Synthesis and evaluation of inhibitory activity in vitro of dihydrostilbenes against human neutrophil elastases

Article abstract of DOI:10.1080/10286020.2019.1571489

Twelve dihydrostilbenes (including five new analogues) were prepared by the Witting-Hornor reaction from appropriate aromatic aldehydes and phosphonate esters, followed by hydrogenation in five steps. The in vitro inhibition activity against human neutrophil elastase (HNE) of these dihydrostilbenes was evaluated, and three 1,2-dihydroxylated dihydrostilbenes (6b, 6j, and 6l) exhibited stronger inhibitory activity against HNE than other analogues.

Full text of DOI:10.1080/10286020.2019.1571489

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Synthesis and evaluation of inhibitory activity in
vitro of dihydrostilbenes against human neutrophil
elastases
Guo-Li Huang, Bo Liu, Xing-Jun Tian & Ye-Gao Chen
To cite this article: Guo-Li Huang, Bo Liu, Xing-Jun Tian & Ye-Gao Chen (2019): Synthesis and
evaluation of inhibitory activity inꢀvitro of dihydrostilbenes against human neutrophil elastases,
Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2019.1571489
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2019.1571489
Synthesis and evaluation of inhibitory activity in vitro of
dihydrostilbenes against human neutrophil elastases
a
a,b
b
a
ꢀ
ꢀ
Guo-Li Huang , Bo Liu , Xing-Jun Tian and Ye-Gao Chen
aSchool of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650050, China;
bSchool of Life Sciences, Nanjing University, Nanjing 210023, China
ABSTRACT
ARTICLE HISTORY
Received 22 November 2018
Accepted 15 January 2019
Twelve dihydrostilbenes (including five new analogues) were pre-
pared by the Witting-Hornor reaction from appropriate aromatic
aldehydes and phosphonate esters, followed by hydrogenation in
five steps. The in vitro inhibition activity against human neutrophil
elastase (HNE) of these dihydrostilbenes was evaluated, and three
1,2-dihydroxylated dihydrostilbenes (6b, 6j, and 6l) exhibited
stronger inhibitory activity against HNE than other analogues.
KEYWORDS
Dihydrostilbenes; synthesis;
human neutrophil
elastase; inhibition
1. Introduction
Dihydrostilbenes have long been considered as an important class of natural products,
which had been isolated from many primitive green plants. Recently, some dihydros-
tilbenes attract increasing interest chiefly due to their various biological activities such
as antimitotic [1], anti-oxidant [2], anti-tumor [3], cancer chemopreventive effect [4],
inhibition of cyclooxygenase [5], inhibition of platelet aggregation [6], nitric oxide
production [7], and melanogenesis in B16F0 melanoma cells [8]. In addition, some
synthetic dihydrostilbenes were found to be tubulin polymerization inhibitors and to
show strong antimitotic activity toward a broad spectrum of human cancer lines [9].
Furthermore, the primary serine proteinase is human neutrophil elastase (HNE) in
the azurophilic granules of neutrophils, which is one of the destructive enzymes with
CONTACT Ye-Gao Chen
ygchen48@126.com; Xing-Jun Tian
tianxj@nju.edu.cn
ꢀ
These authors contributed equally to this work.
Supplemental data for this article can be accessed here.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
G.-L. HUANG ET AL.
optimal activity around neutral pH. The endogenous inhibitors could control the
HNE release in the physiological defense system. This balance between the amount of
HNE and endogenous inhibitors can be upset; however, as a result of intense neutro-
phil infiltration in some pathological conditions [10,11]. The superabundant HNE
leads to the degradation of the tissue matrix and the deterioration of inflammation in
various disease states, including pulmonary emphysema, cystic fibrosis, acute lung
injury (ALI), acute respiratory distress syndrome (ARDS), and other inflammatory
processes. Therefore, HNE plays an important role in a pathogenic agent in various
disease processes, which has aroused interest in searching for appropriate and poten-
tial human neutrophil elastase inhibitors (HNEI) [12,13].
In our previous study, 12 new nigranoic acid esters were synthesized and evaluated
for HNE inhibitory activity. Those phenyls with bromine and trimethoxyls substitu-
tion showed strongly inhibitory activity on HNE [14]. Our continued interest in
searching for new HNEI prompted us to develop an efficient total synthetic route for
the natural dihydrostilbenes, and the activities of these compounds were determined
through HNE inhibitory activity bioassay, and some compounds exhibited consider-
able inhibitory effects on HNE.
