Enhanced Glutathione Peroxidase Activity of Water-Soluble and Polyethylene Glycol-Supported Selenides, Related Spirodioxyselenuranes, and Pincer Selenuranes

Article abstract of DOI:10.1021/acs.joc.6b01593

Diaryl selenides containing o-hydroxymethylene substituents function as peroxide-destroying mimetics of the antioxidant selenoenzyme glutathione peroxidase (GPx), via oxidation to the corresponding spirodioxyselenuranes with hydrogen peroxide and subsequent reduction back to the original selenides with glutathione. Parent selenides with 3-hydroxypropyl or 2,3-dihydroxypropyl groups produced the novel compounds 10 and 11, respectively, with greatly improved aqueous solubility and catalytic activity. The phenolic derivative 28 displayed similarly ameliorated properties and also modest radical-inhibiting antioxidant activity, as evidenced by an assay based on phenolic hydrogen atom transfer to the stable free radical DPPH. In contrast, several selenides that afford pincer selenuranes (e.g., 20 and 21) instead of spiroselenuranes upon oxidation showed inferior catalytic activity. Several selenide analogues were attached to polyethylene glycol (PEG) oligomers, as PEG substituents can improve water solubility and bioavailability, while retarding clearance. Again, the PEG derivatives afforded remarkable activity when oxidation generated spirodioxyselenuranes and diminished activity when pincer compounds were produced. Several such compounds proved to be ca. 10- to 100-fold catalytically superior to the diaryl selenides and their spirodioxyselenurane counterparts investigated previously. Finally, an NMR-based assay employing glutathione in D₂O was designed to accommodate the faster reacting water-soluble mimetics and to more closely duplicate in vivo conditions.

Full text of DOI:10.1021/acs.joc.6b01593

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Article
Enhanced Glutathione Peroxidase Activity of Water-
Soluble and Polyethylene Glycol-Supported Selenides,
Related Spirodioxyselenuranes and Pincer Selenuranes
Nicole M.R. McNeil, David J Press, Don M. Mayder, Pablo Garnica, Lisa M. Doyle, and Thomas G. Back
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01593 • Publication Date (Web): 15 Aug 2016
Downloaded from http://pubs.acs.org on August 21, 2016
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Enhanced Glutathione Peroxidase Activity of Water-Soluble and Polyethylene Glycol-
Supported Selenides, Related Spirodioxyselenuranes and Pincer Selenuranes
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Nicole M. R. McNeil, David J. Press, Don M. Mayder, Pablo Garnica, Lisa M. Doyle and
Thomas G. Back*
Department of Chemistry, University of Calgary, 2500 University Drive N.W., Calgary, Alberta,
Canada, T2N 1N4
Correspondence to: tgback@ucalgary.ca
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Abstract
Diaryl selenides containing oꢀhydroxymethylene substituents function as peroxideꢀ
destroying mimetics of the antioxidant selenoenzyme glutathione peroxidase (GPx), via
oxidation to the corresponding spirodioxyselenuranes with hydrogen peroxide and subsequent
reduction back to the original selenides with glutathione. Parent selenides with 3ꢀhydroxypropyl
or 2,3ꢀdihydroxypropyl groups produced the novel compounds 10 and 11, respectively, with
greatly improved aqueous solubility and catalytic activity. The phenolic derivative 28 displayed
similarly ameliorated properties and also modest radicalꢀinhibiting antioxidant activity as
evidenced by an assay based on phenolic hydrogen atom transfer to the stable free radical DPPH.
In contrast, several selenides that afford pincer selenuranes (e.g. 20 and 21) instead of
spiroselenuranes upon oxidation showed inferior catalytic activity. Several selenide analogues
were attached to polyethylene glycol (PEG) oligomers, as PEG substituents can improve water
solubility and bioavailability, while retarding clearance. Again, the PEG derivatives afforded
remarkable activity when oxidation generated spirodioxyselenuranes and diminished activity
when pincer compounds were produced. Several such compounds proved to be ca. 10ꢀ to 100ꢀ
fold catalytically superior to the diaryl selenides and their spirodioxyselenurane counterparts
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investigated previously. Finally, an NMRꢀbased assay employing glutathione in D O was
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designed to accommodate the faster reacting waterꢀsoluble mimetics and to more closely
duplicate in vivo conditions.
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Introduction
The formation of harmful peroxide byproducts and other reactive oxygen species (ROS)
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is a natural consequence of aerobic metabolism. The oneꢀelectron reduction of dioxygen in the
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mitochondria of cells generates superoxide radical anion, which is converted into hydrogen
peroxide by superoxide dismutase (SOD). Ferrousꢀmediated reduction of hydrogen peroxide then
produces highly reactive hydroxyl radicals. Furthermore, oxidation of lipids results in the
formation of the corresponding lipid hydroperoxides. Both hydrogen peroxide and lipid
hydroperoxides contribute to oxidative stress, which in turn is implicated in a variety of diseases
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,2
and degenerative conditions. Fortunately, homeostasis of ROS is maintained in living
organisms by both exogenous and endogenous antioxidants that catalyze the destruction of
peroxides or inhibit peroxideꢀinitiated radical chain reactions. The exogenous species include,
for example, αꢀtocopherol, ascorbic acid, anthocyanins and other plant pigments, as well as a
wide range of phenolic compounds found in various foods and beverages. The selenoenzyme
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glutathione peroxidase (GPx) is an endogenous antioxidant that catalyzes the reduction of
hydrogen peroxide and lipid peroxides by the tripeptide thiol glutathione. While several
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isozymes of GPx are known, the most common forms are tetrameric structures, in which the
redox properties of each subunit can be attributed to the presence of a selenocysteine residue.
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The catalytic cycle of this process is shown in Scheme 1, where the selenol group (EnzSeH) of
each selenocysteine residue reduces the peroxide and generates the corresponding selenenic acid
(EnzSeOH). The latter is then reduced by two equivalents of glutathione (GSH) via the selenenyl
sulfide (EnzSeSG) to restore the original selenocysteine moiety along with the formation of
glutathione disulfide (GSSG). Finally, the glutathione reductase/NADPHꢀmediated conversion of
the disulfide back to its thiol completes the process.
