5046) is the only
clinically registered synthesised selective HNE inhibitor. However, the use of ONO-5046 is
limited by its poor pharmacokinetics and by the fact that it presents risks of organ toxicity
(Ohbayashi 2002; Huang et al. 2008; Stevens et al. 2011).
Furthermore, nigranoic acid is an A ring-secocycloartene triterpenoid isolated from the
leaves and stems of Schisandra lancifolia and has been reported to show a variety of biological
and medicinal activities, including cytotoxic activity towards leukaemia and HeLa cells, and
inhibition of expression of HIV reverse transcriptase and polymerase (Sun et al. 1996, 2011;
Chen et al. 2001; Xiao et al. 2006; Xu et al. 2010). Recently, nigranoic acid was isolated from
Schisandra propinqua, which inspired us to study the structure–activity relationship of
nigranoic acid analogues by the structural modification. Six new hydroxyl derivatives of
nigranoic acid were obtained by microbial transformation (Dong, Chen, Song, He, et al. 2007;
Dong, Chen, Song, Zhu, et al. 2007; Yang et al. 2012), and only two-hydroxyl analogues
exhibited weak anti-HIV activity (Yang et al. 2012). Following our work on the structural
modification and biological activity of nigranoic acid analogues, we now report the synthesis
and identification of 12 nigranoic acid aromatic ester derivatives by the reaction of nigranoic
acid and benzyl alcohols in the presence of N,N0-dicyclohexylcarbodie (DCC)/4-dimethylami-
nopyridine (DMAP). In addition, the activities of these compounds were determined through
HNE inhibitory activity bioassay, and some compounds exhibited considerable inhibitory
effects on HNE with IC50 values in the range of 2.61–8.95 mM.
2. Results and discussion
2.1. Chemistry
Benzyl alcohols were prepared by stirring aromatic aldehydes with excess NaBH4 in methanol
for 2 h. Twelve nigranoic acid esters were synthesised in the presence of DCC/DMAP with the
nigranoic acid and benzyl alcohols as starting materials. The synthetic route of the compounds is
outlined in Figure 1.
According to the literature (Melzig et al. 2001), in the presence of 2.2 equiv DCC/DMAP,
nigranoic acid aromatic ester derivatives have been synthesised by the reaction of nigranoic acid
(1.0 equiv) and benzyl alcohol (2.2 equiv) in THF at room temperature. The reaction afforded
1
good yield (82–98%) and obtained single regioisomer. With the help of H, 13C NMR and
HMBC spectrum of 3a–l (Figures S1–S12), proton H-31 showed a key HMBC correlation with
the carboxyl carbon C-3, suggesting a linkage between C-31 and C-3 in the product, that is to
say, the esterification regioselectively reacted at C-3 position, not C-27 position, maybe because
of the steric hindrance and links of different carbon chains (saturated aliphatic carbon chain for
C-3 and a,b-unsaturated carbon chain for C-27). The structures of the synthesised compounds
were confirmed by 1H, 13C NMR spectra and HR-MS (Table 1).
Ar
N
HO
O
O
O
O
OH
O
O
NH
DCC/DMAP
THF
+
OH
Ar
1a–l
3a–l
2
Figure 1. Synthesis of nigranoic acid esters 3a–3l.

Natural Product Research
3
Table 1. Synthesis of nigranoic acid esters (3a–3l).
Entry
1
Ar
Producta (yield %)b
Entry
7
Ar
Producta (yield %)b
3a (85)
3g (94)
2
3b (89)
8
3h (88)
3
4
3c (94)
3d (87)
9
3i (82)
3j (91)
10
5
6
3e (85)
3f (89)
11
12
3k (98)
3l (84)
a All the products are confirmed by using NMR.
b Yield refers to pure products after column chromatography.
