Asian Journal of Chemistry; Vol. 24, No.

11 (2012), 4953-4960

Synthesis and Characterization and Anticoagulant Properties of Diethyl Citrate

YAN OU1, JIN HAN1, BIN CHEN2, BAO-SONG GUI1,*, FENG-XIN WANG2, YI-MING DING2, JIN TAN2 and JIAN-MING OUYANG2,*
1
Department of Nephrology, the Second Hospital of Xi'an Jiaotong University, Xi'an 710004, P.R. China
2
Institute of Biomineralization and Lithiasis Research, Jinan University, Guangzhou 510632, P.R. China

*Corresponding authors: E-mail: [email protected]; [email protected]

(Received: 30 August 2011; Accepted: 4 June 2012) AJC-11536

Anticoagulation is an essential for the success of hemodialysis. However, sodium citrate (Na3Cit), a major anticoagulant in current clinical
application, causes hypocalcemia due to its strong binding with Ca2+ ion and the slow Ca2+ release rate from the chelate formed. We have
synthesized a novel anticoagulant: diethyl citrate (Et2Cit). The compound was charcterized by its elemental analysis, infrared spectroscopy,
mass spectrometry and nuclear magnetic resonance. The purity was established by thin layer chromatography and acid titration. The mass
fraction of Et2Cit was 99.27 %. The in vitro anticoagulatory effect of Et2Cit was investigated by measuring the whole blood activated
clotting time. Since the introduction of two ethyl groups to citric acid enhanced the steric hindrance for Ca2+ chelation, it led to a reduced
Ca2+ chelation and accelerated Ca2+ release from Et2Cit in comparison to Na3Cit. Acute toxicity test in mice shows that Et2Cit has no
serious risk of acute poisoning. Therefore, Et2Cit can be a potential and an ideal anticoagulant.

Key Words: Anticoagulant, NMR, Mass spectrometry, Diethyl citrate, Acute toxicity test.

INTRODUCTION tration of free Ca2+ ions in plasma is reduced and in vitro antico-
agulation is achieved5,8,9. This solves the problem of secondary
Hemodialysis is routinely used for toxin clearance in hemorrhage and has attracted a lot of attention in recent years5,8-12.
various acute or chronic renal insufficiency. During hemodi- However, this method can cause hypocalcemia, citric acid
alysis, anticoagulant agent is required to prevent clot formation poisoning, etc.10. The limitations of Na3Cit anticoagulation are
in dialyzer and dialysis pipeline and resulting in failure of mainly attributed to its strong chelating capability with Ca2+
hemodialysis. However, there is no perfect and simple antico- ion and the resulting slow Ca2+ dissociation rate, which restricts
agulation method in clinical appplication as yet. its clinical application.
Nowadays, heparin is a widely used anticoagulant1,2, but Based on the above knowledge, we aim to synthesize and
excessive heparin treatment during dialysis inhibits platelet characterize a new anticoagulant with the advantages of Na3Cit
aggregation, which enhances bleeding tendency with a but reducing Ca2+ chelating capability.
secondary hemorrhage incidence as high as 10-30 %. In That is, by substituting two hydroxyl atoms in -COOH in
addition, it causes several other side effects, such as osteoporo- citric acid with oxyethyl groups (-OC2H5), a new anticoagulant,
sis. Therefore, how to choose appropriate anticoagulant(s) diethyl citrate (Et2Cit), was synthesized. Diethyl citrate can
for blood purification therapy is one of the major clinical chelate with Ca2+ ion to reduce the plasma calcium concentration
problems. to promote anticoagulation. At the same time, the alkyl around
The alternative approaches for heparin treatment include the carboxyl oxygen atoms produces steric hindrance, which
low molecular weight heparin dialysis, no heparin dialysis, reduced the chelation of these ester groups with Ca2+, i.e., the
low dose heparin dialysis, regional protamine-heparin neutra- chelate formed by Ca2+ ions and citrate ester should exhibit
lization anticoagulation dialysis, prostacyclin anticoagulation higher Ca2+ dissociation rate than calcium citrate. Thus,
dialysis, recombinant hirudin anticoagulation dialysis and diethyl citrate can potentially be a novel anticoagulant.
sodium citrate (Na3Cit) anticoagulation dialysis.
However, all these methods exhibit limitations3-7. For EXPERIMENTAL
example, in regional Na3Cit anticoagulation dialysis, Na3Cit All reagents used in experiments were in analytical grade,
can bind calcium ion (Ca2+) to form calcium citrate, which is including citric acid, anhydrous ethanol, benzene, petroleum
difficult to dissociate but water-soluble and thus, the concen- ether (b.p. 60-90 ºC), ethyl acetate and sodium carbonate.
4954 Ou et al. Asian J. Chem.

