Synthesis of ranolazine metabolites and their anti-myocardial ischemia activities

Article abstract of DOI:10.1248/cpb.57.1218

The anti-anginal drug Ranolazine, a partial fatty acid oxidation (pFOX) inhibitor, is thought to modulate the metabolism during myocardial ischemia by activating pyruvate dehydrogenase activity to promote glucose oxidation. Ranolazine and its five principal metabolites: CVT-2512, CVT-2513, CVT-2514, CVT-2738 and CVT-4786, were synthesized. The effect of Ranolazine and its metabolites on the ECG (electrocardiogram) of mice with myocardial ischemia induced by isoprenaline and their effect on alleviating the symptom of myocardial ischemia were tested and compared. The results showed that CVT-2738 and CVT-2513 could be protective against mice myocardial ischemia induced by isoprenaline. Within all the metabolites tested in this study, CVT-2738 exhibited the best potency, however, it was still less potent than Ranolazine.

Full text of DOI:10.1248/cpb.57.1218

1218
Chem. Pharm. Bull. 57(11) 1218—1222 (2009)
Vol. 57, No. 11
Synthesis of Ranolazine Metabolites and Their Anti-myocardial Ischemia
Activities
a
b
b
,b
a
,a
*
*
Zhangyu YAO, Shubo GONG, Teng GUAN, Yunman LI, Xiaoming WU, and Hongbin SUN
a Center for Drug Discovery, College of Pharmacy, China Pharmaceutical University; and b Department of Physiology,
College of Pharmacy, China Pharmaceutical University; 24 Tongjiaxiang, Nanjing 210009, China.
Received April 20, 2009; accepted August 19, 2009; published online August 21, 2009
The anti-anginal drug Ranolazine, a partial fatty acid oxidation (pFOX) inhibitor, is thought to modulate
the metabolism during myocardial ischemia by activating pyruvate dehydrogenase activity to promote glucose
oxidation. Ranolazine and its five principal metabolites: CVT-2512, CVT-2513, CVT-2514, CVT-2738 and CVT-
4786, were synthesized. The effect of Ranolazine and its metabolites on the ECG (electrocardiogram) of mice
with myocardial ischemia induced by isoprenaline and their effect on alleviating the symptom of myocardial is-
chemia were tested and compared. The results showed that CVT-2738 and CVT-2513 could be protective against
mice myocardial ischemia induced by isoprenaline. Within all the metabolites tested in this study, CVT-2738 ex-
hibited the best potency, however, it was still less potent than Ranolazine.
Key words Ranolazine; metabolite; myocardial ischemia; R–R interval; T-wave
Angina pectoris is one of the most frequent clinical syn-
Metabolism modulation is a novel way for the treatment of
dromes associated with ischemic heart diseases. The inci- angina pectoris.1) Ranolazine (Fig. 1) is a piperazine deriva-
dence of this disease is increasing with the improvement of tive developed by Syntex Laboratories, which has been
living standard and the change of the mode of life. It has shown to have anti-ischemic action.2—5) It’s a partial fatty
been generally accepted that an efficient therapeutic ap- acid oxidation (pFOX) inhibitor. Its mechanism has not been
proach to ischemic heart diseases is to improve the myocar- fully understood. The possible mechanism involves modula-
dial oxygen balance between supply and demand in the is- tion of the metabolism of myocardial ischemia by activating
chemic heart, by either increasing coronary blood flow or de- pyruvate dehydrogenase activity to promote glucose oxida-
creasing cardiac mechanical function, or both. The tradi- tion.2,6—9) Ranolazine is thought to switch substrate utiliza-
tional anti-anginal drugs are nitrates, b-blockers and calcium tion from fatty acids to glucose to improve the efficiency
channel blockers, which are thought to improve the myocar-
dial oxygen balance with changes in hemodynamic parame-
ters. But these drugs also bring further injury to the weak
cardiac function. So researchers are trying to find more effi-
cient means with fewer side effects to prevent and treat my-
Fig. 1. Ranolazine
ocardial ischemia.
