Tetrabutylammonium fluoride-assisted rapid N9-alkylation on purine ring: Application to combinatorial reactions in microtiter plates for the discovery of potent sulfotransferase inhibitors in situ

Article abstract of DOI:10.1016/j.bmc.2005.02.066

Tremendous efforts have been invested in the synthesis of purine libraries due to their importance in targeting various enzymes involved in different diseases and cellular processes. The synthesis of N⁹-alkylated purine scaffolds relied mostly

Full text of DOI:10.1016/j.bmc.2005.02.066

Bioorganic & Medicinal Chemistry 13 (2005) 4622–4626
9
Tetrabutylammonium fluoride-assisted rapid N -alkylation
on purine ring: Application to combinatorial reactions in
microtiter plates for the discovery of potent
sulfotransferase inhibitors in situ
Ashraf Brik, Chung-Yi Wu, Michael D. Best and Chi-Huey Wong*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
1
0550 North Torrey Pines Road, La Jolla, CA 92037, USA
Accepted 21 February 2005
Available online 13 June 2005
Abstract—Tremendous efforts have been invested in the synthesis of purine libraries due to their importance in targeting various
enzymes involved in different diseases and cellular processes. The synthesis of N -alkylated purine scaffolds relied mostly on Mits-
9
unobu conditions with a variety of alcohols or strong basic conditions with different organic halides. A more reliable and efficient
way for the synthesis of N -alkylated purine scaffolds is reported. This method uses tetrabutylammonium fluoride (TBAF) to assist
9
such chemistry. In many cases, the reactions were completed within 10 min and gave the desired product in high yield and selectivity.
Moreover, these mild reaction conditions permitted its use in combinatorial reactions in microtiter plates followed by in situ screen-
ing for the discovery of potent sulfotransferase inhibitors.
ꢀ
2005 Elsevier Ltd. All rights reserved.
9
temperature remarkably accelerates the N -alkylation
1. Introduction
of the purine ring with a variety of organic halides.
Moreover, these mild reaction conditions and the effi-
ciency of this chemistry permit its use in combinatorial
reactions in microtiter plates followed by in situ screen-
ing for the discovery of potent sulfotransferase
inhibitors.
Purine derivatives have been used to target various en-
zymes involved in different diseases and cellular pro-
cesses, most notably the kinase family. Thus,
1
tremendous efforts have been invested to develop an effi-
cient chemistry toward the synthesis of purine libraries
that could be used as a source for both drug leads and
1
,2
probes for analyzing complex cellular processes. Pre-
viously, synthesis of a N -alkylated purine library relied
9
2. Results and discussion
on using either a Mitsunobu condition with a variety of
3
alcohols or a strong basic condition (NaH, K CO )
2
3
TBAF, in addition to its wide use as a reagent for desil-
5
4
with a variety of alkyl and benzyl halides. These
reactions require long reaction times (10–48 h), low tem-
peratures for a Mitsunobu condition or elevated temper-
atures for the basic condition, and an inert atmosphere
due to the sensitivity of the reagents in order to give
the product in high yield and higher selectivity for the
ylation reaction, has also been used as an activator for
organic halides to assist the reaction of palladium-cata-
6
lyzed cross-coupling, non-Sonogashira-type palladium-
7
catalyzed coupling of terminal alkynes, alkylation of
8
phenols, and more recently in the catalysis of the syn-
9
thesis of 5-substituted 1H-tetrazoles. Considering the
role of TBAF in these reactions, as well as the basic nat-
9
7
N versus the N -alkylated product. Herein, we report
that tetrabutylammonium fluoride (TBAF) at room
10
ure of the fluoride ion, we wanted to examine the effect
of TBAF alone in the N -alkylation of purine scaffolds,
9
as this type of chemistry would be accelerated if both a
base and activator for the organic halides are present in
the reaction. This mild condition, if achieved, is ideal for
reactions in a microtiter plate for the high-throughput
Keywords: TBAF; Purine-based libraries; N-alkylation; Sulfotransfer-
ase inhibitors.
*
Corresponding author. Tel.: +1 858 784 2487; fax: +1 858 784
409; e-mail: wong@scripps.edu
2
0
968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.066