Instruments used in experiments include CHN-O-Rapid experimental values: C 47.82, H 6.80. FT-IR (KBr, νmax, cm-1):
elemental analyzer (Foss-Heraeus Company), Bruker AM 500 3469 (-OH), 2986-2940 (-CH2, -CH3), 1736 (RO-C=O). 1H
NMR instrument (with CDCl3 as solvent and TMS as internal NMR (CDCl3): δ1 (ppm) 1.24-1.33 (6H, -CH3), δ3 2.75-3.00
standard), FT-IR spectrometer (Nicolet-170 SX-type), high (4H, -OCH2-), δ4 4.05-4.35 (4H, -CH2-). 13C NMR (CDCl3): δ
resolution mass spectrometer (MAT95XP, Finnigan, USA) and (ppm) 14.0-14.2 (-CH3), 43.0-43.4 (-CH2 in -C-CH2-COO-),
UV-VIS-NIR UV-visible spectrophotometer (UV-3100 type, 61.1-62.6 (-CH2 in -OCH2CH3), 73.1-77.1 (C-OH), 173.4-
Shimadzu Corporation). 173.9 (-COO in -CH2COOR), 176.5-176.7 (R-COOH). UV-
Synthesis and purification of diethyl citrate VIS (aqueous solution): 220 nm.
Purity analysis by acid titration: Because the product
Synthesis: Diethyl citrate (Et2Cit) was prepared from citric might contain a small amount of monoethyl citrate, mass
acid and anhydrous alcohol. The reaction scheme is shown in fraction of Et2Cit in the final product was determined by acid
Fig. 1. In a 250 mL three-neck bottle installed with a condenser titration. Certain amount of product was accurately weighed
and a constant pressure dropping funnel filled with anhydrous and diluted in 20 mL distilled water in a 100 mL flask. 3-5
magnesium sulfate particles, 21 g (0.1 mol) citric acid mono- drops of phenolphthalein was added and the solution was
hydrate and 0.2 g catalyst p-toluene sulfonic acid (PTS) were titrated with 0.1 mol/L standard KOH solution, whose concen-
dissolved in 42 mL (0.7 mol) anhydrous ethanol under tration was calibrated by potassium hydrogen phthalate. The
magnetic stirring at room temperature. 70 mL benzene was mass fraction of Et2Cit was calculated based on the consum-
added to the bottle as a water carrier and the reaction was ption of KOH solution.
carried out at 90 ºC for 10 h. Due to the formation of ethanol- Anticoagulatory analysis of diethyl citrate: The efficacy
benzene azeotrope, the temperature within bottle is actually of in vitro anticoagulation for Et2Cit was investigated by measu-
maintained at ca. 72 ºC. After the benzene and the remaining ring activated coagulation time (ACT). 12 mg commercial silica
ethanol were removed by reduced-pressure distillation, a was pre-warmed in a 1-cm diameter glass test tube in 37 ºC water
viscous, light-yellow coloured liquid was finally obtained. bath and then 1 mL venous blood from the experimental dogs
1
was added immediately after extraction. After supplementing
2
CH2 COOH
PTS
CH2 COOC2H5 anticoagulant as shown in Table-1, the test tube was quickly
HO C COOH + 2 C2H5OH 90oC
HO C COOH + 2 H2O plugged with a rubber plug and the solution was mixed upside
CH2 COOH CH2 COOC2H5 down for three times. The tube was placed into 37 ºC water
4 5
(Et2Cit)
bath. After 1 min, the blood clotting was monitored every 5 s
by tilting the tube. The time when the first blood clot(s)
Fig. 1. Synthesis of diethyl citrate (Et2Cit)
appeared was recorded as activated coagulation time.
Purification: To remove triethyl citrate, the viscous liquid
TABLE-1
was diluted with 20 mL distilled water, adjusted to pH 8 with WHOLE BLOOD ACTIVATED CLOTTING TIME (ACT) FOR
Na2CO3 and then extracted with 3 mL × 50 mL petroleum CANINE VENOUS BLOOD SAMPLES IN THE PRESENCE OF
ether and 1 mL × 50 mL ethyl acetate, respectively. The carboxyl 10.9 mmol/L VARIOUS ANTICOAGULANTS
groups (-COOH) in the remaining citric acid and some by- Anticoagulant ACT/s
products (monoethyl citrate and diethyl citrate) were neutra- Control group (group I) 115 ± 10
Na3Cit group (group II) > 14400
lized by Na2CO3 to form sodium salt (-COONa) and left in
Diethyl citrate group (group III) 141 ± 15
aqueous solution, while the triethyl citrate (Et3Cit) was Triethyl citrate group (group IV) 122 ± 13
extracted to the organic layer and removed.
To remove citric acid and monoethyl citrate, the above Twenty whole blood samples from selected experimental
system was adjusted to pH 4 with hydrochloric acid, which dogs were divided into four groups, each group using the fol-
allowed citric acid, its monoethyl ester and the catalyst p- lowing anticoagulant:
toluene sulfonic acid to be dissolved in water phase, while Group I was the negative control group: 0.9 % physiolo-
Et2Cit and the sodium salt residues were extracted to organic gical saline.
phase by ethyl acetate. Then ethyl acetate was distilled in Group II was the positive control group: 10.9 mmol/L of
rotary evaporator to obtain the crude Et2Cit. The carboxylic sodium citrate solution.
acid sodium salt (-COONa) in crude product was converted to Group III was experimental group 1:10.9 mmol/L of
carboxylic acid (-COOH) by adjusting to pH 1 with hydro- diethyl citrate solution.
chloric acid and then the solution was extracted with 3 mL × Group IV was experimental group 2:10.9 mmol/L of
50 mL ethyl acetate. Water in the organic extract was removed triethyl citrate solution.
by anhydrous sodium sulfate and the solvent was distilled in a Acute toxicity test in animals: Animal test was processed
rotary evaporator. About ca. 16.2 g Et2Cit was obtained and by an up-and-down procedure. Each time drug was adminis-
the yield was 65.3 %. The product was analyzed by elemental tered to one animal. If the animal survived, higher dose was
analysis, infrared spectroscopy, mass spectrometry, nuclear administered to the second animal; if the first animal died or
magnetic resonance and thin-layer chromatography and the nearly died, lower dose was administered to the second animal.
purity was determined by acid titration. Method of drug delivery: An intraperitoneal injection
Primary spectral data of Et2Cit: Elemental analysis was used for drug delivery. The details sdfd as follow: seven
(C10H16O7, M = 248.2): calculated values: C 48.35, H 6.50; healthy female mice were chosen, each weighing 18-22 g. All
Vol. 24, No. 11 (2012) Synthesis and Characterization and Anticoagulant Properties of Diethyl Citrate 4955

