Efficient synthesis of cytidine diphosphate choline (CDP-choline) and its analogs

Article abstract of DOI:10.1080/15257770.2015.1004340

An efficient P(V)-N activation approach for the synthesis ofcytidine diphosphate choline (CDP-choline) and related ribo- and deoxyribonucleotide analogs has been established.

Full text of DOI:10.1080/15257770.2015.1004340

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Efficient synthesis of cytidine
diphosphate choline (CDP-choline) and
its analogs
a
a
a
a
Qi Sun , Xiao-Chuan Li , Shan-Shan Gong , Jian Sun , Cheng-Jun
a
a
Wang & Xing-Cong Wang
a
Jiangxi Key Laboratory of Organic Chemistry, Jiangxi Science and
Technology Normal University, Nanchang, Jiangxi, PR China
Published online: 12 May 2015.
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To cite this article: Qi Sun, Xiao-Chuan Li, Shan-Shan Gong, Jian Sun, Cheng-Jun Wang & Xing-
Cong Wang (2015) Efficient synthesis of cytidine diphosphate choline (CDP-choline) and its analogs,
Nucleosides, Nucleotides and Nucleic Acids, 34:6, 379-387, DOI: 10.1080/15257770.2015.1004340
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Nucleosides, Nucleotides and Nucleic Acids, 34:379–387, 2015
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2015.1004340
EFFICIENT SYNTHESIS OF CYTIDINE DIPHOSPHATE CHOLINE
(CDP-CHOLINE) AND ITS ANALOGS
Qi Sun, Xiao-Chuan Li, Shan-Shan Gong, Jian Sun, Cheng-Jun Wang,
and Xing-Cong Wang
Jiangxi Key Laboratory of Organic Chemistry, Jiangxi Science and Technology Normal
University, Nanchang, Jiangxi, PR China
GRAPHICAL ABSTRACT
2
An efficient P(V)–N activation approach for the synthesis of cytidine diphosphate choline (CDP-
choline) and related ribo- and deoxyribonucleotide analogs has been established.
Keywords CDP-choline; phosphoropiperidate; phosphocholine; 4,5-dicyanoimidazole;
P(V)–N activation
INTRODUCTION
Cytidine diphosphate choline, also known as CDP-choline or citicol-
[1]
ine, is an essential biomolecule in cellular metabolism. It has been elu-
cidated that CTP-phosphocholine cytidylyltransferase (CCT)-mediated for-
mation of CDP-choline is the rate-limiting step in phosphatidylcholine (PC)
biosynthetic pathway. In the biosynthesis of one of the key neurotransmit-
[2]
ters, acetylcholine (ACh), CDP-choline serves as the choline donor. Due
Received 25 April 2014; accepted 2 January 2015.
Address correspondence to Professor Qi Sun, Jiangxi Science and Technology Normal University,
Jiangxi Key Laboratory of Organic Chemistry, 605 Fenglin Avenue, Nanchang, 330013 China. E-mail:
sunqi96@tsinghua.org.cn
Color versions of one or more of the figures in the article can be found online at
www.tandfonline.com/lncn.
3
79

