Emission tuning of fluorescent kinase inhibitors: Conjugation length and substituent effects

Article abstract of DOI:10.1021/jo500520x

Fluorescent N-phenyl-4-aminoquinazoline probes targeting the ATP-binding pocket of the ERBB family of receptor tyrosine kinases are reported. Extension of the aromatic quinazoline core with fluorophore "arms" through substitution at the 6- position of the

Full text of DOI:10.1021/jo500520x

Article
pubs.acs.org/joc
Emission Tuning of Fluorescent Kinase Inhibitors: Conjugation
Length and Substituent Effects
Jyothi Dhuguru,† Wenjun Liu,‡ Walter G. Gonzalez,§ W. Michael Babinchak,† Jaroslava Miksovska,§
Ralf Landgraf,‡ and James N. Wilson*,†
†Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146, United States
‡Department of Biochemistry and Molecular Biology, University of Miami, Miami, Florida 33101-6129, United States
§Department of Chemistry and Biochemistry, Florida International University, 11200 South West Eighth Street, Miami, Florida
33199, United States
S
* Supporting Information
ABSTRACT: Fluorescent N-phenyl-4-aminoquinazoline
probes targeting the ATP-binding pocket of the ERBB family
of receptor tyrosine kinases are reported. Extension of the
aromatic quinazoline core with fluorophore “arms” through
substitution at the 6- position of the quinazoline core with
phenyl, styryl, and phenylbutadienyl moieties was predicted by
means of TD-DFT calculations to produce probes with
tunable photoexcitation energies and excited states possessing
charge-transfer character. Optical spectroscopy identified several synthesized probes that are nonemissive in aqueous solutions
and exhibit emission enhancements in solvents of low polarity, suggesting good performance as turn-on fluorophores. Ligand-
induced ERBB2 phosphorylation assays demonstrate that despite chemical modification to the quinazoline core these probes still
function as ERBB2 inhibitors in MCF7 cells. Two probes were found to exhibit ERBB2-induced fluorescence, demonstrating the
utility of these probes as turn-on, fluoroescent kinase inhibitors.
preferentially binding the active kinase conformation, and
lapatinib (Figure 1B,D), a type II inhibitor targeting the inactive
conformation.10 The preference for active and inactive
conformations is addressed through substitution at the 4-
amino position (Figure 1E). While the quinazoline core
conveys binding to the nucleotide pocket, but with limited
specificity for ERBB-type receptors, the 4-amino aryl arm
further increases specificity and contributes discrimination
between activation states. This selectivity reflects increased
access to the hydrophobic pocket adjacent to the nucleotide
binding site in the inactive conformation. Together, the
quinazoline core and N-aryl arm constitute the pharmacophore.
Crystal structures of the kinase domain of EGFR with either
erlotinib (1M17)15 or lapatinib (1XKK)9 (Figure 1A,B) show
that while the pharmacophore arm is oriented deep in the
binding pocket, the 6-position is amenable to chemical
modification without perturbing the key conserved contacts
of the binding pocket. The structure of lapatinib demonstrates
this point as the aromatic core is extended by the addition of a
furan ring. Thus, a potential strategy for synthesizing
fluorescent kinase inhibitors is to modify the 4-aminoquinazo-
line core with fluorphore arms (Figure 1E) at the 6-position. In
principle, probes targeting specific kinases (i.e., IGF1R vs
EGFR) or activation states could be encoded with unique
INTRODUCTION
■
Small molecule kinase inhibitors are cornerstones of advanced
chemotherapy. In their ideal embodiment, they may selectively
target a validated signaling pathway that is uniquely
dysregulated in cancer cells; in reality, multiple factors
complicate this strategy. First, the structural and functional
homology of kinases combined with the abundance of ATP-
binding folds throughout the cell make off-target binding events
unavoidable.1,2 Second, compensatory or alternate modes of
pathway activation can lead to the development of resistance,
requiring additional antineoplastic drugs or a different chemo-
therapy regimen.3,4 Ideally the observation of compensatory
shifts in signaling on a systemic and live cell level could
facilitate the early development of second-generation drugs
with larger barriers or at least anticipated paths to adaptation.
In this context, assaying changes in receptor kinase states (i.e.,
level of expression, oligomerization, localization, conformation,
phosphorylation) remains a challenge, particularly in live cells
or tissue preparations.
In an effort to develop molecular probes capable of
interrogating kinase signaling pathways, we have introduced
the concept of “turn-on” fluorescent ligands that target the
ATP-binding pocket of the EGFR/ERBB family of receptor
tyrosine kinases.5 These molecular probes are built upon the 4-
aminoquinazoline scaffold, which is an established EGFR/
ERBB pharmacophore;6−10 examples include gefitinib or
erlotinib (Figure 1A,C), so-called type I inhibitors that
Received: March 4, 2014
Published: May 2, 2014
© 2014 American Chemical Society
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1a−3a) to strongly electron-withdrawing nitro substitution
(1e−3e).
The 15-membered family of quinazolines, 1a−3e (Figure 1),
was synthesized from a common intermediate, 6-iodo-N-
phenyl-4-quinazolin-amine (4), by Suzuki coupling of the
appropriate arylboronic acid (1a−1e, Scheme 1, route A) or by
Scheme 1. Synthesis Routes to Fluorescent Quinazolines
a
1a−3e
Figure 1. Crystal structures of the EGFR ATP-binding pocket with
(A) erlotinib (PDB ID: 1M17)15 and (B) lapatinib (PDB ID: 1XKK)9
reveal the inhibitor binding modes. The arms at the 6-position of the
quinazoline core (in blue; C, D) may be replaced by fluorophore arms
without disturbing the key binding contacts. (E) General structure and
substituent key for the synthesized fluorescent quinazolines.
optical outputs by tuning the excitation and emission energies
through the fluorophore arm.
Herein, we investigate the effect of conjugation length and
auxochrome substitution on the optical properties of a family of
N-phenyl-4-aminoquinazoline probes. We find that extension of
the π framework effectively lowers the excitation and emission
energies, yielding fluorescent probes that compare favorably
with other fluorescent adenosine analogues.11−14 The intro-
duction of strong electron donors or strong electron-with-
drawing groups generates donor−acceptor systems that are
highly sensitive to solvent polarity. As a result, several probes
exhibit high fluorescence ON/OFF ratios, a feature that is key
to their performance as self-reporting fluorescent kinase
inhibitors. Despite the modifications to the quinazoline core,
we also found that several of these probes inhibit ERBB2
phosphorylation in a live cell setting, demonstrating that
binding to the ATP pocket and cell permeability are preserved.
