99mTc labeled myocardial imaging agents of long-chain fatty acid: An intermediate synthesis process research

Article abstract of DOI:10.14233/ajchem.2013.14866

Myocardial fatty acid metabolic imaging agent can be used for studying the myocardial ischemia positioning, measurements reflect changes in myocardial metabolic function and the detection of myocardial cell survival, provide a reliable basis for the clinical diagnosis of cardiac disease. In this paper, we study the synthesis of fatty brominated products BSE, which is the intermediates of novel ^99mTclabeled fatty acid metabolic imaging agent. The final yield of the product was 38 %, NMR spectroscopy confirmed very consistent the hydrogen peak displacement and integral spectrum area with the goal of formula. This synthetic route is convenient, simple, economic and has a potential value of industrial applications.

Full text of DOI:10.14233/ajchem.2013.14866

Asian Journal of Chemistry; Vol. 25, No. 15 (2013), 8621-8624
http://dx.doi.org/10.14233/ajchem.2013.14866
99mTc Labeled Myocardial Imaging Agents of Long-chain
Fatty Acid: An Intermediate Synthesis Process Research
1
2
QING HUO , JINGQUAN XUE2,* and YU LIU
1Biochemical Engineering College of Beijing Union University, Beijing 100023, P.R. China
2Key Laboratory of NuclearAnalytical Techniques, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, P.R. China
*Corresponding author: Tel: +86 10 88236900; E-mail: jqxue@ihep.ac.cn
(Received: 23 November 2012;
Accepted: 26 August 2013)
AJC-14011
Myocardial fatty acid metabolic imaging agent can be used for studying the myocardial ischemia positioning, measurements reflect
changes in myocardial metabolic function and the detection of myocardial cell survival, provide a reliable basis for the clinical diagnosis
of cardiac disease. In this paper, we study the synthesis of fatty brominated products BSE, which is the intermediates of novel 99mTc-
labeled fatty acid metabolic imaging agent. The final yield of the product was 38 %, NMR spectroscopy confirmed very consistent the
hydrogen peak displacement and integral spectrum area with the goal of formula. This synthetic route is convenient, simple, economic
and has a potential value of industrial applications.
Key Words: 99mTc, Myocardial metabolic agent, Imaging, Radioactivity.
myocardial cell survival, provide reliable clinical diagnosis
of cardiac disease4.
INTRODUCTION
99mTc-labeled imaging agent used to diagnose diseases at
home and abroad, many studies have been carried out. Unlike
the B ultrasound, CT, MRI, it combines form and function as
a whole, it can not only show the organ morphology but also
understand organ function.Although in recent years there have
been many competing diagnostic methods, however, the func-
tion of the nuclear medicine imaging still a popular inspection
method. In 1999,Yamamura1 synthesized 99mTc-MAMA-HA,
this is the first confirmed in vivo fatty acid β-oxidation of the
derivative, which is the development potential imaging agent.
On this basis, Magata2 and Wisneski3 increase the chain length
of fatty acids, synthesis of 99mTc-MAMA-HDA and 99mTc-
MAMA-DA, biological experiment results show 99mTc-
MAMA-HAD has higher heart/blood ratio. Since then, a lot
of exploration and improvement to imaging agent 99mTc
labeled myocardial fatty acid metabolism are made.
99mTc-labeled fatty acids (Fig. 1), after the modification
and exploration of different points, different core and different
chain length, there are not satisfied with the results, the study
stopped at the biodistribution, did not use in the imaging
experiments. The main reason is its biological activity by the
labeled fatty acids change larger.
How to reduce the labeled fatty acids bioactive change,
improve the absorption and retention time of the imaging agent
Fatty acids is the most important material for the normal
myocardial cells. The concentration of the fatty acids in myocar-
dial cells is 20 times higher than in the plasma concentration.
In the fasting, 80 % the energy requirement in myocardial is
from the free fatty acids.When myocardial ischemia, myocardial
fatty acid metabolism and clear have downward trend. There-
fore, myocardial fatty acid metabolism imaging agent can be
used on myocardial ischemia positioning. The measurement
reflect changes in myocardial metabolic function, detect
Fig. 1. Several 99mTc labeled fatty acid metabolic imaging agent