2. Results and discussion
2.1. Chemistry
The general approach to the synthesis of dihydrostilbenes 6 was described in Scheme 1.
After the hydroxy groups were protected by benzyl bromide in acetone under reflux
to give the substituted aldehydes 2, then reduction of 2 by sodium borohydide in
methanol gave the substituted benzyl alcohols (3) in a quantitative yield.
Phosphonate esters 4 were prepared from 3 by treating with phosphonate tribromide
and then by triethyl phosphite in 78–89% yields. A Wittig-Hornor reaction of
phosphonate esters 4 with substituted aldehydes, in the presence of sodium hydride
afforded trans-stilbenes 5 in moderate-good yields. 5 was catalytically hydrogenated
over palladium-charcoal and the benzyl protecting groups were also removed by the
CHO
CHO
CH2OH
a
PO(OC2H5)2
b
c, d
Ar1
Ar1
R
R
R1
R1
OH
OCH2Ph
(R1 = OCH3, OCH2O, OCH2Ph)
(R1 = OCH3, OCH2O, OCH2Ph)
(R = H, OCH3)
(R = H, OCH3)
4
3
1
2
Ar2
Ar2
e
R2
R2
f
R1
R1
Ar1
Ar1
(R1 = OCH3, OCH2O, OCH2Ph)
(R1 = OCH3, OCH2O, OH, OCH2Ph)
(R2 = OCH3, OCH2Ph)
(R2 = OCH3, OH)
5
6
Scheme 1. Synthesis of dihydrostilbenes. Reagents and conditions: (a) PhCH2Br, K2CO3, acetone,
reflux, 12 h, 92–99% yields; (b) NaBH4, CH3OH, rt, 1 h, 98–99% yields; (c) PBr3, THF/benzene, rt, 3 h;
(d) P(OC2H5)3, 140 ꢁC, 3 h, 78–89% yields; (e) Ar2-CHO, NaH, THF, 0 ꢁC, 1 h, rt, 12 h, 53–98% yields;
(f) H2, Pd/C, EtOAc, rt, 12 h, 63–96% yields.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
hydrogenolysis to give the target product 6. Interestingly, under the hydrogenation
for 5b, an unexpected product 6l was obtained, in which the double bond was
reduced but one benzyl protecting group was not removed. The structures of the syn-
1
thesized compounds were confirmed by H and 13C NMR spectra (Table 1).
2.2. HNE activity
The potential inhibitory activity of all newly synthesized dihydrostilbenes was eval-
uated in vitro against HNE according to procedures described in the literature
[14,15]. The inhibitory ratio (%) of the dihydrostilbenes was investigated from the
tested concentration gradients at 1, 3, 10, 30, and 100 lM, respectively, and the results
were displayed in Figure 1. As positive control, sivelestat sodium (ONO-5046) exhib-
ited potent inhibitory activity, with an IC50 value of 87.05 nM. HNE activity was
strongly and directly inhibited by three dihydrostilbenes (6b, 6j, and 6l) with adjacent
dihydroxylated aromatic ring, while the other nine analogues were less effective
against the HNE activity. Compound 6l exhibited a considerable inhibitory effect on
HNE in a dose-dependent manner, with a minimal effective concentration of 1 lM
(26% inhibition), and compounds 6b and 6j showed slightly weak inhibitory potency
than 6s, with the inhibitory ratio values 16 and 17%, respectively. But it was interest-
ing to note that compound 6j displayed the HNE inhibition with more than 59% at
30 lM, and this strongly suggests that adjacent dihydroxylation of dihydrostilbenes
will increase the inhibitory effect on HNE.
In conclusion, a series of dihydrostilbenes prepared in this research were proved to
be a significant inhibitory activity against human neutrophil elastase. The dihydrostil-
benes 6b, 6j, and 6l, bearing adjacent dihydroxyl at 3- and 4-position of the phenyl
Table 1. Synthesis and structure of dihydrostilbenes (6a-l).