the mice were provided by Medical School Animal Experiment of 99.27 %. When calculating Et2Cit mass fraction, monoethyl
Center, Xi'an Jiaotong University. citrate was regarded as the sole remainder, i.e., 1 mol Et2cit
The mice were randomly divided into 7 groups and each consumed 1 mol KOH, whereas 1 mol monoethyl citrate
group contained one mouse. The control group only received consumed 2 mol KOH.
physiological saline. The other six groups received various Thin layer chromatography (TLC): To further confirm
amounts of Et2Cit as indicated in Table-2. After Et2Cit injection, the purity of Et2Cit product. TLC analysis was performed using
each mouse was carefully examined and recorded two times silica gel G plates, with the developing agent of butyl alcohol,
for the first day and then one time per day. This observation acetic acid and water (4:1:5, v/v). After development, silica
continued to the 14th day. The following content was moni- gel G plates were removed and left in air for 2 h to volatilize
tored: the time that toxic effects appeared and disappeared for acetic acid and then stained by bromocresol green. The product
each animal, the breathing, autonomic activity and behaviour showed accumulated spots, with a RT of 3.6, which was signifi-
of the central nervous system, etc. Mice were weighed 1 week cantly higher than that of citric acid (3.0), indicating high
before and after drug administration. All animals were analyzed purity of Et2Cit product. It is worth noting that the acetic acid
by autopsy and the abnormal organ was examined histo-pa- on silica gel G plates needs to be completely volatilized before
thology. staining; otherwise, bromocresol green would react with acetic
acid and make the whole plate yellow, which seriously inter-
RESULTS AND DISCUSSION fered with the observation.
Common physical and chemical properties of Et2Cit: Spectral analyses of diethyl citrate
The final product of diethyl citrate (Et2Cit) is a colourless and FT-IR spectrum of diethyl citrate: Because Et2Cit was
transparent liquid with ester scent. Its boiling point is 355 ºC, a liquid sample, its FT-IR spectrum was recorded by liquid
relative density (25 ºC) is 1.28 g/cm3 and refractive index membrane method. Two thin slices of KBr were prepared. A
(25 ºC) is 1.477. Diethyl citrate can dissolve in water, alcohol, small amount of Et2Cit was evenly spread on one KBr slice
ether and ethyl acetate. The solubility in water is 15.7 g/100 and then covered by the other. FT-IR spectrum was recorded
mL (25 ºC). Et2Cit is light-, heat- and oxygen-stable. The pH using the conventional approach and the results were shown
value of 0.1 mol/L Et2Cit aqueous solution at 25 ºC is 2.22, in Fig. 2.
which is higher than 0.1 mol/L citric acid (pH 2.04) but lower
than 0.1 mol/L acetic acid (pH 2.84).
100
Purity analysis of diethyl citrate
Purity analysis by acid titration: After synthesis reaction, 80
Transmittance / %

