Discovery of quinazoline-based fluorescent probes to α1-adrenergic receptors

Article abstract of DOI:10.1021/ml5004298

α₁-Adrenergic receptors (α₁-ARs), as the essential members of G protein-coupled receptors (GPCRs), can mediate numerous physiological responses in the sympathetic nervous system. In the current research, a series of quinazoline-based small-molecule fluorescent probes to α₁-ARs (1a-1e), including two parts, a pharmacophore for α₁-AR recognition and a fluorophore for visualization, were well designed and synthesized. The biological evaluation results displayed that these probes held reasonable fluorescent properties, high affinity, accepted cell toxicity, and excellent subcellular localization imaging potential for α₁-ARs.

Full text of DOI:10.1021/ml5004298

Letter
pubs.acs.org/acsmedchemlett
Discovery of Quinazoline-Based Fluorescent Probes to α1‑Adrenergic
Receptors
Wei Zhang,† Zhao Ma,† Wenhua Li, Geng Li, Laizhong Chen, Zhenzhen Liu, Lupei Du, and Minyong Li*
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong University, Jinan,
Shandong 250012, China
S
* Supporting Information
ABSTRACT: α1-Adrenergic receptors (α1-ARs), as the
essential members of G protein-coupled receptors (GPCRs),
can mediate numerous physiological responses in the
sympathetic nervous system. In the current research, a series
of quinazoline-based small-molecule fluorescent probes to α1-
ARs (1a−1e), including two parts, a pharmacophore for α1-AR
recognition and a fluorophore for visualization, were well
designed and synthesized. The biological evaluation results
displayed that these probes held reasonable fluorescent
properties, high affinity, accepted cell toxicity, and excellent
subcellular localization imaging potential for α1-ARs.
KEYWORDS: α1-Adrenergic receptors, fluorescent probes, quinazoline, cell imaging, subcellular localization
α1-Adrenergic receptors (α1-ARs), as the important members
of G protein coupled receptors (GPCRs), distribute in a variety
of organs, tissues, and cells, which mediate many crucial
physiological effects in the human body. These receptors are
classified into at least three subtypes (α1A, α1B, and α1D) based
on their differences on the biological structure, tissue
distributions, pharmacological properties, and signaling path-
ways.1 Various studies confirmed that α1-ARs are closely
involved in benign prostatic hyperplasia (BPH), hypertension,
prostate cancer, and other diseases.2−4 Functional experiments
manifested that α1B-AR is mainly in charge of the vasomotion
of small resistance vessels, while α1A- and α1D-ARs take
responsibility for the contraction of the main arteries in animal
species.5
So far, there are many challenges to fully understand the
biological and pharmacological characteristics of each α1-AR
subtype. This dilemma is mainly caused by the difficulties either
to determine their distribution in various organs and tissues or
to define the functional response mediated by each one in the
different species by using classical approaches without their
three-dimensional crystal structure and tissue-selective α1-AR
antagonists.6 Fortunately, with the rapid development of
fluorescence analysis technology in various areas, small-
molecule fluorescent probes with many advantages, including
high sensitivity, selectivity, and visualization, have been widely
applied to track the biological macromolecules, especially
GPCRs.7−10 Small-molecule fluorescent probes for GPCRs
generally consist of two essential components: the pharmaco-
phore moiety that is used to bind with the target through the
receptor−ligand interaction and the fluorophore group that can
trace the target by the fluorescent properties.11 Moreover, good
optical characteristics and high affinity of probe can ensure its
successful labeling of the target. It needs to be noted that in the
case of α1-ARs, only one small-molecule fluorescent probe,
BODIPY-FL-prazosin, is available. However, the application of
this probe is restricted to its complicated preparation. Recently,
some QD-based fluorescent probes have been reported as
well.12,13 Although rational results about α1B-AR study were
given by these QD probes, the studies of probes on purity,
structure, cytotoxicity, and receptor affinity were inadequate.10
Therefore, more small-molecule fluorescent probes with
diversity of structure and fluorescence property are demanded
for the current molecular pharmacology study and drug
discovery of α1-AR.