Overall, these fluorescent adenosine mimics compare favorably
with other nucleobase analogues as they possess tunable optical
properties and high fluorescence turn-on ratios and compete
effectively with the native substrate to inhibit tyrosine
phosphorylation.
a
Representative structures 1a and 3d are shown.
Heck coupling (Scheme1, route B) of the styryl (2a−2e) and
phenyl butadiene (3a−3e) arms. Following reaction workup,
flash chromatography and crystallization from 2-propanol/ethyl
acetate mixtures, the products were isolated in moderate yields
as microcrystals that appeared colorless (e.g., 1c and 3c) to
bright orange−yellow (e.g., 1a and 3e).
Quantum Chemical Calculations. Two photophysical
properties, the fluorescence ON/OFF ratio and the optical
band gap, of the kinase-binding probes can simultaneously be
addressed by varying the donor or acceptor substitution and the
conjugation length. Ideally, the probes should be nonemissive
or off in solution, but upon binding in the solvent excluding and
geometrically confined ATP pocket, emission should be
enhanced or turned on. To achieve this emission behavior, a
probe should possess an excited state with a high degree of
charge-transfer (CT) character and possibly access at twisted
intramolecular charge-transfer (TICT) excited state. TD-DFT
calculations (6-31G*, CHCl3, SMD solvent model)16 show that
both strong electron donors and strong electron acceptors will
lead to an S1 state with CT character. The S1 state is accessible
via the allowed one-electron transition between the HOMO
and LUMO for all 15 members, with energies ranging from 2.5
eV (500 nm) in the case of 3e to 3.6 eV (345 nm) in the case of
1c (Table 1). The addition of each vinylene bridge lowers the
S1 transition energy by approximately 0.25 eV. The
RESULTS AND DISCUSSION
■
Design and Synthesis. The optical properties of kinase-
binding probes of general structure I can be addressed through
the fluorophore arm depicted in Figure 1E. We envisaged
tuning two parameters: (1) the electron-donating ability of the
substituent on the phenyl ring and (2) the conjugation length
between the phenyl ring and the quinazoline core. TD-DFT
calculations, vide infra, guided the selection of the electron-
donating and electron-withdrawing substituents that span the
range from the strongly donating dimethylamino group (e.g.,
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Table 1. Calculated (TDDFT/CAM-B3LYP/6-31G*) S1 Energies and S0 → S1 Oscillator Strength
Article
a
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
ΔE S0 → S1(eV)
f
3.9
0.7
4.0
0.7
4.1
0.7
4.0
0.8
3.9
0.8
3.6
1.7
3.8
1.5
3.8
1.4
3.7
1.6
3.5
1.5
3.3
2.3
3.5
2.1
3.6
2.0
3.4
2.2
3.3
2.0
a
CHCl3, SMD solvent model.16
Figure 2. Frontier molecular orbitals of 2a−2e calculated at the CAM-B3LYP/6-31G* level: the polarization of the HOMO and LUMO shifts across
the series (compare 2a and 2e); strong CT character is expected for both 2a and 2e, although the localization of charge should be reversed.
introduction of a moderately electron-donating or -withdrawing
group (i.e., −OMe or −CN) lowers the energy of the S0 → S1
transition energy by 0.1 to 0.2 eV when compared to that of
isostructural members of the unsubstituted series (i.e., 1c−3c).
The presence of the strong electron-donating dimethylamino
group more effectively lowers the HOMO−LUMO band gap
by 0.5−0.3 eV when comparing 1a−3a and 1a−3c, whereas the
strongly electron-withdrawing nitro group has the greatest
Figure 3. Chloroform solutions of selected probes (5 μM) under UV
effect, with differences of 0.8 to 0.5 eV when comparing 1a−3c
illumination (354 nm); aqueous solutions showed weak or no
emission (vials not shown).
and 1a−3e.
In addition to manipulating the optical band gap, the
presence of an electron-donating or electron-withdrawing
substituent on the pendant phenyl arm influences the CT
character of the excited state; the polarization and spatial
segregation17 of the HOMO and LUMO is directly linked to
the identity of the substituent. In the series of dimethylamino-
substituted compounds (e.g., 2a; Figure 2), the HOMO is
largely localized to the fluorophore arm, whereas the LUMO is
polarized toward the quinazoline core. The presence of a strong
electron-withdrawing group (i.e., −NO2 or −CN) on the
fluorophore arm reverses the frontier molecular orbital
distribution. In the case of 2e (Figure 2), the HOMO is
polarized toward the quinazoline core, whereas the LUMO is
largely localized to the styryl arm; an excited state with high CT
character still results from the promotion of an electron from
the HOMO to the LUMO. Compounds 2b−2d show a gradual
redistribution of the HOMO and LUMO densities between the
two extreme cases, 2a and 2e. Similar polarization of the
frontier molecular orbitals is also observed in the 1a−1e
(Figure S16) and 3a−3e series (Figure S17).
Optical Spectroscopy. Probes 1a−3c were designed as
turn-on fluorescent ligands and thus were expected to be
nonemissive in solvents of high polarity (e.g., water and
methanol) and emissive in less polar solvents. This behavior
can be qualitatively observed by eye as in chloroform (ET(30) =
39.1 kcal/mol)18 solutions; most of the probes appear bright,
with emissions ranging from blue (2b and 3c) to green (1a and
2a) to yellow or orange (3a and 3e), Figure 3, whereas in
aqueous solutions, weak or no emission was observed.
