Synthesis of potential purinoceptor antagonists: Application of P1-tBU phosphazene base for alkylation of adenine in solution and on solid phase

Article abstract of DOI:10.1080/15257770500446857

Alkylation of adenine in solution and on solid phase was accelerated by phosphazene base P1-tBu compared to mineral bases. The reactions in solution afforded regioselectively the appropriate N9-alkylated adenines with high preparative yields while the rea

Full text of DOI:10.1080/15257770500446857

Nucleosides, Nucleotides, and Nucleic Acids, 25:141–157, 2006
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770500446857
SYNTHESIS OF POTENTIAL PURINOCEPTOR ANTAGONISTS:
APPLICATION OF P1-tBU PHOSPHAZENE BASE FOR ALKYLATION
OF ADENINE IN SOLUTION AND ON SOLID PHASE
Erki Enkvist, Gerda Raidaru, and Asko Uri
2
Institute of Organic and Bioorganic
Chemistry, University of Tartu, Tartu, Estonia
Roshni Patel, Catherine Redick, and Jos e´ L. Boyer
2
Inspire Pharmaceuticals
Inc., Durham, North Carolina, USA
Juhan Subbi and Indrek Tammiste
2
Institute of Chemical Physics and Biophysics,
Tallinn, Estonia
2
Alkylation of adenine in solution and on solid phase was accelerated by phosphazene base P1-
tBu compared to mineral bases. The reactions in solution afforded regioselectively the appropriate
N9-alkylated adenines with high preparative yields while the reaction with polystyrene resin-bound
N-bromoacetylated peptides gave three regioisomers (alkylated at the N9, N7, and N3 position of
adenine) in a 4:2:1 molar ratio. Ten novel nonphosphate nucleotide analogues were tested in an
ADP-induced platelet aggregation assay.
Keywords Adenine alkylation; Phosphazene base; Nucleotide analog; Solid phase
synthesis
INTRODUCTION
Adenine nucleosides and nucleotides bind to intracellular proteins,
which participate in important functions such as signalling, metabolism, and
cell proliferation. These adenine derivatives also play important roles in the
extracellular milieu where they interact with almost 20 different membrane
receptor proteins, purinoceptors, and an unknown number of ectoenzymes
[
1]
including kinases and phosphatases. Structure-activity studies have shown
that nucleotide-selective P2X and P2Y receptors require that the ligands con-
tain a negatively charged region (the polyphosphate chain) concentrated
in close proximity to the base moiety. Small differences in the number of
Received 22 July 2005; accepted 1 August 2005.
This work was supported by grants from the Estonian Science Foundation and the Estonian Ministry
of Education and Sciences (0182611s03).
Address correspondence to Asko Uri, Institute of Organic and Bioorganic Chemistry, University of
Tartu, 2 Jakobi St., Tartu, EE 51014, Estonia. E-mail: asko.uri@ut.ee
1
41

142
E. Enkvist et al.
negative charges or the spatial arrangement of these pharmacophore ele-
ments may cause complete loss of affinity or dramatic changes in pharma-
cological activity of the ligand from an agonist to an antagonist. Sequence
conservation of positively charged amino acids in P2 nucleotide receptors
supports the idea of important electrostatic interactions between the recep-
[
2]
tor and the negative charges of the polyphosphate moiety of nucleotides.
Recently, acyclic nucleotide analogues containing no sugar moiety have been
[
2,3]
synthesized and tested as ligands of nucleotide receptors.
Remarkable
antagonistic activity has been demonstrated for several of these compounds.
We and others have shown that phosphate groups of nucleotides can be
replaced with nonhydrolyzable carboxylate groups in antagonists of P2 nu-
cleotide receptors.[
4–6]
To combine two latter approaches we report here the
development of synthetic methods for preparation of acyclic nonphosphate
nucleotide analogues.
Derivatization of adenine has been an important strategy for the devel-
opment of selective ligands for the regulation of the activity of pharmaco-
logically important proteins. The alkylation in neutral medium gives mostly
N3-substituted products while the reaction of adenine anion leads to N9 and
N7 derivatives.[ Mineral hydrides and carbonates are the most frequently
used bases for the activation of adenine in alkylation reactions, giving more
or less regioselectively and with moderate yields the N9 derivatives, the most
common in biological systems. Due to low solubility of adenine and its metal
salts in organic solvents these reactions require long times or high tempera-
tures to proceed. Thus, these reagents are not well applicable for solid phase
synthesis where good swelling of the polymer resin and sufficient solubility
of the reagent in the same solvent are prerequisites for the exploitation of
an organic reaction.
7]
The recent use of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) for the alky-
lation of adenine in dimethylformamide (DMF) led to improvements in
[
8]
yield and regioselectivity of the reactions. Inasmuch as the basicity of
DBU is insufficient for full deprotonation of adenine in non-hydroxylic sol-
vents dimethyl sulfoxide (DMSO), DMF, and acetonitrile (pK = 14.2 for
a
[
9]
[10]
adenine and pK
= 13.9 for DBU
in DMSO), excess of the base,
BH+
higher temperatures, and longer reaction times are required for quantita-
tive alkylation of adenine. The propensity of DBU to alkylation under these
conditions limits its use as an ionizing base for the synthesis on solid phase.
Schwesinger phosphazene bases[ have been successfully used in dif-
ferent applications both in solution (including alkylation of derivatives of
11]
12]
[13]
2
-aminopurine[ ) and on a solid phase.
As a special class of strong or-
ganic nonionic bases, these compounds possess some useful characteristics
for practical applications: their basicity can be varied over a wide pK range,
a
they are not prone to alkylation, and their cationic forms are well soluble in
nonhydroxylic solvents. Commercial availability of phosphazenes with differ-
[
14]
ent basicity (well established in physicochemical studies ) makes it possible

