Preparation of polymer-bound pyrazolone active esters for combinatorial chemistry

Article abstract of DOI:10.1016/j.tetlet.2003.09.044

The preparation of solid-phase active esters from a new pyrazolone linker resin is described. N-Acylation using this resin provides various amide products with a high conversion rate and good purity under mild conditions. The polymer-bound pyrazolone linkers are stable in the reaction conditions and are resistant to hydrolysis. Moreover, this resin can also be reused repeatedly without a loss of reactivity.

Full text of DOI:10.1016/j.tetlet.2003.09.044

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 8063–8067
Preparation of polymer-bound pyrazolone active esters for
combinatorial chemistry
Jang-Woong Byun, Dong-Hoon Lee and Yoon-Sik Lee*
School of Chemical Engineering, Seoul National University, Seoul 151-744, Republic of Korea
Received 8 August 2003; revised 5 September 2003; accepted 8 September 2003
Abstract—The preparation of solid-phase active esters from a new pyrazolone linker resin is described. N-Acylation using this
resin provides various amide products with a high conversion rate and good purity under mild conditions. The polymer-bound
pyrazolone linkers are stable in the reaction conditions and are resistant to hydrolysis. Moreover, this resin can also be reused
repeatedly without a loss of reactivity.
© 2003 Published by Elsevier Ltd.
Polymer-bound reagents and linkers have become
increasingly important tools in organic synthesis due to
the growing use of combinatorial chemistry in various
applications. The polymer-bound linkers have several
advantages over classical solution methods. They can
drive the reactions to a high conversion efficiency with
an excess of reagent which can be removed simply by
filtration, and can be used in multiple step synthesis
and automation. In addition, a reaction using the poly-
mer-bound linkers provides a desirable yield and purity.
Therefore, a wide range of polymer-bound linkers have
been developed for various organic synthesis systems.1
color. These characteristics of pyrazolone active esters
can allow a wide structural diversity in a combinatorial
library synthesis and attractive methods for modifying
biomolecules. This paper reports the preparation of
polymer-bound pyrazolone active esters and their appli-
cations as N-acylating agents in a combinatorial library
synthesis.
As illustrated in Scheme 1, the desired pyrazolone
linker,
1-(4-carboxyphenyl)-3-(4-nitrophenyl)-2-pyra-
zolin-5-one (CNPO), which possesses an anchoring
group to introduce its functionality onto the polymer
support, was synthesized from 4-hydrazinobenzoic acid
Since the preparation of peptide fragments by polymer-
bound active esters have attracted a great deal of
attention, several types of active esters such as nitro-
phenol,2 carbodiimides,3 N-hydroxysuccinimide,4 1-
hydroxybenzotriazole,5 pentafluorophenol,6 and Kaiser
oxime resin7 have been reported. During our continuing
efforts for developing versatile polymer-bound active
ester linkers, we became interested in pyrazolone active
esters for N-acylations in combinatorial chemistry.8
and 4-nitrobenzoylacetate by
a modified Knorr’s
method.9 Due to the poor solubility of 4-hydrazinoben-
zoic acid in most organic solvents, acetic acid was the
only choice for the reaction solvent, which also acted as
an acid catalyst. As the reaction progressed under
refluxing conditions, the reaction mixture was com-
pletely dissolved in a short time due to the formation of
an intermediate adduct. Eventually, the CNPO began
to precipitate as a yellow solid.10 In solution, CNPO
Compared to other reagents, polymer-bound pyra-
zolone active esters are stable in polar solvents such as
alcohols and DMF, and are resistant to hydrolysis.
Furthermore, they can react specifically with amines
under mild conditions when hydroxyl groups are
present in the same solution. The progress of the reac-
tion can be easily monitored by the change in the resin
Scheme 1. Preparation of the pyrazolone active ester linker
* Corresponding author. Tel.: +82-2-880-7073; fax: +82-2-876-9625;
e-mail: yslee@snu.ac.kr
(CNPO).
0040-4039/$ - see front matter © 2003 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2003.09.044

