Efficient synthesis of pyrazolopyrimidine libraries

Full text of DOI:10.1021/cc1001204

J. Comb. Chem. 2010, 12, 807–809
807
stepwise synthesis of intermediates and found that the initial
diversification by amino displacement with anilines 2{1-8}
(Figure 1) proceeded readily in moderate to high yield for
these intermediates (56-98%, see Supporting Information)
to give chemset 3{1-8}, without the need for chromatog-
raphy. After some experimentation, we found that if the
intermediates in chemset 3 were treated with anilines
4{1-16} (Figure 2) under mildly basic conditions, in the
presence of catalytic amounts of phase transfer catalyst, the
substituted diamino intermediates could be obtained, in a
regioselective manner.25 We then investigated conditions for
the final high throughput diversification and cyclization of
the resulting substituted 2,4-diamino-6-chloropyrimidin-5-
carbaldehyde reaction mixture and eventually developed
conditions that cleanly produced pyrazolopyrimidines 6
efficiently and directly upon treatment with hydrazines
5{1-4} (Figure 3).
Efficient Synthesis of Pyrazolopyrimidine
Libraries
P. Jake Slavish, Jeanine E. Price, Parimala Hanumesh,
and Thomas R. Webb*
Department of Chemical Biology & Therapeutics, St. Jude
Children’s Research Hospital, 262 Danny Thomas Place,
Memphis, Tennessee 38105
ReceiVed June 21, 2010
The 1H-pyrazolo[3,4-d]pyrimidine ring system is an
important structural template in anticancer research and in
drug discovery as antibacterial1 and histamine release agents.2
There have been numerous recent literature reports on the
synthesis of relatively small sets of compounds bearing the
pyrazolopyrimidine core that have been shown to inhibit
several oncogenic kinases including: GSK-3,3 mTOR,4-8
Src,9 Abl,10 EGF-R,11 and PI3K.12,13 Because of the preva-
lence of this template in drug discovery, several reports
of pyrazolopyrimidine library synthesis have also ap-
peared. For example, Tan and co-workers have recently
reported traceless solid-phase syntheses of pyrazolo[3,4-d]-
pyrimidines libraries to examine their efficacy against
multidrug resistance protein 4.14 Daniels et al. have
reported conditions that have been used to construct
pyrazolopyrimidine libraries from pyrazole precursors.15 In
addition, Revesz et al. have prepared pyrazolopyrimidine
based libraries targeting novel p38R MAP kinase inhibitors.16
While these previously published library protocols do gener-
ate interesting and useful compounds they have some
limitations. These limitations include (1) only two diversity
points stemming from the bicyclic core are diversified out
of the three possible within this substructure,14-16 (2) at least
four synthetic steps are required,15,16 (3) harsh reagents (e.g.,
sulfuric acid, phosphorus oxychloride, and chromium oxide)
are utilized,15,16 and (4) finally, some require aqueous workup
of the final products,15,16 which is often undesirable in high-
throughput library production.
Considering this background, our programmatic interest
in the identification of inhibitors of oncogenic kinase-driven
pediatric cancers, for example, neuroblastoma17,18 and ana-
plastic large cell lymphoma,19,20 and the recent reports on a
pyrrolopyrimidine series,21,22 which exhibited activity against
anaplastic lymphoma kinase (ALK),23,24 we set out to
develop a method that could provide both diverse and highly
targeted pyrazolopyrimidine-based kinase libraries in a high
throughput mode. Herein, we describe our rapid and produc-
tive process for the parallel synthesis of diverse 1H-
pyrazolo[3,4-d]pyrimidines starting from readily prepared
carbaldehydes 3.
