Synthesis, biological evaluation, and pharmacokinetic study of novel liguzinediol prodrugs

Article abstract of DOI:10.3390/molecules18044561

Liguzinediol (LZDO) ester prodrugs 3-5 were synthesized and evaluated in vitro and in vivo for their potential use in prolonging the half-life of the parent drug LZDO (1a) in vivo. Prodrugs 3-5 were found to display a potent positive inotropic effect on the myocardium, without the risk of arrhythmia. Prodrugs 3-5 rapidly underwent enzymatic hydrolysis to release the parent compound LZDO in 1-3 h in rat liver microsomes and rat plasma. The half-life of the parent compound was prolonged after intragastric administration of prodrug 3, which was found to be a superior prodrug candidate for increasing myocardial contractility.

Full text of DOI:10.3390/molecules18044561

Molecules 2013, 18, 4561-4572; doi:10.3390/molecules18044561
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Biological Evaluation, and Pharmacokinetic Study of
Novel Liguzinediol Prodrugs
Zheng Liu, Wei Li *, Hong-Mei Wen, Hui-Min Bian, Jing Zhang, Lei Chen, Long Chen and
Kun-Di Yang
School of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing 210046,
Jiangsu, China
* Author to whom correspondence should be addressed; E-Mail: liwaii@njutcm.edu.cn;
Tel.: +86-138-0158-8651.
Received: 15 March 2013; in revised form: 8 April 2013 / Accepted: 11 April 2013 /
Published: 18 April 2013
Abstract: Liguzinediol (LZDO) ester prodrugs 3–5 were synthesized and evaluated in vitro and
in vivo for their potential use in prolonging the half-life of the parent drug LZDO (1a) in vivo.
Prodrugs 3–5 were found to display a potent positive inotropic effect on the myocardium,
without the risk of arrhythmia. Prodrugs 3–5 rapidly underwent enzymatic hydrolysis to release
the parent compound LZDO in 1–3 h in rat liver microsomes and rat plasma. The half-life of
the parent compound was prolonged after intragastric administration of prodrug 3, which
was found to be a superior prodrug candidate for increasing myocardial contractility.
Keywords: liguzinediol; liguzinediol prodrugs; synthesis; positive inotropic effect;
pharmacokinetics
1. Introduction
Cardiotonic agents exert their therapeutic activity by inducing positive inotropic effects on the
myocardium. They are either cAMP-dependent or cAMP-independent, based upon their mechanism of
action, but this mechanism of action is inherently responsible for the heart side effects observed in
patients undergoing a long-term treatment. The most common effects associated with cardiotonic agents
therapy are high mortality [1–6], myocardial ischemia aggravation [7,8], arrhythmia, hypotension [9],
and increased heart rate [10].

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Liguzinediol (LZDO, 1a, Figure 1) is a para-dihydroxy derivative of ligustrazine (1), which was
isolated from the traditional Chinese medicine herb Chuanxiong (Ligusticum wallichii Franch).
Ligustrazine (1) has shown cardiac effects in animal experiments and suppressed L-type Ca2+ channel
current in rat ventricular myocytes [11–16]. We recently found that LZDO (which is patented [17])
exerted positive inotropic effects without the risk of arrhythmia, and its positive inotropic effect in
isolated rat hearts was mediated through an elevation of sarcoplasmic reticulum (SR) Ca2+ transient,
which may act on SR Ca2+ ATPase. LZDO (1a) thus has a unique biological mechanism that may prove
effective in clinically treating heart failure [18]. Moreover, a pharmacokinetic study [19] of LZDO (1a)
showed that its half-life was about 2 h and its clearance was approximately 0.9 L/h·kg after intragastric
administration, suggesting that elimination or biotransformation of LZDO was relatively quick in rats,
and that LZDO may be mainly indicated for the treatment of acute heart failure and acute paroxysmal
phase of chronical heart failure. It is necessary to modify molecular structure of LZDO and study
pharmacodynamics of the derivatives, in order to improve treatment compliance among heart failure
patients, satisfy different drug delivery and dosage form clinically. Prodrug formation has been
considered as a useful approach to prolong the half-life of the parent drugs and effective drug duration
in vivo. The general rationale behind the prodrug strategy is to introduce lipophilicity and mask
hydrogen bonding groups of an active compound by the addition of another moiety, most commonly an
ester. Bioreversible esters have received considerable attention because of the presence of enzymes in
the living system capable of hydrolyzing them. In both drug discovery and development, prodrugs have
become an established tool for improving physicochemical, biopharmaceutical, or pharmacokinetic
properties of pharmacologically potent compounds, thereby increasing the developability and usefulness
of a potential drug [20,21].
Figure 1. Chemical structures of LZDO (1a) and LZDO prodrugs 3–5.
O
N
N
OH
O
HO
O
N
N
O
3
1a
O
O
N
N
O
O
O
O
N
N
O
5
O
4
In the present study, prodrugs 3–5 were prepared and evaluated for potential drug application. The
inotropic effects on the myocardium in normal isolated rat hearts, metabolic stability, formation of drug
from prodrug in rat liver microsomes and rat plasma, and pharmacokinetics of the parent compound in
rat plasma after intragastric administration of prodrug 3 have been studied in comparison with the parent
drug LZDO (1a).

