Zwitterionic biphenyl quinone methides in photodehydration reactions of 3-hydroxybiphenyl derivatives: Laser flash photolysis and antiproliferation study

Article abstract of DOI:10.1039/c1pp05338h

In aqueous media, photochemical excitation to S₁ of 3-phenylphenols 4-8 leads to deprotonation of the phenol OH, coupled with protonation of the benzyl alcohol and overall dehydration that delivers zwitterions 17-21. The zwitterions react with

Full text of DOI:10.1039/c1pp05338h

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PAPER
Zwitterionic biphenyl quinone methides in photodehydration reactions of
3-hydroxybiphenyl derivatives: laser flash photolysis and antiproliferation
study†
Nikola Basaric´,*a Nikola Cindro,a Damir Bobinac,a Lidija Uzelac,b Kata Mlinaric´-Majerski,a Marijeta Kraljb
and Peter Wanc
Received 6th October 2011, Accepted 11th November 2011
DOI: 10.1039/c1pp05338h
In aqueous media, photochemical excitation to S1 of 3-phenylphenols 4–8 leads to deprotonation of the
phenol OH, coupled with protonation of the benzyl alcohol and overall dehydration that delivers
zwitterions 17–21. The zwitterions react with nucleophiles (CH3OH, CF3CH2OH and ethanolamine)
converting them in high quantum yields to the corresponding adducts and photosolvolysis products
(for photomethanolysis U ~ 0.1–0.5). Zwitterions 20 and 21 were characterized by laser flash photolysis
in CH3CN–H2O (t~7.5 and 25 ms, respectively) and the associated quenching rate constants with
nucleophiles azide and ethanolamine determined. In vitro studies of antiproliferative activity of the
photochemicaly generated QMs and zwitterions formed from 2-, 3- and 4-phenylphenols were carried
out on three human cancer cell lines HCT 116 (colon), MCF-7 (breast), and H 460 (lung). Irradiation
of cells incubated with 3, 6, and 26 showed enhanced antiproliferative activity compared to the cells
that were not irradiated.
OH, whereas the basic sites usually encountered are heteroatoms
such as nitrogen or oxygen. However, some examples are known
Introduction
where ESIPT takes place from phenol OH to carbon.8 According
to Kasha, ESIPT reactions can be divided in several classes: (I)
those with an intrinsic H-bond between the sites, (II) those where
the proton is far away from the acceptor and requires a mediator,
and (III) those that occur via proton relay mechanisms.9 ESIPT
gives rise to high-energy tautomers that usually rapidly revert to
the corresponding starting molecules. However, certain phenols
substituted with hydroxymethyl groups undergo dehydration re-
actions formally coupled with ESIPT.10 Overall photodehydration
of these phenols gives rise to QMs that are characterized by
longer lifetimes than the usual tautomers obtained in the ESIPT
reactions.11 The other photochemical methods for the generation
of QMs rely on photoextrusion of amines,12 Mannich bases13 or
acetates.14
We became interested in photochemical generation of steri-
cally congested QMs and have found that substitution of the
hydroxymethylphenols with an adamantyl moiety increases the
lifetimes of the corresponding QMs, with corresponding enhanced
selectivity in reaction with nucleophiles.15 We have extended the
study to adamantyl derivatives of 2-phenylphenol16 and found that
in addition to photodehydration or elimination of ammonium salts
giving QM1, these compounds also undergo ESIPT to the carbon
atom of the adjacent phenyl ring giving QM2 (eqn (1)). However,
QM2 cannot be detected by laser flash photolysis (LFP) due to
its short lifetime. To circumvent the problem of competing ESIPT
process that lowers the quantum yield for the formation of QM1,
Quinone methides (QM) are common intermediates in the chem-
istry and photochemistry of phenols.1 They are of particular
importance in biological chemistry because of their reactivity with
DNA and amino acids.2 It is now well established that QMs can
be used as DNA cross-linking agents.3 Some classes of antibiotics
base their antiproliferative action on metabolic formation of
QMs that subsequently alkylate DNA.4 Moreover, Freccero et
al. have recently demonstrated that photogenerated QMs exhibit
antiproliferative activity.5
Excited-state intramolecular proton transfer (ESIPT) has re-
ceived significant scientific interest because of its fundamental
importance in chemistry and biology, as well as in many potential
applications.6,7 ESIPT arises when a molecule with two groups
in close proximity exhibits enhanced acidity and basicity on
photoexcitation. The most common acidic site is the phenolic
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
† Electronic supplementary information (ESI) available: Experimental
procedures for the preparation of compounds, UV-vis and fluorescence
spectra of compounds 4–8, transient absorption spectra obtained by LFP
of 4–8, and 1H and 13C NMR spectra of all prepared compounds, and cross-
linking and cleavage of DNA experiments. See DOI: 10.1039/c1pp05338h
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we prepared a series of 4-hydroxybiphenyl derivatives for which
the ESIPT reaction is impossible. For example, photoexcitation
of 3 gives rise to QM3 that reacts with nucleophiles and gives
addition products 3¢ (eqn (2)). These para-biphenyl QMs formed
in dehydration reactions were characterized by LFP. In addition,
we performed a preliminary antiproliferative investigation on one
human cancer cell line. Irradiation of cells incubated with 3 showed
enhanced antiproliferative activity compared to cells that were not
irradiated, consistent with the activity being due to the formation
of QM3.17
irradiation of cells incubated with alkyl derivatives of 3-biphenyl
or 4-biphenyl system leads to enhanced antiproliferative effect
compared to the cells that were kept in dark.
Results and discussion
Synthesis
Synthesis of 3-hydroxybiphenyls 4–8 was performed according to
the modification of synthetic procedures for 2-hydroxybiphenlys16
and 4-hydroxybiphenlys.17 The synthesis started from the cor-
responding 2-, 3- and 4-bromo-3¢-hydroxybiphenyls, obtained
by Suzuki reaction, which were coupled (Grignard reaction)
with adamantan-2-one to furnish 4a–6a, or with benzophenone,
yielding 7a and 8a (see Experimental and ESI†). Demethylation
by use of CH3CH2SNa or BBr3 gave 4–8.
(1)
Preparative irradiations
Photochemical reactivity of 4–8 and the ability to irreversibly form
stable photoproducts was first investigated by irradiations in UV-
vis cuvettes in CH3CN and CH3CN–H2O (1 : 1) (see the ESI†).
After each exposure to 254 nm light, the UV-vis spectrum was
taken. The experiment indicated that ortho-derivative 4 underwent
a photochemical change giving products absorbing at longer
wavelengths than the starting molecule. The photoconversion
was higher in a solution containing H2O. Irradiations of 5–8 in
both solvents resulted in the broadening of the band at 260 nm,
suggesting that extensive irradiation leads to decomposition of the
material. However, the changes in the spectra were taking place at
a slower rate than for the ortho-derivative.
(2)
Irradiation of the hydroxybiphenyl derivatives substituted with
hydroxymethyl moiety in solvents containing CH3OH gives rise to
photomethanolysis products via biphenyl QM intermediates.16,17,18
To probe for the analogous process in 3-hydroxybiphenyl deriva-
tives, we irradiated 4–8 in CH3OH and CH3OH–H2O. To isolate
the photoproducts, typically 5–100 mg of the compound was
dissolved in CH3OH (or CH3OH–H2O) and irradiated (254 nm,
16 lamps) for 1 min–1h. The composition of the solution was
analyzed by HPLC. Irradiation of the ortho-derivative 4 gave three
photoproducts (4b–19%, 9–43% and 10–21%, eqn (3)). Formation
of fluorenes 9 and 10, which absorb at higher wavelengths than
the starting molecule, is in accordance with the findings from
irradiations in UV-vis cuvettes. Photomethanolysis of 5–8 was
clean, giving only one product (5b–8b, eqn (4) and (5)), and
the photoconversion was more efficient in the systems containing
H2O. The more efficient photoreaction in aqueous systems is in
accordance with more efficient transfer of a proton to H2O-clusters
than to CH3OH.21 It is interesting to note that triphenylmethanol
derivatives 7 and 8 in systems containing more than 10% H2O
achieved conversion of ~25% and continuing irradiation did not
increase the conversion. The products were separated from the
starting material after preparative TLC. However, attempts to
isolate 5b and 6b by chromatography on silica resulted in the
cleavage of the methoxy group and isolation of the starting mate-
rial. Therefore, for the purpose of analytical characterization, 5b
and 6b were prepared by stirring acidic CH3OH solution of 5 and
6, respectively. After 2 days of stirring at rt complete conversion
was achieved and the chromatography was not necessary.
To round off our investigation on QM isomers of biphenyl
systems, we report on photochemical generation of QMs with
zwitterionic character from 3-hydroxybiphenyls 4–8. In these
systems, QMs with neutral quinoidal structures cannot be formed,
and in addition, ESIPT is not possible. Although formation of
biphenyl QMs with zwitterionic structures has been reported,8a,18
this is the first systematic study of photodehydration of 3-
phenylphenol derivatives. The investigation is warranted since
we anticipate higher reactivity of 3-phenylphenols compared to
4-phenylphenols in the photodehydration reactions, due to the
higher acidity expected for 3-phenylphenols,19 as well as operation
of the well-known meta-effect in photochemistry.20 We performed
preparative irradiations in the presence of nucleophiles, character-
ized the nucleophilic adducts, and investigated the mechanisms of
photochemical generation of zwitterions. Moreover, we performed
an in vitro study with the photochemically generated QMs on
three cancer cell lines on a series of 2-, 3- and 4-phenylphenols
to establish a structure–activity relationship for several classes of
biphenyl QMs and probed for the possible selectivity on a specific
cell line. We find that derivatives 4–8 are photoreactive and give
rise to QMs with zwitterionic structures. Furthermore, we find that
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less efficient in basic media. The pKa of the phenolic OH of 3-
phenylphenol in the ground state is 9.62.19 Hence, in the ground
state at pH > 10, most of the compound is in the phenolate form,
so that excitation does not lead to deprotonation.
(3)
(4)
Irradiation of 5–7 was also performed in the presence of 2,2,2-
trifluoroethanol (TFE) which is a more polar but less nucleophilic
solvent than CH3OH. Irradiation in CH3CN-TFE gave rise to
solvolysis products 11–13 which were isolated in the yield of
10–15% after separation from the unreacted starting material
by thin-layer chromatography. Triphenylmethanols 8 and 9 were
also irradiated in the presence of an N-nucleophile ethanolamine.
Photochemical reaction gave adducts 14 and 15 in 35% yield. In
addition, reduction product 16 was formed, probably due to a
secondary photochemical homolytic b-cleavage from amine 15.
(5)
Quantum yields for the photomethanolysis reaction were de-
termined by use of valerophenone actinometer (formation of
acetophenone in aqueous CH3CN, U = 0.65
(6)
0.03).22 The
absorbances of the solutions were measured prior to irradiations,
whereas the composition of the irradiated solution was analyzed
by HPLC. Measurements were performed in triplicate and the
mean values are reported (Table 1).
