Synthesis, Purification, and Selective β2-AR Agonist and Bronchodilatory Effects of Catecholic Tetrahydroisoquinolines from Portulaca oleracea

Article abstract of DOI:10.1021/acs.jnatprod.9b00418

A green, biomimetic, phosphate-mediated Pictet-Spengler reaction was used in the synthesis of three catecholic tetrahydroisoquinolines, 1, 2, and 12, present in the medicinal plant Portulaca oleracea, as well as their analogues 3-11, 13, and 14, with dopa

Full text of DOI:10.1021/acs.jnatprod.9b00418

Article
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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Synthesis, Purification, and Selective β2‑AR Agonist and
Bronchodilatory Effects of Catecholic Tetrahydroisoquinolines from
Portulaca oleracea
Er-Lan Yang,† Bin Sun,‡ Zi-Yi Huang,† Jian-Guang Lin,† Bo Jiao,*,† and Lan Xiang*,†
†Key Laboratory of Chemical Biology (Ministry of Education), Institute of Pharmacognosy, School of Pharmaceutical Sciences,
Shandong University, Jinan, Shandong 250012, People’s Republic of China
‡National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250012, People’s Republic of China
S
* Supporting Information
ABSTRACT: A green, biomimetic, phosphate-mediated Pictet−Spengler reaction was
used in the synthesis of three catecholic tetrahydroisoquinolines, 1, 2, and 12, present in
the medicinal plant Portulaca oleracea, as well as their analogues 3−11, 13, and 14, with
dopamine hydrochloride and aldehydes as the substrates. AB-8 macroporous resin column
chromatography was applied for purification of the products from the one-step high-
efficacy synthesis. It eliminated the difficulties in the isolation of catecholic
tetrahydroisoquinolines from the aqueous reaction system and unreacted dopamine
hydrochloride. Activity screening in CHO-K1/Gα15 cell models consistently expressing
α1B-, β1-, or β2-adrenergic receptors indicated that 12 and 2, compounds that are present
in P. oleracea, possessed the most potent β2-adrenergic receptor agonist activity and 2 was
a selective β2-adrenergic receptor agonist at the concentration of 100 μM. Both 12 and 2
exhibited dose-dependent bronchodilator effects on the histamine-induced contraction of
isolated guinea-pig tracheal smooth muscle, with EC50 values of 0.8 and 2.8 μM,
respectively. These findings explain the scientific rationale of P. oleracea use as an antiasthmatic herb in folk medicine and
provide the basis for the discovery of novel antiasthma drugs.
In our previous phytochemical investigation of the medicinal
plant Portulaca oleracea L., a series of water-soluble catecholic
THIQs were isolated [6,7-dihydroxy-1-methyl-1,2,3,4-tetrahy-
droisoquinoline (1), 6,7-dihydroxy-1-isobutyl-1,2,3,4-tetrahy-
droisoquinoline (2), 1-benzyl-6,7-dihydroxy-1,2,3,4-tetrahy-
droisoquinoline (12), dehydroisoquinoline derivatives of 1-
(furan-2-yl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline
(13), and 6,7-dihydroxy-1-(5-hydroxymethylfuran-2-yl)-
1,2,3,4-tetrahydroisoquinoline (14) (Figure 1)], and most of
them exhibited potent β2-AR agonist activity.16 However, their
selectivity for different AR subtypes and their bronchodilator
effects were not studied. Owing to the low contents in the
medicinal plant, isolation of these compounds from natural
resources is a time- and labor-intensive task. To provide large
amounts of compounds for further pharmacological research,
the chemical synthesis of catecholic THIQs is necessary.
The Pictet−Spengler reaction is an important cyclization
reaction leading to the formation of THIQs as well as other
heterocyclic moieties, including imidazoles, benzoxazoles,
pyrroles, indoles, and tetrahydro-β-carbolines.17,18 Since the
traditional Pictet−Spengler reaction has the disadvantage of
requiring severe reaction conditions, such as high temperature
and strong acid catalysis,19 the environmentally friendly
1,2,3,4-Tetrahydroisoquinoline (THIQ) is one of the “priv-
ileged scaffolds” commonly found in nature.1,2 THIQs exhibit
a variety of pharmacological activities, including antitumor,
antiviral, anti-inflammatory, anticoagulation, and bronchodila-
tion activities and action on the central nervous system.1−3 As
an important skeleton for drug discovery, the structural
diversity and biological diversity of THIQs have attracted
considerable attention in recent years.4−9 Among them,
catecholic THIQs, which possess o-dihydroxy groups on the
benzene ring, as well as their analogues are important sources
for drug discovery targeting the adrenergic receptors (ARs).
ARs belong to the superfamily of G protein-coupled receptors
(GPCRs), and they are categorized into two broad classes: α-
ARs and β-ARs. Of the two α-AR subtypes, α1-ARs are highly
expressed on vascular smooth muscle cells and cardiomyocytes,
whereas α2-ARs are primarily found in the central nervous
system. The three β-AR subtypes, β1-AR, β2-AR, and β3-AR,
are predominantly found in the myocardium, vascular and
bronchial smooth muscle, and adipose tissue, respectively.10
Norepinephrine, epinephrine, dobutamine, and dopamine are
well-known antishock vasoactive drugs that have different
agonist efficacy on α1-AR, β1-AR, and β2-AR.11 Selective β2-AR
agonists such as salbutamol are widely used for the treatment
of asthma.12 The representative catecholic THIQ-derived
drugs include the β2-AR agonist antiasthma compound
trimetolquinol13,14 and the β1-AR agonist higenamine.15
Received: May 4, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 1. Structures of synthesized catecholic THIQs 1−14 and the analogues DH1, DM1, (S)-1, (R)-2, (R)-12, DH13, and DH14 isolated from
the medicinal plant Portulaca oleracea.
synthesis of THIQs was pursued. For example, a mild one-pot
versatile phosphate-mediated Pictet−Spengler reaction has
been exploited in the biomimetic synthesis of THIQs.20
Systematic investigation indicated the uniqueness of phosphate
as the catalyst and necessity of a C-3-OH in the structure of
the substrates. In this mild Pictet−Spengler reaction, dopamine
and an aldehyde form an iminium intermediate under acidic
conditions (pH 6.0) in the first step. Phosphate was
hypothesized to play a role in the following three aspects.