After this research and development on the stepwise
protocol, we then proceeded to explore the possibility of
a two-pot protocol with no chromatographic purification
of any intermediates. This type of procedure would allow
for the preparation of a large number of compounds in a
high-throughput manner to explore the chemistry space
surrounding this interesting substructure. Our goal was
not the production of a few compounds in high yield, but
rather the high throughput preparation of the largest and
most diverse set of compounds possible. Since we
routinely purify all library compounds by HPLC we did
not require each crude reaction mixture to be pure, instead
we only required that we could obtain sufficient material
of high final purity. The overall yields of purified
representative pyrazolopyrimidine examples using the
optimized high-throughput protocol are provided in Table
1
1. Compounds 6{1-4,1-16,1} were characterized by H
and 13C NMR spectroscopy. All other members were
confirmed by LC-MS, using UV and ELSD for additional
purity criteria.
Generally, all anilines used for the second displacement
worked rather well, regardless of the functionality present
on the aromatic ring. In instances where (unsubstituted)
hydrazine was used, we were able to successfully syn-
thesize and purify greater than 1 mg of >85% pure material
(purity based on a combination of UV and ELSD purities)
77% of the time with an average yield of 35%. As for the
alkyl hydrazines, the success rate was not as high (58%)
but the average yield was similar to the yields observed
when hydrazine was used (32%). The major drawback in
cases where alkyl hydrazines were used was lack of
cyclization in the final step, presumably due to steric
interactions. The uncyclized product tended to coelute with
the desired product, which complicated purification and
lowered the success rate or led to failures in some cases.
Regardless, we were pleased with the scope and overall
yield of this reaction sequence. Overall, it should be noted
that a significant cause of failures or low yields was the
Starting from the commercially available 2,4,6-trichloropy-
rimidin-5-carbaldehyde6,8 (1, Scheme 1) we investigated the
* To whom correspondence should be addressed. E-mail:
thomas.webb@stjude.org.
10.1021/cc1001204  2010 American Chemical Society
Published on Web 08/30/2010

808 Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 6
Scheme 1. Synthesis of the Pyrazolopyrimidine Corea
Reports
a Reagents and conditions: (a) R1NH2, KHCO3, TBAI, CH2Cl2, or THF/H2O (1:1) rt; (b) R2NH2, KHCO3, TBAI, CH2Cl2/H2O (1:1) rt; (c) R3NHNH2,
THF reflux (where R3 ) H) or Et3N, EtOH reflux (where R3 ) alkyl).
Figure 1. Anilines 2{1-8} used as building blocks.
Figure 2. Anilines 4{1-16} used as building blocks.
particularly toxic reagents and requires no protecting group
manipulation to provide a source of diversification. The
scope of the chemistry is well-defined and allows for
ample diversification. In addition, this chemistry can easily
be tailored to prepare individual compounds on a larger
Figure 3. Hydrazines 5{1-4} used as building blocks.
scale. Currently the size of this library is >1000 com-
pounds, which could readily be expanded. We envision
these libraries will be used in our HTS process for hit
identification for current and future institutional research
projects.
presence of impurities that overlapped with the product
peak, but since we were interested in rapid high throughput
preparation and purification, we did not make any attempts
at developing special purification protocols for these cases,
and so we present a procedure that is suitable for the
routine and rapid production of libraries.
We have developed a rapid method for the generation
of 1H-pyrazolo[3,4-d]pyrimidine libraries with three points
of diversity in moderate yields and with a relatively high
success rate. This process occurs in two pots without
Acknowledgment. The authors would like to thank the
staff in the High Throughput Analytical Center in our
department; Bing Yan, Andy Lemoff, Cynthia Jefferies, and
Lei Yang for their purification and quality control work for
most of the compounds described in this manuscript. This

Reports
Journal of Combinatorial Chemistry, 2010 Vol. 12, No. 6 809
Table 1. Isolated Overall Yields of the
Displacement-Cyclization Reaction Sequence from 3
(7) Zask, A.; Kaplan, J.; Verheijen, J. C.; Richard, D. J.; Curran,
K.; Brooijmans, N.; Bennett, E. M.; Toral-Barza, L.;
Hollander, I.; Ayral-Kaloustian, S.; Yu, K. J. Med. Chem.
