A concise route for the preparation of nucleobase-simplified cADPR mimics by click chemistry

Article abstract of DOI:10.1016/j.tetlet.2008.05.076

Novel nucleobase-modified cADPR mimics were synthesized by the application of click chemistry. Cu(I)-Huisgen cycloaddition (click reaction) was used to construct 4-amide-1,2,3-triazole nucleobase and connect two building blocks efficiently. A concise protection strategy was used for the synthesis of the corresponding cyclo-pyrophosphate, and the target compounds 6a and 6b were prepared within four steps.

Full text of DOI:10.1016/j.tetlet.2008.05.076

Tetrahedron Letters 49 (2008) 4491–4493
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Tetrahedron Letters
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A concise route for the preparation of nucleobase-simplified
cADPR mimics by click chemistry
*
Lingjun Li, Baichuan Lin, Zhenjun Yang, Liangren Zhang, Lihe Zhang
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel nucleobase-modified cADPR mimics were synthesized by the application of click chemistry. Cu(I)-
Huisgen cycloaddition (click reaction) was used to construct 4-amide-1,2,3-triazole nucleobase and
connect two building blocks efficiently. A concise protection strategy was used for the synthesis of the
corresponding cyclo-pyrophosphate, and the target compounds 6a and 6b were prepared within four
steps.
Received 11 March 2008
Revised 13 May 2008
Accepted 13 May 2008
Available online 21 May 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
cADPR mimics
Click chemistry
Ca2+ signaling
Recently, cyclic ADP-ribose (cADPR) (Fig. 1) has been shown to
control Ca2+-dependent cellular responses in numerous cell sys-
tems.1 The cADPR mimics are valuable tools for the investigation
of the mechanisms of cADPR-mediated Ca2+ signaling.1b,2 As a
complex 18-atom macrocycle molecule, the preparation of cADPR
and its mimics is synthetically challenging.3 cADPR mimics can
be prepared from NAD+ derivatives by enzymatic and chemo-enzy-
matic methods using soluble ADP-ribosyl cyclase from Aplysia cali-
fornica to catalyze the cyclization reaction.4 However, the mimics
obtained by these methods are limited because of the substrate
specificity of ADP-ribosyl cyclase. Shuto et al. developed a method
for the total chemical synthesis of cADPR mimics through building
N-1 substituted purine nucleoside and intra-molecular cyclization
for the formation of pyrophosphate linkage.5 The chemical synthe-
sis method allowed the extensive modifications of both ribose and
nucleobase moieties, and has been well applied for the synthesis of
cADPR mimics.6–9 However, this method has too many steps for
the protection and deprotection strategies in building the substi-
tuted nucleoside and pyrophosphate linkage. Thus, one of the main
objectives resulting from our (and others) previous work was to
develop a concise synthetic pathway to novel cADPR mimics and
at the same time, to reduce the chemical complexity of the natu-
rally occurring cADPR without major loss of biological activity
(Fig. 1).7–9
It was found in our laboratory that cADPR mimics with ribose
moieties replaced by simple ether strand or carbon strand, such
as cIDPRE and cADPRE could act as the membrane-permeant cal-
cium agonist.8,9 Further, Walseth and colleagues have demon-
strated that 3-deaza-cADPR, containing a 3-deazapurine moiety,
is a potent agonist, which is about 70 times more active than
OH
OH
O
Y
X
N
N
NH
O
X
N
N
N
O
O
N
H
N
N
N
-
O
O
N
O P O
O
-O P O
O
O
-O P O
O
O
P
O-
O
N
O
P
O
O
N
O
O-
P
O
O-
OH OH
6a cTDPRC X=CH2,
OH OH
OH OH
cADPR
cIDPRE,X=O, Y=O
cADPRE, X=OCH2, Y=NH
6b cTDPRE X=OCH2
Endogenous Nucleotide
Biologically Active Mimic
Figure 1. cADPR and cADPR mimics with simplified structures.
Still Biologically Active?
* Corresponding author. Tel.: +86 010 82801700; fax: +86 10 82802724.
