Observation of heavy atom effects in the development of water soluble caged 4-hydroxy-trans-2-nonenal

Article abstract of DOI:10.1039/b810954k

During the course of our study on the photochemistry of 1-alkoxy-9,10-anthraquinones, we have developed a second generation of a caged 4-hydroxy-trans-2-nonenal (4-HNE). As we optimized the anthraquinonyl chromophore to achieve water solubility, we studied the photochemistry of various substituents to understand their effect on the photochemistry. We observed a significant heavy atom effect that severely reduced the rate of oxidative cleavage of the alkoxy group. Based on the results of our substituent study, we designed a new caged 4-HNE that is soluble under physiological conditions, and that releases 4-HNE photochemically in high yield.

Full text of DOI:10.1039/b810954k

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Observation of heavy atom effects in the development of water soluble caged
4-hydroxy-trans-2-nonenal†
Paul B. Jones,* Robert G. Brinson,‡ Saurav J. Sarma and Salwa Elkazaz
Received 27th June 2008, Accepted 5th August 2008
First published as an Advance Article on the web 19th September 2008
DOI: 10.1039/b810954k
During the course of our study on the photochemistry of 1-alkoxy-9,10-anthraquinones, we have
developed a second generation of a caged 4-hydroxy-trans-2-nonenal (4-HNE). As we optimized the
anthraquinonyl chromophore to achieve water solubility, we studied the photochemistry of various
substituents to understand their effect on the photochemistry. We observed a significant heavy atom
effect that severely reduced the rate of oxidative cleavage of the alkoxy group. Based on the results of
our substituent study, we designed a new caged 4-HNE that is soluble under physiological conditions,
and that releases 4-HNE photochemically in high yield.
gives zwitterion 3. The zwitterion can be trapped by a nucleophile,
Introduction
usually solvent, to produce acetal 4, which is relatively stable
to hydrolysis.7a However, upon oxidation of hydroquinone 4 to
Development of photolabile “caged” molecules, including car-
bonyl compounds, with biological relevance has received greater
attention over the last few years.1 Ideally a caged molecule is
anthraquinone 5, hydrolysis occurs readily. The result is a 1-
hydroxyanthraquinone 6, and an aldehyde.
inert, can be delivered in a high temporal and spatial manner,
and subsequently can be photolyzed under either aerobic or
anaerobic conditions to release the bioactive molecule. Such
a design would have application in synthesis, biophysics and
photodynamic therapy (PDT), which relies on photosensitization
of porphyrins under aerobic conditions for the production of
singlet oxygen.2 Indeed, the requirement of oxygen for PDT limits
its use, since many tumors operate until hypoxic conditions.3
While several of the caging strategies address this limitation,
many of them to date require ultraviolet light and/or produce
highly toxic by-products.1 To address these issues, our lab recently
developed a strategy for releasing caged bioactive aldehydes,
such as acrolein and 4-hydroxy-trans-2-nonenal (4-HNE),4 that
is oxygen independent. These caged molecules are based on the 1-
alkoxy-9,10-anthraquinonyl chromophore, whose absorption of
light tails out to about 450 nm. 4-HNE, a product of both
enzymatic and non-enzymatic lipid peroxidation,5 in particular
is known to hinder cell functions severely. At high enough
concentrations (>100 ppm), this results in cell death.6
A drawback to the photorelease strategy for 4-HNE described
above is the lack of solubility of the caged molecule under
physiological conditions. Our goal has been to develop a second
generation that is water soluble and photochemically releases 4-
HNE in high yield. The mechanism of this photo reaction for
1-alkoxy-9,10-anthraquinones has been previously described in
detail.7 Briefly, the photochemical oxidative cleavage of 1-alkoxy-
9,10-anthraquinones proceeds via an intramolecular d-hydrogen
abstraction (Scheme 1). Excited triplet 1 initially abstracts a d-
hydrogen to produce 2. This is followed by electron transfer that
Scheme 1
We report below the results of our study of the effect of
substituents on the photochemical oxidative cleavage of 1-alkoxy-
9,10-anthraquinones. The observed effects are explained best by
the influence of the various reaction factors on the fate of the
diradical intermediate 2. The behavior of diradical intermediates
is known to have a significant impact on the outcome of many
photochemical reactions.8,9 The study resulted in the optimization
of a water soluble caged 4-HNE that photochemically generated
4-HNE in high yield at various pHs.
Department of Chemistry, Wake Forest University, Winston-Salem, NC,
USA. E-mail: jonespb@wfu.edu; Fax: 336-758-4656; Tel: 336-758-3708
† CCDC reference number 650843. For crystallographic data in CIF or
other electronic format see DOI: 10.1039/b810954k
‡ Current address: Center for Advanced Research in Biotechnology,
Rockville, MD, USA.
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Results and discussion
Synthesis
In order to increase the water solubility of the anthraquinones,
we planned to introduce multiple carboxylate substituents to
the anthraquinone. Our initial target was the dicarboxylate
corresponding to di-nitrile 10, which we planned to prepare from
8 as shown in Scheme 2. Unfortunately, cyanation of the bromide
in the 2-position of 8 could not be accomplished under any of the
conditions we attempted. In contrast, cyanation of the bromide in
the 4-position proceeded in good yield. The regioselectivity of the
reaction was established by crystallography.10 Hydrolysis of nitrile
9 to give carboxylate 11 went smoothly. The corresponding sodium
salt (12) was quite soluble in water (>10 mM easily achieved).
Scheme 3 Reagents and conditions: (i) Br2, NaOAc, AcOH, 50 ◦C, 100%;
(ii) BnBr, TBAF, DMF, rt, 93%; (iii) CuCN, DMF, 80 ◦C, 99%; (iv) NaOH
(or KOH), EtOH, reflux, 78% (17).