First, a phosphate anion or dianion would effect a nucleophilic
attack onto the iminium intermediate to produce a highly
reactive aminophosphate. Second, phosphate can deprotonate
the C-3-OH and activate the aromatic ring for addition to the
imine at the para-position. Third, phosphate-mediated intra-
(path a) or intermolecular (path b) abstraction of the 8a-H
facilitates rearomatization (Scheme 1).20
with CH2Cl2 followed by preparative HPLC using a gradient of
MeCN−H2O (0.1% trifluoroacetic acid).20 However, some
water-soluble THIQs had short HPLC retention times, which
made them difficult to separate from dopamine. Moreover,
considering the high economic cost, an HPLC method was not
suitable for large-scale preparation of the products. Bonamore
et al. reported an enzymatic stereoselective synthesis of (S)-
norcoclaurine (higenamine) in phosphate buffer, and the
product was purified with Norit, a multipurpose activated
charcoal.21 In this purification procedure, the absorption was
achieved by shaking (30 min) carbon granules that were added
directly to the aqueous phase at room temperature, and the
desorption of (S)-norcoclaurine was achieved by elution with
EtOH at 40 °C in the presence of a slight molar excess of
NaOH.21 However, catecholic THIQs are commonly unstable
under alkaline conditions. Maresh et al. reported that
halogenated derivatives of norcoclaurine and aldehydes
consistently partition into the same organic solvent. With
aldehyde exhaustion, pure target THIQs can be readily
obtained through EtOAc extraction followed by washing with
brine, drying with MgSO4, and solvent evaporation.22
However, in our experiment, in addition to 11, which can be
readily extracted into EtOAc to yield pure powder precipitating
during concentration, most of the catecholic THIQs were
water-soluble. Their Rf values on silica gel thin-layer
chromatography (TLC) were about 0.5 when developed by
n-BuOH−HOAc−H2O (4:1:1) or EtOAc−MeOH−H2O
(4:1:1), and their solubility in EtOAc or CH2Cl2 was low.
Therefore, the introduction of water-soluble phosphate and
antioxidant vitamin C greatly interfered with separation.
Wakchaure et al. and Barbero et al. reported that THIQs
synthesized with a phosphate-mediated method were purified
by flash silica gel column chromatography.23,24 However, it was
found in our experiment that these water-soluble THIQs were
strongly adsorbed when subjected to silica gel column
chromatography, and the products were readily decomposed
during the long separation time due to their low stability.
Unexpectedly, following the one-step synthesis of catecholic
THIQs by the phosphate-mediated Pictet−Spengler reaction,
the purification of these alkaloids became problematic. Pesnot
et al. reported that phosphate-mediated synthesized THIQs,
e.g., norcoclaurine (higenamine), were purified via extraction
Scheme 1. Proposed Phosphate-Mediated Mechanism for
a
20
the Pictet−Spengler Reaction
a
Pi is inorganic phosphate.
B
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Owing to the limitation of the existing purification protocols,
separating water-soluble catecholic THIQs from a water-
soluble reaction system (containing phosphate buffer and
vitamin C) and a dopamine substrate has become a significant
challenge. A facile, benign, and scalable purification method for
catecholic THIQs is urgently needed.
selected as 15 mM by our preliminary screening. Moreover,
distilled water was degassed by boiling before its addition to
the reaction. To avoid exposure to sunlight, protection from
light during the reaction and purification steps was necessary.
Following the reported phosphate-mediated Pictet−Spen-
gler reaction20−22 with some adjustments, 14 catecholic
THIQs (1−14) (Figure 1) were synthesized using inexpensive
and readily available dopamine hydrochloride and aldehydes as
substrates. Their structures were elucidated based on ESIMS,
1H NMR, and 13C NMR data analysis (Supporting
Information). Compounds 1, 2, and 12 represent alkaloids
present in P. oleracea, 14 is a new alkaloid, and 2 and alkaloids
7−14 were synthesized through the phosphate-mediated
Pictet−Spengler reaction for the first time. Pesnot et al.
reported that the highest conversions were achieved when a
solvent mixture of phosphate buffer with MeCN, MeOH, or
DMSO was used in order to improve substrate solubility.20
Since no organic solvent was used in the present experiment,
the low yields of some of the products may be related to the
poor solubility of the aldehydes in water.
RESULTS AND DISCUSSION
■
Synthesis of Catecholic THIQs 1−14. The phosphate-
mediated biomimetic syntheses of 1−14 were performed
according to the route in Scheme 2, and the corresponding
a
Scheme 2. Synthesis Route for Catecholic THIQs
a
a: 1 M of potassium phosphate buffer (pH 6.0)−H2O (1:2, v/v),
vitamin C, 40 °C, 6 h.
Purification of Catecholic THIQs 1−14. The reaction
was completed after 6 h as monitored by TLC, and the mixture
was extracted with EtOAc (3 × 150 mL) to remove unreacted
aldehydes. Except for 11, which can be extracted into EtOAc
and precipitated to yield pure pale yellow product powder after
concentration of the organic layer, other catecholic THIQs
were less soluble in EtOAc and were present in large quantities
in the water layer. The water layer (150 mL) of the reaction
systems 1−10 and 12−14 was subjected to AB-8 macroporous
resin column chromatography (4 × 40 cm), eluted with seven
body volumes (BV) of distilled water at a high flow rate of
1800 mL/h, and then eluted with 1 BV of EtOH at a low flow
rate of 250 mL/h. Identification of the target product in the
eluted fraction was performed on silica gel TLC, developed by
n-BuOH−HOAc−H2O (4:1:1) or EtOAc−MeOH−H2O
(4:1:1), and sprayed with iodine vapor, 5% FeCl3, or 5%
ninhydrin.