3
80
Qi Sun et al.
to its pivotal role in PC and ACh synthesis, CDP-choline has been exten-
sively investigated as a neuroprotective agent in a large number of in vitro
and in vivo studies.[
1b,3]
It has been postulated that CDP-choline restores
the neuronal membrane integrity and functions by increasing the produc-
tion of both PC and ACh and attenuating the formation of reactive oxygen
[
3a]
species (ROS) at the nerve damage site.
Clinical studies have established
that administration of CDP-choline has beneficial effects on central nerve
system disorders (memory impairment, Alzheimer’s disease, and Parkin-
[
4]
[5]
son’s disease)[ and injuries (stroke and brain/spinal cord trauma), sub-
6]
[7]
stance abuse, and eye conditions (glaucoma and amblyopia). Deoxy-
cytidine diphosphate choline (dCDP-choline) has also been isolated from
both human cells and sea urchin eggs, but its biological functions are still
[
8]
elusive.
Currently, a large portion of the commercially available CDP-choline
is produced through biotransformation, which is limited by low reaction
concentration, moderate overall conversion yield, and complicated purifi-
cation procedures.[ The chemical synthesis of CDP-choline is complemen-
tary to microbial fermentation-based methods, and especially useful in the
preparation of modified or labeled CDP-choline analogs. The major chem-
9]
ꢁ
ical synthetic method is the coupling of cytidine 5 -phosphates (CMP) with
phosphocholine using different condensing reagents, such as dicyclohexyl-
carbodiimide (DCC),[ p-toluenesulfonyl chloride,
10]
[11]
and DMF/SOCl2
in low to moderate yields. In addition, CMP has also been converted to
[12]
the activated phosphate[
13]
or phosphoramidate
[14]
to improve the effi-
cacy of the coupling reactions. However, the yields of all these reported
methods rarely exceeded 60%. On the basis of the P(V)–N activation strat-
ꢁ
egy that we established for the synthesis of nucleoside 5 -polyphosphates
[
15]
[16]
(
NPPs),
side polyphosphates,
nucleoside diphosphate sugars (NDP-sugars),
and dinucleo-
[
17]
we report in this paper an efficient approach for the
preparation of CDP-choline and related nucleotide analogs from nucleoside
ꢁ
5
-phosphoropiperidates in high yields.
RESULTS AND DISCUSSION
As shown in Scheme 1, CDP-choline (6) and related nucleotide analogs
ꢁ
were efficiently prepared by treating nucleoside 5 -phosphoropiperidates
(
6
1
1–5) with 2.0 equiv of bis(tetra-n-butylammonium) phosphocholine and
.0 equiv of DCI in anhydrous N -methylpyrrolidone (NMP) at 40 C for
.5 hours. After the reaction solutions were cooled to ambient temperature,
◦
the precipitated crude products were separated by filtration. Ethanol pre-
cipitation of the sodium salts followed by ion exchange chromatography
afforded 6–10 in high isolated yields ranging from 78–84%.

Efficient Synthesis of CDP-Choline and Its Analogs
381
SCHEME 1 The P(V)–N activation method for the synthesis of CDP-choline and related nucleotide
a
analogs (6–10). Isolated yield.
FIGURE 1 Effect of the amount of phosphocholine on the formation of CDP-choline (6).

3
82
Qi Sun et al.
TABLE 1 The effect of the amount of DCI on the formation of CDP-choline (6).
Equiv of DCI
Time (hours)
Yield of 6 (%)a
4.0
5.0
6.0
7.0
22
16
8
81
83
85
80
6
a31P NMR yield.
ꢁ
In the preliminary experiment, cytidine 5 -phosphoropiperidate (1) was
◦
treated with phosphocholine in 1:1 molar ratio in DMF at 20 C with or
31
without DCI activator. P NMR tracing experiments showed that there was
no reaction between the two reactants without DCI after 48 hours, which was
in agreement with our previous observation that the reactivity of monoionic
phosphoramidates toward phosphate nucleophiles was low. In contrast, the
presence of 3.0 equiv of DCI promoted the reaction to complete in 36 hours.
the protonation of the inactive 1
P–O /P–NR ) by DCI generated the reactive zwitterionic phosphoramidate
[15a]
As we proposed in a previous report,
−
(
2
−
+
intermediate (P–O /P–NH R ), and the subsequent P–O coupling with
2
phosphocholine proceeded via an associative pathway. In addition to the
acid catalysis mentioned above, nucleophilic catalysis of the conjugate base
of DCI was also supposed to be involved in this reaction due to the low
nucleophilicity of phosphocholine. However, the 31P NMR yield of CDP-
choline (6) was only 52% with 1 equiv of phosphocholine and 3 equiv of DCI.
The low nucleophilicity of phosphocholine resulted in not only slow reaction
rate but also pronounced side reactions, the hydrolysis (ca. 22%) and self-
condensation (ca. 26%) of 1. To solve this problem, higher equivalents of
phosphocholine (1.5 and 2.0 equiv) were tested in the reaction. As shown
in Figure 1, the self-condensation byproduct (Cp C) was almost completely
2
TABLE 2 The effect of temperature on the formation of CDP-choline (6)
◦
Yield of 6 (%)a
Temperature ( C)
Time (hours)
20
30
40
50
8
85
83
84
80
3.5
1.5
0.7
a31P NMR yield.