Fig. 2. Ranolazine Metabolic Way
© 2009 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: yucaoren@sina.com

November 2009
1219
with water, dried over Na2SO4 and concentrated. The residue was extracted
with hot ether. Concentration of the ether layer gave CVT-2738 (26.2 g,
62%) as a white solid, mp 94—97 °C. H-NMR (300 MHz, CDCl3) d: 7.10
of oxygen utilization and limit the production of lactic
acid.6,10,11) Compared with the traditional anti-anginal drugs,
the main advantage of Ranolazine is that it has little hemody-
namic effect with minimal or no effect on blood pressure or
heart rate.12—14)
1
(s, 1H), 7.09 (s, 1H), 3.19 (s, 2H), 2.98 (t, 4H, Jꢀ5.27 Hz), 2.69 (t, 3H,
Jꢀ4.77 Hz), 2.23 (s, 6H). ESI-MS m/z: 248.3 [MꢁH]ꢁ. The analytical data
of CVT-2738 was identical with the data reported in reference 20. CVT-2738
was dissolved in absolute ethanol and to the solution was added hydrochlo-
ric acid. Then the solvent was removed under reduced pressure and the
residue was recrystallized with acetone to give hydrochloric acid salt of
CVT-2738 as a white solid, mp 194—199 °C.
1-Methoxy-2-(oxiranyl methoxy)benzene (A2) 2-Methoxyphenol
(6.73 ml, 61.2 mmol) and a solution of sodium hydroxide (5.8 g in 12 ml
water) were added to 20 ml of dioxane and the mixture was heated to 45 °C.
Then epichlorohydrin (13.5 ml) was added dropwise. The mixture was
stirred at 45 °C for 3 h. The reaction mixture was cooled, diluted with ethyl
acetate, filtered. The filtrate was washed with water (20 mlꢂ3). The organic
layer was dried over Na2SO4 and concentrated. The crude product was puri-
fied by column chromatography (SiO2, petroleum ether : ethyl acetateꢀ6 : 1)
to give A2 (5.83 g, 53.0%) as a colorless oil. 1H-NMR (300 MHz, CDCl3) d:
6.96—6.89 (m, 4H), 4.25—4.02 (m, 2H), 3.87 (s, 3H), 3.41—3.35 (m, 1H),
2.90—2.72 (m, 2H). EI-MS m/z (%): 180 (38), 124 (59), 109 (47), 95 (14),
77 (25), 58 (100). The analytical data of A2 was identical with the data re-
ported in ref. 20.
Ranolazine is almost completely metabolized after admin-
istration, and less than 5% of the administered dose is ex-
creted unchanged.15) Metabolism of Ranolazine is com-
plex.16,17) Cytochrome P450 (CYP) 3A (mainly in liver and
small intestine) accounts for the major biotransformation of
Ranolazine (70—75%). CYP 2D6 accounts for less than
20% of Ranolazine metabolism. Figure 2 shows the seven
principal metabolic routes of Ranolazine.18,19) Route one: hy-
droxylation of the methyl group in the 2,6-dimethylphenyl
fragment to produce CVT-2551; route two: hydroxylation of
the 2-methoxyphenyl or 2,6-dimethylphenyl group to pro-
duce CVT-5029/CVT-5030/CVT-5031 and CVT-5028/CVT-
3388 respectively; route three and four: N-dealkylation of the
piperazine ring to give the components CVT-2513, CVT-
2535, CVT-2738, CVT-2534; route five: O-dearylation to
produce CVT-2512; route six: O-demethylation to produce
CVT-2514; route seven: hydrolysis of the amide group to
produce CVT-3369. Among those metabolites, there are four
Ranolazine A mixture of CVT-2738 (2 g, 11.10 mmol) and A2 (3 g,
11.1 mmol) in 20 ml of methanol and 40 ml of toluene was stirred at 65 °C
for 9 h. The solvent was removed under reduced pressure. The crude prod-
uct was purified by column chromatography (SiO2, dichloromethane :
methanolꢀ15 : 1) to give Ranolazine (3.31 g, 69.8%) as a yellow oil. 1H-
metabolites should be paid more attention since their blood NMR (300 MHz, DMSO-d6) d: 10.02 (s, 1H), 7.11—6.88 (m, 7H), 4.42 (m,
1H), 4.00 (s, 2H), 4.00—3.93 (m, 2H), 3.94—3.41 (m, 13H), 2.18 (s, 6H);
concentrations exceed 10% of the parent compound concen-
tration. They are CVT-2738 (38%), CVT-2514 (26%), CVT-
4786 (21%, a further metabolite of CVT-2534), and CVT-
ESI-MS m/z: 428.3 [MꢁH]ꢁ. The analytical data of Ranolazine was identi-
cal with the data reported in ref. 20. Ranolazine was dissolved in absolute
ethanol and to the solution was added hydrochloric acid. Then the solvent
2512 (11%). Studies with human liver microsomes indicated
that CVT-2738 and CVT-4786 were formed primarily
through CYP3A; CVT-2514 was formed through CYP2D6;
CVT-2512 was formed through both the pathways. Besides
the four major metabolites, CVT-2537, CVT-2513, CVT-
2535, CVT-3248, CVT-3388/CVT-5028, CVT-2551, and
CVT-5030 have blood concentrations exceeding 1% of
Ranolazine exposure.
