Novel rhynchophylline analogues as microvascular relaxation agents for the treatment of microvascular dysfunction caused by diabetes

Article abstract of DOI:10.1016/j.ejmech.2017.08.026

Dysfunction in vascular reactivity in the micro- and macrocirculation is well established in cardiovascular disease. However, little is known about methods that may improve vascular reactivity in patients likely to develop microvascular dysfunction. One of the racemic analogues of rhynchophylline (G2) and its stereoisomers (G2-a and G2-b) were synthesized to address this knowledge gap. The preliminary pharmaceutical studies on the relaxation of the rat thoracic aorta showed that G2 and its stereoisomers are more potent (at least 30-fold) than the natural product rhynchophylline, which encouraged us to further investigate their functions and mechanisms as treatments for microvascular dysfunction caused by diabetes. G2-a displayed the best microvascular relaxation activity on rat mesenteric arteries among the three compounds, and G2 or G2-a caused relaxation in an endothelium-dependent manner. In ex vivo tests, G2 and G2-a exhibited a weaker potency in inducing microvascular relaxation in mesenteric arteries from diabetic rats than from normal rats, most likely, due to microvascular endothelium damage caused by diabetes. However, based on the animal studies, G2 ameliorated diabetes-induced endothelial dysfunction in rat mesenteric arteries in vivo. Further investigations of the mechanism showed that G2 mainly induced the recovery of endothelial function by upregulating endothelial nitric oxide synthase (eNOS) expression and further increasing the concentration of nitric oxide (NO), which is required for vascular relaxation.

Full text of DOI:10.1016/j.ejmech.2017.08.026

Accepted Manuscript
Novel rhynchophylline analogues as microvascular relaxation agents for the treatment
of microvascular dysfunction caused by diabetes
Wei Guo, Huikun Zhu, Zhijun Wang, Ji-An Chen, Jian Wu, Yizhun Zhu, Xianfeng Gu
PII:
S0223-5234(17)30629-3
10.1016/j.ejmech.2017.08.026
EJMECH 9670
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 May 2017
Revised Date: 8 August 2017
Accepted Date: 9 August 2017
Please cite this article as: W. Guo, H. Zhu, Z. Wang, J.-A. Chen, J. Wu, Y. Zhu, X. Gu, Novel
rhynchophylline analogues as microvascular relaxation agents for the treatment of microvascular
dysfunction caused by diabetes, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.08.026.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel rhynchophylline analogues as microvascular relaxation agents
for the treatment of microvascular dysfunction caused by diabetes
Wei Guo2, Huikun Zhu1, Zhijun Wang2, Ji-An Chen1, Jian Wu2, Yizhun Zhu3,
1
*
Xianfeng Gu
1
Department of Medicinal Chemistry, School of Pharmacy, Fudan University,
Shanghai 201203, China.
2
Shanghai Key Laboratory of Bioactive Small Molecules, Department of
Pharmacology, School of Pharmacy, Fudan University, Shanghai, 201203, China.
3Department of Pharmacology, School of Pharmacy, Macau University of Science &
Technology, Macau, China.
*Corresponding Author: xfgu@fudan.edu.cn
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Abstract
Dysfunction in vascular reactivity in the micro- and macrocirculation is well
established in cardiovascular disease. However, little is known about methods that
may improve vascular reactivity in patients likely to develop microvascular
dysfunction. One of the racemic analogues of rhynchophylline (G2) and its
stereoisomers (G2-a and G2-b) were synthesized to address this knowledge gap. The
preliminary pharmaceutical studies on the relaxation of the rat thoracic aorta showed
that G2 and its stereoisomers are more potent (at least 30-fold) than the natural
product rhynchophylline, which encouraged us to further investigate their functions
and mechanisms as treatments for microvascular dysfunction caused by diabetes.
G2-a displayed the best microvascular relaxation activity on rat mesenteric arteries
among the three compounds, and G2 or G2-a caused relaxation in an
endothelium-dependent manner. In ex vivo tests, G2 and G2-a exhibited a weaker
potency in inducing microvascular relaxation in mesenteric arteries from diabetic rats
than from normal rats, most likely, due to microvascular endothelium damage caused
by diabetes. However, based on the animal studies, G2 ameliorated diabetes-induced
endothelial dysfunction in rat mesenteric arteries in vivo. Further investigations of the
mechanism showed that G2 mainly induced the recovery of endothelial function by
upregulating endothelial nitric oxide synthase (eNOS) expression and further
increasing the concentration of nitric oxide (NO), which is required for vascular
relaxation.
2

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Cardiovascular disease is one of the main causes of disability and even death
throughout the world. Moreover, the prevalence of conditions such as diabetes
mellitus (DM), hypertension, and myocardial infarction are increasing as the
population ages. In particular, DM is often associated with complications of various
organ systems, including ischemic heart disease, stroke, peripheral vascular disease,
renal failure and retinopathy, which are related to microvascular dysfunction. If this
trend is to be reversed, an improved understanding of the biological basis of
cardiovascular disease leading to the identification of new therapeutic methods is
essential. Clearly, endothelial dysfunction, including changes in barrier function,
relaxation ability, inflammation status, and sensitivity to apoptosis, has significant
roles in the development of type 1 diabetes mellitus (T1DM) and type 2 diabetes
mellitus (T2DM).1-3 According to large-scale clinical trials of individuals with T1DM
and T2DM, hyperglycemia is dramatically associated with the pathogenesis of
microvascular complications and is also the main cause of endothelial dysfunction
and endothelial cell apoptosis.4,5
Nitric oxide (NO) is produced by endothelial cells and is a very important
mediator of vasodilation, with anti-apoptosis, anti-inflammatory, and anti-oxidant
properties.6 Several isoforms of NO synthase (NOS) exist: neuronal NOS (nNOS) is
expressed in the brain, inducible NOS (iNOS) is expressed by macrophages and
endothelial NOS (eNOS) is expressed in endothelial cells. NO produced by eNOS
triggers vasodilation by diffusing from endothelial cells to vascular smooth muscle
cells (VSMCs) and, following the release of cyclic GMP, increases evoked vascular
relaxation. Decreased NO availability, including the loss of NO production and the
loss of NO biological activity, is reported to be linked to endothelial dysfunction.7
These two interacting factors lead to microvascular complications.7,8 Uncertainty
regarding the fundamental functions of NO in cardiovascular disease has arguably
contributed to the disappointing lack of progress toward identifying clinical targets for
agents that act via the NO system and the subsequent development of new drugs. A
recent conceptual advance is that these actions are mediated by subtle changes in
intracellular signal transduction mechanisms and may be of therapeutic benefit.
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Uncaria rhynchophylla, a traditional Chinese herbal medicine, has been used
as an anti-hypertension drug in China for thousands of years.9-13 Crude extracts of
Uncaria rhynchophylla have been reported to induce vascular relaxation in both an
endothelium-dependent and an endothelium-independent manner.13-17 The active
ingredients from Uncaria rhynchophylla include indole alkaloids such as
rhynchophylline, isorhynchophylline, hirsutine and corynantheine, which have been
isolated, identified and purified by many laboratories.10 Rhynchophylline, the
ingredient present in the largest amount among all the indole alkaloids extracted from
Uncaria rhynchophylla15 (Figure 1), has been reported to induce vascular relaxant
activity in several animal models.10,13
Our group has long been devoted to extracting, synthesizing and evaluating
indole alkaloids derived from Uncaria rhynchophylla18 and has also been interested in
designing and synthesizing their analogues,19,20 with the intention of developing
additional novel lead compounds with higher vasorelaxant potency for further drug
development studies. We synthesized G2, one of the racemic analogues of
rhynchophylline (Figure 1); this compound exhibited significant vasorelaxant activity
on the rat thoracic aorta in vitro, with even greater potency than rhynchophylline.
After further isolation on a chiral column, two enantiomers, G2-a and G2-b, were
obtained from G2. Most interestingly, G2-a displays approximately 30-fold greater
potency than G2, as shown in an in vitro rat thoracic aorta test (Table 1).
N
C
OH
N
D
C
D
A
B
N
O
H
A
B
O
N
H
O
O
O
O
Rhynchophylline
G2
Figure 1. Structures of rhynchophylline and G2.
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Table 1. The vasorelaxant potency in an in vitro rat thoracic aorta test.
Compounds
IC50
µM
ND
mg/L
ND
Alcohol
G2
G2-a
G2-b
Rhynchophylline
5.8910
0.199
3.686
174.2
2.015
0.0681
1.261
66.893
ND Not detectable ( >1000 µM or 500mg/L )
Based on our preliminary pharmaceutical studies of the relaxation of the rat
thoracic aorta, G2 and its stereoisomers (G2-a and G2-b) have been chosen as the
vascular relaxation agents for further investigations of their functions and mechanisms
as treatments for microvascular dysfunction caused by diabetes.
RESULTS AND DISCUSSION
Synthesis. Compound G2 was prepared using the procedure outlined in
Scheme 1. First, imine 2 was synthesized from tryptamine via the Pictet-Spengler
reaction21 in the presence of 3-dimethoxypropionic acid ethyl ester and trifluoroacetic
acid (TFA). The resulting intermediate 2 was then reacted with benzyloxycarbonyl
chloride and Et3N (TEA) to afford 3, a compound with a carboxybenzyl (Cbz) group
at the N-2 site, where the imino group was further protected by a
tert-butyloxycarbonyl (Boc) group to yield intermediate 4. After the ester group in
compound 4 was reduced to an aldehyde group with diisobutylaluminum hydride
(DIBALH) at -78°C, the resulting intermediate 5 underwent a domino
Knoevenagel-hetero-Diels-Alder reaction in the presence of ethylenediamine
diacetate (EDDA) and dihydrofuran to yield lactone 6. Subsequently, the solvolysis of
compound 6 in a solution of K2CO3/methanol was followed by a hydrogenation
reaction catalyzed by Pd/C (10%) to afford intermediate 7. Finally, G2 was obtained
as a yellow powder by removing the Boc group from compound 7 in the presence of
TFA.
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Scheme 1. The synthesis of G2. Reagents and conditions: a) 3-dimethoxypropionic
acid ethyl ester and TFA; b) Cbz-Cl and EtN3; c) (Boc)2O, 4-dimethylaminopyridine
(DMAP); d) DIBAL-H and THF at -78°C; e) dihydrofuran and EDDA; f) H2, Pd/C,
and K2CO3/methanol; and g) TFA and CH2Cl2.
Stereochemistry of the Enantiomers. Two enantiomers, G2-a and G2-b,
were obtained from G2 on a chiral column. Their absolute configurations were
subsequently determined by obtaining the circular dichroism (CD), nuclear
Overhauser enhancement spectroscopy (NOESY) and heteronuclear single quantum
coherence (HSQC) spectra. G2-a and G2-b are shown as a pair of enantiomers in the
CD spectra (Figure 2b). The optical rotatory dispersion (ORD) curve of G2-a
displayed a negative Cotton effect between 298 and 244 nm, which constituted strong
1
13
evidence for a C-3 β configuration.22 The H- and C-NMR chemical shifts of C-3
(δC 54.8; δH 3.78), C-16 (δC 37.9; δH 2.59 and 2.40) and C-18 (δC 60.0; δH 3.63) were
deduced from the HSQC spectra (Figures S8 and S12). The NOESY correlation of
H-3/H-16 suggested that C-15 was α-oriented, and the correlation of H-18/H-16
indicated that C-20 was also α-oriented (Figures 2a, S7 and S11). Therefore, based on
the above stereochemical analyses, the absolute configurations of G2-a (3β, 15α, 20α)
and G2-b (3α, 15β, 20β) were identified.
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Figure 2. a) Selected NOESY correlation of G2-a, b) CD spectra of G2-a and G2-b.
G2 Induced the Vasorelaxation of Mesenteric Arteries in
Concentration-Dependent Manner. G2, G2-a, and G2-b exerted relaxation effects
on isolated 3rd grade mesenteric arteries in a concentration-dependent manner
compared with the effect of the time-matched vehicle control. The aforementioned
chemicals elicited concentration-dependent relaxation of the mesenteric arteries
reconstructed with KCl or with phenylephrine under an endothelium-intact status, as
shown in Figure 3A and 3C. G2-a was 10-fold more effective than its racemate G2
and was approximately 100-fold more effective than its enantiomer G2-b on
phenylephrine-precontracted mesenteric arteries, but G2, G2-a, and G2-b did not
show significant vasodilatory effects on KCl-precontracted mesenteric arteries,
indicating that the effects of G2, G2-a, and G2-b on receptor-operated calcium
channels (ROCCs) were more potent than the effects on voltage-operated calcium
channels (VOCCs) in isolated 3rd grade mesenteric arteries. The derived IC50 and Emax
values were calculated and are shown in Figure 3E.
a
The vasorelaxation effects of G2, G2-a, and G2-b on both endothelium-intact
and endothelium-denuded mesenteric artery rings pre-contracted with KCl or
phenylephrine (PE) were analyzed to determine whether the vasorelaxation induced
by these compounds depended on the endothelium. Compared with the relaxation
effects on endothelium-intact arteries (Figure 3B and 3D), the relaxation effects of all
chemicals on endothelium-denuded arteries showed a decreased IC50 value, without a
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reduction in the maximum effects (Figure 3E). Thus, G2 and its stereoisomers exerted
their vasodilatory effects in an endothelium-dependent manner.
Tetraethylammonium (TEA, non-selective calcium-activated K+ channel
blocker) and nifedipine (voltage-gated Ca2+ channel blocker, VGCC blocker) were
individually applied to endothelium-intact superior mesenteric arteries for 30 min
prior to pre-contraction with 10 µM PE to determine whether calcium-activated K+
channels and Ca2+ channels in these chemically induced relaxation events. Nifedipine
and TEA promoted a significant rightward shift of the concentration-dependent curve
of G2-a; G2-b induced a slight reduction and G2 had no effect (Figure S13). Based
on these results, G2-a-evoked relaxation was partially mediated by the
calcium-activated K+ channel and VGCC, but these two ion channels were not
substantially involved in the G2-b-induced viscoelastic effects. Interestingly, G2,
which contains a mixture of G2-b and G2-b, was not affected by TEA- or
nifedipine-induced ion channel blockage, indicating that another interaction between
G2-a and G2-b may be responsible for the results we observed.
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Figure 3. Effects of G2, G2-a, and G2-b on KCl- or PE-stimulated vasocontraction in
isolated mesenteric artery rings. (A) Representative traces showing the relaxant
response of KCl-contracted endothelium-intact mesenteric artery rings to G2, G2-a,
and G2-b and log concentration-response curves for G2, G2-a, and G2-b (n=3). (B)
Representative traces showing the relaxant response of KCl-contracted
endothelium-denuded artery rings to G2, G2-a, and G2-b and log
concentration-response curves for G2, G2-a, and G2-b (n=3). (C) Representative
traces showing the relaxant response of phenylephrine-contracted endothelium-intact
mesenteric artery rings to G2, G2-a, and G2-b and log concentration-response curves
for G2, G2-a, and G2-b (n=3). (D) Representative traces showing the relaxant
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response of phenylephrine-contracted endothelium-denuded artery rings to G2, G2-a,
and G2-b and log concentration-response curves for G2, G2-a, and G2-b (n=3). (E)
Table showing the IC50 and Emax values for G2, G2-a, and G2-b. Data are presented
as the means±standard errors of the means (SEM) of at least three replicated
experiments. Statistical analyses were performed using two-way ANOVA with the
Bonferroni post hoc test (A, B, C, and D) and one-way ANOVA with the Dunnett test
a
aa
(E). p≤0.05, p≤0.01 for G2-a compared to the control group; bbp≤0.01 for G2
c
compared to the control group; p≤0.05, ccp≤0.01 for G2-a compared to the control
group; dp≤ 0.05, ddp≤0.01 for G2-a compared to G2; eep≤0.01 for G2-a compared
ff
**
##
to G2-b; p≤0.01 for G2-b compared to G2; p≤0.01, p≤0.01 compared to the
corresponding vehicle.
G2 Ameliorated Diabetes-Induced Endothelial Dysfunction in Rat
Mesenteric Arteries in Vivo. Superior mesenteric arteries were isolated from diabetic
rats and challenged with the chemicals after PE stimulation to further elucidate
whether G2 and its stereoisomers would also exert their vasodilatory effects on a rat
model of diabetes. Hyperglycemia leads to endothelial dysfunction and subsequent
cardiovascular complications. Streptozotocin (STZ, 60 mg/kg) was used to induce a
model of type 1 diabetes (Figure 4A).
We utilized diabetes as a pathological model of endothelial damage to
investigate whether the chemicals still triggered relaxant effects and to further
determine whether endothelium-dependent relaxation was involved in these effects
(Figure 4E). As shown in Figure 4, G2 and G2-a generated vasodilatory effects, with
a rightward shift of curve, indicating that the endothelium was involved in a G2- or
G2-a-induced vasorelaxation.
PE-induced concentration-dependent contractions and acetylcholine-induced
endothelium-dependent relaxations (EDRs) were assessed to determine whether G2
restored endothelial function (Figure S14). The average blood glucose levels were
24.3 mmol/L after a 12-h fast of STZ stimulated DM rats for 8 weeks. After diabetic
rats received G2 (20 mg/kg) by gavage for one week, the EDRs of mesenteric arteries
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from diabetic rats were improved, and the phenylephrine-induced
concentration-dependent contractions were decreased (Figure 4D and F). Based on the
data from immunohistochemical staining and western blots, we deduced that G2
protected vessels from eNOS loss induced by DM, which was vital for artery
relaxation.
Figure 4. A two-week G2 treatment (20 mg/kg) improved PE-induced
endothelium-dependent contraction of rat mesenteric arteries in the DM model. (A)
Blood glucose levels were measured before and 2 months after the STZ treatment
(n=30). (B) Hematoxylin and eosin (H&E) staining and immunostaining for eNOS in
vessels after the G2 treatment. (C) Expression of the eNOS protein in mesenteric
arteries from G2-treated rats. (D) NO concentrations in mesenteric arteries from
G2-treated rats. (E) Traces showing the vasorelaxant effects of G2, G2-a, and G2-b
on mesenteric artery rings from rats with DM and log concentration-response curves
of G2, G2-a, and G2-b (n=3). (F) G2 reduced PE-induced contractions in mesenteric
arteries. ***p≤0.01. Data are presented as the means±SEM of at least three replicate
*
experiments. Statistical analyses were performed using Student’s t test (A). p≤0.05,
**p ≤ 0.01. Data are presented as the means±SEM of at least three replicate
experiments. Statistical analyses were performed using two-way ANOVA with the
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Bonferroni post hoc test. (E). aap≤0.01 for G2-a compared to the control group; bbp≤
0.01 for G2 compared to the control group; ccp≤0.01 for G2-a compared to the
dd
ee
control group; p≤0.01 for G2-a compared to G2; p≤0.01 for G2-a compared to
G2-b; ffp≤0.01 for G2-b compared to G2.
G2 Exerted Relaxation Effects on Mesenteric Arteries through
Endothelium-Derived Vasodilatory Factors. Endothelium-intact arteries were
pre-treated with 100 µM Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME;
an eNOS inhibitor) prior to stimulation with 10 µM PE stimulation to investigate the
mechanisms underlying the vasodilatory effects evoked by G2 and its stereoisomers
on endothelium-dependent pathways (Figure 5A). Pre-incubation with L-NAME
partially reduced the G2-, G2-a-, and G2-b-induced relaxation of the arteries with
intact endothelium (Figure 5C and 5D). Although, the efficacy of all of these
compounds was reduced, the efficacy of G2-a was dramatically decreased to a
magnitude similar to that of G2. Mesenteric arteries from diabetic rats that were
pre-treated with L-NAME were also tested to determine whether an
endothelium-dependent vasorelaxation mechanism was involved (Figure 5B).
Further evidence supporting these results was derived from observations
showing that preparations treated with NO synthase inhibitors such as L-NAME
greatly inhibited G2-, G2-a-, and G2-b-induced vasorelaxation in normal rats.
Mesenteric arteries from both normal and diabetic rats that were pre-treated with
L-NAME were also tested to determine whether an endothelium-dependent
vasorelaxation mechanism was involved (Figure 5E and 5F).
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Figure 5. Effects of G2, G2-b, and G2-b on PE-induced vasoconstriction in
mesenteric arteries isolated from normal and DM rats that had been pre-treated with
L-NAME (100 µM). (A) Traces showing the vasodilatory effects of G2, G2-a, and
G2-b on mesenteric arteries that had been pre-incubated with 100 µM L-NAME and
log concentration-response curves for G2, G2-a, and G2-b (n=3). (B) Log
concentration-response curves showing the effects of G2, G2-a, and G2-b on
mesenteric arteries from rats with DM that had been pre-treated with L-NAME (100
µM) (n=3). (C) Respective log concentration-response curves showing the effects of
the vehicle, G2, G2-a, and G2-b on endothelium-intact and L-NAME pre-treated
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endothelium-intact arteries (n=3). (D) Respective log concentration-response curves
showing the effects of the vehicle, G2, G2-a, and G2-b on endothelium-denuded and
L-NAME pre-incubated endothelium-intact arteries (n=3). (E) Respective log
concentration-response curves showing the effects of the vehicle, G2-, G2-a-, and
G2-b on vasorelaxation in mesenteric artery rings isolated from the L-NAME
pre-treated group and DM model group (n=3). (F) Respective log
concentration-response curves showing the vasodilatory effects of the vehicle, G2-,
G2-a-, and G2-b on the DM group with or without L-NAME pretreatment (n=3). ap≤
aa
bb
0.05, p≤0.01 for G2-a compared to the control group; p≤0.01 for G2 compared
c
cc
d
to the control group; p≤0.05, p≤0.01 for G2-a compared to control group; p≤
dd
ee
ff
0.05, p≤0.01 for G2-a compared to G2; p≤0.01 for G2-a compared to G2-b; p
≤0.01 for G2-b compared to G2. Data are presented as the means±SEM of at least
three replicate experiments. Statistical analyses were performed using two-way
ANOVA and the Bonferroni post hoc test.
METHODS
General. All chemical reagents and solvents used for synthesis were
purchased from commercial suppliers and were used without further purification.
Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 600 MHz
NMR and a Varian 400 MHz spectrometer. Mass spectra were measured on an HP
1100 LC/MS spectrometer. Thin-layer chromatography (TLC) was performed on
silica gel plates, and components were visualized by observation under UV light.
Flash chromatography was conducted on silica gel.
1-Ethoxycarbonyl-Methyl-1,2,3,4
-
Tetrahydrocarboline
(2).
3-Dimethoxypropionic acid ethyl ester (3.9 mL, 20 mmol), TFA (1.2 mL, 15 mmol)
and several drops of H2O were added to a solution of tryptamine (800 mg, 5 mmol) in
CH2Cl2 (40 mL). The reaction mixture was then stirred at room temperature overnight.
Next, the reaction solution was neutralized with a saturated NaHCO3 solution to pH 7
and extracted with CH2Cl2 three times. The combined organic layers were dried over
anhydrous Na2SO4 and evaporated in a rotary evaporator. The crude residue was
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further purified by flash chromatography on silica gel (60:1 dichloromethane
(DCM):methanol (MeOH)) to yield compound 2 (955 mg, yield 74%). C15H18N2O2,
MS (electrospray ionization (ESI)): m/z 259 [M+1]+; 1H NMR (400 Hz, CDCl3): δ:
8.64 (s, 1H), 7.49 (d, J=7.68 Hz, 1H), 7.34 (d, J=7.93 Hz, 1H), 7.18-7.08 (m, 2H),
4.48 (t, J=6.61 Hz, 1H), 4.23 (dd, J1=14.23 Hz, J2=7.09 Hz, 2H), 3.27-3.07 (m, 2H),
2.83 (d, J=6.45 Hz, 2H), 2.75 (t, J=4.94 Hz, 2H), 1.98 (s, 1H), 1.30 (t, J=9.07 Hz, 3H);
13C NMR (CDCl3) δ: 171.304, 135.386, 134.876, 127.008, 121.634, 119.187, 118.036,
110.893, 108.865, 61.052, 48.672, 41.865, 40.721, 22.479, 14.116.