the Et2Cit contained certain by-products, including monoethyl

602
citrate, triethyl citrate and the unreacted citric acid. During 60

858
the purification process, triethyl citrate was completely

794
removed by extraction with petroleum ether and ethyl acetate 40 1600
owing to its low solubility in water (S = 6.5 g). Citric acid
2940

1100
could barely exist in the final product because of its high
1024
20
1374
3469

2986

solubility (S = 60 g). The solubility of monoethyl citrate in

1736

1321
1206

water (S = 31 g) was higher than that of diethyl citrate (S =

0
15.7 g), so did the acidity and thus, most monoethyl citrate
4000 3500 3000 2500 2000 1500 1000 500
was removed by the pH 4 extraction step. In the final product, -1
the major possible impurity is a small amount of monoethyl Wavenumbers / cm
citrate. The purity of Et2Cit was determined by acid titration. Fig. 2. FT-IR spectrum of diethyl citrate
Three Et2Cit samples, weighing 0.2368, 0.2236 and
0.2535 g, was titrated with 0.09231 mol/L standard KOH The stretching vibration absorption peak for carboxyl
solution and the consumed KOH volume was 10.53, 9.78 and (-COOH) of Et2Cit appeared at 3469 cm-1, which exhibited
11.10 mL, respectively. The mass fractions of Et2Cit were ca. 150 cm-1 redshift compared with free -COOH, indicating
98.19, 99.86 and 99.70 %, respectively, with an average value the existence of hydrogen bond in carboxyl group.
TABLE-2
RESULTS OF Et2Cit TOXICITY TEST (n = 1)
Time of drug Et2Cit Et2cit dose/mouse
Group Mouse reaction
administration dose (mg) body weight (g/kg)
1 (control group) 2011.4.23 0 0 Normal activities, no death
2 2011.4.23 94 4.9 No significant reduction of activity; no appearance of unsteady gait or
slowness; no slack or shortness of breath and jump; no death
3 2011.4.24 129 5.9 Ibid
4 2011.4.25 161 6.9 Ibid
5 2011.4.26 190 7.9 Activity reduction, accompanied by unsteady gait, slowness, sluggish,
shortness of breath and jump; no death
6 2011.4.27 208 8.9 Ibid
7 2011.4.28 258 9.9 Ibid
4956 Ou et al. Asian J. Chem.

There were a pair of vibration absorptions at 1374 and 300

1321 cm-1, which result from the coupling of ν(OH) and (d)

2.85
2.91
250
ν(C=O) in interior surface. However, these two absorption
200
peaks were weak because two of the three -COOH groups in
citric acid turned into ester groups (-COOC2H5) and only one 150

2.97

2.80
carboxyl left. The absorption peaks of methyl and methylene
100

2.92
appeared at 2986 and 2940 cm-1.

