Analogues of the allosteric heat shock protein 70 (Hsp70) inhibitor, MKT-077, as anti-cancer agents

Article abstract of DOI:10.1021/ml400204n

The rhodacyanine, MKT-077, has antiproliferative activity against cancer cell lines through its ability to inhibit members of the heat shock protein 70 (Hsp70) family of molecular chaperones. However, MKT-077 is rapidly metabolized, which limits its use as either a chemical probe or potential therapeutic. We report the synthesis and characterization of MKT-077 analogues designed for greater stability. The most potent molecules, such as 30 (JG-98), were at least 3-fold more active than MKT-077 against the breast cancer cell lines MDA-MB-231 and MCF-7 (EC₅₀ values of 0.4 ± 0.03 and 0.7 ± 0.2 μM, respectively). The analogues modestly destabilized the chaperone clients, Akt1 and Raf1, and induced apoptosis in these cells. Further, the microsomal half-life of JG-98 was improved at least 7-fold (t_1/2 = 37 min) compared to MKT-077 (t_1/2 < 5 min). Finally, NMR titration experiments suggested that these analogues bind an allosteric site that is known to accommodate MKT-077. These studies advance MKT-077 analogues as chemical probes for studying Hsp70s roles in cancer.

Full text of DOI:10.1021/ml400204n

Letter
pubs.acs.org/acsmedchemlett
Analogues of the Allosteric Heat Shock Protein 70 (Hsp70) Inhibitor,
MKT-077, As Anti-Cancer Agents
Xiaokai Li,# Sharan R. Srinivasan,⊥ Jamie Connarn,‡ Atta Ahmad,⊥ Zapporah T. Young,∥
Adam M. Kabza,# Erik. R. P. Zuiderweg,§ Duxin Sun,‡ and Jason E. Gestwicki*,†,§,∥,⊥,#
‡
§
∥
⊥
†Departments of Pathology, Pharmaceutical Sciences, Biological Chemistry, Medicinal Chemistry, and Life Sciences Institute,
University of Michigan, Ann Arbor, Michigan 48109-2216, United States
#Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94158, United States
S
* Supporting Information
ABSTRACT: The rhodacyanine, MKT-077, has antiproliferative activity against cancer cell lines
through its ability to inhibit members of the heat shock protein 70 (Hsp70) family of molecular
chaperones. However, MKT-077 is rapidly metabolized, which limits its use as either a chemical
probe or potential therapeutic. We report the synthesis and characterization of MKT-077 analogues
designed for greater stability. The most potent molecules, such as 30 (JG-98), were at least 3-fold
more active than MKT-077 against the breast cancer cell lines MDA-MB-231 and MCF-7 (EC50
values of 0.4 0.03 and 0.7 0.2 μM, respectively). The analogues modestly destabilized the
chaperone clients, Akt1 and Raf1, and induced apoptosis in these cells. Further, the microsomal half-life of JG-98 was improved
at least 7-fold (t1/2 = 37 min) compared to MKT-077 (t1/2 < 5 min). Finally, NMR titration experiments suggested that these
analogues bind an allosteric site that is known to accommodate MKT-077. These studies advance MKT-077 analogues as
chemical probes for studying Hsp70s roles in cancer.
