Concise syntheses of trifluoromethylated cyclic and acyclic analogues of cADPR

Article abstract of DOI:10.3390/molecules15128689

A novel trifluoromethylated analogue of cADPR, 8-CF3-cIDPDE (5) was designed and synthesized via construction of N¹,N⁹- disubstituted hypoxanthine, trifluoromethylation and intramolecular condensation. A series of acyclic analogues of cADPR were also designed and synthesized. These compounds could be useful molecules for studying the structure-activity relationship of cADPR analogues and exploring the cADPR/RyR Ca²⁺ signalling system

Full text of DOI:10.3390/molecules15128689

Molecules 2010, 15, 8689-8701; doi:10.3390/molecules15128689
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Concise Syntheses of Trifluoromethylated Cyclic and
Acyclic Analogues of cADPR
Xiangchen Huang, Min Dong, Jian Liu, Kehui Zhang, Zhenjun Yang, Liangren Zhang *
and Lihe Zhang
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China
* Author to whom correspondence should be addressed; E-Mail: liangren@bjmu.edu.cn;
Tel.: +86 10-82802567; Fax: +86 10-82805063.
Received: 6 November 2010; in revised form: 17 November 2010/ Accepted: 22 November 2010 /
Published: 30 November 2010
Abstract: A novel trifluoromethylated analogue of cADPR, 8-CF3-cIDPDE (5) was
designed and synthesized via construction of N1,N9-disubstituted hypoxanthine,
trifluoromethylation and intramolecular condensation. A series of acyclic analogues of
cADPR were also designed and synthesized. These compounds could be useful molecules
for studying the structure-activity relationship of cADPR analogues and exploring the
cADPR/RyR Ca2+ signalling system.
Keywords: cADPR analogue; acyclic cADPR analogue; trifluoromethylation; synthesis
1. Introduction
Cyclic adenosine diphosphate ribose (cADPR, 1, Figure 1), isolated from sea urchin eggs [1], is a
metabolite of β-nicotinamide adenine dinucleotide (NAD+). It has been proved that cADPR is a
signalling molecule, which regulates calcium mobilization via ryanodine receptor (RyR) in a wide
variety of Ca2+-dependent cellular responses such as fertilization, secretion, contraction, proliferation
and so on [2]. Since the discovery of cADPR, numerous works have been done on the synthesis of
cADPR analogues to search for agonists or antagonists of cADPR/RyR Ca2+ signalling system [3-5].
In our previous work, a series of cADPR analogues in which the southern and/or northern ribose
was replaced by an ether chain were synthesized [6,7]. Most of those compounds, such as cIDPRE (2)
and cIDPDE (3), are membrane permeate agonists in Jurkat T cells.

Molecules 2010, 15
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Figure 1. Structures of cADPR and its analogues.
HO
OH
O
N
NH
O
N
O
O
N
N
N
N
O
N
N
N
N
O
O
N
O
O
O
O
O
O
P
P
O
N
P
HO
HO
HO
O
O
P
O
P
O
O
P
O
O
OH
O
OH
OH
OH
O
OH
HO
HO
2 (cIDPRE)
1 (cADPR)
3 (cIDPDE)
Moreover, it was found that those agonists antagonize the hydrolysis of CD38. Substitution at C-8
of purine affects the agonistic activity of cADPR analogues. For example, 8-Br or 8-Cl substituted
cIDPRE loses activity; however, the activity is retained for 8-N3 or 8-NH2 substituted cIDPRE. These
results indicate that the effect of substitution at 8-position depends on the property of the substituent
group. The trifluoromethyl group, possessing high electronegativity and lipophilicity, usually alters
considerably the overall charge distribution and enhances the membrane permeability of molecules.
Since the trifluoromethyl group imparts a variety of special physical and chemical properties to
molecules, a number of trifluoromethylated compounds exhibit enhanced biological activity [8].
Taking these points into account, we synthesized 8-CF3-cIDPRE (4, Figure 2). We found that this
compound was also a membrane permeate calcium agonist in Jurkat T cells [9]. In this study, the
trifluoromethyl group is introduced to cIDPDE (8-CF3-cIDPDE, 5, Figure 2). This compound provides
a complementary agent for understanding the effect of 8-substitution on calcium signalling property.
Figure 2. Structures of compounds 4-8.
O
O
N
O
N
N
N
O
N
N
CF3
O
O
N
O
CF3
P
O
O
N
O
HO
P
HO
O
P
O
O
P
O
O
OH
OH
O
OH
HO
4 (8-CF3-cIDPRE)
5 (8-CF3-cIDPDE)
O
O
O
N
O
N
O
N
N
X
HO P O
OH
O
N
CF3
HO P O
OH
O
N
N
O
N
HO P O
OH
HO P O
OH
O
O
HO
OH
6
7 (X = H)
8 (X = CF3)
cADPR can be hydrolyzed either in vivo or in vitro [10,11]. The cyclic pyrophosphate moiety, as
one of the most vulnerable linkages in cADPR, can be hydrolyzed by Mn2+-dependent
ADP-ribose/CDP-alcohol pyrophospatase to afford the bisphosphate metabolite [12]. Recently, a series
of acyclic analogues of cADPR, in which the pyrophosphate moiety is cleaved to give a bisphosphate,
have been synthesized [13]. The primary pharmacological research revealed that some of them could
inhibit cIDPRE-induced Ca2+ release. To further explore the Ca2+-modulating activities of this novel

Molecules 2010, 15
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class of cADPR mimics and their mechanism further, we have designed and synthesized acyclic
analogues of cIDPRE and the trifluoromethylated analogues 6-8 (Figure 2).