progress in a clear trend, as λmax,abs increases with increasing
conjugation length. The presence of an auxochrome also serves
to modulate the absorption wavelengths, as is evident in the
lower transition energies for compounds possessing the
dimethylamino (1a−3a) or nitro substituent (1e−3e); these
modifications enhance CT character and lead to a longer
wavelength absorption band lacking vibronic structure. Some
vibronic structure is visible in the absorption spectra of
compounds lacking an auxochrome or possessing the methoxy
or cyano groups; however, these compounds possess some
degree of CT in the excited state, as their emission spectra are
largely devoid of vibronic progressions. Despite the presence of
CT character, the allowability of the S0 → S1 transition is
relatively high for most compounds, as is evident from the good
to moderate molar absorptivities. Increasing the conjugation
length increases the extinction coefficient in a stepwise fashion:
compounds 2a−2e possess molar absorptivities roughly twice
the values observed for 1a−1e, whereas compounds 3a−3c
have molar absorptivities approximately three times greater
than those of 1a−1e. This trend, which reflects the relative
magnitudes of transition dipoles, can be directly linked to the
spatial overlap of the contributing molecular orbitals, in this
case the HOMO and LUMO. In terms of optical compatibility,
all of the synthesized probes are compatible with DAPI,
Hoechst 33342, or blue fluorescent protein filter sets for
epifluorescence microscopy, whereas 2a, 2e, and 3a−3e are
optimally matched to the 405 nm diode laser.
To investigate their optical properties quantitatively, we
obtained their UV−vis and fluorescence spectra in chloroform.
The absorption maxima (Figure 4 and Table 2) correlate very
well with the predicted values both in terms of transition
energies as well as oscillator strength. The transition energies
The emission spectra of 1a−3c in chloroform are shown in
Figure 4; most of the probes exhibit good to moderate
quantum yields (Table 2). Emission intensities are enhanced in
less polar solvents such as toluene and are reduced in more
polar solvents such as acetonitrile, supporting the existence of
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Figure 4. Absorption (solid lines) and emission (dashed lines) spectra of (A) 1a−1e, (B) 2a−2e, and (C) 3a−3e in CHCl3. Emission intensities are
given relative to 3d, which has the highest quantum yield (see Table 2).
Table 2. Photophysical Data for 1a−3e
a
compd
λmax,abs (nm)
ε (M−1 cm−1
)
λmax,em (nm)
ϕem
Δν (cm−1
)
τ1 (ns)
f1
τ2 (ns)
f 2
χ2
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
325
348
344
343
346
381
347
341
358
372
403
369
361
375
391
15 000
14 300
15 400
16 700
19 700
28 800
34 900
21 900
31 700
30 400
53 300
47 500
44 300
51 000
41 200
485
510
502
508
446
495
440
455
436
539
510
443
422
445
561
0.37
0.02
0.03
0.02
0.006
0.28
0.15
0.02
0.09
0.04
0.17
0.42
0.25
0.57
0.11
10 200
9130
9150
9470
6480
6050
6090
7350
5000
8330
5210
4530
4000
4200
7750
1.22
1.39
0.56
0.17
0.11
0.69
0.89
0.05
0.12
0.38
0.80
0.77
0.54
0.23
0.40
0.03
0.70
0.06
0.18
0.74
0.08
0.57
0.11
0.30
0.83
0.77
0.43
0.32
0.21
0.37
6.54
6.76
1.60
1.29
1.56
2.49
1.50
1.75
1.46
1.60
2.48
1.90
1.78
1.57
1.04
0.97
0.30
0.94
0.82
0.26
0.92
0.43
0.89
0.70
0.17
0.23
0.57
0.68
0.26
0.63
2.46
5.89
2.88
1.47
10.2
3.65
2.17
4.86
0.57
1.77
1.47
1.24
1.33
0.86
1.16
a
10%.
excited states with significant CT character (see Supporting
Information, Figure S18). Although many of the probes exhibit
emission on the blue end of the visible spectrum, several probes
show longer-wavelength emission owing to their longer
conjugation length and/or the presence of strong electron-
donating or -withdrawing groups. Of the probes exhibiting
strong emission, 3c has the bluest emission maximum (42 nm),
whereas 3e has the reddest emission maximum (561 nm).
Surprisingly, strong CT character does not necessarily equate to
poor quantum yields, as seen for probes 1a and 2a. Indeed, 1a
is the only member of the 4-phenylquinazoline series (1a−1e)
of compounds that exhibits an appreciable degree of
fluorescence (ϕem = 0.37). In the 4-styrylquinazoline (2a−2e)
series, compounds possessing electron-donating groups (2a and
2b) also exhibit the highest quantum yields. With longer
conjugation lengths, quantum yields of fluorescence are
markedly higher and the identity of the auxochrome influences
ϕem to a lesser degree.
intensities obtained in octanol and water are shown in Figure 5.
Compounds 1a, 2a, and 3e showed the largest on/off ratios
Figure 5. Emission intensities obtained in octanol and water reveal
that several probes are highly responsive to changes in their chemical
microenvironment and possess high ON/OFF ratios.
The overall brightness (ε·ϕem) is one important parameter
when considering the utility of the probes as optical reporters.
An additional parameter, the turn-on ratio, should also be
considered as a measure of the probes’ responsiveness to
changes in their chemical microenvironment.19 Water (ET(30)
= 63.1 kcal/mol, η = 0.89 mPa·s) and octanol (ET(30) = 48.3
kcal/mol, η = 7.24 mPa·s) represent two distinct environments
that can be used to assess the physical properties and
distribution of organic molecules.18,20 The ratios of emission
with enhancements greater than 50-fold; moderate ratios,
between 20 and 40 were observed for 2d, 3a, 3b, and 3d.
Moderate enhancements were found for the remaining
members of the 2 and 3 series, whereas the remaining 6-
arylquinazolines (series 1) showed essentially no emission
enhancements.
Inhibition Studies. The extension of the aromatic system
via the 6-position of the quinazoline core was not anticipated to
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affect the key binding contacts between the probes and the
ATP-binding fold of the ERBB family. The crystallographically
determined binding modes of gefitinib,8 erlotinib,15 and
lapatinib9 show that the 6-position is amenable to substitution
and, in some cases, decreases koff of inhibitors.21 To test this
assumption, we evaluated selected probes as inhibitors of
ERBB2 phosphorylation in MCF7 cells initially using two
probe concentrations, 10 μM and 100 nM (Figure 6). MCF7
phosphorylation at approximately 20 μM, higher concen-
trations of 3d do not lead to complete inhibition. While 3d is a
larger molecule, hydrophobicity does not appear to play a role,
as 1a and 3d have similar octanol/water partition coefficients,
with log Poctanol/water = 1.50 and 1.55, respectively. The lack of
complete inhibition may reflect alternative binding modes for
the inhibitors, as similar partial inhibition resulting from
competing cellular ATP has been described for gefitinib when
compared to the complete inhibition by the type II inhibitor,
lapatinib.23 We are presently evaluating the binding kinetics of
these probes and will report their binding modes separately.