α1-Adrenergic receptor antagonists can be structurally
categorized into several classes, including quinazolines, 1,4-
benzodioxans, dihydropyridines and dihydropyrimidines, fused
pyrimidindiones, pyridazinones, imidazolines, N-arylindoles, N-
aryl, and N-heteroarylpiperazines, and miscellaneous com-
pounds.14 Among all chemotypes, quinazoline compounds,
including prazosin, terazosin, doxazosin, and alfuzosin, are the
most clinically effective α1-AR antagonists (Scheme 1).15 In our
previous study, a pharmacophore model based on quinazoline
derivatives that contribute many effective α1-AR antagonists
was well built, and the proposed interaction model of
quinazoline-based antagonists with α1-ARs was well devel-
oped.16−19 It was found that there is a bulky space around the
piperazine group to accommodate the fluorophore moiety,
which does not influence the affinity of antagonists to receptors.
Therefore, in the current research, we chose the quinazoline
Received: October 22, 2014
Accepted: March 30, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/ml5004298
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Scheme 1. Structures of Quinazoline Derivatives as α1-AR
Antagonists
Figure 1. Proposed docking conformation of prazosin (red sticks) and
1a (white sticks) in the human α1A-AR binding site.
moiety as the pharmacophore to generate fluorescent probes
for selectively binding with α1-ARs. In the meanwhile, coumarin
and fluorescein groups were selected as fluorophores because of
their preferred characteristics, such as high sensitivity, light
stability, small molecular weight, water solubility, and
reasonable cell permeability. The understanding on how α1-
ARs bind with their ligands20 and the development of
fluorescent labeling analysis method can efficiently promote
the emergence of fluorescent probes for α1-ARs. In view of
these evidence, a series of quinazoline derivatives (1a−1e) were
well designed as small-molecule fluorescent probes for α1-ARs
by conjugating pharmacophore (quinazoline) with fluorophores
(coumarin and fluorescein) (Scheme 2).
Scheme 3. Synthetic Routes of Designed Fluorescent Probes
Scheme 2. Designed Quinazoline-Based Fluorescent Probes
for α1-ARs
moiety. More synthetic details can be found in the Supporting
Information.
These probes were first evaluated for in vitro affinities with
three human cloned α1-adrenoceptors subtypes by the
radioligand binding assay using [3H]prazosin in membrane
from transfected CHO cells.21,22 All compounds demonstrated
up to nanomolar affinities with three α1-AR subtypes, which are
close to the positive control, phentolamine (Table 1).
Compound 1e is approximately 100-fold less potent than
compound 1c because of the lack of carbonyl group. In the
absence of the triazole linker, compound 1a shows the similar
affinity with 1b and 1c containing the triazole rings. This
affinity result also proves that a bulky space exists around the
piperazine group to accommodate the fluorophore moiety
To establish a preliminary understanding on if these
quinazoline-based fluorescent probes can be recognized by
α1-ARs, we first developed docking models of molecules 1a−e
with α1A-AR homology model that we developed in 2008.19 As
a result, the docking conformations and orientations of 1a−e
around the active site of α1A-AR were highly consistent with of
prazosin as depicted in Figures 1 and S1−S6.These computa-
tional results clearly propose that compounds 1a−e may be
recognized by α1A-AR. More computational details can be
found in Supporting Information.
The convenient syntheses of these fluorescent probes were
mainly based on CuAAC reaction and amide condensation with
mild conditions, high yield, and easy purification (Scheme 3).
The fluorescein derivative was obtained through the one-step
condensation reaction of fluorescein isocyanate and the
quinazoline parent. Synthesis of coumarin derivatives was
started from the acetylation or azidation of coumarin. And then,
the quinazoline was attached to coumarin by amide or triazole
Table 1. Comparison of the Probes’ Affinity to α1-ARs
a
Ki (nM)
IC50 (nM)
α1B
compd
α1A
α1B
α1D
α1A
α1D
phentolamine
0.6
2.1
1.3
0.3
4.7
29.9
4.8
7.6
1.1
3.9
2.5
0.6
8.8
52.4
10.8
12.5
c
c
b
b
1a
1b
1c
1d
1e
NA
NA
NA
0.4
<5
<5
c
c
b
b
NA
<5
<5
0.1
1.2
2.2
7.3
20.7
27.5
16.3
52.4
34.0
45.2
29.9
a
b
Ki was calculated from IC50 using the Cheng−Prusoff equation. Data
c
was estimated from the unclassical binding-competitive curve. Not
available.
