Reactivity of (E)-4-hydroxy-2-nonenal with fluorinated phenylhydrazines: Towards the efficient derivatization of an elusive key biomarker of lipid peroxidation

Article abstract of DOI:10.1002/ejoc.201200346

4-Hydroxynonenal (4-HNE) is a major product of the oxidation of ε-6-polyunstaturated lipids and an effector of radical-mediated oxidative damage, whose analytical determination requires chemical derivatization. In this work, its reactivity with fluorinated phenylhydrazines was explored both under preparative and analytical settings. A five-step synthesis of 4-HNE on gram-scale with an overall yield of 30% is described. Reaction of 4-HNE with ortho-, meta-, or para-CF₃-phenylhydrazine, as well as with the 3,5-di-CF₃, 2,4-di-CF₃, or pentafluoro analogues, in MeCN with 0.5 mM TFA yields the corresponding hydrazones with rate constants k _f of 2.8±0.4, 1.7±0.1, 3.0±0.2, 0.6±0.1, 0.5±0.1, and 3.5±0.5 M^-1s^-1, respectively at 298 K. At higher temperatures, the hydrazones undergo intramolecular cyclization to form 1,6-dihydropyridazines that, depending on the solvent and temperature, may further react with the hydrazine to yield tetrahydropyridazine adducts and their oxidation products. Other reaction products were isolated, depending on the reaction conditions, and the complex reactivity of 4-HNE with the above nucleophiles is discussed. Due to the good yield and rate of formation of the hydrazone adducts, their stability and favorable UV absorbance, 2-(trifluoromethyl)phenylhydrazine and 2,3,4,5,6-pentafluorophenylhydrazine are the most interesting candidates for the development of rapid and efficient analytical derivatizations of 4-HNE. 4-Hydroxynonenal reacts rapidly at room temperature with 2-, 3-, or 4-CF₃-phenylhydrazine, or with the 3,5-di-CF₃, 2,4-di-CF₃, or pentafluoro analogues, to form hydrazones, which may undergo cyclization to 1,6-dihydropyridazines and other addition/oxidation products. The product distribution can be controlled by solvent and temperature to develop rapid derivatization assays. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Full text of DOI:10.1002/ejoc.201200346

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FULL PAPER
DOI: 10.1002/ejoc.201200346
Reactivity of (E)-4-Hydroxy-2-nonenal with Fluorinated Phenylhydrazines:
Towards the Efficient Derivatization of an Elusive Key Biomarker of Lipid
Peroxidation
Riccardo Matera,[a] Simone Gabbanini,[a] Alice Valvassori,[b] Mathilde Triquigneaux,[c] and
Luca Valgimigli*[b]
Keywords: Synthetic methods / Hydrazones / Cyclization / Lipids / Kinetics
4-Hydroxynonenal (4-HNE) is a major product of the oxi-
dation of ω-6-polyunstaturated lipids and an effector of radi-
cal-mediated oxidative damage, whose analytical determi-
nation requires chemical derivatization. In this work, its reac-
tivity with fluorinated phenylhydrazines was explored both
under preparative and analytical settings. A five-step synthe-
sis of 4-HNE on gram-scale with an overall yield of 30% is
described. Reaction of 4-HNE with ortho-, meta-, or para-
CF3-phenylhydrazine, as well as with the 3,5-di-CF3, 2,4-di-
tures, the hydrazones undergo intramolecular cyclization to
form 1,6-dihydropyridazines that, depending on the solvent
and temperature, may further react with the hydrazine to
yield tetrahydropyridazine adducts and their oxidation prod-
ucts. Other reaction products were isolated, depending on
the reaction conditions, and the complex reactivity of 4-HNE
with the above nucleophiles is discussed. Due to the good
yield and rate of formation of the hydrazone adducts, their
stability and favorable UV absorbance, 2-(trifluoromethyl)-
phenylhydrazine and 2,3,4,5,6-pentafluorophenylhydrazine
are the most interesting candidates for the development of
rapid and efficient analytical derivatizations of 4-HNE.