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2. Results and Discussion
LZDO ester prodrugs 3–5 were synthesized with the aim of obtaining enzymatically labile drugs,
prolonging the half-life of the parent drug 1a in vivo. The prodrugs were evaluated in vitro and in vivo for
their potential use as prodrugs.
2.1. Chemistry
The key intermediate N,N'-dioxotetramethylpyrazine (2), prodrug 3 (34.1% yield), and LZDO (1a)
were prepared as previously reported [22]. The novel LZDO ester prodrugs 4, 5 were synthesized in
18.5% and 36.8% yield, respectively, in a straightforward manner starting from the key intermediate 2,
by treatment of the latter with acetic anhydride, isobutyric anhydride, and benzoyl chloride, respectively,
1
as illustrated in Scheme 1. The structures of all prodrug compounds were established by H-NMR,
13C-NMR, IR, and mass spectrometry, and their purity in excess of 98.0% was confirmed by HPLC analysis.
Scheme 1. Synthesis of LZDO prodrugs 4, 5.
N
N
1
ⅰ
O
N
N
O
2
ⅲ
ⅱ
O
O
N
N
N
O
O
O
O
N
O
O
4
5
Reagents and conditions: (i) 30% H2O2, glacial acetic acid, 98 °C, 12 h; (ii) isobutyric anhydride,
150 °C, 7 h; (iii) benzoyl chloride, 25 °C, 11 h.
2.2. Metabolic Stability
Metabolic stability is an important property of drug candidate, since it affects parameters such as
clearance, half-life, and bioavailability. A successful prodrug candidate is expected to undergo rapid,
complete conversion to the parent compound in the plasma or microsomes within 1–3 h. Prodrugs 3–5
and parent LZDO (1a) were subjected to a metabolic stability study in the presence of rat liver microsomes
and rat plasma. LZDO (1a) was fairly stable in rat liver microsomes and rat plasma, whereas prodrugs
3–5 were highly metabolized and converted to the desired parent compound 1a in 1–3 h (Figure 2).
Chemical degradation was not observed during metabolic stability study. The corresponding loss of
prodrug compounds and formation of parent drug 1a was determined by HPLC.

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Figure 2. Metabolic stability of LZDO and prodrugs in rat liver microsomes and rat plasma.
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Data are presented as the mean ± SD (n = 3). Percentage of prodrug remaining after metabolism was
calculated by the ratio of the peak area at respective time (min) to peak area found at 0 min multiplied
by 100: % remaining = [(peak area at respective time (min))/(peak area at 0 min)] × 100.
2.3. Formation of Parent Compound from Prodrugs in Rat Liver Microsomes and Rat Plasma
The experimental findings proved that all prodrugs were enzymatically labile and converted to parent
compound LZDO as a metabolite by enzymatic hydrolysis of the ester group. The peak areas of the
prodrugs in the HPLC analysis observed below detection level (BDL) after 3 h and correspondingly the
parent drug LZDO peak area were increased (Figure 3).
Figure 3. Formation of LZDO from prodrugs 3–5. Data are presented as the mean ± SD (n = 3).

