Design, synthesis and biological activity of a novel ethylenediamine derivatives as H1 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2019.115127

In this study, a series of novel ethylenediamine compounds were obtained by structural modification of the lead compounds with thonzylamine, and using the principle of modifying by bioisostere formation and modification with alkyl groups. In vitro assay, the biological activities showed that the target compounds have good properties in inhibiting mast cell degranulation and releasing histamine and β-aminohexidase, such as the compounds 5c, 5g, 5k, 5l and 5o, especially of compound 5k to mast cell degranulation is IC₅₀ = 0.0106 ± 0.001 μmol?L^?1, histamine release was IC₅₀ = 0.0192 ± 0.005 μmol?L^?1 and β-hexosaminidase release was IC₅₀ = 0.0455 ± 0.002 μmol?L^?1 in vitro. At the same time, in vivo biological activities assay results showed that have a good Histamie induce bronchospasm effect with relatively long duration and good protective effect in vivo, among which the protective effect of compound 5k was 79.74 ± 0.30%, compounds 5c, 5g, 5k, 5l and 5o could inhibit the capillary permeability of increasing which were caused by histamine.

Full text of DOI:10.1016/j.bmc.2019.115127

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Design, synthesis and biological activity of a novel ethylenediamine deriva-
tives as H1 receptor antagonists
Shiyang Zhou, Gangliang Huang, Guangying Chen
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https://doi.org/10.1016/j.bmc.2019.115127
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Please cite this article as: S. Zhou, G. Huang, G. Chen, Design, synthesis and biological activity of a novel
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Design, synthesis and biological activity of a novel ethylenediamine
derivatives as H1 receptor antagonists
Shiyang Zhou1,2 Gangliang Huang3,* Guangying Chen1,2
1College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou
571158, China;
2Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education, Hainan
Normal University, Haikou 571158, China;
3Active Carbohydrate Research Institute, Chongqing Key Laboratory of Inorganic Functional
Materials, College of Chemistry, Chongqing Normal University, Chongqing 401331, China
E-mail: huangdoctor226@163.com
Abstract: In this study, a series of novel ethylenediamine compounds were obtained by structural
modification of the lead compounds with thonzylamine, and using the principle of modifying by
bioisostere formation and modification with alkyl groups. In vitro assay, the biological activities
showed that the target compounds have good properties in inhibiting mast cell degranulation and
releasing histamine and β-aminohexidase, such as the compounds 5c, 5g, 5k, 5l and 5o, especially
of compound 5k to mast cell degranulation is IC50=0.0106 0.001 μmol.L-1, histamine release was
IC50=0.0192 0.005 μmol.L-1 and β-hexosaminidase release was IC50=0.0455 0.002 μmol.L-1 in
vitro. At the same time, in vivo biological activities assay results showed that have a good
Histamie induce bronchospasm effect with relatively long duration and good protective effect in
vivo, among which the protective effect of compound 5k was 79.74f0.30%, compounds 5c, 5g,
5k, 5l and 5o could inhibit the capillary permeability of increasing which were caused by
histamine.
Keywords: H1 receptor; design; synthesis; biological activity
1. Introduction
Allergies, also known as allergic diseases, were a series of diseases caused by the immune
system's allergy to normally harmless substances in the environment1-3. These include hay fever,
food allergies, atopic dermatitis, allergic asthma and allergic reactions. The symptoms may
include redness of eyes, itching, sneezing, runny nose, shortness of breathing or swelling. In
1

developed countries, about 20% of people have allergic rhinitis, about 6% have allergy to one food
at least, and about 20% have allergic dermatitis at some point in time4. Allergic reactions occur
between 0.05 and 2% of the population in the world, and the incidence of many allergic diseases
appears to be on the rise. The H1 receptor is a histamine receptor that belongs to the G protein
coupled receptor (GPCR) family5. It is expressed in smooth muscle, vascular endothelial cells,
heart and central nervous system. The H1 receptor is link to an intracellular G protein that activates
the phospholipase C and inositol triphosphate signaling pathways. Antihistamines act on the H1
receptor which are use as antiallergic drugs6-8. The crystal structure of the H1 receptor have been
determine that has been use to discover new histamine H1 receptor ligands in structure which
based virtual screening studies9.
The study on H1 receptor (Fig.1) antagonists originate in 1933. Fourneau and Bovet first
reported that Biperoxan could prevent intestinal mice from bronchospasm cause by inhalation of
excessive histamine, which were caused by antagonistic effect on histamine receptors, attracting
great interests in research10-12. However, due to its low activity, piperoxone has not been used in
clinical practice. In the research process, it is found that the introduction of a second aromatic ring
or heterocyclic ring into the structure of such compounds could significantly increase the
antihistamine activities, such as diphenhydramine and chlorpheniramine, which are early classical
antihistamines mainly use to treat allergic diseases13-15. But these drug structures and other drugs
have similarities, usually show different degrees of sympathetic, anti 5-hydroxytryptamine (5-HT),
sedation and similar anti-cholinergic side effects16. It could be cause drowsiness, dry mouth,
blurred vision.Further research work is looking for drugs with small sedative effect and high
antihistamine activity as the main target. llater it founded ketotifen and oxatomide. They could not
only inhibiting the antihistamine activity, but also inhibiting the releasing of histamine by allergic
cell. The true histamine antagonists without sedating side effects were developed in the early
1980s with the discovery of terfenadine. However, the use of terfenadine could cause cardiac side
effects, such as ventricular arrhythmias17-20. In the study, it found that the cardiac side effects for
terfenadine disappeared after the carboxylate metabolites in the body. According to the results of
this study, the risk of cardiac side effects was greatly reduced the polar groups were introduced
into the compound structure and drugs that cetirizine and loratadine were obtain21.
2

Fig. 1 The protein structure of H1 receptor.10-12
In 1942, Halpern based on the transformation of piperoxane structure22-26, simplified
piperidine ring into ethylenediamine group, and substituted benzodiazepine hexachloride ring with
aromatic amine group to obtain the first drug of clinical application value, plcenbenzamine, thus
determining the drug type of ethylenediamine structure containing diaromatic alkyl (Fig.2).
Because the fentanyl was found, the most common method of application is the method of type
derivation27,28. On the basis of the model compound, the functional groups contained in the model
compound were replaced with other groups with similar physical and chemical properties to
obtain the active compound with the biological activity of the model compound, so as to find new
drugs. In the structural modification of fenbenzamide, pyridine ring was used to replace the
benzene ring, and methylamine was introduced into the 4-position of benzylamine to obtain the
more active drug merpyrarnime. The mepiramine could prevent the bronchospasm cause by
histamine in the intestinal experiment of mouse, and could be used for various diseases of allergic
reaction. The effect lasts for a short time, and the sedative effect is more, there were still
drowsiness and gastrointestinal side effects. The successful discovery of mepiramine has
prompted further studies to replace aromatic rings in the structure with aromatic heterocyclic rings.
The tripelennamrine was a drug obtained by substituting a pyridine ring for a phenylamine ring.
The activity of tripelennamrine was similar to that of mepiramine, and it was stronger and longer
lasting than the diphenhydramine later founded. In addition, the methaphenilene and
methapyrilene were prepare with 2-thiophene methyl in place of benzyl and 4-methoxybenzyl in
3