Synthesis of Potential Purinoceptor Antagonists
143
to deprotonate the reagent selectively and quantitatively at the most acidic
site in the presence of the equimolar amount of the base.
Adenine-9-acetic acid is a building block of several biologically active
[
15]
structures like peptide nucleic acid (PNA) monomers
and P2Y recep-
tor antagonists.[ Derivatives of adenine-9-acetic acid are usually prepared
by alkylation of adenine with an ester or an amide of halogenoacetic acid
in a basic medium and further transformation of the intermediates to the
desired products. The present study describes the application of a phos-
phazene base for the solubilization and deprotonation of adenine, which
leads to substantial acceleration of the reaction of adenine with bromoacety-
lated substrates in solution and to the first successful application of adenine
alkylation reaction on solid phase. P1-tBu phosphazene [tert-butylimino-
tris(dimethylamino)phosphorane] was chosen as the ionizing base proceed-
16]
[
10]
ing from its suitable basicity characteristics (pK
= 15.7 in DMSO ):
BH+
only small excess of the base is sufficient for complete deprotonation of
adenine. The method was used for the synthesis of nucleotide analogues,
derivatives of adenine with different positioning of negatively charged car-
boxylate groups. The newly synthesized compounds were tested as potential
inhibitors of platelet aggregation promoted by the activation of adenine nu-
cleotide receptors.
RESULTS AND DISCUSSION
Addition of phosphazene P1-tBu to the suspension of purines in DMF
resulted in complete solubilization of N6-methyladenine and partial sol-
ubilization of adenine. The following reaction with t-butyl bromoacetate
(Scheme 1) was completed within seconds. Esters of adenine-9-acetic acids
1
a and 1b were isolated as single N9-regioisomers with high yields (78% and
9
4%). The conversion of the starting adenines in the presence of the same
molar quantity of DBU was less effective. Alkylations with NaH were slower
and resulted in 1a and 1b with preparative yields 55% and 81%, respectively.
Reactions under the conditions where no base was added required
◦
[7]
higher temperatures (40–65 C, 2–6 h) to proceed and gave expectedly
N3-substituted derivatives 2a–b (2a 46%, 2b 67%) as the main products. Only
traces of the N9-isomer could be detected. The reaction in the presence of di-
isopropylethylamine (trialkylamines are approximately 4–5 pK units weaker
a
[
9]
bases than DBU and adenine anion in DMSO ) yielded a complicated
mixture of products at 70 C in 2 h. 1a–b were treated with trifluoroacetic
◦
acid (TFA) to remove t-butyl groups and the obtained adenine-9-acetic acids
(3a–b) were used in further reactions.
Adenine-9-acetic acid was attached to peptides via two different ap-
proaches (Scheme 2): either 3a and 3b were reacted with polymer-bound
peptides or the peptide was first bromoacetylated by the previously described
[
17]
procedure,
which was followed by the reaction with adenine anion in

144
E. Enkvist et al.
◦
SCHEME 1 Reagents and conditions: (a) P1-tBu phosphazene base, DMF, RT. (b) DMF, 40–65 C, 2–6 h.
c) TFA.
(
DMF in the presence of P1-tBu phosphazene base. Finally, the products were
cleaved from the Wang resin and the protection groups were removed with
9
5% TFA. The alkylation of adenine on solid support was quick and gave
high yields, but the reactions produced mixtures of regioisomers (N9-, N7-,
and N3-alkylated products in a 4:2:1 molar ratio, Figure 1). The application
of DBU afforded worse solubilization of adenines and gave lower yields of
alkylation of adenine due to the reaction of DBU with bromoacetylated pep-
tide. The molar ratio of the obtained adenine regioisomers was similar in
the case of both applied bases. The separation of the target 9-regioisomer
by C18 reverse-phase HPLC (elution with water-acetonitrile gradient in the
presence of 0.1% TFA) was successful for only some of the peptide conjugates
(e.g., 4c). Better separation of the isomers was achieved in the presence of
acetate buffer (AcOH/AcONa, pH = 5.0–5.5) where three peaks could be
distinguished for acetyl-glycine derivative of N6-methyl-adenine (4b). The
adenine moiety of the N3-alkylated derivatives is protonated at this pH while
[
7]
those of N7 and N9 derivatives are predominately neutral. The position
of adenine alkylation was established on the basis of analysis of NMR and
UV spectra of the compounds.[ Absorption maximums in the UV spectra
of the chromatographically separated isomers at pH 5.5 were the following:
18]
2
66 nm (N6-MeAde-9-AcGly) 4b, 278.5 nm (N6-MeAde-7-AcGly), and 283
nm (N6-MeAde-3-AcGly). Two rotamers related to C6-N6 bonds could be
detected by the NMR spectra of N3-, N6-disubstituted adenines (2b and N3-
isomers of 4b–d).[
19]

145

146
E. Enkvist et al.
1
FIGURE 1 Fragments from H NMR (D2O) spectrum of the product of alkylation of adenine on solid
phase (aspartic acid derivative of adenine acetic acid 4a). The signals of three regioisomers (N9-alkyl,
N7-alkyl, and N3-alkyl) are visible.
Derivatives of adenine-9-acetic acid containing two or more carboxy-
late moieties were synthesized according to Scheme 3. Bromoacetylation
of dibenzyl iminodiacetate[20] in the presence of diisopropylcarbodiimide
(DIC) led to the amide 5. Reactions of 5 with adenine or N6-methyladenine
in the presence of bases (NaH, K CO , or phosphazene P1-tBu base) resulted
2
3
in adenine derivatives 6a–b. The application of the organic base gave a higher
yield and the reaction was completed in a shorter time. The following hy-
drogenation of dibenzyl esters 6a–b on Pd/C led to diacids 7a–b that were
coupled with di-t-butyl ester of l-aspartic acid using DIC activation. The prod-
ucts were treated with TFA and, after removal of the solvent, were purified
by HPLC. Two pairs of individual compounds could be separated (8a–b and
9
a–b). DIC activation without auxiliary reagents like 1-hydroxybenzotriazole
was obviously not an appropriate method for derivatives iminodiacetic acid
as it gave low yields. Compounds 9a–b were N-acylurea derivatives of 7a–b
that had formed as side products during the activation of carboxylate groups
with the carbodiimide. The removal of the N-acylurea moiety from 9a–b by
[
21]
hydrolysis in Ca(OH) solution
yielded 10a–b, derivatives of adenine with
2
three carboxylate groups.
The new acyclic nonphosphate nucleotide analogues were evaluated for
activity in human platelet aggregation assay. Compounds 4e, 7a, 8a, and 10b
produced a small inhibition of ADP-induced platelet aggregation (Table 1)
mediated via P2Y and P2Y receptors. The mechanism of the inhibitory
1
12
effect of these compounds has not yet been determined.
EXPERIMENTAL
Materials and Methods
1H and 13C NMR spectra were taken on Bruker AC 200P spectrome-
ter. Masses of all synthesized compounds were measured with MALDI-TOF

