Sterically congested quinone methides in photodehydration reactions of 4-hydroxybiphenyl derivatives and investigation of their antiproliferative activity

Article abstract of DOI:10.1039/c1pp05182b

In aqueous media, photochemical excitation (S₁) of hydroxyadamantyl, diphenylhydroxymethyl, and hydroxypropyl derivatives of 4-phenylphenol 5-9 leads to solvent-assisted deprotonation of the phenol OH, and protonation of the benzyl alcohol coupled with dehydration, that delivers quinone methides (QMs) 14-18. The QMs react with CH₃OH converting them in high quantum yields to the photosolvolysis products (overall Φ ～ 0.1-0.5). QMs were characterized by laser flash photolysis in CH ₃CN-H₂O and TFE. In TFE, the zwitterionic QM 15 has a lifetime of 250 ns, whereas para QMs 16 and 17 have lifetimes of 500 μs and 1.1 s, respectively. Introduction of the steric hindrance to the parent QM structure (with the adamantyl moiety), or additional stabilization by two phenyl rings, results in an increase of QM lifetimes and selectivity in the reactions with nucleophiles. In vitro studies of the antiproliferative activity of photochemically generated QMs 15-17 were carried out on one human cancer cell line. Irradiation of cells incubated with 7 showed enhanced antiproliferative activity compared to cells that were not irradiated, in accordance with the activity being due to the formation of QM 16.

Full text of DOI:10.1039/c1pp05182b

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PAPER
Sterically congested quinone methides in photodehydration reactions of
4-hydroxybiphenyl derivatives and investigation of their antiproliferative
activity †
Nikola Basaric´,*a Nikola Cindro,a Damir Bobinac,a Kata Mlinaric´-Majerski,a Lidija Uzelac,b Marijeta Kraljb
and Peter Wan*c
Received 15th June 2011, Accepted 19th September 2011
DOI: 10.1039/c1pp05182b
In aqueous media, photochemical excitation (S1) of hydroxyadamantyl, diphenylhydroxymethyl, and
hydroxypropyl derivatives of 4-phenylphenol 5–9 leads to solvent-assisted deprotonation of the phenol
OH, and protonation of the benzyl alcohol coupled with dehydration, that delivers quinone methides
(QMs) 14–18. The QMs react with CH3OH converting them in high quantum yields to the
photosolvolysis products (overall U ~ 0.1–0.5). QMs were characterized by laser flash photolysis in
CH3CN–H2O and TFE. In TFE, the zwitterionic QM 15 has a lifetime of 250 ns, whereas para QMs 16
and 17 have lifetimes of 500 ms and 1.1 s, respectively. Introduction of the steric hindrance to the parent
QM structure (with the adamantyl moiety), or additional stabilization by two phenyl rings, results in an
increase of QM lifetimes and selectivity in the reactions with nucleophiles. In vitro studies of the
antiproliferative activity of photochemically generated QMs 15–17 were carried out on one human
cancer cell line. Irradiation of cells incubated with 7 showed enhanced antiproliferative activity
compared to cells that were not irradiated, in accordance with the activity being due to the formation of
QM 16.
QMs can be generated in thermal reactions by oxidation
of phenols,7 extrusion of water and other small molecules
Introduction
from hydroxybenzyl alcohols,8 and fluoride induced desilylation.9
More appealing methods, however, rely on the use of milder
approaches,10 primarily photochemical.11 QMs can be formed
in photochemical reactions from suitably substituted phenols
with appropriate benzylic moieties, such as benzyl alcohols,12
benzylacetates,13 or benzylamines.2,14
Quinone methides (QM) are important intermediates in chemistry
and biology.1 They have attracted considerable interest in the
last decades because of their biological activity. It has been
demonstrated that QMs react with amino acids2 and DNA.3
Particularly interesting is the ability of QMs to alkylate and cross-
link DNA,4 thus making them potent agents in cancer therapy.
For example, the antiproliferative activity of some antibiotics is
based on metabolic formation of QMs and subsequent DNA
alkylation.5 Moreover, Freccero et al. have recently investigated
DNA alkylation ability of QMs derived from BINOL–amino acid
conjugates and the antiproliferative effect of some photogenerated
simple BINOL QMs.6
Recently we reported on the photochemical formation of
long-lived QMs in these systems that were further substituted
by bulky polycyclic adamantyl groups.15 The bulkiness of the
adamantyl polycycle sterically hindered attack of nucleophiles to
the methylene position of the QMs, making them longer-lived
and more selective towards N-nucleophiles, which is a highly
desirable property for use in biological (aqueous) systems as a
DNA alkylating agent. The other approach to achieve selectivity
in targeting biological nucleophiles, including DNA, is by conjuga-
tion of the QM to a selective ligand, such as naphthalene diimide.16
In continuation of the research we prepared a series of adamantyl-
substituted 2-hydroxybiphenyls wherein we observed competition
of two photochemical pathways from their singlet excited state.17
For example, due to an increased acidity of the phenolic OH in
the singlet excited state, 2-hydroxybiphenyl derivative 1 underwent
ESIPT (excited state intramolecular proton transfer), and solvent-
assisted dehydration, both processes giving rise to QMs. QM
2 that was formed by dehydration, reacted with nucleophiles
aDepartment of Organic Chemistry and Biochemistry, Ruąer Bosˇkovic´ Insti-
tute, Bijenicˇka cesta 54, 10 000, Zagreb, Croatia. E-mail: nbasaric@irb.hr;
Fax: 385 1 4680 195; Tel: 385 12 4561 141
bDepartment of Molecular Medicine, Ruąer Bosˇkovic´ Institute, Bijenicˇka
cesta 54, 10 000, Zagreb, Croatia
cDepartment of Chemistry, Box 3065, University of Victoria, Victoria, BC,
V8W 3V6, Canada. E-mail: pwan@uvic.ca; Fax: + 1 (250) 721-7147; Tel: +
1 (250) 721-8976
† Electronic supplementary information (ESI) available: Experimental
procedures for the preparation of compounds, UV-vis and fluorescence
spectra of compounds 5–9, transient absorption spectra obtained by LFP
1
of 5–10, and H and 13C NMR spectra of all prepared compounds. See
DOI: 10.1039/c1pp05182b
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Scheme 1
giving adducts 4, whereas short-lived QM 3 formed by ESIPT,
Results and discussion
rapidly underwent tautomerization to the starting compound 1
(Scheme 1). QM 2 was detected by laser flash photolysis, giving
a transient with a maximum at 430 nm and a band stretching
over longer wavelengths, decaying within 1–6 ms. QM 3 could
not be detected by LFP, and its formation was indicated due
to the regiospecific incorporation of deuterium on photolysis in
CH3CN–D2O.17
Synthesis
The 4-hydroxybiphenyls 5–9 were prepared by modifying
a published procedure.15 The synthetic sequence involved a
Suzuki reaction for the preparation of bromo-substituted 4-
methoxybiphenyls, which were transformed by Grignard reactions
to the corresponding methoxy derivatives 5¢–9¢ (Schemes 2–4).
Subsequent reaction with a Lewis acid, BBr3, gave rise to 6–8
and 11, whereas 5 and 9 were prepared under basic conditions
With the aim of finding precursors of QMs that could be used
in biological systems, we report herein on the photochemistry of
sterically congested 4-hydroxybiphenyl derivatives 5–9, wherein
the above mentioned ESIPT reaction is impossible, and compare
their reactivity with the parent 4-hydroxybiphenyl 10. On excita-
tion, the investigated compounds are expected to undergo only
photosolvolysis, giving rise to QMs that would be long-lived and
selective in reactions with nucleophiles. Therefore, we performed
preparative photolyses in the nucleophilic solvent (CH3OH) and
carried out characterization of the compounds by fluorescence
spectroscopy and laser flash photolysis. We also investigated if
solvent assisted photochemical deuterium incorporation takes
place in the biphenyls 6 and 7, and fluorene 11, to probe for
the possibility of solvent assisted formal proton transfer from
the phenol to the carbon atoms that would decrease quantum
yields in photodehydration reactions. In addition, we performed
a preliminary in vitro antiproliferative study with the photochem-
ically generated QMs on a colon cancer cell line HCT 116 and
showed that the irradiation of cells incubated with 7 leads to an en-
hanced antiproliferative effect compared to cells that were kept in
dark.
Scheme 2
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was cleaner and more efficient in aqueous mixtures containing
more than 10% H2O (see supporting info†), in agreement with
the above described irradiations in UV-vis cuvettes and a higher
probability of transferring a proton from the excited phenol to the
clusters of H2O than CH3OH.20 In aqueous CH3OH solutions the
photolysis of 6–8 gave only photomethanolysis products 6OMe–
8OMe, respectively (eqn (1) and (2)), that were formed in high
yields upon longer irradiation times (16 lamps, 15 min–1 h, yields
50–100%). On the other hand, the dimethyl derivative 9 gave
two products, the methanolysis product 9OMe and the vinyl
derivative 12, in a ratio 1 : 1. Irradiation of 5 gave fluorene 11
and, surprisingly, underwent phototransposition to furnish the
meta derivative 6 (eqn (4)), that was, on prolonged irradiation,
converted to the methoxy derivative 6OMe. Since fluorene 11 and
the meta derivative 6 absorb light at longer wavelengths than 5,
the analytical UV-vis experiments indicated their formation (vide
supra). The photochemical products were isolated after photolyses
by preparative TLC or crystallization wherein they were separated
from the unreacted starting compounds and some unidentified
high molecular weight material.
Scheme 3
(1)
(2)
Scheme 4
by cleavage of the ether by sodium thiolate according to the
modification of a published procedure.18
Preparative irradiations
The photochemical reactivity of biphenyls 5–8 and their ability to
form stable products was first analyzed by irradiations in CH3CN
or CH3OH–H2O (1 : 1) solutions at 254 nm in UV-vis cuvettes.