2.2. HNE activity
The inhibitory effects of the nigranoic acid and its esters were investigated, the concentration
gradients of the tested compounds at 1, 3, 10, 30 and 100 mM. As positive control, ONO-5046
exhibited potent inhibitory activity, with an IC50 value of 87.05 nM (Feng et al. 2012). HNE
activity was strongly and directly inhibited by nigranoic acid, with the IC50 value of 3.77 mM,
whereas nigranoic acid esters having phenyls substituted with bromine and trimethoxyls (3h and
3b) showed slightly more inhibitory potency than that of nigranoic acid, with IC50 values of 2.61
and 3.25 mM, respectively. Compound 3h exhibited HNE inhibition with more than 83.21% at
10 mM ( p , 0.01), and compound 3b imposed a considerable inhibitory effect on HNE in a
dose-dependent manner, with a minimal effective concentration of 1 mM (28.25% inhibition).
The nigranoic acid esters bearing phenyls substituted with dimethoxyls and chlorine (3c and 3i),
and thiophene (3j) showed slightly less potency than that of nigranoic acid, with the IC50 values
ranging from 3.89 to 4.83 mM. The nigranoic acid ester with cinnamenyl group (3g) showed the
least inhibitory potency (IC50 value: 8.95 mM) among the tested compounds (Table 2). The other
six analogues showed no activity.

4
G. Huang et al.

Natural Product Research
5
3. Experimental
3.1. General
TLC analyses were performed on commercial glass plates bearing 0.25-mm layer of Merck
1
Silica gel 60F254. Silica gel (200–300 mesh) was used for column chromatography. H, 13C
NMR and HMBC spectra were recorded on a Bruker AVANCE III 500 MHz spectrometer
(Bruker BioSpin Group, Rheinstetten, Germany) in CDCl3 solution. ESI-MS and HR-MS were
measured on a VG Auto Spec-3000 spectrometer (VG PRIMA, Birmingham, England). Unless
otherwise noted, the starting materials were either commercially available from Aladdin in
Shanghai, China or synthesised according to the references cited. Nigranoic acid (2) was isolated
from the Chinese medicine S. propinqua and identified unambiguously by NMR spectroscopy
and MS, and by comparison with authentic samples from the China Institute for Control of
Pharmaceutical and Biological Products (Beijing, China). The purity was .99% based on high-
performance liquid chromatography and thin-layer chromatography analyses.
3.2. General procedure for the synthesis of nigranoic acid esters (3a–3l)
The mixture of nigranoic acid (0.235 g, 0.5 mmol) and DCC (0.227 g, 1.1 mmol) was dissolved
in dry THF (5 mL) and stirred under 08C for 10 min, to which the solution of benzyl alcohol
(1.1 mmol) and DMAP (0.134 g, 1.1 mmol) in dry THF (5 mL) was added and stirred for 30 min
at the same temperature. The reaction mixture was warmed to room temperature and stirred
overnight. The solvent was removed by vacuum and the residue was purified by column
chromatography over silica gel (ethyl acetate/petroleum ether ¼ 1:4) to afford the target
product.
3.3. HNE activity inhibitory bioassay
HNE (EC 3.4.21.37) from human leucocytes was purchased from Innovative Research Company
(Novi, Michigan). ONO-5046, HNE substrate (MeO–Suc–Ala–Ala–Pro–Val–pNA), soybean
trypsin inhibitor and DMSO were obtained from Sigma (St. Louis, MO, USA) and used without
further purification. All other reagents were of analytical grade.
The assay on the direct inhibition of HNE activity was performed as previously described
(Shelkov et al. 2004), and carried out with ONO-5046 as positive control (Feng et al. 2012).
Briefly, 200 mL substrate solution (1.4 mM MeO–Suc–Ala–Ala–Pro–Val–pNA in Tris-HCl
buffer, 10 mM, pH 7.5) was mixed with different concentration gradients of the test compound
solution; stock solutions of triterpenes were dissolved in DMSO, which was restricted to 0.5%
and diluted with Tris-HCl buffer to produce the final sample concentrations. 0.01 U HNE was
added to the solution, and then the resultant mixture was incubated for 1 h at 378C in the dark.