KEYWORDS: Breast cancer, mortalin, Hsp90, proteostasis, p53
eat shock protein 70 (Hsp70) is an ATP-dependent
molecular chaperone that plays essential roles in protein
MKT-077 involved release of mtHsp70 from the tumor
suppressor, p53.13 Recently, we used NMR to discover that
MKT-077 binds Hsc70 at an allosteric site within the nucleotide-
binding domain.16 Interestingly, this site is conserved in the
major Hsp70 family members, including Hsp72 and mtHsp70,
and it appears to be important in the chaperone’s ATP hydrolysis
cycle because an analogue of MKT-077, YM-01, disrupts nucleotide
turnover and impacts chaperone functions.14,16,17 However,
MKT-077 and YM-01 are not toxic to fibroblasts or normal
epithelial cells.18 Thus, MKT-077 and its analogues only appear
to be toxic to cells that are addicted to Hsp70s, reminiscent of the
mechanisms invoked for Hsp90 and proteasome inhibitors.19,20
MKT-077 was found to be susceptible to rapid metabolism,
with a lifetime of only 4.4 1.0 min in mouse liver microsomes,21
largely because of oxidation at the benzothiazole and pyridinium
rings.22 Guided by those studies, we used a previously reported
synthetic route (Scheme 1)14 to generate MKT-077 analogues
designed to remove these metabolic liabilities. In these
analogues, we retained the cationic character of MKT-077, but
it should be noted that derivatives with neutral pyridines retain
antiproliferative activity.22 Briefly, the syntheses started with
cyclization of substituted anilines with potassium ethyl xanthate,
followed by methylation by iodomethane under mild basic con-
ditions. The resulting benzothiazoles were activated by methyl
p-toluenesulfonate and coupled with a series of N-substituted
H
homeostasis.1,2 There are 13 members of the Hsp70 family
in mammals,3 including the constitutively expressed, cytosolic
heat shock cognate 70 (Hsc70, HSPA8), the stress-inducible,
cytoplasmic heat shock protein 70 (Hsp72, HSP1A1), and the
mitochondrial Hsp70 (mtHsp70, mot-2, HSPA9). These
chaperones bind and stabilize a wide array of proteins, including
many kinases and transcription factors involved in pro-survival
signaling.2,4,5 Accordingly, high levels of Hsp70s are associated
with immortalized cells and poor prognosis in multiple types of
cancer, including breast cancer, endometrial cancer, and cervical
cancer.6 Many cancer cells appear to become addicted to these
high chaperone levels because dual silencing of Hsc70 and Hsp72
leads to cell death in tumor cells, but not normal fibroblasts.7
For these reasons, Hsp70 has emerged as a potential target for
anticancer agents.1 Inhibition of Hsp70s might be especially
powerful in combination with other chemotherapies because the
levels of Hsp72 are dramatically increased after exposure to other
therapies, such as Hsp90 inhibitors,8 proteasome inhibitors,9 and
radiation.10
MKT-077 is a cationic rhodacyanine that was originally developed
as a dye and later found to have promising antiproliferative activity
against numerous cancer cell lines, including those derived from
bladder carcinomas, colon carcinomas, breast carcinomas, melano-
mas, and pancreatic carcinomas.11−13 Wadhwa and colleagues used a
biotinylated version of MKT-077 to show that this compound
derives its anticancer activity by binding to members of the
Hsp70 family, including Hsc70 and mtHsp70.13−15 Subsequent
work showed that at least part of the antiproliferative activity of
Received: May 28, 2013
Accepted: September 12, 2013
© XXXX American Chemical Society
A
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a
Table 1. Summary of the Antiproliferative Activities and Microsomal Stabilities of MKT-077 and Its Derivatives
b
MDA-MB-231 EC50
MCF-7 EC50
MEF EC50
microsome stability
compd
R1
R2
R3
Hy
(μM)
(μM)
(μM)