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2.4. Cardiac Function Screening
The cardiac contractility and safety of prodrugs 3–5 were evaluated on the myocardium in normal
isolated rat hearts with LZDO as a positive control. Prodrugs 3–5 exerted positive inotropic effects in a
dose-dependent manner. The increased rate in left ventricular developed pressure (LVDP, accepted as
contractile force), +dP/dtmax (used as an index of contractility), the maximum rate of pressure decrease
of the left ventricle (−dP/dtmin, used as an index of diastolic function) and the heart rate (HR, used as an
index of heart muscle oxygen consumption) of these compounds vs. control are listed in Table 1.
As shown in Table 1, positive control LZDO and prodrugs 3–5 significantly increased LVDP,
±dP/dtmax at 1 μM, 10 μM, and 100 μM, respectively (p < 0.05). The positive inotropic effect was
reduced when LZDO was esterified. Prodrug 3 was the most potent prodrug for a positive inotropic
effect on the myocardium. None of the compounds had a significant effect on HR (p > 0.05).
2.5. Application to a Pharmacokinetic Study
We selected prodrug 3 for a pharmacokinetic study because its positive inotropic effect in vitro was
the highest among the three compounds. A thorough and complete method validation for determination
of LZDO and prodrug 3 in rat plasma was validated for specificity, sensitivity, matrix effect, recovery,
linearity, precision, accuracy and stability (data not shown).
Table 1. Effects of LZDO and prodrugs 3–5 on cardiac performance in isolated
Langendorff-perfused rat hearts (mean ± SD, n = 6).
Compd
Group
(μM)
0
LVDP
(mmHg)
50 ± 2.8
60 ± 5 b
73 ± 4 bd
88 ± 3 bdf
55 ± 2.8
63 ± 2 b
69 ± 1.1 bd
79 ± 1.6 bdf
54 ± 5
∆%
+dP/dtmax
(mmHg/s)
1,685 ± 147
1,836 ± 121
∆%
−dP/dtmax
(mmHg/s)
∆%
HR
(beat·min−1)
183 ± 22
186 ± 29
209 ± 21
193 ± 15
207 ± 8
1a
−1,058 ± 81
−1,175 ± 125
1
19.4 ± 11.0
45.6 ± 9.0
9.4 ± 8.1
11.1 ± 10.0
30.3 ± 0.3
10
2,125 ± 242 bc 26.5 ± 13.4 −1,371 ± 186 bc
100
0
76.3 ± 12.3 2,702 ± 287 bdf 60.9 ± 17.3 −1,911 ± 200 bdf 82.0 ± 27.9
3
4
5
1,611 ± 27
1,641 ± 29
−1,086 ± 38
−1,139 ± 76
−1,184 ± 75 a
−1,313 ± 70 bdf
−1,044 ± 84
1
15.0 ± 5.5
26.3 ± 7.7
44.7 ± 6.6
1.9 ± 0.5
9.8 ± 7.0
31.8 ± 5.3
4.9 ± 5.9
9.0 ± 5.1
20.9 ± 5.1
203 ± 15
204 ± 14
205 ± 26
196 ± 21
213 ± 46
196 ± 16
191 ± 29
209 ± 47
189 ± 30
196 ± 40
213 ± 41
10
1,768 ± 91 bd
2,123 ± 82 bdf
1,634 ± 100
1,669 ± 103
1,765 ± 52 a
1,952 ± 49 bdf
1,671 ± 93
100
control
1
63 ± 3 b
60 ± 4 a
73 ± 2.3 bdf 20.2 ± 15.2
17.4 ± 9.8
2.2 ± 3.4
4.9 ± 5.4
9.0 ± 8.9
−1,101 ± 102
−1,196 ± 54 bc
−1,399 ± 61 bdf
−1,081 ± 40
5.6 ± 8.3
9.9 ± 9.5
10
14.2 ± 10.0
100
control
1
16.7± 15.2
52 ± 4
55 ± 5
59 ± 2.5 b
64 ± 2.4 bde 24.5 ± 11.2
5.3 ± 3.3
1,688 ± 95
1.0 ± 1.1
9.9 ± 5.9
17.6 ± 6.8 −1,314 ± 63 bde
−1,095 ± 45
−1,245 ± 48 bd
1.3 ± 0.8
10
13.9 ± 7.4
1,831 ± 14 bd
1,962 ± 70 bdf
15.3 ± 2.8
100
21.7 ± 7.2
c
Each value is mean ± SD of six experiments. a p < 0.05, b p < 0.01 significantly different from control; p < 0.05,
d p < 0.01 significantly different from 1 μM; e p < 0.05, f p < 0.01 significantly different from 10 μM.