In this study, Ranolazine and its five principal metabolites,
including CVT-2512, CVT-2513, CVT-2514, CVT-2738 and
CVT-4786 were synthesized and the effect of Ranolazine,
CVT-2512, CVT-2513, CVT-2514, CVT-2738 and CVT-4786
on the ECG (electrocardiogram) of mice with myocardial
ischemia induced by isoprenaline were tested and compared.
was removed under reduced pressure and recrystallization with acetone gave
hydrochloric acid salt of Ranolazine as a white solid, mp 227—229 °C.
2-Benzyloxyphenol (A4) Catechol (2.2 g, 20 mmol), potassium carbon-
ate (3.1 g, 22 mmol) and potassium iodide (0.04 g, 0.6 mmol) were added to
15 ml of acetone and heated to 50 °C. Then chlorobenzyl (2.36 ml, 22 mol)
was added dropwise. The mixture was stirred for 3 h. The solvent was re-
moved and to the residue was added 40 ml of cold water. The mixture was
filtered, and the aqueous layer was neutralized with diluted hydrochloric acid
and extracted with dichloromethane (60 mlꢂ3). The organic layer was dried
over Na2SO4 and concentrated. The crude product was purified by column
chromatography (SiO2, petroleum ether : ethyl acetateꢀ15 : 1) to give A4
1
(0.5 g, 12.5%) as a yellow oil. H-NMR (300 MHz, CDCl3) d: 7.42—7.37
(m, 5H), 6.94—6.83 (m, 4H), 5.64 (s, 1H), 5.10 (s, 2H); EI-MS m/z (%):
200 (3), 91 (100), 65 (10). The analytical data of A4 was identical with the
data reported in ref. 21.
1-(2-Benzyloxyphenoxy)-2,3-propylene Oxide (A5) The procedure
was in accordance with the synthesis of A2. The product A5 was a yellow
oil (62.5%). 1H-NMR (300 MHz, CDCl3) d: 7.47—7.26 (m, 5H), 6.96—
6.91 (m, 4H), 5.14 (s, 2H), 4.30—4.04 (dd, 2H, Jꢀ3.4, 5.4, 11.3 Hz), 3.40—
3.37 (m, 1H), 2.90—2.75 (m, 2H); EI-MS m/z (%): 256 (10), 91 (100), 65
(8). The analytical data of A5 was identical with the data reported in ref. 21.
1-[3-(2-Benzyloxyphenoxy)-2-hydroxypropyl]-4-[N-(2,6-dimethyl-
phenyl)aminocarbonylmethyl]piperazine (A6) The procedure was in ac-
cordance with the synthesis of Ranolazine. The product A6 was a yellow oil
(74.7%). 1H-NMR (300 MHz, CDCl3) d: 8.67 (s, 1H), 7.48—6.92 (m, 12H),
5.11 (s, 2H), 4.16—4.01 (m, 3H), 3.19 (s, 2H), 2.71—2.46 (m, 10H), 2.25
(s, 6H); 13C-NMR (75 MHz, CDCl3) d: 168.3, 149.2, 149.1, 137.3, 134.9,
133.6, 128.4, 128.3, 127.8, 127.3, 127.1, 122.1, 121.8, 115.7, 115.1, 72.3,
71.2, 66.1, 61.6, 60.5, 53.8, 53.6, 53.4, 53.4, 18.6; IR (KBr) cmꢃ1: 3412,
3308, 2937, 2820, 1682, 1592, 1504, 1378, 1257, 1124, 1013, 831, 743,
698; ESI-MS m/z: 504.3 [MꢁH]ꢁ, 526.2 [MꢁNa]ꢁ; FAB-MS m/z:
503.2781 (Calcd for C17H27N3O3: 503.2784).
Experimental
General All commercially available solvents and reagents were used
without further purification. Melting points were determined with a Bü chi
1
capillary apparatus and were not corrected. H- and 13C-NMR spectra were
recorded on an ACF 300Q Bruker, spectrometer in CDCl3, with Me4Si as the
internal reference, or in DMSO-d6. Low-and high-resolution mass spectra
(LR-MS and HR-MS) were recorded in electron impact mode. Reactions
were monitored by TLC on Silica Gel 60 F254 (Qindao Ocean Chemical
Company, China) plates exposed to H2SO4 (10% in EtOH) spray followed by
charring (ꢀ50 °C). Column chromatography was performed with Silica Gel
Geduran Si 60 (Qindao Ocean Chemical Company, China).
