Identification of benzimidazole-based inhibitors of the mitogen activated kinase-5 signaling pathway

Article abstract of DOI:10.1016/j.bmcl.2010.03.033

The MEK-signaling pathways are complex but critical signaling cascades that correlate an extracellular signaling event with internal cell processes. To date at least seven MEK isozymes have been identified. MEK5, in particular, is upregulated in multiple

Full text of DOI:10.1016/j.bmcl.2010.03.033

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2892–2896
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of benzimidazole-based inhibitors of the mitogen activated
kinase-5 signaling pathway
a
a
a
b
b
Patrick T. Flaherty a, , Ishveen Chopra , Prashi Jain , Shuyan Yi , Erika Allen , Jane Cavanaugh
*
a Division of Medicinal Chemistry, School of Pharmacy, Duquesne University, 600 Forbes Ave., Pittsburgh, PA 15282, USA
b Division of Pharmacology, School of Pharmacy, Duquesne University, 600 Forbes Ave., Pittsburgh, PA 15282, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The MEK-signaling pathways are complex but critical signaling cascades that correlate an extracellular
signaling event with internal cell processes. To date at least seven MEK isozymes have been identified.
MEK5, in particular, is upregulated in multiple forms of tumors. Analysis of the EGF-induced MEK5 sig-
naling cascade in cultured HEK cells has identified compounds that can inhibit MEK5 phosphorylation of
ERK5; observed biological activity is dependent on chemical variation.
Received 5 February 2010
Revised 2 March 2010
Accepted 5 March 2010
Available online 10 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
MEK5
ERK5
Inhibitor
Benzimidazole
The mitogen activated protein kinase (MAPK) pathways are a
family of related, and often interconnected, signaling pathways
that relay input from extracellular origins and interpret the resul-
tant outcome in the context of the current biological milieu.1–8
There have been multiple reviews1,3,9 and several critical advances
regarding the categorization,10,11 the intrinsic biochemistry,12,13
and the relevance to human disease states9,14,15 displayed by var-
ious members of the MAPK family.
The sequence of signaling typically originates with ligand bind-
ing or extracellular stressor followed by one of three transduction
mechanisms;3,16 receptor tyrosine kinase activation, G-protein
coupled receptor activation, or hormone receptor transduction.
The MAPK pathways exist as a series of phosphorylation events
where one kinase (mitogen-activated kinase kinase kinase: MEKK)
phosphorylates a second kinase (mitogen-activated kinase kinase:
MEK), which then phosphorylates a third kinase (extracellular sig-
nal-regulated kinase: ERK). This third kinase, or ERK, is typically
translocated to effect modification of cellular function in a specific
subcellular compartment.17 Multiple phosphorylation states of
ERKs have been demonstrated and may direct subcellular traffick-
ing and substrate preference for phospho-ERK15,17 (Fig. 1).
MEKs uniquely display a high degree of substrate specificity.
This selectively is derived from multiple interactions of a MEK’s
D domain with a unique complementary CD domain on the sub-
strate ERK, association with scaffolding proteins,13,18 association
* Corresponding author. Tel.: +1 412 396 1847; fax: +1 412 396 4660.
E-mail address: flahertyp@duq.edu (P.T. Flaherty).
Figure 1. The MEK5 signaling cascade.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.033

P. T. Flaherty et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2892–2896
2893
We recently presented a series of novel benzamidazoles de-
signed as CDK5 inhibitors.30 Given that both CDK5 and MEK5 are
serine/threonine kinases, that similarities between the CDK and
MEK active sites are mentioned in the literature,27 and that 2 and
4 explore a chemical scaffold closely associated with CDK2 inhibi-
tion, screening a select group of our substituted benzimidazoles for
MEK5 inhibitory properties followed logically. Compounds were
screened for the ability to block EGF-initiated MEK5 mediated
ERK5 phosphorylation in human embryonic kidney cells
(HEK293) analyzed by Western blot analysis.