Efficient Synthesis of CDP-Choline and Its Analogs
383
FIGURE 2 31P NMR of the crude reaction mixture of CDP-choline (6) in NMP.
suppressed and the yield of 6 was improved to 83%, when 2.0 equiv of
phosphocholine was applied.
In the following research, the effect of the amount of DCI was inves-
tigated in detail. The data in Table 1 showed that the reaction time was
significantly shortened from 22 to 8 hours, when the amount of DCI was
increased from 4.0 to 6.0 equiv. Though further increment of the amount
of DCI to 7.0 equiv slightly increased the reaction rate (6 hours), the self-
condensation side reaction was also promoted.
To further promote the coupling reaction, the reaction temperature was
◦
gradually elevated from 20 to 50 C. As shown in Table 2, the reaction time
was remarkably shortened to 45 minutes, when the reaction was conducted
◦
at 50 C. But it was also noticed that when the reaction temperature was above
◦
4
0 C, the yield of 6 began to drop due to the self-condensation of 1.
Due to the limited solubility of phosphocholine in DMF, some other
polar aprotic solvents, such as DMSO, pyridine, and NMP, were tested for
this reaction. It was found that NMP exhibited the best solvency for phos-
31
phocholine. The P NMR data (Figure 2) also showed that the yield of 6
was improved to 95% with unexpected suppression of the hydrolysis of 1
(
<4%), when NMP was used as the solvent.
CONCLUSIONS
In summary, we developed an efficient method for the synthesis of CDP-
choline and related ribo- and deoxyribonucleoside analogs. The detailed
reaction conditions such as the ratio of reactants, amount of DCI, tempera-
ture, and solvent were systematically optimized for CDP-choline. Compared
to the known methods, this new approach features easily accessible starting
materials, short reaction time, and high isolated yields, and could be applied
to the synthesis of a variety of nucleoside diphosphate analogs.