t1/2 (min)
MKT-077
H
H
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
allyl
ethyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
2-pyridyl
1.4 0.2
2.0 0.2
4.0 0.4
7.7 0.6
5.6 0.4
6.2 1.0
3.8 0.2
13 0.7
8.3 1.1
2.8 0.2
4.4 0.6
4.6 0.4
8.3 0.7
4.1 0.5
2.8 0.4
4.0 0.4
21 4.5
19 2.9
3.6 0.4
1.8 0.3
1.6 0.2
1.0 0.2
24 3.3
2.2 0.2
5.2 0.8
2.2 0.4
18 4.4
6.2 0.8
8.5 3.0
1.0 0.3
2.4 0.6
0.9 0.3
0.8 0.1
7.8 1.8
28 4.3
9.5 3.1
5.2 1.0
14 2.6
11 2.2
>30
>50
>50
>50
>50
>50
>50
>30
>30
>30
27 3.0
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
<5
<5
15
23
7.7
16
YM-01
1
methyl
methyl
methyl
methyl
methyl
ethyl
3-F
2
4-F
3
5-F
4
6-F
c
5
3-F
NT
6
4-F
ethyl
NT
NT
NT
NT
NT
8.2
7
5-F
ethyl
8
6-F
ethyl
9
3-Cl
4-Cl
5-Cl
6-Cl
4-OMe
5-OMe
4-CF3
5-CF3
H
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
methyl
propyl
benzyl
2-hydro
xyethyl
methyl
methyl
methyl
ethyl
10
11
12
13
14
15
16
17
18
19
20
21
NT
NT
10
NT
NT
NT
NT
NT
NT
NT
>30
8.4 2.4
2.9 0.6
2.8 0.8
1.5 0.2
6.9 2.1
H
benzyl
ethyl
ethyl
ethyl
H
H
H
22
H
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
ethyl
4-pyridyl
1.7 0.1
5.8 0.8
5.1 0.3
2.2 0.2
0.5 0.1
0.4 0.03
0.4 0.02
0.6 0.1
0.4 0.03
0.5 0.1
19 4.9
9.1 1.6
0.7 0.1
0.8 0.1
0.6 0.04
1.0 0.2
0.8 0.2
0.8 0.2
0.7 0.2
1.0 0.8
>50
NT
NT
NT
NT
NT
NT
8.8
23
H
2-pyrimidinyl
2-thiazolyl
2-thiazolyl
2-thiazolyl
2-thiazolyl
2-thiazolyl
2-thiazolyl
2-thiazolyl
2-thiazolyl
>50
24
H
>50
25
H
>30
26
H
benzyl
benzyl
benzyl
benzyl
benzyl
benzyl
6.8 0.4
8.3 0.7
7.0 0.6
>30
27 (JG-83)
28 (JG-84)
29
3-F
3-Cl
4-Cl
5-Cl
6-Cl
NT
37
30 (JG-98)
31
24 1.3
>30
NT
a
Antiproliferative activity was measured using MTT assays. Results are the average of experiments performed in triplicate, and error is SEM. For
stability studies, compounds (1 μM) were incubated with mouse liver microsomes and degradation monitored by LC−MS (see the Supporting
b
c
Information). From C57BL/6 mice. Not tested or poorly soluble.
rhodanines. These products were methylated by methyl
p-toluenesulfonate, followed by another coupling with an
activated heterocycle. The products were dissolved in methanol
and passed through a chloride exchange column to generate the
final compounds 1−31 in overall yields between 20% and 40%
(Table 1).
To compare the antiproliferative activities of these molecules,
MKT-077, YM-01, and compounds 1−31 were evaluated in MDA-
MB-231 and MCF7 breast cancer cells using MTT assays.23 MKT-
077 had EC50 values of 1.4 0.2 and 2.2 0.2 μM against these
cells. YM-01, which differs from MKT-077 only in the methyl
substitution in the R3 position, was only slightly weaker (EC50 values
of 2.0 0.2 and 5.2 0.8 μM, respectively) (Table 1). Consistent
with the literature, neither molecule was toxic to mouse embryonic
fibroblasts (MEFs) (EC50 > 50 μM) and both had short half-lives in
microsomes (t1/2 < 5 min). Introducing fluorine in the 3, 4, 5, or 6
positions of the benzothiazole (R1) of YM-01 (compounds 1−4)
significantly improved stability (t1/2 = 15, 23, 7.7, and 16 min,
respectively). However, these substitutions also reduced potency
(EC50 values between 2.2 and 18 μM). When the same fluorina-
tions were introduced in the context of the ethyl modification at the
R3 position (compounds 5−8), potency was only slightly improved
(EC50 between 0.8 and 13 μM). Replacing fluorines for chlorines in
the YM-01 scaffold (compounds 9−12) did not improve potency
and, in fact, further increased the EC50 values to between 4.1 and
28 μM. Similarly, placing trifluoromethyl or methoxy groups at posi-
tions 4 and 5 (compounds 13−16) decreased activity to between 3
and more than 30 μM (Table 1). Together, these results suggested
that small substitutions on the benzothiazole protected against
metabolism but that larger groups reduced antiproliferative activity.