Beginning with the tri-functional benzimidazole scaffolds,31 5,
10, and 15, chemical variation at the 1-nitrogen, 4-nitrogen, or 6-
carbon permitted generation of hetero or carbon-linked analogs
as presented in Schemes 1–3. Variation at the 1-nitrogen was
found to be accessible via N-alkylation with benzyl bromide
(Scheme 3), but in the case of bulky aliphatic groups, reductive
alkylation at the most nucleophilic nitrogen then cyclization was
found to be the best strategy.31 We envisioned the 4-nitro group
as a protective group that could be converted to an amine when re-
quired. The 6-methoxy group was conveniently converted to the
corresponding 6-akoxy group via acidic demethylation then Mits-
unobu coupling. The intermediate phenol 7 or 12 could be con-
verted to the triflate33 then coupled with
a
benzylic
tetrafluoroborate using a Molander–Suzuki coupling.32 For the ini-
tial screening of compounds we elected to examine compounds
which possessed properties that were synthetically tractable, capa-
ble of additional SAR elaboration, and consistent with prior under-
standing of kinase inhibitors.34,35 Additionally we sought to rapidly
identify which complementary variations were capable of increas-
ing potency and perhaps selectivity.
For all compounds examined reduction with Pd/C and hydrogen
followed by reductive alkylation gave the desired 4-N-benzyl prod-
ucts.36 Monobenzylation was selectively achieved by careful stoi-
chiometry of benzaldehyde and the avoidance of acid to limit
dibenzylation. Demethylation to 8 or 12 required vigorous reaction
conditions: 48% HBr heated to 120 °C under microwave irritation.
Standard Mitsunobu coupling with the known 1-OTBS butane-
1,2-diol37 gave the desired ethers but required addition of the DIAD
generated electrophilic complex in two portions. Alternately, tri-
flate formation then Molander–Suzuki coupling gave the carbon-
linked 6-C variants. Regardless of the nature of the 6-substitution,
subsequent reduction then reductive alkylation was straight
forward.
Figure 2. Examples of known inhibitors of ERK phosphorylation.
with other kinases, and subcellular localization.17 MEK5 has been
demonstrated to uniquely phosphorylate ERK5.19
Activators of signaling cascades utilizing MEK5 mediated phos-
phorylation of ERK5 include nerve growth factor (NGF), endothelial
growth factor (EGF), brain-derived neurotrophic growth factor
(BDNF), oxidative stress, ionizing radiation, or the phorbol esters.
Substrates for ERK5 include Sap1a, c-Myc, and possibly RSK.
ERK5 can also induce MEF2, PPARc
1, c-Fos, and c-Jun15,17 activa-
tion. ERK5 has a role during the G1/S cell-cycle transition for
EGF-induced cell proliferation through a cAMP response element
(CRE) mediated expression of cyclin D1.20 Additionally, the
MEK5/ERK5 pathway has been shown to be significantly upregu-
lated in squamous cell carcinoma,21 prostrate,22 and breast23 can-
cers. Although MEK5 is clearly involved in maintenance of
cardiovascular tissue, has a role in neural plasticity, and displayed
a lethal cardiovascular phenotype in MEK5 knock-out mice,17 three
recent studies examining the role of ERK5 in breast cancer,23 leuke-
mia cells,24 and unique activation by palytoxin or the phorbol es-
ters25 has prompted a re-examination of the potential role of
MEK5 as an anti-cancer therapeutic target.
HEK293 cells were treated with 50 ng/mL of EGF to activate the
MEK/ERK pathway for 30 min then each compound was added to a
final concentration of 10 lM. Activation of ERK1/2 or MEK5 was
examined by western blot analysis for phosphorylated (i.e., acti-
vated) ERK5.38 Of the nine compounds tested, compound 6 signif-
icantly inhibited both ERK1/2 and ERK5 phosphorylation induced
by EGF treatment (Fig. 3 and Table 1).
