Anion-triggered substituent-dependent conformational switching of salicylanilides. New hints for understanding the inhibitory mechanism of salicylanilides

Article abstract of DOI:10.1021/jo701823d

(Chemical Equation Presented) A series of salicylanilides (1a-h) bearing varied substituents at the 3′- or 4′-position of the anilino moiety (substituent = p-OCH₃, p-CH₃, m-CH₃, H, p-Cl, m-Cl, p-CO₂CH₃, and p-CN) were synthesized. In acetonitrile all of the substituted salicylanilides 1a-h predominantly adopt the "closed-ring" conformation facilitated by a strong intramolecular OH...O=C hydrogen bond. In the presence of H₂PO₄ ^-, the conformation of 1a-h was found to be modulated by the substituent. With our proposed proton-transfer fluorescence probing method, we were able to show that the conformation of 1a-f bearing a not highly electron-withdrawing substituent was switched to the "open-ring" form by H₂PO₄^-, whereas 1h bearing a highly electron-withdrawing substituent, p-CN, remained in the "closed-ring" conformation. The significance of these findings for understanding, from a molecular structural point of view, the mechanism of salicylanilide-based inhibitors for inhibiting the protein tyrosine kinase epidermal growth factor receptor was discussed.

Full text of DOI:10.1021/jo701823d

Anion-Triggered Substituent-Dependent Conformational Switching
of Salicylanilides. New Hints for Understanding the Inhibitory
Mechanism of Salicylanilides
Lin Guo, Qiang-Li Wang, Qian-Qian Jiang, Qiu-Ju Jiang, and Yun-Bao Jiang*
Department of Chemistry, College of Chemistry and Chemical Engineering, and The MOE Key Laboratory
of Analytical Sciences, Xiamen UniVersity, Xiamen 361005, China
ybjiang@xmu.edu.cn
ReceiVed August 19, 2007
A series of salicylanilides (1a-h) bearing varied substituents at the 3′- or 4′-position of the anilino
moiety (substituent ) p-OCH3, p-CH3, m-CH3, H, p-Cl, m-Cl, p-CO2CH3, and p-CN) were synthesized.
In acetonitrile all of the substituted salicylanilides 1a-h predominantly adopt the “closed-ring”
-
conformation facilitated by a strong intramolecular OH‚‚‚OdC hydrogen bond. In the presence of H2PO4 ,
the conformation of 1a-h was found to be modulated by the substituent. With our proposed proton-
transfer fluorescence probing method, we were able to show that the conformation of 1a-f bearing a not
-
highly electron-withdrawing substituent was switched to the “open-ring” form by H2PO4 , whereas 1h
bearing a highly electron-withdrawing substituent, p-CN, remained in the “closed-ring” conformation.
The significance of these findings for understanding, from a molecular structural point of view, the
mechanism of salicylanilide-based inhibitors for inhibiting the protein tyrosine kinase epidermal growth
factor receptor was discussed.
Introduction
predominant and closely related to the inhibitory activities of
salicylanilides.1 Meanwhile, an electron-withdrawing substituent
at the anilino moiety appears to be required to promote the
inhibitory activity of salicylanilides.1 Recent calculations,
however, suggested that this would shift the conformational
equilibrium of salicylanilides to the open-ring side,2 not favor-
Salicylanilides have been the subject of intensive interest in
medicinal chemistry, due to their ability to serve as inhibitors
of the protein tyrosine kinase epidermal growth factor receptor
(EGFR PTK) relating to cancer, psoriasis, and restenosis.1 They
are generally suggested to compete with ATP for binding at
the catalytic domain of tyrosine kinase and stop the growth of
tumors.1a,b Salicylanilides exist in a conformational equilibrium
between the “closed-ring” and “open-ring” conformers, Scheme
1. The closed-ring conformation involving a strong intramo-
lecular OH‚‚‚OdC hydrogen bond has been proven to be
(1) (a) Kamatb, S.; Buolamwini, J. K. Med. Res. ReV. 2006, 26, 569-
594. (b) Liechti, C.; Se´quin, U.; Bold, G.; Furet, P.; Meyer, T.; Traxler, P.