the structure of fenbenzanamide, respectively, in accordance with electron isosterism. The
4-methoxybenzyl group in mepiramine was replaced by furan methyl group, and the activity of
methafurilene in cosmetic is equal to that of tripimine, while the toxicity and biological activity of
mesafenaline was lower than that of fenzanamide. However, when 2-thiophene methyl is replace
with 3-thiophene methyl, the thenyldiamine was obtain, in which the activity was about 1.5 times
as high as that of methapyrilene. With the deepening of the research on the biarylalkyl drugs, the
people have gradually realized that there no specific restrictions on the structure of the biarylalkyl
groups, and the ethylenediamine group was an important part of its activity. In the subsequent
studies, the main structural characteristics were the retention of ethylamine. Subsequently,
zolamine was founded to replace pyridine ring with thiazole, and thonzylamine was an analogue
obtained by substituting pyridine ring with pyrimidine ring. The activity of both was similar to
that of mepramine, but the degree of side effects was lower. In this study, a series of novel
ethylenediamine compounds were obtained by structural modification of the lead compounds with
thonzylamine. The lead compound thonzylamine and other H1 receptor inhibitors has an
ethylenediamine structure, and ethylenediamine structure was retained during the design of the
target compounds. At the same time, the structural design of two six-membered rings was retained.
In structural modification using the principle which modified by bioisostere formation and
modification with alkyl groups (Fig.3). These compounds have a molecular spatial structure
similar to that of thonzylamine, and the new design compounds also have an antiallergic effect
according to the structure activity relationship (SAR). The target compounds of the new design
has a similar ethylenediamine antagonists with ethylenediamine, and the different structure of the
target compounds lies in the type of six-element ring and the length of alkyl chain, which has
reached the inhibitor with stronger binding ability to H1 receptor. These target compounds through
a two-steps N-alkylation reaction (Scheme 1), which was characterize by simple operation, high
total yield, mild reaction conditions and cheap reagents and easy reagents.
4

Fig.2 The chemical structure of ethylenediamine antagonists.
Fig.3 Design of ethylenediamine compounds.
2. Results and discussion
2.1. The design and synthesis of target compounds
The thonzylamine (Fig.1) is an ethylenediamine antiallergic drug, which is mainly used for
relieving hypersensitivity diseases, treating nasal congestion, allergic conjunctivitis and other
allergic diseases in clinical. With short duration of action and weak sedative effect, the
thonzylamine has been on the market in the United States, and has been included in the Japanese
pharmaceutical administrationthe of 2015 edition29. In this research, basing on the structure of
5

thonzylamine, a series of novel H1 receptor antagonists were designed with thonzylamine as the
lead compound and using the principle which was modified by bioisostere formation and
modification with alkyl groups (Fig.3). The specific chemical structure design strategy was that
-H, -Cl, -NH2 and -CH3 is selected for R substituents, and the benzene ring structure and pyridine
ring structure are selected, and -CH2- and -CH2CH2- is selected for alkyl chain transformation.
This structure was designed to change the logP, pKa and spatial structure so as to obtain
compounds with higher binding ability to H1 receptor and better antihistamine activity. According
to the structure-activity relationship (SRA) of drugs, this design of ethylenediamine compounds
with ethylenediamine structure, substituent R, modification of the six-member ring structure and
alkyl chain, may have a better effect, up from spatial structure design of the compounds. They
have a similar spatial structure to the thonzylamine, such structural design makes that they have an
antiallergic effect which was possible. In the synthesis strategy, we choose an efficient and
convenient synthesis route (Scheme 1). The first N-alkylation reaction was conducted with aniline
(1a-1h) and 2-chloro-N, N-dimethyl-ethylamineas (2a) as starting material. In this step, toluene
was used as the solvent, anhydrous potassium carbonate (K2CO3) was used as the deacidifying
reagent and a certain amount of potassium iodide (KI) used as a catalyst. The second N-alkylation
reaction, N, N-dimethyl-N’-phenylethane-1, 2-diamine (3a-3h) and 4-methoxybenzylchloride
(4a-4b) as starting material, toluene was used as the solvent, potassium tert-butoxide (t-BuOK)
was used as the alkaline reagent and a certain amount of ammonium chloride (NH4Cl) used as a
catalyst for the reaction. After two steps of N-alkylation, the yield of the synthesized target
compounds reached 73.1%-88.4%. The synthetic route was characterized by simple operation,
high total yield, mild reaction conditions, cheap and easy reagents.
6

Scheme 1 The synthetic route of ethylenediamine compounds.
2.2. The biological activities
2.2.1. The biological activity screening in vitro
The lipid-water distribution coefficient (logp) and dissociation (pKa) were related to the
absorption degree and location of the drugs so as to choose the right way of drug administration.
We synthesized a total of 16 compounds according to the structural design, which tested the logp
and pKa values of these compounds respectively (Tab.1). It could be seen from Tab.1 that the logp
value of these compounds ranges from 2.56 to 4.68 and the pKa value from 9.4 to 9.8. These
compounds were lipid soluble and absorbed in the intestinal tract. The mast cell degranulation,
histamine releases and β-hexosaminidase releases were selected as evaluation indicators in the
screening of antiallergic activity in vitro (Tab.1). As could be seen from Tab.1, the experiment
result shows that these compounds have good properties in inhibiting mast cell degranulation and
releasing histamine and β-aminohexidase. In particular, the compounds 5c, 5g, 5k, 5l and 5o
exhibit strong antiallergic reactions in vitro, which of compound 5k to mast cell degranulation was
IC50=0.0106 0.001 μmol.L-1, histamine release was IC50=0.0192 0.005 μmol.L-1 and
β-hexosaminidase release was IC50=0.0455 0.002 μmol.L-1. At the same time, we conducted
binding assay of compounds 5c, 5g, 5k, 5l and 5o to H1 receptor in vitro (Fig.4). The experimental
results showed that the compounds 5c, 5g, 5k, 5l and 5o had a good binding ability to H1 receptor
7