147

148
E. Enkvist et al.
TABLE 1 Inhibition of Platelet Aggregation (Conditions
in Experimental)
Inhibition (%) of platelet
Compound
aggregation at 100 μM (N = 3)
4
4
4
7
8
1
g, 7b
c, 4d, 8b, 10a
e
a
a
0b
NE
NR
28.7 ± 8.1
11.7 ± 6.0
9.7 ± 4.8
53.3 ± 3.3
NE = No effect, NR = small and not reproducible effect,
N = number of measurements.
mass spectrometer (built at the National Institute of Chemical Physics and
Biophysics, Tallinn, Estonia). UV spectra were taken on Specord M40 (Carl
Zeiss, Jena) or Unicam UV 300 (ThermoSpectronic) spectrometers. ReacR-
ꢀ
tions were monitored by thin-layer chromatography (TLC) on POLYGRAM
SIL G/UV254 plates. UV light was used for the visualization of the products.
ꢀ
R
Langford Sonomatic 375H ultrasonic bath was used for sonication. Column
chromatography was performed on silica gel 60 (0.04–0.063 mm) from Fluka.
The final products were purified with Gilson HPLC system using C18 reverse-
phase column (GL Sciences) Inertsil ODS-3 (5 μm, 25 × 0.46 cm) with mon-
itoring at 260 nm. Elution was performed with water-acetonitrile gradient
(0.1% TFA), flow rate 1 mL/min. The separated products were freeze-dried.
All compounds used in biological tests were >95% pure by HPLC (detected
at 260 nm, Table 2). Adenine, diisopropylethylamine (DIEA), and t-butyl
bromoacetate were purchased from Sigma; celite from Aldrich; derivatives
of amino acids and other chemicals used in peptide synthesis were from
Advanced ChemTech and Nova Biochem; acetone from Merck; DBU, DMF,
bromoacetic acid, sodium hydride, N6-methyladenine, P1-tBu phosphazene,
and DIC from Fluka. All other HPLC grade solvents were from Rathburn
Ltd. Derivatives of
L-aspartic acid were used for the preparation of adenine-
peptide conjugates.
Biological Testing of Compounds
Blood was collected from healthy volunteers into syringes containing 1/6
final blood volume of anti-coagulant ACD (65 mM citric acid, 85 mM sodium
citrate, 110 mM dextrose). Washed platelets were prepared as previously
[
22]
described.
Briefly, ACD-anticoagulated blood was centrifuged at 275 g
for 16 min, the platelet-rich plasma was collected and centrifuged at 2200 g
for 13 min. The supernatant was removed and the platelet pellet resuspended
in Tyrode’s buffer containing 0.35% bovine serum albumin and 0.05 U/mL
potato apyrase. Platelet aggregation was measured using the optical mode of
a ChronoLog aggregometer (Havertown, PA). Five hundred microliters of

Synthesis of Potential Purinoceptor Antagonists
149
TABLE 2 MS and HPLC Data
MWcalc
(g/mol)
MS
M + H
RT
min
HPLC
Compound
Nr
Formula
purity2
60nm
(%)
AdeAcAsp
NMeAdeAcGly
NMeAdeAcAsp
4a
4b
4c
4d
4e
4f
C11H12N6O5
C10H12N6O3
C12H14N6O5
C16H19N7O8
C20H24N8O11
C14H17N7O6
C19H24N8O9
C20H20BrNO5
C25H24N6O5
C26H26N6O5
C11H12N6O5
C12H14N6O5
C19H22N8O11
C20H24N8O11
C22H31N9O8
C23H33N9O8
C15H17N7O8
C16H19N7O8
308.25
264.24
322.28
437.36
552.45
379.33
508.44
434.28
488.5
502.52
308.25
322.28
538.43
552.45
549.54
563.56
423.34
437.36
309
265
323
438
553
380
509
434
489
503
309
323
539
553
550
564
424
438
10.4
13.2
13.3
13.1
13.6
15.4
16.5
—
—
—
9.3
11.5
12.7
15.5
29.7
32.8
11.3
14.6
98
98
96
98
97
99
97
>92
>92
>92
96
96
98
99
95
96
99
95
NMeAdeAcAsp2
NMeAdeAcAsp3
NMeAdeAcAspGly
NMeAdeAcbeetaAlaAsp2
BrAcIdaBn2
AdeAcIdaBn2
NMeAdeAcIdaBn2
AdeAcIda
4g
5
&
&
&
6a
6b
7a
7b
8a
8b
9a
9b
10a
10b
NMeAdeAcIda
AdeAcIda(Asp)Asp
NMeAdeAcIda(Asp)Asp
N-acylurea derivative
N-acylurea derivative
AdeAcIdaAsp
NMeAdeAcIdaAsp
MWcalc = calculated molecular weight.
MS M + H = protonated monoisotopic peak in MALDI-TOF mass spectrum.
RT min = HPLC retention time in minutes using C18 reversed phase column Inertsil ODS-3, 5 μm,
5 × 0.46 cm (GL Sciences) with UV detection at 260 nm. Elution was performed with water-acetonitrile
2
linear gradient (flow rate 1ml/min) from 0% to 30% acetonitrile over 40 min.
&
1
=
purity estimated by H NMR.
8
platelet suspension at a density of 5 × 10 platelets/mL containing 1 mg/mL
◦
fibrinogen were warmed to 37 C and stirred at 1000 rpm. The effect of
inhibitors on platelet aggregation was studied by preincubating the platelet
suspension with the indicated concentration of the inhibitor for 2 min prior
to the addition of a maximally effective concentration of ADP (usually 1–5
μM, determined for each blood donor from concentration-effect curve).
Synthesis
t-Butyl Adenine-9-acetate (1a).
Adenine (27.2 mg, 0.20 mmol) and
phosphazene base P1-t-Bu (65 μL, 0.24 mmol; Fluka) were suspended in
DMF (2.5 mL). The suspension was stirred while bromoacetic acid t-butyl
ester (30 μL, 20.3 mmol) was added. A clear solution formed within seconds
and the solution was evaporated to dryness. The crude mixture was dissolved
in EtOAc and washed with a solution of KH PO /K HPO . Column chro-
2
4
2
4
matography (CHCl /MeOH10 : 2, R = 0.6) yielded 1a (39.3 mg, 77.7%).
3
f
Adenine (50 mg, 0.37 mmol) and 60% NaH (19.5 mg, 0.49 mmol) were
suspended in DMF (1.5 mL). The mixture was stirred for 30 min and then
bromoacetic acid t-butyl ester (66 μL, 0.45 mmol) was added. The stirring
was continued overnight and the solvent was removed in vacuo. The product