Scheme 2 Reagents and conditions: (i) Br2, NaOAc, AcOH, reflux, 97%;
(ii) BnBr, TBAF, DMF, rt, 93%; (iii) CuCN, DMF, 80 ◦C, 74%; (iv) NaOH,
EtOH, reflux, 93%.
Scheme 4 Reagents and conditions: (i) hn (366 nm), MeOH–air, 75%;
(ii) 21, TBAF, DMF, rt, 74%; (iii) KOH, EtOH, reflux, 65% (23).
With a water soluble anthraquinone in hand, we proceeded
to investigate the photochemistry of 11 and 12. Photolysis of
these two compounds in methanol did, indeed, result in cleavage
of the benzyl group. However, this reaction was surprisingly
inefficient, and yields were much lower than expected, presumably
due to the lack of steric bulk in the 2-position. Previous studies
have shown that an alkyl group in this position accelerates the
desired photochemical reaction7b while minimizing unwanted side
reactions.11
Thus, we turned to anthraquinones with a 2-propyl group.
The propyl group is easily installed in the 2-position by Claisen
reaction of 1-allyloxy-9,10-anthraquinone and hydrogenation of
the resulting alkene.4,11 Anthraquinone 13 was brominated in
quantitative yield to give 14, which was alkylated with benzyl
bromide to give 15 (Scheme 3). Benzyl ether 15 was cyanated with
CuCN to give nitrile 16, which was hydrolyzed to afford acid 17.
For synthesis of water soluble caged HNE, the benzyl group
of 16 was removed photochemically to give 20 (Scheme 4). The
phenol was then alkylated with 1-bromonon-2-en-4-ol (21)4 using
TBAF to give nitrile 22. Finally, 22 was hydrolyzed to give
caged 4-HNE 23, which could be converted to the corresponding
potassium salt 24 by treatment with KH in dry THF. This salt was
freely soluble in aqueous systems at concentrations lower than
10 mM.
Photochemistry
In order to compare the effects of the substituents, each an-
thraquinone benzyl ether was photolyzed and the relative rate of
disappearance of starting ether determined. Since measurement of
product formation is inherently unreliable in this system due to a
series of dark reactions subsequent to the primary photochemical
step, the rate of disappearance of starting ether was monitored
by following the reduction of the distinctive benzyl 1H NMR
singlet. Each anthraquinone was photolyzed alongside 2-propyl-1-
benzyloxy-9,10-anthraquinone, 25, which was used as a standard
reaction.7b The photocleavage reaction of 25 has been studied
both by Blankespoor et al. and our laboratory and is well
understood. Photolyses were performed in triplicate in solvent
mixtures of DMSO–methanol or DMSO–water using a 150 W
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Table 1 Relative rate of the disappearance of the benzyl group. The
Hg/Xe lamp in combination with a grating monochromator at a
wavelength of 405 nm ( 5 nm). All compounds were normalized
to the absorbance of a 5 mM solution of 17 at 405 nm. Data
was analyzed by 1H NMR relative to the internal standard
2,4,6-trimethylbenzoic acid or its corresponding sodium salt for
photolyses in D2O. The data was plotted as time (min) against
ln([AQ]o/[AQ]t), where [AQ]o is the integral value at t = 0 and
[AQ]t is the integral value at time point, t. Linear regression was
performed on the data to give a straight line; R2 values were
0.95 or higher. The slope of the linear regression was used as
the relative rate. See the experimental section for complete details
of the photolyses.
In general, electron-withdrawing substituents para to the ben-
zyloxy group slowed the rate of the photoreaction relative to
25. This attenuation is modest, with the relative efficiency only
approximately halved going from 25 to 16, 17 or 18 (Table 1, entries
1, 3–5). The relatively high efficiency exhibited by 29 shows that the
effect of the para electron-withdrawing group is due to a resonance
effect, as the photolysis of a molecule with a carboxylate that is not
electronically tied to the benzyloxy group, but is sterically closer,
actually proceeds more efficiently than in the standard reaction.
The increased relative rate of 29 over 25 (1.55 : 1), is presumably
due to the slight increase in steric bulk of the 2-substituent in 29
(entry 8).
monochromator used was set at 405 nm ( 5 nm)
Entry
Substrate
Solventb
Additive
Relative rate
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
25
15
16
17
18
18
11
29
25
25
25
25
18
18
18
18
18
A
A
A
A
A
B
A
A
A
A
A
A
B
—
—
—
—
—
—
—
—
1.00
0.21
0.50
0.58
0.46
1.02
<0.05a
1.55
0.56
0.25
0.19
1.02
1.08
1.47
1.47
0.39
0
28 mM CHI3
55 mM CHI3
110 mM CHI3
110 mM CHCl3
50 mM LiCl
100 mM LiCl
250 mM LiCl
100 mM LiI
100 mM CsI
B
B
B
B
a 11 was not irradiated in direct comparison with the other compounds
listed, but an estimate of the ratio was made by comparing the rate of 11
and 1-benzyloxy-9,10-anthraquinone (see ref. 11). b For solvent conditions,
see Scheme 5.
Mechanistic rationale
The mechanistic model put forward in Scheme 6 explains the
observations in the photolysis of anthraquinones bearing electron-
withdrawing groups para to the benzyloxy group and the heavy
atom effect. The effect of a para electron-withdrawing group
(entries 3–5) should destabilize the benzylic cation, reducing the
rate of the single electron transfer (SET) (Scheme 6, path a—
the forward, productive path) and, thus, the overall efficiency of
the reaction. However, upon photolysis in D2O, the rate of 18
increased to a rate comparable to the standard reaction (25 in
methanol–DMSO, entry 1). This is presumably due to increased
stabilization of the zwitterion by the more polar solvent D2O
(compare entries 1, 5 and 6). Increasing the ionic strength would
add additional stabilization to the zwitterion, resulting in slightly
increased relative rates compared to the photolysis without salt
(entries 6, 13–17). Thus, the more polar environment and increased
rate of SET can overcome the inhibiting effect of the conjugated
carboxylate.