at low concentration (0.10 μmol.L-1). From the results of structure-activity relationship (SAR)
analysis, it could be seen that the target compounds of the new design has the same linear structure
or spatial structure as the lead compound thonzylamine (Fig.3), so this kind of compounds also
has the ability to bind to H1 receptor. It was found in structure-activity relationship that pyridine
ring had stronger inhibition than benzene ring structure, which should be related to the electron
distribution on six-membered ring. At the same time, the type of substituent R has a great
influence on the inhibition of H1 receptor. We found that when R was Cl and CH3, its inhibition
was significantly higher than that of NH2. Such results should be determined by the spatial
structure of these substituents and their ability to attract electrons. In addition, the length of alkyl
chain also has an effect on H1 receptor inhibitory activity, and its inhibitory activity decreases
with the growth of alkyl chain. All suggest that the compounds may have a good antiallergic
effect.
Tab.1. In vitro the activation of assay compounds on mast cell degranulation, histamine release
and β-hexosaminidase release
IC50fSDa (μmol.L-1)
Compounds
X
R
n
logp
pKa
Mast cell
Histamine
release
β-hexosaminidase
release
degranulation
0.143f0.016
0.306f0.022
0.0181f0.002
0.0892f0.010
2.310f0.193
2.228f0.189
0.0553f0.012
0.153f0.013
0.0815f0.008
0.129f0.018
0.0106f0.001
0.0453f0.002
C
H
1
3.90
4.21
4.47
4.78
3.13
3.43
4.43
4.68
3.32
3.59
3.87
4.16
9.7
0.243f0.021
0.516f0.022
0.411f0.023
1.031f0.121
5a
5b
5c
5d
5e
5f
C
C
C
C
C
C
C
N
N
N
N
H
2
1
2
1
2
1
2
1
2
1
2
9.6
9.6
9.5
9.8
9.8
9.8
9.7
9.7
9.5
9.5
9.4
Cl
Cl
0.0402f0.012 0.0712f0.012
0.141f0.019
4.060f0.261
4.008f0.251
0.261f0.262
8.009f0.0249
7.961f0.0223
NH2
NH2
CH3
CH3
H
5g
5h
5i
0.0891f0.010 0.141f0.029
0.289f0.026 0.561f0.251
0.0926f0.010 0.132f0.022
0.1586f0.011 0.319f0.013
0.0192f0.005 0.0455f0.002
0.0531f0.009 0.0721f0.008
H
5j
Cl
5k
5l
Cl
8

N
N
N
N
NH2
NH2
CH3
CH3
1
2
1
2
2.56
2.79
3.79
4.06
9.4
9.6
9.6
9.5
1.338f0.028
1.206f0.019
0.0289f0.003
0.0839f0.011
0.0318f0.008
0.0533f0.003
iab
2.108f0.028
1.906f0.022
4.092f0.029
4.013f0.026
5m
5n
5o
0.0486f0.008 0.0612f0.009
0.102f0.012 0.211f0.013
5p
Chlorpheniramine
Astermizole
DMSO
0.0536f0.009 0.121f0.015
0.0819f0.012 0.156f0.026
ia
ia
a Data represent the mean values from at least eight independent experiments each in triplicate
(n≥8).
b ia=inactive.
Fig.4. In vitro the binding rate of H1 receptor. The data show the meanfSEM (n=6). The levels of
significance were *p < 0.05 and **p < 0.01.
2.2.2. The evaluation of biological activity in vivo
In vitro screening of antiallergic reactions, the compounds 5c, 5g, 5k, 5l and 5o with better
activity were screened. The antihistamine and capillary permeability were used as evaluation
indexes for antiallergic reaction in vivo. The antihistamine was studied by using the Van Arman
method of application to determine the antihistamine potential of compounds 5c, 5g, 5k, 5l and 5o
screened in vitro (Tab.2). In Tab.2, the results of antihistamine assay showed that this kind of
compounds has a good Histamie induced bronchospasm effect with relatively long duration and
good protective effect in vivo, among which the protective effect of compound 5k a was 79.74f
0.30%, and in oral administration, the dose was 20 mg.kg-1 in 1% Carboxymethylcellulose sodium
9

(CMC-Na). Acute toxicity tests were performed at different doses, intraperitoneal administration
of compounds 5c, 5g, 5k, 5l and 5o to observe any signs of toxicity during short and long term
observation (Tab.2). In Tab.2, the results of acute toxicity test indicated that these five compounds
were belong to low-toxic or non-toxic compounds in vivo, among the LD50 value of compound 5c
was 413.65f2.31 mg.kg-1. After the injection of histamine, the back skin of the mice showed
blue-staining, while the mice without histamine did not show blue-staining, indicating increased
permeability of capillaries at the injection site. The compounds 5c, 5g, 5k, 5l and 5o could
significantly reduce the blue staining of mouse skin which was caused by histamine and reduced
the leakage of Evans blue, indicating that compounds 5c, 5g, 5k, 5l and 5oC can inhibit the
increase of capillary permeability caused by histamine (Tab.3).
Tab.2. In vivo the activation of assay compounds on antihistamine.
Compounds
Histamie-induced bronchospasm a(s) Protectiona (%)
LD50fSDb(mg.kg-1)
413.65f2.31
401.35f2.32
324.21f1.55
334.64f2.14
334.69f1.59
165.13f1.10
5c
5g
5k
5l
376.58f2.73
348.10f2.39
398.69f3.02
351.10f2.32
371.28f2.61
75.32f0.26
69.62f0.31
79.74f0.30
70.22f0.29
74.26f0.28
73.43f0.36
5o
Chlorpheniramine 367.16f2.65
a Data represent the mean values from at least ten independent experiments each in triplicate
(n≥10).
b Data represent the mean values from at least three independent experiments each in triplicate (n
≥ 3).
Tab.3. In vivo the activation of assay compounds on capillary permeability.
Compounds
Evans blue concentration (XfSDa , mg.L-1)
5c
5g
5k
5l
65.34f1.41
53.59f1.38
71.26f1.32
55.67f1.36
62.16f1.35
5o
10

Chlorpheniramine
56.13f1.12
a Data represent the mean values from at least ten independent experiments each in triplicate
(n≥10).
3. Conclusion
We reported that the design synthesis and biological activity evaluation of a novel
ethylenediamine compounds as H1 receptor antagonists. The lead compounds with thonzylamine,
and using the principle of modified by bioisostere formation and modification with alkyl groups.
These target compounds through a two-steps N-alkylation reaction, which was characterized by
simple operation, high total yield, mild reaction conditions and cheap and easy reagents. In vitro
assay, the biological activities showed that the target compounds have good properties in
inhibiting mast cell degranulation and releasing histamine and β-aminohexidase, such as the
compounds 5c, 5g, 5k, 5l and 5o, which of compound 5k to mast cell degranulation was
IC50=0.0106 0.001 μmol.L-1, histamine release was IC50=0.0192 0.005 μmol.L-1 and
β-hexosaminidase release was IC50=0.0455 0.002 μmol.L-1 in vitro. At the same time, in vivo
biological activities assay results showed that the has a good Histamie induced bronchospasm
effect with relatively long duration and good protective effect in vivo, among which the protective
effect of compound 5k a was 79.74f0.30% and compounds 5c, 5g, 5k, 5l and 5o could inhibit the
increasing of capillary permeability caused by histamine. In addition, the results of acute toxicity
tests indicated that these five compounds were belong to low-toxic or non-toxic compounds. In
summary, compound 5k has been developed as an H1 receptor antagonist for the treatment of
allergy.
4. Experimental
4.1. Chemistry section
Chemistry materials and general methods. The reagents (chemicals) are purchase and use
without further purification. Nuclear magnetic resonance (NMR) spectroscopy is perform using a
Bruker AMX-400 spectrometer (IS as TMS). Mass spectroscopy is perform with an Agilent 6460
system. HPLC analysis of all final biologically test compounds is carry out using an Agilent 1260
series HPLC system. The purity is determine by reversed-phase HPLC and is ≥98% for all
11