Subsequently, the reaction was quenched by adding 200 mL soybean trypsin inhibitor at a
concentration of 0.2 mg/mL. The intensity of the colour developed by the digested substrate was
then immediately measured at 405 nm using an automatic microplate reader (Bio-Tek, Synergy
HT, San Diego, CA, USA), and the control background was subtracted. The group in which the
HNE substrate was replaced with 10 mM Tris-HCl buffer solutions of pH 7.5 was used as the
zero alignment of the test group. The inhibitory activity of the nigranoic acid aromatic ester
derivatives was derived using the following equation:
Acontrol 2 ðAsample 2 Azero
Þ
Inhibitory ratio ¼
£ 100%;
Acontrol
where Acontrol is the absorbance of the group containing only human NE and the substrate, Asample

6
G. Huang et al.
is the absorbance of the group containing the test compounds, the substrate and NE, and Azero is
the absorbance of the group containing the test compounds, Tris-HCl buffer solution and HNE.
The absorbance data were expressed as mean ^ standard deviation. The calculation was
carried out in triplicate in three separate experiments. The data were statistically analysed by
one-way ANOVA followed by Dunnett-t test using SPSS 16.0.
4. Conclusions
In conclusion, a series of 12 new nigranoic acid esters 3a–3l were efficiently prepared by
nigranoic acid with benzyl alcohols in excellent yields. Though HNE activity inhibitory bioassay
in vitro, nigranoic acid exhibited significant inhibitory activity against HNE with the IC50 value
of 3.77 mM, whereas nigranoic acid esters having phenyls substituted with bromine and
trimethoxyls (3h and 3b) showed slightly more inhibitory potency than that of nigranoic acid,
with the IC50 values of 2.61 and 3.25 mM, respectively. The nigranoic acid esters bearing
phenyls substituted with dimethoxyls and chlorine (3c and 3i), and thiophene (3j) showed
slightly less potency than that of nigranoic acid, with the IC50 values ranging from 3.89 to
4.83 mM. The nigranoic acid ester with cinnamenyl group (3g) showed the least inhibitory
potency (IC50 value: 8.95 mM) among the tested compounds. The other six analogues showed no
activity.
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1–S24 and
spectral data.
Funding
This work was financially supported by the Yunnan Province of China [grant number 2009CC018] for basic
research in social development and the applied basic research projects of Yunnan Province [grant number
2014FZ042].
Note
1. Guoli Huang and Li Feng contributed equally to this work.
References
Bergin DA, Greene CM, Sterchi EE, Kenna C, Geraghty P, Belaaouaj A, Taggart CC, O’Neill SJ, McElvaney NG. 2008.
Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway. J Biol Chem.
283:31–36.
Caldwell RA, Boucher RC, Stutts M. 2005. Stutts, Neutrophil elastase activates near-silent epithelial Naþ channels and
increases airway epithelial Naþ transport. Am J Physiol Lung Cell Mol Physiol. 288:L813–L819.
Chen YG, Qin GW, Cao L, Leng Y, Xie YY. 2001. Triterpenoid acids from Schisandra propinqua with cytotoxic effect
on rat luteal cells and human decidual cells in vitro. Fitoterapia. 72:435–437.
Dong JY, Chen YG, Song HC, He YP, Li L, Zhong YP, Zhu YH, Cao J, Wang L, Zhang KQ. 2007. Hydroxylation of
nigranoic acid to 6b-hydroxynigranoic acid by Caryospora carllicarpa YMF1.01026. Chin Chem Lett.
18:165–167.
Dong JY, Chen YG, Song HC, Zhu YH, Zhou YP, Li L, He YP, Cao J, Zhang KQ. 2007. Hydroxylation of the
triterpenoid nigranoic acid by the fungus Gliocladium roseum YMF1.00133. Chem Biodivers. 4:112–117.