Eur. J. Med. Chem. 2004, 39, 11-26. (c) Waisser, K.; Buresˇ, O.; Hol’y,
P.; Kunesˇ, J.; Oswald, R.; Jira´skova´, L.; Pour, M.; Klimesˇova´, V.; Kubicova´,
L.; Kaustova´, J. Arch. Pharm. Pharm. Med. Chem. 2003, 1, 53-71. (d)
Macielag, M. J.; Demers, J. P.; Fraga-Spano, S. A.; Hlasta, D. J.; Johnson,
S. G.; Kanojia, R. M.; Russell, R. K.; Sui, Z.-H.; Weidner-Wells, M. A.;
Werblood, H.; Foleno, B. D.; Goldschmidt, R. M.; Loeloff, M. J.; Webb,
G. C.; Barrett, J. F. J. Med. Chem. 1998, 41, 2939-2945.
* To whom correspondence should be addressed. Phone/fax: +86 592
2185662.
10.1021/jo701823d CCC: $37.00 © 2007 American Chemical Society
Published on Web 11/17/2007
J. Org. Chem. 2007, 72, 9947-9953
9947

Guo et al.
SCHEME 1. Conformational Equilibrium in Salicylanilide
able for their inhibitory functions. Although the exact equilib-
rium status is not yet clear, the role of the electron-withdrawing
substituent at the anilino moiety in the inhibitory activity of
salicylanilides remains to be clarified. Another issue untouched
is the possible interaction of salicylanilides with ATP, in addition
to their competition for binding sites in tyrosine kinase. In fact,
salicylanilides, containing conventional hydrogen-bonding donor
groups, phenolic OH and amido NH, are potent anion recep-
tors.3,4 Interaction of them with ATP is indeed expected, and
whether this interaction, if any, influences the conformation of
salicylanilides is unclear.
We thus carried out a systematic investigation of the
conformation in acetonitrile (CH3CN) of a series of salicyla-
nilides bearing substituent X at the 3′- or 4′-position of the
anilino moiety ranging from electron-donating p-OCH3 to highly
electron-withdrawing p-CN (1a-h, Figure 1) in the absence and
presence of H2PO4-, an alternative taken for ATP. Although
infrared (IR) and NMR have been widely employed useful
techniques for clarifying the conformation of amides and the
involved hydrogen bonds,5 we proposed a fluorescence method
for the case of salicylanilides, not only because of the higher
spectroscopy sensitivity and enlarged solvent availability, but
more specifically due to the intriguing photophysical properties
of salicylanilides. In the closed-ring conformer of salicylanilides,
the phenolic OH is intramolecularly hydrogen bonded to the
FIGURE 1. Chemical structures of salicylanilides 1a-h and methoxy
counterparts 2a-g.8a
carbonyl O atom, which could be signaled by the observation
of excited-state intramolecular proton transfer (ESIintraPT)
fluorescence. Phenolic OH in the open-ring conformer, on the
other hand, is not intramolecularly hydrogen-bonded, and
salicylanilides are hence prone to emit excited-state intermo-
lecular proton transfer (ESIinterPT) fluorescence, especially when
a proton acceptor such as an anion is present. As a consequence,
the conformation of salicylanilides could be probed by the
character of the proton-transfer fluorescence. Our investigations
showed fluorescence spectroscopy indeed worked well in this
regard and uncovered that H2PO4- could switch the conforma-
tion of salicylanilides from the closed-ring form to the open-
ring form, and this switching was subject to the electron-
withdrawing ability of substituent X.
Results and Discussion
Suezawa et al.5c in 2000 determined by NMR the conforma-
tion of unsubstituted salicylanilide 1d in CCl4 and suggested
that 1d adopted mainly the closed-ring conformation. It was
thus wondered whether the closed-ring conformation was still
(2) (a) Blanco, S. E.; Ferretti, F. H. Tetrahedron Lett. 2007, 48, 2577-
2581. (b) Palomar, J.; De Paz, J. L. G.; Catala´n, J. J. Phys. Chem. A 2000,
104, 6453-6463. (c) Catala´n, J.; Palomar, J.; De Paz, J. L. G. J. Phys.
Chem. A 1997, 101, 7914-7921.