CF3, or pentafluoro analogues, in MeCN with 0.5 m
M TFA
yields the corresponding hydrazones with rate constants kf
of 2.8Ϯ0.4, 1.7Ϯ0.1, 3.0Ϯ0.2, 0.6Ϯ0.1, 0.5Ϯ0.1, and
3.5Ϯ0.5
M
–1 s–1, respectively at 298 K. At higher tempera-
its electrophilic nature, 4-HNE is also a mutagenic agent
and is able to alter protein function.[7,8]
Introduction
Lipid peroxidation is one of the most important and det-
rimental types of radical-mediated biological damage, a
chain-reaction sustained by the attack of peroxyl radicals
to polyunsaturated lipids.[1] Initially formed lipid hydroper-
oxides undergo enzymatic transformation or further oxi-
dation and spontaneous Hock-type cleavage to yield a vari-
ety of aldehydes.[2] Among them, (E)-4-hydroxy-2-nonenal
(4-HNE) is a major aldehyde product that originates in vivo
during peroxidation of ω-6-polyunsaturated fatty acids
(18:2, 20:4; Scheme 1).[3,4] The importance of 4-HNE is,
however, not limited to being a major oxidation product
but is also related to its primary involvement in inflamma-
tory events, in the regulation of cell signaling, proliferation,
and apoptosis, as well as being a chemotactic factor of
phagocytosis by polymorphonuclear leukocytes.[5,6] Due to
Scheme 1. Formation of 4-HNE during peroxidation of ω-6 poly-
unsaturated lipids.
In other words, 4-HNE is not only a product of lipid
peroxidation but also a key effector of the associated bio-
logical damage[9–12] and/or a mediator of the associated cell
signaling function.[13] Hence its analytical determination in
biological systems as a key biomarker of radical-mediated
damage is of major relevance. Its determination would also
be relevant in food chemistry to assess preservation of fats
and oils and guarantee consumer safety.
Not surprisingly, several investigations have dealt with
the development of analytical methods to address this chal-
lenging need, none of which, however, appears to be ideally
suited to the task. Besides the obvious problem associated
[a] BeC s.r.l., R&D Division,
Via C. Monteverdi 49, 47100 Forlì, Italy
[b] University of Bologna, Department of Organic Chemistry “A.
Mangini”,
Via S. Giacomo 11, 40126 Bologna, Italy
Fax: +39-051-2095688
E-mail luca.valgimigli@unibo.it
[c] Universités Aix-Marseille I, II & III-CNRS,
UMR 6264: Laboratoire Chimie Provence,
13397 Marseille Cedex 20, France
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200346.
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FULL PAPER
with its low concentration (micromolar levels are documen- enol 3 with NaBH4, as originally proposed by Jouanin et
ted in tissues[6]), 4-HNE gives very low sensitivity with the al.[20] Indeed, our revised sequence represents a significant
most common analytical approaches (e.g., HPLC–UV, advancement over the original proposal, where hydroxyl-
HPLC–MS, GC–MS), which makes chemical derivatization ation of the non-2-enonate was obtained in two steps con-
an important preliminary step. Due to the electrophilic na- sisting in oxidation to 3a with SeO2/TBHP (in reported
ture of 4-HNE, the majority of such methods are based on 25% yield after 72 h) followed by reduction.[20] Any subse-
a reaction with a nucleophile having good UV absorbance quent step proceeded with yields close to 90%, including
or yielding a relatively volatile product, such as benzyl- the oxidation of protected alcohol 5 to the desired aldehyde,
amine,[14] O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine,[15] which we accomplished under Swern conditions, at variance
or 2,4-dinitrophenylhydrazine.[16] In most cases, however, with the approach previously reported (Dess–Martin
conversion into the desired imine, oxime, or hydrazone is periodinane) that showed to be less efficient (reported yield
largely incomplete, possibly due to the relevant formation 62%).[20] A lower yield was, however, observed in the last
of side products – a fact that appears to be overlooked in step. Although deprotection of acetal 6 was nearly quantita-
the current literature – resulting in low recoveries in quanti- tive, the product [i.e., 4-HNE (1)] was unstable even under
tative analysis.[17,18] Surprisingly, little is known on the reac- mild purification conditions, affording 4-HNE in 70% yield
tivity of 4-HNE with nucleophiles, partly owing to its insta- after chromatography on silica. Higher yields could be ob-
bility[19] and difficult synthetic accessibility on a suitable tained by using alumina as stationary phase; however, the
scale. Therefore, the vast majority of derivatization methods purification was less reproducible and satisfactory. Interest-
are developed on a trial-and-error basis, limiting the ef- ingly, degradation of 4-HNE was observed also on pro-
ficient and rational optimization of the methods themselves. longed storage at –20 °C; therefore, we preferred to store
In the course of the development of one such analytical protected form 6 and proceed to its rapid hydrolysis/purifi-
approach, based on the quantitation of 4-HNE after cation in small batches shortly before use.
derivatization with fluorinated phenylhydrazines, chosen
because of their favorable chromatographic behavior, we
faced a previously undescribed very complex reactivity of
4-HNE, which we set to explore, by using the nucleophiles
shown in Scheme 2, to fill the gap of knowledge and to
provide a basis for the rational development of improved
analytical methods. We summarize here our results together
with an improved method for the synthesis of 4-HNE.