biologically tested compounds.
4.1.1. A general method for synthesis of compounds 3a to 3h
The aniline (1a, 9.11 mL, 0.10 mol) and 2-chloro-N, N-dimethyl-ethylamine (2a, 11.32 mL,
0.10 mol) were put into 250 mL round bottom flask, then 100 mL of toluene was used as the
solvent, anhydrous potassium carbonate (K2CO3, 6.91 g, 0.05 mL) was used as the deacidifying
reagent and a certain amount of potassium iodide (KI, 3% mol) used as a catalyst for the reaction
were added. After the reactants were added, the reaction lasted for 20 h under refluxing. When the
reflux reaction was completed, the mixture was filtered at a high temperature, removing
unreacted potassium carbonate (K2CO3) and catalyst potassium iodide (KI), the filtrate was
collected, cooled, and left to stand for 12 h, and white crystals were precipitated. The crystals were
filtered and vacuum dried to obtain the crude product of N, N-dimethyl-N’-phenylethane-1,
2-diamine (3a). The crude product ( compound 3a) was recrystallised from absolute ethyl alcohol,
filtered, and vacuum dried to give the pure product of N, N-dimethyl-N’-phenylethane-1,
2-diamine (3a) as white crystals. The general method was used to synthesise compounds 3b and
3h as all were of white crystal.
4.1.2. A general method for synthesis of compounds 5a to 5p
The N, N-dimethyl-N’-phenylethane-1, 2-diamine (3a, 16.41 g, 0.10 mol) and
4-methoxybenzylchloride (4a, 13.56 mL, 0.10 mol) were put into 250 mL round bottom flask,
then 100 mL of toluene was used as the solvent, potassium tert-butoxide (t-BuOK, 11.22 g, 0.10
mL) was used as the alkaline reagent and a certain amount of ammonium chloride (NH4Cl, 3%mol)
used as a catalyst for the reaction were added. After the reactants were added, the reaction lasted
for 24 h under refluxing. When the reflux reaction was completed, the mixture was filtered at a
high temperature, removing unreacted potassium tert-butoxide (t-BuOK) and catalyst ammonium
chloride (NH4Cl), the filtrate was collected, cooled, and left to stand for 12 h, and white crystals
were precipitated. The crystals were filtered and vacuum dried to obtain the crude product of
N-(4-methoxybenzyl)-N, N-dimethyl-N-phenylethane-1, 2-diamine (5a). The crude product
( compound 5a) was recrystallised from butyl alcohol and ethyl acetate (n-C4H9OH:
CH3COOCH2CH3=1:1), filtered, and vacuum dried to give the pure product of
N’-(4-methoxybenzyl)-N, N-dimethyl-N’-phenylethane-1, 2-diamine (5a) as white crystals. The
general method was used to synthesise compounds 5b and 5P as all were of white crystal.
12

N’-(4-methoxybenzyl)-N, N-dimethyl-N’-phenylethane-1, 2-diamine (5a): white crystal, yield
83.2%, m.p. 170-172Ԩ; 1H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3), 2.54 (2H, t, J = 2.7 Hz,
-CH2-), 3.45 (2H, t, J = 2.7 Hz, -CH2-), 3.80 (3H, s, -OCH3), 4.76 (2H, s, -CH2-), 6.83 (1H, tt, J =
8.1, 1.2 Hz, Ph-H), 6.89 (2H, ddd, J = 8.6, 1.7, 0.6 Hz, Ph-H), 6.92 (2H, dtd, J = 8.3, 1.2, 0.5 Hz,
Ph-H), 7.10 (2H, ddd, J = 8.6, 1.1, 0.6 Hz, Ph-H), 7.24 (2H, dddd, J = 8.3, 8.1, 1.4, 0.5 Hz, Py-H);
13C NMR (75MHz, DMSO) δ: 45.5, 50.8, 55.0, 55.6, 57.5, 114.0, 114.4, 124.6, 129.0, 129.5,
+
136.6, 149.2, 160.5; HR-ESI-MS m/z: calcd for C18H24N2O̗˷M+H˹̙284.1887, found 284.4031;
Anal. calcd for C18H24N2O: C, 76.02; H, 8.51; N, 9.85; O, 5.63; found: C, 76.01; H, 8.52; N, 9.86;
O, 5.62%.
N’-(4-methoxyphenethyl)-N, N-dimethyl-N’-phenylethane-1, 2-diamine (5b): white crystal,
1
yield 80.5%, m.p. 173-175Ԩ; H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3), 2.51 (2H, t, J =
6.7 Hz, -CH2-), 2.81 (2H, t, J = 2.7 Hz, -CH2-), 3.45 (2H, t, J = 2.7 Hz, -CH2-), 3.67 (2H, t, J = 6.7
Hz, -CH2-), 3.82 (3H, s, -OCH3), 6.81 (1H, tt, J = 8.1, 1.2 Hz, Ph-H), 6.85 (2H, ddd, J = 8.8, 2.1,
0.5 Hz, Ph-H), 6.95 (2H, dtd, J = 8.3, 1.2, 0.5 Hz, Ph-H), 7.14 (2H, ddd, J = 8.8, 1.0, 0.5 Hz,
Ph-H), 7.22 (2H, dddd, J = 8.3, 8.1, 1.4, 0.5 Hz, Py-H); 13C NMR (75MHz, DMSO) δ: 35.6, 45.5,
49.4, 50.8, 55.4, 57.5, 114.0, 114.5, 123.4, 124.6, 129.4, 131.2, 149.3, 160.5; HR-ESI-MS m/z:
+
calcd for C19H26N2O̗˷M+H˹̙298.2042, found 298.4303; Anal. calcd for C19H26N2O: C, 76.47;
H, 8.78; N, 9.39; O, 5.36; found: C, 76.46; H, 8.79; N, 9.38; O, 5.37%.
N’-(4-chlorophenyl)-N’-(4-methoxybenzyl)-N, N-dimethylethane-1, 2-diamine (5c): white
crystal, yield 76.2%, m.p. 181-183Ԩ; 1H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3), 2.52 (2H,
t, J = 2.7 Hz, -CH2-), 3.45 (2H, t, J = 2.7 Hz), 3.82 (3H, s, -OCH3), 4.81 (2H, s, -CH2-), 6.73 (2H,
ddd, J = 8.3, 1.5, 0.5 Hz, Ph-H), 6.88 (2H, ddd, J = 8.6, 1.7, 0.6 Hz, 7.12 (2H, ddd, J = 8.6, 1.1,
0.6 Hz, Ph-H), 7.17 (2H, ddd, J = 8.3, 1.7, 0.5 Hz, Ph-H); 13C NMR (75MHz, DMSO) δ: 45.5,
51.0, 55.0, 55.7, 57.5, 114.1, 125.5, 129.0, 129.5, 129.9, 136.6, 149.2, 160.5; HR-ESI-MS m/z:
calcd for C18H23ClN2O̗˷M+H˹+̙318.1497, found 318.8453; Anal. calcd for C18H23ClN2O: C,
67.81; H, 7.27; Cl, 11.12; N, 8.79; O, 5.02; O, 4.81; found: C, 67.82; H, 7.26; Cl, 11.14; N, 8.78;
O, 5.01%.
N’-(4-chlorophenyl)-N’-(4-methoxyphenethyl)-N, N-dimethylethane-1, 2-diamine (5d): white
crystal, yield 75.1%, m.p. 185-187Ԩ; 1H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3), 2.51 (2H,
t, J = 6.7 Hz, -CH2-), 2.81 (2H, t, J = 2.7 Hz, -CH2-), 3.45 (2H, t, J = 2.7 Hz, -CH2-), 3.67 (2H,
13