B
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a
Scheme 1. Synthetic Route to MKT-077 and Analogues
a
(A) Chemical structures of MKT-077 and YM-01. (B) General synthetic route to analogues. (a) 1. Potassium ethyl xanthate, DMF, 4 h, 125 °C; 2.
methyl iodide, triethyl- amine, ethanol, 1 h, 80 °C; (b) methyl p-toluenesulfonate, anisole, 125 °C; (c) N-substituted rhodanine, triethylamine,
acetonitrile, 4 h, 25 °C; (d) methyl p-toluenesulfonate, DMF, 3 h, 135 °C; (e) 1. triethylamine, acetonitrile, 3 h, 80 °C, 2. Cl-ion exchange column.
Next, we made substitutions at the central rhodanine ring (R2)
and the N-substitutions of the pyridinium (R3). Replacing the
R2 ethyl group of MKT-077 with an allyl or benzyl group
(compounds 17 and 18) did not substantially affect activity
against either cell line. Likewise, replacing the ethyl moiety of the
pyridinium (R3) with a propyl group (compound 19) did not
significantly improve potency (EC50 values 2.8 0.8 and 1.6
0.2 μM against MCF7 and MDA-MB-231 cells, respectively).
However, increasing the size of this substituent to a benzyl group
(compound 20) improved activity by approximately 2-fold (EC50
values of 1.5 0.2 and 1.0 0.2 μM). The more hydrophilic 2-
hydroxyethyl (compound 21) decreased potency (EC50 values
6.9 2.1 and 24 3.3 μM), suggesting that hydrophobic groups
were favored in this position.
Changing the position of the heteroatom in the pyridinium
heterocycle (Hy) from the 2 to 4 position (compound 22) did
not significantly influence antiproliferative activity in MDA-
MB-231 cells (EC50 1.7 0.1 μM), while the activity was reduced
in MCF-7 cells (EC50 19 4.9 μM). Similarly, adding another
nitrogen to the ring (compound 23) reduced potency by
approximately 2-fold, while replacing the pyridinium with a
thiazolium (compound 24) improved the potency by 3-fold in
MCF-7 cells (EC50 0.7 0.1 μM) and slightly reduced activity in
MDA-MB-231 cells (EC50 5.1 0.3 μM). To test whether the
improvement in activity by the 2-thiazolyl group at R3 would be
Figure 1. Treatment with MKT-077 analogue modestly affects the
stability of chaperone clients and induces apoptosis. (a) Cells were
treated for 24 h with the indicated concentration of compound 27 (JG-
83). Results are representative of experiments performed in triplicate. (b)
Compound 30 (JG-98) incudes cleavage of caspase 3 and PARP in
MDA-MB-231 cells after 48 h. Results are representative of experiments
robust, we also generated analogues with this modification in the
context of ethyl and benzyl substitutions at R2 (compounds 25
and 26, respectively). These compounds followed the structure−
activity trends of the earlier molecules, with the activity of
compound 26 improved by 2-fold in MDA-MB-232 cells and
4-fold in MCF7 cells, with EC50 of 0.5 0.1 and 0.6 0.04 μM,
respectively.
Together, these results suggested that a modest increase in
antiproliferative potency might be gained by switching the
pyridinium for a thiazolium. The results also suggested that
appending a benzyl moiety to this ring might further promote
performed in duplicate. (c) Compound 30 (JG-98) induces oligomeriza-
tion and reduction of p62 in MDA-MB-231 and MCF7 cells after 24 h.
Results are representative of experiments performed in duplicate.
activity. To test these ideas, we combined the thiazolium and
benzyl modifications with halogen replacements on the
benzothiazole ring (compounds 27−31). Each of these
compounds had significantly improved potency compared to
MKT-077, with EC50 values between 1.0 and 0.4 μM (Table 1).