Macroporous (macroreticular) resin is a type of adsorption
material that was developed in the 1960s.25 This resin not only
acts as a molecular sieve depending on its porous structure but
also adsorbs some ingredients with similar polarity through van
der Waals forces and hydrogen-bonding interactions.26
Currently, macroporous resin is widely used in the isolation
and purification of natural products, especially total poly-
phenols,27 total flavonoids,28 total alkaloids,29 and total
saponins.30 When we used AB-8 macroporous resin column
chromatography to remove water-soluble compounds such as
phosphate buffer and vitamin C, we occasionally found that
catecholic THIQs could be separated from the unreacted
substrate dopamine. Using elution with H2O as the first step,
phosphate, vitamin C, and dopamine can be quickly removed.
aldehydes and target products are shown in Table 1. In brief,
dopamine hydrochloride (1.2 equiv), aldehyde (1 equiv), and
vitamin C (0.5 equiv) were added to a mixture of potassium
phosphate buffer (a mixture of KH2PO4 and K2HPO4: 1 M,
pH 6.0) and distilled water (1:2, v/v). The reaction mixture
was maintained at 40 °C in a water bath and stirred under a
nitrogen atmosphere for 6 h.
Phosphate-mediated biomimetic Pictet−Spengler reactions
were successfully applied in the one-pot synthesis of THIQs,
starting from dopamine or amino acids as the substrates.20,22
The reaction conditions in these experiments were slightly
different. Pesnot et al. reported that the highest conversion was
achieved using potassium phosphate buffer.20 Maresh et al.
found that increasing the phosphate buffer concentration
(200−350 mM) led to a decrease in side product formation.22
Therefore, 333 mM potassium phosphate buffer was used in
the present study, through adding 1 M buffer with twice the
volume of H2O. The pH value of the phosphate buffer was
selected as 6, considering that dopamine and catecholic
THIQs are stable under acidic conditions, and a phosphate-
mediated Pictet−Spengler reaction was reported to go
smoothly at pH 6 but failed at pH < 4 or pH > 8.20 The
conversion rate increased when the temperature was elevated
to 50 °C; however, the higher temperature led to the
degradation of dopamine and evaporation of aldehydes.
Therefore, a temperature of 40 °C was used in the present
study. Dopamine and catecholic THIQs were readily
decomposed when exposed to oxygen and sunlight and
produced black pigments. To avoid oxidation, the reaction
was performed under nitrogen, and the antioxidant, vitamin C
was added to the reaction mixture.22 The concentration was
Table 1. Aldehydes and Target Synthesized Catecholic THIQs
R
product
yield (%)
R
product
yield (%)
methyl
isobutyl
phenyl
4-hydroxyphenyl
3-hydroxyphenyl
1
2
3
4
5
6
7
25
28
36
54
78
67
89
4-methoxyphenyl
2-methoxyphenyl
3,4,5-trimethoxyphenyl
1-naphthyl
benzyl
2-furyl
8
9
31
40
42
32
34
53
58
10
11
12
13
14
3,4-dihydroxyphenyl
4-hydroxy-3-methoxyphenyl
2-furyl-5-hydroxymethyl
C
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Figure 2. Agonist effects of 21 compounds (100 μM) on β2-, β1-, and α1B-AR, as expressed by stimulation rate (%) versus the positive control of
100% (n = 4). Note: (1) isoproterenol (1 μM) was used as the β2-AR agonist positive control, and epinephrine (10 μM) was used as the β1-AR
agonist positive control and the α1B-AR agonist positive control. (2) For convenient comparison of the structure−activity relationships, the
reported β2-AR agonist activity16 of the compounds DH1, DM1, (S)-1, (R)-2, (R)-12, DH13, and DH14 isolated from P. oleracea is noted with
blue asterisks.
Using elution with EtOH as the second step, the target
catecholic THIQs can be completely separated from
dopamine, thereby yielding purified product. There are many
kinds of macroporous resin, and they are mainly divided into
three categories according to the resin surface property,
including high-polarity, low-polarity, and no-polarity resin. AB-
8 macroporous resin belongs to a low-polarity resin.26 Since
dopamine hydrochloride possesses higher polarity than the
synthesized catecholic THIQs, it may cause weak adsorption of
dopamine and high adsorption of catecholic THIQs by the AB-
8 resin. This difference may lead to ready desorption of
dopamine by water elution and separation from catecholic
THIQs. Considering that macroporous resin chromatography
consumes a low volume of organic solvent and that
macroporous resin and EtOH are recyclable, the present
purification method for catecholic THIQs by AB-8 macro-
porous resin column chromatography is quite simple,
convenient, economical, and green. We purified products at
the gram scale using this method.
Adrenergic Receptor Agonist Activity of Catecholic
Isoquinolines. In the previous study, the β2-AR agonist
activity of 14 catecholic isoquinolines isolated from P. oleracea
was reported.16 In this study, the effects of 100 μM of 14
synthesized catecholic THIQs and seven compounds isolated
from P. oleracea (Figure 1) were systematically investigated on
β1-, β2-, and α1B-AR, as detected by calcium determination
using CHO-K1/Gα15/ADRB1, CHO-K1/Gα15/ADRB2, or
CHO-K1/Gα15/ADRA1B cell lines that stably express β1-, β2-,
or α1B-AR. The EC50 value of isoproterenol as the β2-AR
agonist was 1.2 × 10−9 M, and EC50 value of epinephrine as the
β1-AR agonist and α1B-AR agonist was 2.9 × 10−7 and 7.5 ×
10−8 M, respectively, which met the internal requirement for
the positive control. Therefore, isoproterenol at the highest
tested concentration of 1 μM was used as the β2-AR agonist
positive control, and epinephrine at the highest tested
concentration of 10 μM was used as the β1-AR agonist
positive control and the α1B-AR agonist positive control. The
AR agonist activity of tested compounds was expressed as the
stimulation rate versus the positive control of 100%.
As illustrated in Figure 2, in addition to the known β2-AR
agonists DH1, DM1, (S)-1, (R)-2, (R)-12, DH13, and
DH14,16 synthesized compounds 2 and 12 were potent β2-
AR agonists; only 12, (R)-12, and DH13 were potent β1-AR
agonists, and the other compounds had no activity. All tested
compounds showed medium (3, 4, 9, 11, and 12) or weak α1B-
AR agonist activity. The structure−activity relationships of
these compounds on β1-, β2-, and α1B-AR activation can be
concluded as follows. (1) Introduction of a C-methyl to simple
dihydroisoquinoline, as in the case of compound DH1 to
DM1, did not affect their weak β2-AR (41.06 3.80%,16 37.70
3.63%16) and α1B-AR agonist activities (24.18
5.82%,
3.74%). Both compounds had no effect on β1-AR
4.64%, −6.31 2.87%). (2) As we previously
15.87
(−6.22
reported, THIQ (S)-1 possessed β2-AR agonist activity (35.47
1.14%)16 equal to its dihydroisoquinoline derivative DM1.