A. Brik et al. / Bioorg. Med. Chem. 13 (2005) 4622–4626
4623
screening against several important enzymes. Initially,
we investigated the alkylation reaction of
,6-dichloropurine with benzyl bromide and methyl io-
dide. In both cases, the reaction was completed within
0 min upon addition of 2 equiv of the organic halide
and 2 equiv of TBAF. The reactions were analyzed by
LC–MS and gave a mixture of the two isomers N /N
in 7/3 ratio and excellent yields (isolated) (entries 1
and 2, Table 1). The reaction proceeded well in different
solvents such as THF, DMF, and DMSO, and was car-
ried out in open air. Similar results were obtained with
2-amino-6-chloropurine (entry 4, Table 1). Interestingly,
using methyl bromoacetate gave better selectivity with
excellent yield. Similarly, we also found that in the case
of 1-bromodecane with 2-amino-6-chloropurine a minor
amount of the N -alkylated product was observed; how-
ever, the reaction in this case required a longer time for
completion (entry 5, Table 1).
2
1
9
7
7
9
Table 1. TBAF assisted N -alkylation
9
N /N
7
Entry
Scaffolds
R–X
Yield (%)
95
Reaction time
10 min
1
70/30
70/30
2
3
CH
3
I
96
98
10 min
10 min
86/14
4
5
90
85
70/30
95/5
10 min
CH
3
3
(CH )
8
–CH
2
–Br
2 h
6
97
98/2
10 min
7
8
9
95
95
70
50
99/1
98/2
98/2
98/2
10 min
10 min
3 h
1
0
12 h

4624
A. Brik et al. / Bioorg. Med. Chem. 13 (2005) 4622–4626
1
9
It has been observed that substitution at the C6 position
scribed. Two wells gave better inhibition activity than
the original core. These two inhibitors were synthesized,
purified, and characterized, and their K values were
9
7
1d
affects the N /N alkylation ratio. Indeed, in all the
cases that we tested with the 2-chloro-6-naphthylpurine
scaffold and a variety of organic halides, only a very
i
determined to be 15 and 9 nM for compounds 3 and
2, respectively (Scheme 1).
7
minute amount of the N -alkylated product was de-
tected (entries 6–10, Table 1). However, sterically hin-
dered primary alkyl halides (entry 9, Table 1) and
secondary alkyl halides (entry 10, Table 1) required
longer reaction times and higher temperatures (60 ꢁC)
and the reaction gave the product with high selectivity,
but with lower yield.
3. Conclusion
In conclusion, the new method described here represents
a useful way toward rapidly accessing N -alkylated pur-
9
ine analogues. The reaction of the purine scaffold with
organic halides in the presence of TBAF is rapid, reli-
able, and gives the product in high yield and selectivity.
The chemistry is also amenable to combinatorial reac-
tions in microtiter plates followed by in situ screening,
as was demonstrated in the discovery of two potent sul-
fotransferase inhibitors. We believe that this chemistry
should ease the preparation of purine-based libraries
and its combination with the in situ screening approach
could represent a useful method for selecting potent
inhibitors against other targets. The scope, limitation,
and further application of the TBAF-assisted N-alkyl-
ation are currently under investigation.
Next, we examined whether this chemistry could be
applied to reactions in microtiter plates followed by in
situ screening of the product for the discovery of potent
inhibitors against a chosen target. To be useful, the
chemistry should be efficient, selective, and free of
protecting group manipulations so that the product
could be used directly for screening in situ without iso-
lation. Since its introduction, this approach was found
to be very successful for the discovery of potent inhibi-
tors against different enzymes, such as HIV prote-
1
1
1
1,12
13
ase,
protease,
b-arylsulfotransferase,
SARS coronavirus
15
1
4
a-1,3-fucosyltransferase,
and a-fucosi-
1
6
dase. As a model system, we have selected the enzyme
b-arylsulfotransferase-IV (b-AST-IV), which represents
an effective model for studying sulfotransferase
4. Experimental
1
7
inhibition.
4
.1. General procedure (entry 3, Table 1)
Our laboratory had previously screened a library of
5,000 purine and pyrimidine analogues against b-
AST-IV, which resulted in the inhibitor 2-chloro-6-
3
2,6-Dichloropurine (100 mg, 0.53 mmol) was dissolved
in 500 lL THF at room temperature and to this was
added 1 mL (1.0 mmol) TBAF (1.0 M solution in THF,
Aldrich). To the clear mixture was added methyl bromo
acetate (100 mL, 1.0 mmol). The reaction was kept for
10 min at room temperature, as at this time TLC analysis
naphthylpurine, 1, (K = 96 nM, Scheme 1) binding to
i
1
8
the aryl site of the enzyme. Thirty organic halides were
reacted in microtiter plates with the 2-chloro-6-naph-
thylpurine core and the reactions were analyzed by
TLC and by LC–MS for completion and selectivity ratio
(CHCl /MeOH, 10:1) indicated complete consumption
3
(
Scheme 1). Most of these reactions were completed
of the purine. The reaction was directly loaded into the
column and the product was eluted with a solution of
hexane/ethyl acetate, 1:1 to yield 120 mg product as a
9
within 20 min and gave the N -alkylated product in high
yield (>95%). Wells were diluted to 100 nM and assayed
directly against b-AST-IV, as was previously de-
1
white solid. H NMR (500 MHz, CDCl ) d 8.19
3
Scheme 1. Combinatorial reactions in microtiter plate for the discovery of potent sulfotransferase inhibitors.