3
84
Qi Sun et al.
EXPERIMENTAL
General Methods. All reactions were performed in anhydrous solvents
under an atmosphere of dry argon. Ion-exchange chromatography em-
ployed DEAE Sephadex A-25 exchanger. NMR spectra were obtained with a
4
00 MHz instrument with chemical shifts reported in parts per million (ppm,
δ). IR spectra were recorded on a FT-IR spectrometer. Low-resolution mass
spectra were obtained with an ion trap mass spectrometer and reported
ꢁ
as m/z. The triethylammonium salts of nucleoside 5 -phosphoropiperidates
ꢁ
were synthesized from the nucleoside 5 -phosphates according to the proce-
dure described in our previous report in over 95% yield.[
15b]
The bis(tetra-
n-butylammonium) phosphocholine was prepared according to a reported
[
18]
method.
ꢁ
General procedure for the synthesis of nucleoside 5 -diphosphate
ꢁ
choline (6–10): To a solution of nucleoside 5 -phosphoropiperidate
(
0.1 mmol) in NMP (2 mL) were added bis(tetra-n- butylammonium) phos-
phocholine (0.2 mmol) and DCI (0.6 mmol). The reaction was stirred at
4
◦
0 C for 1.5 hours. The precipitation was collected by centrifuge. The solid
residue was dissolved in NaOAc aqueous solution (3 M, 1 mL). Then, EtOH
50 mL) was added. The resulting white precipitate was collected by cen-
(
trifuge. The crude product was dissolved in deionized H O (0.5 mL) and
2
loaded on a DEAE Sephadex A-25 ion exchange column (1.6 × 25 cm).
Elution with NH HCO buffer (linear gradient 0.05–0.2 M), combination
4
3
of appropriate fractions, and lyophilization afforded NDP-choline in ammo-
nium salt form. Passage of the solution of the ammonium salt in deionized
+
H O through a bed of Dowex 50W-X8 ion-exchange resin (Na form) and
2
lyophilization afforded NDP-choline as sodium salt, a white solid.
ꢁ
Cytidine 5 -diphosphate choline, sodium salt (6): Starting from cytidine
ꢁ
5
-phosphoropiperidate (49 mg, 0.1 mmol), compound 6 was synthesized ac-
cording to the general procedure. Ion-exchange chromatography afforded
1
6
7
2
9
7
(42 mg, 82%) as a white solid; H NMR (400 MHz, D O): δ 7.90 (d, J =
2
.6 Hz, 1H), 6.07 (d, J = 7.6 Hz, 1H), 5.94 (d, J = 4 Hz, 1H), 4.35−4.30 (m,
H), 4.28−4.27 (m, 4H), 4.25−4.22 (m, 1H), 3.65−3.63 (m, 2H), 3.18 (s,
13
H) ppm; C NMR (100 MHz, D O): δ 165.8, 157.3, 141.1, 96.1, 88.9, 82.2,
2
31
3.7, 68.9, 65.6, 64.4, 59.4, 53.6 ppm; P NMR (162 MHz, D O): δ –11.1
2
(
d, J P-α,P-β = 20.8 Hz, 1P), –11.9 (d, J
= 20.8 Hz, 1P) ppm; IR: ν
P-α,P-β
max
−
1
3
671, 2988, 2899, 1645, 1488, 1392, 1234, 1063, 782, 634, 609, 552 cm ;
−
HRMS (ESI–): m/z calcd for C H N O P [M–H] 487.1001; found 487.
14
25
4
11 2
1
010.
ꢁ
Uridine 5 -diphosphate choline, sodium salt (7): Starting from uridine
-phosphoropiperidate (49 mg, 0.1 mmol), compound 7 was synthesized ac-
ꢁ
5
cording to the general procedure. Ion-exchange chromatography afforded
7
(43 mg, 84%) as a white solid; 7.86 (d, J = 8.0 Hz, 1H), 5.88−5.83 (m, 2H),
13
4.29−4.25 (m, 4H), 4.17−4.09 (m, 3H), 3.59 (m, 2H), 3.12 (s, 9H) ppm; C

