Anti-tumor pyrimidinylpiperazines bind to the prosurvival Bcl-2 protein family member Bcl-XL

Article abstract of DOI:10.1016/j.bmcl.2011.01.054

Overexpression of prosurvival or underexpression of pro-death Bcl-2 family proteins can lead to cancer cell resistance to chemotherapy and radiation treatment. Inhibition of the prosurvival Bcl-2 family proteins has become a strategy for cancer therapy and inhibitors are currently being evaluated in the clinic both as single agents and in combination with established drugs. Here we describe the design, synthesis, and evaluation of pyrimidylpiperazines that were discovered to be inhibitors of the prosurvival Bcl-2 protein family member Bcl-XL. This study identified compound 21 which demonstrated a GI₅₀ value of 8.4 μM against A549 lung adenocarcinoma cells and a binding affinity K_i value for Bcl-XL of 127 nM.

Full text of DOI:10.1016/j.bmcl.2011.01.054

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1325–1328
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-tumor pyrimidinylpiperazines bind to the prosurvival Bcl-2 protein
family member Bcl-XL
Hassan M. Shallal a, Jesika S. Faridi b, Wade A. Russu a,
⇑
a Department of Pharmaceutics & Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA 95211, United States
b Department of Pharmacology & Physiology, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA 95211, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Overexpression of prosurvival or underexpression of pro-death Bcl-2 family proteins can lead to cancer
cell resistance to chemotherapy and radiation treatment. Inhibition of the prosurvival Bcl-2 family pro-
teins has become a strategy for cancer therapy and inhibitors are currently being evaluated in the clinic
both as single agents and in combination with established drugs. Here we describe the design, synthesis,
and evaluation of pyrimidylpiperazines that were discovered to be inhibitors of the prosurvival Bcl-2 pro-
Received 16 December 2010
Revised 12 January 2011
Accepted 14 January 2011
Available online 28 January 2011
tein family member Bcl-XL. This study identified compound 21 which demonstrated a GI50 value of 8.4
against A549 lung adenocarcinoma cells and a binding affinity Ki value for Bcl-XL of 127 nM.
lM
Keywords:
Bcl-2
Bcl-XL
Ó 2011 Elsevier Ltd. All rights reserved.
Apoptosis
Cancer
Pyrimidinylpiperazine
Molecules that can selectively kill tumor cells have great poten-
tial as drug candidates for the treatment of various cancers. One
strategy for the development of potential drug molecules that
can selectively kill tumor cells is the modulation of the apoptosis
process. Apoptosis, sometimes referred to as programmed cell
death, is the process by which cell homeostasis is maintained
and by which devastating genetic abnormalities are dealt.
The B-cell lymphoma-2 (Bcl-2) protein family plays an important
role in the regulation of apoptosis in mammalian cells.1 Apoptosis
control is characterized by a delicate balance between homo- and
hetero-dimerization of pro- and anti-apoptosis members of the pro-
tein family. Inhibiting this protein–protein interaction is one viable
approach to cancer therapy.2 Anti-apoptosis (prosurvival) family
members Bcl-2, Mcl-1, and Bcl-XL are current targets for anti-cancer
drug design.
The Bcl-XL protein is an anti-apoptotic member of the Bcl-2
family and acts by forming a hetero-dimer with, and thus inhibit-
ing the function of pro-apoptosis activator proteins including Bid
and Bim. This association is mediated through the binding of a
BH3 domain helix of Bid and or Bim to a hydrophobic cleft on
the surface of Bcl-XL. The function of pro-apoptosis activator pro-
teins is to form a hetero-dimeric complex with pro-apoptosis
Bcl-2 family members Bak or Bax and initiate the release of cyto-
chrome c (Cyt c) from the inner membrane of the mitochondria.
Cytochrome c induces the formation of the apoptosome with
subsequent activation of caspase-9 followed by caspases-3 and -7
that degrade various cellular targets and cause cell death.3
There are drug leads currently in the developmental pipeline
that target Bcl-2 anti-apoptotic protein family members as well
as drug candidates in current clinical trials. Gossypol,4 other poly-
phenols, and antimycins5 are natural product small molecules
known to bind to Bcl-2 and/or Bcl-XL that are currently being eval-
uated in clinical trials for B-cell malignancies and breast cancer
chemoprevention, respectively. Apogossypol6 and TW-377 are de-
signed small molecule inhibitors of Bcl-2 family proteins based
upon gossypol. Fragment based design methods have produced
the acylsulfonamide class of inhibitors,8 isosteric replacement
based analogs,9 and thiazolidin derivatives.10 Obatoclax11 is in clin-
ical trial for hematological malignancies and ABT-263,12 an acyl-
sulfonamide, is being evaluated in trials for CLL and advanced
small cell lung cancer.
The existence of numerous PDB entries for complexes of Bcl-XL
bound to peptide and various ligands provided an excellent oppor-
tunity for structure-based design of potential new ligands. Bcl-XL
sequesters Bak, Bim, Bid, and other pro-death Bcl-2 protein family
members by binding of their BH3 helix in a groove on the surface of
Bcl-XL. The binding groove on the surface of Bcl-XL is quite large
and may be divided into three regions: (1) A deep narrow binding
site (site 1). (2) A broad shallow binding site (site 2). (3) An aro-
matic ridge that separates site 1 from site 2. Figure 1 depicts the
binding site on Bcl-XL empty (left) and occupied by one of our de-
signed ligands 22 (right). Studies have shown that molecules that
bind tightly to their protein targets are more often ‘needle-like’
⇑
Corresponding author.
E-mail address: wrussu@pacific.edu (W.A. Russu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.054