B
DOI: 10.1021/ml5004298
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
without influencing the affinity to receptors. Certainly, this
space is not infinite. When the fluorophore is changed from
coumarin (1a) to more bulky fluorescein (1d), the affinity
might decrease slightly.
As far as the spectroscopic properties are concerned (Table
2), all target compounds showed excitation and emission
probes and DiD (a cell membrane dye, red, colocalization) at
37 °C for 5 min, in which normal HEK293A cells (without α1-
AR expression) as a negative control, and HEK293A-α1A-AR
and HEK293A-α1D-AR cells incubated with tamsulosin (a
potent α1-AR antagonist) as another negative control. Indeed,
the fluorescence of cells was not strong due to the poor
quantum yield of compounds.24 However, as shown in Figure
1, the incubation of cells with compound 1a allowed
visualization of the α1A- and α1D-ARs in stably transfected
HEK293 cells using conventional fluorescence microscopy
(Figure 2A,B), while a significant reduction in fluorescence
Table 2. Characterization and Properties of Compounds 1a−
1e
compd
λmax (nm)
λex (nm)
λem (nm)
Φ (%)
8.5
42
1a
1b
1c
1d
1e
391
392
420
494
430
391
392
420
485
430
456
454
500
517
505
1
1
7.8 0.2
2.6 0.3
4.7 0.1
wavelengths comparable to that reported in literature for the
parent fluorophores. In addition, the fluorescence quantum
yield was checked in a mixed solvent of methanol and PBS
buffer. All compounds possessed reasonable fluorescence
quantum yields. In particular, compound 1b showed high
quantum yields, which is up to 42%. The solvent effect on
fluorescence property was examined as well (Figure S6). The
hydroxyl coumarin derivatives, 1a and 1b, emitted strong
fluorescence in water; however, their fluorescence emissions
were quenched in methanol and ethanol. Amino coumarin and
fluorescein derivatives behaved in the opposite way.
It should be pointed out that a reasonable fluorescent probe
should be harmless while labeling the target. In view of this
reason, the cytotoxicities of these probes were checked by a
standard sulforhodamine B (SRB) colorimetric assay.23 As
demonstrated in Table 3, the cytotoxicity of compounds 1c and
Figure 2. Confocal fluorescence image of HEK293 cell using
compound 1a. All cells are incubated with 1a at 37 °C for 5 min
and washed immediately, and the exposure time is the same. The
background was adjusted by ImageJ software. (A) Image of HEK293A-
α1A-AR cells incubated with 1a (15 nM) and DiD; (B) image of
HEK293A-α1D-AR cells incubated with 1a (15 nM) and DiD; (C)
image of HEK293A cells incubated with 1a (15 nM) and DID; (D)
image of HEK293A-α1A-AR cells incubated with 1a (15 nM), DID,
and tamsulosin (300 nM); (E) image of HEK293A-α1D-AR cells
incubated with 1a (15 nM), DID, and tamsulosin (300 nM). Scale bar
(yellow) = 20 μm.
Table 3. Cytotoxicity Results of the Synthesized Probes
IC50 (μM)
compd
HEK293A-α1A-AR
HEK293A-α1D-AR
labeling was observed in transfected HEK293 cells upon
competition with 20-fold excess of tamsulosin (Figure 2D,E).
Moreover, very weak fluorescence labeling in negative HEK293
cells showed a spot of nonspecific binding of compound 1a
(Figure 2C). We also proved the application of our probe 1c in
detecting cellular α1-ARs by flow cytometry. As shown in
Figure S20, compound 1c could identify the HEK293 cells
transfected with α1-ARs successfully. Although compound 1c
has weaker affinity to α1d-ARs than α1a-ARs, stronger staining of
α1d-ARs cells than that of α1a-ARs cells was found. This may be
caused by the inconsistent expression levels of α1a-AR and α1d-
AR in cells, and in this case, the expression level of α1d-AR is
higher than α1a-AR.
