Novel derivatives of salicylanilide: Synthesis, characterization, PPO inhibitory activity and cytotoxicity

Article abstract of DOI:10.1016/j.molstruc.2020.128995

Two series of novel salicylanilide analogues (4a-4 g) and (4h-4t) were designed, synthesized, and characterized, especially the structure of compound 4q was further confirmed by single X-ray diffraction. The inhibitory activity on polyphenoloxidase (PPO)

Full text of DOI:10.1016/j.molstruc.2020.128995

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Journal of Molecular Structure 1223 (2020) 128995
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Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Novel derivatives of salicylanilide: Synthesis, characterization, PPO
inhibitory activity and cytotoxicity
Hua Fang a, Honghui Guoa, Hui Chena, Jianyu Zhangb, Zhuan Honga,∗, Meijuan Fang b,∗
a Third Institute of Oceanography, Ministry of Natural Resources, Technical Innovation Center for Utilization of Marine Biological Resources, Ministry of
Natural Resources, Xiamen, 361005, China
b Fujian Provincial Key Laboratory of Innovative Drug Target Research and State Key Laboratory of Cellular Stress Biology, School of Pharmaceutical Sciences,
Xiamen University, Xiamen 361102, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of novel salicylanilide analogues (4a-4 g) and (4h-4t) were designed, synthesized, and char-
acterized, especially the structure of compound 4q was further confirmed by single X-ray diffraction. The
inhibitory activity on polyphenoloxidase (PPO) from the cephalothorax of Chinese white shrimp (Fen-
neropenaeus chinensis) was evaluated. The result indicated that all the synthesized derivatives (except
Received 11 June 2020
Revised 21 July 2020
Accepted 28 July 2020
Available online 29 July 2020
4 g and 4t) exhibited moderate PPO inhibitory properties having IC50 values in the range of 0.21
0.19
to 4.32 0.53 mM, whereas reference inhibitor salicylic acid and 3a have IC50 values 3.93 0.43 mM
Keywords:
Synthesis
and 12.83 0.51 mM, respectively. Specifically, 4-nitro-benzoic acid 3-(2–hydroxy–benzoylamino)- propyl
ester (4q) exhibited the most potent PPO inhibitory activity with an IC50 value of 0.21 0.19 mM. The ki-
netic studies of the compound (4q) demonstrated that the inhibitory effects of the compound on the PPO
were belonging to competitive inhibitors. Meanwhile, the structure-activity relationship was discussed.
Therefore, compound 4q could act as a PPO inhibitor with anti-browning and antimicrobial properties to
be applied in the food industry.
Salicylanilide analogues
Crystal
Ppo inhibitory
Cytotoxicity
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
activities [9–11]. For example, salicylanilide benzoates [12], phenyl
salicylanilide carbamates [13,14], dihalogenated salicylanilide car-
Polyphenoloxidase (PPO) sometimes referred to as phenoloxi-
dase, catecholase, phenolase, catechol oxidase, or even tyrosinase,
is considered to be an o-diphenol. PPO (EC 1.14.18.1) is a multi-
functional copper-containing enzyme that is widely distributed
in microorganisms, plants, and animals [1–5]. PPO catalyzes both
the hydroxylation of monophenols and the oxidation of diphenols
into quinones, followed by non-enzymatic polymerization and
autoxidation of quinones. It plays a crucial role in the formation of
melanins or high molecular weight dark pigments [6,7]. Phenolic
compounds have been investigated widely because of their poten-
tial pharmacological and biological properties. The free phenolic
hydroxyl on the salicylic acid moiety is a functional group and is
required for the activity that might function as proton shuttles that
kill bacterial cells by destroying the cellular proton gradient [8]. 2-
Hydroxy-N-phenylbenzamide, commonly known as salicylanilide,
is a weakly acidic phenolic compound and has been highly no-
ticed due to its significance in vitro antibacterial and antioxidant
bamates [15] have antibacterial activities, acetylcholinesterase
inhibitory activities, and antimycobacterial activities, respectively.
Nowadays, the inhibitory activity of salicylanilide analogues on
PPO from cephalothoraxes of Chinese white shrimp is rarely re-
ported. We herein synthesized a series of salicylanilide analogues
by the reaction of salicylic acid propanolamine with the corre-
sponding acyl chloride in Et3N-contained solvents and evaluated
their inhibitory activity on PPO isolated from cephalothoraxes of
shrimp. Our ongoing research in this study includes the expan-
sion of new salicylanilide derivatives and the evaluation of PPO-
inhibitory activities. The designed salicylanilide compounds mainly
focus on increasing their lipophilicity, which could decrease its cy-
totoxicity in cells and improve biological cell permeability [16].