After each short exposure to light, the UV-vis spectra were taken.
The experiments for the ortho derivative 5 indicated formation of
a new species that had absorption maxima at longer wavelengths,
and the conversion was more efficient in the presence of H2O
(see supporting info†). On the other hand, experiments with 6–
8 indicated no change at shorter irradiation times, whereas on
prolonged irradiation disappearance of the absorption bands at
270 nm and formation of broader bands were observed, indicating
decomposition. However, the decomposition process was much
slower in the presence of H2O (For example of 7, see the supporting
info†).
To probe for the photodehydration reactions that would give
QMs from 5–9, we performed irradiations in the presence of the
nucleophilic solvent CH3OH, wherein photosolvolysis (viz. substi-
tution of the hydroxyl group by methoxy, or photomethanolysis)
would be an indication of the QM formation, as shown in previous
reports.12,15,17,19 The preparative irradiations were performed at
254 nm in CH3OH and CH3OH–H2O solutions with different
H2O content. The course of the photochemical reaction was
followed by HPLC. Control experiments were performed where
the solutions were kept in the dark to check for the possibility
of the thermal methanolysis reactions. Thermal methanolysis was
observed for 7; however, it was significantly slower compared to
the photochemical reaction. Conversion to the photoproducts
(3)
(4)
Formation of the methanolysis products from 6–9 suggested the
presence of QM intermediates in their photochemistry. However,
the photomethanolysis can in principle also take place via
cationic intermediates. In that case, the photolysis of the methoxy
derivatives 6¢–9¢ would also give rise to the methanolysis products.
Hence, we photolyzed 6¢–9¢ to check if the methanolysis products
can be formed with similar quantum efficiencies. However, irra-
diations under the same conditions as for 6–9 gave no observable
methanolysis products, only the starting materials were recovered.
Prolonged irradiations, on the other hand, gave rise to the traces
of the photomethanolysis products (according to NMR of the
crude irradiation mixtures) that were formed in significantly lower
quantum yields.
According to previous reports, the QMs are formed in a solvent-
assisted proton transfer in the singlet excited state from the acidic
phenolic OH to the benzyl alcohol, coupled by dehydration.12,15,17,19
Hence, the efficiency of QM formation and the subsequent
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Table 1 Quantum yields of the photomethanolysis reactiona
to replace the phenolic OD by OH and the extractions with
CH2Cl2 were carried out. The photolysis mixtures were analyzed
by NMR and MS to investigate the position and the extent
of deuterium incorporation, respectively. Photolysis of 6 and 7
gave rise to deuterium incorporation at the biphenyl moiety (MS
indicated 10% of the deuterated species), whereas no deuterium
incorporation was observed on photolysis of fluorene 11. However,
deuterium was not incorporated regiospecifically, as in the case of
2-hydroxybiphenyl. The NMR spectra indicated that deuterium
mostly entered at the phenol ring (not at the adjacent phenyl
ring, Scheme 5), in agreement with some minor deuteration
pathways (presumably via radicals or radical-cations) and not
via the excited state proton transfer. The non-observation of
the regiospecific deuterium incorporation in the adjacent phenyl
ring is in agreement with the lower acidity of 4-phenylphenols
(compared to 2-phenylphenols) and the lower extent of charge
transfer from the phenol to the adjacent ring in the singlet excited
state.24
Comp.
U
5
0.13 0.03b
0.4 0.1
0.11 0.03
0.5 0.2
0.05 0.03
0.026c
6
7
8
9
10
a Photolysis performed in CH3OH–H2O (1 : 1) by use of two actinometers:
photolysis of 2-hydroxybenzyl alcohol in CH3OH–H2O (U = 0.23),12 and
photolysis of valerophenone in aqueous CH3CN (U = 0.65 0.03).21 The
composition of the solution was analyzed by HPLC. b Quantum yield for
the formation of 11 and 6OMe. c Taken from the literature.19b
photomethanolysis should depend on the pH of the solution.19a
Therefore, we performed irradiations of 6 and 7 in CH3OH–H2O
(20%) at different pH (2.4–10.1) and analyzed the composition of
the solution by HPLC (see supporting info†). The change of the
solution pH in the range 4–8 had no effect to the efficiency of the
formation of 6OMe and 7OMe, whereas at pH 10, some weak
base catalysis was observed. Interestingly, at pH 2.4, a significant
acid catalysis was observed for the formation of 7OMe, whereas
acid had no effect to the formation of 6OMe. The observation
parallels the reactivity of the anticipated corresponding QMs,
and is in accordance with the pH-dependant reactivity of QMs
in hydration reactions.13
(5)
Quantum yields for the photochemical transformations of 5–9
were measured by use of two actinometers: photomethanolysis
of 2-hydroxybenzyl alcohol in CH3OH–H2O (U = 0.23),12 and
by use of a primary actinometer, photolysis of valerophenone
giving acetophenone in aqueous CH3CN (U = 0.65 0.03).21
The absorbances of the solutions were measured prior to irra-
diations, whereas the composition of the irradiated solution was
analyzed by HPLC. Measurements were performed in triplicate
and the mean values are reported (Table 1). The efficiency of
the photomethanolysis of the biphenyl derivative 8 was very
much dependant on the CH3OH supplier. Hence, the measured
quantum yield was difficult to reproduce and the reported value
contains a large error. As can be seen from the data, substitution
of the parent molecule with methyl groups increased quantum
yield of the methanolysis 2 times, whereas incorporation of the
adamantyl moiety increased quantum yield 5 times. The observed
quantum yields of the photomethanolysis are also in agreement
with the well-known meta effect in photochemistry,22 since the
meta derivative 6 shows an about 4 times higher quantum yield
than the corresponding para derivative 7.
Irradiation of 2-phenylphenol (13) in the presence of a large
amount of D2O gives rise to the ESIPT from the phenol and
deuterium incorporation at the carbon atom at the 2¢ (ortho)
position of the adjacent phenyl ring, as well as to a solvent assisted
ESPT and deuterium incorporation at the 4¢ (para) position of
the biphenyl (giving 13-D, eqn (5)).23 In the biphenyl systems
5–9 the ESIPT from the phenol to the adjacent phenyl ring is
not possible since the phenol is not in proximity to the adjacent
phenyl ring. However, the question remains if the ESPT assisted
by solvent could lead to deuterium incorporation, as is the case
in the parent system at the 4¢ position. Therefore, we carried
out photolyses (16 lamps, 1 h) of 6, 7 and 11 in CH3CN–
D2O (10%). After photolyses, a large excess of H2O was added
Scheme 5
Fluorescence study
It is well-accepted that proton transfer reactions and formation
of QMs from phenols take place from the singlet excited state.12,19
Hence, we studied the fluorescence of 5–9 to get more insight
into their singlet state reactivity. In CH3CN solutions, 6–8 showed
strong fluorescence with maxima at ~330 nm, whereas the ortho
derivative 5 was very weakly fluorescent. Fluorescence quantum
yields were measured by using fluorescence of fluorene in methanol
as a reference (U = 0.68).25 Lifetimes of the singlet states were
measured by time-correlated single photon timing method. The
results are compiled in Table 2.
The reported photomethanolysis reaction mechanism involves
solvent assisted deprotonation of the phenols due to increased
acidity in the singlet excited state, and protonation of the benzyl
alcohols coupled by dehydration giving QMs.12,15,17,19 Increase of
the H2O content in the CH3CN solutions of the hydroxybiphenyls
5–9 is thus expected to open a new photochemical pathway, the
transfer of the phenolic OH proton to the H2O clusters, that should
give rise to the fluorescence quenching.26 Therefore, in the series
of biphenyls 5–9 we probed for the possibility of solvent assisted
proton transfer by titrating CH3CN solutions with H2O. However,
the initial additions of H2O to the CH3CN solutions (up to 10 M)
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Table 2 Photophysical properties of 4-hydroxybiphenyls 5–9
U
U
t
(CH3CN)a (CH3CN-H2O)a t (CH3CN)/nsb (CH3CN-H2O)/nsb
5
6
7
8
9
~8 ¥ 10-4
~7 ¥ 10-4
0.2 0.1 (13%) 0.2 0.1 (13%)
7.9 0.1 (77%) 7.9 0.1 (77%)
0.22 0.02 0.18 0.01
0.39 0.02 0.44 0.01
0.35 0.02 0.34 0.02
0.36 0.02 0.30 0.02
8.10 0.05
7.75 0.05
7.09 0.01
10.2 0.1
4.5 0.1 phenol
0.4 0.1 phenolate
4.93 0.03 phenol
0.9 0.1 phenolate
2.02 0.02 phenol
0.2 0.1 phenolate
6.1 0.1 phenol
0.6 0.1 phenolate
a Quantum yield of fluorescence determined by use of fluorene in CH3OH
as a reference (U = 0.68).25 b Singlet excited state lifetimes measured by
time-correlated single photon timing method.