In subcellular localization, the fluorescence of α1A-AR was
found on the cell surface, and their location was almost
overlapped with the red area stained by DiD. In the case of α1D-
AR, its fluorescence mainly distributed in cell plasma, as well as
a little bit on the cell surface. These subcellular localization
results are highly in accordance with Piascik’s conclusion, where
α1A-AR fluorescence was detected not only on the cell surface
but also intracellularly, and α1D-AR fluorescence was detected
mainly intracellularly.25 Fluorescence imaging of the other four
compounds 1b−1e presents similar results (Figures S16−S19).
Although the largest emission peak of 1a or 1b was near to
blue, the blue filter did not give a clear imaging result;
therefore, these images were recorded through the green filter.
doxazosin
19
2
22
2
1a
1b
1c
1d
1e
>100
>100
>100
85
2
2
12 0.2
>100
32
>100
26
3
17 0.2
1e in HEK293A (human embryonic kidney) cells transfected
with α1A-AR (HEK293A-α1A-AR) or HEK293A cells trans-
fected with α1D-AR (HEK293A-α1D-AR) was similar to the
positive control, doxazosin, while compounds 1a, 1b, and 1d
had slight cytotoxicity. Obviously, as presented in Table 3, only
compound 1c and 1e had moderate cytotoxicities with IC50
values at the micromolar level. The results showed that cell
viability was not significantly changed upon treatment,
indicating the low cytotoxicity and good biocompatibility of
these probes in living cell study at the nanomolar concentration
without any cell damage.
The above-mentioned results demonstrated that most of the
probes displayed high affinities to α1-ARs and accepted
cytotoxicities that laid a solid foundation for fluorescence
imaging in living cells. Although their fluorescence quantum
yield left something to be desired, cell imaging potential of high
expression of α1-ARs was extensively evaluated. We incubated
HEK293A cells transfected with α1A-AR and α1D-AR with the
C
DOI: 10.1021/ml5004298
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Funding
Additionally, it is a known fact that α1-ARs are overexpressed
in prostate cancer.26 After successful staining of α1A- and α1D-
AR overexpressing HEK293A cells, we tested 1d on PC-3
prostate cancer cells because of its most reasonable
fluorescence property. As shown in Figure 3, PC-3 cells could
The present work was supported by grants from the Fok Ying
Tong Education Foundation (No. 122036), the Program of
New Century Excellent Talents in University (No. NCET-11-
0306), the Shandong Natural Science Foundation (No.
JQ201019), and the Independent Innovation Foundation of
Shandong University, IIFSDU (No. 2010JQ005). Our cell
imaging work was performed at the Microscopy Character-
ization Facility, Shandong University. We also thank Professor
Youyi Zhang from Peking University for her generous gift, the
α1A-AR- and α1D-AR-transfected HEK293A cells.
Notes
The authors declare no competing financial interest.
Figure 3. Fluorescence image of cancer cells using compound 1d. The
background was adjusted by ImageJ software. (A) PC-3 cells were with
incubated with 1d (1 μM) at 37 °C for 30 min, then washed
immediately; (B) PC-3 cells were with incubated with 1d (1 μM) and
doxazosin (10 μM) at 37 °C for 30 min, then washed immediately;
(C) HepG2 cells were incubated with 1d (1 μM) at 37 °C for 30 min,
then washed immediately. Scale bar (yellow) = 20 μm.
ABBREVIATIONS
■
α1-ARs, α1-adrenergic receptors; α1A/B/D-ARs, α1A/B/D-adrener-
gic receptors; CHO, Chinese hamster ovary; CuAAC, Cu-
catalyzed azide−alkyne cycloaddition; GPCRs, G protein
coupled receptors; HEK, Human embryonic kidney; QD,
quantum dot; SRB, sulforhodamine B
REFERENCES
■
be “lighted up” after being incubated with 1d (1 μM), while
HepG2 cells (low α1-AR expression) showed little fluores-
cence.27 Also, 1d shows high displaceable properties in PC-3
prostate cancer cells with doxazosin. These findings confirm
probe 1d as a labeling tool in α1-AR overexpressing cells.