For example, the compound that combines a 3-F group with a
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Figure 2. Biotinylated MKT-077 analogues bind Hsc70. (A) Synthetic route to biotinylated MKT-077 analogues. (B) Biotinylated versions of YM-01
(33) and JG-83 (32) bind human Hsc70 with a similar affinity, as judged by ELISA. Results are the average of at least three experiments performed in
duplicate. The error is SEM. (C) Binding affinities (KD) of compounds 33−45 to Hsc70 by ELISA. See the Supporting Information for details.
benzyl substituted 2-thiazolyl (compound 27; JG-83) was 2-fold
more potent against MCF7 cells (EC50 = 1.0 0.2 μM) and
of Hsp70 and Hsp90 were not elevated by JG-83 in either cell
line (Figure 1a), confirming that MKT-077 analogues do not
elevate a stress response.14 To probe whether these compounds
could induce apoptosis, we treated MDA-MB-231 cells with
JG-98 and measured cleavage of caspase-3 and PARP after 48 h.
Both apoptotic mediators were activated (Figure 1b), suggesting
that JG-98 triggers apoptosis. Finally, we examined whether
JG-98 had an effect on autophagy. Treatment of both MDA-
MB-231 and MCF7 cells with JG-98 strongly affected autophagic
flux, as indicated by an increase in p62 oligomerization and a reduc-
tion in p62 monomer (Figure 1c). Together, these results support
the idea that Hsp70 is a hub for multiple cell survival pathways.1
Next, we wanted to explore whether the MKT-077 derivatives
retained binding to Hsc70 in vitro. To test this idea, we syn-
thesized a biotinylated version of JG-83 (compound 32), using a
modification of the previous synthetic route (Figure 2A). Using
an ELISA,14 the affinity (KD) of 32 for purified, human Hsc70
more than 3-fold improved against MDA-MB-231 cells (EC50
=
0.4 0.03 μM). Likewise, the equivalent compound with a 5-Cl
substitution (compound 30; JG-98) had a potency of 0.4
0.03 μM against MDA-MB-231 cells and an EC50 value of 0.7
0.2 μM for MCF7 cells. Some of these compounds, including JG-
83 and JG-98, had increased activity against MEFs compared to
MKT-077, but the selectivity for cancer cells was still estimated
to be between 8- and 20-fold. Importantly, JG-98 had a micro-
somal lifetime of 37 min, which is at least 7-fold better than
MKT-077 or YM-01.
To explore whether these compounds affected the stability of
known Hsp70 clients, we treated MDA-MB-231 and MCF-7
cells with JG-83 for 24 h and then performed Western blots for
p53, Akt1, and Raf1.24 We found that Akt1 and Raf1 levels were
reduced ∼25% in MDA-MB-231 and MCF7 cells (Figure 1a).
The magnitude of the loss in pro-survival clients is not as
dramatic as is commonly observed for inhibitors of Hsp90.25
It is possible that other clients are more dramatically affected.
For example, in the MCF7 cells, the levels of mutated p5326 were
also significantly elevated at 5 and 10 μM of JG-83, suggesting
that reactivation of the tumor suppressor might be a relevant
mechanism in this cell line. Importantly, we found that the levels
was calculated to be 2.4
0.4 μM (Figure 2B), which was
indistinguishable from the affinity of Hsc70 for a biotinylated
version of YM-01 (KD = 2.0 0.2 μM). Thus, the 3-fold stronger
antiproliferative activity of JG-83 did not seem to arise from
tighter Hsc70 binding in vitro. To explore this relationship
further, we generated biotinylated versions of an additional
subset of MKT-077 analogues (compounds 33−45) (Figure 2A)
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Figure 3. JG-98 binds to a conserved, allosteric site on an Hsp70 family member. (A) Titration of JG-98 into 15N DnaK1−338 revealed chemical shifts,
with a strong cluster in a deep pocket near F148. (B) JG-98 is predicted to bind the same allosteric pocket that accommodates MKT-077. The docked
configuration of MKT-077 is shown (see text). (B) Alignment of DnaK and human Hsp70 family members, highlighting the cluster of residues sensitive
to treatment with JG-98 (bold). Invariant residues are shown in red, conserved residues in orange, and nonconserved residues in white. Note that there is
also high conservation outside the sensitive residues.
and measured their affinities for Hsc70 in the ELISA format.14
The results suggested that modifications at the 4′, 5′, and 6′
positions (compounds 35−37 and 39-41) were generally well
tolerated (KD values between 1.5 and 4.0 μM) and that fluorine
or chlorine at the 3′ position (compounds 34 and 38) only
modestly weakened affinity (KD = 10.9 4.4 and 7.7 1.0 μM,
respectively) (Figure 2C). Thus, Hsc70 binding affinity alone
was not a good predictor of potency in MTT assays, possibly due
to differences in allosteric efficiency27 and/or differences in
cellular permeability.