Scheme
5
Reagents and conditions: (A) hn (405 nm), 4 : 1
CD3OD–DMSO-d6; (B) hn (405 nm), 4 : 1 D2O–DMSO-d6. See Table 1
for relative rates and additional details.
The molecule with a bromide in the para position, 15, reacted
with the least efficiency with a relative rate of 0.21 (entry 2). Along
with the results of photolysis of 11, this suggested a heavy atom
effect. To test this hypothesis, experiments were run with 25 in
the presence of iodoform at concentrations of 0, 28, 55, 110 mM
(entries 1, 9–11). Increasing the concentration of iodoform led to
a proportional decrease in relative efficiency for the reaction. A
control experiment with 110 mM CDCl3 resulted in no decrease
in the relative efficiency (entry 12). Clearly, the forward reaction
rate is decreased in the presence of heavy atoms.
Photolyses of 18 in 1 : 4 DMSO-d6–D2O with 0, 50, 100, 250 mM
LiCl; 100 mM LiI; and 100 mM CsI were carried out to measure
the effect of ionic strength on the rate of the photocleavage (entries
6, 13–17). Increasing the ionic strength of the solution led to
modest increases in reaction rate that leveled off between 100 and
250 mM. When the added salt included a heavy atom (LiI or CsI),
the reaction was slowed. A concentration of 100 mM CsI com-
pletely stopped the reaction over the photolysis time examined,
which is consistent with our heavy atom observations above.
Scheme 6 Mechanistic proposal for the heavy atom effect.
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Table 2 Relative rate of the disappearance of caged HNE
From further analysis of our data, we hypothesized that the
observed effect of heavy atoms increased the rate of intersystem
crossing (ISC) from either T1 anthraquinone (path c) or by a
back hydrogen abstraction from the triplet diradical (path b).
Anthraquinones undergo ISC with a quantum efficiency of 1.12
Thus, heavy atoms should not significantly affect the efficiency
of reaching the T1 excited state from which the initial hydrogen
abstraction occurs. However, back hydrogen abstraction to give
starting material requires a second ISC in going from the
triplet diradical to the singlet diradical. Return to ground state
anthraquinone from the T1 state requires ISC as well. This spin-
flip could be facilitated by a heavy atom effect that would accelerate
paths b and c without affecting path a. The result would be
diminished efficiency in oxidative cleavage of the 1-alkoxy group.
Thus, paths a, b and c are partitioned based on the relative rate of
ISC and SET, with any increase in ISC or decrease in SET favoring
paths b and c, the reverse reaction, in terms of desired product.
To test these possibilities, both racemic and optically active
anthraquinone 31 were prepared by Mitsunobu alkylation using
sec-phenethyl alcohol, which is commercially available as both the
racemate and as an enriched enantiomer (Scheme 7). Compound
31 was prepared without the 2-propyl group to decrease the steric
bulk at this position and to reduce the efficiency of pathway a,
since our goal was to study the possibility of path b. Photolysis
of optically active 31 allowed testing of racemization. If path b
operated, recovered 31 following photolysis should have a lower
optical activity than the starting material. Thus, 31 with 99.8%
ee was photolyzed in a Rayonet reactor using 419 nm lamps in
benzene–methanol containing 250 mM CHI3. At 72% conversion,
recovered 31 had an optical purity of 88% ee. This confirms that
path b does operate in this reaction, although the experiment did
not rule out path c. It seems likely that both pathways b and c are
responsible for the reduction in rate of oxidative cleavage of the
alkoxy group in the presence of heavy atoms.
Entry
Substrate
Solventa
Relative rate
1
2
3
4
25
24
32
24
A
A
A
B
1.00
1.25
2.10
3.96
The monochromator used was set at 405 nm ( 5 nm).a For solvent
conditions, see Scheme 5.
solvent, which is not possible with caged HNE 32, the photorelease
of HNE was rendered more efficient by a factor of approximately
three (3.96–1.25, Table 2, entries 4 and 2, respectively).
The caged 4-HNE 24 was freely soluble at 10 mM in all buffers
studied and the yields of 4-HNE were high in all systems. 4-HNE
was obtained in 84%, 87% and 97% yield in pH 5.0, 7.0, and 9.0
buffers, respectively (Scheme 8). Separation of 4-HNE from 33
was accomplished by simple chemical extraction. In addition, 33
can be recycled to make additional caged aldehydes.
Scheme 8 Reagents and conditions: (i) hn (419 nm), buffer; (ii) petroleum
ether extraction; (iii) 1 N HCl, ether extraction. See text for yields.
Conclusions
Substituent effects were observed on the rate of photocleavage of
1-alkoxy-9,10-anthraquinones with electron-withdrawing groups
in conjugation with the quinone slowing the reaction. A significant
heavy atom inhibition was also observed. The effect of solvent and
ionic strength had a slight accelerating effect on the reaction. These
observations are consistent with the efficiency of the reaction
being determined by the partitioning of the triplet 1,5-diradical
between single-electron transfer (SET) and intersystem crossing
(ISC). Factors that favor SET accelerate the reaction while those
favoring ISC slow the reaction. Using this data, we designed a
water soluble caged 4-HNE that photochemically released 4-HNE
in high yield using visible light. Our previous work has shown
that the photochemical cleavage proceeds under both aerobic and
anaerobic conditions.4 Hence, this second generation of caged
aldehydes represents an important advance in PDT agents that can
be photo-activated with visible light and under hypoxic conditions.