2-Chloro-N-(2,6-dimethylphenyl)acetamide (A1) Toluene (150 ml)
and 2,6-dimethylaniline (9.25 ml, 75.09 mmol) were added to an aqueous so-
lution of sodium bicarbonate (7.95 g in 75 ml water). Chloroacetyl chloride
(7.15 ml, 89.87 mmol) was added slowly at 0 °C. Then the solution was
stirred at room temperature for 2.5 h. The resulting solids were collected by
filtration, washed with water and dried to give A1 (13.18 g, 88.9%) as a
white solid, mp 138—140 °C. The solid was used directly in the next step.
1-[3-(2-Hydroxylphenoxy)-2-hydroxypropyl]-4-[N-(2,6-dimethyl-
phenyl)aminocarbonylmethyl]piperazine (CVT-2514) A suspension of
A6 and palladium on charcoal in ethyl acetate was stirred under a hydrogen
atmosphere for 24 h. The reaction mixture was filtered and the ethyl acetate
was removed under reduced pressure. The crude product was purified by
column chromatography (SiO2, dichloromethane : methanolꢀ1 : 1) to give
N-(2,6-Dimethyl phenyl)-1-piperazine Acetamide (CVT-2738)
A
mixture of A1 (8.0 g, 40 mmol) and piperazine (13.8 g, 160 mmol) in 100 ml
of ethanol was stirred at 85 °C for 3 h. Then ethanol was removed under re-
duced pressure, the residue was neutralized with ammonia water. The mix-
ture was extracted with dichloromethane, and the organic layer was washed
1
CVT-2514 (60.9%) as a yellow oil. H-NMR (300 MHz, CDCl3) d: 8.59 (s,
1H), 7.11—6.77 (m, 7H), 4.14—4.06 (m, 1H), 4.04—3.97 (m, 2H), 3.24 (s,
2H), 2.80—2.54 (m, 10H), 2.23 (s, 6H); 13C-NMR (75 Hz, CDCl3) d: 168.2,

1220
Vol. 57, No. 11
147.9, 146.1, 134.9, 133.6, 128.3, 127.2, 123.7, 119.9, 116.6, 116.1, 73.5,
65.9, 61.6, 60.1, 53.6, 18.6; IR (KBr) cmꢃ1: 3310, 2925, 2822, 1666, 1592,
1503, 1266, 1161, 1011, 834, 771, 745; ESI-MS m/z: 414.3 [MꢁH]ꢁ, 436.2
[MꢁNa]ꢁ; FAB-MS m/z: 413.2316 (Calcd for C17H27N3O3: 413.2315).
CVT-2514 was dissolved in absolute ethanol and to the solution was added
hydrochloric acid. Then the solvent was removed under reduced pressure
and recrystallization with acetone gave hydrochloric acid salt of CVT-2514
as a white solid, mp 247—249 °C.
drochloric acid to acidity and extracted with ethyl acetate (15 mlꢂ3). The
organic layer was dried over Na2SO4 and concentrated. The crude product
was purified by column chromatography (SiO2, petroleum ether : ethyl ac-
1
etateꢀ10 : 1) to give CVT-4786 (40 mg, 37.4%) as a yellow oil. H-NMR
(300 MHz, CDCl3) d: 6.99—6.86 (m, 4H), 4.54 (t, 1H, Jꢀ4.31 Hz), 4.34—
4.28 (m, 2H), 3.83 (m, 3H); ESI-MS m/z: 213.0 [MꢁH]ꢁ, 235.1 [MꢁNa]ꢁ.
The analytical data of CVT-4786 was identical with the data reported in ref.
24.
1-[(2,6-Dimethyl phenyl)aminocarbonylmethyl]-4-[2,3-dihydroxyl-
propyl]piperazine (CVT-2512) A solution of concentrated sulfuric acid
(0.2 g) and epichlorohydrin (1.8 ml) in 12 ml of water was heated to 85 °C,
then heating was stopped. Another 6.1 ml of epichlorohydrin was added
slowly while keeping the solution refluxing. After addition of epichlorohy-
drin the mixture was stirred at 105 °C for 2.5 h. The reaction mixture was
cooled, neutralized with 30% sodium hydroxide aqueous solution. It was pu-
rified by vacuum distillation to give chlorohydrin (6.62 g, 60%) as a color-
less oil (bp 110—114 °C/10 mmHg).