2. Results and Discussion
2.1. Synthesis of 8-CF3-cIDPDE (5)
The synthesis of 8-CF3-cIDPDE is summarized in Scheme 1. Starting from 8-bromoadenine [14],
N9-substitution was carried out with (2-acetoxyethoxy)methyl bromide [15] in the presence of
potassium tert-butoxide (t-BuOK) and 18-crown-6 [16] to afford 10 in 44% yield. It is noteworthy that
when (2-acetoxyethoxy)methyl chloride was employed instead, replacement of the 8-bromo group
1
with a chlorine atom was observed. The structure of compound 10 was confirmed by H-NMR,
13C-NMR, HMBC and HR-ESI-MS spectra. In the HMBC spectrum of 10, the correlation between
H-1′ of the ether chain and C-4 and C-8 of adenine base were observed, which verified that the
substitution was on N-9.
Scheme 1. Synthesis of 8-CF3-cIDPDE (5).
NH2
N
NH2
N
O
N
N
N
N
N
N
N
NH2
N
NH
Br
O
Br
Br
N
a
b
d
c
N
N
Br
H3CCOO
HO
HO
N
H
N
O
O
10
9
11
12
O
O
O
N
N
N
N
N
N
N
O
N
O
N
NH
e
H3CCOO
Br
H3CCOO
CF3
Br
f
g
N
N
TBDPSO
TBDPSO
TBDPSO
H3CCOO
O
O
O
14
13
15
O
N
O
O
N
N
N
N
N
N
O
N
O
N
O
(PhS)2OPO
N
CF3
H3CCOO
CF3
HO
CF3
N
i
h
j
N
HO
(PhS)2OPO
O
O
O
17
18
16
O
O
O
O
HO P O
OH
N
N
N
N
O
O
N
CF3
CF3
O
O
k
P
N
N
N
O
O
PhS P O
OH
HO
O
O
P
O
OH
19
5
Reagents and conditions: (a) t-BuOK, 18-crown-6, BrCH2OCH2CH2OAc, THF, 0 °C; (b) K2CO3,
MeOH, rt; (c) NaNO2, AcOH, rt; (d) TBDPSCl, imidazole, DMF, rt; (e) DBU,
ClCH2OCH2CH2OAc, CH2Cl2, rt; (f) FSO2CF2CO2Me, CuI, HMPA, DMF, 70 °C; (g) 70% HF·Py,
THF; (h) PSS, TPSCl, tetrazole, Py, rt; (i) AcCl, MeOH; (j) i. POCl3/DIPEA, CH3CN, 0 °C; ii. 1 M
TEAB, pH 7.5, rt; (k) I2, 3Å MS, Py, rt.

Molecules 2010, 15
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Deacetylation of 10 with K2CO3/MeOH gave compound 11, and after diazotization, and protection
of the 5′-hydroxyl group with a tert-butyldiphenylsilyl (TBDPS) group, 13 was obtained. An
N1-substitution was carried out on compound 13 with (2-acetoxyethoxy)methyl chloride in the
presence of excess 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to afford 14 in 61% yield. Since both of
the N1 and the O6 have nucleophilicity, the N1-isomer and O6-isomer were obtained (Figure 3). The
structure of 14 was confirmed by 1H-NMR, 13C-NMR, HMBC and HR-ESI-MS spectra. In the HMBC
spectrum of 14, the correlation between H-1′′of the northern ether chain and C-2 of hypoxanthine base,
and that between C-1′′ of the northern ether chain and H-2 of hypoxanthine base were both observed,
which were similar to that of N1-isomer. Corresponding correlations were not found in the HMBC
spectrum of the O6-substituted side product.
Figure 3. Structures of 14 and its O6-isomer.
H1''c H1''d
OOCCH3
H1''a H1''b
O
N
O
O
N
OOCCH3
N
N
O
N
N
N
N
Br
Br
H
2
2
H
TBDPSO
TBDPSO
O
O
O 6-isomer of 14
14
The unstable glycosylic bond in nucleosides is sensitive to certain conditions, which causes great
difficulties in the trifluoromethylation of nucleosides. In our previous work, methyl fluorosulphonyl-
difluoroacetate/copper iodide (FSO2CF2CO2Me/CuI) [17] was initially applied to the synthesis of
8-CF3-purine nucleosides [9]. Adopting this strategy, trifluoromethylation of 14 was achieved
successfully, and optimization of this reaction was carried out (Table 1). Under the optimal reaction
conditions, 15 was obtained in 42% yield, and 14 was recovered in 17% yield. Interestingly, compound
16 was also obtained in a yield of 14%. It is known that the tert-butyldimethylsilyl (TBDMS) group
and TBDPS group could be removed by tetrabutylammonium fluoride/tetrahydrofuran (TBAF/THF),
potassium fluoride and other agents containing fluoride [18]. Accordingly, we deduced it was the
fluoride ion generated in the process of trifluoromethylation [17] that facilitated the removal of the
1
5′-O-TBDPS group. The trifluoromethylated product 15 was characterized by H-NMR,13C-NMR,
19F-NMR and HR-ESI-MS spectra. In the 13C-NMR spectrum of compound 15, signals of the CF3 group
and C-8 were spilt into two quartets, with 1JCF = 270 Hz and 2JCF = 41 Hz, respectively, and the singlet at
−63.358 ppm was observed in the 19F-NMR spectrum. These data strongly support the incorporation of
the trifluoromethyl group.
Table 1. Optimization of the reaction conditions of trifluoromethylation.
Entry
FSO2CF2CO2Me /HMPA
5 equiv
Yield
trace
12%
42%
31%
18%
1
2
3
4
5
10 equiv
15 equiv
20 equiv
30 equiv

Molecules 2010, 15
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The 5′-O-TBDPS group in compound 15 was removed by employing 70% HF·pyridine [19]. The
strong electronegativity of trifluoromethyl group at C-8 of hypoxanthine makes the glycosylic bond
rather sensitive to acid conditions. Hence, 70% HF·pyridine was added dropwise to the reaction
mixture at −20 °C. Compound 16 was successfully converted to 17 by the reaction with
S,S-diphenylphosphorodithioate (PSS) [20] in the presence of triisopropylbenzenesulfonyl chloride
(TPSCl) and tetrazole in pyridine, in a yield of 79%. Considering the instability of phenylthio group
under basic conditions [21], acetyl chloride in methanol (AcCl/MeOH) [22] was applied to the
deacetylation of 17. When 1.2 equivalent of AcCl was utilized, compound 17 was successfully
converted to 18. Phosphorylation of the 5′′-hydroxyl in 18 was carried out in the presence of excess
POCl3 and N,N-diisopropylethylamine (DIPEA) at 0 °C. After being stirred for 14 h, the mixture was
treated with 1 M triethylammonium bicarbonate (TEAB) for 6 h at room temperature [23], which
facilitated the semi-deprotection of the S,S-diphenylphosphate. Purified by high performance liquid
chromatography (HPLC), compound 19 was obtained as its triethylammonium salt.
Following the Matsuda strategy [24], with excess I2 and 3Å molecular sieves as promoters, the
intramolecular cyclization was performed in pyridine by adding a solution of compound 19 slowly
over 20 h utilizing a syringe pump. Purification by HPLC afforded cyclic product 5 as its
1
19
31
triethylammonium salt in 71% yield, which was characterized by H-NMR, F-NMR, P-NMR and
HR-ESI-MS spectra.
2.2. Syntheses of Compounds 6-8
Deacetylation of 16 with K2CO3/MeOH afforded compound 20 (Scheme 2), then both of the free
hydroxyl groups in 20 were phosphorylated by employing POCl3/DIPEA in CH3CN at 0 °C for 16 h,
followed by the treatment with 1 M TEAB for 6 h. Purified by HPLC, the target molecule 6 was
obtained as its triethylammonium salt in 62% yield for two steps.
Scheme 2. Syntheses of compounds 6-8.
O
O
N
O
N
O
P
N
N
N
N
N
N
O
N
O
N
O
N
HO
CF3
CF3
H3CCOO
CF3
HO
O
O
N
OH
a
b
HO
HO
P O
HO
O
O
O
OH
6
16
20
O
O
O
N
O
N
N
N
N
N
N
O
N
O
N
O
N
X
H3CCOO
X
HO
X
HO
P O
O
P
b
a
N
N
OH
HO
HO
HO
O
O
O
O
OH
O
O
O
O
O
O
23 (X = H)
26 (X = CF3)
21 (X = H)
24 (X = CF3)
22 (X = H)
25 (X = CF3)
O
O
N
O
N
X
HO
P
O
Reagents and conditions: (a) K2CO3, MeOH,
rt; (b) i. POCl3/DIPEA, CH3CN, 0 °C; ii. for
20 and 25, 1M TEAB, pH 7.5, rt; for 22, 1 M
NaOH, rt; (c) for 23, 60% HCOOH, rt; for
26, 10% HCOOH, rt.
O
N
N
OH
c
HO
P O
O
OH
HO
OH
7 (X = H)
8 (X = CF3)

Molecules 2010, 15
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Compound 23 was synthesized from 21 [6] in a yield of 71% for two steps by a similar method as
used for the preparation of 6. After removing the 2′,3′-O-isopropylidene group using 60% HCOOH
solution, compound 7 was obtained as its triethylammonium salt in 85% yield. Starting from
compound 24 [9], 26 was synthesized by a similar procedure. Considering the sensitivity of
8-CF3-purine nucleosides to acid conditions, we performed the deprotection of 26 by employing 10%
rather than 60% HCOOH solution, which afforded compound 8, with little de-glycosylated side
product being generated. After purification by HPLC, the target molecule 8 was obtained as its
triethylammonium salt in 68% yield, with 26 recovered in a yield of 15%. The biological activity assay
of all the compounds synthesized is underway.
3. Experimental
3.1. General
1
HR-ESI-MS and ESI-MS were performed with a Bruker BIFLEX III instrument. H-NMR and
13C-NMR were recorded with a Bruker AVANCE III 400; CDCl3, DMSO-d6 or D2O were used as a
solvent. Chemical shifts are reported in parts per million downfield from TMS (1H and 13C). 31P-NMR
spectra were recorded at room temperature by use of a JEOL AL300 spectrometer (121.5 MHz) or
JEOL ECA600 spectrometer (243 MHz). Orthophosphoric acid (85%) was used as external standard.
19F- NMR spectra were recorded on a Varian VXR-500 spectrometer (470 MHz). Chemical shifts of
19F- NMR are reported in ppm with reference to CF3COOH as external standard. Compounds 19, 23,
26, and 5-8 were purified on an Alltech preparative C18 reversed-phase column (2.2 × 25 cm) with a
Gilson HPLC using MeCN/TEAB (pH 7.5) buffer system as eluent.