150
E. Enkvist et al.
was purified by column chromatography (CHCl /MeOH 10:2) to give 1a
3
1
(
(
(
(
50.6 mg, 55%). H NMR (CDCl )δ1.49(9H, s, tBu), 4.87(2H, s, CH ), 5.80
2H, br, NH ), 7.87 and 8.36 (2H, s, A2 and A8). C NMR (CDCl ) δ28.2
3
2
1
3
2
3
3C, tBu), 45.1 (CH ), 83.7 (tBu), 119.4 (A5), 141.1 (A8), 150.5 (A4), 153.4
A2), 155.7 (A6), 166.3 (COO).
2
t-Butyl N6-Methyladenine-9-acetate (1b).
N6-methyladenine (37.5 mg,
0
.25 mmol) and phosphazene base P1-t-Bu (81 μL, 0.32 mmol) were dis-
solved in DMF (1 mL). Bromoacetic acid t-butyl ester (45 μL, 0.30 mmol)
was added and the solution was stirred for 1 h. The solvent was removed
under reduced pressure. The crude mixture was dissolved in EtOAc and
washed with solution of KH PO /K HPO . The residue was purified by col-
2
4
2
4
umn chromatography (CHCl /MeOH 10:1) to give 1b (62 mg, 94%). N6-
3
Methyladenine (19.1 mg, 0.128 mmol) and 60% NaH (7.0 mg, 0.175 mmol)
were suspended in DMF (0.75 mL). The mixture was stirred for 30 min and
then bromoacetic acid t-butyl ester (23 μL, 0.156 mmol) was added. The
stirring was continued overnight and the solvent was removed in vacuo. The
product was purified by column chromatography (CHCl /MeOH 10:2) to
3
1
give 1b (27.2 mg, 81%). H NMR (CDCl ) δ 1.46 (9H, s, tBu), 3.20 (3H, br,
3
NCH ), 4.85 (2H, s, CH ), 6.02 (1H, br, NH), 7.80 and 8.40 (2H, s, A2 and
3
2
1
3
A8). C NMR (CDCl )δ27.9(NCH ), 28.2 (3C, tBu), 45.0 (CH ), 83.6 (tBu),
3
3
2
1
19.6 (A5), 140.3 (A8), 149.5 (A4), 153.6 (A2), 155.8 (A6), 166.4 (COO).
t-Butyl Adenine-3-acetate (2a). Suspension of adenine (40.5 mg, 0.30
mmol) and bromoacetic acid t-butyl ester (55 μL, 0.37 mmol) in DMF (2.1
◦
mL) was stirred for 2 h at 65 C. The solvent was removed under reduced pres-
sure and the residue was partitioned between EtOAc and aqueous K CO3.
2
The organic solution was collected and dried. The residue was purified by
column chromatography (CHCl /MeOH 10:2, R = 0.6) to give 2a (34.2 mg,
3
f
1
4
6%). H NMR (CD SOCD )δ1.41 (9H, s, tBu), 5.09 (2H, s, CH ), 8.03 (2H,
3 3 2
br, NH ), 7.75 and 8.32 (2H, s, A2 and A8). C NMR (CD SOCD )δ27.5
1
3
2
3
3
(
(
3C, tBu), 49.9 (CH ), 82.2 (tBu), 119.9 (A5), 144.0 (A2), 149.9 (A4), 152.3
A8), 154.9 (A6), 166.3 (COO).
2
t-Butyl N6-Methyladenine-3-acetate (2b).
Suspension of N6-methyl-
adenine (65 mg, 0.44 mmol) and bromoacetic acid t-butyl ester (98 μL,
◦
0
.66 mmol) in DMF (3.5 mL) was stirred for 7 h at 40 C. The solvent was
removed under reduced pressure. The residue was purified by column chro-
matography (CHCl /MeOH 10:2, R = 0.7) to give 2b (100.6 mg HBr salt,
3
f
1
6
7%). H NMR (CDCl )δ1.44 (9H, s, tBu), 3.22 (3H, br, NCH ), 5.01 (2H,
3 3
s, CH ), 7.96 and 8.00 (2H, s, A2 and A8), 9.42 (1H, br, NH). C NMR
1
3
2
(
CDCl )δ27.8 (NCH ), 28.1 (3C, tBu), 50.3 (CH ), 84.0 (tBu), 121.2 (A5),
3 3 2
1
43.3 (A2), 149.2 (A4), 153.0 (A8), 154.6 (A6), 165.7 (COO).