Initial investigations on the biological viability of these agents are
underway. Additionally, synthetic work continues to extend the
chromophore of these agents toward longer wavelengths, which
will increase their utility in biological systems.
Scheme
7 Reagents and conditions: (i) Ph3P, DIAD, THF,
1-phenylethanol; (ii) hn, (419 nm), MeOH–PhH–CHI3.
Caged 4-hydroxynonenal (HNE)
Given our observations above, we determined that the optimum
4-HNE releasing molecule would be soluble in aqueous systems
and free of heavy atoms. Despite the deleterious effect of having an
electron-withdrawing group attached to the anthraquinone itself,
the synthetic difficulty in working with 29 led us to 24 as a suitable
water soluble caged 4-HNE candidate. Hence, we studied the
photochemistry of 24 in buffered solutions at pH 5.0, 7.0 and
9.0, respectively. We also carried out a comparison of 24 with the
previously prepared 32 and 25 using the conditions and procedures
described in Scheme 5 and Table 1. The results of this study are
shown in Table 2. By dissolving the caged HNE 24 in aqueous
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(m, 2H, Ar-H), 7.85 (s, 1H, Ar-H), 8.18–8.25 (m, 2H, Ar-H); dC
(75 MHz, CDCl3) 182.5, 182.3, 157.5, 145.9, 142.3, 136.8, 134.1,
133.8, 133.7, 133.6, 130.1, 128.6, 128.5, 128.4, 128.3, 126.8, 126.7,
117.2, 76.9, 32.1, 23.1, 14.0.
Experimental section
General
Unless otherwise indicated, all reagents and solvents were ob-
tained commercially and used without further purification. Melt-
ing points were measured on a Meltemp II apparatus and are
uncorrected. Thin-layer chromatography was performed on silica
gel (250 mm thickness doped with fluorescein) unless otherwise
indicated. The chromatograms were visualized with UV light
(254 nm or 365 nm). Column chromatography was performed
1-(4-Hydroxy-2-nonenyloxy)-4-cyano-2-propyl-9,10-anthraquin-
one 22. Using Method A, 20 and 214 (226 mg, 0.776 mmol) gave
a yellow solid (241.9 mg, 74%). Mp 90.0–92.0 ◦C; found: C, 74.95;
H, 6.9; N, 3.1. Calcd. for C27H29NO4: C, 75.15; H, 6.8; N, 3.25%.
dH (300 MHz, CDCl3, Me4Si) 0.88–0.92 (m, 3H, CH3), 1.01 (t,
J = 7.4 Hz, 3H, CH3), 1.25–1.55 (m, 8H, 4 CH2), 1.64–1.75 (m,
2H, CH2), 2.75–2.80 (m, 2H, CH2), 4.15–4.24 (m, 1H, CHOH),
˚
˚
using silica gel (60 A) or basic alumina (58 A). HPLC analyses
were carried out using a Shimadzu LC-10AT LC with SPD-10AV
UV–Vis detector and a Daicel Chiracel OD-H column with an
eluent of 95 : 5 hexane–iPrOH. 1H and 13C NMR spectra were run
on a Bruker 300 MHz or 500 MHz NMR spectrometer. All photo-
chemical reactions were carried out in Pyrex glassware. Preparative
solutions were stirred by magnetic stirring throughout photolysis.
Anaerobic reactions were degassed by three cycles of freeze–
pump–thaw; the solutions were not backfilled with Ar. Solutions
were photolyzed in borosilicate NMR tubes, unless otherwise
indicated, using a monochromator (Oriel) set to 405 nm with a
10 nm bandpass in conjunction with a focused 150 W Hg/Xe
lamp. CCDC 650843 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/products/csd/request/, by emailing
data request@ccdc.cam.ac.uk, or by contacting The Cambridge
Crystallographic Data Centre, 12, Union Road, Cambridge CB2
1EZ, UK; fax: +44 1223 336033.
=
4.59 (d, J = 5.8 Hz, 2H, OCH2), 6.22 (dd, J = 15.6, 6.0 Hz, CH),
=
6.00–6.09 (m, 1H, CH), 7.79–7.86 (m, 2H, Ar-H), 7.92 (s, 1H,
Ar-H), 8.22–8.27 (m, 1H, Ar-H), 8.28–8.32 (m, 1H, Ar-H). dC
(75 MHz, CDCl3) 181.6, 160.8, 145.7, 141.1, 138.3, 135.6, 134.8,
134.2, 134.0, 132.1, 127.6, 127.2, 126.8, 124.9, 118.0, 106.9, 72.0,
63.7, 37.1, 32.3, 31.7, 25.0, 23.0, 22.6, 14.00, 13.97. HRMS (EI)
found MH+ 454.1981; C27H29O4H+ requires 454.1989.
1-Benzyloxy-2,4-dibromo-9,10-anthraquinone 8. Using Method
A, 713 (26.2 mmol) and benzyl bromide (104.7 mmol) gave 8
(11.52 g, 93%). Mp 172.0–174.0 ◦C (CH3OH); found: C, 53.4; H,
2.6; Br, 33.8. Calcd. for C21H12Br2O3: C, 53.4; H, 2.6; Br, 33.9%. dH
(300 MHz, CDCl3, Me4Si) 5.16 (s, 2H, OCH2), 7.35–7.47 (m, 3H,
Ar-H), 7.69–7.73 (m, 2H, Ar-H), 7.75–7.81 (m, 2H, Ar-H), 8.16–
8.23 (m, 2H, Ar-H), 8.30 (s, 1H, Ar-H); dC (75 MHz, CDCl3)
181.9, 181.4, 155.7, 144.6, 136.0, 134.13, 134.07, 133.7, 133.4,
131.9, 130.0, 128.9, 128.6, 127.3, 127.0, 126.8, 117.6, 76.1. HRMS
(EI) found MNa+ 494.9022; C21H12Br2O3Na+ requires 494.9025.