To further explore binding to Hsp70, JG-98 was titrated into
an aqueous solution of 15N-labeled DnaK nucleotide binding
domain (DnaK1−388) and its interactions with the protein
monitored by NMR. We used DnaK, a prokaryotic orthologue of
Hsp70, in these studies because of its good behavior in solution
and the availability of a large number of NMR resonance
assignments.28 Titrating JG-98 (200 μM) into the nucleotide-
binding domain of 1H−15N DnaK1−388 (160 μM) showed strong
chemical shifts (ΔHz > 30) in the TROSY-HSQC experiment.
Strong shifts appeared in a deep, hydrophobic pocket formed by
Ile142, Phe148, and other residues (Figure 3A), and additional
shifts were seen in nearby surface residues: Gly222, Asp223, and
Thr224. A similar region was previously implicated in the binding
of MKT-077 to Hsc7016 and an overlay of the two sets of
compound-sensitive residues suggested that MKT-077 and JG-
98 interact with the same site (Figure 3B). In addition, treatment
with JG-98 led to scattered chemical shifts in the IIA, IB, and IIB
subdomains of DnaK (see Figure 3A). The major sites perturbed
by JG-98 (residues I142, F148, D222, T223, and H224) are
highly conserved among prokaryotic and eukaryotic Hsp70s, and
they are nearly invariant in the major family members, such as
Hsc70 (HSPA8), Hsp72 (HSPA1A), and mtHsp70 (HSPA9)
(Figure 3C). Thus, compounds of this class will likely have
affinity for nearly all of the family members, as previously
suggested.16
In conclusion, a library of MKT-077 derivatives was
synthesized and evaluated against two breast cancer cell lines.
Compounds such as 27 (JG-83) and 30 (JG-98) had improved
potencies against breast cancer cells and extended lifetimes in
liver microsome studies. Further, JG-98 bound to a conserved,
allosteric site on Hsp70. These studies advance the rhodacya-
nines as potential anticancer agents and improve their utility as
chemical probes for studying Hsp70s roles in cancer.
ASSOCIATED CONTENT
* Supporting Information
Compound characterization and methods for syntheses and
biological studies. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*(J.E.G.) E-mail: jason.gestwicki@ucsf.edu. Tel: (415) 502
7121.
■
Author Contributions
X.L., S.R.S., J.C., A.A., Z.T.Y., and E.R.P.Z. performed experi-
ments. All authors contributed to interpreting the results and
preparing the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from the NIH (NS095690)
and the University of Michigan’s Comprehensive Cancer Center
(CA046592). Z.T.Y. was supported by an NIH Training Grant
(GM007767) and a Rackham Merit Fellowship.
ABBREVIATIONS
■
Hsp70, heat shock protein 70; MEF, mouse embryonic
fibroblast; ELISA, enzyme-linked immunosorbent assay; PARP,
poly ADP ribose polymerase
E
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ACS Medicinal Chemistry Letters
Letter
(18) Wadhwa, R.; Colgin, L.; Yaguchi, T.; Taira, K.; Reddel, R. R.; Kaul,
S. C. Rhodacyanine dye MKT-077 inhibits in vitro telomerase assay but
has no detectable effects on telomerase activity in vivo. Cancer Res. 2002,
62, 4434−4438.
(19) Didelot, C.; Lanneau, D.; Brunet, M.; Joly, A.-L.; De Thonel, A.;
Chiosis, G.; Garrido, C. Anti-cancer therapeutic approaches based on
intracellular and extracellular heat shock proteins. Curr. Med. Chem.
2007, 14, 2839−2847.
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