Although lipophilicity seems to be one of the factors modulating
positively the inhibition of PPO activity, an escalated lipophilic-
ity could hamper in vitro evaluation due to solubility problems
in testing media [17]. It hoped that these findings can lead to
the discovery of potential preservative agents for the food indus-
try and also offer key and useful information for the further de-
sign of highly potent PPO inhibitors. The structures of previously
published salicylanilide carbamates in comparison with novel sal-
∗
Corresponding authors.
E-mail addresses: hfang@tio.org.cn (H. Fang), zhong@tio.org.cn (Z. Hong),
fangmj@xmu.edu.cn (M. Fang).
https://doi.org/10.1016/j.molstruc.2020.128995
0022-2860/© 2020 Elsevier B.V. All rights reserved.

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H. Fang, H. Guo and H. Chen et al. / Journal of Molecular Structure 1223 (2020) 128995
Fig. 1. Design of salicylic acid derivatives as PPO inhibitor.
icylanilide derivatives and the concept of design are depicted in
Fig. 1.
(2.4 mmol) [19]. The reaction was stirred overnight in the ice bath
and concentrated in vacuo. The residue was purified by a silica gel
column chromatography with petroleum ether/ethyl acetate (3:1,
v/v) as eluent. Compounds 4a-4d and 4r are pale yellow oil. The
solid product was then recrystallized from ethyl acetate to give 4e-
4t (except 4r) as colorless crystal in yield of 59–88% (Scheme 1).
Acetic acid 3-(2–hydroxy–benzoylamino)-propyl ester (4a), pale
yellow oil, yield: 77%; 1H NMR (400 MHz, DMSO–d6): δ 12.59 (s,
1H, NH), 8.84 (s, 1H, OH), 7.82–7.80 (m, 1H, Ar), 7.38–7.36 (m, 1H,
Ar), 6.89–6.85 (m, 2H, Ar), 4.06–4.03 (m, 2H, OCH2), 3.38–3.33 (m,
2H, NHCH2), 1.98 (s, 3H, CH3), 1.86–1.83 (m, 2H, CH2), 13C NMR
(100 MHz, DMSO–d6): δ 170.9, 169.5 (C = O), 160.6, 134.1, 128.0,
119.0, 117.8, 115.6 (Ar), 62.3 (OCH2), 36.4 (NHCH2), 28.5, 21.1. HRMS
(ESI, m/z): [M + H]+ for C12H16 NO4+, calculated 238.1074, found
238.1069.
2. Materials and methods
2.1. Chemicals and instruments
L-(3,4-dihydroxylphenyl)-alanine
(L-DOPA),
triethylamine
(Et3N), ammonium sulfate, methyl salicylate, 4-dimethylamino-
pyridine (DMAP), Brij 35 and 3-amino-1-propanol were purchased
from Sigma-Aldrich (St. Louis, MO, USA). Other reagents used in
this study were purchased from the domestic market and were all
analytical purity used without further purification.
Melting points were determined on an SWG X-4 melting point
apparatus and were uncorrected. Visible light absorption was
tested on a UH5300 spectrophotometer (Hitachi, Japan). All NMR
spectra were recorded on a Bruker 400 MHz NMR spectrometer,
and DMSO–d6 was used as the solvent. High-resolution mass spec-
tra (HRMS) data were acquired using a quadrupole-orbitrap (Q-
Exactive) mass spectrometer (Thermo, UK) equipped with a heated
electrospray ionization source (HESI-Ⅱ). X-ray crystallographic data
were collected on the Bruker X-ray diffractometer.
Butyric acid 3-(2–hydroxy–benzoylamino)-propyl ester (4b),
pale yellow oil, yield: 66%; 1H NMR (400 MHz, DMSO–d6): δ 12.77
(s, 1H, NH), 9.00 (s, 1H, OH), 7.99–7.97 (m, 1H, Ar), 7.56–7.52 (m,
1H, Ar), 7.05–7.00 (m, 2H, Ar), 4.23–4.20 (m, 2H, OCH2), 3.52–
3.49 (m, 2H, NHCH2), 2.41–2.32 (m, 2H, CH2), 2.04–1.97 (m, 2H,
CH2), 1.72–1.62 (m, 2H, CH2), 1.04–1.02 (m, 3H, CH3); 13C NMR
(100 MHz, DMSO–d6): δ 174.8, 169.6 (C = O), 160.6, 134.1, 128.0,
118.9, 117.8, 115.5 (Ar), 62.2 (OCH2), 36.4 (NHCH2), 36.0, 28.5,
18.4, 14.0. HRMS (ESI, m/z): [M + H]+ for C14 H20NO4+, calculated
266.1387, found 266.1381.