However, the β2-AR agonist effect of (S)-1 was higher than
that of its racemic counterpart 1 (2.91 13.03%), and both
possessed equally weak agonist activity on α1B-AR (19.20
3.99%, 22.15
5.48%) and no effect on β1-AR (−9.50
3.83%, −11.45 1.75%), reflecting that (R)-1 may have no
effect on β1- and β2-AR and a weak effect on α1B-AR. (3)
Comparing racemic mixtures of 1 and 2, when the C-1 methyl
group was replaced by an isobutyl group, the β2-AR agonist
activity of 2 increased 20-fold to 64.51 13.80%, whereas its
effect on α1B-AR nearly disappeared (7.70
12.96%). (4)
There was no significant difference in β2-AR agonist activity
between (R)-2 (70.89 9.13%)16 and its racemic mixture 2
(65.41 13.80%). The α1B-AR agonist activity of racemic 2
was extremely low (7.70 12.96%), nearly half the value of
(R)-2 (20.49
4.37%), and both had no effect on β1-AR,
indicating that racemic 2 and (S)-2 are selective β2-AR
agonists. (5) When a phenyl or a substituted phenyl group is
located at C-1 of the catecholic THIQs, as in the case of
synthesized racemic compounds 3−10, all compounds had no
agonist effects on β2- and β1-AR. However, 3−10 all showed
D
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Figure 3. Bronchodilator effects of 12 and 2 on histamine-induced contraction in isolated guinea-pig tracheal spiral strips. (A) Representative
tension (g) change diagram of tracheal smooth muscle after adding (↓) histamine (His) then each tested compound 12, 2 or positive control
isoprotenerol (Iso), with increased concentration, in the absence (a, b, c) or presence (d, e, f) of β-AR blocker propranolol hydrochloride (Pro).
(B) Spasmolysis percentage (%) of different concentrations of 12, 2, and Iso in the absence or presence of Pro (n = 5).
medium or weak α1B-AR agonist activity in the order 3 (51.53
13.31%) > 9 (36.31 1.70%) > 4 (31.88 7.61%) > 10
(20.47 5.47%) ≥ 8 (18.50 4.29%) ≥ 6 (16.60 7.04%) ≥
doubled, indicating that racemate 12 had better selectivity for
β1-AR. The difference between (R)-12 and racemic 12 also
reflected that (S)-12 may be a selective β1-AR agonist with a
strong effect on β1-AR but no effect on β2-AR and a weak effect
on α1B-AR. (9) Dihydroisoquinoline DH13 was a potent β2-
AR (91.77 2.78%)16 and β1-AR (73.50 20.12%) agonist.
DH13 had no selectivity for β1 and β2 subtypes but had a very
weak effect on α1B-AR (13.36 4.69%). Its THIQ derivative
5 (14.53
2.29%) ≥ 7 (11.81
13.03%), indicating that
phenyl- or 2-methoxyphenyl-substituted catecholic THIQs
possess more potent agonist activity on α1B-AR than those
connected to a 3- and/or 4-hydroxyphenyl, a 3- and/or 4-
methoxyphenyl, or a 3,4,5-trimethoxyphenyl group. (6) When
the C-1 phenyl group (3) was replaced by a naphthyl group
(11), the α1B-AR activity of 11 (43.75 2.45%) was similar to
3, and it also had no effect on β2- and β1-AR. (7) When the C-
1 phenyl group (3) was replaced by a benzyl group (12),
13 retained a weak effect on α1B-AR (15.61
13.98%);
however, it had no positive activation of β1-AR (−35.29
15.68%) or β2-AR (−9.23 15.89%). (10) As we previously
reported, β2-AR agonist potency of dihydroisoquinoline DH14
racemic 12 exhibited potent β1-AR (99.86
16.50%) and
(70.60
5.18%)16 was slightly lower than that of DH13
medium β2-AR (44.67 17.08%) and α1B-AR (39.42
(91.77 2.78%),16 which lacks a hydroxymethyl group on the
furan ring in comparison with the structure of DH14. Similar
to DH13, DH14 had weak activity on α1B-AR (13.73
4.08%). Interestingly, DH14 had no effect on β1-AR (−10.38
7.33%), which indicated that dihydroisoquinoline DH14
was a potent β2-AR selective agonist. (11) Similar to DH14, its
THIQ derivative 14 retained a weak agonistic effect on α1B-AR
(22.28 6.86%) and no effect on β1-AR (−20.30 9.17%).
Different from DH14, 14 had no activation effect on β2-AR
(−11.10 6.38%).
13.73%) agonist activities, indicating that the introduction of a
benzyl group was necessary for β1- and β2-AR agonist activity.
However, chiral (R)-12 exhibited potent β1-AR (80.34
32.07%) and β2-AR (90.83
5.98%)16 agonist activity and
weak α1B-AR agonist activity (6.10 9.84%), indicating that
(R)-12 is a β-AR agonist, without selectivity for the β1- and β2-
AR subtypes. (8) Compared with chiral (R)-12, racemate 12
decreased the β2-AR activity by half, the β1-AR agonist activity
remained high, and the α1B-AR agonist activity more than
E
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spectra were recorded on a Bruker Avance DRX-600 MHz NMR
spectrometer with tetramethylsilane as the reference. Low-resolution
ESIMS data were recorded on a Tandem 6410 triple quadrupole mass
spectrometer (Agilent Company, USA). High-resolution ESIMS data
were acquired on a Thermo Electron LTQ-Orbitrap XL mass
spectrometer (Thermo Scientific, USA). All reagents and solvents
were purchased from commercial sources and were of analytical grade.
AB-8 macroporous resin was purchased from Hebei Cangzhou Baoen
Chemical Co., Ltd. (China). TLC analysis was performed on
polyamide film (Taizhou Luqiao Siqing Biochemical Plastics Factory,
China) or GF 254 silica gel (Qingdao Marine Chemical Co., China)
sprayed with iodine vapor, 5% ninhydrin, or 5% FeCl3.