t, J = 6.7 Hz, -CH2-), 3.82 (3H, s, -CH3), 6.73 (ddd, J = 8.3, 1.6, 0.5 Hz, Ph-H), 6.85 (ddd, J = 8.8,
2.1, 0.5 Hz, Ph-H), 7.11 (2H, ddd, J = 8.8, 1.0, 0.5 Hz, Ph-H), 7.14 (2H, ddd, J = 8.3, 1.7, 0.5 Hz,
Ph-H); 13C NMR (75MHz, DMSO) δ: 35.6, 45.5, 49.4, 50.8, 55.4, 57.5, 114.2, 123.4, 125.5, 129.3,
+
129.9, 131.2, 149.2, 160.5; HR-ESI-MS m/z: calcd for C19H25ClN2O̗˷M+H˹̙332.1651, found
332.8722; Anal. calcd for C19H25ClN2O: C, 68.56; H, 7.57; Cl, 10.65; N, 8.42; O, 4.81; found: C,
68.57; H, 7.56; Cl, 10.65; N, 8.41; O, 4.82%.
N’-(4-aminephenyl)-N’-(4-methoxybenzyl)-N, N-dimethylethane-1, 2-diamine (5e): white
crystal, yield 88.4%, m.p. 174-176Ԩ; 1H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3), 2.52 (2H,
t, J = 2.7 Hz, -CH2-), 3.45 (2H, t, J = 2.7 Hz, -CH2-), 3.82 (3H, s, -OCH3), 4.81 (2H, s, -CH2-),
6.43 (2H, ddd, J = 8.4, 2.1, 0.5 Hz, Ph-H), 6.65 (2H, ddd, J = 8.4, 2.2, 0.5 Hz, Ph-H), 6.88 (2H,
ddd, J = 8.6, 1.7, 0.6 Hz, Ph-H), 7.13 (2H, ddd, J = 8.6, 1.1, 0.6 Hz, Ph-H); 13C NMR (75MHz,
DMSO) δ: 45.5, 50.8, 55.0, 55.7, 57.65, 114.1, 116.0, 116.8, 129.0, 136.6, 145.4, 149.2, 160.5;
HR-ESI-MS m/z: calcd for C18H25N3O̗˷M+H˹+̙299.1997, found 299.4182; Anal. calcd for
C18H25N3O: C, 72.21; H, 8.42; N, 14.03; O, 5.34; found: C, 72.22; H, 8.41; N, 14.04; O, 5.33%.
N’-(4-aminephenyl)-N’-(4-methoxyphenethyl)-N, N-dimethylethane-1, 2-diamine (5f): white
crystal, yield 86.3%, m.p. 177-179Ԩ; 1H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3), 2.51 (2H,
t, J = 7.1 Hz, -CH2-), 2.81 (2H, t, J = 2.7 Hz, -CH2-), 3.45 (2H, t, J = 2.7 Hz, -CH2-), 3.67 (2H,
t, J = 7.1 Hz, -CH2-), 3.73 (3H, s, -OCH3), 6.43 (2H, ddd, J = 8.4, 2.1, 0.5 Hz, Ph-H), 6.65 (2H,
ddd, J = 8.4, 2.2, 0.5 Hz, Ph-H), 6.85 (2H, ddd, J = 8.8, 2.1, 0.5 Hz, Ph-H ), 7.14 (2H, ddd, J = 8.8,
1.0, 0.5 Hz, Ph-H); 13C NMR (75MHz, DMSO) δ: 35.6, 45.5, 49.5, 50.8, 55.4, 57.5, 114.2, 116.0,
116.7, 123.4, 131.2, 145.4, 149.2, 160.5; HR-ESI-MS m/z: calcd for C19H27N3O̗˷M+H˹+̙
313.2153, found 313.4451; Anal. calcd for C19H27N3O: C, 72.81; H, 8.68; N, 13.41; O, 5.10;
found: C, 72.80; H, 8.69; N, 13.43; O, 5.09%.
N’-(4-methoxybenzyl)-N, N-dimethyl-N’-(p-tolyl)ethane-1, 2-diamine (5g): white crystal,
1
yield 78.1%, m.p. 178-180Ԩ; H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3), 2.33(3H, s,
-CH3), 2.52 (2H, t, J = 2.7 Hz, -CH2-), 3.45 (2H, t, J = 2.7 Hz, -CH2-), 3.82 (3H, s, -OCH3), 4.81
(2H, s, -CH2-), 6.64 (2H, ddd, J = 8.2, 1.3, 0.6 Hz, Ph-H), 6.82 (2H, ddd, J = 8.6, 1.7, 0.6 Hz,
13
Ph-H), 6.96 (2H, ddd, J = 8.2, 1.2, 0.6 Hz, Ph-H), 7.13 (2H, ddd, J = 8.6, 1.1, 0.6 Hz, Ph-H); C
NMR (75MHz, DMSO) δ: 21.4, 45.5, 50.8, 55.0, 55.6, 57.5, 111.4, 114.1, 129.0, 129.8, 132.5,
+
136.6, 149.2, 160.5; HR-ESI-MS m/z: calcd for C19H26N2O̗˷M+H˹̙298.2046, found 298.431;
14