2.2. Synthesis of target compounds 4a-4t
The synthetic strategy of compounds 3a and 4a-4t were de-
picted in Scheme 1.
Hexanoic acid 3-(2–hydroxy–benzoylamino)-propyl ester (4c),
pale yellow oil, yield: 59%, 1H NMR (400 MHz, DMSO–d6): δ 12.70
(s, 1H, NH), 8.47 (s, 1H, OH), 7.68–7.64 (m, 2H, Ar), 7.48–7.44 (m,
1H, Ar), 7.31–7.29 (m, 1H, Ar), 4.21–4.18 (m, 2H, OCH2), 3.49–
3.38 (m, 2H, NHCH2), 2.67–2.63 (m, 2H, CH2), 1.94–1.74 (m, 2H,
CH2), 1.48–1.40 (m, 6H, 3CH2), 1.05–0.99 (m, 3H, CH3); 13C NMR
(100 MHz, DMSO–d6): δ 173.4, 171.8 (C = O), 165.9, 148.2, 131.4,
130.2, 129.2, 123.6 (Ar), 62.1 (OCH2), 36.3 (NHCH2), 33.8, 31.1, 28.7,
24.6, 22.2, 14.2. HRMS (ESI, m/z): [M + H]+ for C16 H24NO4+, calcu-
lated 294.1700, found 294.1694.
The procedure for the synthesis of 2–hydroxy–N-(3–hydroxy–
alkyl)-benzamide 3a was as follows: In a 100 mL round bottom
flask, 3.04 g (0.02 mol) methyl salicylate and 3.00 g (0.04 mol)
3-amino-1-propanol were added [18]. The reaction mixture was
stirred for 24 h at 80 °C, and the completion of the reaction
was monitored by TLC. After completion of the reaction, the mix-
ture was extracted with ethyl acetate and tert–pentyl alcohol
(v/v = 1:1), the combined organic layer was dried over anhy-
drous MgSO4 and concentrated in vacuo. The slurry residue was
further purified by silica gel column chromatography (petroleum
ether/ethyl acetate 1:5) to give 2–hydroxy–N-(3–hydroxy–propyl)-
benzamide 3a as colorless solid in a yield of 80%.
Octanoic acid 3-(2–hydroxy–benzoylamino)-propyl ester (4d),
pale yellow oil, yield: 87%, 1H NMR (400 MHz, DMSO–d6): δ
12.62 (s, 1H, NH), 8.84 (s, 1H, OH), 7.83–7.81 (m, 1H, Ar), 7.39–
7.35 (m, 1H, Ar), 6.88–6.84 (m, 2H, Ar), 4.07–4.04 (m, 2H, OCH2),
3.38–3.33 (m, 2H, NHCH2), 2.28–2.22 (m, 2H, CH2), 1.98–1.88 (m,
2H, CH2), 1.50–1.46 (m, 2H, CH2), 1.26–1.14 (m, 8H, 4CH2), 0.84–
0.81 (m, 3H, CH3); 13C NMR (100 MHz, DMSO–d6): δ 173.4, 169.6
(C = O), 160.6, 134.1, 128.0, 118.9, 117.8, 115.5 (Ar), 62.2 (OCH2),
36.5 (NHCH2), 33.9, 31.6, 28.9, 28.8, 28.5, 24.9, 22.5, 14.3. HRMS
(ESI, m/z): [M + H]+ for C18 H28NO4+, calculated 322.2013, Found
322.2007.