General Method for the Synthesis of Tetrahydroisoquino-
lines 1−14. Dopamine hydrochloride (1.00 g, 5.29 mmol), aldehyde
(4.4 mmol), vitamin C (0.40 g, 2.27 mmol), potassium phosphate
buffer (1 M, pH 6.0, 50 mL), and boiled distilled water (100 mL)
were added sequentially to a round-bottom flask. The mixture was
stirred at 40 °C in a water bath under N2 for 6 h, then extracted with
EtOAc (3 × 150 mL). The water layer was subjected to AB-8
macroporous resin column chromatography (4 × 40 cm), eluting with
7 BV of purified water at a high flow rate of 1800 mL/h, then 1 BV of
EtOH at a low flow rate of 250 mL/h. The EtOH eluate was
concentrated under vacuum, then freeze-dried.
As an important transmembrane protein family, GPCRs play
a significant role in cellular signal transduction and are
important targets for drug design. Structural analysis of GPCRs
has been hindered by their low natural abundance, inherent
structural flexibility, and instability in detergent solutions.31
The crystal structure of human β2-AR was first elucidated in
2007,31 the crystal structure of the β2-AR−Gs protein complex
was elucidated in 2011,32 and the molecular mechanism of β2-
AR−G-protein activation was first elucidated using single-
molecule analysis of ligand efficacy in 2017.33 Considering the
mechanistic complexity of β2-AR−G-protein activation, the
relationship between the absolute configuration of THIQs and
their β2-AR agonist activities needs to be intensively studied.
Bronchodilator Effects of Catecholic THIQs on the
Histamine-Induced Contraction of Isolated Guinea Pig
Tracheal Smooth Muscle. BDTI (1-benzyl-6,7-dihydroxy-
1,2,3,4-tetrahydroisoquinoline HBr) was previously reported to
be a selective β2-AR agonist, and the potency of relaxing effect
on carbachol-induced contraction in isolated canine trachea
was in the order of isoprenaline > BDTI ≈ salbutamol (a
clinically used antiasthma drug).34 Using cell models
expressing the three different ARs, our experiment also
demonstrated that 12, especially enantiomer (R)-12, was a
potent β2-AR agonist; however, 12 also had a strong effect on
β1-AR. It was found for the first time that catecholic THIQ 2
was a β2-AR selective agonist. Since β2-AR selective agonists
have low side effects on the heart, their effect on tracheal
smooth muscle contraction needs to be studied.
6,7-Dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline (1). Ap-
plication of the above method using acetaldehyde (194 μL) gave 1 as
1
a brown powder (196 mg, 25% yield): H NMR (DMSO-d6, 600
MHz) δ 6.49 (s, 1H), 6.40 (1H, s), 3.86 (1H, q, J = 6.6 Hz), 3.06
(1H, m), 2.79 (1H, m), 2.59 (1H, m), 2.46 (1H, m), 1.27 (3H, d, J =
6.6 Hz); 13C NMR (DMSO-d6, 150 MHz) δ 143.99, 143.82,130.56,
125.06, 115.81, 113.22, 50.90, 41.55, 28.46, 21.93; ESIMS m/z 180.2
[M + H]+.
6,7-Dihydroxy-1-isobutyl-1,2,3,4-tetrahydroisoquinoline (2). Ap-
plication of the above method using isobutyraldehyde (480 μL) gave
2 as a green powder (273 mg, 28% yield): 1H NMR (DMSO-d6, 600
MHz) δ 6.49 (1H, s), 6.44 (1H, s), 3.75 (1H, dd, J = 10.2, 3 Hz), 3.02
(1H, m), 2.79 (1H, m), 2.51 (2H, m), 1.91 (1H, m), 1.54 (1H, m),
1.40 (1H, m), 0.97 (3H, d, J = 6.6 Hz), 0.94 (3H, d, J = 6.6 Hz); 13C
NMR (DMSO-d6, 150 MHz) δ 143.62, 143.58, 131.23, 125.78,
115.93, 113.39, 52.75, 46.32, 40.69, 28.97, 24.58, 24.46, 21.93; ESIMS
m/z 222.4 [M + H]+.
As shown in Figure 3A (a, b, c), the smooth muscle tension
of isolated guinea-pig tracheal spiral strips remarkably
increased after addition of histamine, and the tension gradually
decreased with addition of increased concentrations of the
positive control isoproterenol (Iso), 12, or 2, indicating 12 and
2 have bronchodilator effects. Figure 3B further demonstrates
that both racemates 12 and 2 dose-dependently inhibited
histamine-induced guinea-pig tracheal contraction, with EC50
values of spasmolysis at 0.8 and 2.8 μM, respectively, which are
less potent than the positive control Iso (7.9 nM).
To define a potential mechanism for these bronchodilator
effects, a β-AR antagonist propranolol hydrochloride (Pro) was
added.35 As indicated in Figure 3A (d, e, f), after a 15 min
preincubation of Pro, addition of histamine still caused a
significant increase in the tracheal tension. However, as
compared with Figure 3A (a, b, c), the relaxing effects of 12,
2, and Iso, especially at some lower effective concentrations,
reduced or disappeared in the presence of Pro in Figure 3A (d,
e, f), leading to a rightward shift in the dose−effect curve in
Figure 3B, demonstrating that 12 and 2 exert their
bronchodilator effects via activating β2-AR, which is highly
present in the tracheal smooth muscle,36 and their relaxing
actions at lower concentration can be competitively antago-
nized by Pro.
6,7-Dihydroxy-1-phenyl-1,2,3,4-tetrahydroisoquinoline (3). Ap-
plication of the above method using benzaldehyde (449 μL) gave 3 as
1
a yellow powder (386 mg, 36% yield): H NMR (DMSO-d6, 600
MHz) δ 7.36 (2H, m), 7.29 (3H, m), 6.52 (1H, s), 6.05 (1H, s), 4.86
(1H, s), 3.09 (1H, m), 2.88 (1H, m), 2.79 (1H, m), 2.58 (1H, m);
13C NMR (DMSO-d6, 150 MHz) δ 146.08, 144.06, 143.39, 129.51,
129.32, 128.41, 127.28, 126.13, 115.76, 114.94, 61.25, 42.44, 28.91;
ESIMS m/z 242.4 [M + H]+.