In summary, we herein well developed a series of
quinazoline-based small-molecule fluorescent probes with
high sensitivity, high affinity, and low toxicity for convenient
detection of α1-ARs. The probes have up to nanomolar
affinities with three α1-AR subtypes. These fluorescent probes
at the nanomolar level have been successfully used in
visualization and subcellular localization of α1-AR in cell
imaging, including both α1-AR transfected HEK293 cells and
prostate cancer cells, thus supplanting the application of
radioligand for drug screening and providing extra dimensions
of probing receptors that simple competitive radioligands do
not present. The study is a preliminary work to establish that
the strategy can be useful for cell staining. In addition, these
probes have the advantage of easy synthesis from readily
available inexpensive starting materials. It is expected that these
probes now can be added to the armamentarium of fluorescent
ligands that may be utilized as versatile and extremely useful
tools for nowadays molecular pharmacology and drug discovery
in the area of α1-ARs.
(1) Li, W.; Du, L.; Li, M. Alkaloids and flavonoids as α1-adrenergic
receptor antagonists. Curr. Med. Chem. 2011, 18, 4923−4932.
(2) Ruffolo, R. R., Jr; Hieble, J. P. Adrenoceptor pharmacology:
urogenital applications. Eur. Urol. 1999, 36, 17−22.
(3) Forray, C.; Noble, S. A. Subtype selective α1-adrenoceptor
antagonists for the treatment of benign prostatic hyperplasia. Expert
Opin. Invest. Drug. 1999, 8, 2073−2094.
(4) Nagarathnam, D.; Wetzel, J.; Miao, S.; Marzabadi, M.; Chiu, G.;
Wong, W.; Hong, X.; Fang, J.; Forray, C.; Branchek, T. Design and
synthesis of novel α1a adrenoceptor-selective dihydropyridine antag-
onists for the treatment of benign prostatic hyperplasia. J. Med. Chem.
1998, 41, 5320−5333.
(5) Jarajapu, Y. P. R.; Coats, P.; McGrath, J. C.; Hillier, C.;
MacDonald, A. Functional characterization of α1-adrenoceptor
subtypes in human skeletal muscle resistance arteries. Br. J. Pharmacol.
2001, 133, 679−686.
(6) Perez, D. M. Structure-function of α1-adrenergic receptors.
Biochem. Pharmacol. 2007, 73, 1051−62.
(7) Leopoldo, M.; Lacivita, E.; Berardi, F.; Perrone, R. Developments
in fluorescent probes for receptor research. Drug Discovery Today
2009, 14, 706−712.
(8) Kuder, K.; Kiec-Kononowicz, K. Fluorescent GPCR ligands as
new tools in pharmacology. Curr. Med. Chem. 2008, 15, 2132−2143.
(9) Cairo, C. W.; Key, J. A.; Sadek, C. M. Fluorescent small-molecule
probes of biochemistry at the plasma membrane. Curr. Opin. Chem.
Biol. 2010, 14, 57−63.
(10) Ma, Z.; Du, L.; Li, M. Toward fluorescent probes for G-protein-
coupled peceptors (GPCRs). J. Med. Chem. 2014, 57, 8187−8203.
(11) Jacobson, K. A. Functionalized congener approach to the design
of ligands for G protein-coupled receptors (GPCRs). Bioconjugate
Chem. 2009, 20, 1816−35.
(12) Ma, J.; Hou, Z.; Song, Y.; Wang, L.; Guo, E. Visual and
quantitative screening of α1-adrenoceptor antagonists in living cells
using quantum dots. ACS Comb. Sci. 2014, 16, 155−159.
(13) Zhou, G.; Wang, L.; Ma, Y.; Wang, L.; Zhang, Y.; Jiang, W.
Synthesis of a quinazoline derivative: a new α1-adrenoceptor ligand for
conjugation to quantum dots to study α1-adrenoceptors in living cells.
Bioorg. Med. Chem. Lett. 2011, 21, 5905−9.
ASSOCIATED CONTENT
* Supporting Information
Full experimental procedures; analytical and spectral character-
ization data of all compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +86-531-8838-2076. E-mail: mli@sdu.edu.cn.