The majority of inhibitor studies on the MEKs have focused on
MEK1/2 however there is a single paper detailing the identification
of a MEK5 inhibitor.26 Studies examining the ability of compounds
to block in vivo EGF-induced ERK1/2 phosphorylation analyzed by
western blotting identified flavone 1, PD98059,17,19 as a non-com-
petitive ATP site inhibitor of ERK1/2, and 5. The conjugated nitrile
2, U0126,27 blocked phorbol-ester stimulated ERK1/2 phosphoryla-
tion. Examination of various diphenylanalines resulted in the iden-
tification of 3, PD318088,28,29 an inhibitor of MEK1/2, and 5.
Analysis of an X-ray crystal structure of PD318088, 3, bound to
MEK1 (PDB ID: 1S9J) confirms that 3 binds to an allosteric site
on MEK1.5,9,11 A recent survey of oxindole kinase inhibitors identi-
fied BIX02188, 4 as a selective inhibitor of MEK5.26 We sought to
identify new structural classes of inhibitors that could disrupt
the MEK-signaling cascade, specifically MEK5 phosphorylation of
ERK5 (Fig. 2).
Substitution at the 1-ntirogen included benzyl, isopropyl, and
cyclopentyl variations. These groups were selected based on prior
observation39,40 that significant hydrophobic interaction can occur
at a common kinase aromatic binding region proximal to the H-
bond donor/acceptor pair represented respectively by the 4-nitro-
gen proton and the 3-N benzimidazole nitrogen lone pair. Of these
initial variations examined, isopropyl appeared to contribute to-
ward inhibition of ERK5 phosphorylation evidenced by 6, 9b, and
9c. This contribution was dependent on substitution at both the
4-amine, and the 6-carbon. Interestingly, compounds 6 and 14 pos-
sessing the hydroxybutyl ether at the 6-carbon were inactive in
preventing EGF-induced ERK phosphorylation. This result stands
in stark contrast to our observations regarding the role of this
group in potentiating CDK5 inhibition for this benzimidazole scaf-
fold.30 This suggests very different structural requirements for
activity between these two enzymes.

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P. T. Flaherty et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2892–2896
Scheme 1. Reagents and conditions: (a) 50 psi H2, EtOH, 5% Pd/C, 23 °C, 5 h, 80–95%; (b) benzaldehyde (1.1 equiv), NaHB(OAc)3 (2.0 equiv), DCE, 23 °C, 12 h, 30–60%; (c) 48%
HBr, microwave irradiation, 120 °C, 2.5 h, 97%; (d) PNP-OTf, DMF, 23 °C, 2 h; 87%; (e) potassium benzyltrifluoroborate, CsCO3, PdCl2(dppf), H2O/THF, 110 °C, 2 h, 80–99%; (f) 1-
(TBS)butan-2-ol (2.6 equiv), PPh3 (2.6 equiv), DIAD (2.6 equiv), DMF, 0–23 °C, 12 h 65%.
Scheme 2. Reagents and conditions: (a) 48% HBr, microwave irradiation, 120 °C, 2.5 h, 97%; (b) PNP-OTf, DMF, 23 °C, 2 h; 86%; (c) potassium benzyltrifluoroborate, CsCO3,
PdCl2(dppf), H2O/THF, 110 °C, 2 h, 17%; (d) 50 psi H2, EtOH, 5% Pd/C, 23 °C, 5 h, 80–95%; (e) benzaldehyde (1.1 equiv), NaHB(OAc)3 (2.0 equiv), DCE, 110 °C, 2 h, 30–60%; (f) 1-
(TBS)butan-2-ol (2.6 equiv), PPh3 (2.6 equiv), DIAD (2.6 equiv), DMF, 0–23 °C, 12 h, 50%.
Figure 3. Western Blot analysis of EGF-mediated ERK phosphorylation; antibodies
Scheme 3. Reagents and conditions: (a) 50 psi H2, EtOH, 5% Pd/C, 5 h, (b)
benzaldehyde (1.1 equiv), NaHB(OAc)3 (2.0 equiv), DCE, 60% (two steps).
utilized detect all phospho-ERK forms, ERK isofoms are separated with SDS–PAGE
on the basis of molecular weight.