Anal. calcd for C19H26N2O: C, 76.47; H, 8.78; N, 9.39; O, 5.36; found: C, 76.46; H, 8.79; N, 9.38;
O, 5.35%.
N’-(4-methoxyphenethyl)-N, N-dimethyl-N’-(p-tolyl)ethane-1, 2-diamine (5i): white crystal,
1
yield 76.4%, m.p. 181-183Ԩ; H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3), 2.33 (3H, s,
-CH3), 2.52 (2H, t, J = 6.7 Hz, -CH2-), 2.81 (2H, t, J = 2.7 Hz, -CH2-), 3.45 (2H, t, J = 2.7 Hz,
-CH2-), 3.67 (2H, t, J = 6.7 Hz, -CH2-), 3.82 (3H, s, -OCH3), 6.64 (2H, ddd, J = 8.2, 1.4, 0.6 Hz,
Ph-H), 6.85 (2H, ddd, J = 8.8, 2.1, 0.5 Hz, Ph-H), 6.96 (2H, ddd, J = 8.2, 1.2, 0.6 Hz, Ph-H), 7.14
13
(2H, ddd, J = 8.8, 1.0, 0.5 Hz, ); C NMR (75MHz, DMSO) δ: 21.4, 35.6, 45.5, 49.6, 50.9, 55.5,
57.5, 113.4, 114.2, 123.4, 129.7, 131.2, 132.5, 149.2, 160.5; HR-ESI-MS m/z: calcd for
+
C20H28N2O̗˷M+H˹̙312.2203, found 312.4576; Anal. calcd for C20H28N2O: C, 76.88; H, 9.03;
N, 8.97; O, 5.12; found: C, 76.89; H, 9.02; N, 8.97; O, 5.13%.
N’-(4-methoxybenzyl)-N, N-dimethyl-N’-(pyridin-2-yl)ethane-1, 2-diamine (5h) white
crystal, yield 80.1%, m.p. 185-187Ԩ; 1H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3), 2.52 (2H,
t, J = 2.7 Hz, -CH2-), 3.15 (2H, t, J = 2.7 Hz, -CH2-), 3.82 (3H, s, -OCH3), 4.72 (2H, s, -CH2-),
6.64 (1H, ddd, J = 7.9, 4.8, 1.2 Hz, Py-H), 6.74 (1H, ddd, J = 8.2, 1.2, 0.5 Hz, PyH), 6.90 (2H,
ddd, J = 8.6, 1.7, 0.6 Hz, Ph-H), 7.13 (2H, ddd, J = 8.6, 1.1, 0.6 Hz, Ph-H), 7.55 (1H, ddd, J = 8.2,
7.9, 1.9 Hz, Py-H), 8.05 (1H, ddd, J = 4.8, 1.9, 0.5 Hz, Py-H); 13C NMR (75MHz, DMSO) δ: 45.5,
50.8, 53.6, 55.5, 57.5, 114.1, 118.2, 118.9, 129.1, 136.6, 138.5, 149.1, 155.3, 160.5; HR-ESI-MS
+
m/z: calcd for C17H23N3O̗˷ M+H˹̙ 285.1843, found 285.3915; Anal. calcd for C17H23N3O: C,
71.55; H, 8.12; N, 14.72; O, 5.61; found: C, 71.56; H, 8.11; N, 14.71; O, 5.62%.
N’-(4-methoxyphenethyl)-N, N-dimethyl-N’-(pyridin-2-yl)ethane-1, 2-diamine (5j) white
crystal, yield 79.2%, m.p. 191-193Ԩ; 1H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3), 2.52 (2H,
t, J = 2.7 Hz, -CH2-), 3.15 (2H, t, J = 2.7 Hz, -CH2-), 3.82 (3H, s, -OCH3), 4.72 (2H, s, -CH2-),
6.64 (1H, ddd, J = 7.9, 4.8, 1.2 Hz, Py-H), 6.74 (1H, ddd, J = 8.2, 1.2, 0.5 Hz, PyH), 6.90 (2H,
ddd, J = 8.6, 1.7, 0.6 Hz, Ph-H), 7.13 (2H, ddd, J = 8.6, 1.1, 0.6 Hz, Ph-H), 7.55 (1H, ddd, J = 8.2,
7.9, 1.9 Hz, Py-H), 8.05 (1H, ddd, J = 4.8, 1.9, 0.5 Hz, Py-H); 13C NMR (75MHz, DMSO) δ: 35.6,
45.5, 49.5, 50.8, 55.5, 57.5, 114.2, 118.1, 118.9, 123.4, 131.2, 138.5, 149.1, 155.3, 160.5;
HR-ESI-MS m/z: calcd for C18H25N3O̗˷M+H˹+̙ 299.1995, found 99.4186; Anal. calcd for
C18H25N3O: C, 72.21; H, 8.42; N, 14.03; O, 5.34; found: C, 72.23; H, 8.41; N, 14.03; O, 5.33%.
N’-(5-chloropyridin-2-yl)-N’-(4-methoxybenzyl)-N, N-dimethylethane-1, 2-diamine (5k)
15