1
predominant in more polar CH3CN, the solvent used here. H
NMR and NOESY spectra of 1d in CD3CN were recorded. The
phenolic OH signal was observed at 12.01 ppm, which is close
to 11.98 ppm in CCl4 but is shifted to far downfield compared
to that of non-hydrogen-bonded phenolic OH normally observed
at 4-6 ppm,5b indicating the OH‚‚‚OdC intramolecular hydro-
gen bonding in 1d. The NOESY spectrum shows obvious NOE
correlations of the amido NH proton with the aryl CH proton
at the 6- and 6′-positions (Figure 2), providing further evidence
for the preference of the closed-ring to open-ring conformation
in the equilibrium (Scheme 1). Correlation with the Hammett
constant of substituent X of the NMR signals of phenolic OH
and amido NH protons of 1a-h in CD3CN and DMSO-d6
(Figure 3) confirmed that indeed the phenolic OH proton was
involved in an intramolecular hydrogen bond while the amido
NH proton was exposed to solvent molecules to a much higher
extent, and all of them took the closed-ring conformation in
CH3CN and even in highly polar and hydrogen-bonding DMSO.
It was also found in Figure 3 that, with increasing electron-
withdrawing ability of X, the signal of the phenolic OH proton
shifted to high field, suggesting a weakening OH‚‚‚OdC
hydrogen bond. This is in agreement with our molecular
mechanic calculations which showed increasing OH‚‚‚OdC
hydrogen bond length (B3LYP/6-31G* level)6 and decreasing
hydrogen-bonding energy7 (Table 1). This means that the
conformational equilibrium of 1 (Scheme 1) is shifted more
toward the open-ring side when X becomes more electron-
withdrawing.2
(3) (a) Santacroce, P. V.; Davis, J. T.; Light, M. E.; Gale, P. A.; Iglesias-
Sanchez, J. C.; Prados, p.; Quesada, R. J. Am. Chem. Soc. 2007, 129, 1886-
1887. (b) Winstanley, K. J.; Smith, D. K. J. Org. Chem. 2007, 72, 2803-
2815. (c) Winstanley, K. J.; Sayer, A. M.; Smith, D. K. Org. Biomol. Chem.
2006, 4, 1760-1767. (d) Libra, E. R.; Scott, M. J. Chem. Commun. 2006,
1485-1487. (e) Ghosh, S.; Choudhury, A. R.; Guru Row, T. N.; Maitra,
U. Org. Lett. 2005, 7, 1441-1444. (f) Kondo, S.; Suzuki, T.; Toyama, T.;
Yano, Y. Bull. Chem. Soc. Jpn. 2005, 78, 1348-1350. (g) Smith, D. K.
Org. Biomol. Chem. 2003, 1, 3874-3877. (h) Zhang, X.; Guo, L.; Wu,
F.-Y.; Jiang, Y.-B. Org. Lett. 2003, 5, 2667-2670. (i) Kondo, S.; Suzuki,
T.; Yano, Y. Tetrahedron Lett. 2002, 43, 7059-7061. (j) Lee, D. H.; Lee,
K. H.; Hong, J. I. Org. Lett. 2001, 3, 5-8. (k) Lee, D. H.; Lee, H. Y.; Lee,
K. H.; Hong, J. I. Chem. Commun. 2001, 1188-1189. (l) Lee, K. H.; Lee,
H. Y.; Lee, D. H.; Hong, J. I. Tetrahedron Lett. 2001, 42, 5447-5449. (m)
Lee, C.; Lee, D. H.; Hong, J. I. Tetrahedron Lett. 2001, 42, 8665-8668.
(4) (a) Gale, P. A., Ed. Coordination Chemistry ReViews; Elsevier: New
York, 2006; Vol. 250. (b) Gale, P. A. Acc. Chem. Res. 2006, 39, 465-475.
(c) Kang, S. O.; Begum, R. A.; Bowman-James, K. Angew. Chem., Int.
Ed. 2006, 45, 7882-7894. (d) Mart´ınez-Ma´n˜ez, R.; Sanceno´n, F. Chem.
ReV. 2003, 103, 4419-4476. (e) Gale, P. A., Ed. Coordination Chemistry
ReViews; Elsevier: New York, 2003; Vol. 240. (f) Suksai, C.; Tuntulani,
T. Chem. Soc. ReV. 2003, 32, 192-202. (g) Beer, P. D.; Gale, P. A. Angew.
Chem., Int. Ed. 2001, 40, 486-516. (h) Wiskur, S. L.; Ait-Haddou, H.;
Lavigne, J. J.; Anslyn, E. V. Acc. Chem. Res. 2001, 34, 963-972.
