Metathesis reactions of pyrazolotriazinones generate dynamic combinatorial libraries

Article abstract of DOI:10.1021/ol051839s

(Chemical Equation Presented) Reversible metathesis reactions of pyrazolotriazinones and aliphatic aldehydes or ketones proceed in aqueous, phosphate-buffered media at pH 4 and 40-60°C to generate thermodynamically controlled mixtures of heterocycles.

Full text of DOI:10.1021/ol051839s

ORGANIC
LETTERS
2005
Vol. 7, No. 20
4483-4486
Metathesis Reactions of
Pyrazolotriazinones Generate Dynamic
Combinatorial Libraries
Peter Wipf,* S. Graciela Mahler, and Kazuo Okumura
Department of Chemistry, UniVersity of Pittsburgh, Pittsburgh, PennsylVania 15260
pwipf@pitt.edu
Received August 1, 2005
ABSTRACT
Reversible metathesis reactions of pyrazolotriazinones and aliphatic aldehydes or ketones proceed in aqueous, phosphate-buffered media at
pH 4 and 40 60 C to generate thermodynamically controlled mixtures of heterocycles.
−
°
Dynamic combinatorial chemistry (DCC) is a promising
new strategy with potential applications in drug discovery,
host-guest chemistry, and catalysis. The principle of DCC
is based on the use of reversible reactions to generate
compound mixtures that respond to the presence of a
template (enzyme, protein, small molecule, or ion) or a
change in environment. Ideally, these dynamic combinatorial
libraries (DCLs) rapidly evolve toward major products that
possess the highest affinity to the template or that adapt best
to the reaction environment. The concept of DCL has been
widely reviewed, and successful applications have been
described.1
While the chemical literature offers a wide selection of
protocols for essentially irreversible processes, relatively few
reversible reactions are available for DCL generation. These
include transesterification,2 disulfide exchange,3 Schiff base
exchange,4 oxime and hydrazone metathesis,5,6 transpepti-
dation,7 Diels-Alder reaction,8 olefin metathesis,9 and bo-
ronic ester exchange.10 A further expansion of reactions
suitable for DCC is clearly desirable, especially if an aqueous
environment can be tolerated.11
We have been interested in the development of reversible
heterocycle formations suitable for DCL generation and hit
identification. The pyrazolotriazine scaffold was particularly
attractive in this context since it could be constructed from
readily available carbonyl compounds and acylhydrazides.