1-Benzyloxy-2-propyl-9,10-anthraquinone 25 (Method A). To
a yellow-orange solution of 1311 (3.76 mmol) in 1 : 1 DMF–
THF (38 ml) was added benzyl bromide (15.0 mmol) and TBAF
(7.51 mmol). The solution turned a deep purple and was stirred
for eight hours. The reaction was considered finished when the
reaction mixture had turned back to a yellowish color. The crude
mixture was diluted with EtOAc, washed with 1 N HCl, extracted
with EtOAc (2 ¥ 100 ml), washed with water (3 ¥ 200 ml), washed
with brine (200 ml) and concentrated via rotary evaporation. The
crude solid was recrystallized from hexanes to give yellow needles
2-(1-(Benzyloxy)-9,10-anthraquinonyl)-acetic acid 29. Using
Method A, 2-allyl-1-hydroxy-9,10-anthraquinone (1.0 g,
3.79 mmol) and benzyl bromide (15 mmol, 2.56 g) gave 2-allyl-
1-benzyloxy-9,10-anthraquinone (29a) (584 mg, 1.65 mmol,
44%). Crude alkene 29a (250 mg, 0.70 mmol) was dissolved in
dichloromethane (30 mL) and cooled in a dry ice–acetone bath.
Ozone was bubbled through the solution until a persistent blue
color was observed. Dimethyl sulfide (10 mL) was added and
the cold bath removed. The solution was allowed to stir for
6 h. The solution was concentrated in vacuo to give a yellow
solid. The solid was dissolved in CHCl3 (5 mL) and precipitated
by the addition of hexane (10 mL). The solid was collected by
filtration and washed extensively with hexane. The crude aldehyde
(29b) was carried on without further purification. Crude yield:
222 mg, 0.62 mmol, 89%. Aldehyde 29b (154 mg, 0.43 mmol)
and 2-methyl-2-butene (6 mL) were dissolved in tBuOH (25 mL)
and cooled in an ice–water bath. To this solution was added,
dropwise, a solution of NaH2PO4 (415 mg, 3.06 mmol) and
NaClO2 (442 mg, 3.86 mmol) in water (8 mL). The resulting
mixture was allowed to warm to ambient temperature and stir
overnight (14 h). The reaction mixture was poured into 0.1 M
HCl (aq.) (100 mL) and extracted 3 ¥ 50 mL with EtOAc. The
combined organic layers were washed with water (1 ¥ 50 mL),
brine (1 ¥ 50 mL), dried over MgSO4 and concentrated in vacuo
to give a yellow solid. The crude solid was recrystallized from hot
hexane–EtOAc to give the desired acid as yellow crystals (80 mg,
0.22 mmol, 50%). dH (300 MHz, DMSO-d6, Me4Si) 11.3 (bs,
1H, OH), 8.19 (m, 2H, Ar-H), 8.08 (d, J = 8.1 Hz, 1H, Ar-H),
7.72 (m, 2H, Ar-H), 7.62 (d, J = 8.1 Hz, 1H, Ar-H), 7.49 (d,
◦
(1.0 g, 76%). Mp 98.0–99.5 C (hexanes); found C, 80.6; H, 5.7.
Calcd. for C24H20O3: C, 80.9; H, 5.7%; IR (nujol) 3400–2400,
3032, 2924, 1684, 1674, 1593 cm-1; e (405 nm) 1325 M-1 cm-1; dH
(300 MHz, CDCl3, Me4Si) 0.93 (t, J = 7.4 Hz, 3H, CH3), 1.58–1.71
(m, 2H, CH2), 2.67–2.72 (m, 2H, Ar-CH2), 5.05 (s, 2H, OCH2),
7.34–7.47 (m, 3H, Ar-H), 7.60–7.64 (m, 3H, Ar-H), 7.73–7.82 (m,
2H, Ar-H), 8.12 (d, J = 7.9 Hz, 1H, Ar-H), 8.25–8.33 (m, 2H, Ar-
H); dC(125 MHz, CDCl3) 183.2, 182.7, 157.5, 145.5, 137.2, 135.6,
134.9, 134.1, 133.8, 133.4, 132.7, 128.5, 128.3, 128.1, 127.3, 126.6,
125.9, 123.6, 76.2, 32.5, 23.3, 14.0. HRMS (EI): found MNa+
379.1302, C24H20O3Na+ requires 379.1305.
1-Benzyloxy-4-bromo-2-propyl-9,10-anthraquinone 15. Using
Method A, 14 (7.28 g, 21.1 mmol) gave yellow-orange crystals
(8.5 g, 93%). Mp 123.0–125.0 ◦C (hexanes); found C, 66.05; H, 4.3;
Br, 18.65. Calcd. for C24H19BrO3: C, 66.2; H, 4.4; Br, 18.4%. IR
(nujol) 3030, 2954, 1675, 1593, 1568 cm-1; e (405 nm) 792 M-1 cm-1;
d
H (300 MHz, CDCl3, Me4Si) 0.93 (t, J = 7.3 Hz, 3H, CH3), 1.56–
1.68 (m, 2H, CH2), 2.61–2.66 (m, 2H, CH2), 5.04 (s, 2H, OCH2),
7.36–7.46 (m, 3H, Ar-H), 7.55–7.58 (m, 2H, Ar-H), 7.73–7.79
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J = 6.6 Hz, 2H, Ar-H), 7.30 (m, 3H, Ar-H), 5.00 (s, 2H,
OCH2), 3.66 (s, 2H, CH2). dC (75 MHz, DMSO-d6) 183.1, 182.9,
175.3, 158.1, 137.2, 137.1, 136.6, 135.7, 135.2, 134.6, 134.1, 133.0,
129.10, 129.05, 128.5, 127.8, 127.1, 126.3, 124.1, 60.8, 35.9. HRMS
(EI) found MNa+ 395.0884; C23H16O5Na+ requires 395.08990.