(5) (a) Kanamori, D.; Okamura, T.-A.; Yamamoto, H.; Ueyama, N.
Angew. Chem., Int. Ed. 2005, 44, 969-972. (b) Kanamori, D.; Okamura,
T.-A.; Yamamoto, H.; Shimizu, S.; Tsujimoto, Y.; Ueyama, N. Bull. Chem.
Soc. Jpn. 2004, 77, 2057-2064. (c) Suezawa, H.; Hirota, M.; Yuzuri, T.;
Hamada, Y.; Takeuchi, I.; Sugiura, M. Bull. Chem. Soc. Jpn. 2000, 73,
2335-2339. (d) Suezawa, H.; Hirota, M.; Hamada, Y.; Takeuchi, I.;
Kawabe, T. Bull. Chem. Soc. Jpn. 1991, 64, 2863-2864. (e) Endo, H.;
Hirota, M.; Ito, Y.; Takeuchi, I.; Hamada, Y. Bull. Chem. Soc. Jpn. 1982,
55, 1564-1567.
9948 J. Org. Chem., Vol. 72, No. 26, 2007

Conformational Switching of Salicylanilides
the predominant closed-ring conformation in 1a-h remains
despite varying 3′- or 4′-substitution.
Note that the emission maximum of 1 slightly shifts to the
red with increasing electron-withdrawing ability of X (Figures
4b and 5), which is opposite to the intramolecular charge transfer
(ICT) emission of its methoxy counterpart 2 that shifts to the
blue.8a This provides additional support for the intramolecular
proton-transfer nature of the emissive state of 1a-h and hence
their closed-ring conformation.
In the presence of H2PO4-, interaction of 1 with H2PO4- was
expected that may influence the conformation of 1. The phenolic
OH proton of 1 in the closed-ring conformation is hydrogen-
bonded to carbonyl O, and therefore, only the amido NH and
aryl CH protons are available for binding H2PO4-. In contrast,
1 in the open-ring form seems to be more favorable for binding
H2PO4- because the phenolic OH is also available (Scheme 2).3b
To clarify the conformation 1a-h adopt in the presence of
-
H2PO4-, we first carried out 1H NMR titrations of 1d by H2PO4
in CD3CN.
Figure 6 shows that, upon addition of H2PO4-, the signal of
the amido NH proton is broadened and shifted downfield from
8.93 to 11.83 ppm whereas that of the phenolic OH proton
originally at 12.01 ppm rapidly disappears. Meanwhile, down-
field shifts in the NMR signals of CH(6) and CH(6′) protons
are also observed. These facts confirm hydrogen-bonding
FIGURE 2. NOESY spectrum of 1d in CD3CN.
interaction of 1d with H2PO4 .
- 11 1H NMR dilution experiments
on 1d in CD3CN carried out over a 1d concentration range of
0.1-20 mmol L-1 indicated hardly any change in the chemical
shifts of phenolic OH, amido NH, and aryl CH protons (Figures
S1 and S2, Supporting Information), excluding the dimerization
of 1d at 10 mmol L-1, the concentration employed for NMR
titration. Since the pKa of H3PO4 (2.16 in water9) is much lower
than that of 1d (7.68 in water10), deprotonation of 1d by H2PO4
-
was ruled out; otherwise, a signal of the amido NH proton at
ca. 16 ppm would have been observed5b and the signal of the
C-H(6) proton shifted upfield due to an increase in the electron
density in the salicyloyl ring via a through-bond mechanism.11
These, however, are still not enough to make a clear assignment
of the conformation of 1d, because similar spectral changes are
likely to be observed in both the closed-ring and open-ring
conformations upon their interactions with H2PO4-. In principle,
binding H2PO4- by 1d in the closed-ring conformation (Scheme
2) will result in not only downfield shifts of the amido NH and
aryl CH (6 and 6′) signals but also an increase in the electron
density at the carbonyl O atom and hence an enhancement in
the intramolecular OH‚‚‚OdC hydrogen bond. The latter would
bring about a downfield shift and even the disappearance of
the phenolic OH proton signal. Similarly, signals of phenolic
OH, amido NH, aryl CH(6′) and CH(6) protons of 1d in the
open-ring conformation could also be shifted downfield due to
FIGURE 3. Linear relationship against the Hammett constant of
substituent X in 1a-h of the chemical shifts of the OH and NH protons
in CD3CN and DMSO-d6. With 2, the methoxy counterpart of 1, in
DMSO-d6, δ(NH) ) 10.12 + 0.61σX.8a
Due to intramolecular OH‚‚‚OdC hydrogen bonding, sali-
cylanilide 1d, similar to salicylamide, exhibits an absorption
band at 310 nm and an ESIintraPT fluorescence at 470 nm in
CH3CN (Figure 4).8 With introduction of a substituent at the
3′- or 4′-position of the anilino moiety, the characteristic
absorption and fluorescence of salicylanilide at 310 and 470
nm were not much affected (Figures 4 and 5), indicating that
-
hydrogen bonding of 1d with H2PO4 and polarization of the
C-H(6) bond by a through-space effect.11
(6) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.;
Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A.