Fig. 1 Normalized fluorescence spectra of 7 in CH3CN (full line) and
CH3CN–H2O (1 : 4) (dashed line), lex = 265 nm.
resulted in a weak fluorescence increase, that with further H2O
additions (up to 20 M) leveled off (see the supporting info†). Thus,
in CH3CN–H2O solution (1 : 4), fluorescence quantum yields of
5–9 were comparable to the yields in neat CH3CN (Table 2). The
increase of the fluorescence on addition of H2O has already been
reported for the adamantyl phenols, that is probably due to the
unusual solvation of the very lipophilic admantyl molecules in
aqueous media.15 However, comparison of the fluorescence spectra
of 5–9 in CH3CN and CH3CN–H2O clearly showed a presence
of a new shoulder at longer wavelength in the aqueous solvent
(for the example of 7, see Fig. 1). The new band appearing as a
shoulder can be attributed to the fluorescence of phenolates that
were formed in the singlet excited state due to a proton transfer to
the solvent. Fluorescence spectra of 6–9 in basic media (pH > 10)
where phenols are deprotonated showed the presence of the same
band emitting at longer wavelengths, additionally corroborating
the assignment to the phenolate emission (see Fig. 2 and the
supporting info†).
addition, a shorter-lived decaying component contributing more
at longer wavelengths was observed, that was formed during the
decay (negative pre-exponential factor), and was attributed to
phenolates. It is interesting to note that phenolates exhibit 10 times
shorter decay time compared to their corresponding phenols. The
finding indicates that phenolates deactivate from the singlet excited
state by a nonradiative pathway, presumably via a photochemical
pathway giving rise to QMs.
The acid–base properties of 6–8 in the aqueous solution in
the singlet excited state were further investigated by fluorescence
titrations with acid (HClO4) and base (NaOH). The titrations with
acid induced quenching of the fluorescence in the pH range 2.5–
3.0. The Stern–Volmer plots (U/U0 vs. H+ concentration) showed
linear relationship, indicating a dynamic quenching. The estimated
quenching constants for 6, 7 and 8 are kq = 2 ¥ 1010 M-1 s-1,
8.6 ¥ 109 M-1 s-1, and 3.9 ¥ 1010 M-1 s-1, respectively. It should be
mentioned that we observed the fluorescence quenching (without
a change of the shape of the fluorescence spectra), and not a
fluorescence increase, as would be expected if an increase of the
proton concentration blocked the deprotonation of the phenol in
the excited state. The finding suggests that the most basic site in
the molecule in the singlet excited state is not the phenolate but
some other site, presumably the benzyl alcohol moiety. Indeed, the
estimated pKa value in the excited state of 4-phenylphenol is 1.83
The excited-state proton transfer and formation of phenolates
from 6–9 was also indicated by single photon timing (SPT)
measurements. Namely, single exponential fluorescence decays of
6–9 in CH3CN on addition of H2O became double-exponential.
The fluorescence decays in the aqueous media were thus char-
acterized by the presence of a longer-lived decaying component
more present at shorter wavelengths, corresponding to phenols. In
Fig. 2 Quenching of the fluorescence of 6 in CH3CN–H2O (1 : 4) with HClO4 (left, concentration range 0–0.0025 M) and NaOH (right, concentration
range 0–0.012 M).
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0.04,27 so a higher concentration of acid (than in the pH range
2.5–3) would be required to block the deprotonation of the phenol
in the excited state. Fluorescence titration with acid, furthermore,
suggests that in the excited state proton transfer assisted by solvent
from the phenol to the benzyl alcohol can take place. Titration with
base also induced fluorescence quenching. However, in addition to
the quenching, a significant change of the fluorescence spectra was
observed. The band with a maximum at 330 nm corresponding
to the phenol gradually disappeared and only a much weaker
band at 430 nm corresponding to the phenolate was observed. The
finding additionally corroborates the assignment of the observed
shoulder in the fluorescence spectrum in CH3CN–H2O (1 : 4) to
the phenolate emission.
observed transient. Hence, no assignment of the transient was
made.
The transient spectra of 6, 7, 9 and 10 in CH3CN solutions were
similar. In the N2-purged CH3CN solution (and CH3CN–H2O)
we observed a strong transient absorption with a maximum at
390 nm, and a weak shoulder stretching over the whole visible
wavelength region. Its decay was bi-exponential (t ~ 1 ms, k ~ 1.0 ¥
106 s-1, and t ~ 10 ms, k ~ 1 ¥ 105 s-1), and it was strongly quenched
by O2 (in O2-purged solution, t ~ 30–200 ns, k ~ 5.0 ¥ 106–3.3 ¥ 107
s-1). Due to the strong quenching by O2, and in accordance with
the transient spectra of similar phenylphenols,17,19,23 we assigned
the observed transients to the corresponding triplet excited states
of 6, 7, 9 or 10. In the O2-purged CH3CN solutions, after the
decay of the triplet, the short-lived transients were observed with
absorptions at 600–700 nm (t ~ 20 ns, k ~ 4.9 ¥ 107 s-1 and
t ~ 200 ns, k ~ 5 ¥ 106 s-1), and longer-lived transients absorbing at
370 nm, and at 500–600 nm (10–20 ms, k ~ 103–5 ¥ 104 s-1, and 100
ms, k ~ 1 ¥ 102 s-1), that were not affected by O2 or N2O. Hence, the
transients are not due to triplets or solvated electron. Moreover, for
O2-purged CH3CN solution of 7, a rise of the transient absorption
at 550–570 nm was observed that followed the same kinetics as the
decay of the transient at >600 nm (t ~ 240 ns, k ~ 4.2 ¥ 106 s-1).
By comparison to the published transient spectra of aryl radical-
cations28 and phenoxyl radicals,29 these transient absorptions were
assigned to the corresponding radical-cations 20 (at 600–700 nm)
formed by photoionization, and the phenoxyl radicals 21 (500–
600 nm), that were formed by deprotonation of the corresponding
radical-cations. The radical-cations are formed by one-photon
ionization since we observed a linear dependence of the intensity
of the signal with the laser power.
Laser flash photolysis (LFP)
To characterize QMs and other possible long-lived intermediates
in the photochemistry of biphenyls 5–10, we carried out LFP
experiments. Since the formation of QMs from 5–10 is anticipated
only in aqueous media, the transient spectra were recorded in
N2 and O2-purged CH3CN and CH3CN–H2O (1 : 1) solutions,
wherein the difference in the observed transients in these two
solvent systems could in principle be due to QMs or other
intermediates formed by proton transfer reactions.
In the N2- and O2-purged CH3CN–H2O solutions of 5 we
observed transients with the maximum at 350 nm and ~700 nm,
that decayed with the rate constant k ~ 3–5 ¥ 104 s-1 (t ~ 20–30 ms)
and which were not affected by O2 (see supporting info†). However,
we could not assign the observed transient absorption to QM inter-
mediate 14. Due to a steric hindrance of the adamantyl moiety, 14
should probably be better represented as a zwitterion 14Zw, which
is expected to be shorter-lived in CH3CN–H2O solution than the
The transient absorption spectra in O2-purged CH3CN–H2O
solution of 7, 9 and 10 were more complex than the corresponding
spectra in CH3CN solution (see supporting info†), in accordance
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Table 3 Bimolecular quenching constants for QM 17 and cation 22 in
with the formation of new transients from the photochemical
proton transfer pathways. However, transient absorption spectra
of 6 in O2-purged CH3CN–H2O gave no transient absorption that
could be assigned to the presence of the zwitterionic QM 15. This
zwitterion has a dialkylbenzyl cation as a chromophore, so its
absorption is expected in the 350–400 nm region.30 In the aqueous
system it should be characterized by a very short lifetime <20
ns that cannot be detected on our LFP system. In the transient
spectra of O2-purged CH3CN–H2O solutions of 7, 9 and 10, in
the wavelength region at ~350 nm, and at 500–600 nm, in addition
to the above mentioned transient absorptions corresponding to
the phenoxyl radicals, more persistent species were detected that
we tentatively assigned to the corresponding QMs 16, 18, and
19, respectively. They decayed in 100 ms–0.5 ms, but due to
their weak intensity it was difficult to precisely estimate their
decay kinetics, and perform the quenching study. Nevertheless,
substituted biphenyl QMs 16 and 18 showed slower decay kinetics
than the parent biphenyl QM 19 (t ~ 67 ms, k = 1.5 ¥104 s-1).19b
The observed shorter lifetimes than for the monophenyl o- and
p-quinone methides (2–4 ms, and 0.3 s, respectively)2,13 are due to
a loss of aromaticity of two phenyl rings in the biphenyl QMs,
compared to only one in the parent monophenyl QM structure.
It is known that QMs and cations display longer lifetimes
in polar and non-nucleophilic solvents like trifluoroethanol
(TFE).12,15,19,30 Therefore, we performed LFP study of 6 and 7 in
TFE. In the transient absorption spectra of O2-purged solution of
6 in TFE, besides the transients described above corresponding
to the radical-cation and the phenoxyl radical, we observed
a transient at 380–390 nm that decayed with a rate constant
k ~ 4.0 ¥ 106 s-1 (t ~ 250 ns). The transient was quenched by
nucleophiles CH3OH and ethanolamine with the rate constants
kq = 3 ¥ 107 s-1 M-1 and kq = 2 ¥ 108 s-1 M-1, respectively, so we
assign it to the zwitterion 15. The transient could, in principle,
be due to the corresponding cation, but the benzyl cation would
probably be shorter-lived than the zwitterion, and hence not
detectable by the nanosecond LFP. Namely, phenoxide (O-) in
the meta position has an electron donating character (Hammet
constant sm = -0.47), whereas OH in the meta position has electron
withdrawing character (sm = 0.12).25 Although phenoxide in 15 is
not at the same ring as the cation, some stabilization is expected
through conjugation.