6,7-Dihydroxy-1-(4-hydroxyphenyl)-1,2,3,4-tetrahydroisoquino-
line (4). Application of the above method using 4-hydroxybenzalde-
1
hyde (0.538 g) gave 4 as a yellow powder (611 mg, 54% yield): H
NMR (DMSO-d6, 600 MHz) δ 7.03 (2H, d, J = 8.4 Hz), 6.68 (2H, d,
J = 8.4 Hz), 6.43 (1H, s), 6.00 (1H, s), 4.70 (1H, s), 3.03 (1H, m),
2.81 (1H, m), 2.71 (1H, m), 2.47 (1H, m); 13C NMR (DMSO-d6,
150 MHz) δ 156.68, 143.95, 143.32, 136.33, 130.25, 126.01, 115.65,
115.12, 114.95, 60.78, 42.54, 28.91; ESIMS m/z 258.2 [M + H]+.
6,7-Dihydroxy-1-(3-hydroxyphenyl)-1,2,3,4-tetrahydroisoquino-
line (5). Application of the above method using 3-hydroxybenzalde-
hyde (0.538 g) gave 5 as a reddish-brown powder (886 mg, 78%
In this study, the synthesis of catecholic THIQs was green
and the purification was unique and highly efficient. The
finding of the bioassay results of the natural products
demonstrated the scientific rationale for use of P. oleracea as
an antiasthmatic herb in folk medicine, and traditional
medicines are promising sources for drug discovery.
1
yield): H NMR (DMSO-d6, 600 MHz) δ 7.14 (1H, t, J = 7.8 Hz),
6.76 (1H, d, J = 7.2 Hz), 6.69 (2H, m), 6.50 (1H, s), 6.10 (1H, s),
4.76 (1H, s), 3.08 (1H, m), 2.86 (1H, m), 2.76 (1H, m), 2.54 (1H,
m); 13C NMR (DMSO-d6, 150 MHz) δ 157.53, 147.57, 144.03,
143.35, 129.60, 129.25, 126.06, 120.09, 116.13, 115.69, 114.92,
114.25, 61.23, 42.43, 28.97; ESIMS m/z 258.2 [M + H]+.
6,7-Dihydroxy-1-(3,4-dihydroxyphenyl)-1,2,3,4-tetrahydroisoqui-
noline (6). Application of the above method using 3,4-dihydrox-
ybenzaldehyde (0.607 g) gave 6 as a brown-gray powder (809 mg,
67% yield): H NMR (DMSO-d6, 600 MHz) δ 6.71 (1H, d, J = 7.8
Hz), 6.66 (1H, d, J = 1.8 Hz), 6.59 (1H, dd, J = 7.8, 1.8 Hz), 6.48
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
■
1
determined using a Gyromat-Hp digital automatic polarimeter
(Anton Paar Germany GmbH, Germany). H NMR and 13C NMR
1
F
DOI: 10.1021/acs.jnatprod.9b00418
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Journal of Natural Products
Article
(1H, s), 6.10 (1H, s), 4.68 (1H, s), 3.09 (1H, m), 2.84 (1H, m), 2.76
(1H, m), 2.52 (1H, m); 13C NMR (DMSO-d6, 150 MHz) δ 145.26,
144.65, 143.92, 143.28, 137.04, 130.26, 125.95, 120.16, 116.51,
115.58, 115.27, 114.97, 61.02, 42.59, 28.99; ESIMS m/z 274.3 [M +
H]+.
(DMSO-d6, 150 MHz) δ 158.24, 144.45, 143.47, 142.23, 126.56,
126.14, 115.85, 114.60, 110.51, 107.54, 53.67, 40.94, 28.51; ESIMS
m/z 232.2 [M + H]+.
6,7-Dihydroxy-1-(5-hydroxymethylfuran-2-yl)-1,2,3,4-tetrahy-
droisoquinoline (14). Application of the above general method using
5-hydroxymethylfurfural (364 μL) gave 14 as a brick red powder (669
mg, 58% yield): 1H NMR (DMSO-d6, 600 MHz) δ 6.44 (1H, s), 6.30
(1H, s), 6.15 (1H, d, J = 3.0 Hz), 5.93 (1H, d, J = 3.0 Hz), 4.86 (1H,
s), 4.33 (2H, s), 2.94 (1H, m), 2.80 (1H, m), 2.53 (2H, m); 13C
NMR (DMSO-d6, 150 MHz) δ 157.63, 154.66, 144.43, 143.46,
126.67, 126.24, 115.86, 114.67, 108.12, 107.67, 56.20, 53.91, 40.91,
28.33; ESIMS m/z 262.2 [M + H]+; HRESIMS m/z 262.1078 [M +
H]+ (calcd for C14H15NO4, 262.1079).
6,7-Dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-1,2,3,4-tetrahy-
droisoquinoline (7). Application of the above method using 4-
hydroxy-3-methoxybenzaldehyde (0.67 g) gave 7 as a brown powder
1
(1.1 g, 89% yield): H NMR (DMSO-d6, 600 MHz) δ 6.82 (1H, d, J
= 1.8 Hz), 6.70 (1H, d, J = 7.8 Hz), 6.61 (1H, dd, J = 7.8, 1.8 Hz),
6.43 (1H, s), 6.04 (1H, s), 4.71 (1H, s), 3.70 (3H, s), 3.06 (1H, m),
2.83 (1H, m), 2.73 (1H, m), 2.48 (1H, m); 13C NMR (DMSO-d6,
150 MHz) δ 147.72, 145.90, 143.93, 143.27, 136.92, 130.17, 125.96,
121.69, 115.69, 115.17, 114.89, 113.15, 61.26, 56.01, 42.76, 28.94;
ESIMS m/z 288.3 [M + H]+.
β1-, β2-, and α1B-AR Agonist Activity Assay. This assay was
performed according to the protocols of Nanjing GenScript Co. Ltd.