Feng L, Zhu W, Huang C, Li Y. 2012. Direct interaction of ONO-5046 with human neutrophil elastase through 1H NMR
and molecular docking. Int J Biol Macromol. 51:196–200.
Fitch PM, Roghanian A, Howie SE, Sallenave JM. 2006. Human neutrophil elastase inhibitors in innate and adaptive
immunity. Biochem Soc Trans. 34:279–282.

Natural Product Research
7
Huang W, Yamamoto Y, Li Y, Dou D, Alliston KR, Hanzlik RP, Williams TD, Groutas WC. 2008. X-ray snapshot of the
mechanism of inactivation of human neutrophil elastase by 1,2,5-thiadiazolidin-3-one 1,1-dioxide derivatives.
J Med Chem. 51:2003–2008.
Melzig MF, Loser B, Ciesielski S. 2001. Inhibition of neutrophil elastase activity by phenolic compounds from plants.
Pharmazie. 56:967–970.
Ohbayashi H. 2002. Neutrophil elastase inhibitors as treatment for COPD. Expert opinion on investigational drugs.
11:965–980.
Pham CT. 2006. Neutrophil serine proteases: specific regulators of inflammation. Nat Rev Immunol. 6:541–550.
Rubin H. 1996. Serine protease inhibitors (SERPINS): where mechanism meets medicine. Nat Med. 2:632–633.
Shelkov R, Nahmany M, Melman A. 2004. Selective esterifications of alcohols and phenols through carbodiimide
couplings. Org Biomol Chem. 2:397–401.
Siedle B, Hrenn A, Merfort I. 2007. Natural compounds as inhibitors of human neutrophil elastase. Planta Med.
73:401–420.
˚
Stevens T, Ekholm K, Granse M, Lindahl M, Kozma V, Jungar C, Ottosson T, Falk-Hakansson H, Churg A, Wright JL,
et al. 2011. AZD9668: pharmacological characterization of a novel oral inhibitor of neutrophil elastase.
J Pharmacol Exp Ther. 339:313–320.
Sun HD, Qiu SX, Lin LZ, Wang ZY, Lin ZW, Pengsuparp T, Pezzuto JM, Fong HHS, Cordell GA, Farnsworth NR. 1996.
Nigranoic acid, a triterpenoid from Schisandra sphaerandra that inhibits HIV-1 reverse transcriptase. J Nat Prod.
59:525–527.
Sun R, Song HC, Wang CR, Shen KZ, Xu YB, Gao YX, Chen YG, Dong JY. 2011. Compounds from Kadsura
angustifolia with anti-HIV activity. Bioorg Med Chem Lett. 21:961–965.
Williams SE, Brown TI, Roghanian A, Sallenave JM. 2006. SLPI and elafin: one glove, many fingers. Clin Sci (Lond).
110:21–35.
Wright JL, Farmer SG, Churg A. 2002. Synthetic serine elastase inhibitor reduces cigarette smoke-induced emphysema
in guinea pigs. Am J Respir Crit Care Med. 166:954–960.
Xiao WL, Tian RR, Pu JX, Li X, Wu L, Lu Y, Li SH, Li RT, Zheng YT, Zheng QT, Sun HD. 2006. Triterpenoids from
Schisandra lancifolia with anti-HIV-1 activity. J Nat Prod. 69:277–279.
Xu LJ, Peng ZG, Chen HS, Wang J, Xiao PG. 2010. Bioactive triterpenoids from Kadsura heteroclite. Chem Biodivers.
7:2289–2295.
Yang YH, Sun R, Song HC, Xu YB, Yang P, Yang DY, Shen ZK, Wang AR, Chen YG, Dong JY. 2012. Microbial
transformation of the triterpene nigranoic acid in Trichoderma sp. Phytochem Lett. 5:123–127.