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Pharmacokinetic parameters including half-life, clearance, and bioavailability are important
properties of a drug candidate. Figure 4 shows the mean plasma concentrations of LZDO after
intragastric dosing at 10, 20 and 40 mg/kg of prodrug 3 in rats. The major pharmacokinetic parameters of
LZDO were calculated using a noncompartment model based on statistical moment. The
pharmacokinetic parameters of LZDO after intragastric administration of prodrug 3 at 10, 20 and
40 mg/kg in rats are summarized in Table 2. The pharmacokinetic parameters of LZDO after intragastric
administering LZDO at 10, 20 and 50 mg/kg in rats are summarized in Table 3 [19]. Prodrug 3 was
converted completely within 2 h in rats (data not shown).
As illustrated in the time-concentration and pharmacokinetic parameters profiles, prodrug 3 was
highly metabolized and quickly converted to desired parent compound 1a. Compared to LZDO, the Tmax
of prodrug 3 was higher than that of LZDO and the Cmax of prodrug 3 was lower than that of LZDO,
suggesting that prodrug 3 is converted into LZDO in vivo; the AUC of prodrug 3 after intragastric dose is
comparable to that of LZDO, suggesting that the bioavailabilities of the prodrug 3 and LZDO were
equivalent. Moreover, by comparison, the half-life (h) of LZDO increased to 4.01, 4.24, 4.39 for 10, 20,
40 mg/kg, respectively, and the clearance (L/h·kg) of LZDO was reduced to 0.40, 0.52, 0.41 for 10, 20,
40 mg/kg, respectively, suggesting that elimination of LZDO after taking prodrug 3 was relatively
slower than after taking LZDO in rats, Therefore, absorption of LZDO after oral administration of the
prodrug 3 is at least good as that of LZDO.
Figure 4. Time course of mean plasma LZDO concentrations (mean ± SD) after intragastric
administering prodrug 3 at 10, 20 and 40 mg/kg in rats (n = 6).
Table 2. Pharmacokinetic parameters of LZDO after intragastric administering prodrug 3 at
10, 20 and 40 mg/kg in rats (mean ± SD, n = 6).
Dose
10 mg/kg
0.42 ± 0.30
6.50 ± 1.71
4.01 ± 1.36
0.40 ± 0.06
21.51 ± 2.82
25.56 ± 3.82
20 mg/kg
0.42 ± 0.13
10.49 ± 3.14
4.24 ± 1.52
0.52 ± 0.08
33.90 ± 5.71
38.86 ± 5.72
40 mg/kg
0.42 ± 0.13
28.39 ± 9.56
4.39 ± 1.64
0.41 ± 0.06
93.94 ± 13.76
98.30 ± 15.37
Tmax (h)
Cmax (mg/L)
t
1/2 (h)
CL/F (L/h·kg)
AUC0–t (mg·h/L)
AUC0–∞ (mg·h/L)