1
white crystal, yield 73.1%, m.p. 203-205Ԩ; H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3),
2.52 (2H, t, J = 2.7 Hz, -CH2-), 3.15 (2H, t, J = 2.7 Hz, -CH2-), 3.82 (3H, s, -OCH3), 4.72 (2H, s,
-CH2-), 6.83 (1H, dd, J = 8.2, 0.5 Hz, Py-H), 6.90 (2H, ddd, J = 8.6, 1.7, 0.6 Hz, Ph-H), 7.13 (2H,
ddd, J = 8.6, 1.1, 0.6 Hz, Ph-H), 7.78 (1H, dd, J = 8.2, 1.5 Hz, Py-H), 8.21 (1H, dd, J = 1.5, 0.5 Hz,
Py-H); 13C NMR (75MHz, DMSO) δ: 45.5, 50.8, 53.6, 55.5, 57.5, 111.4, 114.1, 123.3, 129.1,
+
136.6, 137.7, 145.1, 155.3, 160.5; HR-ESI-MS m/z: calcd for C17H22ClN3O̗˷M+H˹̙319.1458,
found 319.8337; Anal. calcd for C17H22ClN3O: C, 63.84; H, 6.93; Cl, 11.08; N, 13.14; O, 5.00;
found: C, 63.85; H, 6.92; Cl, 11.06; N, 13.15; O, 5.01%.
N’-(5-chloropyridin-2-yl)-N’-(4-methoxyphenethyl)-N, N-dimethylethane-1, 2-diamine (5l)
1
white crystal, yield 70.8%, m.p. 209-211Ԩ; H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3),
2.52 (2H, t, J = 6.9 Hz, -CH2-), 2.82 (2H, t, J = 2.7 Hz, -CH2-), 3.15 (2H, t, J = 2.7 Hz, -CH2-),
3.38 (2H, t, J = 6.9 Hz, -CH2-), 3.82 (3H, s, -OCH3), 6.83 (1H, ddd, J = 7.9, 4.8, 1.3 Hz, Py-H),
6.85 (2H, ddd, J = 8.8, 2.1, 0.5 Hz, Ph-H), 7.14 (2H, ddd, J = 8.8, 1.0, 0.5 Hz, Ph-H), 7.78 (1H,
ddd, J = 8.2, 1.3, 0.5 Hz, Py-H), 8.21 (1H, ddd, J = 8.2, 7.9, 1.9 Hz, Py-H); 13C NMR (75MHz,
DMSO) δ: 35.6, 45.5, 49.5, 50.8, 55.5, 57.5, 111.5, 114.2, 123.1, 123.9, 131.2, 137.7, 145.1, 155.3,
160.5; HR-ESI-MS m/z: calcd for C18H24ClN3O̗˷M+H˹+̙ 333.1606, found 333.8609; Anal.
calcd for C18H24ClN3O: C, 64.76; H, 7.25; Cl, 10.62; N, 12.59; O, 4.79; found: C, 64.77; H, 7.24;
Cl, 10.64; N, 12.58; O, 4.78%.
N’-(5-aminepyridin-2-yl)-N’-(4-methoxybenzyl)-N, N-dimethylethane-1, 2-diamine (5m)
1
white crystal, yield 84.6%, m.p. 190-192Ԩ; H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3),
2.52 (2H, t, J = 2.7 Hz, -CH2-), 3.15 (2H, t, J = 2.7 Hz, -CH2-), 3.82 (3H, s, -OCH3), 4.72 (2H, s,
-CH2-), 6.65 (1H, dd, J = 7.5, 0.5 Hz, Py-H), 6.80 (1H, dd, J = 7.5, 1.9 Hz, Py-H), 6.88 (2H,
ddd, J = 8.6, 1.7, 0.6 Hz, Ph-H), 7.14 (2H, ddd, J = 8.6, 1.1, 0.6 Hz, Ph-H), 7.18 (1H, dd, J = 1.9,
0.5 Hz, Py-H); 13C NMR (75MHz, DMSO) δ: 45.5, 50.8, 53.6, 55.5, 57.5, 111.5, 114.1, 117.3,
129.1, 136.6, 137.0, 141.1, 155.3, 160.5; HR-ESI-MS m/z: calcd for C17H24N4O̗˷M+H˹+̙
300.1959, found 300.4066; Anal. calcd for C17H24N4O: C, 67.97; H, 8.05; N, 18.65; O, 5.33;
found: C, 67.96; H, 8.06; N, 18.64; O, 5.32%.
N’-(5-aminepyridin-2-yl)-N’-(4-methoxyphenethyl)-N, N-dimethylethane-1, 2-diamine (5n)
1
white crystal, yield 82.9%, m.p. 194-196Ԩ; H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3),
2.51 (2H, t, J = 6.9 Hz, -CH2-), 2.81 (2H, t, J = 2.7 Hz, -CH2-), 3.15 (2H, t, J = 2.7 Hz, -CH2-),
16

3.38 (2H, t, J = 6.9 Hz, -CH2-), 3.82 (3H, s, -OCH3), 6.65 (1H, dd, J = 7.3, 0.5 Hz, Py-H), 6.78
(1H, dd, J = 7.3, 1.9 Hz, Py-H), 6.85 (2H, ddd, J = 8.8, 2.1, 0.5 Hz, Ph-H), 7.14 (2H, ddd, J = 8.8,
1.0, 0.5 Hz, Ph-H), 7.19 (1H, dd, J = 1.9, 0.5 Hz, Py-H); 13C NMR (75MHz, DMSO) δ: 35.7, 45.5,
49.5, 50.9, 55.5, 57.5, 111.5, 114.7, 117.4, 123.4, 131.2, 136.8, 141.1, 155.3, 160.5; HR-ESI-MS
+
m/z: calcd for C18H26N4O̗˷ M+H˹̙ 314.2105, found 314.4338; Anal. calcd for C18H26N4O: C,
68.76; H, 8.34; N, 17.82; O, 5.09; found: C, 68.78; H, 8.33; N, 17.82; O, 5.08%.
N’-(4-methoxybenzyl)-N, N-dimethyl-N’-(5-methylpyridin-2-yl)ethane-1, 2-diamine (5o)
1
white crystal, yield 78.5%, m.p. 201-203Ԩ; H NMR (300MHz, DMSO) δ: 2.23 (6H, s, -CH3),
2.52 (2H, t, J = 2.7 Hz, -CH2-), 3.15 (2H, t, J = 2.7 Hz, -CH2-), 3.82 (3H, s, -OCH3), 4.72 (2H, s,
-CH2-), 6.65 (1H, dd, J = 7.5, 0.5 Hz, Py-H), 6.80 (1H, dd, J = 7.5, 1.9 Hz, Py-H), 6.88 (2H,
ddd, J = 8.6, 1.7, 0.6 Hz, Ph-H), 7.14 (2H, ddd, J = 8.6, 1.1, 0.6 Hz, Ph-H), 7.18 (1H, dd, J = 1.9,
0.5 Hz, Py-H); 13C NMR (75MHz, DMSO) δ: 17.6, 45.5, 50.8, 53.6, 55.5, 57.5, 111.5, 114.1,
127.7, 129.0, 131.5, 136.6, 143.1, 155.3, 160.5; HR-ESI-MS m/z: calcd for C18H25N3O̗˷M+H˹
+
̙ 299.1993, found 299.4186; Anal. calcd for C18H25N3O: C, 72.21; H, 8.42; N, 14.03; O, 5.34;
found: C, 72.23; H, 8.42; N, 14.02; O, 5.34%.
N’-(4-methoxyphenethyl)-N, N-dimethyl-N’-(5-methylpyridin-2-yl)ethane-1, 2-diamine (5p)
1
white crystal, yield 75.3%, m.p. 206-208Ԩ; H NMR (300MHz, DMSO) δ: 2.17 (3H, sˈ-CH3),
2.23 (6H, s, -CH3), 2.51 (2H, t, J = 6.8 Hz, -CH2-), 2.81 (2H, t, J = 2.7 Hz, -CH2-), 3.15 (2H, t, J =
6.8 Hz, -CH2-), 3.38 (2H, t, J = 2.7 Hz, -CH2-), 3.82 (3H, s, -OCH3), 6.55 (1H, dd, J = 8.3, 0.5 Hz,
Py-H), 6.85 (2H, ddd, J = 8.8, 2.1, 0.5 Hz, Ph-H), 7.14 (2H, ddd, J = 8.8, 1.0, 0.5 Hz, Ph-H), 7.32
(1H, dd, J = 1.6, 0.5 Hz, Py-H), 7.91 (1H, dd, J = 8.3, 1.6 Hz, Py-H); 13C NMR (75MHz, DMSO)
δ: 17.6, 35.6, 45.5, 49.5, 50.9, 55.5, 57.5, 111.5, 114.2, 123.4, 127.7, 131.0, 131.6, 143.1, 155.3,
+
160.5; HR-ESI-MS m/z: calcd for C19H27N3O̗˷M+H˹̙ 313.2159, found 313.4451; Anal. calcd
for C19H27N3O: C, 72.81; H, 8.68; N, 13.41; O, 5.10; found: C, 72.82; H, 8.67; N, 13.42; O,
5.09%.
4.2. Biological section
4.2.1. Screening of biological activity of compounds in vitro
(a) Mast cell degranulation assay
The single cell suspension of rat basophilic leukemia cell line (RBL-2H3) was inoculated in
24-wells plates at a concentration of 3×105/mL. Each well was inoculated with 1 mL cell
17

