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Solid Phase Synthesis of Substituted Adenine Phosphates as P2 Receptor Ligands

Ram Chandra Mishra, Diego Dal Ben, Catia Lambertucci, Floriana Rosa Portino, Rosaria Volpini, Sauro Vittori, Gloria Cristalli
Department of Chemical Sciences, University of Camerino,
Via S. Agostino 1, 62032 Camerino (MC) ITALY
Presenting Author E-mail: sauro.vittori@unicam.it

Purinergic (Purine and Pyrimidine) Receptors


Introduction ectonucleotidases
Adenosine kinase

The purinergic receptors are broadly classified into P1 and P2 receptor types. Adenosine is the ATP ADP AMP Adenosine
natural ligand of the P1 receptors, while corresponding nucleotide ATP is natural ligand for P2 class of
receptors (Figure 1). Many derivatives of the 9-ethyl adenine synthesized in our laboratory had shown UTP,UDP,UDP-glucose
to have very good binding affinities towards the P1 receptors.1,2

Other well known antagonists of the P1 receptors are the derivatives of natural xanthines, among
which caffeine and theophilline are of significance.3 These molecular skeletons with substitutions on
various positions have lead to the development of potent and selective P1 receptor antagonists.
P2Y1,2,11,12,13 A1 A2A A2B A3
P2X1-7
Moreover, phosphorylated analogues of these compounds, which are commonly known as ‘mini P2Y2,4,6,14
nucleotides, had shown high affinity for the P2 family of receptors, specifically the P2X subclass.3 P2 receptor family P1 receptors

Figure 1

Present Work The monophosphates derivatives were obtained by reaction with phosphorous oxychloride in
On all the above and for the development of new P2 receptor ligands, we were prompted to trimethylphosphate, under nitrogen stream at room temperature, according to the Yoshikawa method.
synthesize some substituted adenine phosphates, which would be prospective P2 receptor ligands. Phosphorylation on solid phase using Yoshikawa procedure did not work. We are modifying the conditions
to develop a method for solid phase phosphate synthesis. The preparation of the diphosphate and
To begin with, we used the 2,6-dichloropurine as the starting material, which was derivatized at
triphosphate (Hoard-Hott method) is outlined in Scheme 2.
various positions before being phosphorylated. The phosphorylation was done on these derivatives to
obtain the corresponding mono, di and tri phosphates; on the same time attempts were made to
obtain these molecules on solid support.

HN
l
NH2 POC 3 N
NH2 N TMP N
N
O OMe M N N Cl
N /DC HO
Rink Amine Linker R N OH POCl3
NH2 T FA
%
TMP N 30
N SP 3
O
Cross linked Solid Support R N N O P O NH
4
(Polystyrene with DVB) OMe OH

PF 70 & 73
O
Figure 2: Rink Amine Resin Bu3NH H2P2O3 (Bu3NH)2 H2P2O7

N N N N
The use of solid support in synthesis of organic compounds, also known as the SPOS (Solid NH2 NH2
Phase Organic Synthesis) has been beneficial in faster synthesis of various classes of
N N
compounds.4 In the present case we have applied the use of Rink Amine resin for the synthesis of N N
O O O O O
substituted adenine phosphate (mini nucleotides). R N N N
O P O P O R N O P O P O P O
O O O O O
Cl x 3 NH4
PF 72 & 75 x 4 NH4
N PF 71 & 74
N
PF 70, 71, 72 R = Cl,
TMP = Trimethyl Phosphate
Cl N N PF 73, 74, 75 R =-O(CH2)2Ph
Ri n
H kA
NH3 liq. min Scheme 2
1 DI eR
PE esin
NH2 A, HN
85 % DM
N F N Biological Activity
N N
30 % TFA/DCM
Cl N N Cl N N
H H 100
2 SP 2 1uM 50uM 500uM

4-Bromobutyl acetate
4-Bromobutyl acetate
K2CO3, DMF, 80 oC K2CO3, DMF, rt, 24h
75
1uM 50uM

NH2 12 % (3)
viable cells (%)

N 64 % (4)
N 50 500uM

N NH
Cl N OCOCH3 +4
30 % TFA/DCM N N
3 25
Ph Cl N N OCOCH3
en
et
hy SP 3
NH3/MeOH la
98 % m
in 0
e/
al NH3 in Dioxane T PF70 w T PF72 w T PF74 w
NH2 co T PF73 w
ho
l rt, 12h
N N
HN T = Transfected, W = Wild type
Cl N N OH
NH2 N N

N N
Cl N N OH
Figure 3
4
R N N OH

SP 4 Results/Conclusion
30 % TFA/DCM We have synthesized 2,9-disubstituted adenine mono, di and tri phosphates, and developed a method to
load on solid support and derivatize the 2,6-dichlopurine leading to the synthesis of key intermediate for
A 5, R = -O(CH2)2Ph, 79 % B the Solid phase synthesis of mini nucleotides.
6, R = -NH(CH2)2Ph, 77 %
With respect to conventional synthesis, solid phase strategy in the present study needed milder reaction
Scheme 1 conditions, yielded sufficiently pure products without chromatography. All the compounds were
characterized using spectroscopic and chromatographic (TLC, HPLC) techniques.

The synthetic strategy for the preparation of substituted adenines starts with the commercially Preliminary screening of synthesized adenine mininucleotides was performed on P2Y4 receptors,
available 2,6-dichloropurine. The traditional approach, as well as the solid phase approach, overexpressed in human neuroblastoma cells (Figure 3). The activity was compared with the same on wild
are described in Scheme 1. type cells. Compounds PF 70 and PF 74 showed slight cytoxicity, proving the interaction of these
compounds with P2Y4 receptors.

References

[1] Camaioni E; Di Francesco E; Vittori S; Volpini R.; Klotz K-N; Cristalli G. New substituted 9-alkylpurines as adenosine receptor ligands Bioorg. Med. Chem. 1998; 6: 523-533.
[2] Volpini R; Costanzi S; Lambertucci C; Vittori S; Martini C; Trincavelli M L; Klotz K-N; Cristalli G. 2- And 8-alkynyl-9-ethyladenines: Synthesis and biological activity at human and rat adenosines receptors Purinergic Signalling 2005; 1: 173-181.
[3] Fischer B; Yefidoff R; Major D T; Rutman-Halili I; Shneyvays V; Zinman T; Jacobson K A; Shainberg, A. Characterization of "Mini-Nucleotides" as P2X Receptor Agonists in Rat Cardiomyocyte Cultures. An Integrated Synthetic, Biochemical, and
Theoretical Study J. Med. Chem. 1999; 42: 2685-2696.
[4] Dörwald F. Z; Organic Synthesis on Solid Phase: Supports, Linkers, Reactions, Wiley-VCH, March 2000.