8064
J.-W. Byun et al. / Tetrahedron Letters 44 (2003) 8063–8067
might be tautomerized between the enol and keto
forms. However, NMR spectroscopy confirmed that the
enol form was predominant in the polar solvent.
An amino polystyrene (PS) resin was chosen as the
support for the active esters, because it could form a
chemically stable amide bond with the pyrazolone
linker. The amino PS resin was prepared from the
PS-PO resin (BTCore OH resin from BeadTech Inc.) as
reported previously.11 After the CNPO was coupled
with the amino resin (Resin I, 1.47 mmol NH2/g) using
DIC (3.0 equiv.) and DMAP (0.3 equiv.) in DMF, the
pyrazolone containing polymer support (Resin II) was
obtained as a reddish orange resin12 (Scheme 2). From
FT-IR analysis of Resin II, the amide carbonyl band at
1650 cm−1 appeared as a result of coupling (Fig. 1a). A
picric acid titration of Resin II revealed that a trace
amount of amino groups (8.7 mmol NH2/g) remained
unreacted, which shows that the coupling yield of
CNPO linker with the amino resin was approximately
99%. Finally, the degree of substitution of the CNPO
linker on Resin II was 1.01 mmol/g.
Figure 1. FT-IR spectra of (a) CNPO linked amino PS resin
(Resin II), and (b) O-acetylated pyrazolone resin (Resin III).
The polymeric active ester (Resin III) was prepared
from a coupling reaction of Resin II with several kinds
of the acyl derivatives (Fig. 2). The acyl halides such as
benzoyl chloride (1) and phenylacetyl chloride (2) were
reacted in 2% TEA/THF (v/v) for 2 h.13 The carboxylic
acid derivatives such as 4-nitrobenzoic acid (3), and
4-dimethylaminobenzoic acid (4), and amino acids such
as Boc-Gly (5) and Boc-Phe (6) were reacted with DIC
(3.0 equiv.) and DMAP (0.3 equiv.) in DMF for 24 h.14
As a result of the acyl coupling reaction, the color of
the resin changed from reddish orange to a dark brown,
and the carbonyl band at 1790 cm−1 appeared (Fig. 1b).
Figure 2. Acyl derivatives for N-acylation.
The degree of acylation on Resin III was determined by
the benzylamine treatment15 (Table 1). Even if the
acylation onto the CNPO linker was carried out with
an excess of the acyl derivatives for a long reaction
time, approximately 50% of the pyrazolone groups were
available for coupling, because steric hindrance of the
acyl products on the PS backbone prevented further
diffusion of the acylating reagents. The reactivity of the
acyl derivatives also had an effect on the coupling
reactions because the conversion was different accord-
ing to their reactivity. Overall, the acyl chlorides (1, 2)
gave better results than the carboxylic acids due to their
higher reactivity. In the case of compound 4, the elec-
tron donating effect of the p-dimethylamino group
might reduce the reactivity, and showed poor coupling.
When the amino acids (5, 6) were coupled, the bulki-
ness of the side chain appeared to control the reactivity.
Therefore, the less sterically hindered Boc-Gly was
coupled more to the CNPO linker than Boc-Phe.
Several amide derivatives were synthesized by a reac-
tion of Resin III with various organic amines which
have a similar molecular weight.16 The results of the
combinatorial N-acylation using the polymer-bound
pyrazolone active esters are summarized in Table 2.
The progress of the reaction was easily monitored by
the change in the resin color as well as by FT-IR
spectroscopy. As the reaction progressed, the color of
Scheme 2. Preparation of the polymer-bound pyrazolone
active esters and the combinatorial synthesis of amide deriva-
tives using them.

J.-W. Byun et al. / Tetrahedron Letters 44 (2003) 8063–8067
Table 1. Preparation of the acylated CNPO resin (Resin III)
8065
Acyl derivatives Theoretical loading of acyl derivatives on Resin III
Loading of acyl derivatives on Resin III
Conversion of
acylation (%)
(mmol/g)
(mmol/g)c
1a
2a
3b
4b
5b
6b
1.01
1.00
0.88
0.88
0.87
0.81
0.76
0.60
0.43
0.19
0.46
0.28
75.3
59.6
49.2
21.1
52.3
34.9
a The coupling reactions of the acyl chlorides (3.0 equiv.) were carried out in 2% TEA/THF (v/v) at room temperature for 2 h.
b The coupling reactions of the carboxylic acid derivatives (3.0 equiv.) were carried out with a mixture of DIC (3.0 equiv.) and DMAP (0.3 equiv.)
in DMF at room temperature for 24 h.
c The loading was calculated from the amount of N-acyl benzylamine after a treatment with excess benzylamine (10 equiv.) at room temperature
for 1 h.
Table 2. Combinatorial synthesis of the amide derivatives using the polymer-bound pyrazolone active estersa
the resin changed from dark brown to a reddish orange,
and the carbonyl band at 1790 cm−1 completely disap-
peared. The remaining amine was removed by passing
it through a silica gel short column. After evaporating
the solvent, most of the amide products were obtained
as pure solids which were confirmed by NMR. The
product yields were based on the loading of acyl deriva-
tives on the polymer-bound pyrazolone linker (Resin
III).
(R)-a-methylbenzylamine. From the HPLC analysis
data, we found that the degree of racemization was
approximately 14.8% during the acyl coupling with
DIC and DMAP for 24 h.17 However, when we carried
out the acyl coupling with BOP (3.0 equiv.), HOBt (3.0
equiv.) and DIEA (6.0 equiv.) at room temperature for
12 h, the racemizaiton was significantly reduced
(<1.5%).
Repeated tests were performed to examine the stability
of the polymer-bound CNPO linker. Figure 3 shows the
results of the same resin, which was used repeatedly in
the N-acylation five times with the previous benzoyl
chloride and piperidine. These results showed that the
CNPO linker resin (Resin III) could be reused continu-
ously in N-acylation without changing its reactivity.
This study found that the rate of N-acylation was
largely dependent on the reactivity of the amine deriva-
tives. Amines with high nucleophilicity such as pipe-
ridine (entry 1) and cyclohexylamine (entry 2) showed
good results of N-acylation.
However, diisopropylamine (entry 4) and aniline (entry
5) barely reacted with the pyrazolone active esters due
to their poor nucleophilicity.
In conclusion, a polymer-bound pyrazolone active ester
resin can be used successfully with a high reactivity and
good reusability, which makes it suitable candidate as a
new polymeric acylating agent in solid-phase combina-
torial chemistry.
In case of Boc-Phe derivatives, we examined the degree
of racemization during N-acylation after treatment with