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Scheme 1
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Certain diseases and degenerative conditions are accompanied by particularly high levels
of peroxide formation and accompanying oxidative stress that overwhelm the protective effects
of GPx. Thus ischemic reperfusion of heart attack and stroke patients often results in
cardiovascular and neurological injury from the destructive effects of peroxides and other ROS
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released by neutrophils during the reperfusion process. A variety of smallꢀmolecule selenium
compounds has been investigated as possible redox catalysts designed to emulate GPx and afford
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additional protection to such patients. Most notably, ebselen (1), a compound that was first
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reported nearly a century ago, was investigated by DaiichiꢀSankyo Inc. in Phase 3 clinical trials
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for its cardiovascular and neuroprotective effects. More recently, Sound Pharmaceuticals Inc.
has completed a Phase 2 clinical trial (NCT01444846) of SPIꢀ1005 (ebselen) for the treatment of
noiseꢀinduced hearing loss and has begun an investigation of its therapeutic application to bipolar
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disorder, where in each case oxidative stress has been implicated. Moreover, several
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investigations have been reported of the antioxidant properties of ALT 2074 (2), which was the
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subject of a trial by Synvista Inc. for the treatment of plaque psoriasis.
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Ebselen in particular has been widely studied for its catalytic redox properties. However,
its GPxꢀlike catalytic activity is only moderate and its very low aqueous solubility makes
intravenous administration challenging. On the other hand, despite selenosis concerns associated
with many other types of selenium compounds, ebselen has the advantage of proven lack of
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toxicity from its clinical trials. Relatively low toxicity is typically associated with arylseleno
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derivatives that are presumably more resistant than aliphatic analogues toward metabolic
conversion into more toxic inorganic species such as selenite. Thus, there remains a need for the
discovery of novel types of smallꢀmolecule arylseleno GPx mimetics with improved catalytic
activity, enhanced waterꢀsolubility and ease of synthesis.
Our laboratory has investigated three classes of GPx mimetics to date: cyclic seleninate
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18
16c,16d,19
esters 3,
conformationally restricted diselenides 4 and spirodioxyselenuranes 5.
Certain seleninate esters show strong GPxꢀlike catalytic activity, which is however compromised
at increasing thiol:peroxide ratios through the formation of selenenyl sulfides that display
relatively poor activity, thus creating a competing deactivation pathway. Furthermore, the
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seleninate esters 3 catalyze the further oxidation of disulfides to thiolsulfinates which could be
detrimental in vivo if essential native protein and peptide disulfides are similarly affected.
Compounds 3 also catalyze the oxidation of a number of other substrates (e.g. alkenes to
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epoxides, sulfides to sulfoxides and enamines to αꢀhydroxyketones). The diselenides 4 have
been less thoroughly investigated as GPx mimetics, but moderate peroxideꢀdestroying catalytic
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activity was observed. Finally, the spirodioxyselenuranes 5 showed generally high catalytic
activity in the reduction of peroxides with thiols, but a much lower proclivity toward the
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oxidation of the product disulfides or other substrates than in the case of the seleninate esters 3,
and they do not produce the more refractory selenenyl sulfide derivatives.
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The spirodioxyselenuranes 5 function as catalysts via the catalytic cycle shown in
Scheme 2. We have established that the rateꢀdetermining step is the oxidation of the selenides 6
to the selenoxides 7, followed by rapid cyclization and dehydration to afford the spiro
compounds 5. Further reaction with two equivalents of thiol then regenerates the selenide,
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presumably via reductive elimination of the corresponding disulfide from the intermediate 8.
As expected, electronꢀdonating substituents enhance the rate of the oxidation step through
mesomeric stabilization of the positive charge on the selenium atom, which increases from Se(II)
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to Se(IV) during the conversion of 6 to 7.
The cyclic seleninate esters 3 and
conformationally restricted diselenides 4 represent the oxidized and reduced states in their
respective catalytic cycles and are usually introduced in those forms. In contrast, either oxidation
state (spirodioxyselenurane 5 or selenide 6) can be utilized for the process shown in Scheme 2,
as both 5 and 6 are stable and easily handled compounds. However, if it is assumed that there are
typically higher concentrations of glutathione and other thiols than of peroxides within living
organisms, then it follows that the catalyst would exist primarily in its selenide form in vivo. We
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now report the preparation and in vitro assay of a series of more waterꢀsoluble selenide
analogues of 6 containing one aryl and one aliphatic substituent, extra hydroxyl groups on the
aliphatic moiety and/or their polyethylene glycol (PEG) derivatives, along with several other
types of selenuranes not previously investigated.
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Scheme 2
Results and Discussion
In order to increase the aqueous solubility of selenides 6, we prepared a series of
analogues in which one of the aryl substituents was replaced by the 3ꢀhydroxypropyl group. The
other aromatic arylseleno moiety was retained in order to preserve the greater stability and lower
toxicity expected of aryl selenides. Since electronꢀdonating groups para to selenium improved
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catalytic activity in previous studies, and had varied effects at other positions, we prepared
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monoꢀ, diꢀ and trimethoxyaryl selenide derivatives 10bꢀ10g, along with the unsubstituted parent
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selenide 10a for comparison (Scheme 3). The required precursor diselenides 9a,
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d, 9e and 9f were obtained as reported previously, as was the methoxymethyl (MOM)ꢀ
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protected diselenide 12 Diselenides 9aꢀ9f and 12 were then reduced with sodium borohydride
and treated with 3ꢀbromopropanol to afford 10aꢀ10f, as well as 13. Deprotection of the latter
provided selenide 10g. These compounds all displayed modest aqueous solubility, but typically
required the presence of ca. 5% ethanol to attain useful concentrations. In order to improve water
solubility further, a parallel series of 2,3ꢀdihydroxypropyl compounds 11aꢀ11g was prepared by
employing (S)ꢀglycidol in place of 3ꢀbromopropanol (Scheme 3). The presence of the additional
free hydroxyl group on the propyl substituent in compounds 11 resulted in improved solubility,
as expected. All of the selenides 10 and 11 reported here are novel compounds.