The 6,7-dihydro-5H-pyrazolo[1,5-d][1,2,4]triazin-4-one was
first reported by Ainsworth in 1955,12 and this scaffold was
subsequently shown to have anti-inflammatory properties.13
(4) (a) Huc, I.; Lehn, J.-M. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2106.
(b) Leung, K. C. F.; Arico, F.; Cantrill, S. J.; Stoddart, J. F. J. Am. Chem.
Soc. 2005, 127, 5808. (c) Oh, K.; Jeong, K.-S.; Moore, J. S. J. Org. Chem.
2003, 68, 8397.
(5) Polyakov, V. A.; Nelen, M. I.; Nazarpack-Kandlousy, N.; Ryabov,
A. D.; Eliseev, A. V. J. Phys. Org. Chem. 1999, 12, 357.
(6) (a) Cousins, G.; Poulsen, S.-A.; Sanders, J. K. M. Chem. Commun.
1999, 1575. (b) Nguyen; Ivan Huc, R. Chem. Commun. 2003, 942.
(7) Swann, P. G.; Casanova, R. A.; Desai, A.; Frauenhoff, M. M.;
Urbancic, M.; Slomczynska, U.; Hopfinger, A. J.; Le Breton, G. C.; Venton,
D. L. Biopolymers 1996, 40, 617.
(8) Boul, P. J.; Reutenauer, P.; Lehn, J.-M. Org. Lett 2005, 7, 15.
(9) (a) Bra¨ndli, C.; Ward, T. R. HelV. Chim. Acta 1998, 81, 1616. (b)
Nicolaou, K. C.; Hughes, R.; Cho, S. Y.; Winssinger, N.; Smethurst, C.;
Labischinski, H.; Enderman, R. Angew. Chem., Int. Ed. 2000, 39, 3823.
(10) Nakazawa, I.; Suda, S.; Masuda, M.; Asai, M.; Shimizu, T. Chem.
Commun. 2000, 10, 881.
(1) (a) Lehn, J.-M. Science 2002, 295, 2400. (b) Otto, S. Curr. Opin.
Drug Discuss. DeV. 2003, 6, 509. (c) Otto, S.; Furlan, R. L. E.; Sanders, J.
K. M. Curr. Opin. Chem. Biol. 2002, 6, 321. (d) Lehn, J.-M. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 4763. (e) Otto, S.; Furlan, R. L. E.; Sanders, J.
K. M. Drug DiscoV. Today 2002, 7, 117. (f) Furlan, R. L. E.; Otto, S.;
Sanders, J. K. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4801. (g) Vial,
L.; Sanders, J. K. M.; Otto, S. New J. Chem. 2005, 29, 1001. (h) Corbett,
P. T.; Sanders, J. K. M.; Otto, S. J. Am. Chem. Soc. 2005, 127, 9390.
(2) Brady, P. A.; Bonar-Law, R. P.; Rowan, S. J.; Suckling, C. J.; Sanders,
J. K. M. Chem. Commun. 1996, 319.
(3) (a) Ramstro¨m, O.; Lehn, J.-M. ChemBioChem 2000, 1, 41. (b) Otto,
S.; Furlan, R. L. E.; Sanders, J. K. M. J. Am. Chem. Soc. 2000, 122, 12063.
(c) West, K. R.; Bake, K. D.; Otto, S. Org. Lett. 2005, 7, 2615.
(11) Rowan, S. J.; Cantrill, S. J.; Cousins, G. R. L.; Sanders, J. K. M.;
Stoddart, J. F. Angew. Chem., Int. Ed. 2002, 41, 899.
10.1021/ol051839s CCC: $30.25
© 2005 American Chemical Society
Published on Web 09/01/2005