D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi,
M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.;
Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick,
D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.;
Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi,
I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.;
Peng, C. Y.; Nanavakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M.
W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.;
Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98, revision A.9;
Gaussian, Inc.: Pittsburgh, PA, 1998.
Absorption and fluorescence spectroscopies were then ap-
plied. Spectral traces given in Figure 7 for the case of 1d indeed
(8) (a) Zhang, X.; Guo, L.; Jiang, Y.-B. Acta Phys.-Chim. Sin. 2004,
20, 930-935. (b) Woolfe, G. J.; Thistlethwaite, P. J. J. Am. Chem. Soc.
1980, 102, 6917-6923. (c) Thistlethwaite, P. J.; Woolfe, G. J. Chem. Phys.
Lett. 1979, 63, 401-405.
(9) Lide, D. R., Ed. CRC Handbook of Chemistry and Physics, 85th ed.;
CRC Press: Boca Raton, FL, 2004; Section 8.
(10) Natarajan, A.; Sapre, V.; Hadkar, U. B.; Shirodkar, P. Y. Indian
Drugs 1992, 29, 545-552.
(11) Boiocchi, M.; Del Boca, L.; Go´mez, D. E.; Fabbrizzi, L.; Licchelli,
M.; Monzani, E. J. Am. Chem. Soc. 2004, 126, 16507-16514.
(7) Schaefer, T. J. Phys. Chem. 1975, 79, 1888-1890.
J. Org. Chem, Vol. 72, No. 26, 2007 9949

Guo et al.
-
TABLE 1. Structural and Spectral Parameters of 1a-h and 1‚H2PO4 Complexes and Binding Constants in CH3CN
absorption
fluorescence
E
HB,b kJ mol-1
λ, nm
λ, nm
1‚H2PO4
OH‚‚‚O,a
log K,c
log K,c
-
-
Å
CD3CN
DMSO-d6
1
1‚H2PO4
mol-1
L
1
mol-1
L
1a
1b
1c
1d
1e
1f
1.6776
1.6807
1.6804
1.6832
1.6885
1.6867
1.6911
1.6963
23.72
23.38
23.06
22.93
22.29
21.87
21.53
20.84
12.96
12.46
12.21
12.04
11.41
10.99
10.66
10.33
284/309
273/311
267/308
266/310
269/307
265/307
282/309
280/309
351
353
353
353
356
357
366
368
3.81 ( 0.01
3.81 ( 0.01
3.87 ( 0.01
3.85 ( 0.01
3.90 ( 0.02
4.13 ( 0.01
4.01 ( 0.01
4.24 ( 0.02
464
466
468
470
472
476
484
494
405
407
409
410
414
417
484
494
3.77 ( 0.01
3.91 ( 0.02
4.08 ( 0.01
4.15 ( 0.01
4.17 ( 0.02
4.41 ( 0.02
4.34 ( 0.02
4.58 ( 0.03
1g
1h
a OH‚‚‚OdC intramolecular hydrogen bond length calculated at the B3LYP/6-31G* level.6 b Hydrogen-bonding energy calculated in CD3CN and DMSO-
d6 from the difference in the chemical shifts of the phenolic OH proton in 1a-h and phenol (6.928 ppm in CD3CN and 9.32 ppm in DMSO-d6).7 c Binding
constant K obtained by nonlinear fitting assuming a 1:1 stoichiometry.12 Nice fittings support this stoichiometry, which is also confirmed by importing
absorption spectral data, for example, those in Figure 7a, into the Specfit/32 software (SPECFIT/32 Global Analysis System, v. 3.0, Spectrum Software
Associates, Malborough, MA), which only allows for a perfect fitting under 1:1 stoichiometry.