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Table 3. Pharmacokinetic parameters of LZDO after intragastric administering LZDO at 10,
20 and 40 mg/kg in rats (mean ± SD, n = 6) [19].
Dose
10 mg·kg−1 i.g.
0.29 ± 0.10
8.58 ± 2.66
2.21 ± 1.11
0.79 ± 0.51
16.74 ± 8.90
17.08 ± 8.99
20 mg·kg−1 i.g.
0.29 ± 0.10
50 mg·kg−1 i.g.
0.29 ± 0.10
Tmax (h)
Cmax (mg/L)
17.02 ± 5.50
2.57 ± 2.14
49.33 ± 10.50
1.78 ± 0.57
t
1/2 (h)
CL/F (L/h·kg)
AUC0–t (mg·h/L)
AUC0–∞ (mg·h/L)
0.86 ± 0.57
0.98 ± 1.01
34.84 ± 24.64
35.93 ± 24.08
92.85 ± 64.10
94.82 ± 66.88
3. Experimental
3.1. General
All commercial reagents and solvents were used without further purification unless otherwise
specified. Melting points were determined using a model Buchi B-450 apparatus. ESI-MS were recorded
on a TOF mass spectrometer (Applies-Biosystem Mariner). IR spectra were acquired by using a THERMO
IR spectrometer Model Nicolet-100. 1H- and 13C-NMR spectra (500/125 MHz) were recorded on a Bruker
Advance 500 spectrophotometer. Mass spectra were recorded on a ZAB-HS mass spectrometer and are
reported as m/z values. HPLC analysis was conducted on a Waters 2695 HPLC instrument equipped with a
UV detector (model 2489). LZDO and prodrugs 3–5 were analysed by HPLC, and their purity was
confirmed to be greater than 98.0%. In vitro metabolic stability analysis was conducted by a HPLC
method, using stock standard solutions of LZDO and prodrugs that were prepared in water at a
concentration of 30 mM. The positive inotropic effects on the myocardium of LZDO and prodrugs 3–5
in vitro were evaluated on Langendorff-perfused hearts isolated from normal Sprague-Dawley rats. A
HPLC method was applied to measure the rat plasma concentrations of LZDO and prodrug 3 after
intragastric administration of prodrug 3 in the rats.
3.2. Chemistry
2,5-Di-iso-butyryloxymethyl-3,6-dimethylpyrazine (4). Compound 2 (16.8 g, 0.10 mol) was treated with
isobutyric anhydride (200 mL, 0.32 mol) at 150 °C for 7 h. Reaction progress was monitored by TLC.
After reaction completion, the excess propionic anhydride was evaporated. The residue was purified by
silica gel column chromatography (petroleum ether-ethyl acetate = 4:1 v/v) to afford 4 as a yellowish oil
(4.74 g, 15.4% yield, HPLC > 98%). ESI-MS (M+H+) calcd for C16H25N2O4 309.1808, found 309.2153;
UV max (methanol): 279 nm; IR (KBr) cm−1: 2978 (υCH), 1716 (υC=O), 1097 (νC-O), 1461, 1417, 1296
1
(δCH); H-NMR (CDCl3, 500 MHz) δ: 5.29 (s, 4H, 2×CH2-O), 2.71 (s, 6H, 2×CH3), 2.56 (q, 2H,
J = 6.1, 2×CH-C=O), 1.05 (m, 12H, 2×CH(CH3)2) ppm; 13C-NMR (CDCl3, 125 MHz) δ: 173.0 (C=O),
149.8, 147.4 (C=N), 64.5 (C-O), 35.8 (C-C=O), 20.4 (CH3), 18.3 (CH2-CH3) ppm; EI-MS m/z (%): 308
(0.72, M), 237 (36.34, M-O=C-CH2-CH2-CH3), 167 (100, M-2×O=C-CH2-CH2-CH3+H).
2,5-Dibenzoyloxymethyl-3,6-dimethylpyrazine (5). Benzoyl chloride (200 mL) was added dropwise into
a solution of 2 (16.8 g, 0.10 mol) and the mixture was stirred at room temperature for 11 h. Reaction
progress was monitored by TLC. After reaction completion, the reaction mixture was washed with