Synthesis of Potential Purinoceptor Antagonists
151
Synthesis of AdeAcAsp (4a), NMeAdeAcGly (4b), NMeAdeAcAsp (4c),
and NMeAdeAcAsp (4d). Wang-type peptide synthesis resin, Fmoc-Asp(t-
2
Bu)-resin (0.7 mmol/g; Advanced ChemTech) or Fmoc-Gly-resin (0.87
mmol/g; Advanced ChemTech), was swollen in DMF. Fmoc protection was
removed by double treatment of the resin with 20% piperidine in DMF
(
5 + 15 min). The following
L
-aspartic acids (in the form of Fmoc-Asp[t-
Bu]) were attached to the resin with BOP/HOBt activation (0.95 eq.) in the
presence of N-methylmorpholine (5%, v/v) in DMF. The Fmoc protection
of the N-terminal amino group was removed by the double treatment with
2
0% piperidine in DMF (5 + 15 min). The resin-bound peptides or amino
acids were bromoacetylated by adding the solution of 1.2 M bromoacetic
acid (3 eq.) and DIC (3 eq.) in DMF to the resin. The mixture was agi-
tated for 1 h at RT and the resin was washed with DMF. N6-methyladenine
(
(
1.5 eq) and phosphazene base P1-t-Bu (1.5–1.7 eq) were dissolved in DMF
final concentration 0.2–0.4 M) and added to the resin. The mixture was
shaken for 1 h and washed with DMF and DCE. Adenine-peptide conjugates
were cleaved from the resin and the protection groups were removed by 2
h treatment with 95% TFA. Mixtures of three regioisomers of adenine (N9-
alkyl, N7-alkyl, and N3-alkyl with ratios of about 4:2:1) were obtained. The
products were purified on a C18 reversed phase column with water-ACN
gradient (0.1% TFA). Freeze-drying gave the adenine-peptide conjugates in
the form of white solids with 50–60% yields (mixtures of regioisomers alky-
lated at the N9, N7, and N3 positions of adenine in 4:2:1 molar ratio by
NMR).
Synthesis of NMeAde-9-AcAsp (4d), NMeAde-9-AcAsp (4e), NMeAde-9-
2
3
AcAspGly (4f), and NMeAde-9-AcβAlaAsp (4g).
Wang-type peptide syn-
2
thesis resin, Fmoc-Asp(t-Bu)-resin (100 mg, 0.7 mmol/g; Advanced Chem-
Tech) or Fmoc-Gly-resin (100 mg, 0.87 mmol/g; Advanced ChemTech), was
swollen in DMF. Fmoc protection was removed by treatment of the resin
with 20% piperidine in DMF (5 + 15 min). The following
L
-aspartic acids
(
in the form of Fmoc-Asp[t-Bu]) or β-alanine (Fmoc-β-Ala) were attached
to the resin with BOP/HOBt activation (0.95 eq.) in the presence of N-
methylmorpholine (5%, v/v) in DMF. The Fmoc protection of the N-terminal
amino group was removed by the double treatment with 20% piperidine in
DMF (5 + 15 min). N-Me-Ade-9-Ac (3b; 3 eq., compared to N-terminal amino
groups) was suspended in DMF in the presence of DIEA (5%, v/v). BOP and
HOBt (both 0.95 eq.) were added to the suspension. After 3 min, preactiva-
tion the solution was poured on the resin. The reaction vessel was shaken at
room temperature for 1 h. Adenine-peptide conjugates were cleaved from
the resin and the protection groups were removed by 2 h treatment with
9
5% TFA. The products were purified on a C18 reverse-phase column with
water-ACN gradient (0.1% TFA). Freeze-drying gave the adenine-peptide
conjugates in the form of white solids.