8066
J.-W. Byun et al. / Tetrahedron Letters 44 (2003) 8063–8067
8. (a) Lee, K. W.; Lee, Y. S. Bull. Korean Chem. Soc. 1989,
10, 331–335; (b) Kim, J. B.; Lee, Y. S. Bull. Korean
Chem. Soc. 1991, 12, 376–379.
9. Knorr, A. Ber. Deutsch. Chem. Ges. 1950, 16, 2597.
10. A general procedure for the CNPO synthesis. 4-Hydrazi-
nobenzoic acid (6.09 g, 40.0 mmol) and 4-nitrobenzoylac-
etate (10.25 g, 48.0 mmol) were mixed in acetic acid (200
ml), and refluxed for 24 h. After the reaction was com-
pleted, the reaction mixture was cooled to room tempera-
ture. The CNPO was obtained as a brown solid, which
was filtered and washed thoroughly with DCM to the
remove excess acetic acid. Yield, 10.78 g (83%); mp
274°C; NMR (DMSO-d6) l 4.4 (s, -CH2-), 6.6 (s, -OH),
8.2–8.8 (m, aromatic).
Figure 3. Reusability test of the CNPO linker resin.
11. Ryoo, S. J.; Kim, J.; Kim, J. S.; Lee, Y. S. J. Comb.
Chem. 2002, 4, 187–190.
12. A general procedure for the preparation of CNPO linked
amino PS resin (Resin II). Resin I (8.0 g, 1.47 mmol
-NH2/g) was swelled with DMF in a three-necked round-
bottomed flask. The mixture of CNPO (11.4 g, 35.3
mmol), DIC (5.5 ml, 35.3 mmol) and DMAP (422.5 mg,
3.5 mmol) in DMF was then added and stirred for 24 h.
The product resin was filtered and washed three times
each with DMF, MeOH and DCM in turn, and then
dried in a vacuum.
Acknowledgements
We wish to acknowledge the Brain Korea 21 Program
supported by the Ministry of Education and the Nano
Bioelectronics and Systems Research Center of Seoul
National University, which is supported by the Korean
Science and Engineering Foundation (KOSEF).
13. A general procedure for the coupling of the acyl chlorides
to the polymer-bound CNPO linker (Resin III). Resin II
(3.0 g, 1.01 mmol CNPO/g) was treated with benzoyl
chloride (1.2 ml, 9.1 mmol) in 2% TEA/THF (v/v) and
shaken for 2 h. The resin was washed three times each
with DCM and MeOH, and dried under vacuum.
14. A general procedure for the coupling of the caboxylic acid
derivatives to the polymer-bound CNPO linker (Resin III).
Resin II (3.0 g, 1.01 mmol CNPO/g) was treated with a
mixture of 4-nitrobenzoic acid (1.5 g, 9.1 mmol), DIC
(1.6 ml, 10.0 mmol) and DMAP (36.7 mg, 0.9 mmol) in
DMF and shaken for 24 h. The resin was washed three
times each with DCM and MeOH, and then dried in
vacuum.
References
1. For a recent review, see: James, I. W. Tetrahedron 1999,
55, 4855–4946.
2. (a) Fridkin, M.; Hazum, E.; Kalir, R.; Rotman, M.;
Koch, Y. J. Solid-Phase Biochem. 1977, 2, 175–182; (b)
Carpino, L. A.; Cohen, B. J.; Lin, Y. Z.; Stephens, K. E.,
Jr.; Triolo, S. A. J. Org. Chem. 1990, 55, 251–259; (c)
Cohen, B. J.; Karoly-Hafeli, H.; Patchornik, A. J. Org.
Chem. 1984, 49, 922–924; (d) Reichwein, J. F.; Liskamp,
M. J. Tetrahedron Lett. 1998, 39, 1243–1246; (e) Chang,
Y.-T.; Schultz, P. S. Bioorg. Med. Chem. Lett. 1999, 9,
2479–2482; (f) Kim, K.; Le, K. Synlett 1999, 12, 1957–
1959.
15. To determine the degree of substitution, the acylated
CNPO-Resin III (0.1 g) was swelled in THF, and treated
with excess benzylamine (1.0 ml, 9.2 mmol, 10 equiv.).
After reacting for 1 h at room temperature, the resin was
filtered and washed with DCM. The filtrate and the