E-mail address: zdszlh@bjmu.edu.cn (L. Zhang).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.05.076

4492
L. Li et al. / Tetrahedron Letters 49 (2008) 4491–4493
O
O
O
O
N3
PhS
P
O
HO
N
N
X
N
a
O
PhS
P
O
N
H
X
N
SPh
+
H
SPh
HO
O
O
95%, 3a, X=CH2
95%, 3b,X=OCH2
O
1
O
O
2
3
O
P
O
O
O
X
N
O
PhS
O
N
X
N
N
N
N
O
X
N
N
H
H
N
-
c
O
O P O
O
O-
N
d
N
H
O
P
-
N
O
b
O P O
O
P
O
-O
P
O
O
O-
O
O
O-
O
75%, 4a, X=CH2
70%, 4b, X=OCH2
72%, 5a, X=CH2
75%, 5b, X=OCH2
80%, 6a
82.5%, 6b
O-
O
O
OH OH
O
O
6a X=CH2
6b X=OCH2
5
4
Scheme 1. The synthetic route of cTDPRE and cTDPRC. Reagents and conditions: (a) CuI, DIPEA, CH3CN; (b) (1) POCl3, DIPEA, CH3CN, 0 °C, 12 h; (2) 0.1 M TEAB; (c) I2, 3 Å MS,
pyridine; (d) 50% HCOOH.
cADPR itself.10 Thus, starting from the structure of cIDPRE and 3-
deaza-cADPR, we designed and synthesized a new type of tria-
zole-based cADPR analogues, abbreviated cTDPRC 6a and cTDPRE
6b (Fig. 1), in which 1,2,3-triazole-4-amide was constructed in-
stead of the adenine moiety and the northern ribose was replaced
by an ether or carbon strand. We report here the concise synthetic
route for the preparation of this kind of cADPR mimics through
Cu(I)-catalyzed Huisgen [3+2] cycloaddition.
Cu(I)-catalyzed Huisgen [3+2] cycloaddition between terminal
alkyne and azide has significant advantages as an ideal synthetic
reaction such as efficiency, versatility and selectivity.11 It was
applied well in biological conjugation and drug design as a click
reaction.12 In our approach, the efficient Huisgen 1,3-dipolar cyclo-
addition was applied to build the 4-amide-1,2,3-triazole moiety
thereby connecting the northern strand and the southern ribose.
The S,S-diphenylphosphate group, later on used for the formation
of the pyrophosphate ring, was already introduced during the syn-
thesis of building block 1. After phosphorylation of the 50-OH in the
southern ribose, compound 4 was used to construct the cyclo-
pyrophosphate ring by intra-molecular cyclization. Finally, depro-
tection of the 20 and 30 hydroxyl groups on the southern ribose
resulted in the desired target compounds. Thus, the novel cADPR
analogues were obtained by four synthetic steps and the protection
strategy used for synthesizing such cADPR mimics was simplified
significantly (Scheme 1).
For the synthesis of cADPR mimics 6a and 6b, the northern car-
bon and ether strand moiety 1a and 1b and southern 1-b-azido-
2,3-O-isopropylidene-ribose 2 were obtained by the reactions
shown in Scheme 2. Beginning with the commercial reagent 7a
or 7b, compounds 8a and 8b were prepared according to the re-
ported method.13a Compound 8a or 8b was treated with TPSCl
and PSS in the presence of tetrazole in pyridine at room tempera-
ture to obtain 9a or 9b in 92% or 93% yield, respectively. After
deprotection of 9a or 9b by TFA, the intermediate was reacted with
propiolic acid in the presence of DCC to provide the building block
1a in 86% yield or 1b in 87% yield. The total yields for the prepara-
tion of building blocks 1a and 1b were 76% and 80% after three
steps, respectively, and the building block 2 was prepared in 88%
total yield based on the Carrington’ method.13b The ratio of
a:b iso-
mer of 2 was 1:8 and the b isomer was easily separated by silica gel
column chromatography.
Several classical Cu(I) systems were chosen to catalyze the
Huisgen [3+2] cycloaddition reaction between 1a and 2 (Table 1).