Binding-Induced Emission Studies. Emission spectros-
copy identified several probes (1a, 2a, and 3e) that exhibit large
turn-on ratios, and the phosphorylation inhibition studies
demonstrate that the modified N-phenyl quinazolines are
capable of accessing the ATP-binding pocket of ERBB2. To
determine if turn-on emission is observed upon binding to
ERBB2, we obtained the fluorescence spectra of 1a, 2a, and 3e
in PBS in the presence and absence of the soluble ERBB2
kinase domain. In PBS alone, the emission of the probes is
largely quenched, whereas the addition of the ERBB2 kinase
domain produced substantial emission enhancements for both
of the dimethylamino-substituted probes. In the case of 1a, the
emission enhancement was 4-fold when comparing all emission
wavelengths, with a maximum enhancement of 10 at 435 nm
(Figure 7). For 2a, the emission was increased by a factor of 8
Figure 6. (A) Comparative inhibitory properties of selected probes:
the inhibition of NRGβ1-induced ERBB2 tyrosine phosphorylation in
MCF7 cells was evaluated at 0.1 and 10 μM concentrations of the
indicated compounds. For comparison, lapatinib was used at its
clinically used serum concentration of 3 μM. (B) Compounds 3d and
1a show comparable affinity in inhibition studies but a difference in the
maximum achievable inhibition.
cells are a well-established model system for the ligand-induced
activation of ERBB2−ERBB3 heterocomplexes by ligands of
the neuregulin family (NRGβ1 in this case). Compounds 1a,
2a, 2d, 3d, and 3e were selected because they showed the
highest turn-on ratios of their respective series. All five probes
showed inhibition of NRGβ1-induced phosphorylation of
ERBB2 at 10 μM; however, little to no inhibitory action was
observed at 100 nM. These results demonstrate two key
features of these fluorescent quinazoline probes: First, despite
chemical modification, the probes remain membrane permeable
and are able to access the intracellular kinase binding domain of
ERBB2. Second, the pharmacophore remains an inhibitor of
ERBB2 phosphorylation despite the presence of bulky
extensions of the aromatic system.
We next obtained the inhibition curves of 1a, which
possesses the smallest modification at the 6-position, and 3d,
which possesses one of the longest arms. The Ki for these
probes was similar with values of 10 μM for 1a and 9 μM for
3d. A direct comparison with actual binding constants for a
type I inhibitor such as gefitinib in a cellular setting are difficult
to obtain. Recombinant EGFR kinase domains have yielded an
in vitro Kd of approximately 1 nM.22 Equilibrium models in live
cells treated with 10 μM Gefitinib have yielded Kd estimates of
2 to 3 nM after competition with cellular ATP was taken into
account.23 By this standard, the derivatives used in our study
show comparable potency. Although 1a exhibited a typical
inhibition curve profile, with complete inhibition of ERBB2
Figure 7. (A) 1a and (B) 2a exhibit turn-on emission upon binding to
soluble ERBB2 kinase domain. In PBS alone, emission (in black) is
largely quenched, whereas in the presence of ERBB2 kinase, emission
(in green) is significantly enhanced; conditions: [1a] = [2a] = 1 μM;
[ERBB2] = 100 nM, λex = 370 nm.
when comparing all emission wavelengths, with a maximum
enhancement of 12 at 560 nm. No emission enhancement was
observed for 3e despite the fact that it also exhibits solvent
sensitivity and was shown to inhibit ATP binding to ERBB2.
The lack of binding-induced emission enhancement may be a
result of the longer conjugated arm projecting beyond the
binding pocket and being exposed to the polar solvent
environment.
CONCLUSIONS
■
We have synthesized a family fluorescent of quinazoline probes
targeting the ATP-binding pocket of ERBB2 and evaluated the
influence of extended conjugation as well as substituent effects
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Schlenk flask with a stirrer bar under argon purge. The reaction
mixture was degassed for a further 20 min under a slow stream of
argon, at which point 25 mg of a 1:2 mixture of PdCl2(PPh3)2 and
PPh3 was added. The reaction was heated at 80 °C for 24 h, cooled,
poured into 100 mL of H2O, and extracted with EtOAc (3 × 50 mL).
The organic layer was concentrated and purified over silica (100%
CH2Cl2 to 100% EtOAc) followed by crystallization from 2-propanol/
EtOAc to afford 1a (47 mg, 14%); mp 257−259 °C (dec); IR νmax
(cm−1): 2957.1, 1934.3, 1600.9, 1568.8, 1523.8, 1499.1, 1446.5,
through the 6-position of the quinazoline core. Our results
demonstrate that the optical band gap can be manipulated by
varying the conjugation length and auxochrome substitution.
Depending on the auxochrome identity, the quinazoline core
can function either as an electron acceptor or an electron donor
to achieve polar CT excited states. The strongest electron-
donating and -withdrawing groups (e.g., dimethylamine, cyano,
and nitro) yield high on/off ratios, suggesting that they are the
best candidates for designing future turn-on probes. Impor-
tantly, the presence of a fluorophore arm at the 6-position of
the quinazoline does not significantly attenuate the ability of
the probes to function as inhibitors of ERBB2. Two probes, 1a
and 2a, were successfully demonstrated as turn-on probes that
can report binding to the ERBB2 kinase domain in solution.
One limitation of the current family of fluorescent inhibitors is
their relatively low solubility in aqueous solutions, although this
is not unique to their modified structure, as lapatinib shows
significant aggregation at physiologically relevant concentra-
tions.24 Our future work will focus on improving the solubility
of the probes through chemical modification at either the 7-
position of the core (Figure 8) or on the fluorophore arm.