3.2. Synthesis
N9-[(5′-Acetoxyethoxy)methyl]-8-bromoadenine (10). To a stirred suspension of 8-bromoadenine (4.5 g,
21.03 mmol) [14] in anhydrous THF (400 mL) was added potassium tert-butoxide (2.59 g, 23.13 mmol)
and 18-crown-6 (1.11 g, 4.20 mmol). The reaction mixture was stirred at room temperature for 15 min,
and then BrCH2OCH2CH2OAc (3.1 mL, 23.13 mmol) [15] was added dropwise at 0 °C. After being
stirred for 30 min at 0 °C, the mixture was filtered and the filtrate is evaporated under reduced pressure.
The residue was purified by silica gel column chromatography (PE-EA = 1:2) to afford compound 10
(3.02 g, 44%). 1H-NMR (400 MHz, DMSO-d6) δ 1.92 (s, 3H, OAc), 3.69-3.72 (m, 2 H, H4′), 4.04-4.17
(m, 2 H, H5′), 5.51 (s, 2H, H1′), 7.48 (s, 2H, NH2), 8.16 (s, 1H, H2). 13C-NMR (100 MHz, DMSO-d6) δ
170.1, 154.8, 153.2, 151.2, 126.5, 118.7, 72.3, 67.0, 62.7, 20.5. MS (ESI-TOF+): m/z = 330.0 [(M + H)+].
N9-[(5′-Hydroxylethoxy)methyl]-8-bromohypoxanthine (12). Compound 10 (1.43 g, 4.34 mmol) was
dissolved in methanol (120 mL). To the solution was added K2CO3 (73 mg, 0.53 mmol) and stirred for
6 h at room temperature. The mixture was neutralized by addition of 0.1 M HCl solution, and
evaporated under reduced pressure. The residue was dissolved in AcOH (70 mL), and a solution of
NaNO2 (2.52 g, 36.4 mmol) in H2O (17 mL) was added. The resulting mixture was stirred at room
temperature for 24 h. After the mixture was evaporated in vacuo, the residue was partitioned between
CHCl3 and H2O. The aqueous phase was extracted again with CHCl3, the organic layer was combined

Molecules 2010, 15
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and washed with brine, dried (Na2SO4), filtered and concentrated in vacuo. Flash chromatography
1
(CH2Cl2-MeOH = 40:1) afforded 12 (792 mg, 63% for two steps). H-NMR (400 MHz, DMSO-d6) δ
3.46-4.51 (m, 4H, H4′, H5′), 4.63(s, 1H, OH), 5.32 (s, 2H, H1′), 8.14 (s, 1H, H2), 12.56 (s, 1H, NH). MS
(ESI-TOF+): m/z = 289.2 [(M + H)+].
N9-[(5′-tert-Butyldiphenylsilyloxyethoxy)methyl]-8-bromohypoxanthine (13). To a solution of 12
(700 mg, 2.42 mmol) in anhydrous DMF (10 mL) was added imidazole (1.86 g, 24.2 mmol) and
tert-butyldiphenylsilyl chloride (3.4 mL, 12.1 mmol) under argon, and the mixture was stirred at room
temperature for 12 h. And the mixture was evaporated in vacuo, the residue was partitioned between
CH2Cl2 and H2O. The aqueous phase was extracted again with CH2Cl2, the organic layer was combined
and washed with brine, dried (Na2SO4), filtered and concentrated in vacuo. Flash chromatography
1
(PE-acetone = 5:1) afforded compound 13 (1.21 g, 95%). H-NMR (400 MHz, CDCl3) δ 1.06 (s, 9H,
(CH3)3C-), 3.71-3.73 (m, 2H, H4′), 3.82-3.84 (m, 2H, H5′), 5.66 (s, 2H, H1′), 7.37-7.69 (m, 10H, ArH),
8.44 (s, 1H, H2),13.19 (s, 1H, NH). 13C-NMR (100 MHz, CDCl3) δ 157.8, 150.8, 146.3, 135.5, 133.3,
129.6, 127.6, 126.4, 124.6, 77.3, 77.0, 76.7, 73.5, 71.1, 62.9, 26.7, 19.0. HRMS (ESI-TOF+): calcd for
C24H27BrN4O3Si [(M + H)+] 527.1109, [(M + Na)+] 549.0928, [(M + K)+] 565.0662; found, 527.1109,
549.0931, 565.0667.
N1-[(5′′-Acetoxyethoxy)methyl]-N9-[(5′-tert-butyldiphenylsilyloxyethoxy)methyl]-8-bromohypoxanthine
(14). To the solution of 13 (1.23 g, 2.33 mmol) and DBU (3.5 mL, 23.3 mmol) in anhydrous CH2Cl2
(25 mL) was added ClCH2OCH2CH2OAc (1.8 mL, 11.65 mmol) [15] dropwise at 0 °C. After being
stirred for 40 min at room temperature, the solvent was evaporated in vacuo and the residue was
purified by silica gel column chromatography (PE-acetone = 5:1) to afford compound 14 (916 mg,
61%). 1H-NMR (400 MHz, CDCl3) δ 1.04 (s, 9H, (CH3)3C-), 2.03 (s, 3H, OAc), 3.68-3.70 (m, 2H, H4′),
3.79-3.82 (m, 2H, H5′), 3.85-3.87 (m, 2H, H4′′), 4.18-4.20 (m, 2H, H5′′), 5.54 (s, 2H, H1′′), 5.61 (s, 2H,
13
H1′), 7.35-7.66 (m, 10H, ArH), 8.09 (s, 1H, H2). C-NMR (100 MHz, CDCl3) δ 170.7, 155.3, 149.2,
147.8, 135.5, 133.2, 129.7, 127.6, 126.2, 124.3, 75.0, 73.5, 71.0, 68.1, 62.9, 26.7, 20.7, 19.0. HRMS
(ESI-TOF+): calcd for C29H35BrN4O6Si [(M + H)+] 643.1582, [(M + Na)+] 665.1401, [(M + K)+]
681.1135; found, 643.1563, 665.1377, 681.1117.