8622 Huo et al.
Asian J. Chem.
in the myocardium, improving the ratio of heart/blood, heart/
lung and heart/liver is the key. The metallic core and the size
of the spatial structure of the ligand compound, as well as the
fatty acid chain length and structure directly affect the biological
activity of the fatty acid. Small space structure of the metal core
and the ligand compound, as well as modification of the struc-
ture of the fatty acid carbon chain is the development direction
of 99mTc-labeled fatty acid metabolic imaging agent.
times repeatedly with dichloromethane, the organic phase was
separated and placed in a beaker, add the excess of anhydrous
Na2SO4 dried; filtered off Na2SO4, the solvent was evaporated
to dryness under reduced pressure to give the mercaptan
product.
O
O
1.Thiourea
2.NaOH
[
]
[
]
3
3
HO
Br
HO
SH
(1)
(2)
Table-1 shows that the yield of the product with the
addition of an alkali solution and the reaction time has a rela-
tionship: NaOH > Na2CO3 > NaHCO3, the stronger the alkaline,
the higher the yield. Table-2 shows that: 6 h is the optimum
reaction time, the reaction was not complete in short reaction
time, alkali in the solution will continue to react with the product
in long reaction time, therefore, the strong base and the reaction
time was 6 h are the best experimental conditions.
EXPERIMENTAL
Thiourea, sodium hydroxide, potassium hydroxide,
dichloromethane, chloroform, anhydrous ethanol, anhydrous
sodium sulfate, concentrated sulfuric acid, bromo undecanol,
methanol, ethyl acetate, carbon tetrabromide (CP, Beijing
Chemical Plant), triphenylphosphonium (AR, Beijing Chemical
Reagent Company), bromovaleric acid (CP, Tianjin Tiantai
Fine Chemicals Co. Ltd.), n-hexane (AR, Beijing Chemical
Plant), deionized water (Institute of High Energy Physics,
Chinese Academy of Sciences).
TABLE-1
RELATIONSHIP BETWEEN THE YIELD OF THE
PRODUCT AND THE BASICITY
Electronic balance (MP120-1 capacity 120 g/0.001 g);
magnetic stirrer (GSP-79-03, Tai xian Jiangsu province Elec-
tronic Instrument factory; rotary evaporator (R201, Shanghai
Shen Shengsheng Biotechnology Co. Ltd.); heater (R201,
Shanghai Shen Shengsheng Biotechnology Co. Ltd.); silica
gel plates (GF254, Qingdao Marine Chemical Plant) .
Synthesis
Alkali
NaHCO3
Na2CO3
NaOH
Reaction time (h)
Quantity (g)
4.32
5.04
Yield (%)
6
6
6
58
68
78
5.81
TABLE-2
RELATIONSHIP BETWEEN THE YIELD OF
THE PRODUCT AND REACTION TIME
O
O
1.Thiourea
2.NaOH
Br(CH2)11OH
EtOH,KOH
Alkali
NaOH
NaOH
NaOH
Reaction time (h)
Quantity (g)
5.35
5.81
Yield (%)
[
]
[
3
]
3
HO
SH
HO
HO
Br
4
6
8
72
78
74
(1)
(2)
5.52
O
O
EtOH/H+
[
]
[
]
9
3
[
]
S
OH
[
]
9
3
EtO
S
OH
Synthesis of fatty acids: Weigh 2.9 g mercaptan product
of the previous step 5-sulfanyl valeric acid (2) with 2.5 g KOH
solid immiscible in 85 mL of anhydrous ethanol was added
5.4 g bromo undecanol, heat reflux at 60, 100, 135 ºC and
stirred 12 h.After the reaction, mixture was treated with 2.5 M
of H2SO4 acidified.After acidification, the solvent was evapo-
rated to dryness under reduced pressure. 40 mL of distilled
water was added to dissolve, repeatedly washed several times
with chloroform and the organic phase (lower layer) was sepa-
rated using a separatory funnel; separated organic phase was
placed in a beaker and dried over anhydrous Na2SO4, filtered
off Na2SO4, the solvent was evaporated under reduced pressure.
Add 60 mL of methanol, placed at -15 ºC, recrystallization,
the product is a white solid.
(3)
(4)
CBr4 PPh3
O
[
]
[
]
9
3
EtO
S
Br
(5)
5-Bromovaleric acid (1); 5-Sulfanylvaleric Acid (2); 17-Hydroxy-6-
thia-heptadecanoic Acid (3); Ethyl 17- Hydroxy-6-thia-heptadecanoate
(4); Ethyl 17-Bromo-6-thia-heptadecanoate (5)
Fig. 2. Synthetic route
RESULTS AND DISCUSSION
Preparation of thiols: The purity of 5-bromo valerate
has great impact on the follow-up reaction. Adding an appro-
priate amount of n-hexane and recrystallized, 5-bromo-
pentanoic acid change from a pale yellow solid powder into a
white solid powder, so we can get pure product.
O
O
Br(CH2)11OH
EtOH,KOH
[
]
[
]
3
[
]
9
3
HO
SH
10 g of 5-bromo valeric acid with 6.3 g thiourea immiscible
in 100 mL of ethanol, heated to reflux and stirred at 100 ºC
for 16 h until the mixture was cooled to room temperature, the
solvent was evaporated to dryness under reduced pressure;
adding an alkali 7.5 M NaOH (Na2CO3, NaHCO3) and then
the heating was continued under reflux (100 ºC) for 6 h; after
the mixture was cooled to room temperature.
HO
S
OH
(3)
(2)
Table-3 shows that the yield and the reaction temperature
has a relationship. When heated at temperature 100 ºC, yield
relatively highest, the temperature is controlled in ethanol
reflux rate of 60 drops/min. Reflux rate is too fast (the tempe-
rature is too high) or too low (too low temperature) will affect
the progress of the reaction, reducing the yield, temperature
of 100 ºC, the yield is relatively satisfactory.
Acidified with 2.5 M H2SO4 and adding large quantities
of methylene chloride for extraction, the organic phase (lower
layer) was separated using a separatory funnel, washed several