1
1403.8, 1357.4, 1327.4, 1290.7, 1228.1, 810.1, 744.4, 692.5; H NMR
(500 MHz, DMSO-d6): δ 2.98 (s, 6H), 6.87 (d, 2H, J = 8.9 Hz), 7.15
(t, 1H, J = 7.4 Hz), 7.41 (t, 2H, J = 8.3 Hz), 7.78 (t, 3H 8.4 Hz), 7.86
(d, 2H, J = 8.4 Hz), 8.14 (dd, 1H, J = 8.7, 1.8 Hz), 8.53 (s, 1H), 8.73
(s, 1H), 9.88 (s, 1H); 13C NMR (125 MHz, DMSO-d6): δ 40.4, 112.9,
116, 118.6, 123, 124.1, 126.8, 128, 128.6, 128.9, 131.4, 138.7, 139.6,
148.7, 150.5,.154.2, 158.1; HR-ESI (Q-TOF) m/z: calcd for
+
C22H21N4 [M + H]+, 341.1766; found, 341.1770.
Representative Synthesis via Route B: (E)-6-(4-Methoxys-
tyryl)-N-phenylquinazolin-4-amine (2b). Five hundred milligrams
(1.44 mmol) of 6-iodo-N-phenyl-4-quinazolin-amine (4), 289 mg (2.2
mmol) of 4-vinylanisole, 0.38 mL of Et3N (2.87 mmol), and 5 mL of
DMF were placed in a 50 mL Schlenk flask with a stirrer bar under
argon purge. The reaction mixture was degassed for a further 20 min
under a slow stream of argon, at which point 48 mg of Pd(OAc)2 and
56 mg of PPh3 and the subsequent mixture was further degassed and
heated at 80−85 °C for 24 h, cooled, poured into 100 mL of H2O, and
extracted with EtOAc (3 × 50 mL). The organic layer was
concentrated and purified over silica (100% CH2Cl2 to 100%
EtOAc) followed by crystallization from 2-propanol to afford 2b
(111 mg, 22%); mp: 238−241 °C (dec); IR νmax (cm−1): 3034.4,
1600.9, 1568.2, 1524.6, 1512.3, 1495.0, 1445.8, 1409.0, 1358.4, 1272.8,
1
1250.3, 1174.0, 837.0, 751.3, 688.5; H NMR (500 MHz, DMSO-d6):
Figure 8. R1, R2, and R3 are possible sites for chemical modification of
1a and 2a to enhance aqueous solubility.
δ 3.80 (s, 3H), 7.00 (d, 2H, J = 8.7 Hz), 7.15 (t, 1H, J = 7.3 Hz), 7.24
(d, 1H, J = 16.4 Hz), 7.40−7.45 (m, 3H), 7.60 (d, 2H, J = 8.6 Hz),
7.76 (d, 1H, J = 8.6 Hz), 7.86 (d, 2H, J = 7.6 Hz), 8.10 (d, 1H, J = 8.0
Hz), 8.55 (s, 1H), 8.67 (s, 1H), 9.79 (s, 1H); 13C NMR (125 MHz,
DMSO-d6): δ 55.6, 114.8, 115.9, 120.2, 122.8, 124.2, 125.7, 128.3,
128.5, 128.9, 129.9, 130.1, 131.6, 136.0, 139.6, 149.6, 154.5, 158.0,
159.7; HR-ESI (Q-TOF) m/z: calcd for C23H21N3O+ [M + H]+,
354.1606; found,: 354.1621.
EXPERIMENTAL SECTION
General Methods. Reagents and solvents were obtained from
■
1
commercial suppliers and used without further purification. H and
13C NMR spectra were recorded on a 500 MHz spectrometer.
6-(4-Methoxyphenyl)-N-phenylquinazolin-4-amine (1b).
Yield: 52 mg, 16%; mp 245−247 °C; IR νmax (cm−1): 2999.8,
2835.4, 1937.5, 1600.6, 1570.2, 1522.6, 1496.5, 1445.4, 1400.9, 1358.3,
1270.5, 1247.2, 828.7, 818.3 753.3; 1H NMR (500 MHz, DMSO-d6): δ
3.83 (s, 3H), 7.11 (d, 1H, J = 8.7 Hz), 7.15 (t, 2H, J = 7.4 Hz), 7.42 (t,
2H, J = 8.1 Hz), 7.81−7.86 (m, 5H), 8.15 (dd, 1H, J = 8.7, 1.5 Hz),
8.57 (s, 1H), 8.79 (s, 1H), 9.93 (s, 1H); 13C NMR (125 MHz, DMSO-
d6, TFA) δ 55.7, 114.6, 115.0, 120.8, 122.1, 124.9, 126.8, 128.8, 129.2,
130.7, 134.3, 137.4, 139.7, 140.1, 151.7, 159.8, 160.2; HRMS HR-ESI
(Q-TOF) m/z: calcd for C21H18N3O+ [M + H]+, 328.1449; found,:
328.1453.
Absorbance spectra were obtained using probe concentrations of 10
μM. Fluorescence studies were performed using probe concentrations
of 1 μM. For determination of ϕem, solutions were prepared to an
optical density of 0.05 or less in order to minimize inner filter effects.
Perylene in cyclohexane was used as a reference for quantum yields.25
Computational Methods. Quantum chemical calculations were
carried out utilizing the Gaussian ′09 suite of electronic structure
modeling software.26 Ground-state geometries of the dyes were
optimized by DFT with the B3LYP/6-31G(d) method using Truhlar’s
SMD solvation model.16 Vertical transition energies were obtained by
TD-DFT calculations with the B3LYP/6-31G(d) with the SMD
model. Molecular orbitals were visualized using the GaussView 5
program. The coordinates of optimized geometries are provided in the
Supporting Information.
Inhibition of Ligand-Induced Receptor Activation. MCF cells
were seeded with equal quantity (200 000/well) in six-well plates.
After 48 h, cells were pretreated with small molecule inhibitors of
various concentrations for 1 h before induction by neuregulin
(NRGβ1, 20 nM, 15 min). Cell lysates were generated immediately
by SDS lysis. Equal aliquots were subjected to SDS-PAGE and western
blot analysis. ERBB2 phosphorylation was evaluated for Tyr1239
located close to the extreme cytoplasmic C-terminus of the receptor
(validated by pan-TyrP detection (4G10)). The signal obtained for
pTyr1239 relative to the ERBB2 receptor levels was determined as the
relative receptor phosphorylation.