P. T. Flaherty et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2892–2896
2895
Table 1
22. Dudderidge, T. J.; McCracken, S. R.; Loddo, M.; Fanshawe, T. R.; Kelly, J. D.; Neal,
D. E.; Leung, H. Y.; Williams, G. H.; Stoeber, K. Br. J. Cancer 2007, 96, 1384.
23. Montero, J. C.; Ocana, A.; Abad, M.; Ortiz-Ruiz, J. M.; Pandiella, A.; Esparis-
Ogando, A. PLoS One 2009, 4.
24. Charni, S.; Aguilo, J. I.; Garaude, J.; de Bettignies, G.; Jacquet, C.; Hipskind, R. A.;
Singer, D.; Anel, A.; Villalba, M. J. Immunol. 2009, 182, 3398.
25. Charlson, A. T.; Zeliadt, N. A.; Wattenberg, E. V. Toxicol. Appl. Pharmacol. 2009,
241, 143.
26. Tatake, R. J.; O’Neill, M. M.; Kennedy, C. A.; Wayne, A. L.; Jakes, S.; Wu, D.;
Kugler, S. Z., Jr.; Kashem, M. A.; Kaplita, P.; Snow, R. J. Biochem. Biophys. Res.
Commun. 2008, 377, 120.
27. Favata, M. F.; Horiuchi, K. Y.; Manos, E. J.; Daulerio, A. J.; Stradley, D. A.; Feeser,
W. S.; Van Dyk, D. E.; Pitts, W. J.; Earl, R. A.; Hobbs, F.; Copeland, R. A.; Magolda,
R. L.; Scherle, P. A.; Trzaskos, J. M. J. Biol. Chem. 1998, 273, 18623.
28. Ohren, J. F.; Chen, H.; Pavlovsky, A.; Whitehead, C.; Zhang, E.; Kuffa, P.; Yan, C.;
McConnell, P.; Spessard, C.; Banotai, C.; Mueller, W. T.; Delaney, A.; Omer, C.;
Sebolt-Leopold, J.; Dudley, D. T.; Leung, I. K.; Flamme, C.; Warmus, J.; Kaufman,
M.; Barrett, S.; Tecle, H.; Hasemann, C. A. Nat. Struct. Mol. Biol. 2004, 11, 1192.
29. Han, S.; Zhou, V.; Pan, S.; Liu, Y.; Hornsby, M.; McMullan, D.; Klock, H. E.;
Haugen, J.; Lesley, S. A.; Gray, N.; Caldwell, J.; Gu, X.-j. Bioorg. Med. Chem. Lett.
2005, 15, 5467.
Inhibition of EGF-induced ERK phosphorylation in HEK 293 cells
Compound p-ERK1/2 Relative
p-ERK5 Relative
phsophorylation (%)a
phsophorylation (%)a
6
7
35.98
12.35
110.05
129.49
70.84
96.27
135.22
78.00
131.53
135.59
100
260.57
461.55
309.67
346.25
331.95
521.18
609.86
436.18
100
9a
9b
9c
10
13
14
16
DMSO
a
Values are means of duplicate experiments, standard deviation is 10%.
Of the compounds examined, the relatively simple compound 6
30. Jain, P.; Chopra, I.; Yi, S.; Flaherty, P. T.; Madura, J. D. Abstracts of Papers, 238th
ACS National Meeting, Washington, DC, United States, August 16–20, 2009,
MEDI.
was shown to display inhibition of EGF-induced phosphorylation
of ERK. Some modest selectivity was observed for the preferential
inhibition of ERK5 phosphorylation relative to ERK1/2 phosphory-
lation. Interestingly, compound 6 was unique among the com-
pounds examined in that no upregulation of ERK1/2
phosphorylation was observed. This compound was submitted
for the NCI 60-cell line screen and was shown to selectively inhibit
the growth of MCF-7 cells in the single dose survey. However, com-
pound 6 was not selected for the subsequent log dose–response
analysis. It has been noted that the MCF-7 cell line displays unique
properties regarding cytosolic effects of ERK5 activity.17
31. Moore, M. D.; Jain, P.; Flaherty, P. T.; Wildfong, P. L. D. Acta Crystallographica,
Section E: Structure Reports Online 2008, E64, o1336.