washing solution were combined and passed through a
silica gel short column to remove the remaining benzyl-
amine. After evaporating the solvent, the degree of
acylation on th polymer-bound CNPO linker was calcu-
lated from the amount of the isolated N-acyl benzyl-
amine. The quantitative conversion was confirmed by
FT-IR spectroscopy, which showed that the carbonyl
3. (a) Weinshenker, A.; Shen, C. M. Tetrahedron Lett. 1972,
14, 3281–3284; (b) Desai, M. C.; Stephens Straminello, L.
M. Tetrahedron Lett. 1993, 34, 7685–7688.
4. Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. J.
Am. Chem. Soc. 1963, 85, 3039.
5. (a) Kalir, R.; Warshawsky, A.; Fridkin, M.; Patchornic,
A. Eur. J. Biochem. 1975, 59, 55–61; (b) Mokotoff, M.;
Patchornic, A. Int. J. Pept. Protein Res. 1983, 21, 145–
154; (c) Mokotoff, M.; Zhao, M.; Roth, S. M.; Slavosky,
J. N.; Shelley, J. A. J. Med. Chem. 1990, 33, 354–360; (d)
Pop, I. E.; Deprez, B. P.; Tartar, A. L. J. Org. Chem.
1997, 62, 2594–2603; (e) Dendrinos, K. G.; Kalivretenos,
A. G. Tetrahedron Lett. 1998, 39, 1321–1324; (f) Dendri-
nos, K.; Jeong, J.; Kalivrenos, A. G. Chem. Commun.
1998, 499–500; (g) Schiemann, K.; Showalter, H. D. H. J.
Org. Chem. 1999, 64, 4972–4975.
band of active esters (1790 cm−1
disappeared.
) had completely
16. A general procedure for the combinatorial synthesis of the
amide derivatives. Benzoyl-CNPO-Resin III (0.3 g, 0.76
mmol/g) was swelled with DCM in a filtered vial. Pipe-
ridine (77.4 ml, 0.78 mmol) in THF (5 ml) was transferred
into the reaction vessel and the reaction mixture was
shaken for 1 h at room temperature. The resin was
filtered and washed with DCM. The filtrate and the
washing solution were combined and passed through a
silica gel short column to remove the excess amine deriva-
tives. After evaporating the solution, the amide product
6. Kovacs, J.; Kisfaludy, L.; Ceprini, M. Q. J. Am. Chem.
Soc. 1967, 89, 183.
7. (a) Smith, R. A.; Bobko, M. A.; Lee, W. Bioorg. Med.
Chem. Lett. 1998, 8, 2369–2374; (b) Golebiowski, A.;
Klopfenstein, S. Tetrahedron Lett. 1998, 39, 3397–3400;
(c) Scialdone, M. A.; Shuey, S. W.; Soper, P.; Hamuro,
Y.; Burns, D. M. J. Org. Chem. 1998, 63, 4802–4807.

J.-W. Byun et al. / Tetrahedron Letters 44 (2003) 8063–8067
8067
was obtained as a white solids (43.2 mg) and the yield was
99.7% based on the amount of the loaded benzoyl active
ester groups.
remove the remaining (R)-a-methylbenzylamine. After
evaporating the solvent, the product was dissolved in
MeOH and analyzed by HPLC using a Supelcosil LC-18
column (4×150 mm). The elution condition was as follows:
0 to 5 min, CH3CN/H2O (50/50); 5 to 25 min, up to
CH3CN/H2O (90/10, gradient); 25 to 40 min, CH3CN/H2O
(90/10); 1.0 ml/min, 210 nm UV. The retention time of
(S,R)-Boc-Phe-a-methylbenzylamide and (R,R)-Boc-Phe-
a-methylbenzylamide were 13.7 and 13.2 min, respectively.
17. To determine the degree of racemization, Boc-L-Phe-
CNPO-Resin III (0.1 g, 0.28 mmol/g) was swelled in THF,
and treated with excess of (R)-a-methylbenzylamine (40 ml,
0.28 mmol, 10 equiv.). After reacting for 1 h at room
temperature, the filtrate and the washing solution were
combined and passed through a silica gel short column to