SCHEME 2. Conformational Equilibria of 1d in the
-
Presence of H2PO4
FIGURE 4. Absorption (a) and fluorescence (b) spectra of 1a-h in
CH3CN.
shifted to 410 nm, which is continuously enhanced with
-
increasing H2PO4 concentration. This new emission is blue-
shifted by 60 nm from the ESIintraPT fluorescence, excluding
the closed-ring conformation of 1d, since no enhancement was
observed in the ESIintraPT fluorescence that would have been
-
expected from binding of H2PO4 with 1d in the closed-ring
conformation (Scheme 2), which shall increase the electron
density of the carbonyl O atom and in turn facilitate ESIintraPT.
The open-ring conformation was therefore assumed as the
-
predominant conformation of 1d in the presence of H2PO4
.
1d in the open-ring conformation could form a hydrogen-
bonding complex with H2PO4- via its phenolic OH, amido NH,
and aryl CH protons (Scheme 2). Upon photoexcitation, the
acidity of phenolic OH proton is dramatically enhanced so that
it is transferred to H2PO4-, leading to the observed ESIinterPT
fluorescence of 1d (Figure 7b). To support this assumption, a
control experiment was carried out by using an organic base,
triethylamine (TEA; pKb ) 3.25 in water9), that is able to
deprotonate phenolic OH (pKa ) 7.68 in water10) and to result
in an ESIinterPT fluorescence emission.3h Spectral responses of
1d toward TEA in CH3CN (Figure S3, Supporting Information)
show a profile similar to that observed toward H2PO4-. This
supports the occurrence of ESIinterPT of 1d in the presence of
FIGURE 5. Linear correlation against the Hammett constant of
substituent X of absorption and emission energies of 1 and its complex
with H2PO4- in CH3CN. With 2, the methoxy counterpart of 1, hνflu
2.42 + 0.378σX.8a
)
point to the interaction of it with H2PO4-. The absorption
spectrum of 1d originally peaking at 266 nm splits, and a new
band at 353 nm appears. During this course two clear isosbestic
points at 290 and 327 nm are observed, establishing the
formation of a well-defined ground-state hydrogen-bonding
-
H2PO4 and the preference of the open-ring to closed-ring
conformation of 1d (Scheme 2). It is therefore made clear that
1
complex, as also suggested by the H NMR titrations. Mean-
the conformation of 1d is switched from the closed-ring to the
-
while, the fluorescence of 1d originally peaking at 470 nm is
open-ring in the presence of H2PO4
.
9950 J. Org. Chem., Vol. 72, No. 26, 2007

Conformational Switching of Salicylanilides
FIGURE 6. Portions of 1H NMR spectra of 1d in CD3CN in the presence of increasing equivalents of H2PO4- as an n-Bu4N+ salt. [1d] ) 10 mmol
L-1. For the numbering of the protons in 1d see Figure 1.
FIGURE 8. Absorption (a) and fluorescence (b) spectra of 1g (X )
p-CO2CH3) in CH3CN in the presence of increasing concentrations of
H2PO4-. [1g] ) 3.5 × 10-5 (a) and 1.8 × 10-5 (b) mol L-1. The
excitation wavelength employed to record the spectra in (b) was 287
nm, an isosbestic wavelength seen in (a).
FIGURE 7. Absorption (a) and fluorescence (b) spectra of salicyla-
nilide 1d in CH3CN in the presence of increasing concentrations of
H2PO4-. [1d] ) 2.0 × 10-5 (a) and 1.0 × 10-5 (b) mol L-1. The
excitation wavelength employed to record the spectra in (b) was 290
nm, an isosbestic wavelength seen in (a).