N1-[(5′′-Acetoxyethoxy)methyl]-N9-[(5′-tert-butyldiphenylsilyloxyethoxy)methyl]-8-trifluoromethyl-
hypoxanthine (15). To a solution of compound 14 (576 mg, 0.895 mmol) and CuI (206 mg, 1.074
mmol) in anhydrous DMF (33 mL), hexamethylphosphoric triamide (2.39 mL, 13.425 mmol) and
FSO2CF2CO2Me (1.71 mL, 13.425 mmol) were added successively. The reaction mixture was stirred
for 20 h at 70 °C under argon, then cooled to room temperature, 22 mL of saturated aq. NH4Cl was
added and the mixture was extracted with 200 mL of EA-hexanes (7:3). The organic layer was washed
successively with sat. aq. NaHCO3, water and brine, dried (Na2SO4), filtered, and concentrated under
reduced pressure. The residue was purified by silica gel column chromatography (PE-acetone = 9:2) to
afford compound 15 (238 mg, 42%) and compound 16 (48 mg, 14%), with compound 14 recovered
(96 mg, 17%). 1H-NMR (400 MHz, CDCl3) δ 1.03 (s, 9H, (CH3)3C-), 2.04 (s, 3H, OAc), 3.68-3.70 (m,
2H, H4′), 3.79-3.81 (m, 2H, H5′), 3.87-3.89 (m, 2H, H4′′), 4.20-4.22 (m, 2H, H5′′), 5.56 (s, 2H, H1′′),
13
5.76 (s, 2H, H1′), 7.35-7.66 (m, 10H, ArH), 8.18 (s, 1H, H2). C-NMR (100 MHz, CDCl3) δ 170.7,

Molecules 2010, 15
8696
156.3, 149.4, 149.3, 138.6 (d, 2JCF = 41 Hz), 135.5, 133.2, 129.7, 127.7, 122.7, 118.2 (q, 1JCF = 270 Hz),
19
75.1, 73.5, 71.4, 68.3, 63.1, 63.0, 26.7, 20.8, 19.1. F-NMR (470 MHz, CDCl3) δ -63.4 (s). HRMS
(ESI-TOF+): calcd for C30H35F3N4O6Si [(M + Na)+] 655.2170, [(M + K)+] 671.1904; found, 655.2169,
671.1913.
N1-[(5′′-Acetoxyethoxy)methyl]-N9-[(5′-hydroxylethoxy)methyl]-8-trifluoromethylhypoxanthine (16). A
solution of 15 (182 mg, 0.288 mmol) in anhydrous THF (35 mL) was added 70% HF·Py 1.3 mL at −20 °C.
The mixture was stirred at 0 °C for 1 h and at room temperature over night. The reaction mixture was
quenched with saturated aq. NaHCO3 at 0 °C and diluted with ethyl acetate, then partitioned and the
water layer was washed with ethyl acetate again. The organic layer was combined, washed with brine,
dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was purified by silica
1
gel column chromatography (PE-EA = 1:5) to afford the compound 16 (91 mg, 82%). H-NMR (400
MHz, CDCl3) δ 2.05 (s, 3H, OAc), 3.68-3.74 (m, 4H, H4′, H5′), 3.87-3.89 (m, 2H, H4′′), 4.19-4.22 (m,
2H, H5′′), 5.56 (s, 2H, H1′′), 5.76 (s, 2H, H1′), 8.23 (s, 1H, H2). 13C-NMR (100 MHz, CDCl3) δ 170.8,
2
1
156.2, 149.6, 149.3, 138.4 (q, JCF = 41 Hz), 122.7, 118.2 (q, JCF = 270 Hz), 75.1, 73.2, 71.2, 68.3,
62.9, 61.4, 20.8；19F-NMR (470 MHz, CDCl3) δ-63.4 (s). HRMS (ESI-TOF+): calcd for C14H17F3N4O6
[(M + Na)+] 417.0992, [(M +K)+], 433.0726; found, 417.0991, 433.0730.
N1-[(5′′-Acetoxyethoxy)methyl]-N9-[[5′-bis(phenylthio)phosphoryloxyethoxy]-methyl]-8-trifluoro-meth
ylhypoxanthine (17). To a solution of 16 (66 mg, 0.167 mmol) in anhydrous pyridine (5 mL) was
added TPSCl (302 mg, 1.00 mmol), PSS (571 mg, 1.50 mmol) [20], and tetrazole (105 mg,
1.50 mmol), and the mixture was stirred at room temperature for 12 h. The mixture was evaporated,
and the residue was purified by silica gel column chromatography (PE-EA = 1:2) to give compound 17
(86 mg, 79%). 1H NMR (400 MHz, CDCl3) δ 2.05 (s, 3H, OAc), 3.82-3.84 (m, 2H, H4′), 3.86-3.88 (m,
2H, H5′), 4.18-4.21 (m, 2H, H4′′), 4.31-4.35 (m, 2H, H5′′), 5.56 (s, 2H, H1′′), 5.68 (s, 2H, H1′),
7.33-7.52 (m, 10H, ArH), 8.22 (s, 1H, H2). 13C-NMR (100 MHz, CDCl3) δ 170.8, 156.3, 149.7, 149.3,
2
1
138.5 (q, JCF = 41 Hz), 135.3, 129.7, 129.4, 125.9, 122.7, 118.2 (q, JCF = 269 Hz), 75.2, 73.0, 69.0,
19
31
68.4, 66.2, 62.9, 20.8. F NMR (470 MHz, CDCl3) δ-63.4 (s). P-NMR (D2O, 243 MHz, decoupled
with H) δ50.41 (s). HRMS (ESI-TOF+): calcd for C30H35F3N4O6Si [(M + H)+], 659.1005; found,
1
659.1006.