32. Ishiyama, T.; Itoh, Y.; Kitano, T.; Miyaura, N. Tetrahedron Lett. 1997, 38, 3447.
33. Molander, G. A.; Yun, C.-S.; Ribagorda, M.; Biolatto, B. J. Org. Chem. 2003, 68,
5534.
34. Briem, H.; Guenther, J. Chem.Bio.Chem 2005, 6, 558.
35. Fabian, M. A.; Biggs, W. H.; Treiber, D. K.; Atteridge, C. E.; Azimioara, M. D.;
Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M.; Ford, J. M.;
Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.;
Lai, A. G.; Lelias, J.-M.; Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L.
M.; Patel, H. K.; Zarrinkar, P. P.; Lockhart, D. J. Nat. Biotechnol. 2005, 23, 329.
36. 1-Isopropyl-6-methoxy-4-nitro-1H-benzo[d]imidazole (5).31
A
well stirred
mixture of 5-methoxy-3-nitrobenzene-1,2-diamine (5.0 g, 27.3 mmol),
NaHB(OAc)3 (17.36 g, 81.9 mmol), and 110 mL of THF was cooled to an
internal temperature of 0 oC. Formic acid (3.77 g, 81.9 mmol) was added with
dropwise addition maintaining the internal temperature below 10 oC. After
stirring an additional 15 min at 0 oC, acetone (7.93 g, 137 mmol, 5 equiv) was
added in one portion. The mixture was stirred overnight. The solvent was
removed in vacuo, and the dark red residue was dissolved in formic acid
(31 mL, 0.85 mol). Butylated hydroxytoluene, BHT, (20 mg, 0.09 mmol) was
added, and the mixture was cooled to 0 °C. Concd HCl (87 mL, 0.95 mol) was
added, and the mixture was brought directly to reflux. After maintaining reflux
for 15 min, the solvent was removed in vacuo at 80 °C. The residue was
Further studies are under way to analyze the biological contri-
bution of and structure variations from compound 6 for inhibition
of EGF-mediated ERK5 phosphorylation.
Acknowledgments
Ms. Darlene Monlish, NIH (NINDS): 1R15NS057772, NIA:
AG025848, and NSF equipment grant: NMR: CHE 0614785.
adjusted to
a pH of 8 with 50% aqueous NaOH then extracted with EA
(4 Â 50 mL). The combined EA extracts were washed (3 Â 10 mL brine) and
dried (Na2SO4). Evaporation of the solvent gave a brown solid, which after
silica gel column chromatography (hexanes/EtOAc 1:1) afforded 4.5 g (74%) of
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.033.
the desired product as
a yellow solid. Rf 0.32 (CH2Cl2/MeOH/NH4OH
100:10:0.1). mp 127.2–128.1 °C. 1H NMR (400 MHz, CDCl3): d 8.12 (s, 1H),
7.21 (d, J = 2.3 Hz, 1H), 7.79 (d, J = 2.3 Hz, 1H), 4.58–4.65 (m, 1H), 3.95 (s, 3H),
1.65 (d, J = 6.8 Hz, 6H). Anal. Calcd for C11H13N3O3: C, 56.16; H, 5.57; N, 17.86.
Found: C, 56.45; H, 5.62; N, 17.55.
References and notes
1-Isopropyl-6-methoxy-1H-benzo[d]imidazol-4-aminium chloride. 1-Isopropyl-6-
methoxy-4-nitro-1H-benzo[d]imidazole (833 mg, 3.54 mmol) was dissolved in
EtOH (42 mL) and CHCl3 (8 mL) containing 10% Pd/C (150 mg). The
hydrogenation flask was evacuated and backfilled with H2 three times, and
then shaken under 55 PSI of H2 atmosphere for 12 h at 23 oC. The mixture was
filtered through Celite and the Celite pad was washed with 20 mL additional
EtOH. Evaporation of the solvent gave 841 mg (98%) of the salt as a light green
solid. This material was used directly in the next step.