(Nanjing City, China).16 A total of 14 synthesized catecholic THIQs
as well as their analogues isolated from the medicinal plant P. oleracea
(Figure 1) were screened for their β1-, β2-, and α1B-AR agonist
activities, at the concentration of 100 μM, according to intracellular
calcium fluorescence changes in CHO-K1/Gα15/ADRB1 (Gen-
Script, M00269), CHO-K1/Gα15/ADRB2 (GenScript, M00308),
and CHO-K1/Gα15/ADRA1B (GenScript, M00260) cell lines that
consistently express β1-, β2-, and α1B-AR. A 1 μM concentration of
isoproterenol (Sigma) was used as the β2-AR agonist positive control,
and 10 μM epinephrine (Sigma) was used as the β1-AR agonist
positive control as well as the α1B-AR agonist positive control. Briefly,
cells were cultured in Ham’s F12 medium supplemented with 10%
fetal bovine serum, 200 μg/mL Zeocin, and 100 μg/mL Hygromycin
B and incubated in a CO2 incubator at 37 °C. After reaching 80%
confluency in a 10 cm dish, cells were further subcultured in a 384-
well plate (20 μL/well, 1.5 × 104 cells/well). After 18 h of incubation,
20 μL of FLIPR Calcium 4 assay kit solution (Molecular Devices,
R8141) was added to the cells, which were further incubated at 37 °C
for 1 h. The plate was removed from the CO2 incubator and
equilibrated to room temperature for 15 min. The FLIPR Tetra
apparatus (Molecular Devices) was used to determine the relative
calcium fluorescence units (RFU values). The detection time was set
for 120 s, and 10 μL of the positive control or the tested compound
was automatically added to the plate at 21 s. The tested compounds
were dissolved in DMSO (Sigma-Aldrich) at concentrations of 100
mM and diluted with Hank’s balanced salt solution containing 20 mM
HEPES (pH 7.4) before the assay. AR agonist activity was calculated
according to the following equation: stimulation rate (%) =
6,7-Dihydroxy-1-(4-methoxyphenyl)-1,2,3,4-tetrahydroisoquino-
line (8). Application of the above method using 4-methoxybenzalde-
1
hyde (534 μL) gave 8 as a brown powder (369 mg, 31% yield): H
NMR (DMSO-d6, 600 MHz) δ 7.20 (2H, d, J = 8.4 Hz), 6.92 (2H, d,
J = 8.4 Hz), 6.50 (1H, s), 6.05 (1H, s), 4.81 (1H, s), 3.79 (3H, s),
3.08 (1H, m), 2.87 (1H, m), 2.77 (1H, m), 2.53 (1H, m); 13C NMR
(DMSO-d6, 150 MHz) δ 158.60, 143.99, 143.34, 138.22, 130.28,
130.00, 126.11, 115.71, 114.94, 113.76, 60.69, 55.47, 42.55, 28.98;
ESIMS m/z 272.4 [M + H]+.
6,7-Dihydroxy-1-(2-methoxyphenyl)-1,2,3,4-tetrahydroisoquino-
line (9). Application of the above method using 2-methoxybenzalde-
1
hyde (0.598 g) gave 9 as a yellow powder (474 mg, 40% yield): H
NMR (DMSO-d6, 600 MHz) δ 7.30 (1H, td, J = 7.8, 1.2 Hz), 7.10
(1H, d, J = 7.8 Hz), 7.00 (1H, dd, J = 7.8, 1.2 Hz), 6.91 (1H, td, J =
7.8, 1.2 Hz), 6.53 (1H, s), 6.08 (1H, s), 5.34 (1H, s), 3.89 (3H, s),
3.03 (1H, m), 2.91 (1H, m), 2.75 (1H, m), 2.64 (1H, m); 13C NMR
(DMSO-d6, 150 MHz) δ 157.30, 143.98, 143.41, 134.01, 129.96,
129.26, 128.21, 126.50, 120.36, 115.75, 114.83, 111.27, 55.92, 53.65,
41.83, 29.06; ESIMS m/z 272.4 [M + H]+.
6,7-Dihydroxy-1-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydroiso-
quinoline (10). Application of the above method using 3,4,5-
trimethoxybenzaldehyde (0.86 g) gave 10 as a yellow powder (617
mg, 42% yield): 1H NMR (DMSO-d6, 600 MHz) δ 6.63 (2H, s), 6.49
(1H, s), 6.13 (1H, s), 4.79 (1H, s), 3.76 (6H, s), 3.69 (3H, s), 3.12
(1H, m), 2.88 (1H, m), 2.81 (1H, m), 2.52 (1H, m); 13C NMR
(DMSO-d6, 150 MHz) δ 152.97, 144.04, 143.32, 136.75, 129.63,
125.91, 115.81, 114.69, 106.37, 61.85, 60.40, 56.24, 43.01, 28.89;
ESIMS m/z 332.5 [M + H]+.
6,7-Dihydroxy-1-(naphthalen-1-yl)-1,2,3,4-tetrahydroisoquino-
line (11). 1-Formylnaphthalene (297 μL) was used in the synthesis of
11. Different from the above method, after the reaction mixture was
extracted with EtOAc, the organic layer was concentrated under
vacuum and a pale yellow powder precipitated to yield 11 (414 mg,
(ΔRFUcompound
− ΔRFUbackground)/(ΔRFUpositive control −
ΔRFUbackground) × 100%. In this equation, the average in the
fluorescence units from 1 to 20 s was used as the baseline, and the
ΔRFU value, i.e., the relative fluorescence units, was calculated as the
maximum fluorescence units from 21 to 120 s minus the baseline.
EC50 values of the positive control isoproterenol and epinephrine
1
32% yield): H NMR (DMSO-d6, 600 MHz) δ 8.29 (1H, d, J = 8.4
Hz), 7.89 (1H, d, J = 8.4 Hz), 7.82 (1H, d, J = 8.4 Hz), 7.44 (2H, m),
7.39 (1H, m), 7.32 (1H, m), 6.52 (1H, s), 5.92 (1H, s), 5.47 (1H, s),
3.07 (1H, m), 2.91 (1H, m), 2.85 (1H, m), 2.61 (1H, m); 13C NMR
(DMSO-d6, 150 MHz) δ 144.05, 143.50, 141.26, 134.40, 131.88,
129.93, 128.72, 128.04, 126.35, 126.04, 125.74, 125.69, 125.53,
115.96, 114.26, 60.22, 43.17, 29.14; ESIMS m/z 292.4 [M + H]+.