The reaction of 1a with 2 catalyzed by CuI/DIPEA in CH3CN at
20 °C for 3 h afforded compound 3a in the yield of 95%, which
was higher than the yields by using other catalytic systems. The
b-configuration in ribofuranoside moiety of 3a was retained during
this reaction condition. Moreover, the click reaction is highly regio-
selective and only 1,2,3-triazole-4-amide derivative 3a was
formed. The structure of compound 3a was confirmed by HMBC
O
O
S
P
S
O
S
P
S
O
iii
HO
ii
HO
i
HN
X
NHBoc
X
X NHBoc
X NH
2
86% a
88% b
92% a
93% b
97% a
98% b
O
9a, X=CH
9b, X=OCH ,
2
8a, X=CH
7a, X=CH
7b, X=OCH
2
2
2
8b, X=OCH
2
2
1a, X=CH
2
1b, X=OCH
2
HO
N
O
3
AcO
AcO
HO
OAc
N
3
O
O
OH
O
vi
v
iv
a
97%
O
99%
92%
O
OAc
10
OAc
11
AcO
AcO
OH OH
2
Scheme 2. Synthetic route of building blocks 1 and 2. Reagents and conditions: (i) (Boc)2O, (CH3CH2)3N; (ii) PSS, TPSCl, Tetrazole; (iii) (a) CF3COOH, CH2Cl2; (b) propiolic acid,
DCC, CH2Cl2; (iv) Ac2O, Py, 80 °C; (v) (a) HCl (gas), (CH3CH2)2O, 0 °C; (b) NaN3, CH3CN, reflux; (vi) (a) K2CO3, CH3OH; (b) H+/acetone. a For b and
isomer.
a

L. Li et al. / Tetrahedron Letters 49 (2008) 4491–4493
4493
Table 1
2. (a) Dodd, A. N.; Gardner, M. J.; Hott, C. T.; Hubbard, K. E.; Dalchau, N.; Love, J.;
Assie, J. M.; Robertson, F. C.; Jakobsen, M. K.; Goncalves, J.; Sanders, D.; Webb,
A. A. R. Science 2007, 318, 1789–1792; (b) Guse, A. H. FEBS J. 2005, 272, 4590–
4597; (c) Potter, B. V. L.; Walseth, T. F. Curr. Mol. Med. 2004, 4, 303–311.
3. Shuto, S.; Matsuda, A. Curr. Med. Chem. 2004, 11, 827–845.
4. (a) Wagner, G. K.; Black, S.; Guse, A. H.; Potter, B. V. L. Chem. Commun. 2003,
1944–1945; (b) Wagner, G. K.; Guse, A. H.; Potter, B. V. L. J. Org. Chem. 2005, 70,
4810–4819.
5. (a) Shuto, S.; Shirato, M.; Sumita, Y.; Ueno, Y.; Matsuda, A. J. Org. Chem. 1998,
63, 1986–1994; (b) Fukuoka, M.; Shuto, S.; Minakawa, N.; Ueno, Y.; Matsuda, A.
J. Org. Chem. 2000, 65, 5238–5248.
6. (a) Kudoh, T.; Fukuoka, M.; Ichikawa, S.; Murayama, T.; Ogawa, Y.; Hashii, M.;
Higashida, H.; Kunerth, S.; Weber, K.; Guse, A. H.; Potter, B. V. L.; Matsuda, A.;
Shuto, S. J. Am. Chem. Soc. 2005, 127, 8846–8855; (b) Shuto, S.; Fukuoka, M.;
Manikowsky, A.; Ueno, Y.; Nakano, T.; Kuroda, R.; Kuroda, H.; Matsuda, A. J. Am.
Chem. Soc. 2001, 123, 8750–8759.
Cu(I)-catalyzed Huisgen 1,3-cycloaddition reaction between
different catalyst systems
1 and 2a to 3a in
Entry Catalyst system
Temperature (°C) Time (h) Yield of
3a (%)
1
2
CuSO4ꢁ5H2O, Cu, H2O/tBuOH
20
CuSO4ꢁ5H2O, sodium ascorbate 20
H2O/tBuOH
24
24
81a
65a
3
4
5
CuI, TEA, CH3CN
CuI, DIPEA, CH3CN
None catalyst, toluene
20
20
80
24
3
48
85a
95a
50b
a
Separated yield.