17 in THF with 1 equiv. of the corresponding metal hydride,
stirring and concentrating. Found C, 74.60; H, 4.89. Anal. calcd.
for C22H20O5: C, 74.99; H, 5.03%. IR (nujol) 3500–2400, 3034,
2922, 1695, 1592 cm-1; e (405 nm) 870 M-1 cm-1; dH (300 MHz,
CDCl3, Me4Si) 0.95 (t, J = 7.3 Hz, 3H, CH3), 1.59–1.72 (m, 2H,
CH2), 2.68–2.73 (m, 2H, Ar-CH2), 5.06 (s, 2H, OCH2), 7.36–7.48
(m, 3H, Ar-H), 7.57–7.61 (m, 2H, Ar-H), 7.71 (s, 1H, Ar-H),
7.76–7.85 (m, 2H, Ar-H), 8.24–8.32 (m, 2H, Ar-H). dC (75 MHz,
CDCl3) 182.7, 182.2, 173.5, 158.9, 145.8, 136.8, 134.6, 134.4, 134.2,
133.8, 132.4, 131.4, 130.0, 128.6, 128.4, 127.4, 127.0, 126.2, 76.8,
32.4, 23.1, 14.1. HRMS (EI) found MH+ 401.13822; C25H20O5H+
requires 401.138351.
1-Benzyloxy-2-bromo-4-cyano-9,10-anthraquinone 9 (Method B).
To a suspension of 8 (2.12 mmol) in DMF (21 ml) was added
cuprous cyanide (4.24 mmol). The reaction mixture was then
heated to 75 ◦C and stirred for 22 hours. The reaction was
considered complete by TLC (1 : 1 EtOAc–hexanes). The crude
mixture was diluted with ethyl acetate (250 ml), washed with 1 N
HCl (250 ml), water (2 ¥ 250 ml), brine (100 ml), dried with
MgSO4, and concentrated via rotary evaporation. The crude solid
was then preabsorbed onto basic alumina and purified through a
basic alumina plug (3 : 1 CHCl3–hexanes to neat CHCl3) to give a
yellow solid (571.3 mg, 74%). Mp 238.0–240.0 ◦C. dH (300 MHz,
DMSO-d6, Me4Si) 5.12 (s, 2H, OCH2), 7.35–7.48 (m, 3H, Ar-
H), 7.62–7.65 (m, 2H, Ar-H), 7.92–8.00 (m, 2H, Ar-H), 8.16–
8.22 (m, 2H, Ar-H), 8.76 (s, 1H, Ar-H). dC (75 MHz, DMSO-d6)
180.6, 180.5, 158.4, 143.5, 136.9, 136.1, 135.0, 134.4, 133.7, 131.8,
128.6, 128.39, 128.36, 128.3, 126.8, 126.5, 126.3, 116.6, 107.1, 75.9.
HRMS (EI) found MNa+ 439.99065; C22H12BrNO3Na+ requires
439.989273.
1-(4-Hydroxy-2-nonenyl)oxy-2-propyl-9,10-anthraquinone-4-car-
boxylic acid (water soluble, caged 4-HNE) 23. Using Method
C, 22 (358 mg, 0.834 mmol) gave a yellow powder (244.8 mg,
◦
65%). Mp 163.0–170.0 C dec (CH3OH). IR (nujol) 3420–2550,
3030, 3020, 2930, 1695, 1677, 1592, 1557 cm-1; dH (300 MHz,
CDCl3, Me4Si) 0.90 (m, 3H, CH3), 1.00 (t, J = 7.4 Hz, 3H, CH3),
1.27–1.58 (m, 8H, C4H4), 1.63–1.76 (m, 2H, CH2), 2.74–2.79 (m,
2H, CH2), 4.19–4.25 (m, 1H, CHOH), 4.55 (d, J = 6.0 Hz, 2H,
=
OCH2), 5.93 (dd, J = 6.0, 15.4 Hz, 1H, CH), 6.04–6.13 (m, 1H,
=
CH), 7.61 (s, 1H. Ar-H), 7.73–7.83 (m, 2H, Ar-H), 8.19–8.27 (m,
2H, Ar-H), 10.23 (bs, 1H, CO2H). dC (75 MHz, CDCl3) 182.7,
182.2, 172.8, 158.8, 145.6, 137.7, 134.5, 134.8, 134.2, 133.8, 132.4,
131.2, 130.0, 127.3, 127.0, 126.1, 125.6, 74.8, 72.2, 37.1, 32.5, 31.7,
25.0, 23.2, 22.6, 14.1, 14.0. HRMS (EI) found MH+ 451.20948;
C27H30O6H+ requires 451.21152.
1-Benzyloxy-4-cyano-2-propyl-9,10-anthraquinone 16. Using
Method B, 15 (2.30 mmol) gave 16 as a pure yellow solid (864.1 mg,
99%). Mp 158.0–160.0 ◦C (hexanes); found: C, 78.26; H, 4.85; N,
3.62. Calcd for C24H19BrO3: C, 78.72; H, 5.02; N, 3.67%. IR (nujol)
3058, 3032, 2943, 1676, 1592, 1528 cm-1; e (405 nm) 438 M-1 cm-1;
dH (300 MHz, CDCl3, Me3Si) 0.92 (t, J = 7.3 Hz, 3H, CH3),
1.54–1.67 (m, 2H, CH2), 2.64–2.69 (m, 2H, Ar-CH2), 5.09 (s, 2H,
OCH2), 7.36–7.46 (m, 3H, Ar-H), 7.51–7.54 (m, 2H, Ar-H), 7.82–
7.85 (m, 2H, Ar-H), 7.92 (s, 1H, Ar-H), 8.28–8.33 (m, 2H, Ar-
H). dC (75 MHz, CDCl3) 181.5, 180.8, 160.9, 145.9, 141.1, 136.3,
135.6, 134.7, 134.2, 134.0, 132.0, 128.64, 128.56, 138.4, 127.3,
127.2, 126.8, 118.0, 106.9, 77.4, 32.2, 22.9, 13.9. HRMS (EI) found
MNa+ 404.12579; C25H19NO3Na+ requires 404.125712.