N1-[(5′′-Phosphonoxyethoxy)methyl]-N9-[[5′-(phenylthio)phosphoryloxyethoxy]methyl]-8-trifluoro-
methylhypoxanthine (19). Compound 17 (54 mg, 0.082 mmol) was dissolved in MeOH (4 mL), and a
solution of acetyl chloride (7 μL, 0.098 mmol) in anhydrous CH2Cl2 (1 mL) was added at −20 °C. The
mixture was stirred at 0 °C for 30 min and raised to room temperature for 24 h, then neutralized by sat.
aq. NaHCO3 solution. The mixture was evaporated, and the residue was partitioned between CH2Cl2
and H2O. The aqueous phase was extracted again with CH2Cl2, the organic layers were combined and
washed with brine, dried (Na2SO4), filtered and concentrated in vacuo. The residue was purified by
silica gel column chromatography (PE-EA =1:10) to give compound 18 (31 mg). The deacetylated
product 18 (31 mg, 0.050 mmol) was dissolved in anhydrous CH3CN (8 mL). DIPEA (65 μL, 0.375 mmol )
and POCl3 (28 μL, 0.300 mmol) were added successively to the solution at −20 °C, and the mixture
was stirred at 0 °C for 14 h, and then added 5mL of TEAB (1 M, pH 7.5) at 0 °C and stirred at room

Molecules 2010, 15
8697
temperature for 6 h. After evaporation under reduced pressure, the residue was partitioned between
H2O and CHCl3, and the aqueous layer was washed with CHCl3 and evaporated in vacuo. The residue
was dissolved in 5 mL of TEAB buffer (0.05 M, pH 7.5), then applied to a C18 reversed-phase column
(2.2 × 25 cm). The column was developed using a linear gradient of 0-40% CH3CN in TEAB buffer
(0.05 M, pH 7.5) within 30 min to afford 19 (27 mg, 41% for two steps) as its triethylammonium salt.
1H-NMR (400 MHz, D2O) δ 3.70-3.73 (m, 4H, H4′, H5′), 3.85-3.89 (m, 2H, H4′′), 3.94-3.98 (m, 2H,
H5′′), 5.45 (s, 2H, H1′′), 5.64 (s, 2H, H1′), 7.09-7.29 (m, 5H, ArH), 8.41 (s, 1H, H2). 13C-NMR
2
(100 MHz, D2O) δ 157.6, 150.9, 149.5, 138.8 (q, JCF = 41 Hz), 132.7, 129.6, 128.9, 127.7, 122.1,
117.8 (q, 1JCF = 270 Hz), 76.4, 73.3, 69.3, 64.9, 64.3, 46.6, 8.2. 19F-NMR (470 MHz, D2O) δ-63.0 (s).
1
31P-NMR (D2O, 243 MHz, decoupled with H) δ 1.10 (s), 17.80 (s). HRMS (ESI-TOF−) calcd for
C18H21N4O10P2SF3 [(M − H)−], 603.0333; found, 603.0331.
N1-[(5′′-O-Phosphorylethoxy)methyl]-N9-[(5′-O-phosphorylethoxy)methyl]-8-trifluoromethylhypo-
xanthine-cyclic pyrophosphate (5). A solution of 19 (5 mg, 6.1 μmol) in anhydrous pyridine (4.5 mL)
was added slowly over 20 h, utilizing a syringe pump, to a mixture of I2 (36 mg, 142 μmol) and 3 Å
molecular sieves (0.36 g), in pyridine (40 mL) at room temperature in the dark. The molecular sieves
were filtered off with Celite and washed with H2O. The combined filtrate was evaporated, and the
residue was partitioned between CHCl3 and H2O. The aqueous layer was evaporated, and the residue
was dissolved in 0.05 M TEAB buffer, which was applied to C18 reversed-phase column (2.2 × 25 cm).
The column was developed using a linear gradient of 0-20% CH3CN in TEAB buffer (0.05 M, pH 7.5)
1
within 30 min to give 5 as its triethylammonium salt (3.0 mg, 71%). H-NMR (400 MHz, D2O) δ
3.70-3.78 (m, 4H, H4′, H5′), 3.81-3.83 (m, 2H, H4′′), 3.88-3.90 (m, 2H, H5′′), 5.54 (s, 2H, H1′′), 5.75 (s,
2H, H1′), 8.49 (s, 1H, H2). 19F-NMR (470 MHz, D2O) δ-62.5 (s). 31P-NMR (D2O 121.5 MHz,
decoupled with 1H) δ −10.07 (d, JP,P = 18.2 Hz), −10.42 (d, JP,P = 18.2 Hz). HRMS (ESI-TOF−) calcd
for C12H15N4O10P2F3 [(M − H)−], 493.0143; found, 493.0146.
N1-[(5′′-Phosphonoxyethoxy)methyl]-N9-[(5′-Phosphonoxyethoxy)methyl]-8- trifluoromethylinosine (6).