2-Hydroxy-N-(3–hydroxy–propyl)-benzamide (3a), m.p. 83.7–
85.2 °C; 1H NMR (400 MHz, DMSO–d6): δ 12.84 (br, 1H, NH), 8.81
(s, 1H, OH), 7.83–7.80 (m, 1H, Ar), 7.37–7.35 (m, 1H, Ar), 6.89–
6.84 (m, 2H, Ar), 4.02 (s, 1H, OH), 3.47–3.44 (m, 2H, OCH2), 3.34–
3.31 (m, 2H, NHCH2), 1.72–1.66 (m, 2H, CH2); 13C NMR (100 MHz,
DMSO–d6): δ 169.4 (C = O), 160.6, 134.0, 128.0, 118.9, 117.8, 115.6
(Ar), 59.0 (OCH2), 36.9 (NHCH2), 32.5. HRMS (ESI, m/z): [M + H]+
for C10H14 NO3+, calculated 196.0968, found 196.0966.
Dodecanoic acid 3-(2–hydroxy–benzoylamino)-propyl ester (4e),
white solid, m.p. 34.3–34.9, yield: 64%, 1H NMR (400 MHz, DMSO–
d6): δ 12.78 (s, 1H, NH), 8.98 (s, 1H, OH), 7.98–7.96 (m, 1H, Ar),
7.55–7.51 (m, 1H, Ar), 7.04–7.00 (m, 2H, Ar), 4.23–4.19 (m, 2H,
OCH2), 3.52–3.49 (m, 2H, NHCH2), 2.42–2.32 (m, 2H, CH2), 2.03–
1.98 (m, 2H, CH2), 1.66–1.62 (m, 2H, CH2), 1.38–1.32 (m, 16H,
The general procedure for the synthesis of target compounds
4a-4t was as follows: In a 50 mL round bottom flask, 975 mg
(5 mmol) of intermediate 3a, DMAP (2 mmol, 244 mg) and 0.8 mL
Et3N were dissolved in 20 mL of dried CH2Cl2 solution in the
ice bath, following by addition of the corresponding acyl chloride

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3
Scheme 1. General synthesis of compounds 4a-4t.
(ESI, m/z): [M + H]+ for C28H48NO4+, calculated 462.3578, found
462.3570.
8CH2), 1.00–0.97 (m, 3H, CH3); 13C NMR (100 MHz, DMSO–d6): δ
173.3, 169.6 (C = O), 160.7, 134.1, 128.0, 118.9, 117.8, 115.5 (Ar), 62.2
(OCH2), 36.5 (NHCH2), 33.9, 31.8, 29.4, 29.3, 29.3, 29.2, 29.1, 28.+9,
4-Fluoro-benzoic acid 3-(2–hydroxy–benzoylamino)-propyl es-
ter (4 h), white solid, m.p. 63.1–64.3, yield: 72%, 1H NMR
(400 MHz, DMSO–d6): δ 12.64 (s, 1H, NH), 8.93 (s, 1H, OH), 8.05–
8.00 (m, 2H, Ar), 7.84–7.82 (m, 1H, Ar), 7.41–7.37 (m, 1H, Ar),
7.35–7.30 (m, 2H, Ar), 6.89–6.85 (m, 2H, Ar), 4.35–4.32 (m, 2H,
OCH2), 3.50–3.45 (m, 2H, NHCH2), 2.05–1.98 (m, 2H, CH2); 13C
NMR (100 MHz, DMSO–d6): δ 169.6, 165.3 (C = O), 160.6, 134.1,
132.5, 132.4, 128.0, 126.9, 126.8, 118.9, 117.9, 116.4, 116.1, 115.6 (Ar),
63.3 (OCH2), 36.6 (NHCH2), 28.5. HRMS (ESI, m/z): [M + H]+ for
C17 H17 FNO4+, calculated 318.1136, found 318.1130.
28.5, 24.9, 22.5, 14.4. HRMS (ESI, m/z): [M + H]+ for C22H36NO4
,
calculated 378.2639, found 378.2649.
Hexadecanoic acid 3-(2–hydroxy–benzoylamino)-propyl ester
(4f), white solid, m.p. 35.3–36.0, yield: 69%, 1H NMR (400 MHz,
DMSO–d6): δ 12.78 (s, 1H, NH), 8.99 (s, 1H, OH), 7.98–7.96 (m,
1H, Ar), 7.55–7.51 (m, 1H, Ar), 7.04–7.00 (m, 2H, Ar), 4.23–4.20
(m, 2H, OCH2), 3.53–3.48 (m, 2H, NHCH2), 2.42–2.30 (m, 2H, CH2),
2.03–1.97 (m, 2H, CH2), 1.66–1.62 (m, 2H, CH2), 1.42–1.37 (m, 24H,
12CH2), 0.98–0.96 (m, 3H, CH3); 13C NMR (100 MHz, DMSO–d6):
δ 173.3, 169.6 (C = O), 160.7, 134.1, 128.0, 118.9, 117.8, 115.5 (Ar),
62.2 (OCH2), 36.5 (NHCH2), 33.9, 31.8, 29.5, 29.5, 29.5, 29.4, 29.4,
29.4, 29.3, 29.2, 29.1, 28.9, 28.5, 24.9, 22.6, 14.4. HRMS (ESI, m/z):
[M + H]+ for C26H44NO4+, calculated 434.3265, found 434.3258.