2.82
2.88
2.88

2.81
2.98
The characteristic absorption peak of carbonyl [ν(C=O)]

2.77
2.76
50
in ester appeared at 1736 cm-1, which was consistent with the
0
published value for free fatty ester13,14. In addition, absorption
3.00 2.95 2.90 2.85 2.80 2.75
peaks of esters for asymmetric [(νas(C-O-C)] and symmetric
ppm
stretching vibration [νs(C-O-C)] were observed at 1206 and
1100 cm-1, respectively and the intensity of as C-O-C absor-
ption peak was stronger than that of νs(C-O-C). The simulta-

4.15
4.18
neous appearance of as C-O-C and νs(C-O-C) absorption peaks 300 (e)
is a significant characteristic for esters in comparison to other
carbonyl compounds (such as carboxylic acid).

4.13
1
H NMR spectra of Et2Cit: The 1H NMR spectra of Et2Cit 200

were shown in Fig. 3.

4.13
4.20
4.26
4.29
100

4.31

4.24

4.11
4.08
600
(a)
0
500
4.35 4.30 4.25 4.20 4.15 4.10 4.05

400
a
b ppm
de
300
cd 50
200
(f) 45

7.256

7.254
7.26

bc 40 30
100 ef
15

0 30
0
7 6 5 4 3 2 1 7.26 7.25
ppm 20
6.28

10
600
(b)
1.26

500 0
7.5 7.2 6.9 6.6 6.3 6.0 5.7
400
1.28

ppm
1.24

300 Fig. 3. 1
H NMR spectra of diethyl citrate. (a) Whole spectrum; (b) δ =
1.24-1.33; (c) δ = 2.02-2.12; (d) δ = 2.75-3.00; (e) δ = 4.05-4.35;
1.30

200 (f) δ = 5.70-7.50

1.33

100
A group of peaks appeared at δ = 1.24-1.33 ppm (Fig. 3b,
0
the relative integral peak area is 44.42), indicating the presence
1.35 1.30 1.25 1.20
ppm of hydrogen in methyl (-CH3). This methyl is connected with
rich charged atoms or groups since the peak position is slightly
100 higher than that of -CH3 in a straight-chain alkane (ca. 0.9
(c) ppm). The molecular structure of Et2Cit (Fig. 1) revealed that
80 the rich charged group was -OCH2-.
A series of peaks (Fig. 3e, with the relative peak area
2.05

60
of 29.97) appeared at δ = 4.05-4.35 ppm, indicating the
existence of hydrogen in methylene (-CH2-). These hydrogen
40
2.11

atoms were potentially located on the carbons that were

connected with the charge-rich atoms or groups. It is concluded
20
that it was a -COOCH2CH3 group because typical -OCH2CH3
0
group has a triplet peak at ca. 1.25 ppm with the integral
2.12 2.10 2.08 2.06 2.04 number of 3 and a quartet peak at ca. 4.0 ppm with the integral
ppm number of 2.
Vol. 24, No. 11 (2012) Synthesis and Characterization and Anticoagulant Properties of Diethyl Citrate 4957

According to the coupling and splitting as shown in Fig. 100

3e, the peak series include two groups of quartet peaks: one is (a)
larger (δ = 4.22-4.34 ppm) and the other is smaller (δ = 4.06- 80
b
4.22 ppm). Since the peaks at δ = 1.24-1.33 ppm are not triplet d c
(Fig. 3b), there may be two types of -OCH2CH3 groups in 60
e
different chemical environments: 1,3-diethyl citrate and 1,5-
40
diethyl citrate. However, further HLPC-MS analysis is required f
to depict the ratio of these two compounds. 20
A series of peaks (Fig. 3d, with the relative peak area of
180 160 140 120 100 80 60 40 20
30.61) at δ = 2.75-3.00 ppm was attributed to the hydrogen ppm
atoms of two methylene groups (on C2 and C4, Fig. 1) in citric
acid. It included multiple peaks with irregular shapes, indicating 100
the presence of hydrogen atoms of methylene groups in various (b) 14.0
chemical environments. 80
When comparing the relative peak areas and choosing
60
the peak number at δ = 2.75-3.00 ppm (relative peak area
30.61) as 4, the peak number at δ = 1.24-1.33 (area 44.42) is 4 40 14.1
× 44.42/30.61 = 5.80 ≈ 6 and the peak number at δ = 4.05-
4.35 (area 29.97) is 4 × 29.97/30.61 = 3.92 ≈ 4. Therefore, 20 14.2
this product is Et2Cit.
0
In the 1H NMR spectra of Et2Cit, no normal signal for 14.3 14.2 14.1 14.0 13.9 13.8 13.7
hydrogen atoms of -COOH or -OH group was detected, except ppm
a weak signal at 7.256 ppm (Fig. 3f). This is because the
carboxyl hydrogen atom is active and easily dissociated in 100
polar solvent and they are easily deuterated in CDCl3 solvent. (c)
43.0
80
Relative area of this peak is ca. 0.8, only one tenth of a normal
proton signal, indicating that most hydrogen atoms in -COOH 60
and -OH groups were deuterated during spectrum measurement.
In addition, a proton signal with peak area of 1.44 40
appeared at δ = 2.02-2.12 ppm. It is not a sharp and narrow 43.4 43.1
20 43.3
peak like other proton spectra, but a wide and short "slope"
(Fig. 3c), which should be due to the presence of small amount
0
of water molecule. 43.5 43.4 43.3 43.2 43.1 43.0 42.9 42.8
It is worth noting that, despite Et2Cit is a mixture of two ppm
isomers, they share similar physical and chemical properties
and the anticoagulatory potential. Therefore, there is no need 100
to further separate them. (d) 61.3
13
C NMR spectra of Et2Cit: The 13C NMR spectra of 80