TFE solution
Transient
species
CH3OH
Ethanolamine
kq/s-1 M-1
kq/s-1 M-1
17
22
3.6
4.0 ¥ 103
5.9 ¥ 104
4.9 ¥ 103
it to the triplet state of 8. In addition, transient absorptions were
observed with a broad band at 400–500 nm decaying with k ~ 1.0 ¥
104 s-1 (100 ms), which in analogy to previous transients we assigned
to the phenoxy radical 21. Addition of H2O to the CH3CN solution
changed the appearance of the transient absorption spectra. A new
broad band appeared at 500–700 nm with a maximum at 580 nm
(Fig. 3). The transient was not quenched by O2. In O2-purged
CH3CN–H2O (1 : 1) the decay of the transient absorption was bi-
exponential with k ~ 1.0 ¥ 104 s-1 (100 ms) and k ~ 8.3 ¥ 102 s-1
(t ~ 1.2 ms). The longer-lived transient that is formed only in the
aqueous media (t ~ 1.2 ms) could in principle be due to QM 17, or
cation 17H+. However we assigned it to the QM since triarylmethyl
cations in aqueous media decay faster (for trityl cation k = 1.5 ¥
105 s-1).31 In addition, we performed quenching with sodium azide,
an ubiquitous cation quencher and observed slower quenching
kinetics (kq = 1.2 ¥ 106 s-1 M-1) than reported for the triarylmethyl
cations (for trityl cation kq = 9.4 ¥ 109 s-1 M-1).32
LFP of TFE solution of 8 and 8¢ was performed to characterize
QM 17 and cation 22 (Fig. 4). In O2-purged TFE solutions
of 8 and 8¢, similar transient absorption spectra were observed
with maxima at 390 and 580 nm, in accordance with the similar
structure of both chromophores. However, in the spectra of 8, an
additional transient absorption band was present at >650 nm.
The transients in O2-purged TFE solution at 500–600 nm for both
compounds decayed with similar bi-exponential kinetics that were
not affected by O2, k ~ 2.3 ¥ 103 s-1 (t ~ 430 ms) and k ~ 0.9
s-1 (t ~ 1.1 s) for 8, and k ~ 2–10 ¥ 103 s-1 (t ~ 100–500 ms),
k = 1.15 s-1 (t ~ 0.9 s) for 8¢. Similar decay kinetics for both
compounds suggested that we detected the same type of transient
species from both compounds, the cation. However, quenching
experiments with nucleophiles CH3OH and ethanolamine revealed
very different quenching kinetics (Table 3) in accordance with the
different nature of the transient species. Consequently, long-lived
transients with lifetimes of 1.1 s, and 0.9 s we assigned to QM 17
and cation 22, respectively.
In O2-purged TFE solution of 7, the transient was observed at
650–750 nm (t ~ 35 ns, k ~ 2.8 ¥ 107 s-1, and t ~ 800 ns, k ~ 1.2 ¥
106 s-1) corresponding to radical-cation 20. It was quenched by
CH3OH and ethanolamine with the rate constants kq = 3–4 ¥ 107
s-1 M-1 and kq > 5 ¥ 109 s-1 M-1, respectively. At 500–650 nm, a
more persistent transient absorption was observed that decayed
with the rate constants k ~ 1 ¥ 104 s-1 (t ~100 ms), and k ~ 2 ¥
103 s-1 (t ~500 ms). The longer-lived transient with the lifetime of
500 ms was assigned to QM 16. The transient was quenched by
CH3OH and ethanolamine with the rate constants kq = 3 ¥ 104 s-1
M-1 and kq = 2 ¥ 104 s-1 M-1, respectively. However, the estimated
quenching constant probably represents only a lower limit, since
the quencher also reacts with the transient radical-cations.
Photochemical reaction mechanisms
From the results of preparative photolyses as well as fluorescence
and LFP studies, the mechanisms of photochemical reactions
of 5–9 can be proposed. Upon excitation of 5, one of the
deactivation channels from the singlet excited state leads to the
phototransposition product 6. The phototransposition is a well-
known reaction for polyalkylated benzene derivatives, particularly
with bulky substituents.33 Our finding is in accordance with a
deactivation pathway giving rise to overall phototransposition
generating 6 in the ground state. However, to the best of our
knowledge the phototransposition has not been reported for
phenols. Furthermore, our finding suggests that the excitation of
5 resides more on the adjacent phenyl ring than at the phenol,
The transient absorption spectra of 8 were different from those
described above. In N2-purged CH3CN solution, a strong transient
absorption was observed with a maximum at 400 nm that decayed
with a bi-exponential law with k ~ 8.3 ¥ 105 s-1 (t ~ 1.2 ms), and
k ~ 8.3 ¥ 104 s-1 (t ~ 12 ms). It was quenched by O2, so we assigned
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Fig. 3 Transient absorption spectra in N2-purged solution of 8 in CH3CN (left) and CH3CN–H2O (right).
Fig. 4 Transient absorption spectra in O2-purged TFE solution of 8 180 ns after the laser pulse (left) and 8¢ 1.6 ms after the laser pulse (right).
which can also be deduced from the UV-vis spectrum wherein
the maximum is significantly hypsochromically shifted compared
to the spectra of 6 and 7. In addition, the deactivation of the
singlet excited state giving rise to phototransposition results in
low fluorescence of 5. Besides phototransposition, an important
photochemical pathway from the singlet excited state in aqueous
solvent involves proton transfer from the phenol and formation
of the phenolate, as indicated by fluorescence measurements.
Formation of the phenolate in the excited state, coupled with
solvent assisted protonation of the benzyl alcohol group and
dehydration leads to the formation of QM 14. However, due to
the steric hindrance of the adamantane, the QM is probably more
represented by its zwitterionic form that very quickly cyclizes to
fluorene 11 (Scheme 6). Due to the very fast cyclization, QM 14
has a short lifetime and we were not able to characterize it by LFP.
Due to its short lifetime QM 14 does not give photomethanolysis
products.
The meta derivative 6 is characterized by a significantly higher
quantum yield of fluorescence, suggesting that it cannot undergo
efficient deexcitation, giving phototransposition as the ortho
derivative. In the aqueous solvent, the phenol moiety, which
becomes more acidic in the S1 state, undergoes deprotonation,
giving phenolate. The excited-state formation of phenolates,
coupled with protonation of the benzylic alcohol and dehydration
gives rise to zwitterionic QM 15. The zwitterionic species reacts
with nucleophiles giving photosolvolysis products (Scheme 7).
Photomethanolysis reaction could principally take place also via
a cationic intermediate. However, a significantly lower quantum
yield for the photomethanolysis of the methoxy derivative 6¢
strongly indicates the importance of the free phenolic OH in the
reaction mechanism, and additionally corroborates the mechanis-
tic scenario via zwitterion 15. Zwitterion 15 was detected by LFP
in TFE (t~ 250 ns).
The important photochemical pathway for the deactivation of
the para derivatives 7–10 from the singlet excited state involves
deprotonation of the phenol, as indicated from the steady-
state and time-resolved fluorescence measurements. Formation of
phenolates and acid-catalyzed heterolytic cleavage of the benzyl
alcohols coupled with dehydration converts these molecules to
QMs 16–19 (Scheme 8). Although these QMs can be represented
by quinoidal structural formulas (compared to 15), quantum yield
of their formation is probably lower than for the corresponding
meta derivative, as suggested from the quantum efficiency for the
photomethanolysis reaction (the meta effect). The acid catalyzed
photosolvolysis reaction at pH 2.4 suggests that, in acidic media,
formation of QMs and photomethanolysis may take place also
via the cationic species 16H+–19H+. However, at neutral pH
this represents only a minor pathway, since photomethanolysis
of the methoxy derivatives 7¢–10¢ takes place with significantly
lower quantum yields than for the free phenolic species. QMs
16–19 were detected by LFP in CH3CN–H2O and in TFE.
These are relatively stable species reacting slowly and selectively
with nucleophiles (e.g. lifetime of 17 in aqueous CH3CN is
1.2 ms). Inefficient incorporation of deuterium on photolysis in
CH3CN–D2O suggests that only one type of QMs are formed,
that is, formal solvent-assisted proton transfer from the phenol
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Scheme 6
Scheme 7
Scheme 8
to the carbon atoms giving QM 23 probably does not take
place.
exhibit antiproliferative activity on photoactivation.6a The results
demonstrated that without the irradiation the compounds exert
antiproliferative activity towards this cell line (IC50 ª 18–68 mM),
predominantly at the maximal tested concentration (100 mM).
Interestingly, the least active compound 7, which did not cause the
50% growth inhibition even at the maximal tested concentration,
was the only compound that exhibited a significant activity
enhancement upon irradiation at 300 nm (3 ¥ 1 min), resulting
in an IC50 in the low micromolar range (9 mM) (Fig. 5). This
result cannot be compared to the reference compound trioxsalen,
which was significantly more potent after the irradiation. However,
it is comparable to some of the binol–amino acid conjugates
Antiproliferative study
To investigate if the photochemically generated QMs could lead to
antiproliferative activity, compounds 1 and 7–9 were subjected to
preliminary antiproliferative testing on a colon carcinoma cell line
HCT 116 (Table 4 and Fig. 5). The series of compounds was chosen
to probe if the structure of the biphenyl QM could be correlated to
the activity. Trioxsalen (2,5,9-trimethyl-7H-furo[3,2-g]chromen-7-
one) was used as a positive reference compound that is known to
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Table 4 IC50 values (in mM)a
species potentially useful in biological systems for the alkylation
of DNA. Preliminary investigation of the antiproliferative activity
of photogenerated QMs was investigated for phenols 1, and 7–9
on the HCT 116 human cancer cell line. It was shown that 7 on
irradiation exhibits a 10 ¥ increase in the antiproliferative activity.
Compound
HCT 116 cell line
1
19
1
7
>100
7c
9
18
68
1
8
9
1
7
Experimental
trioxsalen
trioxsalenb
trioxsalenc
>100
1
0.2
General
0.3 0.2
1H and 13C NMR spectra were recorded on a Bruker AV- 300,
500 or 600 MHz. The NMR spectra were taken in CDCl3 or
DMSO-d6 at rt using TMS as a reference and chemical shifts
were reported in ppm. Melting points were determined using an
original Ko¨fler Mikroheiztisch apparatus (Reichter, Wien) and
were not corrected. UV-vis spectra were recorded on a Varian
Cary 100 Bio spectrophotometer at rt. IR spectra were recorded
on a FT-IR-ABB Bomem MB 102 spectrophotometer in KBr.