Vol. 25, No. 15 (2013)
99mTc Labeled Myocardial Imaging Agents of Long-chain Fatty Acid 8623
7500
7000
6500
6000
5500
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
-500
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm)
Fig. 3. Fatty acid bromide spectrum
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5 -1.0
TABLE-3
RELATIONSHIP BETWEEN THE YIELD OF THE
PRODUCT AND TEMPERATURE
and molecular corresponding relationship has been marked
with italics and boldface. The results are as follows: 1H NMR
(CDCl3): δ = 1.24-1.32 (m, 17H, 7 × CH2, COOCH2CH3), 1.53-
1.77 (m, 8H, CH2CH2OH, CH2CH2SCH2CH2, CH2CH2
COOCH2CH3), 2.32 (t, J = 7.3, 2H, CH2COOCH2CH3), 2.48-
2.53 (m, 4H, CH2SCH2), 3.65 (t, J = 6.2, 2H, CH2OH), 4.12
(q, J = 7.1, 2H, COOC H2CH3).
Temperature
Reaction time (h)
Quantity (g)
Yield (%)
60
12
12
12
0.9
1.7
1.1
31
59
38
100
135
From Fig. 3 we can see, this product is indeed the desired
product of a fatty acid bromide (yield ~ 59 %).
Esterification reaction of fatty acids: Weigh 2 g fatty
acids product of the previous step 17-hydroxy-6-thia-hepta-
decanoic acid (3) and 2 mL of concentrated H2SO4 placed in
150 mL of absolute ethanol, heated stirred at reflux 100 ºC,
after the reaction, heating was stopped and stirring was conti-
nued overnight. The solvent was evaporated to dryness under
reduced pressure; which was dissolved in 100 mL of ethyl
acetate, washed with 100 mL of deionized water once, the
organic phase (upper layer) was separated using a separatory
funnel and the separated organic phase was placed in a beaker,
dried with anhydrous Na2SO4, filtered off Na2SO4, solvent was
evaporated under reduced pressure and purified by flash column
chromatography (3:1 hexane/ethyl acetate) obtain a fatty acid
esterified product as a white solid.
Synthesis of the imaging agents of the long chain fatty
acid intermediates-BSE: Weigh 0.332 g product of the previ-
ous step Ethyl 17-Hydroxy-6-thia-heptadecanoate (4), add CBr
40.996 g, dichloromethane reagent 50 mL, 0 ºC (ice-water)
nitrogen stirring, add triphenyl phosphorus (PPh3) 0.788 g and
allowed to react overnight. After drying under reduced pressure
and purified by flash column chromatography (10:1 hexane/ethyl
acetate), we can get the crude product as a pale yellow oil.
O
O
CBr4 PPh3
[
]
[
]
[
]
9
3
[
]
9
3
EtO
S
OH
EtO
S
Br
(5)
(4)
1H NMR chemical shifts and integrated area of analysis,
the hydrogen atoms in the chemical shifts and integral area
and molecular corresponding relationship has been marked
with italics and boldface. The results are as follows: 1H NMR
(CDCl3): δ = 1.20-1.35 (m, 17H, 7 × CH2, COOCH2CH3), 1.55-
1.75 (m, 8H, CH2CH2Br, CH2CH2SCH2CH2, CH2CH2COOCH2
O
O
EtOH/H+
[
[
]
3
]
[
]
9
[
]
3
9
EtO
S
OH
HO
S
OH
(3)
(4)
1H NMR chemical shifts and integrated area of analysis,
the hydrogen atoms in the chemical shifts and integral area