Animal Experiments. Drugs and Reagents Drugs were dissolved in
5% CMC-Na (carboxymethyl cellulose sodium) to form clear solutions
which were adjusted to the expected concentrations before use. Isoprenaline
injection: Shanghai Harvest Pharmaceutical Co., Ltd., batch number:
6E20008, specification: 2 mg/ml. Composite Salvia Dropping Pills (pro-
duced by Tianshili Pharmaceutical Co., Ltd., batch number: 20040906) were
dissolved in distilled water before use (concentration: 3 dropping pills/10
ml). Sodium chloride injection (0.9%) was provided by Shandong Hualu
Pharmaceutical Co., Ltd., batch number: A07012001. Urethane (20%) was
provided by Shanghai Jinxi Chemical Co., Ltd. The hydrochloric acid salts
of Ranolazine and its metabolites: CVT-2738, CVT-2514, CVT-2512 and
CVT-2513, and CVT-4786 in free acid form were used in the animal experi-
ments.
A mixture of CVT-2738 (1.00 g, 4 mmol), chlorohydrin (0.44 g, 4 mmol)
and sodium bicarbonate (0.50 g, 6 mmol) in 25 ml of ethanol was stirred at
65 °C overnight. The solvent was removed and to the residue was added ace-
tone. The mixture was filtered and the filtrate was concentrated to give a yel-
low solid (1.2 g). The crude product was purified by column chromatography
(SiO2, dichloromethane : methanolꢀ60 : 1) to give CVT-2512 (0.86 g,
Apparatus Electrocardiograph: ECG-6511, Shanghai Photoelectric
Medical Electronic Instrument Co., Ltd. JA1003 electronic balance: Shang-
hai Balance Instrument Factory.
1
66.2%) as a white solid, mp 128—131 °C. H-NMR (300 MHz, CDCl3) d:
8.58 (s, 1H), 7.10 (s, 3H), 3.90—3.74 (m, 2H), 3.62—3.50 (m, 1H), 3.23 (s,
2H), 2.80—2.43 (m, 10H), 2.23 (s, 6H); 13C-NMR (75 MHz, CDCl3) d:
168.2, 134.9, 133.6, 128.3, 127.2, 66.9, 64.8, 61.6, 60.2, 53.9, 53.5, 18.6; IR
(KBr) cmꢃ1: 3395, 3308, 2819, 1655, 1509, 1453, 1338, 1151, 1040, 942,
830, 767, 534; ESI-MS m/z: 322.3 [MꢁH]ꢁ; FAB-MS m/z: 321.2050 (Calcd
for C17H27N3O3: 321.2052). CVT-2512 was dissolved in absolute ethanol
and to the solution was added hydrochloric acid. Then the solvent was re-
moved under reduced pressure and recrystallization with methanol/acetone
gave hydrochloric acid salt of CVT-2512 as a white solid, mp 247—249 °C.
1-[3-(2-Methoxyphenoxy)-2-hydroxypropyl]piperazine (CVT-2513)
A solution of A2 (0.5 g, 2.8 mmol) and piperazine (1.0 g, 11.1 mmol) in
15 ml of absolute ethanol was stirred at 65 °C for 1.5 h. Then the solvent was
removed and the residue was dissolved in 40 ml of dichloromethane. The or-
ganic layer was washed with water (25 mlꢂ2), dried over Na2SO4 and con-
centrated. The crude product was purified by column chromatography (SiO2,
dichloromethane : methanolꢀ50 : 1) to give CVT-2513 (0.44 g, 59.5%) as a
Experimental Animals Animal studies were performed in adherence
with the guidelines established in the Guide for the Care and Use of Labora-
tory Animals (NIH publication 85-23, revised 1985). All the experiments
were carried out with Kunming mice which were half male and half female
weighing 20—30 g.
Statistic Analysis All date were represented as meanꢄstandard error of
mean. Data were analyzed by t-test for comparisons between groups. Values
of pꢅ0.05 and pꢅ0.01 were considered statistically significant.
Effect on ECG (Electrocardiogram) of Myocardial Ischemic Mice In-
duced by Isoprenaline The animal experimentation was carried out based
on the methodology described by Xu et al.25) A total of 64 mice were used.