N,6-Diphenylquinazolin-4-amine (1c). Yield: 41 mg, 14%; mp
258−262 °C; IR νmax (cm−1): 3067.3, 1939.0, 1600.5 1567.8, 1528.9,
1488.6, 1445.3, 1405.6, 1359.8, 1325.9, 1295.0, 840.7, 748.5, 689.2; 1H
NMR (500 MHz, DMSO-d6): δ 7.16 (t, 1H, J = 7.3 Hz), 7.40−7.46
(m, 3H), 7.56 (t, 2H, J = 7.6 Hz), 7.85−7.91 (m, 5H), 8.20 (d, 1H, J =
8.7 Hz), 8.59 (s, 1H), 8.86 (s, 1H), 9.96 (s, 1H); 13C NMR (125
MHz, DMSO-d6) δ 115.8, 120.9, 123.1, 124.3, 127.6, 128.4, 128.8,
128.9, 129.5, 132.2, 138.4, 139.5, 139.6, 149.5, 155.0, 158.3; HR-ESI
+
(Q-TOF) m/z: calcd for C C20H16N3 [M + H]+, 298.1344; found,
298.1352.
4-(4-Phenylamino)quinazolin-6-yl)benzonitrile (1d). Yield: 48
mg, 15%; mp 242−245 °C; IR νmax (cm−1): 3107.2, 2228.4, 1624.1,
1596.7, 1571.4, 1527.8, 1496.9, 1443.8, 1403.4, 1326.6, 1255.8, 1106.2,
829.1, 747.3, 690.4; 1H NMR (500 MHz, DMSO-d6): δ 7.31 (t, 1H, J
= 7.4 Hz), 7.57 (t, 2H, J = 8.3 Hz), 7.98 (d, 2H, J = 7.8 Hz), 8.02 (d,
1H, J = 8.7 Hz), 8.19 (d, 2H, J = 8.5 Hz), 8.26 (d, 2H, J = 8.4 Hz),
8.41 (dd, 1H, J = 8.7, 1.9 Hz), 8.75 (s, 1H), 9.09 (s, 1H), 10.15 (s,
1H); 13C NMR (125 MHz, DMSO-d6): δ 110.8, 115.8, 118.4, 119.2,
121.9, 123.2, 124.5, 128.3, 129.0, 132.0, 133.3, 136.3, 139.3, 144.0,
Representative Synthesis via Route A: 6-(4-(Dimethyl-
amino)phenyl)-N-phenylquinazolin-4-amine (1a). Three hun-
dred forty seven milligrams (1.0 mmol) of 6-iodo-N-phenyl-4-
quinazolin-amine (4), 248 mg (1.5 mmol) of boronic acid, 425 mg
of CsCO3 (1.3 mmol), and 3 mL of DMF were placed in a 25 mL
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The Journal of Organic Chemistry
Article
+
150.2, 155.5, 158.4; HR-ESI (Q-TOF) m/z: calcd for C21H15N4+ [M +
H]+, 323.1296; found, 323.1305.
154.4, 158.0; HR-ESI (Q-TOF) m/z: calcd for C26H25N4 [M + H]+,
393.2079; found, 393.2088.
6-(1E,3E)-4-(4-(Methoxyphenyl)buta-1,3-dienyl)-N-phenyl-
quinazolin-4-amine (3b). Yield: 70 mg, 13%; mp 245−248 °C
(dec); IR νmax (cm−1): 3029.9, 1939.7, 1599.1, 1568.7 1525.3, 1498.5,
1446.4, 1410.1, 1385.1, 1357.7, 1300.1, 1248.8, 988.5, 806.7, 740.2,
690.7; 1H NMR (500 MHz, DMSO-d6): δ 3.84 (s, 3H), 6.77 (d, 1H, J
= 15.5 Hz), 6.85 (d, 1H, J = 15.5 Hz), 6.95 (d, 2H, J = 8.6 Hz), 7.07
(dd, 1H, J = 15.4, 10.7 Hz), 7.15 (t, 1H, J = 7.3 Hz), 7.29 (dd, 1H, J =
15.5, 10.7 Hz), 7.42 (t, 2H, J = 7.9 Hz), 7.51 (d, 2H, J = 8.5 Hz), 7.73
(d, 1H, J = 8.6 Hz), 7.86 (d, 2H, J = 7.7 Hz), 8.04 (d, 1H, J = 8.6 Hz),
8.54 (s, 1H), 8.59 (s, 1H), 9.84 (s, 1H); 13C NMR (125 MHz, DMSO-
d6): δ 55.6, 114.7, 115.9, 120.4, 122.9, 124.2, 127.5, 128.3, 128.6,
128.9, 130.0, 130.8, 131.3, 131.7, 133.7, 135.9, 139.6, 149.6, 154.6,
158.0, 159.6; HR-ESI (Q-TOF) m/z: calcd for C25H22N3O+ [M +
H]+, 380.1763; found, 380.1766.
N-Phenyl-6-((1E,3E)-4-phenylbuta-1,3-dienyl)quinazolin-4-
amine (3c). Yield: 70 mg, 14%; mp 258−260 °C (dec); IR νmax
(cm−1): 3259.1, 3055.1, 1924.2, 1604.2, 1569.2, 1528.3, 1496.2,
1440.3, 1406.5, 1388.2, 1363.4, 1329.5, 1310.3, 1253.8, 978.0, 901.4,
750.3, 686.5; 1H NMR (500 MHz, DMSO-d6): δ 6.81 (d, 1H, J = 15.2
Hz), 6.91 (d, 1H, J = 15.2 Hz), 7.11 (t, 1H, J = 7.2 Hz), 7.19−7.32 (m,
3H), 7.38 (d, 2H, J = 7.4 Hz), 7.39 (d, 2H, J = 7.4 Hz), 7.56 (d, 2H, J
= 7.6 Hz), 7.68 (d, 1H, J = 8.5 Hz), 7.80 (d, 2H, J = 7.6 Hz), 8.01 (d,
1H, J = 8.5 Hz), 8.47 (s, 1H), 8.58 (s, 1H), 9.84 (s, 1H); 13C NMR
(125 MHz, DMSO-d6): δ 116.1, 121.1, 123.0, 123.8, 126.9, 128.2,
128.4, 128.8, 129.2, 129.8, 131.0, 132.3, 133.7, 135.3, 137.4, 140.7,
149.9, 154.9, 158.1; HR-ESI (Q-TOF) m/z: calcd for C24H20N3+ [M +
H]+, 350.1657; found, 350.1673.