Octadecanoic acid 3-(2–hydroxy–benzoylamino)-propyl ester
(4 g), white solid, m.p. 39.3–41.0, yield: 77%, 1H NMR (400 MHz,
DMSO–d6): δ 12.78 (s, 1H, NH), 8.98 (s, 1H, OH), 7.98–7.96 (m,
1H, Ar), 7.55–7.51 (m, 1H, Ar), 7.04–7.00 (m, 2H, Ar), 4.23–4.20
(m, 2H, OCH2), 3.53–3.48 (m, 2H, NHCH2), 2.42–2.30 (m, 2H, CH2),
2.03–1.97 (m, 2H, CH2), 1.66–1.62 (m, 2H, CH2), 1.38–1.34 (m, 28H,
14CH2), 1.00–0.98 (m, 3H, CH3); 13C NMR (100 MHz, DMSO–d6):
δ 173.3, 169.6 (C = O), 160.7, 134.1, 128.0, 118.9, 117.8, 115.4 (Ar),
62.2 (OCH2), 36.5 (NHCH2), 33.9, 31.8, 29.5, 29.5, 29.5, 29.4, 29.4,
29.4, 29.3, 29.3, 29.3, 29.2, 29.1, 28.9, 28.5, 24.9, 22.6, 14.4. HRMS
4-Chloro-benzoic acid 3-(2–hydroxy–benzoylamino)-propyl es-
ter (4i), white solid, m.p. 60.3–61.2, yield: 70%, 1H NMR (400 MHz,
DMSO–d6): δ 12.62 (s, 1H, NH), 8.93 (s, 1H, OH), 7.97–7.94 (m, 2H,
Ar), 7.84–7.81 (m, 1H, Ar), 7.58–7.55 (m, 2H, Ar), 7.42–7.37 (m, 1H,
Ar), 6.90–6.85 (m, 2H, Ar), 4.36–4.33 (m, 2H, OCH2), 3.50–3.46 (m,
2H, NHCH2), 2.05–1.98 (m, 2H, CH2); 13C NMR (100 MHz, DMSO–
d6): δ 169.7, 165.4 (C = O), 160.6, 138.7, 134.1, 131.4, 129.3, 129.1,
128.0, 118.9, 117.9, 115.6 (Ar), 63.5 (OCH2), 36.5 (NHCH2), 28.5.
HRMS (ESI, m/z): [M + H]+ for C17 H17 ClNO4+, calculated 334.0841,
found 334.0834.
4-Bromo-benzoic acid 3-(2–hydroxy–benzoylamino)-propyl es-
ter (4j), white solid, m.p. 78.6–80.9, yield: 63%, 1H NMR (400 MHz,
DMSO–d6): δ 12.61 (s, 1H, NH), 8.93 (s, 1H, OH), 7.89–7.81 (m,
3H, Ar), 7.72–7.70 (m, 2H, Ar), 7.42–7.38 (m, 1H, Ar), 6.90–6.85

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H. Fang, H. Guo and H. Chen et al. / Journal of Molecular Structure 1223 (2020) 128995
(m, 2H, Ar), 4.36–4.33 (m, 2H, OCH2), 3.50–3.45 (m, 2H, NHCH2),
2.05–1.98 (m, 2H, CH2); 13C NMR (100 MHz, DMSO–d6): δ 169.6,
165.5 (C = O), 160.6, 134.1, 132.3, 131.6, 129.4, 128.0, 127.8, 118.9,
117.9, 115.6 (Ar), 63.5 (OCH2), 36.5 (NHCH2), 28.5. HRMS (ESI, m/z):
[M + H]+ for C17 H17 BrNO4+, calculated 378.0335, found 378.0329.