152
E. Enkvist et al.
1
Ade-9-AcAsp (4a).
H NMR (D O) δ 2.99 (2H, d, J = 5.9 Hz, β Asp),
2
5
.22 (2H, s, CH Ac), 8.34 and 8.43 (2H, s, A2 and A8), signals of α Asp were
2
hidden by HDO.
1
NMeAde-9-AcGly (4b).
H NMR (CD SOCD )δ3.03 (3H, s, NCH ),
3
3
3
4
.55 (2H, d, J = 5.6 Hz, α Gly), 5.01 (2H, s, CH Ac), 8.29 and 8.38 (2H, s,
2
A2 and A8), 8.75 (1H, t, J = 5.6 Hz, C(O)NH), 9.0 (1H, br, NH A).
1
NMeAde-9-AcAsp (4c).
H NMR (CD SOCD )δ2.67 (1H, dd, J = 16.9
3
3
and 6.4 Hz, β Asp), 2.69 (1H, dd, J = 16.9 and 5.8 Hz, β Asp), 3.03 (3H,
s, NCH ), 4.55 (1H, ddd, J = 8.0, 6.4 and 5.8 Hz, α Asp), 4.99 (2H, s, CH
3
2
Ac), 8.27 and 8.37 (2H, s, A2 and A8), 8.83 (1H, d, J = 8.0 Hz, C(O)NH),
1
3
8
4
1
.95 (1H, br, NH A). C NMR (CD SOCD )δ27.9 (NCH ), 36.0 (β Asp),
3 3 3
5.0 (CH Ac), 48.8 (α Asp), 118.1 (A5), 142.8 (A8), 148.7 (2C, A2 and A4),
52.1 (A6), 165.8 (C(O)NH), 171.3 and 171.7 (2C, COOH).
2
1
NMeAde-9-AcAsp (4d).
H NMR (CD SOCD ) δ 2.4–2.8 (4H, m, β
3 3
2
Asp), 3.00 (3H, s, NCH ), 4.45–4.7 (2H, m, α Asp), 4.93 (2H, s, CH Ac),
3
2
8
.19 and 8.32 (2H, s, A2 and A8), 8.28 (1H, d, J = 8.2 Hz, NH Asp), 8.26
13
(2H, br, NH ), 8.76 (1H, d, J = 8.0 Hz, NH Asp). C NMR (CD SOCD )
2
3
3
δ28.3 (NCH ), 35.8 and 36.3 (2C, β Asp), 45.0 (CH Ac), 48.6 and 49.4 (2C,
3
2
α Asp), 118.3 (A5), 142.2 (A8), 149.9 (2C, A4 and A2), 153.1 (A6), 166.0 and
1
70.0 (2C, C(O)NH), 171.3, 171.4 and 171.9 (3C, COOH).
1
NMeAde-9-AcAsp (4e).
H NMR of Na salt (D O) δ 2.42–2.85 (6H, m,
2
3
β Asp), 3.08 (3H, NCH ), 4.35 (1H, dd, J = 8.5 and 4.9 Hz, α Asp), 4.63–4.75
3
(
2H, m, α Asp), 5.94 (2H, s, CH Ac), 8.08 and 8.20 (2H, s, A2 and A8).
2
1
NMeAde-9-AcAspGly (4f).
H NMR of Na salt (D O) δ2.64 (1H, dd,
2
J = 16.2 and 8.7 Hz, β Asp), 2.76 (1H, dd, J = 16.2 and 4.9 Hz, β Asp), 3.05
2
(
3H, s, NCH ), 3.75 and 3.77 (2H, d, J = 17.3 Hz, α Gly), 4.72 (1H, dd,
3
J = 8.7 and 4.9 Hz, α Asp), 4.91 (2H, s, CH Ac), 8.03 and 8.14 (2H, s, A2
2
1
3
and A8). C NMR of Na salt (D O) δ28.1 (NCH ), 39.5 (β Asp), 44.1 (α Gly)
2
3
4
5.9 (CH Ac), 52.6 (α Asp), 119.1 (A5), 142.8 (A8), 148.8 (A4), 153.5 (A2),
2
1
55.9 (A6), 169.3 and 173.4 (2C, C(O)NH), 176.8 and 178.3 (2C, COO-).
1
NMeAde-9-Ac βAlaAsp (4g).
H NMR (CD SOCD ) δ2.30 (2H, t,
3 3
2
J = 7.1 Hz, CH2 β Ala), 2.37–2.76 (4H, m, β Asp), 3.01 (3H, s, NCH ),
3
3
.28 (2H, m, J = 2 × 7.1 and 5.5 Hz, βAla), 4.45–4.68 (2H, m, α Asp), 4.89
(
2H, s, CH Ac), 8.13 (1H, d, J = 8.1 Hz, C(O)NH), 8.23 and 8.34 (2H, s, A2
2
and A8), 8.27 (1H, d, J = 8.3 Hz, C(O)NH), 8.36 (1H, t, J = 5.5 Hz), 8.80
1
3
(1H, br, NH A). C NMR (CD SOCD ) δ28.0 (CH , 34.9, 35.4, 35.8, and
3 3 3
3
6.0 (4C, βAla and β Asp), 45.1 (CH Ac), 48.5, and 49.1 (2C, α Asp), 118.3
2

Synthesis of Potential Purinoceptor Antagonists
153
(
(
A5), 142.6 (A8), 149.2 (2C, A2 and A4), 152.6 (A6), 165.8, 170.2, and 170.4
3C, C(O)NH), 171.5 (2C, COOH), 172.0 (COOH).
N-(Bromoacetyl) iminodiacetic Acid Dibenzylester (5). BrAcIdaBn2.
Iminodiacetic acid dibenzyl ester (161 mg, 0.514 mmol) was dissolved in
DCE (0.5 mL) and solution of bromoacetic anhydride (synthesized sepa-
rately in situ by coupling with DIC) (146 mg, 0.56 mmol) in DCE (1.8 mL)
was added. The solution was stirred for 1 h and then the solvent was removed.
The product 5 (168.5 mg, 75% yield) was separated by column chromatogra-
1
phy (CHCl /EtOAc, 4:1, R = 0.75). H NMR (CDCl ) δ3.86 (2H, s, CH Br),
3
f
3
2
4
.25 and 4.27 (4H, s, CH N), 5.15 and 5.19 (4H, s, CH Ph), 7.3 - 7.4 (10H,
2 2
m, Bn). C NMR (CDCl ) δ25.4 (CH Br), 48.8 and 51.2 (2C, CH N), 67.3
1
3
3
2
2
and 67.9 (2C, CH Ph), 128.4–129.1 (10C, Bn), 135.0 and 135.4 (2C, Bn1),
2
1
67.4 (C(O)N), 168.3 and 168.6 (2C, COO).
N-(Adenine-9-yl-acetyl)iminodiacetic Acid Dibenzylester (6a). AdeAc-
IdaBn2.
Adenine (10.5 mg, 0.078 mmol) and 60% NaH (2.3 mg, 0.058
mmol) were suspended in DMF (0.5 mL). The mixture was stirred for 30
min and then BrAcIdaBn (5) (28 mg, 0.064 mmol) in DMF (0.9 mL)
2
was added. The stirring was continued overnight and the solvent was re-
moved in vacuo. The mixture was acidified with acetic acid and purified
by column chromatography (CHCl and CHCl /MeOH, 11:2, R = 0.6) to
3
3
f
1
give 6a (23.4 mg, 74% yield). H NMR (CDCl ) δ4.26 and 4.36 (4H, s,
3
CH N), 5.06 (2H, s, CH Ac), 5.13 and 5.19 (4H, s, CH Ph), 5.63 (2H, br,
2
2
2
1
3
NH ), 7.25–7.5 (10H, m, Bn), 7.86 and 8.32 (2H, s, A2 and A8). C NMR
2
(
CDCl ) δ43.8 (CH Ac), 49.2 and 50.5 (2C, CH N), 67.5 and 68.1 (2C,
3 2 2
CH Ph), 118.6 (A5), 128.2–129.4 (10C, Bn), 134.9 and 135.2 (2C, Bn1), 141.5
2
(
(
A8), 150.1 (A4), 152.8 (A2), 155.8 (A6), 167.1 (C(O)N), 168.2 and 168.4
COO).
N-(N6-Methyladenine-9-yl-Acetyl)iminodiacetic Acid Dibenzylester (6b).
NMeAdeAcIdaBn2. N6-methyladenine (23.3 mg, 0.156 mmol) and 60%
NaH (7.2 mg, 0.18 mmol) were suspended in DMF (0.95 mL). The mixture
was stirred for 30 min and then BrAcIdaBn (56.5 mg, 0.130 mmol) (5) in
2
DMF (0.3 mL) was added. The stirring was continued overnight. Acetic acid
(60 μL) was added and the solvents were removed in vacuo. The product
was purified by column chromatography (CHCl and CHCl :MeOH, 10:2.
3
3
R = 0.68) to give 6b (41.8 mg, 64% yield). N6-methyladenine (22.2 mg,
f
0
.149 mmol) and phosphazene base P1-t-Bu (42 μL, 0.165 mmol) were dis-
solved in DMF (0.4 mL). The solution was added to BrAcIdaBn (53.9 mg,
2
0
.124 mmol) and stirred for 1 h. The solvent was evaporated to dryness. The
crude mixture was partitioned between EtOAc and an aqueous solution of
KH PO /K HPO . The product 6b (43.5 mg, 70% yield) was purified by
2
4
2
4