Photosolvolysis products 5b–8b can in principle be formed via
cationic intermediates formed by heterolytic cleavage of the OH
group and not via zwitterions (vide infra). In that case, irradiation
of the corresponding methoxy derivatives 5a–8a in CH3OH would
give the photomethanolysis products with similar quantum yields.
However, under the same irradiation conditions 5a–8a gave no
trace of the product indicating that the free phenolic OH is
essential for the photomethanolysis.
Further indication that dissociation of the phenolic OH in the
excited state is important in the photomethanolysis mechanism of
5–8 was obtained by irradiation in CH3OH–H2O (1 : 1) at different
pH. In principle, if only heterolytic cleavage of the OH group takes
place on excitation, one would expect acid-catalyzed formation
of 5b–8b. The acid catalysis is anticipated if the benzyl alcohol
becomes more basic in the S1 state. However, irradiation of 5–8
in the pH range 2–10 indicated that increased acidity in this pH
range did not catalyze the reaction. Studying the reaction below
pH 2 was problematic since thermal methanolysis was observed,
particularly for 6 and 8. On the contrary, irradiation of 5 and
8 in basic solutions above pH 10 showed that photolysis was
2-Phenylphenols undergo ESIPT and formal solvent-assisted
ESPT, giving rise to ortho- and para-biphenyl QMs, respectively,
by a transfer of the OH proton to the carbon atoms of the
adjacent phenyl ring.8c,d,16,23 3-Phenylphenols cannot undergo
ESIPT, however, the question remains if solvent-assisted formal
transfer of the proton to the carbon atoms can take place. To probe
for the formal ESPT and possibility of deuterium incorporation
at the adjacent phenyl ring, we performed photolyses of 5 and 6
in CH3CN–D2O (9 : 1). After 1 h photolysis and aqueous work-
up, the composition of the solution was analyzed by NMR
and MS to determine the position and extent of deuteration,
respectively. MS analysis revealed that irradiation furnished 15%
of monodeuterated 5 and 7% of monodeuterated 6. However,
in 5 the deuterium was not regiospecifically incorporated in the
phenyl ring bearing the adamantyl substituent. NMR indicated
formation of 5-D1 and 5-D2 (eqn (7)) suggesting that solvent-
assisted ESPT via QMs is not the predominant pathway for the
deuterium incorporation. In the para-derivative 6 deuterium was
incorporated at the phenyl ring bearing the adamantane (6-D, eqn
(8)), but the efficiency of the reaction is very low, much lower than
the solvolysis in CH3OH giving 6b.
Table 1 Quantum yields of the photomethanolysis reactiona
Comp.
U
4
5
6
7
8
0.10 0.05b
0.15 0.03
0.50 0.05
0.06 0.02
0.20 0.03
(7)
(8)
a Photolysis performed in CH3OH–H2O (1 : 1) by use of valerophenone
actinometer (photolysis in aqueous CH3CN U = 0.65
0.03).22 The
composition of the solution was analyzed by HPLC. b Quantum yield
for the formation of 4b, 9, and 10.
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Table 2 Photophysical parameters of 4–8
U (CH3CN– t (CH3CN)/
at 450 nm. For example, fluorescence spectra of 8 in CH3CN and
CH3CN–H2O (1 : 4) are shown in Fig. 2. The fluorescence band
at 450 nm seen only in the aqueous solvent is assigned to the
fluorescence of the corresponding phenolates formed by adiabatic
ESPT. The assignment is corroborated by observation of the same
fluorescence band in basic media (pH>10) where the phenols are
deprotonated (see the ESI†).
t (CH3CN–
U (CH3CN)a H2O)
nsb
H2O)/ns
4
5
6
7
8
0.041 0.001 0.033 0.003 0.24 0.01 (13%) 0.26 0.03 (35%)
4.1 0.1 (55%)
5.02 0.01
2.8 0.1 (65%)
0.66 0.08
0.58 0.05
0.53 0.03
0.69 0.05
0.13 0.03
0.12 0.01
0.12 0.02
0.11 0.02
0.67 0.02 phenol
2.3 0.1 phenolate
0.46 0.02 phenol
2.4 0.1 phenolate
0.42 0.02 phenol
3.1 0.1 phenolate
0.34 0.02 phenol
2.8 0.1 phenolate
4.47 0.01
5.10 0.01
4.69 0.01
a Quantum yield of fluorescence determined by use of fluorene in CH3OH
as a reference (U = 0.68).24 b Singlet excited state lifetimes measured by
time-correlated single photon timing method.
Fluorescence study
It is generally accepted that ESIPT and ESPT reactions of phenols
take place from the singlet excited state.8,10,18,21 Therefore, we
studied fluorescence of 4–8 to gain more insight into their singlet
excited state properties and reactivity. Fluorescence quantum
yields were measured by using fluorene as a reference compound
(U = 0.68).24 Singlet excited state lifetimes were determined by
time-correlated single photon timing (SPT) (compiled in Table
2). Fluorescence spectra of 4–8 in CH3CN are characterized by
structureless bands with the maxima at 330 nm and relative high
quantum yields of fluorescence (except for the ortho-derivative
4). In addition, fluorescence of 5–8 displayed single-exponential
decays giving singlet-excited state lifetimes of ~4.5–5.1 ns.
Addition of H2O to the CH3CN solution resulted in pronounced
fluorescence quenching for 5–8. An example of the fluorescence
spectra for 7 and the corresponding Stern–Volmer plot are shown
in Fig. 1. The quenching of fluorescence by addition of H2O is in
accordance with an opening of a new deactivation pathway from
the singlet excited state due to the presence of a protic solvent,
that is, transfer of the phenolic OH proton to the H2O clusters. A
small addition of H2O (< 3 M) resulted in bathochromic shifts of
~10 nm, but significant quenching occurred on further addition of
H2O. The observed non-linear dependence in the Stern–Volmer
plot of quenching by H2O is typical for systems where ESPT
takes place.25 In the presence of a higher concentration of H2O
emission spectra of 5–8 are characterized by dual fluorescence
with a band at 350 nm and additional band (not seen in CH3CN)
Fig. 2 Fluorescence spectra of 8 in CH3CN (full) and CH3CN–H2O
(dash) (lexc = 260 nm).
In aqueous media ESPT for phenols 5–8 was also indicated by
SPT measurements. Whereas in the CH3CN solution fluorescence
decays were single exponential, in CH3CN–H2O they were best
fitted to two-exponential functions. At shorter wavelengths a short
decay component associated to the fluorescence of phenols has
stronger contribution. On the contrary, at longer wavelength where
phenolates emit, fluorescence decay is characterized by predomi-
nant long decay component with a negative pre-exponential factor,
due to the formation of fluorescent species in the excited state.
Acid–base properties of the singlet excited states of 4–8 were
investigated by fluorescence titrations with acid (HClO4) and base
(NaOH). The titrations with acid resulted in quenching of the
fluorescence in the pH range 2.5–3.0 for 5–8 (see the ESI†). The
Stern–Volmer plots (U/U0 vs. H+ concentration) showed a linear
relationship, indicating a dynamic quenching with quenching
constants in the range kq = (1–7) ¥ 1010 M-1 s-1. However, the
addition of acid resulted in only quenching of the fluorescence
but no change in the appearance of the spectra. The ratio of the
intensity corresponding to the emission from phenol vs. phenolate
remained the same. The finding suggests that the most basic site in
the excited state of 3-phenylphenols is not the phenolate anion, but
rather benzyl alcohol or an aromatic ring. The estimated pKa value
Fig. 1 Fluorescence titration of CH3CN solution of 7 with H2O (left) and Stern–Volmer plot of fluorescence quenching by H2O (right).
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of 3-phenylphenol in the singlet excited state is 1.36,19 so a higher
concentration of acid (pH<2) would be required to block the
deprotonation in the excited state. Furthermore, the finding is in
accordance with the operation of a formal solvent-assisted transfer
of the proton from the phenol OH to the benzyl alcohol. Addition
of base to the solutions of 4–8 induced significant fluorescence
quenching and changed the appearance of the spectra. At higher
pH the band at 350 nm gradually disappeared, whereas the band
at 450 increased, in accordance with the assignment of the bands
to the emission from phenol and phenolate, respectively.
The photophysical properties of the ortho-derivative 4 are
quite different from 5–8. The molar absorption coefficient in
the maximum of the absorption is lower than for the other 3-
phenylphenols, and the fluorescence quantum yield in CH3CN
solution is much lower. The fluorescence decay of 4 is not a single
exponential function. Similar behavior has also been observed for
the ortho-adamantyl derivative of 4-phenylphenol wherein it was
rationalized by a steric hindrance of the adamantane resulting
in a very twisted geometry of the biphenyl chromophore.17 The
addition of H2O does not quench the fluorescence but induces
an increase, indicating that protic solvent blocks a non-radiative
deactivation pathway from the singlet excited state. However, in
very aqueous media (CH3CN–H2O 1 : 4) phenolate emission was
observed indicating that 4 undergoes ESPT. Interestingly, contrary
to 5–8, titration with HClO4 performed under identical conditions
induced an increase of fluorescence (see ESI†). The finding may
be rationalized by a higher pKa value in S1 for the ortho-derivative
compared to 5–8 due to more twisted geometry.
Photolysis of 3-hydroxybiphenyls 7 and 8 in aqueous media
is expected to give rise to triphenylmethyl cations or zwitterions
that should be longer-lived in aqueous solvent (compared to 17–
19) and detectable by nanosecond LFP. Hence, we conducted
measurements in CH3CN and CH3CN–H2O wherein differences
in the transient absorptions should be due to zwitterions, or
other transients from the ESPT pathways that are possible only
in protic media. In N2-purged CH3CN solutions (similar to the
TFE solutions of 4–6) we observed transient absorptions of the
corresponding triplets with maxima at 370 nm decaying within
10–15 ms that were quenched by O2. However, on addition of
H2O, a new band appeared in the spectra at 420 nm for 7 and
500 nm for 8 (Figs. 3 and 4). The decay of the new band was not
affected by O2, in accordance with the assignment as zwitterions
(20 and 21) or cations (20H+ and 21H+). In both N2 and O2 purged
CH3CN–H2O solution the decay was mono-exponential with the
rate constants: k = 1.3 ¥ 105 s-1(t = 7.5 ms) for 7 and k = 4.0 ¥ 104
s-1 (t = 25 ms) for 8. The assignment of the transient absorption
at 420 nm obtained by LFP of 7 to 20 or 20H+ is in accordance
with the published spectra of triphenylmethy cations.27 On the
other hand, absorption maximum at 500 nm obtained by LFP
of 8 was somewhat surprising at first glance. However, it is in
accordance with the published maxima in the fluorescence spectra
of triarylmethyl cations.28 In 21 or 21H+ the positive charge should
be more delocalized over the biphenyl system than in 20 or 20H+.