Scheme 3
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16e
We recently reported the preparation and further oxidation with hydrogen peroxide of
selenides 15a,b, thus affording the corresponding pincer selenuranes 17a,b via initially formed
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selenoxides 16a,b. The absence of products derived from the [2,3]sigmatropic rearrangement
of 16b was noteworthy, as such rearrangements typically occur rapidly. The preferential
formation of 17b indicates that the dehydrative cyclization of the selenoxide to the pincer
compound is exceptionally facile (Scheme 4). In order to improve the waterꢀsolubility of these
selenides, we prepared the novel 3ꢀhydroxypropyl analogue 20 from the easily obtained
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diselenide 18 via the protected selenide 19. As expected, oxidation with hydrogen peroxide
produced the corresponding pincer selenurane 21. The formation of the alternative
spiroselenurane oxidation/cyclization product 22 was ruled out by the NMR spectra of the
product, which were consistent with the more symmetrical structure 21 (Scheme 5).
The phenolic derivative 28 was synthesized via the diselenide 26, as shown in Scheme 6.
The selenide 28 is of special interest as it has the potential to fill a dual role as an antioxidant by
not only catalyzing the reduction of peroxides with thiols, but also by serving as a radical
inhibitor through hydrogen atom donation from the phenolic moiety to chainꢀpropagating
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radicals in lipid peroxidation and other radicalꢀmediated processes. By analogy, many phenols,
such as αꢀtocopherol, possess radicalꢀinhibiting properties, while various phenolic selenides and
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tellurides have been previously investigated in this context by Engman et al.
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Scheme 6
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The poor bioavailability due to high clearance rates, poor waterꢀsolubility or untargeted
distributions of certain smallꢀmolecule drugs can sometimes be mitigated by attachment to a
suitable support. The use of polyethylene glycol (PEG) polymers or oligomers has proven
particularly effective for this purpose, as PEG derivatives tend to be waterꢀsoluble, nontoxic and
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nonimmunogenic.
Thus, in order to further refine the properties of the above GPx mimetics,
their attachment to polyethylene glycol supports was investigated. In order to produce
compounds with uniform molecular weights, we chose oligomers with only three or four PEG
units, instead of higher polymers of PEG. The preparation of these compounds is illustrated in
Scheme 7. Thus, diselenide 18 was reduced and alkylated with the monotosylated PEG reagents
39a and 39b to afford PEGylated selenides 30a and 30b, respectively, after deprotection. Similar
treatment of 18 with the ditosylated PEG reagents 40a and 40b produced the dimeric species 32a
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and 32b. Furthermore, dibenzoylation of the free alcohol functionalities of 27, followed by
deprotection of the phenolic hydroxyl group provided selenide 34. PEGylation followed by
saponification then furnished the PEGylated compound 36. The conversion of 34 to the dimeric
product 37 was achieved similarly by employing the bistosylate 40a and subsequent
saponification provided 38. An attempt was also made to prepare 38 directly from the
unprotected trihydroxy precursor 28 by exploiting the greater acidity of its phenolic moiety
relative to the aliphatic alcohols. However, selective formation and alkylation of the phenoxide
of 28 by treatment with potassium carbonate and bistosylate 40a afforded at best only 16% of the
desired product, even in the presence of 18ꢀcrownꢀ6.
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Scheme 7
OH
Se
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. NaBH , EtOH
OMOM
OMOM
4
AmberliteR
2. K CO3
2
Me
Se
Me
Se ]2
39a or 39b
for n = 3
[
O]n
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HCl - MeOH
for n = 4
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OH
OMOM
29a (n = 3) 23%
OMOM
1
8
30a (n = 3) 57%
30b (n = 4) 76%
29b (n = 4) 74%
1. NaBH4,
EtOH
OMOM
Se
MOMO
Se
OH
Se
HO
2. K CO3
HCl
MeOH
2
Se
40a or 40b
[
O ]n
[
O ]n
1
8
OMOM
OMOM
OH
OH
32a (n = 2) 27%
31a (n = 2) 47%
31b (n = 3) 71%
32b (n = 3) 62%
BzCl
pyridine
HCl
MeOH
Se
OH
Se
OBz
Se
OBz
MOMO
MOMO
HO
OH
OBz
OBz
27
33 80%
34 79%
K CO , 39a
2
3
K CO
2
3
Se
OBz
Se
OH
MeOH
[ O
[ O
Me
]3 O
]3 O
35 61%
OBz
Me
OH
OBz
36 51%
Se
OBz
Se
K CO
2
3
K CO , 40a
[ O
2
3
]3
O
MeOH
3
4
OBz
BzO
37 54%
Se
OH
OH
Se
[ O
]3 O
OH
HO
38 40%
Me
Ts [ O
] OTs
n
TsO
[
O ]n
3
3
9a (n = 3)
9b (n = 4)
40a (n = 3)
40b (n = 4)
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6
28
With the desired suite of waterꢀsoluble selenides in hand, the next stage was their
1
6,18ꢀ20,29
evaluation as GPx mimetics. In previous work,
we had employed an HPLCꢀbased assay
that measured the rate of disulfide formation when hydrogen peroxide or tꢀbutyl hydroperoxide
were reduced with benzyl thiol in the presence of catalytic amounts of the selenium compound.
This thiol was chosen because it possesses both an aromatic chromophore for UV detection and a
wellꢀseparated methylene NMR signal to aid in identifying products and intermediates.
Furthermore, the aliphatic benzyl thiol more closely resembles glutathione than do aromatic
thiols such as benzenethiol, due to the considerably greater acidity of the latter and the greater
ease of oxidation of the resulting thiolate anion compared to its conjugate acid. The assay was
performed in dichloromethaneꢀmethanol (95:5) because this solvent was capable of dissolving all
of our previously investigated catalysts, which were generally highly insoluble in water. In order
to compare the catalytic activity of various types of GPx mimetics, we typically measured the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
time (t ) required for oxidation of 50% of the thiol. The use of rate constants for this purpose
1/2
was avoided because of kinetic irregularities resulting from anomalously rapid initial reaction
rates when the catalysts were introduced in their oxidized form in all three classes of compounds
3ꢀ5. This can be attributed to the rapid reduction of the oxidized catalysts with the thiol
accompanied by disulfide formation, compared to the considerably slower and rateꢀlimiting
subsequent oxidation of Se(II) to Se(IV). Moreover, in the case of seleninates 3, the
accumulation of less reactive selenenyl sulfide byproducts in the later stages of the catalytic
cycle suppressed the reaction rate considerably. However, it should be noted that the thiol
(
benzyl thiol) and solvent (dichloromethaneꢀmethanol) employed in this assay are poorly
representative of in vivo conditions.