6-(4-Nitrophenyl)-N-phenylquinazolin-4-amine (1e). Yield:
55 mg, 16%; mp 262−264 °C; IR νmax (cm−1): 3089.6, 1934.3,
1597.5, 1569.0, 1531.1, 1514.6, 1488.0, 1449.5, 1401.4, 1417.5, 1341.8,
1
1271.0, 834.7, 752.1, 693.2; H NMR (500 MHz, DMSO-d6): δ 7.32
(t, 1H, J = 7.4 Hz), 7.57 (t, 2H, J = 8.1 Hz), 7.98 (d, 2H, J = 7.6 Hz),
8.05 (d, 1H, J = 8.6 Hz), 8.33 (d, 2H, J = 8.8 Hz), 8.44 (dd, 1H, J =
8.7, 1.7 Hz), 8.55 (d, 2H, J = 8.8 Hz), 8.76 (s, 1H), 9.14 (d, 1H, J = 1.3
Hz), 10.20 (s, 1H); 13C NMR (125 MHz, DMSO-d6): δ 115.8, 122.3,
123.2, 124.5, 128.6, 128.9, 129.1, 132.1, 135.8, 139.3, 145.9, 147.3,
+
150.3, 155.7, 158.5; HR-ESI (Q-TOF) m/z: calcd for C20H15N4O2
[M + H]+, 343.1195; found, 343.1194.
(E)-6-(4-Dimethylaminostyryl)-N-phenylquinazolin-4-amine
(2a). Yield: 130 mg, 25%; mp 220 °C (dec); IR νmax (cm−1): 3206.0,
2940.8, 1646.0, 1599.4, 1564.6, 1521.6, 1493.6, 1443.9, 1403.9, 1382.3,
1
1363.2, 1254.4, 957.2, 752.1, 691.3; H NMR (400 MHz, DMSO-d6):
δ 2.94 (s, 6H), 6.76 (d, 2H, J = 8.7 Hz), 7.10−7.14 (m, 2H), 7.34−
7.42 (m, 3H), 7.48 (d, 2H, J = 8.7 Hz), 7.73 (d, 1H, J = 8.7 Hz), 7.87
(d, 2H, J = 7.8 Hz), 8.07 (d, 1H, J = 8.7 Hz), 8.52 (s, 1H), 8.60 (s,
1H), 9.78 (s, 1H); 13C NMR (125 MHz, DMSO): δ 112.3, 115.5,
119.1, 122.4, 123.8, 124.6, 127.7, 128.0, 128.5, 130.5, 131.1, 136.2,
139.3, 148.7, 150.3, 153.8, 157.6; HR-ESI (Q-TOF) m/z: calcd for
+
C24H23N4 [M + H]+, 367.1923; found, 367.1929.
(E)-N-Phenyl-6-styrylquinazolin-4-amine (2c). Yield: 74 mg,
16%; mp 248−252 °C (dec); IR νmax (cm−1): 3048.8, 1949.9, 1622.7,
1600.8, 1570.5, 1523.4, 1494.9, 1446.5, 1409.5, 1385.9, 1360.5, 1332.6,
1
1254.5, 827.7, 747.8, 687.8; H NMR (500 MHz, DMSO-d6): δ 7.16
(t, 1H, J = 7.2 Hz), 7.32 (t, 1H, J = 7.2 Hz), 7.38−7.51 (m, 6H), 7.66
(d, 2H, J = 7.4 Hz), 7.78 (d, 1H, J = 8.6 Hz), 7.87 (d, 2H, J = 7.6 Hz),
8.14 (d, 1H, J = 8.3 Hz), 8.56 (s, 1H), 8.72 (s, 1H), 9.81 (s, 1H); 13C
NMR (125 MHz, DMSO-d6): δ 115.8, 120.8, 122.9, 124.2, 127.0,
128.0, 128.4, 128.6, 128.9, 129.3, 130.3, 131.7, 135.6, 137.2, 139.6,
149.8, 154.8, 158.1; HR-ESI (Q-TOF) m/z: calcd for C22H18N3+ [M +
H]+, 324.1500; found, 324.1516.
4-((1E,3E-4-(4-(Phenylamino)quinazolin-6-yl)buta-1,3-
dienyl)benzonitrile (3d). Yield: 134 mg, 25%; mp 242−245 °C
(dec); IR νmax (cm−1): 3365.8, 3028.7, 2224.5, 1598.1, 1568.6, 1525.6,
1496.8, 1443.5, 1402.4, 1387.6, 1361.7, 1252.8, 983.0, 746.2, 848.0,
1
687.4; H NMR (500 MHz, DMSO-d6): δ 7.02 (d, 1H, J = 15.2 Hz),
7.16 (d, 1H, J = 15.1 Hz), 7.30 (t, 1H, J = 7.4 Hz), 7.46−7.60 (m, 4H),
7.90 (d, 3H, J = 8.5 Hz), 7.96 (d, 2H, J = 8.35 Hz), 8.00 (d, 2H, J =
7.65 Hz), 8.22 (dd, 1H, J = 8.7, 1.0 Hz), 8.69 (s, 1H), 8.79 (s, 1H),
9.98 (s, 1H); 13C NMR (125 MHz, DMSO-d6): δ 109.9, 115.8, 119.4,
121.4, 122.9, 124.2, 127.5, 128.7, 128.9, 130.6, 131.3, 131.8, 133.0,
133.5, 134.4, 135.2, 139.5, 142.1, 150.1, 154.9, 158.1; HR-ESI (Q-
4-((E-2-(4-(Phenylamino)quinazoline-6-yl)vinyl)benzonitrile
(2d). Yield: 98 mg, 20%; mp 250−254 °C; IR νmax (cm−1): 3071.4,
2221.6, 1599.8, 1571.8, 1498.4, 1441.7, 1403.6, 1362.3, 964.8, 838.4,
1
752.4, 693.8; H NMR (500 MHz, DMSO-d6): δ 7.15 (t, 1H, J = 7.4
+
Hz), 7.42 (t, 2 H, J = 8.3 Hz), 7.56 (d, 1H, J = 16.4 Hz), 7.59 (d, 1H, J
= 16.4 Hz), 7.79−7. 89 (br m, 7H), 8.17 (dd, 1H, J = 8.7, 1.7 Hz), 8.57
(s, 1H), 8.76 (s, 1H), 9.88 (s, 1H); 13C NMR (125 MHz, DMSO-d6):
δ 110.2, 115.8, 119.3, 121.8, 122.9, 124.2, 127.5, 128.5, 129.1, 129.2,
131.9 133.1, 134.7, 139.5, 141.9, 150.2, 155.1, 158.2; HR-ESI (Q-
TOF) m/z: calcd for C25H19N4 [M + H]+, 375.1609; found,
375.1610.