Therefore, the lowest-energy absorption at 500 nm becomes more
intense. Furthermore, better resonance stabilization of the positive
charge in the para-derivatives (21 or 21H+) is also demonstrated
through its slower decay kinetics.
Laser flash photolysis
To characterize zwitterions and other transient species in the
photochemistry of 4–8, we carried out a LFP study. The transient
spectra for 7 and 8 were taken in CH3CN, CH3CN–H2O and TFE,
whereas for 4–6, the measurements were performed only in TFE.
The anticipated zwitterions 17–19 probably have shorter lifetimes
than 20 ns in CH3CN–H2O, and hence, cannot be detected by
nanosecond LFP. In TFE, which is less nucleophilic, cations (and
zwitterions) have significantly longer lifetimes.26 However, in N2-
purged TFE solution of 4 we detected only the presence of a triplet
characterized by a maximum of the absorption at 370 nm, decaying
with k = 4 ¥ 104 s-1(t = 25 ms). The assignment is based on the sim-
ilarity with spectra detected for other hydroxybiphenyls8c,d,16,17,18
and quenching by O2. In O2-purged solution, after the decay of
the negative transient absorbance due to the absorption of the
laser flash and fluorescence, only negligible weak transients were
detected that were not assigned. Similarly, in the N2-purged TFE
solution of 5 and 6 the transients were detected associated to the
triplet–triplet absorption with a maximum at 370 nm. The decay
of the transients could be fitted with two exponential functions
with k = 2.5 ¥ 105 s-1(t = 3.8 ms) and k = 6.5 ¥ 104 s-1(t = 15 ms) for
5, and k = 2.8 ¥ 105 s-1(t = 3.5 ms) and k = 1.5 ¥ 105 s-1(t = 6.7 ms) for
6. In O2-purged solution the triplets were quenched, and decayed
with k = 1.5 ¥ 107 s-1(t = 65 ns). After decay of the triplet in
O2-purged solution only weak transient absorptions were detected
which precluded further study. The anticipated zwitterions 17–19
have phenylbenzyl cations as chromophores. Therefore, they are
expected to absorb in the same wavelength as triplets, at 300–400
nm.26 Hence, due to their short lifetimes and overlapping with the
signal of the triplets their detection was not possible.
The experiment that enabled us to probe whether the observed
transients correspond to zwitterions 20 and 21 or the protonated
cations 20H+ and 21H+, respectively, was obtained by LFP of
the corresponding methoxy derivatives 7a and 8a that can give
only cations 22 and not the zwitterions. The transient absorp-
tion spectra of the methoxy derivatives recorded in O2-purged
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Fig. 3 Transient absorption spectra recorded for 8 in N2-purged CH3CN (left) and CH3CN–H2O 1 : 1 solution (right).
Fig. 4 Transient absorption spectra of zwitterions 20 (left) and 21 (right) in O2-purged CH3CN–H2O (1 : 1) solution taken at 790 ns and 1.4 ms after the
laser flash, respectively (Insets: decays at 420 nm (left) and 500 nm (right)).
Table 3 Bimolecular quenching constants for QM 20 and cation 21 in
CH3CN–H2O did not show the characteristic bands at 420 nm and
500 nm. Instead, the broader transient absorption was observed at
500–700 nm decaying with the rate constant k = 5 ¥ 104 s-1(t = 20 ms)
that we tentatively assigned to radical cations 23. The assignment
is based on similarity with published spectra of styrene radical-
cations and quenching with nucleophile ethanolamine (kq = 3 ¥
108 M-1s-1).29 Consequently, the observed transient absorptions at
420 and 500 nm (Fig. 4) were undoubtedly assigned to zwitterions
20 and 21.
LFP of 7 and 8 was further conducted in TFE wherein the
observed transients are expected to show slower decay kinetics.
However, in TFE, which is a more acidic solvent, the intensity
of the observed transients was 3–5 times weaker which precluded
further studies. Lower intensity of the transient absorption in more
acidic media is in line with the importance of photodissociation
of the phenolic OH in the mechanism of zwitterion formation.
After assignment of the transients, we performed a study of their
kinetics with nucleophiles. Because of the relevance in biological
systems, we were interested in the kinetics with N-nucleophiles. As
examples, we chose ethanolamine and azide, the ubiquitous cation
quenchers. The estimated quenching constants are compiled in
Table 3. It is interesting to note that azide quenched the transients
with a rate constant that is roughly an order of magnitude lower
than the diffusion limit, contrary to previous reports.27 On the
other hand, the observed quenching constant with ethanolamine
CH3CN–H2O solution
Transient
species
Azide
Ethanolamine
kq/s-1M-1
kq/s-1 M-1
20
21
8.5 ¥ 108
3.5 ¥ 108
1.4 ¥ 107
3.5 ¥ 106
is in line with the published values for the quenching of trityl
cations by primary amines.27 However, the data in Table 3 clearly
points to stabilization of the cationic center, by conjugation with 3-
phenoxyl in 21, compared to 20, inducing slower kinetics with both
nucleophiles, with the effect being larger for ethanolamine. This
further suggests that the reactivity of these biphenyl zwitterion
derivatives depends on their structure. Their reactivity cannot be
expressed by N+,30 in line with the findings obtained by McClelland
et al. for trityl cations.27
Photochemical reaction mechanisms
From the results of preparative photolyses, fluorescence and
LFP studies, the mechanisms of photochemical reactions of
4–8 can be proposed. Excitation of 4 in aqueous media leads
to ESPT, as demonstrated by fluorescence measurements wherein
the phenolate emission was detected. The ESPT is coupled with
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Scheme 1
a loss of the hydroxyl group from the benzylic position (overall
dehydration) delivering zwitterion 17 (Scheme 1). Although 17 has
not been detected by LFP due to its short lifetime, it is inferred
from the formation of fluorenes 9 and 10, and solvolysis product
4b.
Excitation of 5 and 6 to the S1 in the aqueous media also leads
to ESPT, as indicated by detection of the phenolate emission.
Although the exact mechanism of the photodehydration still
remains unsolved, it is probably the deprotonation in the excited
state that triggers the elimination of the hydroxyl group from the
benzylic position, resulting in the formation of zwitterions 18 and
19, respectively (Scheme 2). The importance of the ESPT in the
mechanism is indicated due to the inefficient solvolysis reaction
of the methoxy derivatives 5a and 6a, no apparent acid-catalysis
in the photosolvolysis reactions, and less efficient solvolysis in the
basic media where phenols are deprotonated in the ground state.
In addition, formal solvent assisted ESPT leading to zwitterions
is also indicated by fluorescence titrations with acid wherein it
was demonstrated that the most basic site in the excited state of
the phenolate is not the anion, but some other site, presumably
the benzylic alcohol. Therefore, the mechanistic scenario for the
formation of zwitterions can probably be best described as type
II or III (vide supra).9 The fact that the reaction is less efficient
at high pH indicates that the dissociated proton at neutral pH
plays a role in the departure of the benzylic OH, either by proton
transfer to the basic site, or simply by increasing the effective pH
within the solvent cage. Zwitterions 18 and 19 were not detected by
LFP due to their short lifetimes and overlapping of the absorption
spectra with the spectra of triplets. However, their intermediacy is
strongly indicated by the formation of solvolysis products 5b and
Scheme 2
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6b. Zwitterions react with nucleophiles (e.g. solvent) leading to
adducts or solvolysis products with high quantum yields. Higher
quantum yield for the photomethanolysis reaction of 6 than 5 is
in line with the activation of 3 and 4¢ positions in the biphenyl
system due to the meta-effect.20
Photolyses of 5 and 6 in the presence of D2O indicated that pho-
todehydration leading to zwitterions 18 and 19 is probably not in
competition with the solvent-assisted formal proton (deuterium)
transfer from the phenol OH (OD) to the carbon atoms leading to
QMs 24 and 25, respectively (Scheme 2). The finding is logical since
carbon atoms of the aromatic ring are probably less basic than the
benzyl alcohol oxygen. In addition, formation of 24 and 25 leads
to the loss of aromaticity in one phenyl ring, whereas in 18 and
19 both phenyls remain aromatic. However, we cannot completely
rule out a solvent-assisted ESPT pathway with protonation of
the carbon atoms, particularly for derivative 6 wherein the cation
would be less destabilized by the hydroxyadamantyl moiety. In any
case, the quantum yield for the protonation of C atoms is very low,
much lower than for the photodehydration.
Photochemical reactions of triphenylmethanols 7 and 8 are sim-
ilar to the reactions of adamantyl derivatives 5 and 6. Excitation to
S1 leads to ESPT which triggers the expulsion of the benzylic OH
resulting in the dehydration and formation of zwitterions 20 and
21. The zwitterions were detected by LFP in CH3CN–H2O wherein
they have lifetimes of 7.5 and 25 ms respectively. It is interesting to
note that LFP of 5 and 6 in TFE, which is more acidic, gave
weaker signal of the corresponding zwitterions, in accordance
with lower quantum yield for the formation of zwitterions in
more acidic media when phenols are less deprotonated in the
excited state. The zwitterions react with nucleophiles giving
adducts and/or solvolysis products. Interestingly, zwitterions 20
and 21 react with azide with bimolecular quenching constants
that are by one order of magnitude lower than those reported
for triphenylmethyl cations. The finding is in line with better
stabilization of the zwitterions through additional conjugation in
the biphenyl system and stabilization by the phenoxide anion.
Namely, the phenoxide (O-) at the meta-position has electron
donating character (Hammet constant sm = -0.47), whereas OH
Table 4 IC50 values (in mM)a
Comp.
log(P)d
4.6
HCT 116
19 1c
18
11 0.7
14
>100c
9
36
26
19 0.7
18
68 7c
64
18 1c
MCF-7
H 460
1
17
5
16 0.5
1b
2
1
19 0.4
17
4
2
>5
11
15
2
4
1
2b
3
3
12 0.4
>100
>100
4.6
>100
>100
29 13
3b
6
1c
5
5
4.6
29
25
20
3
3
1
6b
8
19
20
17
50
13
13
16
5
5
1
2
3
4
2
3
6.1
8b
26
26b
27
27b
28
28b
0
18 0.2
3.3
20
16
2
3
3
6.1
18 0.8
16
18
3
1
2.3
>100c
1
>100
0.07 0.04
0.3 0.2c
0.2 0.05
a IC50; the concentration that causes 50% growth inhibition. b Irradiated
3 ¥ 1 min at 300 nm with 6 lamps. c From reference 17 d Logarithm of
the partition coefficient [n-octanol/water] calculated by ChemDraw 10
software.
that causes 50% inhibition of the cell growth) are compiled in
Table 4. Without the irradiation all compounds except 3 showed
antiproliferative effect in micromolar concentration range. The
most active compound (in dark) is the ammonium derivative of
2-phenylphenol 2. The highest activity may be correlated with the
highest solubility of that compound in H2O. However, solubility in
H2O and lipophylicity (expressed as logP, Table 4) are not the only
parameters that determine the activity, since structural isomers 1,
3 and 6 that have the same logP exhibit quite different activity; the
structure of the molecule probably plays an important role.
in the meta-position has electron withdrawing character (sm
=
0.12).24 The additional stabilization contributes more in 21 than
in 20, resulting in its slower decay kinetics and lower values of the
quenching constants with nucleophiles (Table 3).