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1
2
3
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9
In order to circumvent these limitations and to take advantage of the aqueous solubility of
1
30
the present compounds, we devised a H NMRꢀbased assay in D O for the present work. This
2
employed the reduction of hydrogen peroxide with glutathione (GSH) with accompanying
formation of the disulfide (GSSG) in the presence of the selenide catalyst. A typical reaction
with catalyst 11d is shown in Figure 1, where spectrum A was recorded prior to the addition of
hydrogen peroxide (t ), spectrum B represents ca. 50% completion (t ) and spectrum C was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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41
42
43
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47
48
49
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51
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55
56
57
58
59
60
0
1/2
obtained from an authentic sample of GSSG. These spectra reveal that the signal for Hꢀ2 (GSH)
and Hꢀ2' (GSSG) remains essentially unchanged in this process, while that for Hꢀ4 (GSH)
gradually disappears and the corresponding peak Hꢀ4' (GSSG) appears further downfield, where
it overlaps with the residual water peak (not shown). Furthermore, the signal for Hꢀ5'a is wellꢀ
separated from its diastereotopic counterpart Hꢀ5'b (GSSG) as well as from Hꢀ5 (GSH). Thus, it
is possible to integrate either the disappearance of the Hꢀ4 or the appearance of the Hꢀ5'a peak
against the constant Hꢀ2 plus Hꢀ2' signal to determine the extent of completion of the process at
any given time.
The assays were carried out in phosphateꢀbuffered D O solution at a pD of 2.3 and are
2
corrected for the uncatalyzed background reaction of hydrogen peroxide with glutathione, which
is considerably slower under these conditions. However, attempts to perform the assays at
30a,31
physiological pH produced a much faster control reaction
that resulted in poor
reproducibility of the corrected results with the catalyst present. Overall, advantages of the NMR
assay include a medium that more closely resembles biological conditions, despite the low pD,
and faster acquisition of data, which proved useful for the most active catalysts. The results of
both the HPLC and NMR assays are provided in Table 1.
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2
2
2
2
2
2
2
2
3
3
3
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3
3
3
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0
1
2
3
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9
0
1
2
3
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0
1
2
3
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8
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Conversion of GSH to GSSG with H O in the presence of 10 mol % of selenide 11d,
2
2
1
as monitored by H NMR (400 MHz) spectroscopy. Spectrum A: at t . Spectrum B: at t .
0
1/2
Spectrum C: authentic GSSG.
The presence of electronꢀdonating methoxy groups at various positions of the aromatic
rings of 10aꢀ10g had a significant effect on the reaction rates. Thus, in the HPLC assay in Table
1
, the pꢀmethoxy (i.e. para to the Se substituent) derivative 10d (entry 4; t1/2 = 35 min) was more
active than the unsubstituted derivative 10a (entry 1; t1/2 = 55 min), as expected because of
resonance stabilization by the electronꢀdonating group of the increasing positive charge on the
selenium atom during the rateꢀdetermining oxidation step. The ortho and meta isomers 10b and
1
0c (entries 2 and 3, respectively), however, showed only slight rate enhancements relative to
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6
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9
1
0a and provided inferior activity compared to 10d. The o,pꢀdimethoxy derivative 10e (entry 5)
produced essentially the same results as 10d, while the m,pꢀanalogue 10f catalyzed a remarkably
rapid consumption of the thiol (entry 6, t1/2 = 10.0 min). These results indicate that the presence
of a single methoxy group in the para position is more effective than its ortho or metaꢀ
substituted isomers in promoting the reduction of the peroxide. Furthermore, the ineffectiveness
of a second methoxy substituent in the ortho position suggests a balance between resonance
enhancement and steric suppression of the rate of the process. In contrast, a second methoxy
group at the metaꢀposition provided a strong further promotive effect compared to 10d. The
relatively poor performance of the trimethoxy analogue 10g (entry 7; t1/2 = 102 min) was
unexpected, but clearly shows that the introduction of a third methoxy substituent is
counterproductive.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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26
27
28
29
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37
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The analogous series of selenides 11aꢀ11g, containing an additional free hydroxyl group,
provided generally comparable activity in the HPLC assay, but with some differences. Thus, the
pꢀmethoxy derivative 11d (entry 11; t1/2 = 36 min) was considerably more active than the
unsubstituted analogue 11a (entry 8; t1/2 = 79 min), again attributed to mesomeric effects. In
contrast, the oꢀmethoxy substituent in 11b (entry 9; t1/2 = 103 min) suppressed reactivity
considerably, presumably because of dominant steric effects. The mꢀmethoxy compound 11c
(
entry 10; t1/2 = 49 min) showed a modest increase in reaction rate compared to 11a (entry 8). No
further improvement relative to the para derivative 11d was observed when multiple methoxy
groups were introduced in 11eꢀ11g (entries 12ꢀ14). As in the previous series of selenides 10, the
trimethoxy product 11g provided the slowest rate of all (entry 14; t1/2 = 150 min) of the
compounds 11. It is noteworthy that all of the compounds 10 and 11 exhibited considerably
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6
superior catalytic activity to the variously substituted diaryl selenides 6 reported previously (t1/2
1
9c
16d
=
5.5 – 21 h ), or to ebselen (t = 24 h) under similar conditions.
1/2
Table 1. Assays of catalytic activity of GPx mimetics.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
a,b
b,c
HPLC assay
t1/2 (min)
55
NMR assay
t1/2 (min)
ꢀ
entry
1
compound
10a
2
3
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
11e
11f
11g
15a
15b
20
48
44
ꢀ
ꢀ
4
35
12.7
ꢀ
5
37
6
10.0
102
79
4.7
ꢀ
7
8
61
241
81
9
103
49
10
d
11
36
22 (31)
12
43
58
24
13
70
14
150
627
ꢀ
e
15
570
e
16
1260
ꢀ
17
306
ꢀ
18
28
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
10.5
1086
1008
19
30a
30b
32a
32b
36
20
f
21
921
22
924
12.5
6.9
23
24
38
a
Footnotes to table 1: Conducted with 35 mM hydrogen peroxide, 31 mM benzyl thiol and 3.1
mM (10 mol %) of the catalyst in dichloromethaneꢀmethanol (95:5) at 18 °C. Each t1/2 value is
the average of at least two runs and is corrected for the uncatalyzed background oxidation of
thiol with H O . For more detail, including kinetic plots, see the Experimental Section.