Et2Cit were shown in Fig. 4. (1) A group of peaks appearing at

60
δ = 14.0, 14.1 and 14.2 ppm (Fig. 4b) indicate the presence of
carbon atom in methyl (-CH3). (2) There are two groups of 40
peaks in the range of δ (ppm) = 42-44 (Fig. 4c). A series of 61.2
62.6
peaks appeared at δ = 43.0, 43.1, 43.3 and 43.4), indicating 20 61.1
62.4
the existence of carbon of -CH2- connecting to carbonyl
(-CO) in -C-CH2-CO-. The strongest peak at δ (ppm) = 43.0 0
62.8 62.4 62.0 61.6 61.2 60.8 60.4
was attributed to the carbon of -CH2- in two symmetric ppm
-C-CH2-COOR in 1,5-diethyl citrate (Fig. 5a). For the two
-CH2- groups connecting to carbonyl in the 1,3-diethyl citrate,
100
one was ester carbonyl (-C-CH2-COOR), the other was the (e)
carboxyl carbonyl (-C-CH2-COOH), therefore, the δ (ppm)
77.1

80
appears at 43.3 and 43.4, respectively.
The multiple peaks appearing in δ (ppm) = 61.1, 61.2, 60
73.1

61.3, 62.4 and 62.6 were attributed to the carbon in methylene

(-CH2-) in -OCH2CH3 (Fig. 4d). The strongest peak at δ (ppm) 40

= 61.3 was the carbon in -CH2- in the two symmetry -OCH2CH3

73.2

20
in 1,5-diethyl citrate (Fig. 5a). In the 1,3-diethyl citrate, the
chemical environment of the two -OCH2CH3 is not exactly 0
the same, therefore, one appears in δ (ppm) = 61.1 and 61.2 78 77 76 75 74 73 72
ppm
and the other appears in δ (ppm) = 62.4 and 62.6.
4958 Ou et al. Asian J. Chem.

are two kinds of ester with different chemical environments,

60
(f) that is, 1,3-diethyl citrate and 1,5-diethyl citrate. However,

170.1
the proportion between them is still difficult to be judged.
Mass spectra of diethyl citrate: The mass spectra of
40

173.4
Et2Cit were shown in Fig. 6, according to which the molecular
176.7

173.5

169.9
fragments were shown in Fig. 7. In Fig. 7a, the M/z peaks of
173.7
173.9
203, 185, 156, 114 and 70 represented the molecular fragments
176.5

20
of 1,3-diethyl citrate. In Fig. 7b, the M/z peaks of 203, 158,
141, 116 and 74 represented the molecular fragments of 1,5-
diethyl citrate. Because the molecular fragment of MW
0 (molecular weight) 277 did not appear in the spectrum, the
177 176 175 174 173 172 171 170 169 sample does not contain triethyl citrate. Because the molecular
ppm
13
fragment of MW 175 also did not appear in the spectrum, the
Fig. 4. C NMR spectra of diethyl citrate. (a) Whole spectrum; (b) δ = 13-
sample does not contain monoethyl citrate.
15; (c) δ = 42-44; (d) δ = 60-63; (e) δ = 72-78; (f) δ = 169-177 ppm