On exposure of cells incubated with compounds to 300 nm
irradiation, an enhanced activity was observed for derivatives
3, 6 and 26. On the contrary, irradiation of the cells that were
incubated with 2, diminished the activity. The finding is logical
since the photolysis of 2 gave 1,16 which is (in dark) less active than
2. It is interesting to note that compounds that are derivatives
of triphenylmethanol, 8 and 27, exhibit no enhancement of
the antiproliferative activity, suggesting that the corresponding
zwitterion 21 and the QM exhibit no activity. The enhancement
of the activity for the compounds 3, 6, and 26 on irradiation
is in line with the activity being due to the formation of the
corresponding QMs (or zwitterion from 6) in the photochemical
reaction. Furthermore, the finding suggests that enhancement
of the activity can be accomplished by substitution of the QM
methylene position with alkyl groups, rather than phenyl. A further
interesting finding was obtained by investigation of the activity
for compound 3. On excitation, 3 showed selective activity only
Antiproliferative study
Our preliminary results indicated that cells incubated with the
adamantyl derivative of 4-phenylphenol 3 showed enhanced
antiproliferative activity compared to the cells that were not irra-
diated, in accordance with the activity being due to the formation
of QM 3.17 Herein we investigate further the antiproliferative
effect of the photogenerated QMs on three cancer cell lines
HCT 116 (colon carcinoma), MCF-7 (breast carcinoma), and
H 460 (lung carcinoma), with a series of 7 compounds. The
tested compounds were derivatives of 2-phenylphenol 1 and 2, 3-
phenylphenol 6 and 8, and 4-phenylphenol 3, 26, and 27. Psoralen
28 was used as a positive reference compound that is known to
exhibit antiproliferative activity on photoactivation.5 The cells
were incubated with the compounds and kept in dark, or irradiated
at 300 nm 3 ¥ 1min. The activities expressed as IC50 (concentration
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to HCT 116 cell line. On the other hand, compounds 6 and
26 showed more pronounced activity/selectivity (up to 4-fold
increase in the activity for compound 26) towards MCF-7 cell
line. Although at the moment, we cannot give rational explanation
for the observed selectivity, similar cell line-selectivity was also
observed by Freccero et al.5b
In order to check the DNA-alkylation ability of 3, 6 and 26,
as well as their abilities to unwind, or cleave the supercoiled
plasmid DNA, we performed irradiations of DNA in the presence
of compounds followed by alkaline and native DNA gel elec-
trophoresis, respectively. However, although we observed certain
differences in the plasmid migration performance, we cannot draw
straightforward conclusions about the DNA-damaging ability of
these compounds. We are currently performing further, more
sensitive methods to check the DNA-alkylation ability of 3, 6 and
26. Nevertheless, these preliminary data are encouraging enough
for further study of 3, 6 and 26 as potential leads and for further
derivatization and investigation of the antiproliferative activity on
exposure to light.
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
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, 3-bromoanisol, 2-
adamantanone, benzophenone, solution of BBr3) were purchased
from the usual commercial sources and were used as received.
Solvents for chromatographic separations were purified by distil-
lation.
General procedure for the Grignard reaction
Conclusion
The reaction was carried out in a two-neck round bottom flask
(100 mL) under 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
magnesium reacted (1–1.5 h). The solution of the Grignard reagent
was cooled to rt and a THF solution (20 mL) of 2-adamantanone
or benzophenone (10 mmol) was added dropwise during 1 h. After
the addition was completed, the reaction mixture was refluxed for 4
h and stirred at rt over night. The next day, to the reaction mixture a
saturated solution of ammonium chloride (100 mL) was added, the
layers were separated, and the aqueous layer additionally acidified
with 1 M HCl until all solid dissolved. The acidified aqueous layer
was extracted with diethyl ether (3 ¥ 30 mL), the 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 eluent.
We prepared new derivatives of 3-phenylphenol 4–8 and investi-
gated their photochemistry and photophysics. In aqueous solvent,
upon excitation to the S1, they undergo deprotonation of the
phenolic OH that is coupled with the protonation of the benzylic
alcohol and dehydration, delivering zwitterions 17–21. The zwit-
terions react with nucleophiles giving adducts. Triphenylmethyl
zwitterions 20 and 21 were detected by LFP. In the aqueous
solvent they have lifetimes of 7.5 and 21 ms, respectively. The
reactivity of zwitterions with azide and ethanolamine was studied
by LFP. The corresponding quenching constants are lower than
the reported for trityl cations and dependent on the zwitterion
structure. Antiproliferative activity was investigated on a series
of 2-, 3-, and 4-phenylphenols, with and without exposure to
300 nm light, on three human cancer cell lines HCT 116, MCF-
7, and H 460. The enhancement of the activity on exposure to
light was observed for compounds 3, 6, and 26 suggesting that
QMs and zwitterions with alkyl substituents at the methylene
position exhibit higher activity than QMs with phenyl substituents.
The observed selectivity of 3, 6 and 26 on antiproliferation cells
classifies them as potential leads for further derivatizations and
investigations.
Experimental
General
2-(2-Hydroxyadamantan-2-yl)-3¢-methoxybiphenyl (4a)
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. Deuterium content was determined by MS recorded
on an Agilent 6410 Triple Quadrupole Mass Spectrometer by
use of the electrospray ionization technique. The spectra were
The Grignard reagent was prepared from 2-bromo-3¢-
methoxybiphenyl (1.03 g, 3.8 mmol) and magnesium (0.11 g,
4.56 mmol), and reacted with 2-adamantanone (0.57 g, 3.8 mmol)
to afford 1.53 g of the crude product that was purified on a column
of silica gel to give the product (0.72 g, 57%) in a form of colorless
oil.
Colorless oil, IR (KBr) n/cm-1: 3451 (OH), 2899 (CH), 2852
(CH); 1H NMR (300 MHz; CDCl3) d (ppm) 7.62 (1H, d, J = 7.8
Hz), 7.35 (1H, dt, J = 1.5 Hz, J = 8.0 Hz), 7.22–7.30 (2H, m), 7.12
(1H, dd, J = 1.5 Hz, J = 7.5 Hz), 6.92–6.98 (2H, m), 6.86 (1H, dd,
J = 1.8 Hz, J = 7.8 Hz), 3.80 (3H, s, OCH3), 2.27 (2H, br s), 2.18
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(1H, br s), 2.14 (1H, br s), 1.86 (1H, s, OH), 1.45–1.75 (10H, m);
13C NMR (75 MHz; CDCl3) d (ppm) 158.83 (s), 146.15 (s), 141.89
(s), 141.68 (s), 133.17 (d), 128.61 (d), 127.13 (d), 126.84 (d), 126.38
(d), 121.40 (d), 114.42 (d), 112.48 (d), 77.68 (s), 55.14 (q), 37.44
(t), 34.94 (2C, d), 37.71 (2C, t), 32.85 (2C, t), 27.17 (d), 26.30 (d);
HRMS (MALDI) calculated for C23H26O2 (+H+-H2O) 317.1900,
found 317.1891.
Hz), 7.36 (1H, t, J = 7.7 Hz), 7.22–7.32 (12H, m), 7.09 (1H, dd, J =
1.5 Hz, J = 7.7 Hz), 7.04 (1H, t, J = 2.0 Hz), 6.85 (1H, dd, J = 0.8
Hz, J = 8.1 Hz), 3.82 (3H, s, OCH3), 2.83 (1H, s, OH); 13C NMR
(150 MHz; CDCl3) d (ppm) 159.80 (s), 147.29 (s), 146.71 (2C, s),
142.52 (s), 140.58 (s), 129.59 (d), 128.16 (d), 127.88 (4C, d), 127.84
(4C, d), 127.23 (2C, d), 127.02 (d), 126.65 (d), 125.98 (d), 119.62
(d), 112.91 (d), 112.57 (d), 82.00 (s), 55.17 (q); HRMS (MALDI)
calculated for C26H22O2 (+K) 405.1251, found 405.1239.
3-(2-Hydroxyadamantan-2-yl)-3¢-methoxybiphenyl (5a)
(4¢-Methoxybiphenyl-3-yl)diphenylmethanol (8a)
The Grignard reagent was prepared from 3-bromo-3¢-
methoxybiphenyl (1.64 g, 6.22 mmol) and magnesium (0.16 g,
6.90 mmol), and reacted with 2-adamantanone (0.93 g, 6.22 mmol)
to afford 2.27 g of the crude product that was purified on a column
of silica gel to give the product (1.80 g, 86%) in a form of colorless
oil.
The Grignard reagent was prepared from 4-bromo-3¢-
methoxybiphenyl (0.80 g, 3.04 mmol) and magnesium (0.090 g,
3.60 mmol), and reacted with benzophenone (0.55 g, 3.04 mmol)
to afford 1.56 g of the crude product that was purified on a column
of silica gel to give the product (0.60 g, 54%) in the form of colorless
oil.
Colorless oil, IR (KBr) n/cm-1: 3054, 3051, 3025, 2938 (CH),
2833 (CH); 1H NMR (600 MHz; CDCl3) d (ppm) 7.52 (2H, d, J =
8.5 Hz), 7.26–7.36 (13H, m), 7.17 (1H, dd, J = 0.8 Hz, J = 7.6
Hz), 7.11 (1H, t, J = 2.0 Hz), 6.88 (1H, dd, J = 1.6 Hz, J = 8.0
Hz), 3.84 (3H, s, OCH3), 2.82 (1H, s, OH); 13C NMR (150 MHz;
CDCl3) d (ppm) 159.85 (s), 146.71 (2C, s), 145.97 (s), 142.07 (s),
139.81 (s), 129.64 (d), 128.22 (2C, d), 127.88 (4C, d), 127.82 (4C,
d), 127.21 (2C, d), 126.56 (2C, d), 119.50 (d), 112.72 (d), 112.68
(d), 81.80 (s), 55.17 (q); HRMS (MALDI) calculated for C26H22O2
(+K) 405.1251, found 405.1237.