Conducted with the same concentrations as the HPLC assay in D O containing a phosphate
b
2
2
c
2
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1
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9
d
e
buffer (pD 2.3) at 22 °C. Value in parentheses was obtained in unbuffered D O. Value taken
from ref. 16e. Value obtained in unbuffered DMSOꢀd due to poor solubility in D O.
2
f
6
2
The NMR assay was only run on two compounds, 10d and 10f, from the 3ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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26
27
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29
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35
36
37
38
39
40
41
42
43
44
45
46
47
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49
50
51
52
53
54
55
56
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59
60
hydroxypropyl selenides 10 due to solubility constraints in the D O solvent, but revealed further
2
improvement in catalytic activity in the aqueous medium (10d, entry 4, t1/2 = 12.7 min; 10f, entry
6, t1/2 = 4.7 min) compared to the HPLC assay, and barely within the time limitations for
monitoring by NMR. The NMR assay again confirmed that the strongest catalytic activity of
compounds 11 was obtained with the pꢀmethoxy derivative 11d (entry 11; t1/2 = 22 min),
although the m,pꢀdimethoxy analogue 11f was comparable (entry 13; t = 24 min). In this assay,
1
/2
oꢀmethoxy groups suppressed the catalytic activity of 11b, 11e and 11g (entries 9, 12 and 14,
respectively), relative to the pꢀmethoxy selenide 11d, although the o,pꢀdimethoxy compound 11e
was comparable to the unsubstituted selenide 11a.
The selenide precursors 15a and 15b of the corresponding pincer selenuranes 17a and
16e
1
7b were reported previously and their corresponding HPLC assay data are included in Table
1
for comparison (entries 15 and 16). These compounds and the related derivative 20 (entry 17)
all showed greatly inferior catalytic activity compared to the selenides 10aꢀ10g and 11aꢀ11f in
this assay.
The phenolic selenide 28 (entry 18) proved sufficiently waterꢀsoluble to be subjected to
the NMR assay and produced a remarkably short t of only 10.5 min. Since other phenols have
1
/2
3
2
been reported to undergo oxidation to quinones with benzeneseleninic acid and anhydride, we
performed several control reactions to investigate its behaviour further. When oxidized with
hydrogen peroxide in the absence of a thiol, it afforded the corresponding spiroselenurane 41 in
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0
1
2
3
4
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
high yield in ca. 15 min (Scheme 8). The product was isolated and then treated with two
equivalents of benzyl thiol in the absence of hydrogen peroxide, resulting in the immediate
precipitation of the original selenide 28. These experiments establish that 41 is a plausible
intermediate in the catalytic cycle of 28, which we postulate is analogous to that shown in
Scheme 2. They also demonstrate that the thiolysis of 41 is even faster than the oxidation of 28,
by at least an order of magnitude. Although there was no evidence of quinone formation during
the oxidation step under these conditions, prolonged exposure to excess hydrogen peroxide, or to
MCPBA, produced more complex mixtures that could not be separated. Furthermore, control
experiments with selenide 11d, chosen as a representative of the aryl 3ꢀhydroxy and 2,3ꢀ
dihydroxypropyl selenide series 10 and 11, similarly revealed a relatively slow oxidation with
hydrogen peroxide in the absence of a thiol that required ca. 1 h to go to completion. A much
faster reduction of the resulting spirodioxyselenurane 42 with benzyl thiol was observed in the
absence of the peroxide, where conversion back to the original selenide 11d was complete in less
than 2 min (Scheme 8). It therefore appears that the oxidation of Se(II) to Se(IV) is again the
rateꢀdetermining step in the catalytic cycles of 28 and 11d, as had been observed previously for
GPx mimetics 3ꢀ5.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In a simple but preliminary test to determine whether phenol 28 could also serve as an
antioxidant radical inhibitor, we allowed it to react with the stable radical 2,2ꢀdiphenylꢀ1ꢀ
3
3
picrylhydrazyl (DPPH) while monitoring the UV/visible absorption of the latter at 515 nm. A
decrease in the absorbance of DPPH then indicates that a test substance has the ability to transfer
a hydrogen atom to it, thus producing the corresponding hydrazine (DPPHꢀH), which is
essentially transparent at this wavelength, along with the radical species derived from the
hydrogen donor (Scheme 9). The results are shown in Figure 2 at two different molar ratios.
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1
1
2
3
4
5
6
7
8
9
While a significant decrease in the absorbance of DPPH was observed at a DPPH:28 ratio of 1:1,
the reaction did not go to completion even after a relatively prolonged period of 4 h. In the
presence of only 10 mol % of the phenol, the reaction barely proceeded. This suggests that a
hydrogen transfer equilibrium may be gradually established between DPPH and the phenol, or
that other reactions consume DPPH under these conditions. Thus, on the basis of this assay, 28
does not appear to be especially wellꢀsuited for suppressing radical chain reactions such as lipid
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
34
oxidations.
Scheme 8
Scheme 9
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2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
5
2
0
1
9
:1 ratio
:1 ratio
48
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
6
44
4
4
2
0
0
50
100
150
200
250
Time (min)
Figure 2. Decrease of [DPPH] with time as measured by the decrease in its absorption at 515 nm.
Upper line (green) represents a DPPH:28 ratio of 9:1; lower curve (blue) corresponds to a
DPPH:28 ratio of 1:1.