The mice were divided into 8 groups with 8 mice in each group (4 male
mice and 4 female mice). Group one was positive control group treated with
composite Salvia Dropping Pills; Group two was model control group
treated with equal volume of distilled water; Group three—eight were goups
treated with Ranolazine, CVT-2512, CVT-2513, CVT-2514 and CVT-2738
and CVT-4786 (dose: 100 mg/kg) respectively. The medication administra-
tion volume of each group was 0.4 ml/20 g. 30 min after first intragastric ad-
ministration of the drugs, the mice were anesthetized by intraperitoneal in-
jection with 1.2 g/kg 20% ethyl carbamate. Then the mice were fixed on the
breast position to record normal II lead ECG and observe R–R interval
(heart rate), height of T-waves. Fifteen minutes after subcutaneous bolus in-
jection of isoprenaline 0.5 ml/20 g, ECG was recorded again. T-test was ap-
plied to compare the difference values between pre and post administration
of the drugs between groups.
Effect on Myocardial Water Content and Cardiac Index of Myocar-
dial Ischemic Mice Induced by Isoprenaline Two hours after the injec-
tion of isoprenaline the mice were sacrificed and the hearts were separated.
The hearts were weighed, and the wet hearts were baked for 8 h at 110 °C
and then the dry hearts were weighed. The myocardial water content ((wet
heart weightꢃdry heart weight)/wet heart weight) and cardiac index (wet
heart weight/body weight) were calculated and compared among groups.
1
yellow oil. H-NMR (300 MHz, CDCl3) d: 6.95—6.87 (m, 4H), 4.17—4.13
(m, 1H), 4.02—3.97 (m, 2H), 3.85 (s, 3H), 2.91—2.42 (m, 10H); ESI-MS
m/z: 267.1 [MꢁH]ꢁ. The analytical data of CVT-2513 was identical with the
data reported in ref. 22. CVT-2513 was dissolved in absolute ethanol and to
the solution was added hydrochloric acid. Then the solvent was removed
under reduced pressure and recrystallization with methanol/acetone gave hy-
drochloric acid salt of CVT-2513 as a white solid, mp 172—177 °C.
3-(2-Methoxyphenoxy)-1,2-propylene Glycol (A3) A mixture of A2
(5.6 g, 30.9 mmol) and concentrated sulfuric acid (20 ml) in 100 ml of water
was stirred at 70 °C for 2 h. The reaction mixture was extracted with di-
chloromethane (150 mlꢂ3). The organic layer was dried over Na2SO4 and
concentrated. The crude product was purified by column chromatography
(SiO2, dichloromethane : ethyl acetateꢀ1 : 1) to give A3 (3.64 g, 59.1%) as a
yellow solid, mp 75—78 °C. 1H-NMR (300 MHz, CDCl3) d: 7.01—6.89 (m,
4H), 4.20—4.06 (m, 3H), 3.87 (s, 3H), 3.81—3.77 (m, 2H); ESI-MS m/z:
199.2 [MꢁH]ꢁ, 221.1 [MꢁNa]ꢁ. The analytical data of A3 was identical
with the data reported in ref. 23.
2-Hydroxyl-3-(2-methoxyphenoxy)propionic Acid (CVT-4786) A3
(0.1 g, 0.5 mmol), TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy free radi-
cal, 5.5 mg, 0.035 mmol), 3 ml of acetonitrile, and 1.87 ml of 0.67 M sodium
phosphate buffer (the sodium phosphate buffer consisted of a 1 : 1 mixture
of 0.67 M NaH2PO4 and 0.67 M Na2HPO4, pHꢀ6.7) were added to a flask.
The reaction mixture was heated to 35 °C with stirring and approximately
0.05 ml of sodium hypochlorite solution was added via one addition funnel
followed by 0.1 ml of sodium chlorite solution via another funnel (the solu-
tion of sodium chlorite (NaClO2) was prepared by dissolving 80% NaClO2
(9.14 g, 80.0 mmol) in 40 ml of water and the solution of diluted sodium
Results and Discussion
Chemistry As shown in Chart 1, Ranolazine, CVT
2738, and CVT 2512 were synthesized following a literature
procedure.20) 2,6-Dimethylaniline reacted with chloroacetyl
chloride in the presence of sodium bicarbonate to afford 2-
chloro-N-(2,6-dimethylphenyl) acetamide (A1), which could
be used in the next step without further purification. Treat-
ment of compound A1 with excess piperazine formed com-
hypochlorite (NaOCl) was prepared by diluting a sodium hypochlorite solu- pound CVT-2738. N-Alkylation of CVT-2738 with chlorohy-
tion (5.25%, 1.06 ml, ca. 2.0 mol%) with 19 ml of water). Other 0.4 ml of
sodium chlorite solution and 0.2 ml of sodium hypochlorite solution were
then added simultaneously over 3 h. The resulting mixture was stirred at
35 °C for 2 d. After cooling the reaction mixture, 5 ml of water was added
drin in the presence of sodium bicarbonate gave compound
CVT-2512. Condensation of CVT-2738 with epoxide A2,
which could be easily accessible by treating 2-methoxyphe-
nol with epichlorohydrin in the presence of sodium hydrox-
ide, afforded Ranolazine.