154
E. Enkvist et al.
1
column chromatography (CHCl and CHCl /MeOH, 10:1. R = 0.60). H
3
3
f
NMR (CDCl )δ3.20 (3H, br, NCH ) 4.25 and 4.36 (4H, s, CH N), 5.04 (2H,
3
3
2
s, CH Ac), 5.12 and 5.17 (4H, s, CH Ph), 5.95 (1H, br, NH), 7.25–7.5 (10H,
2
2
1
3
m, Bn), 7.80 and 8.37 (2H, s, A2 and A8). C NMR δ27.9 (NCH ), 43.7
3
(
(
(
CH Ac), 49.1 and 50.5 (2C, CH N), 67.4 and 68.0 (2C, CH Ph), 119.4
A5), 128.3–129.2 (10C, Bn), 134.9 and 135.3 (2C, Bn1), 140.6 (A8), 149.3
A4), 153.4 (A2), 155.8 (A6), 167.4 (C(O)N), 168.3 and 168.5 (COO).
2 2 2
N-(Adenine-9-yl-acetyl)iminodiacetic Acid (7a). AdeAcIda.
AdeAcI-
daBn (23 mg, 0.053 mmol) was dissolved in EtOH (3 mL). Pd/C as a
2
catalyst was added and hydrogen was passed through the solution until
the starting material had completely disappeared. The mixture was filtered
1
through celite and the solvent was removed (12.6 mg, 77% yield). H NMR
(
(
CD SOCD ) δ 4.03 and 4.39 (4H, s, NCH ), 5.26 (2H, CH Ac), 8.29 and 8.38
3 3 2 2
1
3
2H, s, A2 and A8), 8.72 (2H, br, NH ). C NMR (CD SOCD ) δ44.4(C H
2
2
3
3
Ac), 48.4 and 49.2 (2C, NCH ), 117.4 (A5), 144.6, 145.2, 148.9, and 150.5
2
(4C, A2, A4, A6 and A8), 166.6 (C(O)N), 169.7 and 170.0 (2C, COOH).
N-(N6-Methyladenine-9-yl-acetyl)iminodiacetic Acid (7b). NMeAd-
eAcIda.
NMeAdeAcIdaBn2 (41 mg, 0.081 mmol) was dissolved in
n-butanol (3.6 mL) and acetic acid (2.1 ml). Pd/C (33 mg) was added
and hydrogen was bubbled through the solution for 3.5 h. The mixture
was filtered through celite and the solvents were removed (20.7 mg, 78.7%
1
yield). H NMR (CD SOCD ) δ3.01 (3H, s, CH ), 4.03 and 4.39 (4H,
3
3
3
s, NCH ), 5.23 (2H, CH Ac), 8.20 and 8.34 (2H, s, A2 and A8), 8.80
2
2
(1H, br, NH).
Coupling of N-(Adenine-9-yl-acetyl)iminodiacetic Acid (7a) and As-
ꢁ
partic Acid Di-tert-butyl Ester with N,N -Diisopropylcarbodiimide (DIC).
AdeAcIda (7a) (17.9 mg, 58 μmol) and aspartic acid di-t-butyl ester (35
mg, 142.7 μmol) were dissolved in DMF (0.6 mL). The solution was cooled
◦
to 0 C and DIC (20 μL, 129 μmol) was added. The solution was stirred
◦
overnight at 0–4 C and then for 10 h at room temperature. The solvents
were removed and the mixture was purified by column chromatography
(
CHCl /MeOH, 10:2). The fraction with R = 0.55–0.60 was collected and
3
f
the solvents were removed. The residue was treated with 95% TFA for 1–3 h
and then the solvents were removed. The separation of the mixture by HPLC
gave AdeAcIda(Asp)Asp 8a (4.0 mg, 12.8% yield) and AdeIdaAcIda(Asp)N-
1
acyl urea of DIC 9a (4.6 mg, 14.4% yield). (8a) H NMR (CD SOCD ) δ
3
3
2
.5–2.87 (4H, m, β Asp), 3.99 and 4.28 (4H, s, NCH ), 4.48–4.71 (2H, m, α
2
Asp), 5.20 (2H, s, CH Ac), 8.16 and 8.29 (2H, s, A2 and A8), 8.51 (1H, d,
2
J = 8.0 Hz, C(O)NH), 8.99 (1H, d, J = 7.9 Hz, C(O)NH).