MS were recorded on an Agilent 6410 Triple Quadrupole Mass
Spectrometer by use of the electrospray ionization technique. The
spectra were obtained in the positive and negative mode in the
presence of formic acid. HRMS were obtained on an Applied
Biosystems 4800 Plus MALDI TOF/TOF instrument (AB, Foster
City, CA). For the sample analysis a Shimadzu HPLC equipped
with a Diode-Array detector and a Phenomenex Luna 3u C18(2)
column was used. Mobile phase was CH3OH-H2O (20%). For
the chromatographic separations silica gel (Merck 0.05–0.2mm)
was used. Analytical thin layer chromatography was performed
a IC50; the concentration that causes 50% growth inhibition. b Irradiated 1
min at 300 nm with 6 lamps. c Irradiated 3 ¥ 1 min at 300 nm with 6 lamps.
Fig. 5 Concentration-response profiles for compound 7 tested on HCT
116 cell line, without irradiation, or after the irradiation at 300 nm (1 min,
or 3 ¥ 1 min).
R
on Polygramꢀ SILG/UV254 (Machery-Nagel) plates. Irradiation
experiments were performed in a Rayonet reactor equipped with
16 lamps with the output at 254 nm or a Luzchem reactor
equipped with 8 lamps. During irradiations in the Rayonet, the
irradiated solutions were continuously purged with Ar and cooled
by a tap-water finger-condenser. Solvents for irradiation were
of HPLC purity. Chemicals (dibromobenzenes, bromoanisoles,
2-adamantanone, solution of BBr3) were purchased from the
usual commercial sources and were used as received. Solvents for
chromatographic separations were purified by distillation.
(p-BQMPs) described by Freccero et al. that are similarly not
cytotoxic towards another colon carcinoma cell line (HT29),
but upon irradiation the IC50 concentrations reach 9–30 mM
values.6a Since 8 did not show increased activity on irradiation, the
preliminary result suggests that the activity cannot be correlated
only to the lifetimes of the generated QMs and their reactivity
with nucleophiles. Probably, the structure of the QM, as well as the
interplay between the lipophilicity (and membrane permeability)
and solubility of the phenols in H2O play an important role that
is, however, yet to be investigated.
General procedure for the Grignard reaction
The reaction was carried out in a two-neck round bottom flask
(100 mL) under a N2 inert atmosphere equipped with a condenser
and a dropping funnel. Magnesium (10 mmol), which was freshly
activated prior to reaction, was placed in the flask and suspended
in THF (10 mL). In the dropping funnel was placed THF solution
(20 mL) of bromobiphenyl (10 mmol). A few drops of the solution
were added to the suspension in the flask and the reaction was
initiated by adding a crystal of iodine and heating. The remaining
solution in the funnel was added over 30 min at rt. After the
addition was completed, the reaction mixture was refluxed until
all the magnesium reacted (1–1.5 h). The solution of the Grignard
reagent was cooled to rt and a THF solution (20 mL) of ketone
(10 mmol) was added dropwise during 1 h. After addition was
completed, the reaction mixture was refluxed for 4 h and stirred
at rt over night. The next day, saturated solution of ammonium
chloride (100 mL) was added to the reaction mixture, the layers
were separated, and the aqueous layer additionally acidified with
1 M HCl until all solid dissolved. The acidified aqueous layer was
Conclusion
We have synthesized 4-phenylphenol derivatives 5–9 substituted
with the sterically congested adamantyl moiety or two phenyl
groups. In aqueous media in the singlet excited state, 5–9 undergo
solvent assisted deprotonation of the phenol and protonation of
the benzyl alcohol moiety, giving rise to QMs. The QMs react
with nucleophilic solvent (except the ortho derivative 14, which
cyclizes to fluorene) giving rise to photosolvolysis products with
high quantum yields (U ~ 0.1–0.5). The zwitterionic QM 15
formed from 6 in TFE was characterized by LFP (t ~ 250 ns).
The para QM derivatives 16–19 were detected by LFP in CH3CN–
H2O and TFE solutions. In TFE, the adamantyl QM derivative
16 has a lifetime of 500 ms, whereas QM 17, which has additional
stabilization by two phenyl rings, has a particularly long lifetime
of 1.1 s. The long lifetimes and selectivity in the reaction with
N-nucleophiles renders this class of sterically congested transient
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extracted with diethyl ether (3 ¥ 30 mL), organic extracts combined
and dried over anhydrous MgSO4. After filtration and removal of
the solvent, the crude product was obtained that was additionally
purified by chromatography on silica gel using CH2Cl2 or CH2Cl2–
EtOAc as an eluent.
(t), 35.61 (d, 2C), 34.84 (t, 2C), 32.88 (t, 2C), 27.36 (d), 26.86 (d);
HRMS (MALDI) calculated for C23H26O2 (+H+-H2O) 317.1900,
found 317.1912.
(4¢-methoxybiphenyl-4-yl)diphenylmethanol (8¢)34
The Grignard reagent was prepared from 4-bromo-4¢-
methoxybiphenyl (1.16 g, 4.41 mmol) and magnesium (0.12 g,
4.8 mmol), and reacted with benzophenone (0.80 g, 4.41 mmol) to
afford 1.57 g of the crude product that was purified on a column of
silica gel to give the product (0.82 g, 51%) in the form of colorless
crystals.
2-(2-hydroxyadamantan-2-yl)-4¢-methoxybiphenyl (5¢)
The Grignard reagent was prepared from 2-bromo-4¢-
methoxybiphenyl (1.65 g, 6.3 mmol) and magnesium (0.17 g, 7.0
mmol), and reacted with 2-adamantanone (1.02 g, 6.8 mmol) to
afford 2.6 g of the crude product that was purified on a column of
silica gel to give the product (1.69 g, 79%) in the form of colorless
crystals that were crystallized from CCl4/hexane.
Colorless crystals, mp 86–87 ◦C; 1H NMR (CDCl3, 600 MHz) d
(ppm): 7.51 (d, 2H, J = 8.8 Hz), 7.48 (d, 2H, J = 8.5 Hz), 7.30–7.34
(m, 10H), 7.28 (dd, 2H, J = 8.1 Hz, J = 8.5 Hz), 6.95 (d, 2H, J =
8.8 Hz), 3.83 (s, 3H), 2.81 (br s, 1H OH); 13C NMR (CDCl3, 125
MHz) d (ppm): 159.11 (s), 146.78 (s), 145.17 (s), 139.54 (s), 133.06
(s), 128.23 (d, 2C), 127.96 (d, 2C), 127.85 (d, 4C), 127.82 (d, 4C),
127.17 (d, 2C), 126.05 (d, 2C), 114.13 (d, 2C), 81.80 (s), 55.21 (q);
HRMS (MALDI) calculated for C26H22O2 (-e-) 366.1625, found
366.162.
mp 125–130 ◦C; IR (KBr) n/cm-1: 3448, 3396, 2897; 1H NMR
(CDCl3, 600 MHz) d (ppm): 7.61 (d, 1H, J = 8.0 Hz), 7.32 (t, 1H,
J = 8.0 Hz), 7.28 (d, 2H, J = 8.5 Hz), 7.25 (t, 1H, J = 8.0 Hz), 7.10
(d, 2H, J = 8.5 Hz), 3.84 (s, 3H), 2.27 (s, 2H), 2.17 (br s, 1H), 2.15
(br s, 1H), 1.81 (s, 1H), 1.73 (br s, 1H), 1.62–1.69 (m, 3H), 1,60
(br s, 2H), 1.52–1.57 (m, 2H), 1.49 (d, 2H, J = 12.4 Hz); 13C NMR
(CDCl3, 75 MHz) d (ppm): 158.31 (s), 141.99 (s, 2C), 137.16 (s),
133.77 (d), 129.90 (d, 2C), 126.88 (d), 126.82 (d), 126.40 (d), 112.99
(d, 2C), 77.77 (s), 55.12 (q), 37.50 (t), 34.94 (d, 2C), 34.71 (t, 2C),
32.92 (t, 2C), 27.20 (d), 26.33 (d); HRMS (MALDI) calculated for
C23H26O2 (+H+-H2O) 317.1900, found 317.1891.
4-(2-hydroxypropan-2-yl)-4¢-methoxybiphenyl (9¢)35
The Grignard reagent was prepared from 4-bromo-4¢-
methoxybiphenyl (1.0 g, 3.81 mmol) and magnesium (0.10 g, 4.2
mmol), and reacted with acetone (5 mL) to afford 1.05 g of the
crude product that was purified on a column of silica gel to give
the product (0.50 g, 54%) in the form of colorless crystals.
Colorless crystals, mp 122–124 ◦C; 1H NMR (CDCl3, 300 MHz)
d (ppm): 7.48–7.54 (m, 6H), 6.96 (d, 2H, J = 8.8 Hz), 3.83 (s, 3H),
1.81 (brs, 1H, OH), 1.61 (s, 6H); 13C NMR (CDCl3, 75 MHz) d
(ppm): 159.01 (s), 147.43 (s), 139.12 (s), 133.29 (s), 127.93 (d),
126.40 (d), 124.72 (d), 114.11 (d), 72.29 (s), 55.21 (q), 31.64 (q);
HRMS (MALDI) calculated for C16H18O2 (-e-) 242.1312, found
242.1314.
3-(2-hydroxyadamantan-2-yl)-4¢-methoxybiphenyl (6¢)
The Grignard reagent was prepared from 3-bromo-4¢-
methoxybiphenyl (1.67 g, 6.4 mmol) and magnesium (0.17 g, 7.0
mmol), and reacted with 2-adamantanone (1.05 g, 7.0 mmol) to
afford 2.3 g of the crude product that was purified on a column of
silica gel to give the product (1.66 g, 78%) in the form of colorless
crystals.