The 5-triazole amide-isomer was given in 25% yield from 1H NMR.
b
7. Guse, A.; Gu, X.; Zhang, L.-R.; Weber, K.; Zhang, L.-H. J. Biol. Chem. 2005, 280,
15952–15959.
8. Gu, X.-F.; Yang, Z.-J.; Zhang, L.-R.; Kunerth, S.; Fliegert, R.; Weber, K.; Guse, A.
H.; Zhang, L.-H. J. Med. Chem. 2004, 47, 5674–5682.
9. Xu, J.-F.; Yang, Z.-J.; Dammermann, W.; Zhang, L.-R.; Guse, A. H.; Zhang, L.-H. J.
Med. Chem. 2006, 49, 5501–5512.
10. Wong, L.; Aarhaus, R.; Lee, H. C.; Walseth, T. F. Biochim. Biophys. Acta 1999,
1472, 555–564.
11. Bock, V. D.; Hiemstra, H.; Maarseveen, J. H. Eur. J. Org. Chem. 2006, 51–58.
12. (a) Kolb, H. C.; Sharpless, K. B. Drug Discov. Today 2003, 8, 1128–1137; (b) Tron,
G. C.; Pirali, T.; Billington, R. A.; Canonico, P. L.; Sorba, G.; Genaz-zani, A. A.
Med. Res. Rev. 2008, 28, 178–308.
13. (a) Ponnusamy, E.; Fotadar, U.; Spisni, A.; Fait, D. Synthesis 1986, 48; (b)
Carrington, R.; Shaw, G.; Wilson, D. V. J. Chem. Soc. 1965, 6864–6879.
14. Spectroscopic data of compound 3a–6a. Compound 3a 1H NMR (500 MHz,
CDCl3), d 8.09 (s, 1H), 7.32–7.56 (m, 10H), 5.82 (d, J = 8.5 Hz, 1H), 5.41 (d,
J = 7 Hz, 1H), 4.76–4.78 (m, 1H), 4.68–4.72 (m, 1H), 4.45–4.48 (m, 1H), 4.30–
4.37 (m, 1H), 4.20–4.27 (m, 1H), 4.05 (dd, J = 3, 13 Hz, 1H), 3.78–3.81 (dd, J = 3,
13 Hz, 1H), 3.40–3.48 (m, 1H), 3.23–3.29 (m, 1H), 1.62–1.78 (m, 7H,), 1.43 (s,
3H). 13C NMR (125 MHz, CDCl3), 160.3, 141.8, 135.3, 129.5, 126.4, 126.2, 110.6,
86.2, 74.1, 73.4, 68.1, 66.8, 65.3, 38.5, 27.7, 26.5, 25.3, 25.0. 31P NMR (CDCl3,
121.5 Hz, decoupled with 1H), 51.1 (s). Anal. Calcd for C27H33N4O7P1S2: C,
52.25; H, 5.36; N, 9.03. Found: C, 52.02; H, 5.54; N, 9.06. Compound 4a 1H NMR
(500 MHz, DMSO-d6), d 8.87 (s, 1H), 8.48 (s, 1H), 7.11–7.51 (m, 5H), 4.96–4.97
(m, 1H), 5.89 (d, J = 8.5, 1H), 4.94 (d, J = 7 Hz, 1H), 4.46 (d, J = 7.5 Hz, 1H), 4.17
(d, J = 12.5 Hz, 1H), 3.70–3.72 (m, 2H), 3.62 (d, J = 12.5 Hz, 1H), 3.19–3.20 (m,
2H), 1.46 (m, 7H), 1.31 (s, 3H). 13C NMR (75 MHz, DMSO-d6), 159.6, 142.8,
135.4, 131.5, 128.1, 125.3, 108.8, 84.3, 73.2, 72.4, 68.6, 64.5, 57.3, 38.1, 27.7,
26.3, 25.8, 24.9. 31P NMR (D2O, 121.5 Hz, decoupled with 1H), d 20.8 (s), 3.5 (s).