4-Iodo-benzoic acid 3-(2–hydroxy–benzoylamino)-propyl ester
(4k), white solid, m.p. 83.9–84.8, yield: 69%, 1H NMR (400 MHz,
DMSO–d6): δ 12.61 (s, 1H, NH), 8.92 (s, 1H, OH), 7.90–7.88 (m, 2H,
Ar), 7.83–7.81 (m, 1H, Ar), 7.71–7.69 (m, 2H, Ar), 7.42–7.38 (m, 1H,
Ar), 6.90–6.85 (m, 2H, Ar), 4.35–4.32 (m, 2H, OCH2), 3.50–3.45 (m,
2H, NHCH2), 2.04–1.98 (m, 2H, CH2); 13C NMR (100 MHz, DMSO–
d6): δ 169.6, 165.9 (C = O), 160.6, 138.2, 134.1, 131.3, 129.7, 128.0,
119.0, 117.8, 115.6, 102.2 (Ar), 63.4 (OCH2), 36.5 (NHCH2), 28.5.
HRMS (ESI, m/z): [M + H]+ for C17 H17 INO4+, calculated 426.0197,
found 426.0189.
(NHCH2), 28.4. HRMS (ESI, m/z): [M + H]+ for C18 H17 N2O4+, calcu-
lated 325.1183, found 325.1177.
4-Nitro-benzoic acid 3-(2–hydroxy–benzoylamino)-propyl ester
(4q), white solid, m.p. 123.0–125.1, yield: 78%, 1H NMR (400 MHz,
DMSO–d6): δ 12.59 (s, 1H, NH), 8.92 (s, 1H, OH), 8.32–8.30 (m, 2H,
Ar), 8.19–8.17 (m, 2H, Ar), 7.83–7.80 (m, 1H, Ar), 7.41–7.37 (m, 1H,
Ar), 6.91–6.85 (m, 2H, Ar), 4.42–4.38 (m, 2H, OCH2), 3.52–3.47 (m,
2H, NHCH2), 2.08–1.99 (m, 2H, CH2); 13C NMR (100 MHz, DMSO–
d6): δ 169.6, 164.8 (C = O), 160.6, 150.7, 135.7, 134.1, 131.0, 128.0,
118.9, 117.8, 115.6 (Ar), 64.1 (OCH2), 36.5 (NHCH2), 28.4. HRMS
(ESI, m/z): [M + H]+ for C17 H17 N2O6+, calculated 345.1081, found
345.1074.
2-Trifluoromethyl-benzoic acid 3-(2–hydroxy–benzoylamino)-
propyl ester (4r), pale yellow oil, yield: 53%, 1H NMR (400 MHz,
DMSO–d6): δ 12.59 (s, 1H, NH), 8.90 (s, 1H, OH), 7.89–7.77 (m,
5H, Ar), 7.42–7.38 (m, 1H, Ar), 6.91–6.86 (m, 2H, Ar), 4.38–4.34 (m,
2H, OCH2), 3.47–3.42 (m, 2H, NHCH2), 2.03–1.98 (m, 2H, CH2); 13C
NMR (100 MHz, DMSO–d6): δ 169.7, 166.6 (C = O), 160.6, 134.1,
133.3, 132.3, 130.5, 128.1, 127.2, 127.1, 119.0, 117.9 (Ar), 115.6 (CF3),
64.2 (OCH2), 36.3 (NHCH2), 28.4. HRMS (ESI, m/z): [M + H]+ for
C18 H17 F3NO4+, calculated 368.1104, found 368.1097.
4-Methoxy-benzoic acid 3-(2–hydroxy–benzoylamino)-propyl
ester (4l), white solid, m.p. 71.6–72.3, yield: 71%, 1H NMR
(400 MHz, DMSO–d6): δ 12.65 (s, 1H, NH), 8.94 (s, 1H, OH), 7.93–
7.90 (m, 2H, Ar), 7.85–7.83 (m, 1H, Ar), 7.42–7.38 (m, 1H, Ar), 7.02–
7.00 (m, 2H, Ar), 6.91–6.86 (m, 2H, Ar), 4.32–4.29 (m, 2H, OCH2),
3.86 (s, 3H, OCH3), 3.50–3.45 (m, 2H, NHCH2), 2.03–1.98 (m, 2H,
CH2); 13C NMR (100 MHz, DMSO–d6): δ 169.6, 165.9 (C = O),
163.6, 160.7, 134.1, 131.7, 128.0, 122.5, 119.0, 117.9, 117.6, 114.4 (Ar),
62.8 (OCH2), 56.0 (OCH3), 36.6 (NHCH2), 28.7. HRMS (ESI, m/z):
[M + H]+ for C18 H20NO4+, calculated 330.1336, found 330.1331.