The parent pyrazolotriazinone heterocycles were synthe-
sized in a four-step sequence as shown in Scheme 1.
Table 1. Optimization of Pyrazolotriazinone Formation
Scheme 1. Synthesis of Pyrazolotriazinones
starting
material
entry
reaction conda
time (h)
6a/6b (ratio)b
1
2
3
4
5
6a
6a
6b
6a
6a
pH 4, rt
48
72
72
120
48
97:3
1:1
48:52
52:48
1:0
pH 4, 40 °C
pH 4, 40 °C
pH 4, 40 °C
pH 5, 36 °C
a The concentration of each component was 1 mM in a buffered
phosphate solution. b The ratio was determined by GC, using 1,4-
dimethoxybenzene as an internal standard, and confirmed by preparative
isolation.
Similar results could be observed with either 7a or 7b as
starting materials using the same equilibrating conditions,
as shown in Table 2 (entries 1 and 2).16
Table 2. Equilibration Condition for Compounds 7a and 7b
Ketoesters 3 were obtained by aldol condensation of acetone
(1) and diethyloxalate (2) in EtOH/EtONa at room temper-
ature. The cyclocondensation of 3 with hydrazine in basic
aqueous media led the pyrazole carboxylic acid 4,14 which
could be converted into pyrazolecarboxylic hydrazide 5 by
acylation of methyl hydrazine using DCC as an activating
agent. Ring closure of 5 was achieved with a range of
aliphatic aldehydes to give the corresponding pyrazolotri-
azinone derivatives 6.15 However, attempts to synthesize
pyrazolotriazinones from aromatic aldehydes or ketones
failed.
starting
material
entry
reaction conda
time (h)
7a/7b (ratio)b
1
2
7a
7b
pH 4, 40 °C
pH 4, 40 °C
72
72
3:7
3:7
a The concentration of each component was 1 mM in a buffered
phosphate solution. b The ratio was determined by GC, using 1,4-
dimethoxybenzene as an internal standard, and confirmed by preparative
isolation (7a, 25%; 7b, 67%).
Several conditions were screened to identify a thermody-
namically controlled cyclocondensation-carbonyl exchange
process in aqueous media (Table 1). The reaction of 6a with
equimolar amounts of hydrocinnamaldehyde and acetalde-
hyde at room temperature and pH 4 over 2 days proceeded
only slowly (entry 1). Furthermore, at pH 5 and 36 °C only
starting material could be detected (entry 5).
Thermodynamic equilibration was achieved at pH 4 after
3 d at 40 °C (entries 2 and 3). After 5 d, these compounds
were still stable in the aqueous environment, and the
distribution was maintained (entry 4). We also probed the
reversibility of the system by starting from products 6a or
6b (entries 2 and 3, respectively) and obtained an identical
product ratio either way. The yields of recovered 6a and 6b
were quantitative.
These results demonstrate that despite the acidic aqueous
reaction conditions, heterocycles 6 and 7 were not irreversibly
hydrolyzed to the acyl hydrazide precursors. In all cases,
pyrazolotriazinones proved stable to the mildly acidic aque-
ous enviroment, but the yield and rate of heterocycle
formation were dependent on pH, concentration, and tem-
perature. As further proof of principle, a small DCL was
generated by incubating pyrazolotriazinone 6c with aldehydes
8-11 (Scheme 2). The starting concentrations of aldehydes
were kept at 1 mM each, and a 1 mM concentration of 6c
was established at reaction onset. The product distribution
was established by GC analysis using independently prepared
(12) Ainsworth, C. J. Am. Chem. Soc. 1955, 77, 1148.
(13) Tihanyi, E.; Ga´l, M.; Feher, O¨ .; Janaky, J. HU 42771, 1983.
(14) Baraldi, P. G.; Cacciari, B.; Romagnoli, R.; Spalluto, G. Synthesis
1999, 453.
(15) (a) Tihanyi, E.; Ga´l, M.; Feher, O¨ .; Dvortsa´k, P. J. Heterocycl. Chem.
1989, 26, 1045. (b) Tihanyi, E.; Ga´l, M.; Feher, O¨ .; Janaky, J. HU 42771,
1987.
(16) Compounds 7a and 7b were synthesized using the same four-step
sequence as shown in Scheme 1, starting with 3-methyl-2-butanone: (1)
EtOH/EtONa, 87%; (2) NH2NH2/KOH, 80%; (3) BOP, DIPEA, THF, 45%,
(4) 7a, EtOH, rt, OHCCH(CH3)2, 70%; 7b, EtOH, rt, OHC(CH2)2Ph, 90%.
4484
Org. Lett., Vol. 7, No. 20, 2005