■
Author Contributions
†These authors contributed equally to this work. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
(14) Rosini, M.; Bolognesi, M. L.; Giardina, D.; Minarini, A.;
Tumiatti, V.; Melchiorre, C. Recent advances in α1-adrenoreceptor
antagonists as pharmacological tools and therapeutic agents. Curr. Top.
Med. Chem. 2007, 7, 147−62.
D
DOI: 10.1021/ml5004298
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(15) Desiniotis, A.; Kyprianou, N. Advances in the design and
synthesis of prazosin derivatives over the last ten years. Expert Opin.
Ther. Targets 2011, 15, 1405−18.
(16) Li, M. Y.; Tsaib, K. C.; Xia, L. Pharmacophore identification of
a1A-adrenoceptor antagonists. Bioorg. Med. Chem. Lett. 2005, 15, 657−
664.
(17) Du, L.; Li, M. Modeling the interactions between 1-adrenergic
receptors and their antagonists. Curr. Comput.-Aided Drug Des. 2010, 6,
165−178.
(18) Zhang, W.; Chen, L.; Ma, Z.; Du, L.; Li, M. Design, synthesis
and biological evaluation of naphthalimide-based fluorescent probes
for α1-adrenergic receptors. Drug Discovery Ther. 2014, 8, 11−17.
(19) Li, M.; Fang, H.; Du, L.; Xia, L.; Wang, B. Computational
studies of the binding site of α1A-adrenoceptor antagonists. J. Mol.
Model. 2008, 14, 957−66.
(20) Jain, K. S.; Bariwal, J. B.; Kathiravan, M. K.; Phoujdar, M. S.;
Sahne, R. S.; Chauhan, B. S.; Shah, A. K.; Yadav, M. R. Recent
advances in selective α1-adrenoreceptor antagonists as antihyperten-
sive agents. Bioorg. Med. Chem. 2008, 16, 4759−4800.
(21) Greengrass, P.; Bremner, R. Binding characteristics of 3H-
prazosin to rat brain α-adrenergic receptors. Eur. J. Pharmacol. 1979,
55, 323−326.
(22) Nagatoma, T.; Tsuchihashi, H.; Sasaki, S.; Nakagawa, Y.;
Nakahara, H.; Imai, S. Displacement by α-adrenergic agonists and
antagonists of 3H-prazosin bound to the α-adrenoceptors of the dog
aorta and the rat brain. Jpn. J. Pharmacol. 1985, 37, 181−187.
(23) Vichai, V.; Kirtikara, K. Sulforhodamine B colorimetric assay for
cytotoxicity screening. Nat. Protoc. 2006, 1, 1112−1116.
(24) Baker, J. G.; Adams, L. A.; Salchow, K.; Mistry, S. N.;
Middleton, R. J.; Hill, S. J.; Kellam, B. Synthesis and characterization of
high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-labeled fluores-
cent ligands for human β-adrenoceptors. J. Med. Chem. 2011, 54,
6874−6887.
(25) Chalothorn, D.; McCune, D. F.; Edelmann, S. E.; García-
Cazarín, M. L.; Gozoh, T.; Piascik, M. T. Differences in the cellular
localization and agonist-mediated internalization properties of the α1-
adrenoceptor subtypes. Mol. Pharmacol. 2002, 61, 1008−1016.
(26) Shi, T.; Gaivin, R. J.; McCune, D. F.; Gupta, M.; Perez, D. M.
Dominance of the alpha1B-adrenergic receptor and its subcellular
localization in human and TRAMP prostate cancer cell lines. J. Recept.
Signal Transduction 2007, 27, 27−45.
(27) Spector, M.; Nguyen, V.-A.; Sheng, X.; He, L.; Woodward, J.;
Fan, S.; Baumgarten, C. M.; Kunos, G.; Dent, P.; Gao, B. Activation of
mitogen-activated protein kinases is required for α 1-adrenergic
agonist-induced cell scattering in transfected HepG2 cells. Exp. Cell
Res. 2000, 258, 109−120.
E
DOI: 10.1021/ml5004298
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX