Synthesis and biological evaluation of novel leonurine-SPRC conjugate as cardioprotective agents

Article abstract of DOI:10.1016/j.bmcl.2010.09.135

The synthesis and biological evaluation of novel leonurine-SPRC conjugate, 3,5-dimethoxy-4-(2-amino-3-prop-2-ynylsulfanyl-propionyl)-benzoic acid 4-guanidino-butyl ester (1) is reported in this Letter. It is designed to improve the pharmacology efficiency by combining leonurine with S-propargyl-l-cysteine (SPRC), a cysteine analog, via a phenolic hydroxyl ester bond, which could be readily hydrolyzed to release bioactive leonurine and SPRC. Pharmacological evaluation has shown that 1 possesses potent cardioprotective effect against hypoxia-induced neonatal rat ventricular myocytes damage at lower molar concentration (10-fold less than leonurine required and 100-fold less than SPRC required). The mechanism is in partial related to improve hydrogen sulfide production, anti-oxidative stress and anti-apoptosis.

Full text of DOI:10.1016/j.bmcl.2010.09.135

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6942–6946
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel leonurine–SPRC conjugate
as cardioprotective agents
Chunhua Liu a, Xianfeng Gu b, , Yi Zhun Zhu a,
⇑
⇑
a Department of Pharmacology, School of Pharmacy and Institute of Biomedical Sciences, Fudan University, Shanghai 200032, China
b Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2010
Revised 26 September 2010
Accepted 28 September 2010
Available online 7 October 2010
The synthesis and biological evaluation of novel leonurine–SPRC conjugate, 3,5-dimethoxy-4-(2-amino-
3-prop-2-ynylsulfanyl-propionyl)-benzoic acid 4-guanidino-butyl ester (1) is reported in this Letter. It is
designed to improve the pharmacology efficiency by combining leonurine with S-propargyl-L-cysteine
(SPRC), a cysteine analog, via a phenolic hydroxyl ester bond, which could be readily hydrolyzed to
release bioactive leonurine and SPRC. Pharmacological evaluation has shown that 1 possesses potent car-
dioprotective effect against hypoxia-induced neonatal rat ventricular myocytes damage at lower molar
concentration (10-fold less than leonurine required and 100-fold less than SPRC required). The mecha-
nism is in partial related to improve hydrogen sulfide production, anti-oxidative stress and anti-
apoptosis.
Keywords:
Leonurine–SPRC conjugate
Leonurine
S-propargyl-
L-cysteine (SPRC)
Ó 2010 Elsevier Ltd. All rights reserved.
Cardioprotective effect
Leonurine, a plant alkaloid present in Chinese motherwort
(Leonurus artemisia and Leonurus heterophyllus), has long been
known that it possesses uterotonic effect (Fig. 1).1,2 Recently our
group have reported that leonurine has cardioprotective effects
both in vivo and in vitro3–5 and that it is able to relax vascular
smooth muscle.6 These beneficial effects are at least partially re-
lated to its abilities on anti-oxidation, anti-apoptosis, regulating
the Ca2+ concentration and protecting mitochondrial function.5,6
So leonurine has been considered as a good drug candidate in
our laboratory for further studies. However, several issues have
to be overcome on the therapeutic development of leonurine. For
example, it is difficult to isolate and purify leonurine from natural
resources due to the low content in Chinese motherwort and vari-
ety of other ingredients as impurities. In addition, high-dosage
used implicates its low potency, which may hinder clinical trials
in future.4 Besides, the syntheses of leonurine and its derivatives
are rare.1,7 Therefore, we spontaneously considered that structural
modification of leonurine may be a potential way to improve its
pharmacological efficacy.
the antimalarial activity of artemisinin and the antiplasmodial
properties of chloroquine. Moreover, the trioxaquine PA1103-
SAR116242 is one of the promising antimalarial agents and has been
selected as candidate for preclinical development.9,10 Inspired by
these paradigms, we were intrigued to assume whether combining
leonurine with another cardioprotective compound to form new
compound will have a dual mode of cardioprotective action (a
‘double-edged sword’) via different mechanisms and be able to
improve cardioprotective efficiency of the precursor. Fortunately,
‘another cardioprotective compound’ is just at hand. A cysteine
Incorporation of two mutually complementary biological active
entities into one by conjugation has been widely used in drug de-
sign. For examples, this symbiotic approach has been applied to the
design of the vasodilator/b-adrenoceptor antagonist 2-[3-(tert-
butylamino)-2-hydroxypropoxy]-3-cyanopyridine,8 and trioxaqu-
ines, a series of synthetic hybrid molecules containing a trioxane
motif and an aminoquinoline entity which have reserved both of
⇑
Corresponding authors. Tel.: +86 21 51980112; fax: +86 21 51980008.
E-mail addresses: xfgusky@yahoo.com (X. Gu), zhuyz@fudan.edu.cn (Y.Z. Zhu).
Figure 1. Structure of leonurine (LEO), S-propargyl-
compound 1.
L-cysteine (SPRC), and
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.135

C. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6942–6946
6943
Scheme 1. The synthetic route of the key intermediate 3,5-dimethoxy-4-hydroxy-benzoic acid 4-(N,N0-Boc-guanidino)-butyl ester (6). Reagents and conditions: (i) in DMF, rt,
3.5 h; (ii) triethylamine, acetyl chloride in dichloromethane, rt, 2 h; (iii) DIC, DPTS, rt, 5 h; (iv) sodium methoxide, 4 °C, rt, 1 h.
analog, S-propargyl-
L
-cysteine (SPRC) has been synthesized as a no-
hydrolysis etc. Then, it has been evaluated on neonatal rat ventric-
ular myocytes (NRVM) and showed potent protective effect against
hypoxia-induced NRVM damage at lower molar concentration (10-
fold less than leonurine required and 100-fold less than cysteine
analogues required).
vel endogenous hydrogen sulfide (H2S) modulator in our laboratory
(Fig. 1). And we have reported that this cysteine analog has potent
protective effects on acute myocardial ischemia through regulating
endogenous H2S production by the pyridoxal-5-phosphate-depen-
dent enzyme cystathionine-
c
-lyase (CSE) passageway.11,12 As we
In our studies, the designed compound 1 was prepared as out-
lined in Schemes 1 and 2. To prepare the key intermediate 6, caryo-
phyllic acid (3) was chosen as the starting material, and two
approaches were studied. In the first strategy, 4-hydroxy-3,5-dime-
thoxy-benzoic acid 4-amino-butyl ester was synthesized according
to the literature method,14 and then condensed with N,N0-Boc-
methyl-isothiourea in DMF at 60 °C to afford compound 6 with very
low yield (less than 10%). Thus, to improve the yield of the key inter-
mediate 6, another approach was employed. Firstly, the hydroxyl
group of caryophyllic acid was protected by the esterification of
acetyl chloride in the presence of triethylamine to provide the
corresponding product 4. The resulting carboxylic acid 4, in the
presence of N,N0-diisopropylcarbodiimide (DIC) and 4-(dimethyl-
amino)-pyridinium-4-toluene sulfonate (DPTS), subsequently
all know, hydrogen sulfide (H2S) has recently been discovered to
be the third gasotransmitter and received much attention as impor-
tant biological mediator.13
With the general approach and results in mind, we designed
and synthesized leonurine–SPRC conjugate. Two molecules, leonu-
rine and SPRC, are bound together by a phenolic hydroxyl ester
bond. The structural modification of leonurine was expected to
exhibit similar pharmacological activity with leonurine and SPRC,
because the phenolic hydroxyl ester group is liable to be hydro-
lyzed and then readily release bioactive leonurine and SPRC. In
present report, compound 1, a leonurine–SPRC conjugate, has been
studied ( Fig. 1). Starting from caryophyllic acid, 1 has been
synthesized via seven steps, including acetylation, esterification,
Scheme 2. The synthetic route of 3,5-dimethoxy-4-(2-amino-3-prop-2-ynylsulfanyl-propionyl)-benzoic acid 4-guanidino-butyl ester (1). Reagents and conditions: (i) di-tert
butyl dicarbonate in tetrahydrofuran and Na2CO3, rt, 5 h; (ii) DIC, DPTS, rt, 5 h; (iii) TFA.

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C. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6942–6946
Figure 2. (A) Effect of 1 on hypoxia-induced neonatal rat ventricular myocytes. (B) Effects of 1, LEO, and SPRC on hypoxia-induced neonatal rat ventricular myocytes. (C)
Effects of 1, LEO, and SPRC on LDH leakage in hypoxia-induced neonatal rat ventricular myocytes. (D) Effects of 1, LEO, and SPRC on CK activity in hypoxia-induced neonatal
rat ventricular myocytes. Values are expressed as means SE from six individual samples. denotes P <0.05 denotes P <0.01 versus vehicle group; #denotes P <0.05,
##denote P <0.01 versus control group; $denotes P <0.05 versus 1 group.
*
**
condensated with 2, which synthesized from 4-hydroxyl butyl-
amine and methyl-isothiourea in two steps in Scheme 1, to afford
5 in 70% yields. Finally, the acetyl group was hydrolyzed by catalytic
amount of sodium methoxide at 4 °C to give the key intermediate 6
in 98% yields.
drug-treated groups significantly increased cell viability (Fig. 2B)
and decreased LDH and CK leakage (Fig. 2C and D) (P <0.05); At
0.1 lmol/L, compound 1 decreased CK leakage much lower than
To build up the other fragment of designed compound 1, S-
propargyl-L-cysteine (7) was obtained from the reaction of cysteine
with propargyl bromide based on the literature method.11 And the
amino group of 7 was protected by Boc anhydride under basic con-
dition to afford 8 in good yield which subsequently condensed
with intermediate 6 in the presence of DPTS and DIC to afford 9.
Finally the Boc groups in 9 were removed by trifluoroacetic acid
(TFA) to give target compound 1 in good yield (78%).
Compound 1 was evaluated on neonatal rat ventricular myo-
cytes in vitro. The protective effect of 1 was investigated by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay,15 the lactate dehydrogenase (LDH) leakage and crea-
tine kinase (CK) leakage assay. As we all know, MTT assay demon-
strates cell viability, LDH leakage and CK activity are the markers of
cell membrane integrity.12 Cells were incubated with 1 for 12 h be-
fore exposure to hypoxia for 5 h. Three concentrations (1 nmol/L,
10 nmol/L, and 100 nmol/L) were tested in MTT assay. It showed
that the hypoxic condition significantly decreased cell viability
Figure 3. Effects of 1, LEO, and SPRC on H2S content in hypoxia-induced neonatal
(P <0.01). At 0.1
viability (Fig. 2A). Thus 0.1
quent experiments. Compared with the vehicle group, all of the
lmol/L, 1 caused a significantly increase in cell
rat ventricular myocytes. Values are expressed as means SE from four individual
samples. denotes P <0.05 versus vehicle group; #denote P <0.05 versus control
lmol/L was employed for the subse-
*
group.

C. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6942–6946
6945
Figure 4. (A) Effects of 1, LEO and SPRC on SOD activity in hypoxia-induced neonatal rat ventricular myocytes. (B) Effects of 1, LEO, and SPRC on MDA level in hypoxia-
*
**
induced neonatal rat ventricular myocytes. Values are expressed as means SE from four individual samples. denotes P <0.05, denotes P <0.01 versus vehicle group;
#denotes P <0.05 versus control group; $denotes P <0.05 versus 1 group.
Figure 5. Effects of 1, LEO, and SPRC on morphological features of cultured neonatal rat ventricular myocytes. Fluorescence photomicrographs of cells stained with Hoechst
33258 (400ꢀ). Each photograph is a representative of three independent observations. Percentage of cardiomyocytes with nuclear fragmentation from three independent
experiments based on Hoechst stain. Means SE; denotes P <0.05, denotes P <0.01 versus vehicle group; ##denotes P <0.01 versus control group.
*
**
their precursors did in Fig 2D (P <0.05), The results indicated 1 pos-
sessed potent cardioprotective effects against hypoxia-induced
neonatal rat ventricular myocytes damage at lower molar
concentration (10-fold less than leonurine required and 100-fold
less than SPRC required). As most conjugates contain a metaboli-
cally stable linker, ‘cleavable conjugates’ employ a linker that is

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C. Liu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6942–6946
designed to be metabolized to release two ligands that interact
independently with each target.16 Compound 1 might be easily
hydrolyzed and readily release bioactive leonurine and SPRC to
show the synergism at the same time. In that way, there would
be a lower risk of drug-drug interactions compared to the mixture
of leonurine and SPRC.16
consistent with MTT results. In recent years, oxidative stress has
been proven to be a powerful inducer and a common mediator of
programmed cell death,22 and reactive oxygen species are directly
implicated in the initiation of apoptosis.23 Our study demonstrated
compound 1 was able to alleviate oxidative damage which could
decrease the probability of apoptosis.
It has been reported that H2S content would be down-regulated
In conclusion, we have designed and successfully synthesized a
novel leonurine–SPRC conjugate. Pharmacological evaluation has
shown its potent cardioprotective effect and we assume the mech-
anism is in partial related to modulate hydrogen sulfide produc-
tion, anti-oxidative stress and anti-apoptosis. These preliminary
results demonstrate that 1 is worth to be further investigated as
potential anti-myocardial ischemia drug.
in myocardial ischemia.11 In our studies, it was significantly in-
creased to 21.49 1.33
the level of H2S to 20.18 2.80
13.58 0.97 mol/L in the vehicle group after hypoxia (Fig. 3). In
lmol/L in 1 group, while SPRC ameliorated
l
mol/L. but it was only
l
this study, 1 and SPRC groups had significant difference compared
with the vehicle group (P <0.05). However, leonurine group and
leonurine + SPRC group had no significant difference on modulat-
ing H2S production (P >0.05). As we know that SPRC improved
hydrogen sulfide level by CSE pathway,11,12 and 1 was able to
up-regulate hydrogen sulfide level at lower molar concentration,
So we infer that 1 may regulate H2S production by the same path-
way, due to it could be easily hydrolyzed and readily release bioac-
tive leonurine and SPRC.
Myocardial ischemia and reperfusion is characterized by a de-
crease in the endogenous anti-oxidant species, particularly super-
oxide dismutase (SOD).17 As shown in Fig 4A, the superoxide
dismutase (SOD) activity was significantly decreased in the vehicle
Acknowledgments
This study was supported by National Basic Research Program
of China (973 Program) (No. 2010CB912600), National Natural Sci-
ence Foundation of China (No. 30772565, No. 30888002) and New
Teacher Foundation from Ministry of Education in China (No.
20090071120).
References and notes
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l
mol/L of 1 signif-
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