◦
mp 103–105 C; IR (KBr) n/cm-1: 3517, 3049, 2909, 2846; H
NMR (CDCl3, 300 MHz), d (ppm): 7.71 (br s, 1H), 7.51 (d, 2H,
J = 8.8 Hz), 7.27–7.49 (m, 3H), 6.97 (d, 2H, J = 8.8 Hz), 3.84 (s,
3H), 2.62 (br s, 2H), 2.45 (br s, 1H), 2.41 (br s, 1H), 1.91 (br s, 1H),
1.68–1.80 (m, 10H); 13C NMR (CDCl3, 75 MHz) d (ppm): 159.07
(s), 145.74 (s), 141.16 (s), 133.93 (s), 128.96 (d), 128.12 (d, 2C),
125.65 (d), 123.94 (d), 123.69 (d), 114.10 (d, 2C), 75.63 (s), 55.24
(q), 37.60 (t), 35.67 (d, 2C), 34.87 (t, 2C), 32.90 (t, 2C), 27.36 (d),
26.86 (d). HRMS (MALDI) calculated for C23H26O2 (+H+-H2O)
317.1900, found 317.1894.
1
General procedure for the removal of the methoxy groups from
phenols with BBr3
The reaction was carried out under N2-atmosphere in a three-
neck round bottom flask (250 mL) equipped with a septum and
a syringe. In the flask was placed methoxybiphenyl derivative (10
mmol), dissolved in dry CH2Cl2 (100 mL) and the solution cooled
to 0 ◦C by ice-bath. To the cold solution, a solution of BBr3 in
CH2Cl2 (1 M, 30 mL) was added dropwise over 1 h using a syringe.
After the addition was completed, the stirring was continued for
1 h at 0 ◦C, and at rt over night. The next day 100 mL of cold
water was added to the reaction mixture, the layers were separated
and the aqueous layer extracted with CH2Cl2 (3 ¥ 50 mL). The
combined organic extracts were dried over anhydrous MgSO4,
solid was removed by filtration and solvent removed on a rotary
evaporator. The obtained crude product was purified on a column
filled with silica gel by use of CH2Cl2 and CH2Cl2–EtOAc (4 : 1)
as eluent, and crystallization.
4-(2-hydroxyadamantan-2-yl)-4¢-methoxybiphenyl (7¢)
The Grignard reagent was prepared from 4-bromo-4¢-
methoxybiphenyl (1.71 g, 6.5 mmol) and magnesium (0.18 g, 7.5
mmol), and reacted with 2-adamantanone (1.14 g, 7.6 mmol) to
afford 2.38 g of the crude product that was purified on a column of
silica gel to give the product (1.62 g, 65%) in the form of colorless
crystals.
◦
mp 186–187 C; IR (KBr) n/cm-1: 3563, 2928, 2897, 2854; H
NMR (CDCl3, 300 MHz) d (ppm): 7.58 (d, 2H, J = 8.8 Hz), 7.54
(d, 2H, J = 8.8 Hz), 7.53 (d, 2H, J = 8.8 Hz), 6.97 (d, 2H, J =
8.8 Hz), 3.84 (s, 3H), 2.59 (br s, 2H), 2.45 (br s, 1H), 2.40 (br s,
1H), 1.91 (br s, 1H), 1.70–1.80 (m, 10H); 13C NMR (CDCl3, 75
MHz) d (ppm): 159.07 (s), 143.67 (s), 139.54 (s), 133.12 (s), 127.90
(d), 126.79 (d), 125.74 (d), 114.11 (d), 75.41 (s), 55.20 (q), 37.60
1
3-(2-hydroxyadamantan-2-yl)-4¢-hydroxybiphenyl (6)
From 3-(2-hydroxyadamantan-2-yl)-4¢-methoxybiphenyl (6¢, 1.23
g, 3.7 mmol) and solution of BBr3 (1 M, 12 mL), the reaction gave
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1.55 g of the crude product that was purified by crystallization
from mixture of ethyl acetate and hexane to afford 0.94 g (79%) of
the pure produ◦ct in a form of colorless crystals.
(d, 2H, J = 8.6 Hz), 6.41 (br s, 1H, OH);13C NMR (CDCl3, 125
MHz) d (ppm): 157.05 (s), 147.75 (s, 2C), 145.84 (s), 138.47 (s),
130.52 (s), 128.23 (d, 2C), 127.72 (d, 4C), 127.62 (d, 2C), 127.50
(d, 4C), 126.59 (d, 2C), 125.03 (d, 2C), 115.66 (d, 2C), 80.38 (s);
HRMS (MALDI) calculated for C25H20O2 (-e-) 352.1469, found
352.1461.
mp 155–157 C; IR (KBr) n/cm-1: 3499, 3462, 3360, 2908, 2853;
1H NMR (DMSO-d6, 300 MHz) d (ppm): 9.50 (br s, OH), 7.63 (br
s, 1H), 7.46 (d, 2H, J = 8.6 Hz), 7.35–7.44 (m, 3H), 6.85 (d, 2H, J =
8.0 Hz), 2.52 (br s, 2H), 2.42 (br s, 1H), 2.38 (br s, 1H), 1,81 (br s,
1H), 1.55–1.70 (m, 10H); 13C NMR (DMSO-d6, 75 MHz) d (ppm):
156.45 (s), 146.10 (s), 139.52 (s), 131.07 (s), 128.07 (d), 127.22 (d,
2C), 123.61 (d), 123.33 (d), 122.93 (d), 115.19 (d, 2C), 73.09 (s),
36.93 (t), 34.29 (d, 2C), 33.85 (t, 2C), 32.08 (t, 2C), 26.46 (d),
26.07 (d); HRMS (MALDI) calculated for C22H24O2 (+H+-H2O)
303.1743, found 303.1754.
General procedure for the removal of the methoxy groups from
phenols with thiolate
The reaction was carried out under a N2-atmosphere in a round-
bottom flask (100 mL) equipped with a condenser and a dropping
funnel, and the outlet of the N2 from the condenser was purged
through a solution of sodium ethoxide in ethanol. In the flask
was placed sodium hydride (0.46 g, 20 mmol) and suspended in
5 mL of dry DMF. The suspension was cooled by ice-bath and
a solution of ethyl mercaptane (1.4 mL, 20 mmol) in DMF (5
mL) was added dropwise. When the addition was completed, the
reaction mixture was stirred for 10 min and the ice-bath was
removed. Methoxybiphenyl derivative (1.7 g, 5.08 mmol) was
dissolved in DMF (10 mL) and added to the reaction mixture.
After addition, the mixture was refluxed over 4 h, cooled, and
poured into 100 mL of H2O. The aqueous mixture was washed
with hexane (2 ¥ 50 mL) and acidified by saturated solution of
ammonium chloride. Extraction with ethyl acetate was carried out
(3 ¥ 100 mL), extracts dried over anhydrous MgSO4, solid removed
by filtration, and solvent removed on the rotary evaporator.
The reaction furnished the crude product that was purified by
chromatography and crystallization.
4-(2-hydroxyadamantan-2-yl)-4¢-hydroxybiphenyl (7)
From 4-(2-hydroxyadamantan-2-yl)-4¢-methoxybiphenyl (7¢,
1.40 g, 4.2 mmol) and solution of BBr3 (1 M, 13 mL), the
reaction gave 1.35 g of the crude product that was purified by
crystallization from ethyl acetate to afford 0.9 g (67%) of the pure
product in a form of colorless crystals.
◦
1
mp 225–228 C; IR (KBr) n/cm-1: 3431, 3190, 2924, 2856; H
NMR (DMSO-d6, 300 MHz) d (ppm): 9.48 (br s, OH), 7.42–7.61
(m, 6H), 6.84 (d, 2H, J = 8.5 Hz), 2.47 (br s, 2H), 2.41 (br s, 1H),
2.38 (br s, 1H), 1.81 (br s, 1H), 1.53–1.73 (m, 10H); 13C NMR
(DMSO-d6, 75 MHz) d (ppm): 156.94 (s), 144.26 (s), 138.14 (s),
130.77 (s), 127.49 (d, 2C), 126.05 (d, 2C), 125.59 (d, 2C), 115.67 (d,
2C), 73.34 (s), 37.44 (t), 34.80 (d, 2C), 34.34 (s, 2C), 32.55 (s, 2C),
26.97 (d), 26.57 (d); HRMS (MALDI) calculated for C22H24O2
(+H+-H2O) 303.1743, found 303.1740.
2-(2-hydroxyadamantan-2-yl)-4¢-hydroxybiphenyl (5)
Spiro[adamantane-2,9¢-(2¢-hydroxy)fluorene] (11)
From 2-(2-hydroxyadamantan-2-yl)-4¢-methoxybiphenyl (5¢,
0.90 g, 2.67 mmol), sodium hydride (0.5 g, 21 mmol) and ethyl
mercaptane (780 mL, 11 mmol) in DMF (10 mL) the reaction gave
900 mg of the crude product that was purified by crystallization
from mixture of ethyl acetate and hexane to afford 600 mg (69%)
of the pure pro◦duct in a form of colorless crystals.