As absorption and fluorescence spectral variation profiles of
1a-f bearing a substituent from electron-donating p-OCH3 to
electron-withdrawing m-Cl (Figures 5 and S4-S8, Supporting
Information) are similar to those of 1d (Figure 7), it is concluded
that the conformation of 1a-f is switched as is that of 1d by
H2PO4-. Note in Figure 5 that the emission energy of 1a-f in
in the ESIintraPT fluorescence at 494 nm. Following analysis
previously given for 1d, 1h in the presence of H2PO4 was
-
concluded to remain predominantly in the closed-ring conforma-
-
tion (Scheme 3), opposite to 1d which was switched by H2PO4
to the open-ring conformation (Scheme 2).
Although the phenolic OH proton of 1a-f is strongly bonded
to the carbonyl O atom, they still take an open-ring conformation
in the presence of H2PO4-. It thus appears that the NH‚‚‚O
hydrogen bond between the amido NH proton and the phenolic
O atom in the open-ring conformation plays a more important
role in controlling the conformation of substituted salicylanilides.
In case the acidity of the NH proton is relatively low, this
NH‚‚‚O hydrogen bond would be weak.5c The amido NH proton
is relatively more accessible, which allows for a cooperation of
phenolic OH, aryl CH, and NH protons in their hydrogen
binding to H2PO4-. The conformational equilibrium of 1a-f
-
the presence of H2PO4 has a dependence on the Hammett
constant of X similar to that of the ESIintraPT fluorescence of
1a-f, again supporting the intermolecular proton-transfer
-
character of the emission of 1a-f in the presence of H2PO4
.
1g and 1h, substituted by p-CO2CH3 and p-CN of stronger
electron-withdrawing ability, however, displayed differing
-
spectral responses to H2PO4 (Figures 8 and 9). With 1g the
ESIinterPT fluorescence at 410 nm increased very weakly upon
adding H2PO4-, at the expense of the ESIintraPT fluorescence
at 484 nm. In the case of 1h, H2PO4- induced an enhancement
J. Org. Chem, Vol. 72, No. 26, 2007 9951

Guo et al.
in agreement with the recently reported calculations on C6H6-
anion complexes by Hay et al.13 It was revealed that simple
aryl CH protons form hydrogen bonds with anions that can be
as strong as over 50% of those formed by acidic OH and NH
protons. Moreover, when substituted by an electron-withdrawing
substituent, the aryl CH protons could become more powerful
hydrogen bond donors to form stronger hydrogen-bonding
complexes even than those from conventional OH and NH
protons, as observed here.
Conclusions
We showed that in CH3CN all of the substituted salicylanil-
ides 1a-h adopted predominantly the closed-ring conformation
involving a strong intramolecular OH‚‚‚OdC hydrogen bond.
In the presence of H2PO4-, however, the conformation of
salicylanilides 1a-h was subject to the substituent. The observed
switching of intramolecular to intermolecular proton-transfer
FIGURE 9. Absorption (a) and fluorescence (b) spectra of 1h (X )
p-CN) in CH3CN in the presence of increasing concentrations of
H2PO4-. [1h] ) 2.0 × 10-5 (a) and 1.0 × 10-5 (b) mol L-1. The
excitation wavelength employed to record the spectra in (b) was 283
nm, an isosbestic wavelength seen in (a).
-
fluorescence of 1a-f by H2PO4 directly pointed to the open-
ring conformation of them in the presence of H2PO4-. 1h
bearing a highly electron-withdrawing substituent, p-CN, how-
ever, was shown to remain in the closed-ring conformation in
the presence of H2PO4-. The observed enhancement in the
excited-state intramolecular instead of the intermolecular proton-
SCHEME 3. Conformational Equilibria of Salicylanilide 1h
-
in the Presence of H2PO4
-
transfer fluorescence of 1h in the presence of H2PO4 served
as a direct indication. The character of the proton-transfer
fluorescence, inter- or intramolecular, clearly probes the
conformation of salicylanilides, which also enables direct
fluorescence imaging of the conformations by strongly con-
trasted colors.