8624 Huo et al.
Asian J. Chem.
X-20121012-TS-3
5500
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
-500
-0.6
5.4
5.0
4.6
4.2
3.8
3.4
3.0
2.6
2.2
1.8
1.4
1.0
0.6
0.2
-0.2
f1 (ppm)
Fig. 4. Fatty acid of brominated products spectrum
CH3), 2.32 (t, J = 7.3,2 H, CH2COOCH2CH3), 2.45-2.53 (m,
4H, CH2SCH2), 3.64 (t, J = 6.6, 2H, CH2Br), 4.14 (q, J = 7.1,
2 H, COOCH2CH3).
reaction time is 6h, the highest yield is 55 %. In synthesis of
ethyl 17-bromo-6-thia-heptadecanoate, the order of addition
should be noted.
Inferred from Fig. 4 that this product is the intermediates
of the final product 99mTc labeled myocardial fatty acid meta-
bolism imaging agent of BSE, the yield is 38 %.
ACKNOWLEDGEMENTS
This work was supported by the Introduction Talent
Project of Chinese Academy of Sciences.
Conclusion
The purpose of this experiment is to synthesize a fatty
acid bromide-BSE. This fatty acid bromide can promote meta-
bolism, likely to be marked, in addition, the carbon chain structure
help to simulate in vivo myocardial fatty acid behaviour. Its
carbon chain structure contributes to the absorption and
retention of the myocardium. Preparation of thiol should add
7.5M alkali NaOH, the reaction time is 6h, the highest yield is
78 %. Fatty acid synthesis in the reflux temperature is 100 ºC,
the highest yield is 59 %. In Fatty acids esterification, the
REFERENCES
1. N.Yamamura,Y. Magata,Y.Arano, T. Kawaguchi, K. Ogawa, J. Konishi
and H. Saji, Bioconjug. Chem., 10, 489 (1999).
2. Y. Magata, T. Kawaguchi, M. Ukon, N. Yamamura, T. Uehara, K.
Ogawa, Y. Arano, Y. Temma, T. Mukai, E. Tadamura and H. Saji,
Bioconjug. Chem., 15, 389 (2004).
3. J.A. Wisneski, E.W. Gertz, R.A. Neese, M. Mayr, J. Clin. Invest., 79,
359 (1987).
4. L.P. Han, Y. Fan and Y.M. Li, China Medical University (2002).