From 2-(2-hydroxyadamantan-2-yl)-4¢-methoxybiphenyl (5¢,
0.18 g, 0.54 mmol) and solution of BBr3 (1 M, 4 mL), the
reaction gave 0.17 g of the crude product that was purified by
crystallization from mixture of ethyl acetate and hexane to afford
100 mg (59%) of the pure product in a form of colorless crystals.
mp 254–256 ◦C; IR (KBr) n/cm-1: 3302, 2973, 2888; 1H NMR
(CDCl3, 300 MHz) d (ppm): 8.06 (d, 1H, J = 8.0 Hz), 7.67 (dd, 1H,
J = 7.5 Hz, J = 0.9 Hz), 7.60–7.65 (m, 2H), 7.33 (dt, 1H, J = 7.5
Hz, J = 0.8 Hz), 7.21 (dt, 1H, J = 7.9 Hz, J = 1.3 Hz), 6.85 (dd, 1H,
J = 8.1 Hz, J = 2.3 Hz), 4.76 (br s, 1H, OH), 2.83–2.97 (m, 4H),
2.15–2.22 (m, 2H), 1.98 (br s, 2H), 1.73–1.83 (m, 4H), 1.55–1.64
(m, 2H); 13C NMR (CDCl3, 125 MHz) d (ppm): 153.79 (s), 152.82
(s), 150.33 (s), 140.62 (s), 133.94 (s), 129.16 (d), 126.78 (d), 124.71
(d), 119.80 (d), 118.48 (d), 117.14 (d), 113.84 (d), 58.42 (s), 41.38
(t), 34.75 (d, 2C), 34.15(t, 2C), 33.95 (t, 2C), 27.26 (d), 27.18 (d);
HRMS (MALDI) calculated for C22H20O (+H+) 303.1743, found
303.1735.
mp 160–163 C; 1H NMR (DMSO-d6, 300 MHz) d (ppm): 9.29
(s, 1H, OH), 7.55 (d, 1H, J = 7.7 Hz), 7.29 (t, 1H, J = 7.7 Hz),
7.16–7.26 (m, 3H), 6.98 (dd, 1H, J = 1.0 Hz, J = 7.2 Hz), 6.70 (d,
2H, J = 8.4 Hz), 4.73 (s, 1H, OH), 2.10–2.30 (m, 4H), 1.20–1.70
(m, 10 H); 13C NMR (DMSO-d6, 75 MHz) d (ppm): 158.82 (s),
142.53 (s), 142.26 (s), 135.54 (s), 133.40 (d), 130.12 (d, 2C), 127.11
(d), 126.27 (d), 126.00 (d), 114.16 (d, 2C), 75.70 (s), 37.34 (t), 34.28
(t, 2C), 34.08 (d, 2C), 32.62 (t, 2C), 26.78 (d), 26.13 (d); HRMS
(MLADI) calculated for C22H24O2 (+H+-H2O) 303.1743, found
303.1754.
4-hydroxy-4¢-(2-hydroxypropan-2-yl)biphenyl (9)36
From 4-methoxy-4¢-(2-hydroxypropan-2-yl)biphenyl (9¢, 0.40 g,
1.65 mmol), sodium hydride (0.3 g, 6.61 mmol) and ethyl
mercaptane (480 mL, 6.61 mmol) in DMF (10 mL) the reaction
gave a crude product that was purified by chromatography on silica
gel using dichlormethane–ethyl acetate (7 : 3) to afford 0.30 g (80%)
of the pure product in the form of colorless crystals.
mp 178–180 C; H NMR (DMSO-d6, 300 MHz), d (ppm):
9.48 (s, 1H, OH), 7.48 (s, 4H), 7.45 (d, 2H, J = 8.6 Hz), 6.83 (d,
2H, J = 8.6 Hz), 4.98 (br s, 1H, OH), 1.44 (s, 6H); 13C NMR
4¢-(hydroxydiphenylmethyl)biphenyl-4-ol (8)34
From (4¢-methoxybiphenyl-4-yl)diphenylmethanol (8¢, 0.82 g, 2.4
mmol) and solution of BBr3 (1 M, 10 mL), the reaction gave 0.67 g
of the crude product that was purified by crystallization from a
mixture of carbon tetrachloride, toluene and ether to afford 0.57
g (83%) of the pure product in a form of colorless crystals.
◦
1
◦
1
mp 274–276 C; H NMR (CDCl3, 300 MHz) d (ppm): 9.50
(br s 1H, OH), 7.49 (t, 4H, J = 8.7 Hz), 7.18–7.34 (m, 12H), 6.83
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(DMSO-d6, 75 MHz) d (ppm): 156.87 (s), 148.60 (s), 137.86 (s),
130.94 (s), 127.52 (d, 2C), 125.31 (d, 2C), 124.98 (d, 2C), 115.64
(d, 2C), 70.47 (s), 31.90 (q, 2C); HRMS (MALDI) calculated for
C15H16O2 (-e-) 228.1156, found 228.1156.
(d, 2C), 115.82 (d, 2C), 79.08 (s), 47.65 (q), 37.25 (t), 34.18 (t,
2C), 32.45 (t, 2C), 32.11 (d, 2C), 27.18 (d), 26.35 (d); HRMS
(MALDI) calculated for C23H26O2 (+H+-OCH3) 303.1743, found
303.1742.
Irradiation of 8. (100 mg, 0.28 mmol) in 100 mL CH3OH for
30 min gave, after evaporation of the solvent and chromatographic
separation using CH2Cl2–EtOAc (30%) as eluents, 10 mg (10%) of
product 8OMe and 90 mg of the starting material.
Photochemical experiments, general
In a quartz vessel was placed a CH3OH, or CH3OH–H2O (3 : 1
or 4 : 1), solution (100 mL, c ~ 10-3 M) of biphenyl 5–8 (20–100
mg) and irradiated in a Rayonet reactor using 16 lamps at 254 nm
for 1 min–1 h. Prior to and during the irradiation, the solution
was continuously purged with a stream of Ar and cooled by a
cold-finger condenser. After irradiation, CH3OH was removed on
a rotary evaporator and the residue (if irradiated in CH3OH–
H2O) extracted with EtOAc (3 ¥ 75 mL). Extracts were dried over
anhydrous MgSO4, filtered and solvent was removed on a rotary
evaporator. The residue was chromatographed on a thin layer of
silica gel using CH2Cl2–EtOAc (30%) as eluents.
4¢-(methoxydiphenylmethyl)biphenyl-4-ol (8OMe). Colorless
◦
1
crystals, mp 178–182 C; H NMR (CDCl3, 500 MHz) d (ppm):
7.43–7.48 (m, 8H), 7.26–7.32 (m, 4H), 7.20–7.25 (m, 2H), 7.14 (d,
2H, J = 8.1 Hz), 6.86 (d, 2H, J = 8.7 Hz), 5.04 (br s, 1H, OH), 3.08
(s, 3H, OCH3); 13C NMR (CDCl3, 125 MHz) d (ppm): 155.02 (s),
143.84 (s), 142.25 (s), 139.05 (s), 133.27 (s), 129.02 (d, 2C), 128.61
(d, 4C), 128.14 (d, 2C), 127.69 (d, 4C), 126.83 (d, 2C), 125.86 (d,
2C), 115.52 (d, 2C), 52.00 (q); HRMS (MALDI) calculated for
C26H22O2 (+H+-OCH3) 335.1441, found 335.1455.
Irradiation of 5. (80 mg, 0.16 mmol) in 100 mL CH3OH for
2 h gave, after evaporation of the solvent and chromatographic
separation using CH2Cl2–EtOAc (30%) as eluents, 10 mg (12%) of
product 11, 30 mg (38%) of product 6 and 30 mg of the starting
material.
Irradiation of 9. (60 mg mmol) in 100 mL CH3OH for 1 h gave,
after evaporation of the solvent and chromatographic separation
using CH2Cl2–EtOAc (2%), 10 mg of product 12, and 10 mg of
product 9OMe, as well as 40 mg of the starting material.
4-hydroxy-4¢-(prop-1-en-2-yl)biphenyl (12)37. Colorless crys-
Irradiation of 6. (20 mg, 0.31 mmol) in 100 mL CH3OH–
H2O (3 : 1) for 15 min gave, after evaporation of the solvent
and chromatographic separation using CH2Cl2–EtOAc (30%) as
eluents, 20 mg (95%) of product 6OMe.
1
tals, H NMR (CDCl3, 500 MHz) d (ppm): 7.48–7.52 (m, 4 H),
7.47 (d, 2H, J = 8.7 Hz), 6.88 (d, 2H, J = 8.7 Hz), 5.39–5.40 (m,
1H), 5.08 (q, 1H, J = 1.5 Hz), 4.85 (br s, 1H, OH), 2.16 (dd, 3H,
J = 0.7 Hz, J = 1.5 Hz); 13C NMR (CDCl3, 125 MHz) d (ppm):
155.02 (s), 142.69 (s), 139.64 (s), 139.46 (s), 133.46 (s), 128.11 (d,
2C), 126.34 (d, 2C), 125.76 (d, 2C), 115.56 (d, 2C), 112.11 (t), 23.91
(q); RMS (MALDI) calculated for C15H14O (-e-) 210.1050, found
210.1047.
3-(2-methoxyadamantan-2-yl)◦-4¢-hydroxybiphenyl
(6OMe).
Colorless crystals, mp 174–178 C; 1H NMR (CDCl3, 300 MHz)
d (ppm): 7.66 (br s, 1H), 7.35–7.50 (m, 5H), 6.89 (d, 2H, J = 8.6
Hz), 5.65 (br s, 1H, OH), 2.88 (s, 3H, OCH3), 2.71 (br s, 2H),
2.37 (br s, 1H), 2.33 (br s, 1H), 1.91 (br s, 1H), 1.60-1.80 (m,
9H); 13C NMR (CDCl3, 75 MHz) d (ppm): 155.28 (s), 141.18
(s), 140.55 (s), 133.84 (s), 128.30 (d, 2C), 128.14 (d), 125.72 (d),
125.59 (d), 125.46 (d), 115.56 (d, 2C), 80.36 (s), 40.03 (q), 37.56
(t), 34.46 (t, 2C), 32.74 (t, 2C), 27.59 (d), 26.73 (d); HRMS
(MALDI) calculated for C23H26O2 (+H+-OCH3) 303.1743, found
303.1757.