Finally, we tested the PEGylated compounds in the NMR assay. Selenides 30a and 30b
(entries 19 and 20 in Table 1), as well as the dimeric species 32a and 32b (entries 21 and 22),
contain the PEG chains attached to the selenium atom. They were therefore expected to react via
pincer selenuranes upon oxidation, analogous to selenuranes 17a and 17b (Scheme 4) and 21
(
Scheme 5). Compound 32a proved too insoluble in either D O or dichloromethaneꢀmethanol to
2
permit analysis via either assay. The NMR assay was therefore run in DMSOꢀd and produced a
6
3
5
relatively long t1/2 of 921 min (15.4 h). Its homologue 32b, as well as the selenurane monomers
0a and 30b, also proved to be poor catalysts, with t1/2 values of ca. 17ꢀ18 h. Thus, like the nonꢀ
3
PEGylated compounds 15a, 15b and 20, these analogues are relatively poor GPx mimetics. In
contrast, the remaining selenides 36 and 38 are derivatives of the phenolic compound 26, with
attachment of the PEG chain at the phenolic hydroxyl group. They were therefore expected to
generate the more reactive spirodioxyselenuranes upon oxidation. Both the monomer 36 (entry
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2
3
4
5
6
7
8
9
2
3; t1/2 = 12.5 min) and the dimer 38 (entry 24; t1/2 = 6.9 min) displayed excellent catalytic
activity, approaching the limit of monitoring ability, even by the more rapid NMR method. It is
interesting to note that the dimeric species 38 exhibited a t1/2 roughly half that of the monomer
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
36
3
6, consistent with the presence of two redox centers in 38 instead of a single one in 36. In this
way, 38 more closely emulates the multivalent selenoenzyme GPx, which it will be recalled
possesses four redoxꢀactive selenocysteine moieties.
Summary and Conclusions
These results show that the waterꢀsoluble selenides 10aꢀ10g and 11aꢀ11g are highly
active catalysts for the reduction of hydrogen peroxide with thiols, as evidenced by both HPLC
and NMRꢀbased assays. In both series 10 and 11, the single p-methoxy substituents in 10d and
1
1d, as well as the m,pꢀanalogue 10f, provided the fastest reaction rates compared to their
unsubstituted or otherwise methoxyꢀsubstituted analogues. We attribute this to resonance
stabilization of increased positive charge on the selenium atom during the rateꢀdetermining
oxidation step. On the other hand, when introduced ortho to the selenium atom, a single methoxy
group strongly suppressed reactivity in 11b, but had little effect in 10b, presumably due to a
balance between mesomeric and steric effects. The incorporation of a mꢀmethoxy substituent
afforded less predictable results, with a slightly faster rate for 10c and 11c compared to 10a and
1
1a, respectively, observed in the HPLC assay in dichloromethaneꢀmethanol (95:5), and a slower
rate recorded for 11c compared to 11a in buffered D O. The incorporation of multiple methoxy
2
groups provided no further benefit compared with a single pꢀmethoxy substituent, except in the
case of the remarkably active m,pꢀderivative 10f. In contrast to selenides that produce
spirodioxyselenuranes upon oxidation, analogues 15a, 15b, 20, 30a, 30b, 32a and 32b that
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2
2
2
2
2
2
2
2
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3
3
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3
3
3
3
3
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instead afford the corresponding pincer selenuranes proved far less effective as catalysts,
regardless of the presence or absence of a PEG support. However, we were pleased to discover
that the PEGylated compounds 36 and 38, as well as the phenol 28 and the m,pꢀdimethoxy
derivative 10f, revealed excellent catalytic activity, with t1/2 values significantly shorter than
those of any other organoselenium compound previously investigated in our laboratory and more
than two orders of magnitude faster than ebselen, which could only be subject to the HPLC
protocol. These waterꢀsoluble compounds therefore merit further investigation as potentially
useful biological antioxidants.
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Experimental
General Experimental
1
6c
16f
16f
16d
16f
16f
16f
16e
10
11
12
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49
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59
60
Diselenides 9a, 9b, 9c, 9d, 9e, 9f, 12 and 18 were obtained as reported
1
13
77
previously. H NMR spectra were recorded at 400 MHz, while C and Se NMR spectra were
7
7
obtained at 101 and 76 MHz, respectively. Chemical shifts of Se NMR spectra were measured
37
with diphenyl diselenide in CDCl at 463.0 ppm as the standard, relative to dimethyl selenide at
3
0
.0 ppm. High resolution mass spectra were obtained with a time of flight (TOF) analyzer and
electron impact (EI) ionization or with a quadrupole TOF analyzer and electrospray ionization
ESI).
(
The HPLCꢀbased assay for catalytic activity was performed by adding the catalyst (0.031
mmol; 10 mol % relative to benzyl thiol) to a mixture of hydrogen peroxide (ca. 30%; 35 mM)
and benzyl thiol (31 mM) in 10.0 mL of dichloromethaneꢀmethanol (95:5) while maintaining the
temperature at 18 ºC. The HPLC instrument employed a UV detector at 254 nm and a reversed
phase column (Novapak C18; 3.9 x 150 mm). The eluent was acetonitrileꢀwater (isocratic or
gradient as required), with a flow rate of 0.9 mL/min. Naphthalene (8.0 mM) was employed as
38
an internal standard. Benzyl thiol was redistilled and hydrogen peroxide was titrated prior to
use. At least two replicates were performed for each compound and each run was performed in a
clean, new vial.
For the NMR assay, a buffer solution was prepared by dissolving NaH PO (105 mg,
2
4
0
.875 mmol) and phosphoric acid (111 µL, 85%, 0.162 mmol) in 25 mL of D O to afford a
2
39
solution with pD = 2.3. A fresh solution of GSH (31 mM) and the catalyst (3.1 mM) in the
buffered D O was prepared in a clean, new vial for each run. Sufficient aqueous hydrogen
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peroxide (ca. 30%) was added to produce a final concentration 35.4 mM, thereby initiating the
reaction. The solution was pipetted into a clean, dry NMR tube and immediately placed in the
spectrometer. Acquisition of data typically commenced within 2ꢀ3 min of the addition of
hydrogen peroxide and data collection continued until nearly complete conversion of GSH to
GSSG had occurred. The NMR spectrometer parameters were set to a 90 degree pulse program
with a relaxation delay of 3 seconds and a minimum of 8 scans per spectrum. At least two
replicates were performed for each compound.
0
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0
The DPPH assay of phenolic selenide 28 was conducted in methanol at 22 °C. The DPPH
concentration was obtained from a calibration plot of absorbance at 515 nm vs. concentration.
After the DPPH and the desired amount of 28 were mixed and placed in a cuvette, the reaction
mixture was scanned four times per min and the DPPH concentration thus obtained was plotted
vs. time as shown in Figure 2.