Entry
1
Structure
Producta (yield %)b [ref.]c Entry
Structure
Producta (yield %)b [ref.]c
OH
6a (83) [16]
7
6g (72) [19]
H3CO
H3CO
OH
OH
OCH3
OH
OH
OH
2
6b (65)
8
6h (73) [20]
H3CO
H3CO
OH
OH
OH
OCH3
OCH3
OH
3
4
6c (77) [17]
6d (78)
9
6i (86) [21]
6j (90)
OH
H3CO
OH
10
O
O
OH
OH
OH
H3CO
OCH3
OH
5
6
6e (90)
11
12
6k (84) [17]
6l (24)
O
O
OH
H3CO
OCH3
OH
OH
OH
6f (88) [18]
O
O
O
aAll the products are confirmed by NMR.
bYields refer to pure products after column chromatography.
cRated references.

4
G.-L. HUANG ET AL.
70
60
50
40
30
20
10
0
6b
6j
6l
1
3
10
30
100
Concentration (μM)
Figure 1. HNE inhibitory activity of 6b, 6j, and 6l. The values of the inhibitory ratio are presented
as mean standard deviation (SD).
ring, were found to be the most potent compounds. Compound 6s was found to be
the most potent derivative against human neutrophil elastase. The bearing adjacent
dihydroxyls of dihydrostilbenes 6b, 6j, and 6l can be considered as promising leads
for further structural modifications guided by the valuable information from our
detailed HNE inhibitors.
3. Experimental
3.1. General experimental procedures
1H and 13C NMR spectra were recorded on a Bruker AVANCE III 500 MHz spec-
trometer (Bruker BioSpin Group, Rheinstetten, Germany) in CDCl3 solution. ESI-MS
and HRESIMS were measured on Finnigan Mat TSQ 7000 instruments (VG PRIMA,
Birmingham, UK). TLC analyses were performed on commercial glass plates bearing
0.25-mm layer of Merck Silica gel 60F254. Silica gel (200–300 mesh) was used for col-
umn chromatography. Unless otherwise noted, the starting materials were either com-
mercially available from Aladdin in Shanghai, China or synthesized according to the
references cited. Solvents were freshly distilled prior to use. All the products (except
6b, 6d, 6e, 6j, and 6l) are known compounds [16–21], which were characterized by
NMR and HRESIMS data.
3.2. General procedure for the synthesis of dihydrostibene (6a-l)
A solution of 5 (0.5 mmol) in EtOAc (10 ml) and MeOH (2 ml) was stirred under a
hydrogen atmosphere in the presence of 10% Pd/C (0.03 g) at room temperature
overnight. The catalyst was removed by filtration and washed with EtOAc. The fil-
trate was concentrated in vacuo and the crude product was purified by column chro-
matography to give the product (6l was the side product of hydrogenation of 5b).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
3.2.1. 4-(2-Hydroxyphenethyl)benzene-1,2-diol (6b)
1
White solid. Yield: 65%. H NMR (500 MHz, CDCl3) d 7.10–7.07 (m, 2H, Ar-H),
6.86 (td, J ¼ 7.5, 1.0 Hz, 1H, Ar-H), 6.78 (d, J ¼ 8.0 Hz, 1H, Ar-H), 6.76–6.73 (m, 1H,
Ar-H), 6.71 (d, J ¼ 2.0 Hz, 1H, Ar-H), 6.63 (dd, J ¼ 8.0, 2.0 Hz, 1H, Ar-H), 5.21 (s,
1H, –OH), 5.08 (s, 1H, –OH), 4.63 (s, 1H, –OH), 2.90–2.84 (m, 2H, –CH2CH2–),
2.83–2.77 (m, 2H, –CH2CH2–). 13C NMR (125 MHz, CDCl3) d 153.7, 143.6, 141.8,
135.3, 130.5, 128.0, 127.5, 121.1, 121.0, 115.8, 115.6, 115.6, 35.7, 32.6. HRESIMS: m/z
229.0898 [M ꢂ H]ꢂ (calcd for C14H13O3, 229.0870).