4-hydroxy-4¢-(2-methoxypropan-2-yl)biphenyl (9OMe). Col-
orless crystals, mp 126–132 ◦C; IR (KBr) nmax/cm-1: H NMR
1
(CDCl3, 500 MHz) d (ppm): 7.50 (d, 2H, J = 8.6 Hz), 7.47 (d, 2H,
J = 8.6 Hz), 7.43 (d, 2H, J = 8.6 Hz), 6.89 (d, 2H, J = 8.6 Hz),
5.14 (br s, 1H, OH), 3.09 (s, 3H, OCH3), 1.55 (s, 6H, CH3); 13C
NMR (CDCl3, 125 MHz) d (ppm): 155.04 (s), 144.13 (s), 139.18
(s), 133.42 (s), 128.16 (d, 2C), 126.35 (d, 2C), 126.15 (d, 2C), 115.54
(d, 2C), 50.58 (q), 27.84 (q, 2C), one singlet is covered by CDCl3;
HRMS (MALDI) calculated for C15H14O (+Na+) 265.1199, found
265.1205.
Irradiation of 7. (70 mg, 0.31 mmol) in 500 mL CH3OH–H2O
(4 : 1) for 1 h gave mixture of 7 and 7OMe. After evaporation of
the CH3OH, the aqueous residue was extracted with EtOAc (3 ¥
75 mL). The extracts were dried, filtered and solvent was removed.
The residue was analyzed by HPLC. It contained 7 and 7OMe in
a ratio 1 : 3.
Quantum yields for the photomethanolysis reaction
Solutions of 5–8 and a solution of 2-hydroxybenzyl alcohol
in CH3CN–H2O (1 : 1), as well as a solution of valerophenone
in CH3CN–H2O (1 : 1) were freshly prepared and their con-
centrations adjusted to have absorbances at 254 nm 0.4–0.8.
After adjustment of the concentration and measurement of the
corresponding UV-vis spectra the solutions were put in quartz
cuvettes (20 mL), purged with a stream of N2 (20 min each), and
sealed with a septum. The cuvettes were irradiated at the same time
in a Luzchem reactor equipped with a carousel and 8 lamps with
the output at 254 nm for 0.5, 1, and 4 min. After each irradiation,
the samples were taken from the cuvettes by use of a syringe
and analyzed by HPLC. Quantum yields for the methanolysis
were calculated using the quantum yield for the methanolysis
of 2-hydroxybiphenyl (U = 0.23)12 and the Norrish-II cleavage
The pure product 7OMe was obtained by stirring 7 (50 mg) in
CH3OH (50 mL) in the presence of H2SO4 (500 mg) overnight.
The next day, to the solution NaHCO3 (860 mg) was added and
stirring was continued over 4 h. The mixture was filtered and
solvent evaporated to afford 7OMe for the analysis by NMR.
4-(2-methoxyadamantan-2-yl)-4¢-hydroxybiphenyl
(7OMe).
Colorless crystals, mp 180–181 ◦C; 1H NMR (DMSO-d6, 300
MHz) d (ppm): 9.48 (br s, 1H, OH), 7.57 (d, 2H, J = 8.5 Hz),
7.49 (d, 2H, J = 8.6 Hz), 7.46 (d, 2H, J = 8.5 Hz), 6.84 (d,
2H, J = 8.6 Hz), 2.71 (s, 3H, OCH3), 2.61 (br s, 2H), 2.23 (br
s, 1H), 2.19 (br s, 1H), 1.82 (br s, 1H), 1.50–1.72 (m, 9H); 13C
NMR (DMSO-d6, 75 MHz) d (ppm): 157.20 (s), 138.83 (s),
138.65 (s), 130.49 (s), 127.72 (d, 2C), 127.68 (d, 2C), 125.52
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(formation of acetophenone) of valerophenone in aqueous media
(U = 0.65 0.03).21
Antiproliferative investigation
The experiments were carried out on a human colon carcinoma cell
line HCT 116. Cells were cultured as monolayers and maintained
in Dulbecco’s modified Eagle medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 U
mL-1 penicillin and 100 mg mL-1 streptomycin in a humidified
atmosphere with 5% CO2 at 37 ◦C.
Irradiation in CH3CN–D2O
To a solution of 6, 7, or 11 (10 mg, 0.018 mmol) in CH3CN (90 mL)
was added 10 mL of D2O. The solution was purged with Ar for
30 min and irradiated in a Rayonet recator at 254 nm (16 lamps)
for 1 h. During irradiation, the solution was continuously purged
with Ar and cooled by a cold-finger condenser. After irradiation,
50 mL of H2O was added and extractions with EtOAc (3 ¥ 75 mL)
were carried out. The extracts were dried over anhydrous MgSO4.
After filtration and evaporation of the solvent, the crude mixture
was analyzed by NMR and MS.
The cells were inoculated in parallel on three 96-well microtiter
plates on day 0, at 1.5 ¥ 104 cells mL-1. Test agents were added in
ten-fold dilutions (10-8 to 10-4 M) on the next day and incubated
for a further 72 h. Working dilutions were freshly prepared on
the day of testing. One of the plates was left in the dark, while the
other was irradiated in a Luzchem reactor (6 lamps 300 nm, 1 min)
four hours after the addition of the compounds. The third plate
was additionally irradiated (6 lamps 300 nm, 1 min) 24 and 48 h
after the first irradiation. After 72 h of incubation the cell growth
rate was evaluated by performing the MTT assay38,39 which detects
dehydrogenase activity in viable cells. The absorbance (A) was
measured on a microplate reader at 570 nm. The absorbance is
directly proportional to the number of living, metabolically active
cells. The percentage of growth (PG) of the cell lines was calculated
according to one or the other of the following two expressions:
Steady state and time-resolved fluorescence measurements
The steady state measurements were performed on a Photon
Technology International (PTI) Quanta-Master QM-2 lumines-
cence spectrometer. The samples were dissolved in CH3CN, or
CH3CN–H2O (4 : 1) and the concentrations were adjusted to have
absorbances at the excitation wavelength (270 nm) < 0.1. Solutions
were purged with nitrogen for 30 min prior to analysis. The
measurements were performed at 20 ◦C. Fluorescence quantum
yields were determined by comparison of the integral of the
emission bands with the one of fluorene in methanol (U = 0.68).25
Typically, three absorption traces were recorded (and averaged)
and three fluorescence emission traces, exciting at three different
wavelengths. Three quantum yields were calculated and the mean
value reported.
If (mean Atest - mean Atzero) ≥ 0, then PG = 100 ¥ (mean Atest
mean Atzero)/(mean Actrl - mean Atzero).
-
If (mean Atest - mean Atzero) < 0, then: PG = 100 ¥ (mean
Atest - mean Atzero)/Atzero, where the mean Atzero is the average
of optical density measurements before exposure of cells to the
test compound, the mean Atest is the average of optical density
measurements after the desired period of time and the mean
Actrl is the average of optical density measurements after the
desired period of time with no exposure of cells to the test
compound. In the experiments where the cells were irradiated,
Fluorescence titrations were performed by adding aliquots
(60 mL) of aqueous HClO4 (pH = 2.10) to 2 mL of the CH3CN–
H2O (4 : 1) solution. The pH of aqueous HClO4 was measured by
a pH meter. The titration was performed at 20 ◦C in air-saturated
solutions.
Actrl represents irradiated control cells, which typically showed 25%
growth inhibition compared to Actrl without irradiation.
Fluorescence decay histograms were obtained on an Edin-
burgh instrument OB920, equipped with a light emitting diode
(excitation wavelength 265 nm), using time-correlated single
photon counting technique in 1023 channels. Histograms of the
instrument response functions (using LUDOX scatterer), and
sample decays were recorded until they typically reached 3 ¥ 103
counts in the peak channel (except for 5 where only 103 counts were
collected). The half width of the instrument response function was
typically ~1.5 ns. The time increment per channel was 0.049 or
0.098 ns. Obtained histograms were fitted as sums of an expo-
nential using Gaussian-weighted non-linear least-squares fitting
based on Marquardt–Levenberg minimization implemented in the
software package of the instrument. The fitting parameters (decay
times and pre-exponential factors) were determined by minimizing
The results are expressed as IC50, which is the concentration
necessary for 50% of inhibition. The IC50 values for each com-
pound are calculated from concentration-response curves using
linear regression analysis by fitting the test concentrations that
give PG values above and below the reference value (i.e. 50%). If,
however, all of the tested concentrations produce PGs exceeding
the respective reference level of effect (e.g. PG value of 50), then
the highest tested concentration is assigned as the default value,
which is preceded by a “>“ sign. Each test was performed in
quadruplicate in at least two individual experiments.
Acknowledgements
These materials are based on work financed by the National
Foundation for Science, Higher Education and Technological
Development of the Republic of Croatia (HRZZ grant no.
02.05/25), the Ministry of Science Education and Sports of the
Republic of Croatia (grant No. 098-0982933-2911), the Natural
Sciences and Engineering Research Council (NSERC) of Canada
and the University of Victoria.
2
the reduced chi-square c . An additional graphical method was
used to judge the quality of the fit that included plots of the
weighted residuals vs. channel number.
Laser flash photolysis (LFP)
All LFP studies were conducted at the University of Victoria LFP
facility employing a YAG laser, with a pulse width of 10 ns and
excitation wavelength 266 nm. Static cells (0.7 cm) were used and
solutions were purged with nitrogen or oxygen for 20 min prior to
measurements. Absorbances at 266 nm were ~0.4–0.6.
Notes and references
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