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saturated sodium bicarbonate solution (3 × 100 mL), extracted with ethyl acetate, dried over anhydrous
sodium sulphate, filtered, and concentrated under vacuum. The residue was purified by silica gel column
chromatography (petroleum ether-ethyl acetate = 3:1 v/v) affording 5 as white needle crystals (13.84 g,
36.8% yield, HPLC > 98%). mp 152–154 °C; ESI-MS (M+H+) calcd for C22H21N2O4 377.1496, found
377.2237; UV max (methanol): 306 nm; IR (KBr) cm−1: 2947 (νCH), 1728 (νO=C), 1458 (δCH), 1206
1
(νC-O), 1102 (νC-C), 886, 825 (δ=CH); H-NMR (CDCl3, 500 MHz) δ: 8.06 (d, 4H, J = 7.2 Hz,
2×Ph-H-2,6), 7.55 (t, 2H, J = 4.4 Hz, Ph-H-4), 7.44 (t, 4H, J = 7.2 Hz, 2×Ph-H-3,5), 5.26 (s, 4H,
2×CH2-O), 2.60(s, 6H, 2×CH3) ppm; 13C-NMR (CDCl3, 125 MHz) δ: 165.9 (C-O), 149.8, 147.4 (C=N),
133.1 (Ph-C-4), 131.0 (Ph-C-1), 129.9 (Ph-C-2,6), 128.8 (Ph-C-3,5), 64.2 (C-O), 19.8 (CH3) ppm;
EI-MS m/z: 376 (M), 272 (M+H+-Ph-C=O), 168 (M+2H+-2×Ph-C=O), 105 (Ph-C=O+H+).
3.3. Metabolic Stability Analysis
Analysis was performed by using a Waters Alliance HPLC system (2695 pump, and 2489 PDA
detector) with the following chromatographic parameters: wavelength, 278 nm; injection volume, 20 μL;
run time, 20 min. The chromatographic separation was performed on an Phecda C18 (4.6 mm × 250 mm,
5 μm) column with a gradient elution of methanol and deionized water. Flow rate was 1.0 mL/min at 30 °C.
3.3.1. Rat Plasma
Rat plasma was harvested from in-house rats. Fresh blood was collected from the male rat using a
retro-orbital bleeding method in a tube containing heparin (100 IU/mL blood). After the collection of
blood, plasma was separated from the blood by centrifugation at 5,000 rpm for 10 min. The supernatant
plasma was separated and utilized for the further experiment.
In Vitro Physiological Stability of Prodrugs 3–5 and LZDO (1a) in Rat Plasma
The test compound in aqueous solution 30 mM (20 μL) was dissolved in rat plasma (980 μL).
Immediately after addition (0 min), aliquots (100 μL) were removed and added to ice cold dissolvent
(ethanol-ethyl acetate = 1:4 v/v, 1 mL) and mixed well by vortexing for 2 min. The mixture was
centrifuged at 12,000 rpm for 10 min, and the supernatant was diluted with methanol and analyzed by
HPLC. After 0 min, the remaining sample was incubated at 37 °C for 1, 2, and 3 h. After 1, 2, and 3 h, the
sample (100 μL) was treated with ice cold blending dissolvent (ethanol-ethyl acetate = 1:4 v/v, 1 mL),
the mixture was vortex-mixed for 5 min, and then centrifuged at 5,000 rpm for 5 min. The upper organic
layer (0.9 mL) was transferred to another tube and evaporated to dryness in a rotary evaporator
(Centrivap console, Labconco Company, Kansas, MO, USA) at 40 °C. The supernatant was dissolved
with 200 μL of water : methanol (30:70, v/v), vortex-mixed for 3 min, centrifuged at 12,000 rpm for 5 min,
and a 20 μL of the supernatant was then injected into HPLC instrument.
(1)
% remaining = peak area at respective time (min)/peak area at 0 min × 100%

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3.3.2. Rat Liver Microsomes
Rat liver microsomes were prepared in house by a previously published method [23] and used
immediately in the experiments. Protein concentrations were determined by a Lowry protein assay [24].
3.3.2.1. Materials
Rat liver microsomes (2.0 mg/mL protein concentration), NADPH (1.0 mM solution, Sigma,
Shanghai, China), Tris-HCl buffer (50 mM, pH 7.4, Sigma, Shanghai, China), test compound in
aqueous solution (30 mM, 20 μL).
3.3.2.2. Assay Procedure
Tris-HCl buffer (780 μL), 1.0 mM NADPH solution (100 μL), and 100 μL of rat liver microsomes
were mixed and vortexed at 37 °C for 5 min. To this mixture, 30 mM drug in aqueous solution (20 μL)
was injected and vortexed well. Sample (100 μL) was immediately taken out (0 min) and transferred to
the centrifuge tube containing ice cold dissolvent (ethanol-ethyl acetate = 1:4 v/v, 1 mL). The assay
mixture was incubated in a water bath at 37 °C for 3 h and at specific time points (1 h, 2 h, 3 h). The assay
mixture (100 μL) was taken out and added to the centrifuge tube containing an equal volume of cold
dissolvent. Then all the tubes were vortexed and centrifuged at 12,000 rpm for 10 min. Aliquots of the
supernatant were separated and used for analysis by HPLC. The percentage of prodrug remaining was
calculated according to the following equation:
% remaining = peak area at respective time (min)/peak area at 0 min × 100%
(2)
3.4. Ex Vivo Langendorff-Perfused Rat Heart [25]
Sprague-Dawley adult male rats weighing 260–320 g were anaesthetised by intraperitoneal
administration of 20% urethane (1.2 g/kg). The hearts were quickly removed and placed into an ice-cold
modified buffer until contractions ceased. The heart was cleaned of surrounding tissues, mounted on the
aortic cannula of the Langendorff perfusion system apparatus, and perfused with oxygenated buffer
(mM): NaCl 117, KCl 5.7, CaCl2 1.8, MgCl2 1.7, NaHCO3 4.4, NaH2PO4 1.5, HEPES 20 and glucose 11.
The buffer was equilibrated with 95% O2 and 5% CO2 and adjusted to pH 7.4 with NaOH before it
entered the heart. An epicardial electrogram was registered using two stainless steel electrodes, one
attached to the apex of the heart and the other to the aortic cannula. To obtain an isovolumetrically
beating preparation, a latex balloon filled with water and connected by a catheter to a transducer was
inserted through the left atrium into the left ventricle and inflated to provide an end-diastolic pressure
between 8 and 12 mm Hg. Before each experimental protocol was initiated, the isolated hearts were set
at a mean arterial pressure of 60–80 mm Hg and allowed to stabilise at 37 °C for 40–60 min. The isolated
rat hearts were then perfused in sequence with 1, 10, and 100 μM of LZDO and prodrugs 3–5 in each
group. A RM6240B/C four channel physiological recording instrument (Chengdu Instrument Factory,
Chengdu, China) was used for continuous recording of LVSP, LVEDP, HR (derived from electrogram),
+dP/dtmax and −dP/dtmax throughout the experiments. The criteria for established stability were LVDP >
50 mmHg, +dP/dtmax > 1,600 mmHg·s−1, heart rate > 180 beats·min−1 and normal sinus rhythm. The