A. Brik et al. / Bioorg. Med. Chem. 13 (2005) 4622–4626
4625
a
b
Figure 1. Inhibition of b-AST-IV with compound 2: (a) reciprocal rate versus reciprocal MUS concentration at 0, 50, 100, 150, 200, and 250 nM
inhibitor; (b) slop replot.
1
3
(
1
s, 1H), 5.06 (s, 2H), 3.84 (s, 3H); C NMR 166.57,
53.3, 153.1, 152.0, 146.02 130.3, 53.3, 44.39; ESI-MS
Calcd for (C H Cl N O )H 260.99. Found 261.0 (entry
sion plate reader. Inhibitor concentrations were chosen
such that enzymatic rates were linear. For K measure-
ments, inhibitors were studied at 0, 50, 100, 150, 200,
and 250 nM concentrations. The dissociation constants
were obtained as the absolute value of the x-axis intercept
on plotting K/V versus inhibitor concentration. Multiple
i
+
8
1
6
2
4
2
1
7
1
1
(
1
, Table 1): H NMR (500 MHz, CDCl ) d 8.06 (s, 1H),
3
13
.27–7.3 (m, 5H), 5.4 (s, 2H); C NMR 153.07, 151.75,
45.53, 133.92, 133.91, 130.55, 129.32, 129.29, 128.98,
28.04,
128.00,
47.95;
ESI-MS
Calcd
for
K values were determined and the results were averaged
i
+
C H Cl N )H 279.01. Found 279.0, (entry 7, Table
4
). H NMR (500 MHz, DMSO) d 8.9 (br s, 1H), 8.3
s, 1H), 8.28 (d, 8 Hz, 2H), 7.96–7.79 (m, 7H), 7.56–
7.43 (m, 8H), 5.54 (s, 2H), 5.14 (d, 2H, 5.9 Hz);
NMR 155.97, 154.18, 150.89, 135.04, 134.15, 133.64,
to yield the final reported values. The reactions were com-
pleted after 10 min. Those that still show starting material
were heated to 60 ꢁC for several hours. The wells were
then assayed at 100 nM, in which compounds 2 and 3
showed better inhibition activity. These two compounds
1
2
1
8
2
(
1
3
C
1
1
1
4
33.26 132.26, 131.69, 129.4 128.63, 128.26, 127.35,
27.12, 127.07, 126.94, 126.64, 126.44, 125.93, 124.33,
19.15, 47.39, 42.20; ESI-MS Calcd for (C H ClN )H
were synthesized on a large scale and their K values were
i
determined (see Fig. 1).
+
2
7
20
5
50.14. Found 450.0.
Acknowledgments
4
.2. Library preparation for in situ screening
We thank the National Institutes of Health and the
Skaggs Institute for Chemical Biology for funding.
We thank the National Science Council of Taiwan
and the Genomic Research Center, Academia Sinica,
for the financial support (C.-Y.W). We are also very
thankful to Sheng-Kai Wang for the useful discussion.
Thirty milligrams of 2-chloro-6-naphthylpurine was dis-
solved in 195 lL of 1 M solution of TBAF in DMF and
to this was added 105 lL anhydrous DMF. From this
stock solution 10 lL was taken and added to 30 wells
of the microtiter plate. To each well was added 2 equiv
of different alkyl bromide and the plate was kept at
room temperature. The reactions were analyzed by
TLC and LC–MS (C8 column). Most of the reactions
at this time were completed. The wells were then diluted
to 100 nM ready for the assay.
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