Colorless oil; IR (KBr) n/cm-1: 3449 (OH), 2907 (CH), 2853
1
(CH); H NMR (300 MHz; CDCl3) d (ppm) 7.74 (1H, s), 7.46–
7.56 (2H, m), 7.43 (1H, t, J = 7.5 Hz), 7.35 (1H, t, J = 7.9 Hz),
7.17 (1H, d, J = 7.7 Hz), 7.11 (1H, t, J = 2.0 Hz), 6.89 (1H, dd,
J = 2.3 Hz, J = 8.1 Hz), 3.86 (3H, s, OCH3), 2.62 (1H, br s), 2.45
(1H, br s), 2.41 (1H, br s), 1.92 (1H, br s), 1.68–1.82 (10H, m);
13C NMR (75 MHz; CDCl3) d (ppm) 159.80 (s), 145.73 (s), 142.90
(s), 141.41 (s), 129.62 (d), 128.99 (d), 126.10 (d), 124.44 (d), 124.39
(d), 119.68 (d), 113.00 (d), 112.41 (d), 75.62 (s), 55.21 (q), 37.55
(t), 35.61 (2C, d), 34.82 (2C, t), 32.86 (2C, t), 27.32 (d), 26.81 (d);
HRMS (MALDI) calculated for C23H26O2 (+H+-H2O) 317.1900,
found 317.1912.
General procedure for the removal of the methyl ether groups from
phenols with BBr3
4-(2-Hydroxyadamantan-2-yl)-3¢-methoxybiphenyl (6a)
The Grignard reagent was prepared from 4-bromo-3¢-
methoxybiphenyl (1.35 g, 5.13 mmol) and magnesium (0.16 g,
6.90 mmol), and reacted with 2-adamantanone (0.95 g, 6.3 mmol)
to afford 1.85 g of the crude product that was purified on a column
of silica gel to give the product 6a (1.42 g, 83%) in a form of
colorless crystals.
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 5a–8a
(10 mmol), dissolved in dry CH2Cl2 (100 mL) and the solution
was cooled to 0 ◦C by ice-bath. To the cold solution, a solution of
BBr3 in CH2Cl2 (1 M, 30 mL) was added dropwise for 1 h by use
of a syringe. After the addition was completed, the stirring was
continued for 1 h at 0 ◦C, and then at rt over night. The next day to
the reaction mixture 100 mL of cold water was added, 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
rotary evaporator. The crude product obtained was purified on a
column filled with silica gel by use of CH2Cl2 and CH2Cl2-EtOAc
(4 : 1) as eluent, or by crystallization.
Colorless crystals, mp 65–68 ◦C; IR (KBr) n/cm-1: 3398 (OH),
2904 (CH), 2852 (CH); 1H NMR (300 MHz; CDCl3) d (ppm) 7.60
(4H, s), 7.35 (1H, t, J = 7.8 Hz), 7.18 (1H, d, J = 7.7 Hz), 7.13
(1H, t, J = 2.0 Hz), 6.89 (1H, dd, J = 1.8 Hz, J = 8.1 Hz), 3.86
(3H, s, OCH3), 2.59 (2H, br s), 2.45 (1H, br s), 2.41 (1H, br s),
1.92 (1H, br s), 1.68–1.82 (10H, m); 13C NMR (75 MHz; CDCl3)
d (ppm) 159.79 (s), 144.45 (s), 142.09 (s), 139.77 (s), 129.61 (d),
127.28 (2C, d), 125.74 (2C, d), 119.43 (d), 112.64 (d), 112.55 (d),
75.39 (s), 55.15 (q), 37.55 (t), 35.55 (2C, d), 34.79 (2C, t), 32.83 (2C,
t), 27.30 (d), 26.81 (d); HRMS (MALDI) calculated for C23H26O2
(+H+-H2O) 317.1900, found 317.1912.
3-(2-Hydroxyadamantan-2yl)-3¢-hydroxybiphenyl (5)
From 3-(2-hydroxyadamantan-2-yl)-3¢-methoxybiphenyl (5a, 1.40
g, 4.2 mmol) and a solution of BBr3 (1 M, 22 mL), the reaction
gave 1.60 g of the crude product that was purified by crystallization
from a mixture of carbon tetrachloride and hexane to afford 1.12
g (83%) of the pure product 5 in the◦form of colorless crystals.
Colorless crystals, mp 181–183 C; IR (KBr) n/cm-1: 3565
(3¢-Methoxybiphenyl-3-yl)diphenylmethanol (7a)
The Grignard reagent was prepared from 3-bromo-3¢-
methoxybiphenyl (1.20 g, 4.60 mmol) and magnesium (0.13 g,
5.60 mmol), and reacted with benzophenone (0.84 g, 4.60 mmol)
to afford 1.90 g of the crude product that was purified on a column
of silica gel to give the product 7a (0.98 g, 58%) in the form of
colorless oil.
1
(OH), 3379 (OH), 2913 (CH), 2853 (CH); H NMR (300 MHz;
DMSO-d6) d (ppm) 9.49 (1H, br s, OH), 7.65 (1H, s), 7.36–7.50
(3H, m), 7.25 (1H, t, J = 7.8 Hz), 7.00–7.08 (2H, m), 6.76 (1H, dd,
J = 1.2 Hz, J = 7.8 Hz), 2.50 (2H, br s), 2.42 (1H, br s), 2.38 (1H,
br s), 1.82 (1H, br s), 1.52–1.76 (10H, m); 13C NMR (75 MHz;
Colorless oil, IR (KBr) n/cm-1: 3474 (OH), 3057 (CH), 3025
1
(CH), 2936 (CH), 2834 (CH); H NMR (600 MHz; CDCl3) d
(ppm) 7.56 (1H, t, J = 1.6 Hz), 7.50 (1H, d, J = 1.6 Hz, J = 7.7
390 | Photochem. Photobiol. Sci., 2012, 11, 381–396
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DMSO-d6) d (ppm) 157.81 (s), 146.71 (s), 142.24 (s), 140.11 (s),
129.92 (d), 128.69 (d), 124.78 (d), 124.69 (d), 124.05 (d), 117.46 (d),
114.26 (d), 113.50 (d), 73.59 (s), 37.43 (t), 34.84 (2C, d), 34.35 (2C,
t), 32.58 (2C, t), 26.98 (d), 26.58 (d); HRMS (MALDI) calculated
for C22H24O2 (+H+-H2O) 303.1743, found 317.1750.
147.66 (s, 2C), 146.94 (s), 141.20 (s), 138.58 (s), 129.85 (d), 128.30
(2C, d), 127.74 (4C, d), 127.54 (4C, d), 126.64 (2C, d), 125.72 (2C,
d), 117.38 (d), 114.38 (d), 113.39 (d), 80.41 (s); HRMS (MALDI)
calculated for C25H20O2 (+H+-H2O) 335.1430, found 335.1445.
2-(2-Hydroxyadamantan-2-yl)-3¢-hydroxybiphenyl (4)
4-(2-Hydroxyadamantan-2-yl)-3¢-hydroxybiphenyl (6)
The reaction was carried out under N2-atmosphere in a two
neck flask (100 mL) equipped with a dropping funnel, and a
condenser. The outlet of the N2 from the condenser was passed
through a solution of sodium ethoxide in ethanol. In the flask
was placed sodium hydride (0.400 g, 16.7 mmol) and suspended
in dry DMF (5 mL). The suspension was cooled in an ice-bath
and a solution of ethyl mercaptane (1.3 mL, 16.7 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. 2-(2-Hydroxyadamantan-2-yl)-3¢-methoxybiphenyl (4a,
1.75 g, 5.2 mmol) was dissolved in DMF (10 mL) and added to
the reaction mixture. After the addition, the mixture was refluxed
over 4 h, cooled, and poured to 100 mL of H2O. The aqueous
mixture was washed with hexane (2 ¥ 50 mL) and acidified by a
saturated solution of ammonium chloride. Extraction with ethyl
acetate was carried out (3 ¥ 100 mL), the extracts were dried over
anhydrous MgSO4, the solid was removed by filtration, and the
solvent removed on a rotary evaporator. Reaction gave the crude
product that was purified by column chromatography on silica gel
by use of CH2Cl2 as an eluent to furnish 370 mg (22%) of the
product in a form of colorless crystals.
From 4-(2-hydroxyadamantan-2-yl)-3¢-methoxybiphenyl (6a, 1.14
g, 3.5 mmol) and solution of BBr3 (1 M, 15 mL), the reaction gave
1.60 g of the crude product that was purified by crystallization
from a mixture of carbon tetrachloride and hexane to afford 0.38
g (34%) of the pure product 6 in the◦form of colorless crystals.
Colorless crystals, mp 201–203 C; IR (KBr) n/cm-1: 3406
1
(OH), 3144 (OH), 2908 (CH), 2856 (CH); H NMR (300 MHz;
DMSO-d6) d (ppm) 9.50 (1H, br s, OH), 7.56 (2H, d, J = 8.6 Hz),
7.53 (2H, d, J = 8.6 Hz), 7.24 (1H, t, J = 7.6 Hz), 7.06 (1H, d, J =
7.6 Hz), 7.02 (1H, s), 6.75 (1H, dd, J = 1.2 Hz, J = 7.9 Hz), 2.50
(2H, br s), 2.41 (1H, br s), 2.38 (1H, br s), 1.82 (1H, br s), 1.54–
1.72 (10H, m); 13C NMR (75 MHz; DMSO-d6) d (ppm) 157.80
(s), 145.40 (s), 141.42 (s), 138.24 (s), 129.88 (d), 126.27 (2C, d),
126.17 (2C, d), 117.29 (d), 114.25 (d), 113.29 (d), 73.38 (s), 37.44
(t), 34.79 (2C, d), 34.36 (2C, t), 32.56 (2C, t), 26.98 (d), 26.58 (d);
HRMS (MALDI) calculated for C22H24O2 (+H+-H2O) 303.1743,
found 303.1740.
(3¢-Hydroxybiphenyl-3-yl)diphenylmethanol (7)
From (3¢-methoxybiphenyl-3-yl)diphenylmethanol (7a, 0.80 g,
2.2 mmol) and a solution of BBr3 (1 M, 10 mL), the reaction gave
1.00 g of the crude product that was purified by crystallization from
carbon tetrachloride to afford 0.75 g (96%) of the pure product in
a form of colorless crystals.
Mp 119–120 ◦C; IR (KBr) n/cm-1: 3430 (OH), 3364 (OH), 2901
(CH), 2855 (CH); 1H NMR (600 MHz; DMSO-d6) d (ppm) 9.26
(1H, s, OH), 7.56 (1H, d, J = 7.9 Hz), 7.32 (1H, dt, J = 1.4 Hz, J =
8.1 Hz), 7.22 (1H, t, J = 7.3 Hz), 7.08 (1H, t, J = 7.8 Hz), 6.98 (1H,
dd, J = 1.4 Hz, J = 7.5 Hz), 6.80–6.86 (2H, m), 6.67 (1H, dd, J =
2.2 Hz, J = 8.1 Hz), 4.68 (1H, s, OH), 2.24 (2H, br s), 2.19 (1H, br
s), 2.17 (1H, br s), 1.63 (1H, br s), 1.59 (1H, br s), 1.40–1.55 (6H,
m), 1.28–1.36 (2H, m); 13C NMR (150 MHz; DMSO-d6) d (ppm)
156.15 (s), 146.15 (s), 142.22 (s), 142.09 (s), 132.84 (d), 128.02 (d),
126.98 (d), 126.55 (d), 125.91 (d), 120.13 (d), 116.23 (d), 113.35
(d), 75.66 (s), 37.27 (t), 34.31 (2C, t), 34.16 (2C, d), 32.56 (2C,
t), 26.74 (d), 26.08 (d); HRMS (MALDI) calculated for C22H24O2
(+H+-H2O) 303.1743, found 317.1750.