43.0 173.6 61.3 14.1

CH2 COOCH2CH3
77.1 176.6
HO C COOH
CH2 COOCH2CH3
43.0 173.6 61.3 14.1
(a)

43.4 173.8
CH2 COOH
73.2 170.0 61.1 14.1
HO C COOCH2CH3
CH2 COOCH2CH3
43.4 170.0 61.5 14.1
(b)
Fig. 5. Two isomers of diethyl citrate. (a) 1,5-diethyl citrate; (b) 1,3-diethyl
citrate. The figures are the d (ppm) values of corresponding C atoms

From this discussion, it is suggested that there is

-CH2COOCH2CH3 in the molecule being detected. Because
there are two groups of peaks, one group is relatively strong
and the other group is relatively weak, it indicates the existence Fig. 6. Mass spectrum of diethyl citrate
of two different chemical environments of -CH2COOCH2CH3
group. The strong peaks are attributed to the two symmetrical Based on the results of elemental analysis, mass spectro-
-CH2COOCH2CH3 groups in 1,5-diethyl citrate and the weaker metry, 1H NMR, FT-IR and chromatography, it can be concluded
peaks are attributed to two asymmetric -CH2COOCH2CH3 that the product was Et2Cit as expected.
groups in 1,3-diethyl citrate. Anticoagulation analysis of Et2Cit: Reduction of plasma
As -OH group is an electron-withdrawing group, the Ca2+ concentration (generally to 0.4 mmol/L) is essential for
carbon peak in HO-C(s) (s, showing quaternary carbon atom) successful anticoagulation. However, hypocalcemia occurs
shifts to low field. Because of the existence of two different when serum Ca2+ concentration is below 0.8 mmol/L.
chemical environments of OH-C(s) groups, there have been When using Na3Cit as anticoagulant, the strong chelating
two groups of peaks in Fig. 4e. The peaks at δ (ppm) = 73.1 capability of Na3Cit with Ca2+ leads to dramatic reduction of
and 73.2 are attributed to the carbon in HO-C of 1,3-diethyl plasma Ca2+ concentration and causes hypocalcemia, which
citrate; and peaks at δ (ppm) = 77.1 is attributed to the carbon requires timely calcium supplement. In addition, after dialysis,
in HO-C of 1,5-diethyl citrate. the slowly release of Ca2+ ion from calcium citrate and the
The peaks at chemical shift δ (ppm) in the range of 169- supplemented calcium results in hypercalcemia in patients.
177 are typical carbonyl carbon atoms (-CO-) peaks (Fig. 4f). After substituting two carboxyl groups (-COOH) in citric
The two peaks at δ (ppm) = 176.5 and 176.7 are the carbon acid with ester groups (-COOC2H5), the new anticoagulant
peaks of carboxyl (R-COOH, R is a saturated group). The four Et2Cit displays the following advantages: Et2Cit still has oxygen
peaks appeared at δ (ppm) = 173.4, 173.5, 173.7 and 173.9 atoms (in ester and hydroxyl, as shown in Fig. 1) that can
(average 173.6) are attributed to the carbons of esters in the chelate with Ca2+ ion, leading to reduced plasma calcium concen-
two symmetrical -CH2COOR. While the peaks appeared at δ tration and achieving anticoagulation. During to the introduction
(ppm) = 169.9 and 170.1 are attributed to the carbons of esters of two ethyl groups in citric acid, steric hindrance augmented
in the two asymmetrical -CH2COOR in 1,3-diethyl citrate. for Ca2+ chelation, which leads to reduced chelating ability of
In summary, 13C NMR spectra also showed that the syn- Et2Cit in comparison to Na3Cit. However, it is easier for Ca2+
thesized product is the target product Et2Cit. Furthermore, there ion to release from the chelate formed by Et2Cit and Ca2+ than
Vol. 24, No. 11 (2012) Synthesis and Characterization and Anticoagulant Properties of Diethyl Citrate 4959