Compound 16 (20 mg, 0.051 mmol) was dissolved in methanol (2 mL). To the solution was added
K2CO3 (1 mg, 7.24 μmol) at room temperature and stirred for 6h. The mixture was neutralized by
addition of 0.01 M HCl solution, and removed of the solvent in vacuo. The residue was partitioned
between CHCl3 and H2O, and the organic layer was washed with brine, dried (Na2SO4), and
evaporated, affording compound 20 (16 mg). Compound 20 (16 mg, 0.045 mmol) was dissolved in
anhydrous CH3CN (5 mL). DIPEA (94 μL, 0.54 mmol ) and POCl3 (42 μL, 0.45 mmol) were added
successively to the solution at −20 °C. The mixture was stirred at 0 °C for 16 h, and then added 5 mL
of TEAB (1 M, pH 7.5) at 0 °C and stirred for 6 h at room temperature. After evaporation under
reduced pressure, the residue was partitioned between H2O and CHCl3, and the aqueous layer was
washed with CHCl3 and evaporated in vacuo. The residue was dissolved in 5 mL of TEAB buffer (0.05 M,
pH 7.5), and applied to a C18 reversed-phase column (2.2 × 25 cm). The column was developed using a
linear gradient of 0-40% CH3CN in TEAB buffer (0.05 M, pH 7.5) within 30 min to give 6 (22.3 mg,
62% for two steps) as its triethylammonium salt. 1H-NMR (400 MHz, D2O) δ 3.73-3.79 (m, 4H, H4′, H5′),
3.85-3.92 (m, 4H, H4′′, H5′′), 5.45 (s, 2H, H1′′), 5.77 (s, 2H, H1′), 8.51 (s, 1H, H2). 13C-NMR (100 MHz,
2
1
D2O) δ 157.8, 151.0, 149.7, 138.6 (q, JCF = 41 Hz), 122.2, 117.9 (q, JCF = 270 Hz), 76.4, 73.3, 69.5,

Molecules 2010, 15
8698
69.2, 64.1, 63.8. 19F-NMR (470 MHz, D2O) δ -62.9 (s). 31P-NMR (D2O, 243 MHz, decoupled with 1H) δ
0.19 (s), 0.22 (s). HRMS (ESI-TOF−) calcd for C12H17N4O11P2F3 [(M − H)−], 511.0248; found, 511.0246.
N1-[(5′′-Phosphonoxyethoxy)methyl]-5′-O-phosphoryl-2′,3′-O-isopropylidene-inosine(23). Compound
21 (49 mg, 0.116 mmol) [6] was dissolved in 24 mL of methanol. To the solution was added K2CO3 (2 mg,
14.5 μmol) and stirred at room temperature for 6h. The mixture was neutralized by addition of 0.1 M
HCl solution, and removed of the solvent in vacuo. The residue was partitioned between CHCl3 and
H2O, and the organic layer was washed with brine, dried (Na2SO4), and evaporated, affording
compound 22 (38mg). Compound 22 (38 mg, 0.099 mmol) was dissolved in anhydrous CH3CN (5 mL).
DIPEA (0.21 mL, 1.19 mmol) and POCl3 (91 μL, 0.99 mmol) were added successively to the solution
at −20 °C. The mixture was stirred at 0 °C for 16 h, and then was neutralized by addition of 1 M NaOH
solution. And the resulting mixture was stirred at room temperature for 2 h. After evaporated under
reduced pressure, the residue was partitioned between H2O and CHCl3, and the aqueous layer was
washed with CHCl3 and evaporated in vacuo. The residue was dissolved in 5 mL of TEAB buffer (0.05 M,
pH 7.5), and applied to a C18 reversed-phase column (2.2 × 25 cm). The column was developed using a
linear gradient of 0-40% CH3CN in TEAB buffer (0.05 M, pH 7.5) within 30 min to give 23 (61 mg,
71% for two steps) as its triethylammonium salt. 1H-NMR (400 MHz, D2O) δ 1.31, 1.53 (each s, each 3H,
2 × CH3), 3.72-3.74 (m, 2H, H5′), 3.85-3.89 (m, 2H, CH2O), 3.91-3.94 (m, 2H, CH2OP), 4.52-4.56 (m, 1H,
H4′), 5.06 (dd, 1H, JH3′,H4′ = 1.6 Hz, JH3′, H2′ = 6.0 Hz, H3′), 5.29 (dd, 1H, JH2′,H1′= 2.8 Hz, JH2′,H3′ = 6.0 Hz,
H2′), 5.47 (d, 1H, JH1″b, H1″a = 10.8 Hz, H1″b), 5.51 (d, 1H, JH1″a, H1″b = 10.8 Hz, H1″a), 6.17 (d, 1H, JH1′,H2′
= 2.8 Hz, H1′), 8.23, 8.34 (each s, each 1H, H8, H2). 31P-NMR (D2O, 121.5 MHz, decoupled with 1H) δ
1.79 (s), 1.91 (s). HRMS(ESI-TOF−): calcd for C14H24N4O13P2 [(M − H)−], 541.0742; found, 541.0733.