◦
Colorless crystals, mp 141–142 C; IR (KBr) n/cm-1: 3364
(OH), 3217 (CH); 1H NMR (600 MHz; DMSO-d6) d (ppm) 9.47
(1H, s, OH), 7.50 (1H, t, J = 1.4 Hz), 7.48 (1H, dd, J = 1.7 Hz, J =
7.7 Hz), 7.36 (1H, t, J = 7.7 Hz), 7.32 (4H, dd, J = 7.2 Hz, J = 7.6
Hz), 7.23–7.28 (6H, m), 7.21 (1H, t, J = 7.8 Hz), 7.13 (1H, dd, J =
1.5 Hz, J = 7.7 Hz), 6.95 (1H, dd, J = 1.3 Hz, J = 7.7 Hz), 6.92
(1H, t, J = 2.0 Hz), 6.73 (1H, dd, J = 8.0 Hz, J = 2.3 Hz), 6.51
(1H, s, OH); 13C NMR (150 MHz; DMSO-d6) d (ppm) 157.75
(s), 148.36 (s), 147.62 (2C, s), 141.70 (s), 139.54 (s), 129.89 (d),
128.02 (d), 127.73 (4C, d), 127.55 (4C, d), 126.96 (d), 126.67 (2C,
d), 126.10 (d), 124.93 (d), 117.33 (d), 114.39 (d), 113.38 (d), 80.60
(s); HRMS (MALDI) calculated for C25H20O2 (+Na+) 375.1355,
found 375.1345.
Photochemical experiments, general
In a quartz vessel was placed a CH3OH, or CH3OH–H2O (3 : 1),
solution (100 mL, c ~ 10-3 M) of biphenyl 4–8 (20 mg–100 mg)
and irradiated in a Rayonet reactor using 16 lamps at 254 nm for
1–30 min. Prior to, and during the irradiation, the solution was
continuously purged with a stream of Ar and cooled by a cold-
finger condenser. After the irradiation, CH3OH was removed on a
rotary evaporator and the residue (if irradiated in CH3OH–H2O)
extracted with EtOAc (3 ¥ 75 mL). The extracts were dried over
anhydrous MgSO4, filtered and the solvent was removed on a
rotary evaporator. The residue was chromatographed on a thin
layer of silica gel using CH2Cl2-EtOAc (30%) as eluent.
(3¢-Hydroxybiphenyl-4-yl)diphenylmethanol (8)
From (3¢-methoxybiphenyl-4-yl)diphenylmethanol (8a, 0.548 g,
1.5 mmol) and a solution of BBr3 (1 M, 5 mL), the reaction gave
0.60 g of the crude product that was purified by crystallization
from a mixture of carbon tetrachloride toluene and ether to afford
0.46 g (98%) of the pure product in the form of colorless crystals.
◦
Colorless crystals, mp 168–170 C; IR (KBr) n/cm-1: 3561
(OH), 3363 (OH); 1H NMR (600 MHz; DMSO-d6) d (ppm) 9.49
(1H, s, OH), 7.54 (2H, d, J = 8.4 Hz), 7.32 (4H, dd, J = 7.2 Hz,
J = 7.7 Hz), 7.22–7.30 (9H, m), 7.06 (1H, d, J = 7.6 Hz), 7.02
(1H, t, J = 2.0 Hz), 6.75 (1H, dd, J = 1.6 Hz, J = 8.0 Hz), 6.46
(1H, s, OH); 13C NMR (150 MHz; DMSO-d6) d (ppm) 157.76 (s),
Irradiation of 4. (100 mg, 0.31 mmol) in 400 mL CH3OH–
H2O (3 : 1) for 30 min gave after evaporation of the methanol,
extraction, drying, and removal of the solvent crude mixture that
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was separated on a thin layer of silica using CH2Cl2-EtOAc (5%)
as eluent.
analysis, to the solution NaHCO3 was added and stirring was
continued over 1 h. The mixture was filtered and the solvent
evaporated. The remaining solid was extracted by ethyl acetate
to afford the pure product for the analysis.
Spiro[adamantane-2,9¢-(1¢-hydroxy)fluorene] (9)
40 mg (43%); colorless crystals, mp 209–210 ◦C; IR (KBr) n/cm-1:
3578 (OH), 2903 (CH), 2874 (CH), 1H NMR (600 MHz; CDCl3)
d (ppm) 8.12 (1H, d, J = 8.0 Hz), 7.76 (1H, dd, J = 1.2 Hz, J = 7.5
Hz), 7.44 (1H, dd, J = 1.2 Hz, J = 7.5 Hz), 7.33 (1H, dt, J = 1.1 Hz,
J = 7.3 Hz), 7.27 (1H, dt, J = 1.4 Hz, J = 7.3 Hz), 7.21 (1H, t, J =
7.5 Hz), 6.62 (1H, dd, J = 1.0 Hz, J = 7.8 Hz), 5.09 (1H, s, OH),
3.42 (2H, dd, J = 2.6 Hz, J = 13.8 Hz), 2.98 (2H, dd, J = 2.9 Hz, J =
13.8 Hz), 2.18 (1H, br s), 2.07 (1H, br s), 2.00 (2H, br s), 1.73–1.81
(6H, m); 13C NMR (150 MHz; CDCl3) d (ppm) 152.08 (s), 150.66
(s), 144.27 (s), 139.98 (s), 135.80 (s), 129.54 (d), 128.42 (d), 126.56
(d), 125.88 (d), 119.31 (d), 117.54 (d), 112.56 (d), 64.20 (s), 42.34
(t), 36.51 (2C, d), 36.26 (2C, t), 35.29 (2C, t), 27.93 (d), 27.63 (d);
HRMS (MALDI) calculated for C22H22O (-e-) 302.1676, found
302.1667.
3-(2-Methoxyadamantan-2-yl)-3¢-hydroxybiphenyl (5b)
Colorless crystals, mp 179–182 ◦C; IR (KBr) n/cm-1: 3448 (OH),
2923 (CH), 2906 (CH), 2852 (CH); 1H NMR (300 MHz; DMSO-
d6) d (ppm) 9.60 (1H, br s, OH), 7.60 (1H, br s), 7.42–7.54 (3H,
m), 7.25 (1H, t, J = 7.8 Hz), 7.05 (1H, d, J = 7.8 Hz), 7.03 (1H, br
s), 6.76 (1H, dd, J = 1.8 Hz, J = 7.8 Hz), 2.73 (3H, s, OCH3), 2.67
(2H, br s), 2.26 (1H, br s), 2.22 (1H, br s), 1.84 (1H, br s), 1.52–
1.78 (9H, m); 13C NMR (75 MHz; DMSO-d6) d (ppm) 158.04 (s),
141.84 (s), 141.14 (s), 140.00 (s), 129.93 (d), 128.66 (d), 126.25 (d),
125.47 (d), 125.34 (d), 117.35 (d), 114.45 (d), 113.60 (d), 79.20 (s),
47.64 (q), 37.15 (t), 34.11 (2C, t), 32.38 (2C, t), 32.11 (2C, d), 27.08
(d), 26.24 (d).
Irradiation of 6. (18 mg, 0.28 mmol) in 100 mL CH3OH for
1 min gave after evaporation of the methanol crude mixture that
contained 90% of the product (according to NMR). An attempt
at separation on a thin layer of silica using CH2Cl2-EtOAc (5%)
afforded only starting material.
Spiro[adamantane-2,9¢-(3¢-hydroxy)fluorene] (10)
20 mg (21%); colorless crystals, mp 221–222 ◦C; IR (KBr) n/cm-1:
1
3529 (OH), 3360 (OH), 2906 (CH), 2883 (CH); H NMR (600
The pure product 6b was obtained by stirring 6 (30 mg) in
CH3OH (50 mL) in the presence of H2SO4 (500 mg) over 2
days. After the conversion was completed, as indicated by HPLC
analysis, to the solution NaHCO3 was added and stirring was
continued over 1 h. The mixture was filtered and the solvent
evaporated. The remaining solid was extracted by ethyl acetate
to afford the pure product for the analysis.
MHz; CDCl3) d (ppm) 8.08 (1H, d, J = 7.9 Hz), 7.94 (1H, d, J =
8.4 Hz), 7.70 (1H, dd, J = 0.8 Hz, J = 7.9 Hz), 7.34 (1H, dt, J = 0.9
Hz, J = 7.4 Hz), 7.27 (1H, dt, J = 1.5 Hz, J = 7.4 Hz), 7.22 (1H,
d, J = 2.6 Hz), 6.74 (1H, dd, J = 2.6 Hz, J = 8.4 Hz), 4.80 (1H, s,
OH), 2.90 (2H, dd, J = 2.7 Hz, J = 13.6 Hz), 2.85 (2H, dd, J = 2.7
Hz, J = 13.6 Hz), 2.18 (2H, br s), 1.97 (2H, br s), 1.79 (2H, br s),
1.76 (2H, br s), 1.56–1.59 (2H, m); 13C NMR (150 MHz; CDCl3)
d (ppm) 154.64 (s), 151.79 (s), 143.53 (s), 142.55 (s), 140.38(s),
129.99 (d), 129.15 (d), 126.72 (d), 126.00 (d), 119.34 (d), 112.88
(d), 105.84 (d), 57.88 (s), 41.39 (t), 35.06 (2C, d), 34.32 (2C, t),
34.27 (2C, t), 27.32 (d), 27.30 (d); HRMS (MALDI) calculated for
C22H22O (-e-) 302.1676, found 302.1669.
4-(2-Methoxyadamantan-2-yl)-3¢-hydroxybiphenyl (6b)
Colorless crystals, mp 177–179 ◦C; IR (KBr) n/cm-1: 3280 (OH),
2928 (CH), 2856 (CH); 1H NMR (300 MHz; DMSO-d6) d (ppm)
9.64 (1H, br s, OH), 7.61 (2H, d, J = 8.3 Hz), 7.52 (2H, d, J = 8.3
Hz), 7.25 (1H, t, J = 7.8 Hz), 7.09 (1H, d, J = 7.8 Hz), 7.05 (1H,
t, J = 1.8 Hz), 6.76 (1H, dd, J = 1.8 Hz, J = 7.8 Hz), 2.72 (3H, s,
OCH3), 2.64 (2H, br s), 2.25 (1H, br s), 2.21 (1H, br s), 1.84 (1H,
br s), 1.52–1.77 (9H, m); 13C NMR (75 MHz; DMSO-d6) d (ppm)
157.95 (s), 141.07 (s), 139.74 (s), 138.85 (s), 129.90 (d), 127.70 (2C,
d), 126.10 (2C, d), 117.21 (d), 114.47 (d), 113.35 (d), 79.00 (s),
47.61 (q), 37.14 (t), 34.09 (2C, t), 32.36 (2C, t), 32.02 (2C, d), 27.07
(d), 26.24 (d).