2009, 52, 7942–7945.
(8) Zask, A.; Verheijen, J. C.; Curran, K.; Kaplan, J.; Richard, D. J.;
Nowak, P.; Malwitz, D. J.; Brooijmans, N.; Bard, J.; Svenson, K.;
Lucas, J.; Toral-Barza, L.; Zhang, W.-G.; Hollander, I.; Gibbons,
J. J.; Abraham, R. T.; Ayral-Kaloustian, S.; Mansour, T. S.; Yu, K.
J. Med. Chem. 2009, 52, 5013–5016.
(9) Manetti, F.; Santucci, A.; Locatelli, G. A.; Maga, G.; Spreafico,
A.; Serchi, T.; Orlandini, M.; Bernardini, G.; Caradonna, N. P.;
Spallarossa, A.; Brullo, C.; Schenone, S.; Bruno, O.; Ranise,
A.; Bondavalli, F.; Hoffmann, O.; Bologna, M.; Angelucci,
A.; Botta, M. J. Med. Chem. 2007, 50, 5579–5588.
(10) Schenone, S.; Brullo, C.; Bruno, O.; Bondavalli, F.; Mosti,
L.; Maga, G.; Crespan, E.; Carraro, F.; Manetti, F.; Tintori,
C.; Botta, M. Eur. J. Med. Chem. 2008, 43, 2665–2676.
(11) Traxler, P.; Bold, G.; Frei, J.; Lang, M.; Lydon, N.; Mett, H.;
Buchdunger, E.; Meyer, T.; Mueller, M.; Furet, P. J. Med.
Chem. 1997, 40, 3601–3616.
(12) Gilbert, A. M.; Nowak, P.; Brooijmans, N.; Bursavich, M. G.;
Dehnhardt, C.; Santos, E. D.; Feldberg, L. R.; Hollander, I.;
Kim, S.; Lombardi, S.; Park, K.; Venkatesan, A. M.; Mallon,
R. Bioorg. Med. Chem. Lett. 2010, 20, 636–639.
(13) Link, W.; Oyarzabal, J.; Serelde, B. G.; Albarran, M. I.; Rabal,
O.; Cebria, A.; Alfonso, P.; Fominaya, J.; Renner, O.;
Peregrina, S.; Soilan, D.; Ceballos, P. A.; Hernandez, A.-I.;
Lorenzo, M.; Pevarello, P.; Granda, T. G.; Kurz, G.; Carnero,
A.; Bischoff, J. R. J. Biol. Chem. 2009, 284, 28392–28400.
(14) Tan, T. M. C.; Yang, F.; Fu, H.; Raghavendra, M. S.; Lam,
Y. J. Comb. Chem. 2007, 9, 210–218.
(15) Daniels, R. N.; Kim, K.; Lebois, E. P.; Muchalski, H.; Hughes,
M.; Lindsley, C. W. Tetrahedron Lett. 2008, 49, 305–310.
(16) Revesz, L.; Blum, E.; Di Padova Franco, E.; Buhl, T.; Feifel,
R.; Gram, H.; Hiestand, P.; Manning, U.; Neumann, U.;
Rucklin, G. Bioorg. Med. Chem. Lett. 2006, 16, 262–6.
(17) Chen, Y.; Takita, J.; Choi, Y. L.; Kato, M.; Ohira, M.; Sanada,
M.; Wang, L.; Soda, M.; Kikuchi, A.; Igarashi, T.;
Nakagawara, A.; Hayashi, Y.; Mano, H.; Ogawa, S. Nature
(London, U. K.) 2008, 455, 971–974.