4-Ethoxy-benzoic acid 3-(2–hydroxy–benzoylamino)-propyl es-
ter (4 m), white solid, m.p. 90.3–91.4, yield: 80%, 1H NMR
(400 MHz, DMSO–d6): δ 12.65 (s, 1H, NH), 8.92 (s, 1H, OH), 7.91–
7.89 (m, 2H, Ar), 7.85–7.83 (m, 1H, Ar), 7.42–7.38 (m, 1H, Ar), 7.00–
6.98 (m, 2H, Ar), 6.91–6.86 (m, 2H, Ar), 4.31–4.28 (m, 2H, OCH2),
4.12–4.07 (m, 2H, NHCH2), 3.50–3.45 (m, 2H, OCH2), 2.03–1.96 (m,
2H, CH2), 1.36–1.33 (m, 3H, CH3); 13C NMR (100 MHz, DMSO–d6):
δ 169.6, 165.9 (C = O), 162.9, 160.7, 134.1, 131.7, 128.0, 122.3, 118.9,
117.9, 115.6, 114.8 (Ar), 64.0, 62.8 (OCH2), 36.6 (NHCH2), 28.7, 14.9.
HRMS (ESI, m/z): [M + H]+ for C19 H22NO5+, calculated 344.1493,
found 344.1485.
5-Fluoro-2-methyl-benzoic acid 3-(2–hydroxy–benzoylamino)-
propyl ester (4 s), white solid, m.p. 105.4–106.9, yield: 59%, 1H
NMR (400 MHz, CDCl3): δ 10.71 (s, 1H, NH), 7.84–7.81 (s, 1H,
OH), 7.50–7.46 (m, 1H, Ar), 7.21–7.18 (m, 1H, Ar), 7.10–7.01 (m,
3H, Ar), 6.99–6.85 (m, 1H, Ar), 6.15 (m, 1H, Ar), 4.53–4.50 (m, 2H,
OCH2), 3.63–3.60 (m, 2H, NHCH2), 2.42 (s, 3H, CH3), 2.17–2.11 (m,
2H, CH2); 13C NMR (100 MHz, DMSO–d6): δ 170.3, 161.7 (C = O),
160.6, 136.0, 132.7, 132.6, 129.8, 119.3, 116.9, 113.8, 112.1 (Ar), 62.9
(OCH2), 36.8 (NHCH2), 28.9, 19.1. HRMS (ESI, m/z): [M + H]+ for
C18 H19 FNO4+, calculated 332.1293, found 332.1287.
Benzoic acid 3-[(2-benzoyloxy)-benzoylamino]-propyl ester (4t),
white solid, m.p. 71.6–72.3, yield: 65%, 1H NMR (400 MHz, DMSO–
d6): δ 8.50 (s, 1H, NH), 8.10–8.08 (m, 2H, Ar), 7.92–7.90 (m, 2H, Ar),
7.69–7.62 (m, 3H, Ar), 7.60–7.57 (m, 3H, Ar), 7.55–7.48 (m, 2H, Ar),
7.39–7.33 (m, 2H, Ar), 4.21–4.18 (m, 2H, OCH2), 3.27–3.26 (m, 2H,
NHCH2), 1.98–1.80 (m, 2H, CH2); 13C NMR (100 MHz, DMSO–d6): δ
166.1, 165.8, 164.8 (C = O), 148.3, 134.3, 133.7, 131.6, 130.4, 130.3,
129.6, 129.4, 129.3, 129.3, 129.1, 126.5, 123.7 (Ar), 62.8 (OCH2), 36.2
(NHCH2), 28.7. HRMS (ESI, m/z): [M + H]+ for C24H22NO5+, calcu-
lated 404.1493, found 404.1485.
4-Methyl-benzoic acid 3-(2–hydroxy–benzoylamino)-propyl es-
ter (4n), white solid, m.p. 60.0–61.5, yield: 65%, 1H NMR (400 MHz,
DMSO–d6): δ 12.64 (s, 1H, NH), 8.92 (s, 1H, OH), 7.87–7.83 (m,
3H, Ar), 7.42–7.38 (m, 1H, Ar), 7.32–7.30 (m, 2H, Ar), 6.91–6.86 (m,
2H, Ar), 4.34–4.30 (m, 2H, OCH2), 3.50–3.45 (m, 2H, NHCH2), 2.38
(s, 3H, CH3), 2.04–1.97 (m, 2H, CH2); 13C NMR (100 MHz, DMSO–
d6): δ 169.7, 166.2 (C = O), 144.0, 134.1, 129.7, 129.6, 128.0, 127.6,
119.0, 117.9, 117.6 (Ar), 63.0 (OCH2), 36.6 (NHCH2), 28.6, 21.6. HRMS
(ESI, m/z): [M + H]+ for C18 H20NO5+, calculated 314.1387, found
314.1381.