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N-Benzyl-1-isopropyl-6-methoxy-1H-benzo[d]imidazol-4-amine (6) NaHB(OAc)3
(608 mg, 2.87 mmol, 1 equiv) was added to
a
solution of 1-isopropyl-6-
methoxy-1H-benzo[d]imidazol-4-aminium
chloride (155a, 832 mg,
2.87 mmol) in 1,2-dichloroethane (10 mL) at 23 oC. After stirring for 2 min,
benzaldehyde (457 mg, 4.31 mmol) was added. After stirring for 1 min, a
second amount of NaHB(OAc)3 (914 mg, 4.31 mmol) was added and stirring
was continued for an additional 12 h. Saturated aqueous 8 mL of NaHCO3 (satd,
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aq)
extracted with Et2O (10 mL Â 3). The combined extracts were washed with
NaCl(satd,
and then dried over Na2SO4. Evaporation of the solvent gave a
aq)
brown solid, which was subjected to silica gel column chromatography
(hexanes/EtOAc 2:1 to 1:2, 0.5% Et3N) to afford 790 mg (93%) of the product
as a white solid. Rf 0.58 (CH2Cl2/MeOH/NH4OH 100:10:0.1). mp 81.8 – 83.9 °C.
1H NMR (400 MHz, CDCl3): d 7.72 (s, 1H), 7.25–7.42 (m, 5H), 6.20 (d, J = 2.1 Hz,
1H), 6.03 (d, J = 2.1 Hz, 1H), 5.29 (t, J = 5.4 Hz, 1H), 4.47–4.53 (m, 1H), 3.80 (s,
3H), 1.59 (d, J = 6.8 Hz, 6H). Anal. Calcd for C18H21N3O: C, 73.19; H, 7.17; N,
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38. (a) HEK293 (ATCC, Manassas, VA) cells were grown on 60 mm culture plates
(Sarstedt, Newton, NC) in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco,
Carlsbad, CA) with 10% heat-inactivated FBS (Atlanta Biological, Lawrenceville,
GA), and 0.5% penicillin/streptomycin (Gibco, Carlsbad, CA). Cells were

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P. T. Flaherty et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2892–2896
maintained at 37 °C with 5% CO2. The cells were treated with 50 ng/mL
epidermal growth factor (EGF; Sigma) 30 min before treatment with the
putative MEK5 inhibitors (10 M of each). Cells were lysed 30 min following
treatment with the inhibitors. The cells were washed with 1x PBS and then
lysed in 1% Triton X-100 buffer containing 20 mM Tris (pH 6.8), 137 mM NaCl,
25 mM beta glycerophosphate, 2 mM NaPPi, 2 mM EDTA, 1 mM Na3 VO4, 10%
NaN3 for 1 h and then incubated overnight at 4 °C in primary antibody (1:1000
pERK1/2, 1:500 pERK5, Cell Signaling). Blots were washed with 1x TBS/0.1%
Tween for 30 min and then incubated with secondary antibody (1:1000,
horseradish peroxidase-conjugated goat anti-rabbit, Upstate). Proteins were
visualized with enhanced chemiluminescence (Upstate). Films were scanned
and quantified using MATLAB, v.7.1 (Mathworks, Natick, MA).
l
glycerol, 5
lg/mL leupeptin, 5
lg/mL aprotinin, 2 mM benzamidine, 0.5 mM
39. Kufareva, I.; Abagyan, R. J. Med. Chem. 2008, 51, 7921.
DTT, and 1 mM PMSF. The lysates were then centrifuged at 10,000 rpm for 10
min at 4 °C. Determination of protein in the supernatant was done using a
Bradford assay (Biorad, Hercules, CA).; (b) Western Blot Analysis. Equal
amounts of protein (60 lg) from each treatment were separated on 10% SDS
gels and transferred to PVDF membrane [Millipore, Danvers, MA] for Western
blot analysis. Blots were blocked in 5% nonfat milk in 1x TBS/0.1% Tween/0.02%
40. Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge, C. E.;
Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.; Edeen, P. T.; Faraoni, R.;
Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.;
Patel, H. K.; Pritchard, S.; Wodicka, L. M.; Zarrinkar, P. P. Nat. Biotechnol. 2008,
26, 127.