and the solution was neutralized with sodium bicarbonate to pHꢀ8. Then
the solution was washed with 10 ml of ethyl acetate, neutralized with hy-

November 2009
1221
Chart 1. Synthesis of Compounds CVT-2512, CVT-2738, Ranolazine
Table 1. Effect of the Test Drugs on the T-Waves
T-wave
Dose
Pre-
Post-
Groups
(mg/kg)
administration
of isoprenaline
(mV)
administration
of isoprenaline
(mV)
Change rate
Model control
3.1000ꢄ0.2878 5.8625ꢄ0.8700 0.9026ꢄ0.3198
Positive control 6 pills/kg 2.9500ꢄ0.3546 4.0250ꢄ0.6756 0.3595ꢄ0.0993**
Chart 2. Synthesis of Compounds CVT-2513, CVT-4786
Ranolazine
CVT-2512
CVT-2513
CVT-2514
CVT-2738
CVT-4786
100
100
100
100
100
100
3.2500ꢄ0.6279 3.4063ꢄ0.6951 0.0572ꢄ0.1506**
3.2875ꢄ0.6446 4.8250ꢄ0.6296 0.5189ꢄ0.3416*
2.9375ꢄ0.1996
4.000ꢄ0.5182 0.3644ꢄ0.1757**
The syntheses of metabolites CVT-2513 and CVT-4784
are shown in Chart 2. Treatment of epoxide A2 with piper-
azine in absolute ethanol afforded CVT-2513. In this step,
excess piperazine should be added to avoid the double N-
alkylation of the piperazine ring. Hydrolysis of A2 in aque-
ous sulfuric acid solution provided glycol derivative A3. Se-
lective oxidation of the terminal hydroxyl group of A3 with
sodium hypochlorite and TEMPO afforded CVT-4784.26)
As shown in Chart 3, the preparation of metabolite CVT-
2514 started from pyrocatechol. One hydroxyl group was se-
lectively protected with benzyl to afford compound A4. Then
A6 was prepared according to the method for preparation of
Ranolazine. Finally, deprotection of A6 led to CVT-2514.
Anti-myocardial Ischemia Activities It is well-known
that subcutaneous bolus injection of isoprenaline should in-
troduce myocardial ischemia injury to mice so as to be mim-
icking the acute myocardial ischemia injury in human. We
therefore employed isoprenaline-induced myocardial is-
chemia in mice as an animal model to evaluate the anti-
myocardial ischemia activities of the test drugs.27) Ischemic
alterations such as T-waves elevation and R–R interval in-
creasing are known to occur in ECG upon the injection of
isoprenaline. Moreover, elevation of the myocardial water
2.2130ꢄ0.4998 2.6375ꢄ1.6483 0.1880ꢄ0.7004*
2.9000ꢄ0.2619
2.2750ꢄ0.5365
3.000ꢄ0.9442 0.0445ꢄ0.3437**
2.725ꢄ1.9558 0.1812ꢄ0.7686*
∗ pꢅ0.05, ∗∗ pꢅ0.01, vs. model group.
Table 2. Effect of the Test Drugs on the R–R Interval
R–R interval
Dose
Pre-
Post-
Groups
(mg/kg) administration
administration
Change rate
of isoprenaline of isoprenaline
(s) (s)
Model control
0.1343ꢄ0.0200 0.0985ꢄ0.0105 ꢃ0.2554ꢄ0.1143
0.2119ꢄ0.1144**
Positive control 6 pills/kg 0.0868ꢄ0.0053 0.1048ꢄ0.0066
Ranolazine
CVT-2512
CVT-2513
CVT-2514
CVT-2738
CVT-4786
100
100
100
100
100
100
0.1060ꢄ0.0200 0.1023ꢄ0.0088 ꢃ0.0097ꢄ0.1749**
0.1188ꢄ0.0141 0.1050ꢄ0.0101 ꢃ0.1052ꢄ0.1356*
0.1063ꢄ0.0163 0.0963ꢄ0.0110 ꢃ0.0764ꢄ0.1718*
0.1300ꢄ0.0156 0.0923ꢄ0.0111 ꢃ0.2839ꢄ0.0998***
0.1023ꢄ0.0173 0.0915ꢄ0.0089 ꢃ0.0872ꢄ0.1549*
0.1393ꢄ0.0304 0.0935ꢄ0.0083 ꢃ0.3021ꢄ0.1471***
∗ pꢅ0.05, ∗∗ pꢅ0.01, ∗∗∗ pꢅ0.05, vs. model group.