1326
H. M. Shallal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1325–1328
Figure 1. Close-up view of X-ray derived crystal structure of Bcl-XL binding site alone (left) and docked with 22 (magenta, right).
Figure 2. General structural formula of pyrimidinylpiperazines.
and bind to deep pockets.13 These observations led us to reason
that most of the binding affinity of a drug molecule bound to
Bcl-XL will likely come from targeting the lower deep narrow bind-
ing site (site 1).
Scheme 2. Chemical synthesis of intermediate 2.
Based on these observations and molecular docking experi-
ments we designed a series of molecules with a pyrimidinylpiper-
azine core which primarily target site 1 (Fig. 2). The 5-position of
the pyrimidine is substituted with either an ethyl or benzyl group.
The site 1 binding group was varied with N-benzylic substituents.
The piperazine ring system is attractive for its symmetry, nitrogen
atom content, and low molecular weight. From a synthetic point of
view the binucleophilic character of piperazine makes it easy to
derivatize, and to design multiple series of molecules. Schemes 1
and 2 exhibit the synthesis of these basic core molecules 1 and 2.
Commercially available starting materials were converted to 1
and 2 in two steps. A nucleophilic aromatic substitution reaction
followed by removal of the Boc group provided 1 (Scheme 1). A Su-
zuki cross-coupling reaction followed by removal of the Boc group
provided 2 (Scheme 2).14
Molecules 1 and 2 were derivatized to provide the N-benzyl ser-
ies. Reductive amination with aromatic aldehydes gave molecules
5–22 (Scheme 3).15 Transformation of the nitro derivatives 10,
11, 19, and 20 to the corresponding amino derivatives 23–26 was
problematic. Attempted hydrogenation with H2 and Pd/C resulted
in significant debenzylation of the piperazine nitrogen. Reduction
using the SnCl2 method was messy and difficult to workup.16 Ulti-
mately reduction with iron(II) sulfide provided clean conversion to
Scheme 3. Chemical synthesis of final compounds 5–22.
the desired products (Scheme 4).17 Molecules 12, 13, 21, and 22 re-
quired the aromatic aldehydes 3 and 4 which were prepared via
Scheme 1. Chemical synthesis of intermediate 1.