NMR. A coupling peak between 5C in the 1,2,3-triazole ring and 10H
in southern ribose was observed, which supported the formation of
1,2,3-triazole-4-amide of compound 3a. However, 1,2,3-triazole-5-
amide, an isomer of compound 3a, was obtained with 25% yield in
Huisgen dipolar cycloaddition reaction when the reaction was car-
ried out in toluene at 80 °C without Cu+ as catalyst (Table 1). Com-
pound 3b was obtained under the same condition in 95% yield.
The precursor 4a for the formation of pyrophosphate was
obtained from compound 3a in 75% yield by using POCl3/DIPEA
in CH3CN at 0 °C for 12 h, followed by treatment of 0.1 M TEAB.
More interestingly, the phosphorylation of 50-OH of the southern
ribose and the partial deprotection of S,S-diphenylphosphate were
completed by a one-pot reaction. The same procedure was applied
for the synthesis of 4b resulting in the yields of 70%.
The intramolecular cyclization was performed in the presence
of excess I2 and 3 Å molecular sieves in pyridine by adding a solu-
tion of compound 4a over 20 h using a syringe.9 The cyclic product
5a was purified by HPLC as its triethylammonium salt in 75% yield.
The structure of 5a was confirmed by ESI-MS+, 31P NMR and 1H
NMR. Finally, the removal of isopropylidene group of 5a was car-
ried out with 50% HCOOH in water at room temperature for 2 h
to obtain the target compound 6a, cTDPRC. 6a was purified by
HPLC as its triethylammonium salt in 80% yield. The total yield of
cTDPRC 6a was 42.7% from building blocks 1a and 2.14
Similarly, compound cTDPRE 6b was prepared by the same
strategy. The total yield of cTDPRE 6b is 41.5% from building blocks
1b and 2.15
In summary, novel nucleobase-modified cADPR mimics 6a and
6b were synthesized. Cu(I)-Huisgen cycloaddition (click reaction)
was used to construct 4-amide-1,2,3-triazole nucleobase and con-
nect two building blocks efficiently. A concise protection strategy
was used for the synthesis of the corresponding cyclo-pyrophos-
phate, and the target compounds 6a and 6b were prepared within
four steps in more than 41.5% total yields.
HRMS (ESI) m/z calcd for
C21H31N4O11P2S1: 609.1180; found, 609.1179.
Compound 5a 1H NMR (500 MHz, CD3OD) d 9.13 (s, 1H), 6.09 (d, J = 9.5 Hz,
1H), 5.25 (dd, J = 3, 7 Hz, 1H), 5.37–5.41 (m, 1H), 4.77–4.80 (m, 1H), 4.51–4.57
(m, 3H), 4.12 (d, J = 12.5 Hz, 1H), 3.75–3.80 (m, 2H), 1.72–1.89 (m, 4H), 1.39,
1.55 (each s, each 3H). 31P NMR (D2O, 121.5 Hz, decoupled with 1H), d ꢀ6.78 (d,
Jp,p = 13.5 Hz), ꢀ8.69 (d, Jp,p = 13.5 Hz). HRMS (ESI) m/z calcd for
C
15H25N4O11P2: 499.0989; found, 499.0984. Compound 6a 1H NMR
(500 MHz, D2O) d 8.68 (s, 1H), 6.04 (d, J = 9 Hz, 1H), 4.53–4.57 (m, 2H), 4.11–
4.17 (m, 1H), 3.94–4.03 (m, 2H), 3.82–3.88 (m, 1H), 3.67–3.73 (m, 1H), 3.54–
3.5 (m, 1H), 3.37–3.42 (m, 1H), 1.71–1.90 (m, 4H). 31P NMR (81 MHz, D2O) d
ꢀ10.64 (br s), ꢀ11.59 (br s). HRMS (ESI) m/z calcd for
C12H19N4O11P2:
457.0531; found, 457.0526.