Typical procedure for the preparation of selenides 10a-10f. Preparation of 2-
(
hydroxymethyl)phenyl 3-hydroxypropyl selenide
was dissolved in 35 mL of THFꢀethanol (3:1) and cooled to 0 °C. Sodium borohydride (261 mg,
.90 mmol) was added and after 5 min 1ꢀbromoꢀ3ꢀpropanol (0.271 mL, 417 mg, 3.00 mmol) was
(10a). Diselenide 9a (515 mg, 1.38 mmol)
6
added. The mixture was warmed to room temperature, left for an additional 2 h and quenched
with 25 mL of water. The mixture was extracted with ethyl acetate, washed with brine, dried and
concentrated under reduced pressure. The resulting oil was purified by flash chromatography
(
hexanesꢀethyl acetate, 2:1) to afford 488 mg (72%) of the product as a colourless solid, mp 42ꢀ
ꢀ1 1
4
3 °C (from ethyl acetate); IR (solid) 3376, 3303, 2911, 2838, 1436, 1180, 1051 cm ; H NMR
(
400 MHz; CDCl ) δ 7.54 (dd, J = 7.6, 1.2 Hz, 1 H), 7.39 (dd, J = 7.6, 1.2 Hz, 1 H), 7.26 (td, J =
3
7.5, 1.5 Hz, 1 H), 7.21 (td, J = 7.4, 1.6 Hz, 1 H), 4.76 (d, J = 5.6 Hz, 2 H), 3.70 (q, J = 5.3 Hz, 2
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H), 2.98 (t, J = 7.2 Hz, 2 H), 2.62 (t, J = 6.0 Hz, 1 H), 1.95 (t, J = 4.4 Hz, 1 H), 1.91 (p, J = 6.7
1
3
Hz, 2 H); C NMR (101 MHz; CDCl ) δ 142.5, 133.8, 130.0, 128.7, 128.6, 127.7, 65.5, 62.2,
3
7
7
3
2.7, 24.6; Se NMR (76 MHz; CDCl ) δ 230.7; mass spectrum m/z (EI, relative intensity) 246
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
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36
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
+
(
100, M ), 229 (27), 187 (36), 170 (38), 107 (90), 91 (26), 78 (90); HRMS (EIꢀTOF) m/z: [M]
8
2
0
Calcd for C H O Se: 246.0159; found: 246.0163. Anal. Calcd for C H O Se: C, 48.99; H,
1
0
14
10 14
2
5
.76; found: C, 49.02; H, 5.73.
Compounds 10bꢀ10f and 13 were prepared similarly from diselenides 9bꢀ10f and 12,
respectively.
3
-Hydroxypropyl 6-methoxy-2-(hydroxymethyl)phenyl selenide (10b). From 200 mg
(0.463 mmol) of 9b. Yield: 213 mg, 84%. Colourless solid; mp 49ꢀ50 °C (from ethyl acetate); IR
ꢀ1
1
(
solid) 3362, 3267, 2933, 1567, 1462, 1262, 1038 cm ; H NMR (400 MHz; CDCl ) δ 7.29 (t, J
3
=
8.0 Hz, 1 H), 7.04 (dd, J = 7.6, 1.2 Hz, 1 H), 6.84 (dd, J = 8.0, 1.2 Hz, 1 H), 4.80 (s, 2 H), 3.88
(
s, 3 H), 3.68 (t, J = 6.0 Hz, 2 H), 2.94 (t, J = 7.0 Hz, 2 H), 2.83 (br s, 1 H), 2.03 (br s, 1 H), 1.81
13
(
tt, J = 7.0, 6.0 Hz, 2 H); C NMR (101 MHz; CDCl ) δ 160.2, 145.9, 129.9, 121.2, 117.3,
3
77
110.5, 66.1, 62.3, 56.3, 32.9, 24.5; Se NMR (76 MHz; CDCl ) δ 127.3; mass spectrum m/z (EI,
3
+
relative intensity) 276 (92, M ), 217 (28), 200 (40), 137 (92), 108 (100); HRMS (EIꢀTOF) m/z:
+
80
[
M] Calcd for C H O Se: 276.0265; found: 276.0275. Anal. Calcd for C H O Se: C, 48.01;
11 16 3 11 16 3
H, 5.86; found: C, 47.99; H, 5.99.
3-Hydroxypropyl 5-methoxy-2-(hydroxymethyl)phenyl selenide (10c). From 200 mg
0.463 mmol) of 9c. Yield: 215 mg, 84%. Colourless solid; mp 58ꢀ59 °C (from ethyl acetate); IR
(
(
ꢀ1 1
solid) 3352, 3271, 2930, 1594, 1468, 1279, 1221, 1041 cm ; H NMR (400 MHz; CDCl ) δ
3
7
.26 (d, J = 8.4 Hz, 1 H), 7.07 (d, J = 2.8 Hz, 1 H), 6.75 (dd, J = 8.4, 2.4 Hz, 1 H), 4.67 (s, 2 H),
.78 (s, 3 H), 3.67 (t, J = 6.0 Hz, 2 H), 2.96 (t, J = 7.2 Hz, 2 H), 2.83 (br s, 1 H), 2.44 (br s, 1 H),
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13
1
.89 (tt, J = 7.0, 6.0 Hz, 2 H); C NMR (101 MHz; CDCl ) δ 159.3, 134.7, 131.5, 130.0, 119.2,
3
7
7
1
12.5, 64.9, 62.0, 55.5, 32.6, 24.5; Se NMR (76 MHz; CDCl ) δ 228.5; mass spectrum m/z (EI,
3
+
relative intensity) 276 (100, M ), 217 (82), 199 (24), 135 (50), 108 (62); HRMS (EIꢀTOF) m/z:
0
1
2
3
4
5
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7
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9
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80
[
M] Calcd for C H O Se: 276.0265; found: 276.0267. Anal. Calcd for C H O Se: C, 48.01;
11 16 3 11 16 3
H, 5.86; found: C, 47.95; H, 5.87.
3-Hydroxypropyl 4-methoxy-2-(hydroxymethyl)phenyl selenide (10d). From 310 mg
0.717 mmol) of 9d. Yield: 265 mg, 67%. Colourless solid; mp 44ꢀ45 °C; IR (film) 3329, 2929,
(
1
595, 1476, 1295, 1 Prepared according to the same procedure as for 187. Yield: 67%.