4-Bromo-1-hydroxy-2-propyl-9,10-anthraquinone 14. To a so-
lution of 13 (22.5 mmol) in glacial acetic acid (11.2 ml) was
added sodium acetate (67.4 mmol) and bromine (67.4 mmol). The
reaction mixture was stirred under Ar at 50 ◦C for 4.5 hours and
was considered complete when no starting material was visible
1
by H NMR. Distilled water (100 ml) was added to the reaction
mixture, and it was cooled to 0 ◦C. The product was concentrated
via vacuum filtration and dried in vacuo to give a yellow-orange
◦
powder (7.28 g, 94%). Mp 134.0–136.0 C (CH3OH). Found: C,
59.07; H, 3.76; Br, 23.07. Calcd. for C17H13BrO3: C, 59.15; H, 3.80;
Br, 23.15%. IR (nujol) 3300–2900, 3086, 3052, 2964, 2932, 1670,
1627, 1591, 1584 cm-1; dH (300 MHz, CDCl3, Me4Si) 1.02 (t, J =
7.3 Hz, 3H, CH3), 1.63–1.77 (m, 2H, CH2), 2.69–2.74 (m, 2H,
Ar-CH2), 7.75 (s, 1H, Ar-H), 7.75–7.85 (m, 2H, Ar-H), 8.26–8.31
(m, 2H, Ar-H), 13.71 (s, 1H, Ar-OH). dC (75 MHz, CDCl3) 188.4,
181.1, 161.6, 143.3, 140.3, 134.9, 134.2, 133.8, 132.2, 127.8, 127.6,
126.5, 116.9, 113.2, 31.6, 22.0, 13.9.
1-Benzyloxy-2-bromo-9,10-anthraquinone-4-carboxylic acid 11.
(Method C). To a suspension of 9 (2.06 mmol) in 74% EtOH
(18 ml) was added a 2.5 M solution of NaOH (8.24 mmol). The
reaction mixture, which reddened over the course of the reaction,
was refluxed for 35 minutes and was considered complete via TLC
(1 : 1 EtOAc–hexanes). The reaction mixture was poured into
3% H2SO4 (4 ml), forming a yellow precipitate. The crystals were
◦
cooled to 0 C, filtered, and washed with water (2 ¥ 30 ml). The
yellow crystals were then dried in vacuo (768.7 mg, 93%). Mp
214.0–216.0 ◦C (CH3OH). IR (nujol) 3300–2450, 3033, 2924, 1682,
1674, 1594, 1538 cm-1; e (405 nm) 1380 M-1 cm-1; dH (300 MHz,
DMSO-d6, Me4Si) 5.07 (s, 2H, OCH2), 7.37–7.48 (m, 3H, Ar-H),
7.66–7.68 (m, 2H, Ar-H), 7.88–7.99 (m, 2H, Ar-H), 8.10–8.21 (m,
2H, Ar-H), 8.21 (s, 1H, Ar-H). dC (75 MHz, DMSO-d6) 181.7,
180.9, 168.6, 155.4, 136.4, 136.3, 134.8, 134.2, 133.9, 133.3, 132.0,
131.3, 128.5, 128.3, 128.2, 127.6, 126.9, 126.7, 126.2, 75.1. HRMS
(EI) found MH+ 436.99912; C22H13BrO5H+ requires 437.001912.
4-Cyano-1-hydroxy-2-propyl-9,10-anthraquinone 20. A solu-
tion of 16 (1.70 g, 4.46 mmol) in 12 : 7 : 1 AcOH–MeOH–H2O
(1.0 L) was photolyzed (MPL with UO2 filter, hv > 340 nm)
in a 1 L immersion well. The reaction was considered complete
after 2 hours via TLC (alumina, CH2Cl2). The reaction solution
was concentrated in vacuo and purified via flash chromatography
(silica, 3 : 1 CH2Cl2–hexanes). The fractions containing product
were combined and concentrated in vacuo to give a yellow solid
(980.5 mg, 75%). Mp 185.0–186.0 ◦C (CH3OH). Found: C, 74.22;
H, 4.43; N, 4.87. Calcd. for C18H13NO3 requires C, 74.22; H, 4.50;
N, 4.81%. IR (nujol) 3250–2800, 3072, 3055, 2964, 2932, 2218,
1674, 1638, 1589, 1580 cm-1; dH (300 MHz, CDCl3, Me4Si) 1.02 (t,
J = 7.3 Hz, 3H, CH3), 1.67–1.79 (m, 2H, CH2), 2.73–2.80 (m, 2H,
1-Benzyloxy-2-propyl-9,10-anthraquinone-4-carboxylic acid 17.