2-(2-Methoxyadamantan-2-yl)-3¢-hydroxybiphenyl (4b)
20 mg (19%), colorless crystals, mp 181–182 ◦C; IR (KBr) n/cm-1:
3392 (OH), 2922 (CH), 2854 (CH); 1H NMR (300 MHz; CDCl3)
d (ppm) 7.68 (1H, br s), 7.38–7.50 (3H, m), 7.30 (1H, t, J = 7.8
Hz), 7.16 (1H, dd, J = 1.5 Hz, J = 7.8 Hz), 7.07 (1H, t, J = 2.2 Hz),
6.81 (1H, ddd, J = 0.8 Hz, J = 2.2 Hz, J = 7.8 Hz), 5.21 (1H, s,
OH), 2.85 (3H, s, OCH3), 2.69 (2H, br s), 2.36 (1H, br s), 2.32 (1H,
br s), 1.91 (1H, br s), 1.65–1.80 (9H, m); 13C NMR (150 MHz;
DMSO-d6) d (ppm) 155.89 (s), 143.19 (s), 141.52 (s), 140.32 (s),
129.83 (d), 128.27 (d), 126.48 (d), 126.13 (d), 125.79 (d), 119.62 (d),
114.10 (d), 114.03 (d), 80.02 (s), 48.07 (q), 37.61 (t), 34.48 (2C, t),
32.85 (2C, d), 32.77 (2C, t), 27.65 (d), 26.79 (d); HRMS (MALDI)
Irradiation of 7. (70 mg, 0.20 mmol) in 100 mL CH3OH for 5
min gave, after evaporation of the methanol, crude mixture that
was separated on a thin layer of silica using CH2Cl2 as eluent.
3¢-(Methoxybiphenylmethyl)-3-hydroxybiphenyl (7b)
calculated for C23H26O2 (+H+-OCH3 ) 303.1754, found 303.1753.
-
30 mg (41%), colorless crystals mp 147–151 ◦C, IR (KBr) n/cm-1:
3384 (OH), 3057 (CH), 3028 (CH), 2980, 2955, 2930, 2826, 2359,
1H NMR (500 MHz; CDCl3) d (ppm) 7.62 (1H, t, J = 1.8 Hz),
7.39 (4H, d, J = 8.0 Hz), 7.35 (2H, tt, J = 1.5 Hz, J = 8.0 Hz),
7.27 (1H, t, J = 7.7 Hz), 7.24 (4H, t, J = 8.0 Hz), 7.15–7.20 (3H,
m), 7.03 (1H, dd, J = 1.5 Hz, J = 7.7 Hz), 6.91 (1H, t, J = 2.2
Hz), 6.70 (1H, dd, J = 0.8 Hz, J = 8.0 Hz), 4.92 (1H, br s, OH),
3.02 (3H, s, OCH3); 13C NMR (125 MHz; CDCl3) d (ppm) 155.68
(s), 144.61 (s), 143.63 (2C, s), 142.91 (s), 140.00 (s), 129.79 (d),
Irradiation of 5. (22 mg, 0.068 mmol) in 100 mL CH3OH
for 1 min gave after evaporation of the methanol, crude mixture
containing 8% of product 5b (according to NMR). Attempt of
separation on a thin layer of silica using CH2Cl2-EtOAc (5%)
afforded only starting material.
The pure product 5b was obtained by stirring 5 (40 mg) in
CH3OH (50 mL) in the presence of H2SO4 (500 mg) over 2
days. After the conversion was completed, as indicated by HPLC
392 | Photochem. Photobiol. Sci., 2012, 11, 381–396
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128.71 (4C, d), 128.11 (d), 127.72 (4C, d), 127.12 (d), 126.91 (2C,
d), 125.55 (d), 119.66 (d), 114.02 (d), 113.97 (d), 87.03 (s), 52.02
(q); HRMS (MALDI) calculated for C26H22O2 (+K+) 405.1251,
found 405.1253.
JCF = 277 Hz), 119.50 (d), 114.26 (d), 113.84 (d), 81.07 (s), 59.34 (q,
JCCF = 34 Hz), 37.41 (t), 34.42 (2C, t), 33.17 (2C, d), 32.49 (2C, t),
27.40 (d), 26.57 (d); HRMS (MALDI) calculated for C24H25F3O2
(+H+-OCH2CF3) 303.1754, found 303.1747.
Irradiation of 8. (50 mg, 0.14 mmol) in 100 mL CH3OH for
5 min gave after evaporation of the methanol, the crude mixture
was separated on a thin layer of silica using CH2Cl2 as eluent.
3-[(2,2,2-Trifluoroethoxy)diphenylmethyl]-3¢-hydroxybiphenyl (13)
10 mg (40%) colorless oil, IR (KBr) n/cm-1: 3343, 3063, 2927,
1
2359; H NMR (500 MHz; CDCl3) d (ppm) 7.60 (1H, t, J = 1.8
4¢-(Methoxybiphenylmethyl)-3-hydroxybiphenyl (8b)
Hz), 7.40 (4H, d, J = 8.0 Hz), 7.27 (4H, t, J = 8.0 Hz), 7.16–7.24
(5H, m), 7.08 (1H, dd, J = 1.7 Hz, J = 7.7 Hz), 7.02 (1H, dd, J =
2.4 Hz, J = 7.7 Hz), 6.90 (1H, t, J = 2.1 Hz), 6.73 (1H, ddd, J = 0.9
Hz, J = 2.3 Hz, J = 7.7 Hz), 4.87 (1H, br s, OH), 3.43 (2H, q, J =
8.4 Hz); 13C NMR (125 MHz; CDCl3) d (ppm) 155.72 (s), 143.24
(s), 142.59 (s), 142.33 (2C, s), 140.38 (s), 129.90 (d), 129.34 (d),
128.47 (4C, d), 128.07 (4C, d), 127.52 (2C, d), 127.42 (d), 126.91
(d), 126.14 (d), 124.20 (q, JCF = 277 Hz), 119.63 (d), 114.21 (d),
113.92 (d), 87.32 (s), 62.02 (q, JCCF = 34 Hz); HRMS (MALDI)
calculated for C27H21F3O2 (+K+) 473.1125, found 473.1133.
10 mg (20%); colorless crystals, mp 170–173 ◦C, IR (KBr) n/cm-1:
1
3562 (OH), 3365 (OH), 3056 (CH), 3026 (CH), 2926 (CH); H
NMR (500 MHz; CDCl3) d (ppm) 7.48 (4H, s), 7.46 (4H, d, J =
8.4 Hz), 7.26–7.33 (5H, m), 7.22–7.26 (2H, m), 7.13 (1H, dd, J =
2.0 Hz, J = 7.9 Hz), 7.02 (1H, t, J = 2.1 Hz), 6.78 (1H, ddd, J =
0.8 Hz, J = 2.5 Hz, J = 8.0 Hz), 5.36 (1H, br s, OH), 3.08 (3H, s,
OCH3); 13C NMR (125 MHz; CDCl3) d (ppm) 155.83 (s), 146.60
(s), 143.69 (2C, s), 142.29 (s), 139.03 (s), 129.81 (d), 128.94 (2C, d),
128.63 (4C, d), 127.70 (4C, d), 126.88 (2C, d), 126.32 (2C, d), 119.40
(d), 114.09 (d), 113.83 (d), 86.85 (s), 52.01 (q); HRMS (MALDI)
calculated for C26H22O2 (+K+) 405.1251, found 405.1249.
Irradiation in the presence of ethanolamine. Phenylphenol
derivative (50 mg, 0.14 mmol) was dissolved in CH3CN–H2O (3 : 1,
100 mL). To the solution, ethanolamine (500 mL, 8.28 mmol) was
added. The solution was purged with Ar for 30 min and irradiated
in a Rayonet reactor at 254 nm for 30 min. After the irradiation,
H2O (100 mL) was added and extractions with ethyl acetate (3 ¥
75 mL) were carried out. The extracts were dried over anhydrous
MgSO4, the solution was filtered and the solvent was removed on
a rotary evaporator. The residue was chromatographed on a thin
layer of silica using CH2Cl2/ethyl acetate (20%) as eluent.
Irradiation in the presence of TFE. Phenol derivative (20
mg) was dissolved in CH3CN (50 mL). To the solution, 2,2,2-
trifluoroethanol (TFE, 20 mL) was added. The solution was
purged with Ar for 30 min and irradiated in a Rayonet reactor
at 254 nm for 30 min. After evaporation of the solvent the residue
was chromatographed on a thin layer of silica using CH2Cl2 as
eluent.
3-[2-(2,2,2-Trifluoroethoxy)adamantan-2-yl]-3¢-hydroxybiphenyl
(11)
3-[(2-Hydroxyethylamino)diphenylmethyl]-3¢-hydroxybiphenyl (14)
Yellowish oil, 20 mg (35%); IR (KBr) n/cm-1: 3329, 3057, 2928,
10 mg (38%), colorless oil, IR (KBr) n/cm-1: 3425, 2918, 2858,
2359; H NMR (500 MHz; CDCl3) d (ppm) 7.60 (1H, s), 7.44
1
1
2361; H NMR (500 MHz; CDCl3) d (ppm) 7.72 (1H, t, J = 1.8
(1H, dt, J = 1.8 Hz, J = 6.7 Hz), 7.34–7.40 (2H, m), 7.25 (1H, t,
J = 7.8 Hz), 7.09 (1H, ddd, J = 0.8 Hz, J = 1.6 Hz, J = 7.6 Hz),
6.98 (1H, t, J = 2.2 Hz), 6.76 (1H, ddd, J = 0.7 Hz, J = 2.5 Hz, J =
8.0 Hz), 4.92 (1H, br s, OH), 3.20 (2H, dq, J = 2.2 Hz, J = 8.0 Hz),
2.60 (1H, br s), 2.56 (1H, br s), 2.31 (1H, br s), 2.28 (1H, br s),
1.86 (1H, br s), 1.58–1.74 (9H, m); 13C NMR (125 MHz; CDCl3)
d (ppm) 155.78 (s), 142.86 (s), 140.75 (s), 140.30 (s), 129.94 (d),
128.64 (d), 126.51 (d), 126.34 (d), 126.00 (d), 124.28 (q, JCF = 280
Hz), 7.39 (4H, d, J = 8.0 Hz), 7.29–7.33 (2H, m), 7.22 (1H, t, J =
7.7 Hz), 7.19 (4H, t, J = 8.0 Hz), 7.15 (1H, t, J = 8.0 Hz), 7.11
(2H, tt, J = 1.10 Hz, J = 8.0 Hz), 6.99 (1H, ddd, J = 0.9 Hz, J =
1.5 Hz, J = 7.4 Hz), 6.95 (1H, t, J = 2.4 Hz), 6.71 (1H, ddd, J =
0.9 Hz, J = 2.5 Hz, J = 8.0 Hz), 3.81 (3H, br s), 3.62 (2H, t, J =
5.3 Hz), 2.32 (2H, t, J = 5.3 Hz); 13C NMR (125 MHz; CDCl3) d
(ppm) 156.15 (s), 145.60 (s), 145.52 (s), 142.65 (2C, s), 140.29 (s),
129.85 (d), 128.50 (4C, d), 128.17 (d), 128.00 (d), 127.84 (4C, d),
126.71 (d), 126.42 (2C, d), 125.07 (d), 119.17 (d), 114.30 (d), 113.85
(d), 70.63 (s), 62.38 (t), 45.49 (t); HRMS (MALDI) calculated for
C27H25NO2 (+H+-2H) 394.1801, found 394.1814.