2.3. Preparation of PPO from the cephalothoraxes of chinese white
shrimp
Chinese white shrimps with an average weight of 10.0
0.5 g
4-Butyl-benzoic acid 3-(2–hydroxy–benzoylamino)-propyl ester
(4o), white solid, m.p. 51.9–52.6, yield: 78%, 1H NMR (400 MHz,
DMSO–d6): δ 12.64 (s, 1H, NH), 8.94 (s, 1H, OH), 7.88–7.82 (m,
3H, Ar), 7.42–7.38 (m, 1H, Ar), 7.32–7.29 (m, 2H, Ar), 6.90–6.86
(m, 2H, Ar), 4.34–4.31 (m, 2H, OCH2), 3.50–3.45 (m, 2H, NHCH2),
2.66–2.62 (m, 2H, CH2), 2.04–1.97 (m, 2H, CH2), 1.60–1.52 (m, 2H,
CH2), 1.32–1.26 (m, 2H, CH2), 0.91–0.87 (m, 3H, CH3); 13C NMR
(100 MHz, DMSO–d6): δ 169.7, 166.2 (C = O), 160.7, 148.7, 134.1,
129.7, 129.0, 128.0, 127.8, 118.9, 117.9, 115.6 (Ar), 63.0 (OCH2), 36.6
(NHCH2), 35.2, 33.2, 28.6, 22.2, 14.2. HRMS (ESI, m/z): [M + H]+ for
C21H26NO4+, calculated 356.1856, found 356.1848.
and an average body length of 10.5 0.5 cm were purchased from
the Xiashang supermarket in Xiamen, Fujian province, China. The
cephalothoraxes of shrimps were immediately separated, pooled,
and powdered by grinding with liquid nitrogen in a blender (DFT-
200, China). All subsequent procedures for the extraction of PPO
were conducted at 4 °C. Cephalothorax pulpy (150 g) was mixed
with 450 mL of 0.05 M sodium phosphate buffer (pH 7.2) contain-
ing 1.0 M NaCl, and 0.2% Brij 35. The mixture was stirred continu-
ously for 30 min, followed by centrifugation at 8000 g for 30 min
using a refrigerated centrifuge (Hitachi, Japan). Solid ammonium
sulfate (82.0 g) was added into the supernatant fraction to obtain
30% saturation. The precipitate was removed by centrifugation at
8000 g for 30 min using a refrigerated centrifuge (Hitachi, Japan).
Another 43.0 g solid ammonium sulfate was added into the super-
natant fraction to obtain 45% saturation. The precipitate was col-
lected by centrifugation at 10,000 g for 30 min using a refrigerated
centrifuge (Hitachi, Japan). The pellet obtained was dissolved in
5 mL 0.05 M sodium phosphate buffer, pH 7.2, and dialyzed against
1.0 L of the same buffer with three changes of dialysis buffer [20].
Finally, the insoluble materials were removed by centrifugation at
4-Cyano-benzoic acid 3-(2–hydroxy–benzoylamino)-propyl es-
ter (4p), white solid, m.p. 93.8–95.2, yield: 61%, 1H NMR (400 MHz,
DMSO–d6): δ 12.60 (s, 1H, NH), 8.92 (s, 1H, OH), 8.11–8.08 (m,
2H, Ar), 7.99–7.97 (m, 2H, Ar), 7.82–7.80 (m, 1H, Ar), 7.41–7.37
(m, 1H, Ar), 6.91–6.85 (m, 2H, Ar), 4.39–4.36 (m, 2H, OCH2), 3.51–
3.46 (m, 2H, NHCH2), 2.06–1.99 (m, 2H, CH2); 13C NMR (100 MHz,
DMSO–d6): δ 169.6, 165.0 (C = O), 160.6, 134.2, 134.1, 133.2, 130.2,
128.0, 119.0, 118.5, 117.9, 115.9 (Ar), 115.6 (CN), 63.9 (OCH2), 36.5