1-Ethoxycarbonylmethyl-2-Benzyloxycarbonyl-1,2,3,4
-
tetrahydrocarboline (3). Et3N (4.31 mL, 30.8 mmol), 4-dimethylaminopyridine
(DMAP; 238 mg, 1.95 mmol) and benzyl chloroformate (Cbz-Cl; 2.91 mL, 20.7
mmol) were added to a solution of compound 2 (2.65 g, 10.3 mmol) in CH2C12 (100
mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction
solution was washed with water three times. The organic layers were combined and
dried with Na2SO4. The solvent was removed under a vacuum. Then, the residue was
purified by flash chromatography on silica gel (10:1 PE:ethyl acetate (EtOAC)) to
1
yield compound 3 (3.20 g, yield 80%). C23H24N2O4, MS (ESI): m/z 392 [M+1]+; H
NMR (400 Hz, CDCl3) δ: 8.92,8.76 (2×s, 1H), 7.45 (dd, J1= 7.1 Hz, J2=9.1 Hz, 1H),
7.40-7.30 (m, 6H), 7.15 (t, J=7.0 Hz, 1H), 7.07 (t, J=7.0 Hz, 1H), 5.80-5.60 (m, 1H),
5.20 (s, 2H), 4.65-4.45 (m, 1H), 4.30-4.17 (m, 2H), 3.23-3.07 (m, 1H), 3.01-2.70 (m,
4H), 1.28 (m, 3H); 13C NMR (CDCl3) δ: 172.9, 155.0, 136.5, 135.6, 133.6, 128.5,
128.0, 127.8, 122.0, 119.3, 118.0, 111.0, 108.1, 67.4, 61.1, 47.5, 39.7, 39.3, 21.0, 14.6,
14.0 ppm.
1-Ethoxycarbonylmethyl-2-Benzyloxycarbonyl-12-Tert-Butoxycarbonyl-1
,2,3,4 - Tetrahydrocarboline (4). Di-tert-butylpyrocarbonate (3.37 g, 15.42 mmol)
and DMAP (245 mg, 2.00 mmol) were added to a solution of compound 3 (4.03 g,
10.28 mmol) in CH2C12 (150 mL). After stirring at room temperature for 2 hours, the
reaction was washed with H2O (100 mL) and extracted with CH2Cl2. The organic
layers were combined, dried over anhydrous Na2SO4, and the solvent was removed
under reduced pressure. The crude residue was purified by flash chromatography on
15

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silica gel (15:1 PE:EtOAC) to yield compound 4 (4.57 g, yield 90%). C28H32N2O6,
MS (ESI): m/z 493 [M+1]+; 1H NMR (400 Hz, CDCl3) δ: 8.20 (d, J=7.0 Hz, 1H),
7.43-7.19 (m, 8H), 6.35, 6.25 (2×d, J=9.9 Hz, 1H), 5.18, 5.15 (2×s, 2H), 4.52, 4.40
(2×dd, J=14.4, 6.0, 1H), 4.17-4.01 (m, 2H), 3.39-3.30 (m, 1H), 3.10-2.60 (m, 4H),
13
1.73, 1.60 (2×s, 9H), 1.18 (m, 3H); C NMR (CDCl3) δ: 170.1, 169.8, 155.3, 149,6,
136.5, 136.2, 133.8, 128.5, 128.4, 128.3, 128.1, 124.7, 124.5, 122.9, 122.8, 118.0,
117.9, 115.8, 115.3, 84.7, 67.7, 67.2, 60.6, 49.8, 39.0, 38.9, 36.7, 36.2, 28.1, 28.0,
21.2, 20.5, 14.0 ppm.
1-Carbonylmethyl-2-Benzyloxycarbonyl-12-Tert-Butoxycarbonyl-1,2,3,4–
Tetrahydrocarboline (5). Compound 4 (2.22 g, 4.52 mmol) was dissolved in dry
tetrahydrofuran (THF; 50 mL), then DIBALH (12.56 mL, 18.07 mmol) was added
drop-wise at -78°C. The reaction mixture was stirred at room temperature for 1 hour,
after which the reaction mixture was quenched with an aqueous HCl solution (1 N),
and then extracted with CH2C12 three times. The organic layers were dried over
anhydrous Na2SO4 and filtered. The solvent was evaporated, and the residue was
purified by flash chromatography on silica gel (8:1 PE:EtOAc) to yield compound 5
1
as a yellow oil (1.83 g, yield 87%). C26H28N2O5, MS (ESI): m/z 405 [M+1]+; H
NMR (400 Hz, CDCl3) δ: 9.95, 9.80 (2×d, J1=4.3 Hz, J2=3.1 Hz, 1H), 8.10 (d, J=8.2
Hz, 1H), 7.56 (m, 2H), 7.50-7.20 (m, 8H), 6.58, 6.43 (2×d, J=9.78 Hz, 1H), 5.16 (dd,
J=12.52, 12.92 Hz, 2H), 4.53, 4.40 (2×dd, J1=6.2 Hz, J2=13.1 Hz, 1H) 3.15-2.60 (m,
5H), 1.73, 1.60 (2×s, 9H); 13C NMR (CDCl3) δ: 201.1, 200.3, 155.5, 149.9, 136.2,
135.8, 133.6, 128.5, 128.5, 128.2, 128.1, 127.8, 124.7, 124.6, 122.9, 118.1, 118.0,
116.1, 115.9, 115.5, 84.7, 67.8, 67.5, 48.2, 47.7, 36.8, 36.5, 28.0, 21.1, 20.5 ppm.