N1-[(5′′-Phosphonoxyethoxy)methyl]-5′-O-phosphorylinosine (7). A solution of 23 (25 mg, 33.6 μmol)
in 60% HCOOH (6 mL) was stirred for 8 h, and then 14 mL of TEAB (1M, pH 7.5) was added. The
solution was evaporated under reduced pressure. The residue was dissolved in 0.05 M TEAB buffer
(4.0 mL), which was applied to C18 reversed-phase column (2.2 × 25 cm). The column was developed
using a linear gradient of 0-40% CH3CN in TEAB buffer (0.05 M, pH 7.5) within 30 min to afford 7 as
1
its triethylammonium salt (20.2 mg. 85%). H-NMR (400 MHz, D2O) δ 3.72-3.74 (m, 2H, H5′),
3.85-3.88 (m, 2H, CH2O), 3.98-4.01 (m, 2H, CH2OP), 4.23-4.26 (m, 1H, H4′), 4.34-4.37 (m, 1H, H3′),
4.59-4.61 (m, 1H, H2′), 5.46-5.52 (m, 2H, H1″), 6.01 (d, 1H, JH1′,H2′ = 5.6 Hz, H1′), 8.31, 8.36 (each s,
1
each 1H, H8, H2). 31P-NMR (D2O, 243 MHz, decoupled with H) δ 0.81 (s), 0.92 (s). HRMS
(ESI-TOF−): calcd for C14H24N4O13P2 [(M − H)−], 501.0429; found, 501.0426.
N1-[(5′′-Phosphonoxyethoxy)methyl]-5′-O-phosphoryl-2′,3′-O-isopropylidene-8-trifluoromethylinosine
(26). By a similar procedure that described for 6, 26 was synthesized from 24 [9], as its
triethylammonium salt, in 57% yield for two steps. 1H-NMR (400 MHz, D2O) δ 1.29, 1.50 (each s, each
3H, 2×CH3), 3.71-3.73 (m, 2H, H5′), 3.82-3.96 (m, 4H, CH2O, CH2OP), 4.35-4.39 (m, 1H, H4′), 5.19 (dd,
1H, JH3′,H4′ = 4.0 Hz, JH3′,H2′ = 6.8 Hz, H3′), 5.40 (d, 1H, JH1″a,H1″b=10.8 Hz, H1″a), 5.55-5.60 (m, 2H,
13
H1″b, H2′), 6.23 (d, 1H, JH1′,H2′ = 2.0 Hz, H1′), 8.44 (s, 1H, H2). C-NMR (100 MHz, D2O) δ 157.8,
150.6, 148.9, 138.3 (q, 2JCF = 40Hz), 122.9, 117.8 (q, 1JCF = 270 Hz), 115.4, 90.0, 86.7, 86.6, 83.7, 81.1,
19
31
76.4, 69.2, 64.2, 63.8, 25.9, 24.2. F-NMR (470 MHz, D2O) δ -61.9 (s). P-NMR (D2O, 243 MHz,

Molecules 2010, 15
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1
decoupled with H) δ 0.54 (s), 0.70 (s). HRMS(ESI-TOF−): calcd for C17H23F3N4O13P2 [(M − H)−],
609.0612; found, 609.0615.
N1-[(5′′-Phosphonoxyethoxy)methyl]-5′-O-phosphoryl-8-trifluoromethylinosine (8). A solution of 26
(15 mg, 18.47 μmol) in 10% HCOOH (7.5 mL) was stirred at room for 60 h, and then 11 mL of TEAB
(1 M, pH 7.5) was added. The solution was evaporated in vacuo. The residue was dissolved in 0.05 M
TEAB buffer (2.0 mL), which was applied to C18 reversed-phase column (2.2 cm × 25 cm). The
column was developed using a linear gradient of 0-40% CH3CN in TEAB buffer (0.05 M, pH 7.5)
within 30 min to afford compound 8 (9.7 mg, 68%) as its triethylammonium salt, with the compound 26
(2.2 mg, 15%) recovered. 1H-NMR (400 MHz, D2O) δ 3.76-3.78 (m, 2H, H5′), 3.87-3.91 (m, 2H, CH2O),
4.02-4.13 (m, 2H, CH2OP), 4.21-4.25 (m, 1H, H4′), 4.55-4.57 (m, 1H, H3′), 5.20-5.23 (m, 1H, H2′), 5.46
(d, 1H, JH1″b, H1″a = 10.6 Hz,H1′b), 5.59 (d, 1H, JH1″a, H1″b = 10.6 Hz, H1″a), 5.99 (d, 1H, JH1″,H2′ = 5.6 Hz,
H1′), 8.47 (s, 1H, H2). 13C-NMR (100 MHz, D2O) δ 157.9, 150.3, 149.4, 138.9 (q, 2JCF = 40Hz), 123.2,
1
19
117.8 (q, JCF = 269 Hz), 89.8, 84.4, 76.3, 72.0, 70.0, 69.3, 64.4, 64.1. F-NMR (470 MHz, D2O) δ
-61.7 (s). 31P-NMR (D2O, 121.5 MHz, decoupled with H) δ 6.25(s), 6.27 (s). HRMS(ESI-TOF−):
calcd for C14H19F3N4O13P2 [(M − H)−], 569.0303; found, 569.0315.
1
4. Conclusion
In conclusion, we have successfully synthesized 8-CF3-cIDPDE (5) via construction of N1,
N9-disubstituted hypoxanthine, trifluoromethylation and intramolecular condensation. A series of
novel acyclic analogues of cADPR, compounds 6-8, were also synthesized by concise synthetic routes.
With the special properties of trifluoromethyl, 8-CF3-cIDPDE and the acyclic derivatives are expected
to provide useful agents to explore the cADPR/RyR Ca2+ signalling system and illuminate the
structure-activity relationship of cADPR analogues.
Acknowlegements
This study was supported by the National Natural Sciences Foundation of China (Grant no.
90713005, 20832008) and the Ministry of Education of China (Grant no. 200800010078).
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