Scheme 2. Reversible Exchange Reactions between
Pyrazolotriazinone 6c and the Aldehydes 8-11
Scheme 3. Metathesis of Pyrazolotriazinonesa
a The yields in parentheses reflect the equilibrium distribution.
We further investigated the scope of this new DCC
methodology by varying the substituents on the hydrazine
segment and using ketones as exchange components. At-
tempts to equilibrate the system composed of 5,6-disubsti-
tuted pyrozolotriazinones 12 and 13 under the standard
conditions failed; no exchange product was detected (Figure
1). Heterocycle 12 was further incubated with 1 equiv of 6e
6b-f as reference compounds for the identification of each
peak. In addition to the metathesis products, only traces of
hydrazide 5 were detected by 1H NMR and GC-MS in the
reaction mixture.17 It is interesting to note that the thermo-
dynamic distribution of these heterocycles is not exactly
even. This could be due to differential steric interactions of
side chain substituents with the heterocyclic cores or variation
in the free energy of aqueous solvation which is expected to
change considerably depending on the exact substitution
pattern of the pyrazolotriazinones.18 Heterocycles 6 and 7
are stable in acidic, neutral, and basic aqueous media over
several days; however, after several weeks exposure to air,
small amounts of the oxidized 5H-pyrazolo[1,5-d][1,2,4]-
triazin-4-ones are detectable by LC-MS.
For the screening of DCLs in biological assays, it is
important to minimize the presence of free aldehydes which
are capable of covalently modifying active site residues.
Therefore, we tested the possibility for direct side-chain
metathesis of pyrazolotriazinones. An equimolecular mixture
of 6c and 7a, which differ by their substitutions at the
2-positions of the heterocycles, was equilibrated during 3 d
at pH 4 and 40 °C (Scheme 3). As expected, a mixture of
four products (the original starting materials and two
crossover derivatives) was formed. The equilibration distri-
bution was identical when 6b and 7b were used as starting
materials.19
Figure 1. N(5),N(6)-Disubstituted pyrazolotriazinones are stable
under the metathesis reaction conditions.
in the aqueous buffer solution for 4 d, but only starting
materials were recovered. Heterocycle 13 was allowed to
equilibrate with cylohexanecarboxaldehyde in a 1 mM buffer
solution, and exchange product was not detected either. We
hypothesized that substitution at N(6) of the pyrazolotriazi-
none scaffold stabilizes the ring system sufficiently to prevent
reversible ring opening.
In contrast, the equilibration of ketone-derived C(7)-
disubstituted pyrazoltriazinones was feasible but required a
prolonged equilibration time and slightly harsher conditions
(Scheme 4). After a 4 d exposure to pH 4 phosphate buffer
at 60 °C, a 1 mM solution of 14a20 and ketones 15, 16, and
17 was fully equilibrated. LC-MS analyses detected a small
amount of an unidentified byproduct; otherwise, the reaction
was clean.
(17) Typical Procedure. A mixture of compound 6c (6.4 mg, 0.031
mmol) and aldehydes 8-11 (0.031 mmol each) in a phosphate buffer
solution at pH 4 was stirred for 3 d (72 h) at 40 °C. The pH was raised to
7 with NaHCO3, and the reaction mixture was extracted with AcOEt. The
combined organic layers were dried (MgSO4), filtered, and concentrated in
vacuo. The residue was analyzed by GC using dimethoxyphenol as an
internal standard, or alternatively, by LC-MS.
(18) See, for example: (a) Jorgensen, W. L.; Tirado-Rives, J. Persp.
Drug Discuss. Design 1995, 3, 123. (b) Perrin, C. L.; Fabian, M. A.; Rivero,
I. A. J. Am. Chem. Soc. 1998, 120, 1044.
In summary, we developed a new exchange reaction
between pyrazolotriazinone heterocycles and carbonyl com-
(19) We currently do not know the exact mechanism of the exchange
process. Both a direct metathesis as well as an exchange reaction mediated
by small amounts of hydrolyzed intermediates are feasible.
(20) Compound 14a was synthesized from hydrazide 5 and cyclohex-
anone in EtOH at rt in 70% yield.
Org. Lett., Vol. 7, No. 20, 2005
4485

sents a significant extension of current DCC methodologies.
Structural diversity of the core scaffolds can be accomplished
by modifications at the 2- and 7-positions of the heterocycles.
The pyrazolotriazinones are stable in buffered media over
several days, and these conditions are suitable for the
generation of DCLs as well as for the direct biological
screening of these libraries.21 Moreover, the exchange
reaction can be stopped by raising the pH to 7, thus providing
a convenient way to analyze the compound distribution
patterns.
Scheme 4. Metathesis of C(7)-Disubstituted
Pyrazolotriazinones
Acknowledgment. This work was supported in part by
the NIH P50 program (GM067082) and by the National
Cancer Institute (CA78039).
Supporting Information Available: Experimental pro-
cedures, GC and LC-MS traces, and spectral data for
compounds 6b-f, 7a,b, and 12-14. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
OL051839S
(21) The optimal pH stability of many biological targets ranges from
pH 4 to 8; therefore, DCLs of pyrazolotriazinones can be directly exposed
to proteins in order to discover new binding agents. Schaefer, M.; Sommer,
M.; Karplus, M. J. Phys. Chem. B 1997, 101, 1663.
pounds. This dynamic exchange proceeds in a reversible
fashion in a mildly acidic aqueous environment and repre-
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