15-Methoxycarbonylmethyl-20-Hydroxyethyl-1-Tertbutoxycarbonyl-Indo
le (2,3-c)Octahydroquinolizine (7). EDDA (89.9 mg, 0.450 mmol), dihydrofuran
(766.4 µl, 6.744 mmol) and Meldrum’s acid (328.6 mg, 2.248 mmol) were added to
the solution of compound 5 (1.01 g, 2.248 mmol) in toluene (40 mL) and heated at
50-60°C under an argon atmosphere. Next, the reaction was stimulated with ultrasonic
energy for 6-8 hours and monitored with TLC until full conversion was achieved.
Then, the solvent was removed under a vacuum. The residue was purified by
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chromatography on silica gel (10:1 PE:EtOAc) to yield compound 6 as a solid yellow
powder (407 mg, yield 30%). Compound 6 was dissolved in MeOH (20 mL), K2CO3
(44.5 mg) was added, and the mixture was stirred at room temperature for 1 hour.
Subsequently, a hydrogenation reaction was catalyzed by 5% Pd/C under an H2
atmosphere at room temperature for 8 hours. The reaction mixture was filtered with
celite, and the filtrate was concentrated under a vacuum. The crude residue was
purified by flash chromatography on silica gel (4:1 PE:EtOAC) and afforded
1
compound 7 (229 mg, yield 80%). C25H34N2O5 H NMR (400Hz, CDCl3) δ: 7.90 (d,
J=5.6, 1H), 7.41(d, J=4.0 Hz, 1H), 7.28-7.21 (m, 2H), 3.96 (d, J=8.0 Hz, 1H),
3.76-3.66 (m, 5H), 3.01-2.91 (m, 3H), 2.80-2.62 (m, 5H), 2.35 (m, 1H), 2.18 (d,
13
J=12.0 Hz, 1H), 1.94-1.79 (m, 4H), 1.69 (s, 9H); C NMR (CDCl3) δ: 173.0, 149.9,
135.9, 128.6, 123.2, 122.0, 117.4, 116.6, 114.5, 83.1, 58.8, 55.9, 55.5, 50.9, 50.5, 37.6,
37.2, 35.0, 32.4, 31.6, 27.5, 21.1 ppm. High-resolution MS (HR-MS) calculated mass
of C25H34N2O5 443.2540, found 443.2544.
15-Methoxycarbonylmethyl-20-Hydroxyethylindole(2,3-c)Octahydroquin
olizine (G2). TFA (7 mL) was added to a mixture of compound 7 (0.88 g, 2.0 mmol)
in CH2Cl2 (20 mL) and stirred at room temperature for 30 min. Then, the solvent was
removed under reduced pressure, and the crude residue was purified by flash
chromatography on silica gel (10:1 DCM:MeOH) to yield a yellow powder
1
designated compound G2 (0.49 g, yield 72%). H NMR (400 Hz, CDCl3) δ: 7.98
(s,1H), 7.44 (d, J=8.0 Hz, 1H), 7.31 (d, J=8.0 Hz, 1H), 7.14-7.05 (m, 2H), 3.72-3.68
(m, 4H), 3.66-3.63 (m, 2H), 3.07 (m, 1H), 3.01-2.93 (m, 1H), 2.83-2.77 (m, 1H),
2.70-2.58 (m, 5H), 2.45-2.40 (m, 1H), 2.12-2.08 (m, 1H), 1.85-1.74 (m, 3H), 1.67 (s,
1H); 13C NMR (CDCl3) δ:173.4, 136.0, 133.5, 127.3, 121.4, 119.4, 118.0, 110.9,
108.3, 59.8, 54.6, 53.8, 52.6, 51.7, 37.7, 36.4, 36.2, 32.3, 32.0, 20.0 ppm. HR MS
calculated mass of C20H26N2O3 343.2016, found 343.2019.
Artery Preparation and Functional Assay. All animal experiments reported
in the article were performed in compliance with the principles of laboratory animal
care for medical research from Fudan University and the Guide for the Care and Use
of Laboratory Animals. Male Sprague-Dawley rats (180-300 g) were euthanized with
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an intraperitoneal (i.p.) injection of chloral hydrate (300 mg/kg). Third grade
mesenteric arteries was excised and transferred to an oxygenated Krebs solution
containing (in mM): 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 25 NaHCO3, 1 MgCl2, 2.5
CaCl2, and 11 D-glucose. Arteries were cleaned of the adhering connective tissues and
cut into rings (approximately 2 mm in length) for the functional study. For the
endothelium-denuded arteries, the endothelium was removed by rubbing the interior
layer of the rat vessels with a board. Successful removal of endothelium was shown
by the loss of acetylcholine (ACh)-induced relaxation in vessels that had been
pre-constricted with PE.