Using Method C, 16 (0.865 mmol) gave 17 as a yellow solid
◦
(268.6 mg, 78%). Mp 153.0–156.0 C (CH3OH). The sodium or
potassium salt (18 or 19, respectively) was obtained by treating
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Ar-CH2), 7.81 (s, 1H, Ar-H), 7.84–7.91 (m, 2H, Ar-H), 8.30–8.39
(m, 2H, Ar-H), 13.62 (s, 1H, Ar-OH). dC (75 MHz, CDCl3) 188.6,
180.1, 163.9, 141.1, 139.7, 135.5, 134.7, 133.7, 133.1, 133.3, 132.3,
128.0, 127.1, 118.2, 115.9, 102.3, 31.6, 21.9, 13.9.
petroleum ether (3 ¥ 60 ml). The petroleum ether was concentrated
in vacuo to give 4-HNE. The aqueous layer was then diluted
with diethyl ether and acidified with 1 N HCl. NaCl (s, 1 g) was
added, and the aqueous layer was extracted with diethyl ether (2 ¥
60 ml). The organic extracts were combined, dried with MgSO4,
and concentrated in vacuo to give 33. 1-Hydroxy-2-propyl-9,10-
1-(1-Phenylethyl)-9,10-anthraquinone 31. To a solution of 6
(1.32 g, 5.9 mmol) in dry THF (120 mL) under Ar in an oven
dried flask was added, sequentially, Ph3P (1.94 g, 7.4 mmol), 1-
phenylethanol (1.07 g, 8.8 mmol) and DIAD (2.50 g, 10.3 mmol).
The solution was stirred at 60 ◦C for 1 h. The solvent was removed
in vacuo and the residue subjected to column chromatography
over silica (hexanes–EtOAc). Compound 31 thus obtained was
recrystallized from methanol to give yellow crystals (1.25 g,
3.8 mmol, 64%). (S)-31 could be prepared using the same
procedure but starting with (R)-1-phenylethanol instead of the
racemate. Optical purity was determined by analytical HPLC. Mp
92.0–93.0 ◦C; found C, 80.34; H, 4.86. Calcd. for C22H16O3: C,
80.46; H, 4.91%. IR (nujol) 3064, 3052, 3044, 2987, 2935, 1668,
1662, 1584, 1495 cm-1; dH (300 MHz, CDCl3, Me4Si) 1.82 (d, J =
6 Hz, 3H), 5.53 (q, J = 6 Hz, 1H), 7.19 (d, J = 9 Hz, 1H), 7.28 (d,
J = 9 Hz, 1H), 7.35 (t, J = 8 Hz, 2H), 7.51 (q, J = 7.5 Hz, 3H),
7.77 (dtd, J = 1.5 Hz, 3.9 Hz, 8 Hz, 2H), 7.89 (d, J = 6 Hz, 1H),
8.24 (dd, J = 1.5 Hz, 6 Hz, 1H), 8.34 (dd, J = 1.5 Hz, 4 Hz, 6 Hz,
1H); dC (300 MHz, CDCl3) 183.7, 182.4, 158.9, 142.5, 135.9, 135.3,
134.6, 134.4, 133.3, 132.7, 129.0, 128.0, 127.4, 126.7, 125.9, 122.5,
121.5, 120.0. HRMS (EI) found MNa+ 351.0982; C22H16O3Na+,
requires 351.0992.
◦
anthraquinone-4-carboxylic acid 33. Mp 243.0–250.0 C dec. dH
(300 MHz, DMSO-d6, Me4Si) 0.93 (t, J = 7.4 Hz, 3H, CH3), 1.56–
1.68 (m, 2H, CH2), 2.65–2.70 (m, 2H, Ar-CH2), 7.51 (s, 1H, Ar-H),
7.91–7.96 (m, 2H, Ar-H), 8.09–8.12 (m, 1H, Ar-H), 8.19–8.23 (m,
1H, Ar-H), 13.14 (bs, 1H, Ar-OH). dC (75 MHz, DMSO-d6) 188.8,
181.1, 170.2, 159.9, 137.9, 135.2, 135.0, 134.5, 133.2, 132.4, 127.1,
126.8, 126.5, 114.7, 31.0, 21.8, 13.8. HRMS (EI) found MNa+
333.07492; C18H14O5Na+ requires 333.07334.
Acknowledgements
This work was supported in part by NIH (R15GM070468–01)
and NSF (CHE-0514576). The authors thank Drs Marcus Wright
and Cynthia Day for spectroscopic assistance.
Notes and references
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Relative quantum yields (rates) of 25, 15, 16, 17, 18, 29. UV–
VIS spectra were taken of all compounds at 250 mM. Absorbances
were normalized to the absorbance of 17 at 405 nm at a concentra-
tion of 5 mM. The molar absorptivities of 25, 15, 16, 17, 18, and 29
were 1325, 792, 438, 870, 1876, 1345, respectively. The necessary
concentrations of 25, 15, 16, 18, and 29 were calculated to be
2.75 mM, 4.60 mM, 8.31 mM, 4.0 mM, and 2.5 mM, respectively,
using Beer’s law. Experiments probing the heavy atom effect used
0, 28, 55, 110 mM iodoform, and 110 mM chloroform against 25.
All substrates were dissolved in 1 : 4 DMSO-d6–CD3OD or 1 : 4
DMSO-d6–D2O. Due to the lack of solubility of 16 in this solvent
system at 8.31 mM, this substrate was diluted to 4.16 mM. 2,4,6-
Trimethylbenzoic acid (1 equiv.) was added to each sample as an
internal standard; the sodium salt of 2,4,6-trimethylbenzoic acid
(1 equiv.) was used for the D2O sample. Stock solutions of each
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to an NMR tube. Then, a 0 minute 1H NMR was performed with
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were run with and rates referenced to 25.
Release of 4-HNE in aqueous solution. 24 (0.0819 mmol) was
dissolved in a buffered solution (pH 5.0, 7.0, or 9.0; 32.7 mL) and
irradiated with a Rayonet reactor (16 lamps with peak emission at
419 nm) for 2.5 hours. The aqueous solution was extracted with
4210 | Org. Biomol. Chem., 2008, 6, 4204–4211
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