15. Spectroscopic data of compound 3b–6b. Compound 3b 1H NMR (500 MHz,
CDCl3), d 8.15 (s, 1H), 7.34–7.55 (m, 10H), 5.82 (d, J = 8.5 Hz, 1H), 4.75 (dd,
J = 3.5, 7 Hz, 1H), 4.56–4.59 (m, 1H), 4.52–4.48 (m, 1H), 4.33–4.36 (m, 2H), 3.00
(dd, J = 13, 3 Hz, 1H), 3.82 (dd, J = 3, 13 Hz, 1H), 3.69–3.71 (m, 2H), 3.55–3.65
(m, 4H), 1.43, 1.61 (each s, each 3H). 13C NMR(75 MHz, CDCl3), 160.2, 142.1,
135.3, 129.5, 126.3, 126.0, 121.5, 110.6, 86.2, 73.9, 73.3, 69.6, 67.2, 67.0, 66.9,
65.2, 39.0, 26.6, 25.0. 31P NMR (CDCl3, 121.5 Hz, decoupled with 1H), 51.05 (s).
Anal. Calcd for C27H33N4O8P1S2: C, 50.93; H, 5.22; N, 8.80. Found: C, 51.08; H
5.42; N, 8.71. Compound 4b 1H NMR (500 MHz, D2O), d 8.57 (s, 1H), 7.30–7.55
(m, 5H), 6.03 (d, J = 8 Hz, 1H), 5.08 (dd, J = 3, 8 Hz, 1H), 4.94–4.98 (m, 1H), 4.69
(d, J = 8 Hz, 1H), 4.26 (dd, J = 13.5, 1 Hz, 1H), 4.07–4.23 (m, 2H), 3.87 (dd, J = 1,
13.5 Hz, 1H), 3.72–3.74 (m, 2H), 3.70 (t, J = 11 Hz, 2H), 3.56 (t, J = 11 Hz, 2H),
1.46–1.60 (each s, each 3H). 31P NMR (D2O, 81 Hz), d 18.31 (br s), 2.75 (br s).
Primary pharmacological research showed that 6a cTDPRC and
6b cTDPRE could introduce Ca2+ release in intact human Jurkat T
cell,16 and the further pharmacological study will be discussed
elsewhere.
Acknowledgements
HRMS (ESI) m/z calcd for
C21H29N4O12P2S1: 623.0983; found, 623.0984.
Compound 5b 1H NMR (300 Hz, D2O), d 8.48 (s, 1H), 5.97 (d, J = 8 Hz, 1H),
4.81–4.89 (m, 2H), 4.57–4.60 (m, 1H), 4.23 (d, J = 13 Hz, 1H), 3.85 (d, J = 13 Hz,
1H), 3.39–3.60 (m, 8H), 1.30, 1.45 (each s, each 3H). 31P NMR (D2O, 81 Hz), d
ꢀ9.11 (br s), ꢀ11.4 (br s). HRMS (ESI) calcd for C15H25N4O12P2 m/z: 515.0939;
found, 515.0925. Compound 6b 1H NMR (500 MHz, D2O), d 8.67 (s, 1H), 6.04 (d,
J = 9 Hz, 1H), 4.54–4.56 (m, 2H), 4.14–4.18 (m, 1H), 3.93–4.02 (m, 2H), 3.80–
3.87 (m, 2H), 3.69–3.73 (m, 2H), 3.53–3.66 (m, 4H). 31P NMR (D2O, 81 Hz)
ꢀ8.91 (br s), ꢀ11.39 (br s). HRMS (ESI) m/z calcd for C12H19N4O12P2: 473.0480;
found, 473.0477.
This study was supported by the National Natural Sciences
Foundation of China (Grant no. 20332010) and the Ministry of
Science and Technology of China (Grant no. 2005BA711A04).
References and notes
1. (a) Guse, A. H. Curr. Mol. Med. 2004, 4, 239–248; (b) Lee, H. C. In Cyclic ADP-
Ribose and NAADP: Structures, Metabolism and Functions; Kluwer Academic
Publisher, 2002; pp 217–444.
16. The biological activity of cTDPRC 6a and cTDPRE 6b was assessed in intact
human Jurkat T-lymphocytes as described previously.8,9