Arterial rings were mounted in myograph chambers (Danish Myo Technology,
Aarhus, Denmark) with two stainless wires passing through the vessel lumens, which
were fixed to the two jaws of the chamber and were oxygenated (95% O2-5% CO2) in
Krebs solution maintained at 37°C (pH 7.4) to monitor the changes in isometric
tension. The mesenteric arterial rings were stretched to their optimal baseline tension
of 3 mN. After an equilibration period at the baseline tension (60 min), a 60 mM KCl
solution (an equimolar amount of KCl was used to replace the NaCl in the Krebs
solution) was applied to the vessels to validate the viability of the arteries.
A solution of 60 mM KCl and 10 µM PE were used to pre-constrict
endothelium-intact or endothelium-denuded arterial rings, respectively, and to
determine the vasodilatory effect of G2. After a sustained plateau was obtained, the
cumulative concentration-dependent responses to G2, G2-a, and G2-b (from 10-10 to
10-4 M) were examined. A parallel vehicle control (ethanol) group was also analyzed.
Establishment of the rat model of DM. DM was induced in Sprague-Dawley
rats through an intrapulmonary injection of 60 mg/kg STZ followed by gavage with a
high-glucose solution. After 8 weeks, DM was confirmed by measuring the animals’
blood glucose levels, and experiments using the vessel wire myograph were
performed.
Western Blot Analysis. Diabetic rats were administered 20 mg/kg G2 (gavage)
for 2 weeks, and then the mesenteric arteries were harvested. Next, vessels were lysed
in radioimmunoprecipitation (RIPA) lysis buffer, and centrifuge for 20 min at 12,000
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rpm at 4°C in a microcentrifuge. After protein concentrations were determined using
the bicinchoninic acid (BCA) protein assay kit (Thermo), equal amounts of protein
samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
and the proteins were then transferred from the gel to polyvinylidene difluoride
(PVDF) membranes. After blocking in 5% nonfat milk for 1 h, membranes were
incubated with antibodies against eNOS and iNOS (Cell Signaling Technology). The
membranes were subsequently incubated with horseradish peroxidase
(HRP)-conjugated secondary antibodies. The protein bands were identified using an
enhanced chemiluminescence system (Thermo Fisher Scientific, Shanghai, China).
Histology and Immunohistochemical Staining. Cross sections of the 2nd and
3rd mesenteric arteries from diabetic rats treated with or without G2 (20 mg/kg,
gavage) were used for histology and immunohistochemical staining. After
decalcification, the slides were blocked with 10% goat serum (v/v) diluted in PBS for
1 h and incubated overnight with antibodies against eNOS and iNOS. For the
histological study, sections were incubated with biotin-conjugated secondary
antibodies for 30 min at ambient temperature. Immunization was detected with an
avidin-biotin-immunoperoxidase system with diaminobenzidine (DAB), and sections
were counterstained with hematoxylin.
Measurement of NO Concentrations. NO concentrations were detected by
measuring nitrite accumulation in the tissue using a commercially available kit
(Beyotime Biotechnology, Haimen, China) according to the manufacturer’s
instructions. Artery rings were incubated with the indicated concentrations of G2 for 1
h. The tissues were homogenized, and protein concentrations were quantified with a
BCA protein assay kit (Thermo). NO concentrations were measured in equal amounts
of protein.
Statistical Analysis. Functional experiments were performed at least three
times. Data are presented as means±SEM and were analyzed with GraphPad Prism 6
(GraphPad Software, San Diego, CA, USA).
CONCLUSIONS
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In this paper, G2, the racemic analogue of rhynchophylline, and a pair of
enantiomers derived from G2 (G2-a and G2-b) were synthesized, and their structures
1
13
and stereochemistry were identified from H NMR, C NMR, NOESY, HSQC and
CD spectra. Based on the results of the pharmaceutical studies, G2-a showed the best
microvascular relaxation activity on rat mesenteric arteries among the three analogues,
and G2- or G2-a-induced relaxation depended on the endothelium. In ex vivo tests,
G2 and G2-a exhibited a weaker potency in inducing microvascular relaxation in
mesenteric arteries from diabetic rats than in arteries from normal rats, most likely
due to the DM-induced microvascular endothelial damage. However, according to the
animal studies, G2 ameliorated DM-induced endothelial dysfunction in rat mesenteric
arteries in vivo. As shown in the mechanistic investigations, G2 restored the
endothelial function mainly by upregulating eNOS expression and further increasing
the NO concentrations, which was required for vascular relaxation.
ACKNOWLEDGMENT
We gratefully acknowledge the financial support from STCSM (no. 15401901900)
and NSFC (no. 21572039).
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Hightlights
1. An analogue of rhynchophylline (G2) and its stereoisomers (G2-a and G2-b) were
synthesized.
2. The three compounds displayed the significant microvascular relaxation activity
on rat mesenteric arteries.
3. G2 ameliorated diabetes-induced endothelial dysfunction in rat mesenteric arteries
in vivo.
4. G2 could induce the recovery of endothelial function by upregulating eNOS
expression.
1