(18) George, R. E.; Sanda, T.; Hanna, M.; Froehling, S.; Luther,
W., II; Zhang, J.; Ahn, Y.; Zhou, W.; London, W. B.;
McGrady, P.; Xue, L.; Zozulya, S.; Gregor, V. E.; Webb,
T. R.; Gray, N. S.; Gilliland, D. G.; Diller, L.; Greulich, H.;
Morris, S. W.; Meyerson, M.; Look, A. T. Nature (London,
U. K.) 2008, 455, 975–978.
(19) Mosse, Y. P.; Wood, A.; Maris, J. M. Clin. Cancer Res. 2009,
15, 5609–5614.
(20) Palmer, R. H.; Vernersson, E.; Grabbe, C.; Hallberg, B.
Biochem. J. 2009, 420, 345–361.
(21) Chamberlain, S. D.; Redman, A. M.; Patnaik, S.; Brickhouse,
K.; Chew, Y.-C.; Deanda, F.; Gerding, R.; Lei, H.; Moorthy,
G.; Patrick, M.; Stevens, K. L.; Wilson, J. W.; Shotwell, J. B.
Bioorg. Med. Chem. Lett. 2009, 19, 373–377.
(22) Chamberlain, S. D.; Wilson, J. W.; Deanda, F.; Patnaik, S.;
Redman, A. M.; Yang, B.; Shewchuk, L.; Sabbatini, P.;
Leesnitzer, M. A.; Groy, A.; Atkins, C.; Gerding, R.; Hassell,
A. M.; Lei, H.; Mook, R. A., Jr.; Moorthy, G.; Rowand, J. L.;
Stevens, K. L.; Kumar, R.; Shotwell, J. B. Bioorg. Med. Chem.
Lett. 2009, 19, 469–73.
(23) Li, R.; Morris, S. W. Med. Res. ReV. 2008, 28, 372–412.
(24) Webb, T. R.; Slavish, J.; George, R. E.; Look, A. T.; Xue,
L.; Jiang, Q.; Cui, X.; Rentrop, W. B.; Morris, S. W. Expert
ReV. Anticancer Ther. 2009, 9, 331–356.
(25) Beingessner, R. L.; Deng, B.-L.; Fanwick, P. E.; Fenniri, H.
J. Org. Chem. 2008, 73, 931–939.
Chemset yield Chemset yield Chemset yield Chemset yield
6{1,1,1}
6{1,2,1}
6{1,3,1}
6{1,4,1}
6{1,5,1}
6{1,6,1}
6{1,7,1}
6{1,8,1}
6{1,9,1}
30 6{3,1,1}
56 6{3,2,1}
28 6{3,3,1}
49 6{3,4,1}
44 6{3,5,1}
60 6{3,6,1}
45 6{3,7,1}
49 6{3,8,1}
18 6{3,9,1}
38 6{4,1,2}
17 6{4,2,2}
27 6{4,3,2}
50 6{4,4,2}
38 6{4,5,2}
45 6{4,9,2}
33 6{4,12,2} 32 6{6,4,3}
46 6{5,1,2}
25 6{5,2,2}
28 6{5,5,3}
20 6{5,9,3}
40
42
0
6{5,12,3} 40
39 6{6,1,3}
13 6{6,2,3}
44 6{6,3,3}
57
50
74
49
54
52
51 6{6,5,3}
33 6{6,9,3}
30 6{6,12,3} 46
46 6{5,1,4}
47 6{5,2,4}
41 6{5,3,4}
6{1,10,1} 21 6{3,10,1} 19 6{5,3,2}
6{1,11,1} 33 6{3,11,1} 38 6{5,4,2}
6{1,12,1} 51 6{3,12,1} 44 6{5,5,2}
6{1,13,1} 47 6{3,13,1} 47 6{5,9,2}
6{1,14,1} 51 6{3,14,1} 36 6{5,12,2} 38 6{5,4,4}
6{1,15,1} 28 6{3,15,1} 28 6{6,1,2}
6{1,16,1} 26 6{3,16,1} 25 6{6,2,2}
24
17
20
10