H. M. Shallal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1325–1328
1327
Table 1
Growth inhibition of A549 lung adenocarcinoma cells by N-benzyl pyrimidinylpip-
erazine analogs
Compound
R
X
Y
GI50 SEMa
(lM)
5
6
7
Ethyl
Ethyl
Ethyl
H
H
F
H
Br
Br
>100
70.9 2.57
>100
8
9
Ethyl
Ethyl
Ethyl
Ethyl
Ethyl
Ethyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Ethyl
OH
OH
NO2
NO2
H
F
H
H
F
OH
OH
NO2
NO2
H
H
Br
H
Cl
>100
57.6 48.7
88.0 4.25
67.2 4.99
51.4 2.01
49.2 0.95
49.6 4.85
86.5 42.3
>100
51.1 0.70
23.7 1.78
61.2 15.5
>100
8.43 2.02
9.91 1.18
>100
62.9 1.61
75.4 4.16
15.1 2.06
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Scheme 4. Chemical synthesis of final compounds 23–26.
1H-Pyrazol-4-yl
1H-Pyrazol-4-yl
H
Br
Br
H
Br
H
Suzuki cross-coupling reaction according to Scheme 5. Synthetic
intermediates and final products were characterized by NMR (1H
and 13C) and mass spectrometry, and the purity of final products
was determined by analytical HPLC (see Supplementary data).
The molecules were screened against human non-small cell
lung tumor A549. The A549 cell line expresses significant amounts
of Bcl-XL protein and is moderately sensitive to Bcl-XL inhibitors.18
In order to identify active molecules, we initially screened at a sin-
Cl
1H-Pyrazol-4-yl
1H-Pyrazol-4-yl
H
Cl
H
F
NH2
NH2
NH2
NH2
Ethyl
Benzyl
Benzyl
gle high dose of 100 lM using the sulfarhodamine B (SRB), 48-h
growth inhibition assay.19 The SRB assay identified several mole-
cules as having significant activity at this concentration (data not
shown). This initial screen was followed up with determination
of these molecules’ GI50 values. The GI50 values against the cell line
are shown in Table 1. Additionally, we confirmed that the cell line
used for screening expressed Bcl-XL by immunoprecipitation. Rep-
resentative active molecules were then evaluated in a competitive
protein binding assay. Molecules 17, 21, and 26 were subjected to a
binding competition for Bcl-XL with a fluorescent BAK BH3 pep-
tide.20 The inhibition constants (Ki) for these molecules are re-
ported in Table 2.
The prepared pyrimidinylpiperazines exhibit inhibitory activity
against the A549 lung adenocarcinoma cell line. The GI50 values for
this cell line make it evident that the benzyl series is more potent
than the ethyl series. From this observation it is surmised that the
hydrophobic aromatic group is necessary at the 5-position of the
pyrimidine ring in the molecule for either binding to the biological
target or efficient penetration of the cell. The most active mole-
cules bear a hydrogen bond donor/acceptor group and a lipophilic
(halogen or pyrazole ring) group on the common N-benzylic sub-
stituent in both series.
Cl
a
Values are the average of two experiments done in triplicate.
Table 2
Binding affinities of select pyrimidinylpiperazines for Bcl-XL
Compound
Ki SEMa
(lM)
0.52 0.21
0.127 0.04
0.170 0.07
The pyrimidinylpiperazines have a relatively small ethyl or ben-
zyl group that accesses but does not fill site 2, yet these molecules
still bind to Bcl-XL in the mid-nM range. Figure 1 demonstrates the
predicted binding geometry of 22 in the binding site of Bcl-XL.
From the predicted docking pose, it is hypothesize that the pyri-
midinylpiperazine core of 22 adopts a conformation that comple-
ments the contour of the binding site and that the benzyl group
at the 5-position of the pyrimidine just accesses binding site 2.
a
Values are average of three experiments.
The favorable shape complementarity of 22 and Bcl-XL allows
the N-benzyl substituent to penetrate deep into the site 1 binding
Scheme 5. Chemical synthesis of intermediates 3 and 4.