Synthesis of Potential Purinoceptor Antagonists
155
CouplingofN-(N6-Methyladenine-9-yl-acetyl)iminodiaceticAcid(7b)and
ꢁ
Aspartic Acid Di-tert-butyl Ester with N,N -Diisopropylcarbodiimide (DIC).
NMeAdeAcIda (7b) (20.7 mg, 64.2 μmol) and aspartic acid di-t-butyl es-
ter (58.8 mg, 240 μmol) were dissolved in DMF (0.5 mL). The solution was
◦
cooled to 0 C and DIC (25 μL, 161 μmol) was added. The mixture was
◦
◦
stirred for 20 h at 0 C and then for 12 h at 10–12 C. The solvent was re-
moved and the mixture was purified by column chromatography (CHCl3,
CHCl /MeOH, 15:2), the fraction with R = 0.5–0.6 was collected and the
3
f
solvents were removed. The residue was treated with 95% TFA for 3h. The
solvents were removed and the separation of the mixture by HPLC gave
NMeAdeAcIda(Asp)Asp 8b (8.7 mg, 19.3% yield) and NMeIdaAcIda(Asp)N-
1
acyl urea of DIC 9b (4.9 mg, 13.5% yield). (8b) H NMR (CD SOCD ) δ
3
3
2
.5–2.86 (4H, m, β Asp), 3.00 (3H, s, NCH ), 3.99 and 4.28 (4H, s, NCH ),
3
2
4
.49–4.70 (2H, m, α Asp), 5.19 (2H, s, CH Ac), 8.10 and 8.31 (2H, s, A2
2
and A8), 8.50 (1H, d, J = 8.0 Hz, C(O)NH), 8.62 (1H, br, NHMe), 8.98 (1H,
1
3
d, J = 8.1 Hz, C(O)NH). C NMR (CD SOCD ) δ27.7 (NCH ), 35.8 and
3
3
3
3
6.0 (2C, β Asp), 43.8 (CH2 Ac), 48.4 and 48.7 (2C, α Asp), 50.1 and 50.8
(
2C, CH Ac), 118.2 (A5), 142.2 (A8), 148.5 (A4), 149.7 (A2), 153.1 (A6),
2
1
67.3, 167.9 and 167.9 (3C, amides), 171.2, 171.4, 171.76 and 171.82 (4C,
COOH).
Hydrolysis of N-Acylureas.
A derivative of N-acylurea (9a and 9b) was
dissolved in the suspension of Ca(OH) (5 mg of the compound in 1 mL
2
of a suspension). The suspension was initially mixed by sonication and then
stirred at room temperature for 7–9 h. The mixture was acidified with 1
M HCl and evaporated to dryness. The products were purified by HPLC.
Yields: 10a 42% and 10b 76%. Slow rotations of unsymmetrical secondary
amide bonds of the compounds give unequal signals from two rotamers into
the NMR spectra. The resonances of the major rotamers are labeled with
asterisks.
1
AdeAcIdaAsp (10a) H NMR (CD SOCD ) δ 2.5–2.85 (2H, m, β Asp),
3
3
∗
∗
4
.00 and 4.02 (2H, s, NCH ), 4.30 and 4.33 (2H, s, NCH ), 4.47–4.70 (1H,
m, α Asp), 5.16 and 5.21 (2H, s, CH Ac), 7.80 (2H, br, NH ), 8.09 and
2
2
∗
∗
2
2
∗
8
.11 (1H, s, A), 8.21 (1H, s, A), 8.40 and 8.76 (1H, d, J = 8.0 Hz, NH).
1
NMeAdeAcIdaAsp (10b) H NMR (CD SOCD ) δ 2.5–2.85 (2H, m, β Asp),
3
3
∗
∗
3
.02 (3H, br, NCH ), 3.99 and 4.02 (2H, s, NCH ), 4.30 and 4.34 (2H, s,
3
2
∗
NCH ), 4.47–4.71 (1H, m, α Asp), 5.20 and 5.26 (2H, s, CH Ac), 8.17 (1H,
2
2
∗
∗
br s, A), 8.34 (1H, s, A), 8.40 and 8.77 (1H, d, J = 8.3 Hz and 8.0 Hz , NH).
1
3
∗
∗
C NMR (CD SOCD ) δ27.9 (1C, br, NCH ), 35.8 and 36.0 (β Asp), 43.98
3
3
3
and 44.04 (1C, CH Ac), 48.36–49.27 (2C, NCH ), 50.5 (α Asp), 118.2 (A5),
2
2
∗
1
1
42.5 (A8), 149.5 (2C, A2, and A4), 152.8 (A6), 167.1 and 167.3 , 167.5 and
67.7 (2C, amides), 170.1 and 170.3, 171.3 and 171.4 (2C, COOH), 171.9
∗
∗
∗
(1C, COOH).

156
E. Enkvist et al.
CONCLUSIONS
We have demonstrated the effectiveness of P1-tBu phosphazene base for
the deprotonation and alkylation of adenine. The reactions of adenine anion
with bromoacetic acid derivatives were fast in solution and on solid phase
and adenine-peptide conjugates could be prepared in good yields. Unexpect-
edly, the alkylation reactions on polystyrene Wang resin led to a mixture of
regioisomers that were difficult to separate with reverse-phase HPLC. Some
of the prepared adenine derivatives containing negatively charged carboxy-
late groups inhibited platelet aggregation caused by the activation of adenine
nucleotide receptors with ADP.
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