Hz), 119.68 (d), 114.19 (d), 114.02 (d), 81.31 (s), 59.38 (q, JCCF
=
35 Hz), 37.40 (t), 34.46 (2C, t), 33.35 (d), 33.19 (d), 32.52 (2C, t),
27.39 (d), 26.56 (d); HRMS (MALDI) calculated for C24H25F3O2
(+H+-OCH2CF3) 303.1754, found 303.1762.
4-[2-(2,2,2-Trifluoroethoxy)adamantan-2-yl]-3¢-hydroxybiphenyl
4-Diphenylmethyl-3¢-hydroxybiphenyl (16)
(12)
◦
10 mg (%), colorless crystals, mp 129–131 C; IR (KBr) n/cm-1:
15 mg (60%),colorless oil, IR (KBr) n/cm-1: 3383, 2924, 2856,
2358; 1H NMR (500 MHz; CDCl3) d (ppm) 7.52 (2H, d, J = 8.4
Hz), 7.45 (2H, d, J = 8.4 Hz), 7.25 (1H, t, J = 7.8 Hz), 7.12 (1H,
ddd, J = 0.9 Hz, J = 1.0 Hz, J = 7.7 Hz), 7.02 (1H, t, J = 1.7
Hz), 6.76 (1H, ddd, J = 0.9 Hz, J = 2.5 Hz, J = 8.0 Hz), 4.92
(1H, br s, OH), 3.20 (2H, q, J = 8.7 Hz), 2.57 (2H, br s), 2.31
3358, 3080, 3053, 3024, 2923, 2853; 1H NMR (300 MHz; CDCl3)
d (ppm) 7.48 (2H, d, J = 8.2 Hz),7.12–7.34 (14H, m), 7.04 (1H, t,
J = 2.2 Hz), 6.79 (1H, ddd, J = 1.0 Hz, J = 2.2 Hz, J = 8.2 Hz), 5.58
(1H, s), 4.81 (1H, br s, OH); 13C NMR (75 MHz; CDCl3) d (ppm)
155.70 (s), 143.67 (s), 143.20 (s), 142.54 (s), 129.84 (d), 129.72
(2C, d), 129.34 (4C, d), 128.26 (4C, d), 126.86 (2C, d), 126.27 (2C,
d), 119.54 (d), 113.96 (d), 113.81 (d), 56.43 (d), one singlet was
not seen due to a small amount of substance; HRMS (MALDI)
calculated for C27H20O (+Na+) 359.1406, found 359.1395.
(1H, br s), 2.28 (1H, br s), 1.86 (1H, br s), 1.60–1.75 (9H, m); 13
C
NMR (CDCl3, 125 MHz) d (ppm) 155.80 (s), 142.07 (s), 139.83
(s), 138.96 (s), 129.92 (d), 127.57 (2C, d), 126.85 (2C, d), 124.30 (q,
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4-[(2-Hydroxyethylamino)diphenylmethyl]-3¢-hydroxybiphenyl (15)
Yellowish oil, 20 mg (35%); IR (KBr) n/cm-1: 3342, 3062, 2937,
H2O (1 : 4) solution. The pH of aqueous HClO4 was measured by
a pH meter. The titration was performed at 20 ◦C in air-saturated
solutions.
1
2359; H NMR (500 MHz; CDCl3) d (ppm) 7.39–7.44 (6H; m),
Fluorescence decay histograms were obtained on an Edin-
burgh instrument OB920, equipped with a light emitting diode
(excitation wavelength 265 nm), using a 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 4 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 exponentials
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
7.37 (2H, d, J = 8.6 Hz), 7.21 (4H, t, J = 8.1 Hz), 7.17 (1H, t, J =
8.0 Hz), 7.12 (2H, tt, J = 1.1 Hz, J = 7.3 Hz), 7.04 (1H, ddd, J =
0.9 Hz, J = 1.3 Hz, J = 7.7 Hz), 6.94 (1H, t, J = 2.1 Hz), 6.70 (1H,
ddd, J = 0.9 Hz, J = 2.4 Hz, J = 8.0 Hz), 3.63 (2H, t, J = 5.1 Hz),
3.20 (3H, br s), 2.31 (2H, t, J = 5.1 Hz); 13C NMR (125 MHz;
CDCl3) d (ppm) 155.92 (s), 145.50 (s), 144.86 (s), 142.19 (s, 2C),
138.53 (s), 129.80 (d), 128.84 (2C, d), 128.49 (4C, d), 127.83 (4C,
d), 126.40 (2C, d), 126.37 (2C, d), 119.26 (d), 114.10 (d), 113.78
(d), 70.35 (s), 62.51 (t), 45.46 (t); HRMS (MALDI) calculated for
C27H25NO2 (+H+-2H) 394.1801, found 394.1815.
Quantum yields for the photomethanolysis reaction
Solutions of 4–8 in CH3OH-H2O (1 : 1), as well as a solution
of valerophenone in CH3CN–H2O (1 : 1) were freshly prepared
and their concentrations adjusted to have absorbances 0.4–0.8 at
254 nm. After adjustment of the concentration and measurement
of the corresponding UV-vis spectra, the solutions were filled 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 merry-go-
round and 6 lamps with the output at 254 nm for 0.5, 1, and
2 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 valerophenone
actinometer (formation of acetophenone in aqueous media, U =
0.65 0.03).22
2
reduced chi-square c . An additional graphical method was used
to judge the quality of the fit that included plots of surfaces
(“carpets”) 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.
Antiproliferative investigation
The experiments were carried out on three human carcinoma
cell lines HCT 116, MCF-7 and H 460. 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.
The cells were inoculated in parallel on two 96-well microtiter
plates on day 0, at 1.5 ¥ 104 cells mL-1. Test agents were added in
ten-fold dilutions (108 to 104 M) on the next day and incubated
for 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) 4, 24, and 48 h after the addition of the compounds. After 72
h of incubation the cell growth rate was evaluated by performing
the MTT assay31 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:
Irradiation in CH3CN–D2O. To a solution of 5 or 6 (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, crude mixture was analyzed by NMR
and MS.
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 (1 : 4) 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).24
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 absorbance measurements before exposure of the cells to
the test compound, the mean Atest is the average of absorbance
measurements after the desired period of time and the mean Actrl
is the average of the optical density measurements after the desired
Fluorescence titrations were performed by adding aliquots
(60 mL) of aqueous HClO4 (pH = 2.10) to 2 mL of the CH3CN–
394 | Photochem. Photobiol. Sci., 2012, 11, 381–396
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period of time with no exposure of cells to the test compound. In
the experiments where the cells were irradiated, Actrl represents
irradiated control cells, which typically showed the 25% growth
inhibition compared to Actrl without irradiation.
photoinducible DNA cross-linking agent, J. Am. Chem. Soc., 2003,
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13979.
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antitumor drug menogaril to 2¢-deoxyguanosine through reductive
activation, J. Am. Chem. Soc., 1989, 111, 8291–8293; (b) G. Gaudiano,
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5892.
The results are expressed as IC50, which is the concentration nec-
essary for 50% of inhibition. The IC50 values for each compound
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,
for 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.
5 (a) F. Doria, S. N. Richter, M. Nadai, S. Colloredo-Mels, M. Mella,
M. Palumbo and M. Freccero, BINOL-amino acids conjugates as
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Percivalle, M. Di Antonio, M. Palumbo, S. N. Richter and M. Freccero,
Photogeneration and reactivity of naphthoquinone methides as purine
selective DNA alkylating agents, J. Am. Chem. Soc., 2010, 132, 14625–
14637.
Cross-Linking of plasmid DNA. DMSO-stock solutions of
compounds 3, 6 and 26, as well as psoralen 28 were diluted in H2O
and used for reactions. Plasmid pCI (0.8 mg sample-1) was mixed
with compound dilutions. Reaction mixtures were irradiated for 3
min at 300 nm in a Luzchem reactor. Irradiated solutions were
added to alkaline agarose gel loading buffer [50 mM NaOH,
1 mM ethylenediaminetetraacetic acid (EDTA), 3% Ficoll, and
0.02% bromophenol blue] and loaded on a 1% alkaline agarose
gel containing 50 mM NaOH and 1 mM EDTA. Gels were run
in 50 mM NaOH and 1 mM EDTA at 25 V constant voltage in
horizontal electrophoresis system (BIO-RAD, USA), stained with
ethidium bromide (0.5 mg mL-1) for 10 min, after neutralization
of the gel with 30 mM Tris-HCl (pH 7.5). Resulting products
were visualized and documented with UV light at 254 nm (Image
Master VDS, Pharmacia Biotech, Sweden).
6 Hydrogen-transfer reactions, ed. J. T. Hynes, J. P. Klinman, H.-H.
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Intramolecular proton transfer to carbon in electronically excited states,
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P. Wan, Excited state intramolecular proton transfer (ESIPT) in 2-
phenylphenol: an example of proton transfer to a carbon of an aromatic
ring, Chem. Commun., 2001, 1004–1005; (d) M. Lukeman and P. Wan,
A new type of excited-state intramolecular proton transfer: Proton
transfer from phenol OH to a carbon atom of an aromatic ring for
2-phenylphenol, J. Am. Chem. Soc., 2002, 124, 9458–9464; (e) M.
Lukeman and P. Wan, Excited-state intramolecular proton transfer
in o-hydroxybiarils: A new route to dihydroaromatic compounds, J.
Am. Chem. Soc., 2003, 125, 1164–1165; (f) M. Flegel, M. Lukeman,
L. Huck and P. Wan, Photoaddition of water and alcohols to
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Cleavage of plasmid DNA was analysed using the same pro-
cedure as described above, except that the gel electrophoresis
experiments were carried out with using TAE buffer (45 mM Tris,
1 mM EDTA, pH 7.5) and the gel loading buffer contained 0.25%
bromphenol blue, 0.25% xylene cyanol, 60% glycerol, 150 mM Tris
pH 7.6.
Acknowledgements
These materials are based on work financed by the Croatia
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.
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