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H. M. Shallal et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1325–1328
site. The docking experiment did not predict the formation of
hydrogen bonds between Bcl-XL and either 21 or 22. However, it
should be noted that the flexibility of Bcl-XL may allow hydrogen
bonding between the pyrazolyl group of 21 and 22 to a main-chain
carbonyl as well as the side chain of Ser145 near the bottom of site
1, whereas in silico docking experiments performed with the rigid
X-ray derived structure of Bcl-XL may be incapable of predicting
these interactions.
In summary, based on available structural information of pep-
tide and acylsulfonamide complexes with Bcl-XL, we have de-
signed, synthesized, and conducted preliminary biological
evaluations of pyrimidinylpiperazine based inhibitors of the Bcl-2
prosurvival protein family member Bcl-XL. Two series of pyrimid-
inylpiperazines were synthesized in few steps from readily avail-
able starting materials. The two series are distinguishable by the
substituent on the pyrimidine ring which is either an ethyl or a
benzyl group. The syntheses of both series are relatively straight
forward and afford reasonable yields of final products. These pyri-
midinylpiperazines showed promising activities on both the cyto-
toxic and Bcl-XL binding levels. These molecules conform to the
standard Lipinski rules for druglikeness.21,22 This new class of
Bcl-XL inhibitors represent valuable new lead molecules for further
development. Based on in silico docking experiments these mole-
cules may take advantage of favorable shape complementary to
the BH3 helix binding site, a matter which warrants further
investigation.
References and notes
1. Adams, J. M.; Cory, S. Science 1998, 281, 1322.
2. Huang, Z. Chem. Biol. 2002, 9, 1059.
3. Green, D. R.; Evan, G. I. Cancer Cell 2002, 1, 19.
4. Qui, J.; Levin, L. R.; Buck, J.; Reidenberg, M. M. Exp. Biol. Med. 2002, 227, 389.
5. Tzung, S.-P.; Kim, K. M.; Basanez, G.; Giedt, C. D.; Simon, J.; Zimmerberg, J.;
Zhang, K. Y. J.; Hockenbery, D. M. Nat. Cell Biol. 2001, 3, 183.
6. Becattini, B.; Kitada, S.; Leone, M.; Monosov, E.; Chandler, S.; Zhai, D.; Kipps, T.
J.; Reed, J. C.; Pellecchia, M. Chem. Biol. 2004, 11, 389.
7. Wang, G.; Nikolovska-Coleska, Z.; Yang, C.-Y.; Wang, R.; Tang, G.; Guo, J.;
Shangary, S.; Qiu, S.; Gao, W.; Yang, D.; Meagher, J.; Stuckey, J.; Krajewski, K.;
Jiang, S.; Roller, P. P.; Abaan, H. O.; Tomita, Y.; Wang, S. J. Med. Chem. 2006, 49,
6139.
8. Bruncko, M.; Oost, T. K.; Belli, B. A.; Ding, H.; Joseph, M. K.; Kunzer, A.;
Martineau, D.; McClelen, W. J.; Mitten, M.; Ng, S.-C.; Nimmer, P. M.; Oltersdorf,
T.; Park, C.-M.; Petros, A. M.; Shoemaker, A. R.; Song, X.; Wang, X.; Wendt, M.
D.; Zhang, H.; Fesik, S. W.; Rosenberg, S. H.; Elmore, S. W. J. Med. Chem. 2007, 50,
641.
9. Dömling, A.; Antuch, W.; Beck, B.; Schauer-Vukašinovic´, V. Bioorg. Med. Chem.
Lett. 2008, 18, 4115.
10. Wang, L.; Sloper, D. T.; Addo, S. N.; Tian, D.; Slaton, J. W.; Xing, C. Cancer Res.
2008, 68, 4377.
11. Trudel, S.; Li, Z. H.; Rauw, J.; Tiedermann, R. E.; Wen, X. Y.; Stewart, A. K. Blood
2007, 109, 5430.
12. Park, C.-M.; Bruncko, M.; Adickes, J.; Bauch, J.; Ding, H.; Kunzer, A.; Marsh, K. C.;
Nimmer, P.; Shoemaker, A. R.; Song, X.; Tahir, S. K.; Tse, C.; Wang, X.; Wendt, M.
D.; Yang, X.; Zhang, H.; Fesik, S. W.; Rosenberg, S. H.; Elmore, S. W. J. Med. Chem.
2008, 51, 6902.
13. Kallblad, P.; Mancera, R. L.; Todorov, N. P. J. Med. Chem. 2004, 47, 3334.
14. Flaherty, A.; Trunkfield, A.; Barton, W. Org. Lett. 2005, 7, 4975.
15. Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. J.
Org. Chem. 1996, 61, 3849.
16. Difficulties with SnCl2 reductions have been noted previously. See note 25 in:
Song, A.; Zhang, J.; Lebrilla, C. B.; Lam, K. S. J. Comb. Chem. 2004, 6, 604.
17. Desai, D. G.; Swami, S. S.; Dabhade, S. K.; Ghagare, M. G. Synth. Commun. 2001,
31, 1249.
Acknowledgments
18. Wesarg, E.; Hoffarth, S.; Wiewrodt, R.; Kroll, M.; Biesterfeld, S.; Huber, C.;
Schuler, M. Int. J. Cancer 2007, 121, 2387.
19. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.
20. O’Neill, J. W.; Manion, M. K.; Maguire, B.; Hockenbery, D. M. J. Mol. Biol. 2006,
356, 367.
21. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
1997, 23, 3.
The authors thank Mr. Matthew Curtis for obtaining HRMS data
and the National Science Foundation for grant support of the UOP
NMR facility (NSF-MRI-0722654). W.A.R. is grateful for support in
the form of the Burroughs Wellcome Fund-AFPE-AACP New Inves-
tigator Grant for Pharmacy Faculty.
Supplementary data
22. Rishton, G. M. DDT 2003, 8, 86.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.054.