(a) (b)
Fig. 7. Schemes of fragment peaks for two diethyl citrate isomers. (a) 1,3-diethyl citrate; (b) 1,5-diethyl citrate

from calcium citrate. This helps to overcome the Na3Cit-caused For experimental mice, no significant reduction of activities
hypocalcemia and other problems. The appropriate ethyl size or death occurred when Et2Cit was less than 6.9 g/kg. When
add little molecular weight after substitution of two H in citric the dose of Et2Cit increased 9.9 g/kg, activity reduction, ataxia,
acid and Et2Cit can easily go through the dialysis membrane. slowness and sluggish and other adverse effects immediately
Therefore, Et2Cit is a better anticoagulant occurred, but still no animal died. These side effects can gradually
Table-1 showed the whole blood activated clotting time disappear in 1 h.
(ACT) for canine venous blood samples in the presence of There were no significant difference of female mice
various anticoagulants. Twenty canine blood samples were weight between experimental animals and control animals.
divided into four groups, each containing five. The ACT for No significant lesions of the body tissues or organs were
Et2Cit group was 141 ± 15 s, which was significantly higher observed by macroscopic analysis at the end of experiment.
than the control group (115 ± 10 s) and triethyl citrate group According to British Toxicology Society Working Party
(122 ± 13 s), but markedly lower than the Na3Cit group, in on Toxicity (1984)15, when LD50 (Lethal Dose, 50 %) value
which blood did not coagulate within 4 h (14400 s). These > 2.0 g/kg, it indicate this drug has no serious risk of acute
data suggest that the chelating capability of Et2Cit is weaker poisoning. Therefore, Et2Cit has no serious risk of acute
than Na3Cit, but stronger than triethyl citrate. Therefore, Et2Cit poisoning according to the data in Table-1.
may potentially be an ideal anticoagulant due to the efficient
Conclusion
anticoagulation and rapid calcium dissociation.
The experimental data was analyzed by inter-group t test By substituting two hydroxyl atoms in -COOH in citric
between group I and group III (P1 = 0.001), group I and group acid with oxyethyl groups (-OC2H5), a new anticoagulant,
IV (P2 = 0.115), group II and group III (P3 = 0.001), group III diethyl citrate (Et2Cit), was synthesized and charcterized by
and group IV (P4 = 0.001), respectively. its elemental analysis, infrared (IR) spectroscopy, mass spectro-
Acute toxicity test in mice: Acute toxicity test in mice metry (MS) and nuclear magnetic resonance (NMR). Its purity
was carried out to observe the safety of Et2Cit. The experi- was established by thin layer chromatography (TLC) and acid
mental results for each group were shown in Table-2. No signi- titration. Since the introduction of two ethyl groups to citric
ficant adverse effect was observed in control mice, which had acid enhanced the steric hindrance for Ca2+ chelation, this led
normal activity and no mortality. to a reduced Ca2+ chelation and accelerated Ca2+ release from
4960 Ou et al. Asian J. Chem.

Et2Cit in comparison to Na3Cit, which overcomes Na3Cit- 6. B.-S. Gui, G.-B. Zhao, Y. Zan, X.-Q. Ma, L. Zhao and X.-H. Wei, Chin.
caused hypocalcemia and other problems. Acute toxicity test J. Pract. Internal Med. (Chin)., 25, 328 (2005).
7. L.-C. Chang, H.-F. Lee, M.-J. Chung and V.-C. Yang, Bioconj. Chem.,
in mice showed Et2cit has no serious risk of acute poisoning. 16, 147 (2005).
Therefore, diethyl citrate can be a potential and an ideal 8. M. Antonic, J. Gubenšek, J. Buturovic-Ponikvar and R. Ponikvar, Ther.
anticoagulant. Apher. Dial., 13, 322 (2009).
9. H.M. Oudemans-van Straaten, R.J. Bosman, M. Koopmans, P.H. van der
ACKNOWLEDGEMENTS Voort, J.P. Wester, J.I. van der Spoel, L.M. Dijksman and D.F. Zandstra,
Crit. Care Med., 37, 545 (2009).
This work was supported by the National Natural Science 10. J. Kozik-Jaromin, V. Nier, U. Heemann, B. Kreymann and J. Bohler,
Nephrol. Dial. Transplant., 24, 2244 (2009).
Foundation of China (30871164) and the Scientific Research 11. J. Gubensek, J. Buturovic-Ponikvar, N. Skofic and R. Ponikvar, Ther.
Foundation for the Returned Overseas Chinese Scholars, State Apher. Dial., 13, 306 (2009).
Education of China. 12. C. Morath, N. Miftari, R. Dikow, C. Hainer, M. Zeier, S. Morgera,
M.A. Weigand and V. Schwenger, Nephrol. Dial. Transplant., 23, 421
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