content and cardiac indexes are other alterations of myocar- tablished.
dial ischemia. Therefore, T-waves, R–R interval, myocardial
The results of T-waves test (Table 1) showed that 15 min
water content and cardiac indexes were recorded in this study after the subcutaneous bolus injection of isoprenaline, Ra-
to evaluate anti-myocardial ischemia activities of the test nolazine, CVT-2513 and CVT-2738 could very significantly
drugs.
inhibit the elevation of T-waves (pꢅ0.01), and on the other
Fifteen minutes after the subcutaneous bolus injection of hand, CVT-2512, CVT-2514 and CVT-4786 could also re-
isoprenaline, the height of T-waves, R–R interval, the my- markably inhibit the elevation of T-waves (pꢅ0.05). When
ocardial water content and cardiac index significantly in- comparing CVT-2513 and CVT-2738 with Ranolazine, no re-
creased. Composite Salvia Dropping Pill was used as a posi- markable difference was found between CVT-2738 and Ra-
tive control since it had been widely used in China as a plant nolazine (pꢆ0.05), but very remarkable difference was
medicine with anti-myocardial ischemic activity. In this found between CVT-2513 and Ranolazine (pꢅ0.01). Ac-
study, the injection of Composite Salvia Dropping Pills could cording to the present data, the beneficial effect of CVT-2513
significantly inhibit the alternations induced by isoprenaline on T-waves is much less potent than that of Ranolazine.
(pꢅ0.01), indicating that the models were successfully es-
The results of heart rate test (Table 2) showed that 15 min

1222
Vol. 57, No. 11
Chart 3. Synthesis of Compound CVT-2514
Table 3. Effect of the Test Drugs on Myocardial Water Content and Car- China (No. 111-2-07).
diac Index
References and Notes
Dose
(mg/kg)
Myocardial water
content
Cardiac
index
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Model control
0.7867ꢄ0.0213
0.0045ꢄ0.0004
Positive control 6 pills/kg 0.5689ꢄ0.1095**
0.0020ꢄ0.0003**
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Ranolazine
CVT-2512
CVT-2513
CVT-2514
CVT-2738
CVT-4786
100
100
100
100
100
100
0.7791ꢄ0.0183*** 0.0044ꢄ0.0005***
0.7324ꢄ0.03823** 0.0038ꢄ0.0003**
0.7667ꢄ0.0220*** 0.0039ꢄ0.0003**
0.7503ꢄ0.0142**
0.7707ꢄ0.0157*** 0.0039ꢄ0.0004**
0.7525ꢄ0.0377* 0.0037ꢄ0.0005**
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after the subcutaneous bolus injection of isoprenaline, Ra-
nolazine could inhibit the extension of R–R interval very re-
markably (pꢅ0.01), and CVT-2512, CVT-2513 and CVT-
2738 could also inhibit the extension of R–R interval
(pꢅ0.05), but in a less extent. On the other hand, CVT-2514
and CVT-4786 had no remarkable effect (pꢆ0.05). There-
fore, CVT-2512, CVT-2513 and CVT-2738 had moderate ef-
fect on R–R interval, but less potent than that of Ranolazine.
Table 3 illustrates that CVT-2512 and CVT-2514 could in-
hibit the elevation of myocardial water content very remark-
ably (pꢅ0.01), and CVT-4786 could inhibit the elevation of
myocardial water content remarkably (pꢅ0.05). On the other
hand, Ranolazine, CVT-2738 and CVT-2513, which showed
remarkable beneficial effect on T-waves and R–R interval,
had no significant inhibition of the elevation of myocardial
water content (pꢆ0.05). While Ranolazine showed no re-
markable effect on cardiac index (pꢆ0.05), all of its metabo-
lites had very remarkable effect on cardiac index (pꢅ0.01).
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may have moderate anti-myocardial ischemia activity in my-
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best potency, however, it was still less potent than the mother
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Acknowledgments This program is financially supported by program
for New Century Excellent Talents in University (NCET-05-0495). This
work is partially supported by the “111 Project” from the Ministry of Educa-
tion of China and the State Administration of Foreign Expert Affairs of
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