6-((1E,3E)-4-(4-Nitrophenyl)buta-1,3-dienyl)-N-phenylquina-
zolin-4-amine (3e). Yield: 113 mg, 20%; mp 273−275 °C (dec); IR
νmax (cm−1): 3421.3, 3020.7, 1927.9, 1603.3, 1585.6, 1571.5, 1557.7,
1535.3, 1494.4, 1444.3, 1331.5, 1253.8, 1109.3, 987.1, 743.6, 804.3; 1H
NMR (500 MHz, DMSO-d6): δ 6.92 (d, 1H, J = 15.4 Hz), 7.03 (d,
1H, J = 15.3 Hz), 7.13 (t, 1H, J = 7.2 Hz), 7.32−7.41 (m, 3H), 7.48
(dd, 1H, J = 15.4, 10.7 Hz), 7.73 (d, 1H, J = 8.6 Hz), 7.81 (d, 2H, J =
8.8 Hz), 7.83 (d, 2H, J = 8.8 Hz), 8.08 (d, 1H, J = 8.6 Hz), 8.20 (d,
2H, J = 8.6 Hz), 8.53 (s, 1H), 8.64 (s, 1H), 9.86 (s, 1H); 13C NMR
(125 MHz, DMSO-d6): δ 115.8, 121.5, 123.0, 124.3, 124.4, 127.6,
128.7, 128.9, 130.6, 131.4, 132.0, 134.5, 135.1, 135.2, 139.5, 144.2,
146.6, 150.1, 155.0, 158.1; HR-ESI (Q-TOF) m/z: calcd for
+
TOF) m/z: calcd for C23H17N4 [M + H]+, 349.1453; found,
349.1453.
(E)-6-(4-Nitrostyryl)-N-phenylquinazolin-4-amine (2e). Yield:
128 mg, 24%; mp 240 °C (dec); IR νmax(cm−1): 3085.1, 1932.2,
1602.1, 1591.5, 1571.4, 1527.7, 1501.7, 1328.8, 1404.1, 1443.2, 1365.5,
1255.4, 1105.2, 968.0, 749.5, 684.1, 694.9; 1H NMR (500 MHz,
DMSO-d6): δ 7.30 (t, 1H, J = 7.3 Hz), 7.56 (t, 2H, J = 7.8 Hz), 7.72
(d, 1H, J = 16.5 Hz), 7.76 (d, 1H, J = 16.5 Hz), 7.93 (d, 1H, J = 8.6
Hz), 8.01 (t, 4H, J = 7.8 Hz), 8.30, (d, 1H, J = 8.6 Hz), 8.40 (d, 2H, J
= 8.4 Hz), 8.71 (s, 1H), 8.91(s, 1H), 9.98 (s, 1H); 13C NMR (125
MHz, DMSO-d6): δ 115.8, 122.1, 122.9, 124.3, 124.6, 127.8, 128.1,
128.7, 128.9, 131.9, 132.7, 134.7, 139.4, 144.1, 146.8, 150.3, 155.2,
+
C24H19N4O2 [M + H]+, 395.1508; found, 395.1503.
ASSOCIATED CONTENT
■
+
158.2; HR-ESI (Q-TOF) m/z: calcd for C22H17N4O2 [M + H]+,
S
* Supporting Information
1H and 13C NMR spectra, emission spectra in toluene and
acetonitrile, frontier molecular orbitals, and Cartesian coor-
dinates of optimized geometries. This material is available free
of charge via the Internet at http://pubs.acs.org.
369.1351; found, 369.1346.
6-(1E,3E)-(4-(4-Dimethylaminophenyl)buta-1,3-dienyl)-N-
phenylquinazolin-4-amine (3a). Yield: 90 mg, 16%; mp 255−258
°C (dec); IR νmax (cm−1): 2980.6, 1596.3, 1570.0, 1521.7, 1496.4,
1
1445.6, 1409.3, 1385.4, 1354.0, 1186.4, 987.4, 801.0, 746.0, 689.8; H
NMR (500 MHz, DMSO-d6): δ 2.94 (s, 6H), 6.71 (d, 2H, J = 8.7 Hz),
6.77 (d, 2H, J = 15.6 Hz), 6.95 (dd, 1H, J = 15.2, 11.7 Hz), 7.15 (t,
1H, J = 7.0 Hz), 7.27 (dd, 1H, J = 15.0, 11.1 Hz), 7.38−7.43 (m, 4H),
7.72 (d, 1H, J = 8.4 Hz), 7.87 (d, 2H, J = 7.3 Hz), 8.00 (d, 1H, J = 8.3
Hz), 8.54 (d, 2H, J = 15.6 Hz), 9.8 (s, 1H); 13C NMR (125 MHz,
DMSO-d6): δ 112.6, 115.9, 119.9, 122.9, 124.2, 125.0, 125.2, 128.1,
128.5, 128.9, 129.2, 131.2, 132.2, 134.7, 136.2, 139.6, 149.5, 150.5,
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: jnwilson@miami.edu.
Notes
The authors declare no competing financial interest.
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The Journal of Organic Chemistry
Article
(26) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,
K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega,
N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;
Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.;
ACKNOWLEDGMENTS
■
This work was supported by the National Cancer Institute
Innovative Molecular Analysis Technologies Program,
CA182341-01 (J.N.W. and R.L.). J.N.W. also acknowledges
the support of the Bankhead-Coley New Investigator Research
Program, 3BN08. R.L. also acknowledges the support of
National Cancer Institute, CA98881-05, and the Braman
Family Breast Cancer Institute.
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