12
28
50 6{5,5,4}
52 6{5,9,4}
58 6{5,12,4} 24
6{2,1,1}
6{2,2,1}
6{2,3,1}
6{2,4,1}
6{2,5,1}
6{2,6,1}
6{2,7,1}
6{2,8,1}
6{2,9,1}
30 6{4,1,1}
29 6{4,2,1}
17 6{4,3,1}
38 6{4,4,1}
43 6{4,5,1}
40 6{4,6,1}
23 6{4,7,1}
37 6{4,8,1}
42 6{4,9,1}
45 6{6,3,2}
34 6{6,4,2}
17 6{6,5,2}
58 6{6,9,2}
0
6{7,1,4}
9
0
24
7
25
27
57 6{7,2,4}
59 6{7,3,4}
53 6{6,12,2} 51 6{7,4,4}
56 6{4,1,3}
46 6{4,2,3}
48 6{4,3,3}
29 6{4,4,3}
0
6{7,5,4}
31 6{7,9,4}
10 6{7,12,4} 26
46 6{8,1,4}
39 6{8,2,4}
9
8
7
27
14
10
6{2,10,1} 23 6{4,10,1} 39 6{4,5,3}
6{2,11,1} 26 6{4,11,1} 29 6{4,9,3}
6{2,12,1} 15 6{4,12,1} 15 6{4,12,3} 33 6{8,4,4}
6{2,13,1} 25 6{4,13,1} 30 6{5,1,3}
6{2,14,1} 30 6{4,14,1} 27 6{5,2,3}
6{2,15,1} 23 6{4,15,1} 46 6{5,3,3}
6{2,16,1} 29 6{4,16,1} 16 6{5,4,3}
0
6{8,3,4}
50 6{8,5,4}
44 6{8,9,4}
50 6{8,12,4} 60
42
work is supported by the American Lebanese Syrian As-
sociated Charities (ALSAC), St. Jude Children’s Research
Hospital.
Supporting Information Available. General procedures
for the preparation of all compounds and spectroscopic data
(1H, 13C NMR spectra and HPLC chromatograms) for all
products. This material is available free of charge via the
Internet at http://pubs.acs.org.
References and Notes
(1) Kreutzberger, A.; Burgwitz, K. Eur. J. Med. Chem. 1979, 14,
539–41.
(2) Quintela, J. M.; Peinador, C.; Moreira, M. J.; Alfonso, A.;
Botana, L. M.; Riguera, R. Eur. J. Med. Chem. 2001, 36, 321–
332.
(3) Smalley, T. L.; Peat, A. J.; Boucheron, J. A.; Dickerson, S.;
Garrido, D.; Preugschat, F.; Schweiker, S. L.; Thomson, S. A.;
Wang, T. Y. Bioorg. Med. Chem. Lett. 2006, 16, 2091–2094.
(4) Curran, K. J.; Verheijen, J. C.; Kaplan, J.; Richard, D. J.;
Toral-Barza, L.; Hollander, I.; Lucas, J.; Ayral-Kaloustian,
S.; Yu, K.; Zask, A. Bioorg. Med. Chem. Lett. 2010, 20, 1440–
1444.
(5) Kaplan, J.; Verheijen, J. C.; Brooijmans, N.; Toral-Barza, L.;
Hollander, I.; Yu, K.; Zask, A. Bioorg. Med. Chem. Lett. 2010,
20, 640–643.
(6) Verheijen, J. C.; Richard, D. J.; Curran, K.; Kaplan, J.;
Lefever, M.; Nowak, P.; Malwitz, D. J.; Brooijmans, N.;
Toral-Barza, L.; Zhang, W.-G.; Lucas, J.; Hollander, I.; Ayral-
Kaloustian, S.; Mansour, T. S.; Yu, K.; Zask, A. J. Med.
Chem. 2009, 52, 8010–8024.
CC1001204