Molecules 2013, 18
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hearts were not studied if they had weak contractility (+dP/dtmax < 1,600 mmHg·s−1) and severe
arrhythmia. Diastolic balloon pressure was maintained at 8 mmHg. LVDP was calculated as the
difference between the systolic and diastolic pressures. The results are expressed as the mean value of
hemodynamic variables (n = 6). Statistical analysis was performed using SPSS for Windows (version 13.0).
Data were analyzed by One-way ANOVA. Values are given as mean ± SD and a p value less than 0.05
was considered as statistically significant.
3.5. Application to a Pharmacokinetic Study in Rats
Sprague-Dawley rats (male, weighing 200 ± 20 g) were obtained from Slack Laboratory Animal Site
(Shanghai, China) and kept in an environmentally controlled breeding room for at least five days before
experimentation. The rats were fasted overnight but with free access to water before the test. Animal
welfare and experimental procedures were strictly in accordance with the guide for the care and use of
laboratory animals [26]. After intragastric administration of prodrug 3 at 10, 20 and 40 mg/kg in rats,
100 μL of heparinized blood samples were collected at 0.08, 0.17, 0.25, 0.50, 1, 2, 3, 4, 6, 8, 10, 12 and
24 h from the ocular veins and immediately centrifuged at 5,000 rpm for 10 min. The supernatant plasma
were collected and immediately frozen at −20 °C for not more than 7 days until analysis. The collected
blood samples were pretreated by the aforementioned method (sample dilutions were made when
necessary) and the concentrations of LZDO and prodrug 3 in each sample were finally determined
based on the validated HPLC method. Pharmacokinetic parameters in rats were estimated by a
noncompartmental method using DAS 2.0 software package (Mathematical Pharmacology Professional
Committee of China, Shanghai, China).
4. Conclusions
The positive inotropic effect on the myocardium of LZDO and prodrugs 3–5 were evaluated by
known experimental techniques. Prodrugs 3–5 exerted positive inotropic effects in normal isolated rat
hearts, and prodrugs were transformed enzymatically to the parent drug 1a in both rat liver chromosome
and rat plasma. Pharmacokinetic studies showed that the half-life of the parent compound was extended
to 4 h after intragastric administering prodrug 3 and absorption of LZDO after oral administration of the
prodrug 3 wasat least good as for LZDO. On the basis of in vitro and in vivo studies, prodrug 3 was
found to be a superior prodrug candidate for increasing myocardial contractility.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/4/4561/s1.
Acknowledgments
This work was financially supported by National Natural Science Foundation of China (81072542)
and The Major Scientific and Technological Special Project (2009ZX09103-081).

Molecules 2013, 18
4571
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Sample Availability: Samples of the compounds (1a, 3, 4, 5) are available from the authors.
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(http://creativecommons.org/licenses/by/3.0/).