ꢀ
1 1
Colourless solid; mp 44ꢀ45 °C; IR (film) 3329, 2929, 1595, 1476, 1295, 1229, 1057 cm ; H
NMR (400 MHz; CDCl ) δ 7.49 (d, J = 8.4 Hz, 1 H), 7.00 (d, J = 2.8 Hz, 1 H), 6.74 (dd, J = 8.6,
3
3
7
1
.0 Hz, 1 H), 4.76 (s, 2 H), 3.80 (s, 3 H), 3.67 (t, J = 6.0 Hz, 2 H), 2.99 (br s, 1 H), 2.87 (t, J =
13
.2 Hz, 2 H), 2.24 (br s, 1 H), 1.85 (tt, J = 7.2, 6.0 Hz, 2 H); C NMR (101 MHz; CDCl ) δ
3
77
59.9, 145.0, 137.2, 119.0, 114.1, 113.9, 65.5, 62.0, 55.3, 32.6, 25.4; Se NMR (76 MHz;
+
CDCl ) δ 212.5; mass spectrum m/z (EI, relative intensity) 276 (100, M ), 200 (38), 137 (45),
3
+
80
1
08 (90); HRMS (EIꢀTOF) m/z: [M] Calcd for C H O Se: 276.0265; found: 276.0270. Anal.
11 16 3
Calcd for C H O Se: C, 48.01; H, 5.86; found: C, 48.24; H, 5.69.
1
1
16
3
4
,6-Dimethoxy-2-(hydroxymethyl)phenyl 3-hydroxypropyl selenide (10e). From 100
mg (0.203 mmol) of 9e. Yield: 66 mg, 53%. Colourless solid; mp 66ꢀ67 °C; IR (solid) 3352,
ꢀ1
1
3
243, 2933, 1581, 1310, 1167, 1029 cm ; H NMR (400 MHz; CDCl ) δ 6.64 (d, J = 2.4 Hz, 1
3
H), 6.41 (d, J = 2.8 Hz, 1 H), 4.79 (s, 2 H), 3.85 (s, 3 H), 3.81 (s, 3 H), 3.69 (t, J = 6.2 Hz, 2 H),
.84 (t, J = 7.2 Hz, superimposed on br s, 3 H), 2.02 (br s, 1 H), 1.79 (tt, J = 7.2, 6.0 Hz, 2 H);
2
13
C NMR (101 MHz; CDCl ) δ 161.6, 161.3, 147.0, 107.7, 105.4, 98.2, 66.3, 62.4, 56.3, 55.5,
3
7
7
3
2.7, 24.6; Se NMR (76 MHz; CDCl ) δ 109.3; mass spectrum m/z (EI, relative intensity) 306
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(
100, M ), 230 (32), 166 (96), 138 (66); HRMS (EIꢀTOF) m/z: [M] Calcd for C H O Se:
12 18 4
3
06.0370; found: 306.0367. Anal. Calcd for C H O Se: C, 47.22; H, 5.94; found: C, 47.25; H,
12 18 4
5
.76.
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49
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60
4
,5-Dimethoxy-2-(hydroxymethyl)phenyl 3-hydroxypropyl selenide (10f). From 210
mg (0.427 mmol) of 9f. Yield: 247 mg, 95%. Colourless solid; mp 38ꢀ39 °C (from ethyl acetate);
1
IR (solid) 3300, 2924, 1505, 1452, 1252, 1152; H NMR (400 MHz; CDCl ) δ 7.11 (s, 1 H), 6.97
3
(
s, 1 H), 4.75 (d, J = 5.6 Hz, 2 H), 3.88 (s, 3 H), 3.87 (s, 3 H), 3.70 (q, J = 5.3 Hz, 2 H), 2.91 (t, J
1
3
=
7.0 Hz, 2 H), 2.56 (t, J = 6.0 Hz, 1 H), 1.92ꢀ1.83 (m 3 H); C NMR (101 MHz; CDCl ) δ
3
7
7
149.4, 148.5, 136.6, 119.4, 119.0, 112.1, 65.6, 62.2, 56.3, 56.1, 32.8, 25.8; Se NMR (76 MHz;
+
CDCl ) δ 228.3; mass spectrum m/z (EI, relative intensity) 306 (22, M ), 247 (15), 165 (20), 138
3
+
80
(
100), 123 (22), 95 (25); HRMS (EIꢀTOF) m/z: [M] Calcd for C H O Se: 306.0370; found:
12 18 4
3
06.0372. Anal. Calcd. for C H O Se: C, 47.22; H, 5.94; found: C, 47.46; H, 5.89.
12 18 4
3
-Hydroxypropyl 2,3,4-trimethoxy-2-[(methoxymethoxy)methyl]phenyl selenide
(
13). From 347 mg (0.542 mmol) of 12. Yield: 329 mg, 80%. Colourless oil; IR (neat) 3457,
ꢀ1 1
2943, 1576, 1486, 1319, 1262, 1105, 1033, 724 cm ; H NMR (400 MHz; CDCl ) δ 6.87 (s, 1
3
H), 4.77 (s, 2 H), 4.73 (s, 2 H), 3.91 (s, 3 H), 3.88 (s, 3 H), 3.86 (s, 3 H), 3.71 (t, J = 6.2 Hz, 2
1
3
H), 3.43 (s, 3 H), 2.90 (t, J = 7.0 Hz, 2 H), 1.91 (br s, 1 H), 1.81 (tt, J = 7.0, 6.2 Hz, 2 H); C
NMR (101 MHz; CDCl ) δ 155.2, 154.1, 142.0, 137.5, 115.4, 108.3, 96.1, 70.2, 62.2, 61.2, 61.1,
3
77
5
6.1, 55.7, 32.9, 25.2; Se NMR (76 MHz; CDCl ) δ 146.1; mass spectrum m/z (EI, relative
3
+
intensity) 380 (100, M ), 317 (40), 289 (50), 197 (80), 181 (40), 168 (25); HRMS (EIꢀTOF) m/z:
+
80
[
M] Calcd for C H O Se: 380.0738; found: 380.0729.
15 24 6
3
-Hydroxypropyl 2,3,4-trimethoxy-6-(hydroxymethyl)phenyl selenide (10g). Selenide
1
3 (214 mg, 0.564 mmol) was dissolved in 10 mL of methanol along with 6 drops of
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