These conclusions would be of considerable significance for
understanding the inhibitory mechanism of salicylanilides. As
more hydrophobic salicylanilide is preferred in its action as an
inhibitor,1 the binding site in the catalytic domain shall be less
polar. One issue then that deserves attention is the occurrence
of interaction with ATP of the salicylanilide-based inhibitors,
in addition to their competition for binding to the catalytic
domain. This also helps to understand the experimental observa-
tions that the inhibitory activity of salicylanilides is positively
related to the electron-withdrawing ability of the substituent at
the anilino moiety.1 On the basis of our model investigations,
this substituent of higher electron-withdrawing ability facilitates
the closed-ring conformation of salicylanilides required to be
functional. Introduction of a strongly electron-withdrawing
substituent at the anilino ring is thus suggested, and it is even
better if hydrophobicity can also be enhanced by doing so.
with a weakly acidic amido NH proton is thus shifted to the
open-ring conformation (Scheme 2). With 1h, however, the
highly electron-withdrawing substituent p-CN at the anilino
phenyl ring enhances the acidity of the amido NH proton. The
NH‚‚‚O hydrogen bond in the open-ring conformation is
relatively strong so that the amido NH proton is now less
- 3b
accessible for H2PO4
.
The closed-ring conformation of 1h
Experimental Section
-
therefore remains preferable during its binding to H2PO4
(Scheme 3). The acidity of the amido NH proton in 1g is
between those of 1f and 1h, and its conformation in the presence
of H2PO4- could be reasonably assumed as a mixture of open-
ring and closed-ring conformations, as indeed suggested by its
fluorescence titration traces.
1H NMR and 13C NMR spectra were acquired on a 400 or 500
MHz NMR spectrometer in DMSO-d6 or CD3CN using TMS as
an internal standard. Absorption and fluorescence spectral titrations
for anion binding were carried out by adding an aliquot of anion
solution to a bulk salicylanilide solution at a given concentration.
Chemicals used for syntheses were commercially available, were
of AR grade, and were used as received. Solvents used for spectral
investigations were further purified by redistillations so that no
fluorescent impurity could be detected at the employed excitation
wavelength.
Binding constant analysis did show strong interaction of
-
H2PO4 with 1a-h in CH3CN. Data obtained from nonlinear
fitting assuming a 1:1 stoichiometry12 (Table 1) clearly indicate
that 1h exhibits a higher binding affinity toward H2PO4- despite
the participation in anion binding of the aryl CH instead of the
more acidic phenolic OH proton as in, for example, 1d. This is
Salicylanilides 1a-h are known compounds in the literature,1b,c
but synthesis procedures and characterization data are provided as
modified procedures were employed in this work.
(12) (a) Kato, R.; Nishizawa, S.; Hayashita, T.; Teramae, N. Tetrahedron
Lett. 2001, 42, 5053-5056. (b) Madrid, J. M.; Mendicuti, F. Appl. Spectrosc.
1997, 51, 1621-1627.
(13) (a) Bryantsev, V. S.; Hay, B. P. Org. Lett. 2005, 7, 5031-5034.
(b) Bryantsev, V. S.; Hay, B. P. J. Am. Chem. Soc. 2005, 127, 8282-8283.
9952 J. Org. Chem., Vol. 72, No. 26, 2007

Conformational Switching of Salicylanilides
General Procedure for Preparation of Salicylanilides 1a-h.
A mixture of salicylic acid (1.0 g, 7.0 mmol) and thionyl chloride
(2.5 mL, 35 mmol) was heated at 50 °C for 2 h. The excess SOCl2
was removed by distillation in vacuo at room temperature. The
residue was dissolved in dry CH2Cl2 and the solvent removed as
before; this procedure was repeated twice. The freshly formed
salicyloyl chloride dissolved in dry CH2Cl2 was added dropwise
to a solution of the substituted aniline, pyridine, and DMAP in dry
CH2Cl2. The resultant solution was stirred at room temperature for
2 h, and the solvent was removed. The residue was washed with 1
mol L-1 HCl and dissolved in 2 mol L-1 NaOH. The filtrate was
neutralized with 3 mol L-1 HCl, and a white solid was precipitated,
which was collected by filtration and purified by recrystallization
from CH3CN/H2O (9:1, v/v). 1a-h were fully characterized, and
detailed spectral data are supplied in the Supporting Information.
Acknowledgment. This work was supported by the NSF of
China though Grants 20425518 and 20675069.
Supporting Information Available: NMR dilution experiment
data of 1d in CD3CN, fluorescence spectra of 1d in CH3CN in the
presence of triethylamine, absorption and fluorescence spectra of
1a-f in CH3CN in the presence of H2PO4-, characterization data,
and 1H NMR and 13C NMR spectra of 1a-h. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO701823D
J. Org. Chem, Vol. 72, No. 26, 2007 9953