1-Benzyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (12). Ap-
plication of the above general method using phenylacetaldehyde (515
were calculated according to the four-parameter equation Y = Bottom
50/IC50
+ (Top − Bottom)/(1 + 10(logEC
−X)HillSlope), where X is the
log(concentration) and Y is the stimulation activity.
Evaluation of Effects of Catecholic THIQs on the Histamine-
Induced Contraction of Isolated Guinea-Pig Tracheal Smooth
Muscle. The experiment was performed according to the published
protocol with some modifications.35 All procedures were conducted in
compliance with the animal use regulations and were approved by the
Shandong University Animal Care and Use Committee. Healthy
guinea pigs (SCXK(Lu)20150001) weighing 200−350 g were
stunned, and the whole trachea was cut from the subthyroid cartilage
to the bifurcation at the lower end of the trachea. Tracheas were
immersed in cold Krebs-Henseleit solution with a mixture of 5% CO2
and 95% O2. After gently removing the connective tissue around the
trachea, the trachea was spirally cut from one end to the other into a
spiral strip approximately 3−5 mm wide and approximately 20−30
mm long. Every 2 or 3 cartilage rings were sheared with a helix. Each
end of the trachea spiral strip was threaded using a suture needle. One
end of the spiral strip was tied to an L-shaped hook, and the spiral
strip was carefully placed into a two-chamber organ bath with 20 mL
of Krebs-Henseleit solution at 37 °C. The L-shaped hook was fixed on
1
μL) gave 12 as a yellow powder (384 mg, 34% yield): H NMR
(DMSO-d6, 600 MHz) δ 7.28 (4H, m), 7.20 (1H, m), 6.63 (1H, s),
6.42 (1H, s), 3.91 (1H, dd, J = 10.2, 3.6 Hz), 2.99 (2H, m), 2.71 (2H,
m), 2.50 (1H, m), 2.45 (1H, m); 13C NMR (DMSO-d6, 150 MHz) δ
143.89, 143.57, 140.37, 130.16, 129.84, 128.57, 126.33, 126.08,
115.91, 113.78, 56.70, 42.73, 40.62, 29.21; ESIMS m/z 256.3 [M +
H]+.
1-(Furan-2-yl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline
(13). Application of the above general method using furfural (364 μL)
1
gave 13 as a reddish-brown powder (536 mg, 53% yield): H NMR
(DMSO-d6, 600 MHz) δ 7.61 (1H, dd, J = 1.8, 0.6 Hz), 6.51 (1H, s),
6.42 (1H, dd, J = 3, 1.8 Hz), 6.36 (1H, s), 6.11 (1H, d, J = 3 Hz), 4.98
(1H, s), 3.00 (1H, m), 2.88 (1H, m), 2.60 (2H, m); 13C NMR
G
DOI: 10.1021/acs.jnatprod.9b00418
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Journal of Natural Products
Article
the iron bracket with a double concave clamp. Oxygen was
continuously injected into the bath through the ventilator. The
other end of the spiral strip was connected to the muscle tension
transducer. At the beginning of the experiment, the initial load tension
of the spiral strip was set to approximately 2 g and was balanced for 60
min. After each test, the spiral strip was washed with Krebs-Henseleit
solution three times and balanced for 3 min. The balanced tension
was recorded as Tensionbaseline for each test. Histamine (200 μL, 1 ×
10−2 M) was added into the bath at a final concentration of 1.0 × 10−4
M, and the tension reaching the plateau was recorded as
Tensionhistamine. A total of 20 μL of tested compound dissolved in
DMSO was added to the organ bath (accumulative DMSO
concentration was less than 0.5%), and the tension reaching the
plateau was recorded as Tensiontested compounds. The bronchodilator
effect of the compounds on histamine-induced tracheal contraction in
guinea pigs was expressed by spasmolysis percentage and calculated
according to the equation: spasmolysis percentage (%) =
(Tensionhistamine − Tensiontested compounds)/(Tensionhistamine − Ten-
sionbaseline) × 100%. The effects versus concentration curves of the
tested compounds were plotted. To explore the potential target of
tracheal relaxation of the compounds, propranolol (with a final
concentration of 1.0 × 10−7 M in the bath), a β-AR antagonist, was
added into the bath for 15 min before the addition of histamine, and
the compounds were added as described above. The bronchodilator
effect of the compounds after the addition of propranolol was also
calculated according to the above equation.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.9b00418.
■
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Figures S1.1−S14.4: ESIMS, H NMR, and 13C NMR
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AUTHOR INFORMATION
Corresponding Authors
*(B. Jiao) Tel: +86-531-88382542. Fax: +86-531-88382548. E-
mail: jiaob@sdu.edu.cn.
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Sanishvili, R.; Fischetti, R. F.; Schertler, G. F.; Weis, W. I.; Kobilka, B.
K. Nature 2007, 450, 383−387.
(32) Rasmussen, S. G.; DeVree, B. T.; Zou, Y.; Kruse, A. C.; Chung,
K. Y.; Kobilka, T. S.; Thian, F. S.; Chae, P. S.; Pardon, E.; Calinski, D.;
Mathiesen, J. M.; Shah, S. T.; Lyons, J. A.; Caffrey, M.; Gellman, S.
H.; Steyaert, J.; Skiniotis, G.; Weis, W. I.; Sunahara, R. K.; Kobilka, B.
K. Nature 2011, 477, 549−555.
*(L. Xiang) Tel: +86-531-88382028. Fax: +86-531-88382548.
E-mail: xianglan02@sdu.edu.cn.
ORCID
Lan Xiang: 0000-0002-7149-8235
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for the financial support from Shandong
Natural Science Foundation (ZR2017MH093), National
Natural Science Foundation of China (81973200), and
China-Australia Centre for Health Sciences Research (2017,
2019).
(33) Gregorio, G. G.; Masureel, M.; Hilger, D.; Terry, D. S.; Juette,
M.; Zhao, H.; Zhou, Z.; Perez-Aguilar, J. M.; Hauge, M.; Mathiasen,
S.; Javitch, J. A.; Weinstein, H.; Kobilka, B. K.; Blanchard, S. C. Nature
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DOI: 10.1021/acs.jnatprod.9b00418
J. Nat. Prod. XXXX, XXX, XXX−XXX