3.2.2. 2-(2-(Benzo[d][1, 3]dioxol-5-yl)ethyl)phenol (6d)
1
White solid. Yield: 78%. H NMR (500 MHz, CDCl3) d 7.09 (t, J ¼ 7.1 Hz, 2H, Ar-H),
6.86 (t, J ¼ 7.4 Hz, 1H, Ar-H), 6.77–6.68 (m, 3H, Ar-H), 6.64 (dd, J ¼ 7.9, 1.1 Hz, 1H,
Ar-H), 5.92 (s, 2H, –OCH2O–), 4.63 (s, 1H, -OH), 2.89–2.81 (m, 4H, –CH2CH2–).
13C NMR (125 MHz, CDCl3) d 153.6, 147.7, 145.8, 136.0, 130.5, 127.9, 127.5, 121.3,
121.0, 115.5, 109.1, 108.3, 100.9, 36.1, 32.7. HRESIMS: m/z 241.0873 [M ꢂ H]ꢂ (calcd.
for C15H13O3, 241.0870).
3.2.3. 4-(2-(Benzo[d][1, 3]dioxol-5-yl)ethyl)benzene-1,2-diol (6e)
1
White solid. Yield: 90%. H NMR (500 MHz, CDCl3) d 6.76 (d, J ¼ 8.0 Hz, 1H, Ar-
H), 6.71 (d, J ¼ 7.9 Hz, 1H, Ar-H), 6.67 (d, J ¼ 8.0 Hz, 2H, Ar-H), 6.60 (d, J ¼ 8.0 Hz,
2H, Ar-H), 5.92 (s, 2H, –OCH2O–), 5.19 (s, 1H, –OH), 5.07 (s, 1H, –OH), 2.82–2.71
(m, 4H, –CH2CH2–). 13C NMR (125 MHz, CDCl3) d 147.6, 145.8, 143.6, 141.7, 135.8,
135.0, 121.4, 121.0, 115.7, 115.4, 109.1, 108.2, 100.9, 37.9, 37.6. HRESIMS: m/z
257.0853 [M ꢂ H]ꢂ (calcd. for C15H13O4, 257.0819).
3.2.4. 4-(4-Methoxyphenethyl)benzene-1,2-diol (6j)
1
White solid. Yield: 90%. H NMR (500 MHz, CDCl3) d 7.07 (d, J ¼ 8.5 Hz, 2H, Ar-
H), 6.82 (d, J ¼ 8.5 Hz, 2H, Ar-H), 6.76 (d, J ¼ 8.0 Hz, 1H, Ar-H), 6.68 (d, J ¼ 1.9 Hz,
1H, Ar-H), 6.60 (dd, J ¼ 8.1, 1.9 Hz, 1H, Ar-H), 5.21 (s, 1H, –OH), 5.10 (s, 1H,
–OH), 3.79 (s, 3H, –OCH3), 2.85–2.79 (m, 2H, –CH2CH2–), 2.79–2.73 (m, 2H,
–CH2CH2–). 13C NMR (125 MHz, CDCl3) d 157.9, 143.5, 141.7, 135.2, 134.1, 129.5,
129.5, 121.0, 115.8, 115.4, 113.9, 113.9, 55.4, 37.6, 37.2. HRESIMS: m/z 243.1048
[M ꢂ H]ꢂ (calcd for C15H15O3, 243.1027).
3.2.5. 4-(2-(Benzyloxy)phenethyl)benzene-1,2-diol (6l)
White solid. Yield: 24%. 1H NMR (500 MHz, CDCl3) d 7.47 (d, J ¼ 7.1 Hz, 2H),
7.43–7.40 (m, 2H), 7.35 (t, J ¼ 7.2 Hz, 1H), 7.21–7.14 (m, 1H), 7.13 (dd, J ¼ 7.4,
1.5 Hz, 1H), 6.96–6.86 (m, 2H), 6.74 (d, J ¼ 7.9 Hz, 1H), 6.62–6.55 (m, 2H), 5.09 (s,
2H), 5.01 (s, 1H), 4.99 (s, 1H), 2.90 (m, 2H), 2.78 (m, 2H). 13C NMR (125 MHz,
CDCl3) d 156.8, 143.3, 141.6, 137.6, 135.8, 130.8, 130.2, 128.7, 128.7, 127.9, 127.5,
127.3, 127.3, 121.1, 120.9, 115.8, 115.3, 111.8, 70.1, 35.8, 33.2. HRESIMS: m/z
319.1383 [M ꢂ H]ꢂ (calcd. for C21H19O3, 319.1340).