suspension, cultured overnight in 5% CO2 and 37 Ԩ incubator for adherent. Add 20 μL of
compound solution with different concentration to each well, Chlorpheniramine and Astermizole
were taken as the positive control, and Dimethylsulfoxide (DMSO) was taken as the blank control.
After 30 min, add mast cell activator compound 48/80 (C48/80) with 20 μL and final
concentration of 10 μg·mL -1, and discard culture solution after 1 h, add 0.5% neutral red dye for 3
minutes. The number of degranulated cells per 100 mast cells was randomly counted. The
concentration required for the degranulation of a half of mast cells was used as the evaluation
index (IC50), and the lower IC50 value, the better the bioactivity of the compound.
(b) Histamine release assay
The single cell suspension of rat basophilic leukemia cell line (RBL-2H3) was inoculated in
24-wells plates at a concentration of 3×105/mL. Each well was inoculated with 1 mL cell
suspension, cultured overnight in 5% CO2 and 37 Ԩincubator for adhere. Add 20 μL of compound
solution with different concentration to each well, Chlorpheniramine and Astermizole were taken
as the positive control, and Dimethylsulfoxide (DMSO) was taken as the blank control. After 30
min, the C48/80 with 20 μL a final concentration of 10 μg·mL-1, which was used for 1 h. The 50
μL of cell culture supernatant was taken and the histamine content was determined according to
the following steps. In the cell culture supernatant, the 50 μL 0.1 mol.L-1 HCl, 25 μL 0.4 mol.L-1
NaOH solution and 5 μL 0.1% phthalaldehyde/methanol solution were added. The reaction was
fully mixed and terminated after 10 minutes reaction at room temperature, the 25 μL 0.5 mol.L-1
HCl was added to terminate the reaction. The excitation wavelength was 347 nm and the emission
wavelength was 442 nm. The cell lysates were collected to detect the histamine levels in cells. The
concentration required for the degranulation of a half of histamine release was used as the
evaluation index (IC50), which the lower IC50 value, the better the bioactivity of the compound.
(c) β-hexosaminidase release assay
The single cell suspension of rat basophilic leukemia cell line (RBL-2H3) was inoculated in
24-wells plates at a concentration of 3×105/mL and were sensitized overnight with
anti-dinitrophenylated specific immunoglobulin E (IgE) at 150 ng.mL-1. The IgE sensitized cells
were washed twice with piperazine-N, N’-bis [2-ethanesulfonic acid] buffer (20 mmol.L-1 PIPES,
pH 7.0, 100 mmol.L-1 NaCl, 0.5 mmol.L-1 MgCl, 5 mmol.L-1 KCl, 1 mmol.L-1 CaCl2, 40 mmol.L-1
HCl, 5.8 mmol.L-1 glucose and 0.2% bull serum albumin). At 5% CO2 37Ԩ, the synthesized
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compounds was treated for 30 min, Chlorpheniramine and Astermizole were taken as the positive
control, and Dimethylsulfoxide (DMSO) was taken as the blank control. The cells were then
stimulated with 20 ng.mL-1 dinitrophenyl human serum albumin at 5% CO2 37Ԩ for 15 min and
frozen on ice to stop the stimulation. The cell culture supernatant from the IgE/Ag-stimulated cells
in pipes buffer and β-hexosaminidase substrate (P-nitrophenyl-N-acetyl-β-D-glucosaminide) in 0.2
mol.L-1 citrate buffer (pH 4.6) were mixed in 96-well plates and incubated at 5% CO2 37Ԩfor 2 h
to measure β-hexosaminidase release. This reaction was terminated with the addition of 0.2
mol.L-1 pH 10.2 sodium carbonate buffer and the absorbance of 405 nm was measured at
wavelength. The concentration required for the degranulation of a half of β-hexosaminidase
releases was used as the evaluation index (IC50), which the lower IC50 value, the better the
bioactivity of the compound.
4.2.2. In vivo animal study
(a) Antihistaminic activity
The antihistamine potential of synthetic compounds was determined by the modified Van
Arman method. The male guinea pigs (220-260 g)that were given water every 8 hours fasted for
24 hours, and the six guinea pigs were randomly assi gned to each group. The assay compounds
was taken orally at a dose of 20 mg.kg-1 in 1% Carboxymethylcellulose sodium (CMC-Na),
histamine aerosol (5 mL 0.3% histamine chloride solution) was added to the steam epinephrine
bag atomizer sprayed into the closed transparent cage, and Chlorpheniramine was taken as the
positive control. The respiratory conditions reflecting increased bronchoconstriction was recorded,
and the duration of convulsions (preeclampsia) was recorded. Animals that remain stable for more
than 10 min are thought to prevent histamine-induced bronchospasm. Chlorpheniramine maleate
was injected at a dose of 25 mg.kg -1 for the recovery of assay animals. The mean preeclampsia
time of the animals treated with the test compound was compared with that of the control group
and expressed as protection percentage.
(b) Capillary permeability assay
The mice (20-22 g) were randomly divided into three groups according to their weight, with
10 mice in each group, half male and half female. The mice in the assay compound groups were
given different compounds suspensions by gavage with a dose of 20 mg·kg-1 for one week. The
mice in the model groups were given 0.5% of Carboxymethylcellulose sodium (CMC-Na). One
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day before the assay, the mice were depilated with 8% sodium sulfide. Subcutaneous injection of
0.1% 50 μL of histamine phosphate. The injection by tail vein, 0.5% of Evans blue normal saline
solution (25mg· kg-1), after 15 min was put to death without head. The blue-stained part of the
skin was cutted into four holes, and then soaked in 1 mL acetone-normal saline solution for 48
hours, the 3000 r.min-1 supernatant was centrifuged for 10 min, and the absorbance value (OD)
was measured at the wavelength of 610 nm using an ultraviolet spectrophotometer. Calculate the
concentration according to the Evans blue standard curve.
Acknowledgements
The project was sponsored by the Scientific Research Foundation for the
Returned Overseas Chinese Scholars, State Education Ministry (No. 2015-1098). The
work was also supported by Chongqing Key Research Project of Basic Science &
Frontier Technology (No. cstc2017jcyjBX0012), Foundation Project of Chongqing
Normal University (No. 14XYY020), Chongqing General Research Program of Basic
Research and Frontier Technology (No. cstc2015jcyjA10054), Chongqing Normal
University Postgraduate's Research and Innovation Project (No. YKC17004), the
National Natural Science Foundation of China (21662012, 41866005), and Postgraduate Research
and Innovation Project of Hainan Normal University (Hsyx2018-8)ˈand Open Foundation Project
of Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education
(RDZH2019002), China.
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Graphical abstract
Conflicts of interest
The authors declare that they have no conflict of interest.
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