Efficient Synthesis of CDP-Choline and Its Analogs
385
NMR (100 MHz, D O): δ 166.0, 151.6, 141.1, 102.2, 88.0, 82.8, 73.3, 69.2,
2
31
6
2
1
5.4, 64.5, 59.4, 53.5 ppm; P NMR (162 MHz, D O): δ –11.6 (d, J
=
2
P-α,P-β
3673, 2983, 2904,
1.0 Hz, 1P), –12.3 (d, J P-α,P-β = 21.0 Hz, 1P) ppm; IR: ν
max
−
1
689, 1463, 1395, 1237, 1082, 770, 608, 536 cm ; HRMS (ESI–): m/z calcd
−
for C H N O P [M–H] 488.0841; found 488.0853.
14
24
3
12
2
ꢁ
Adenosine 5 -diphosphate choline, sodium salt (8): Starting from adeno-
ꢁ
sine 5 -phosphoropiperidate (52 mg, 0.1 mmol), compound 8 was synthe-
sized according to the general procedure. Ion-exchange chromatography
1
afforded 8 (42 mg, 78%) as a white solid; H NMR (400 MHz, D O): δ 8.40 (s,
2
1
4
H), 8.15 (s, 1H), 6.05 (d, J = 4 Hz, 1H), 4.54−4.44 (m, 2H), 4.43−4.42 (m,
13
H), 4.30−4.28 (m, 2H), 3.55 (s, 2H), 3.10 (s, 9H) ppm; C NMR (100 MHz,
D O): δ 155.2, 152.5, 148.7, 139.3, 118.1, 86.4, 83.4, 73.7, 69.9, 65.4, 64.8,
2
31
5
1
1
9.4, 53.4 ppm; P NMR (162 MHz, D O): δ –11.5 (d, J
= 20.9 Hz,
2
P-α,P-β
P), –12.3 (d, J P-α,P-β = 20.9 Hz, 1P) ppm; IR: νmax 3332, 2981, 2491, 1651,
−
1
477, 1409, 1331, 1235, 1118, 944, 873, 817, 784, 710, 646, 561 cm ; HRMS
−
(ESI–): m/z calcd for C H N O P [M–H] 511.1113; found 511.1126.
1
5
25
6
10 2
ꢁ
Guanosine 5 -diphosphate choline, sodium salt (9): Starting from guano-
ꢁ
sine 5 -phosphoropiperidate (53 mg, 0.1 mmol), compound 9 was synthe-
sized according to the general procedure. Ion-exchange chromatography
1
afforded 9 (43 mg, 78%) as a white solid; H NMR (400 MHz, D O): 8.10
2
(
2
1
s, 1H), 5.93 (s, 1H), 4.51 (s, 1H), 4.25−4.40 (m, 4H), 4.20 (s, 2H), 3.62 (s,
13
H), 3.17 (s, 9H); C NMR (100 MHz, D O): δ 159.9, 154.7, 151.9, 137.4,
2
31
16.4, 86.7, 83.7, 73.6, 70.4, 66.0, 65.3, 59.8, 53.9 ppm; P NMR (162 MHz,
= 20.8 Hz, 1P), –12.1 (d, J = 20.8 Hz, 1P)
D O): δ –11.3 (d, J
ppm; IR: νmax 3668, 2986, 2313, 1667, 1530, 1481, 1237, 1080, 1047, 939, 917,
7
2
P-α,P-β
P-α,P-β
−
1
−
78, 642, 571 cm ; HRMS (ESI–): m/z calcd for C H N O P [M–H]
15 25 6 11 2
5
27.1062; found 527.1077.
ꢁ
ꢁ
2
-Deoxycytidine 5 -diphosphate choline, sodium salt (10): Starting from
ꢁ
ꢁ
2
-deoxycytidine 5 -phosphoropiperidate (48 mg, 0.1 mmol), compound 10
was synthesized according to the general procedure. Ion exchange chro-
1
matography afforded 10 (40 mg, 81%) as a white solid; H NMR (400 MHz,
D O): δ 7.81 (d, J = 8.1 Hz, 1H), 6.21 (t, J = 7.3 Hz, 1H), 5.98 (d, J = 7.2 Hz,
2
1
2
1
H), 4.45 (s, 1H), 4.26 (s, 2H), 4.07−4.05 (m, 3H), 3.55 (s, 2H), 3.09 (s, 9H),
13
.32−2.26 (m, 1H), 2.21−2.14 (m, 1H) ppm; C NMR (100 MHz, D O): δ
2
31
66.3, 157.7, 141.7, 96.6, 86.1, 85.5, 70.9, 66.1, 65.5, 60.0, 54.1, 39.5 ppm; P
NMR (162 MHz, D2O): δ –10.9, –11.7 ppm; IR: ν
3680, 2979, 2909, 1682,
max
−
1
1
498, 1402, 1248, 1078, 794, 653, 628, 564 cm ; HRMS (ESI–): m/z calcd
−
for C H N O P [M–H] 471.3172; found 471.3165.
14
25
4
10 2
FUNDING
The authors thank the National Natural Science Foundation of China
No. 21262014), Major Science and Technology Project of Jiangxi Province
(

3
86
Qi Sun et al.
(
No. 20143ACB21014), and Research Funds from JXSTNU (Nos. ky2012zy08
and 2013QNBJRC001) for financial support.
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