6
G.-L. HUANG ET AL.
Acknowledgments
The authors thank Prof. Yiming Li and Dr. Li Feng at Shanghai University of Traditional
Chinese Medicine for providing HNE activity tests.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This investigation was financially supported by a grant (Nos 31260085 and 21861043) from
the Natural Science Foundation of China, the Key Laboratory (No. 2016-5), and Science and
Technology Innovation Team of Higher Education (No. 2014-6) in Yunnan, China.
References
[1] M. Cushman, D. Nagarathnam, D. Gopal, A.K. Chakraborti, C.M. Lin, and E. Hamel,
J. Med. Chem. 34, 2579 (1991).
[2] X. Zhang, J.K. Xu, J. Wang, N.L. Wang, H. Kurihara, S. Kitanaka, and X.S. Yao, J. Nat.
Prod. 70, 24 (2007).
[3] G.R. Pettit, S.B. Singh, E. Hamel, C.M. Lin, D.S. Alberts, and D. Garcia-Kendal,
Experientia 45, 209 (1989).
ꢀ
ꢀ
[4] P. Waffo-Teguo, M.E. Hawthorne, M. Cuendet, J.M. Merillon, A.D. Kinghorn, J.M.
Pezzuto, and R.G. Mehta, Nutr. Cancer 40, 173 (2001).
[5] K. Likhitwitayawuid, K. Sawasdee, and K. Kirtikara, Planta Med. 68, 841 (2002).
[6] C.R. Pace-Asciak, O. Rounova, S.E. Hahn, E.P. Diamandis, and D.M. Goldberg, Clin.
Chim. Acta 246, 163 (1996).
[7] J.S. Hwang, S.A. Lee, S.S. Hong, X.H. Han, C. Lee, S.J. Kang, D. Lee, Y. Kim, J.T.
Hong, M.K. Lee, and B.Y. Hwang, Bioorg. Med. Chem. Lett. 20, 3785 (2010).
[8] C. Oode, W. Shimada, Y. Izutsu, M. Yokota, T. Iwadate, and K. Nihei, Eur. J. Med.
Chem. 87, 862 (2014).
[9] F. Mu, E. Hamel, D.J. Lee, D.E. Pryor, and M. Cushman, J. Med. Chem. 46, 1670
(2003).
[10] B. Gooptu, U.I. Ekeowa, and D.A. Lomas, Eur. Respir. J. 34, 475 (2009).
[11] F. von Nussbamum, and V.M.-J. Li, Bioorg. Med. Chem. Lett. 25, 4370 (2015).
[12] H. Yamamoto, T. Koizumi, T. Kaneki, M. Hanaoka, and K. Kubo, Eur. J. Pharmacol.
409, 179 (2000).
[13] S.D. Shapiro, Am. J. Respir. Cell Mol. Biol. 26, 266 (2002).
[14] G. Huang, L. Feng, B. Liu, Y. He, Y. Li, and Y. Chen, Nat. Prod. Res. 29, 1650 (2015).
[15] L. Feng, W. Zhu, C. Huang, and Y. Li, Int. J. Biol. Macromol. 51, 196 (2012).
[16] R.B. Bates and T.J. Siahaan, J. Org. Chem. 51, 1432 (1986).
[17] M. Yoshikatsu, K. Shigehiko, K. Hiroyuki, T. Chiyozo, N. Akira, and S. Shinichi, Chem.
Senses 14, 781 (1989).
[18] D.R. Benedicto, C.R. Angel, C. Esther, I. Alba, Y. Gloria, M. Manuel, D.A. Antonieta
Rojas, and S.F. Arturo, Bioorg. Med. Chem. Lett. 9, 2711 (1999).
[19] H. Li, S. Jean, D. Webster, G.A. Robichaud, L.A. Calhoun, J.A. Johnson, and C.A. Gray,
J. Nat. Prod. 78, 2837 (2015).
[20] M. Manuel, P.-L.C. Rafael, L. Jose Luis, and A. San Feliciano, J. Nat. Prod. 57, 1136
(1994).
[21] T. Naoki, G. Riki, I. Rie, H. Atsuhiro, O. Taketoshi, A. Fumitoshi, and F. Koichi,
Chem. Pharm. Bull. 48, 1729 (2000).