Scheme 2. Fluorinated phenylhydrazines investigated as reagents in
this study.
Scheme 3. Synthesis of (E)-4-(R/S)-hydroxy-2-nonenal.
Results and Discussion
Other synthetic routes where comparatively tested; how-
Synthesis of (E)-4-Hydroxy-2-nonenal
ever, they were generally less satisfactory. For instance, the
Synthesis of 4-HNE was accomplished on gram-scale by two-step sequence consisting in the epoxidation of 3-nonen-
using the five-step sequence summarized in Scheme 3, with 1-ol with 3-chloroperbenzoic acid, followed by oxidation
~30% overall yield. The first step, consisting in the regiose- with pyridinium chlorochromate, described by Spiteller et
lective selenium dioxide mediated allylic hydroxylation of al.,[21] yielded overall 16% of 4-hydroxy-2-nonenal selec-
commercially available methyl non-2-enonate, proceeded in tively in the (unwanted) Z-isomer, whereas the five-step se-
ca. 60% yield (in product 3), affording also as much as 10% quence from furan described by Grée et al.,[22] in our hands,
of corresponding ketone 3a. However, the unreacted start- yielded overall less than 3% of the desired product after a
ing material was easily recovered by flash chromatography very difficult final purification step. Additional methods for
and could be recycled in subsequent product batches, the synthesis of 4-HNE have been proposed in recent
whereas ketone 3a can, in principle, be reduced to desired years;[23,24] however, they where not comparatively tested.
2
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Derivatization of an Elusive Key Biomarker of Lipid Peroxidation
Scheme 4. Summary of products isolated in the reaction of 4-HNE (1) with hydrazine 2c.
Reactivity of 4-HNE with hydrazines 2a–f
in Ͼ50% yield by treating isolated 8c with 1 equiv. of 2c
and 0.3 equiv. of TFA (in MeCN, 70 °C, N2 atmosphere)
envisioning a mechanism of 1,4-addition of the second hy-
drazine on dihydropyridazine 8c (Scheme 5). On standing
in solution at room temperature, 9c was rapidly oxidized by
atmospheric oxygen to diazenyl derivative 10c.
Whereas the reaction of 4-HNE with 4-trifluoromethyl-
phenylhydrazine (2c) in dry ethanol proceeded inefficiently
and very slowly in the absence of acids, addition of 1 equiv.
of sulfuric acid at 50 °C for 1 h yielded a complex multitude
of uncharacterized products, of which the expected hydraz-
one represented only trace amounts. Replacing sulfuric acid
with 0.1 to 0.3 equiv. of 4-toluensulfonic acid (TsOH) or
trifluoroacetic acid (TFA) afforded a somewhat simpler sce-
nario, as depicted in Scheme 4. The nature and yield of the
reaction products significantly depended on the reaction
conditions, particularly the solvent and temperature, and
allowed some degree of control, as summarized in Table 1.
Table 1. Experimental conditions and associated product yields for
the reaction of 4-HNE with 4-trifluomethylphenylhydrazine (2c).
Solvent
T / °C
t
Product (% yield)[a]
Scheme 5. Proposed mechanism leading to the formation of prod-
ucts 8c–10c.
EtOH
EtOH/H2O (99:1)
MeCN
tBuOH
EtOH
tBuOH
MeCN
r.t.
r.t.
r.t.
25
50
50
50
70
70
10 min 7c (65) + 11c (trace)
10 min 7c (70)[b]
15 min 7c (80)[b]
It is worth noting that with any of the tested hydrazines,
the 1,4-addition proceeded in a diastereoselective fashion
to furnish predominantly the anti adducts (i.e., for 9–10c,
10 min 7c (73)[b]
6 h
3 h
3 h
5 h
7 h
8c (30) + 11c (40)
7c (32) + 8c (38)
8c (32) + 9c (15) +10c[c]
8c (64)[b]
1
anti/syn = 8:1dr, from H NMR spectroscopy), as nucleo-
tBuOH
MeCN
philic attack is more favorable from the less hindered side.
8c (3) + 9c (50) + 10c[c]
From racemic 8c, major anti adducts were obtained as a
4
[a] Yield of isolated product. [b] Yield appeared quantitative by
TLC analysis of the reaction mixture. [c] Formed at the expense of
9c during workup and purification.
couple of enantiomers. A J coupling can be observed in
the COSY spectrum between the H3 and H5 protons of the
pyridazine ring due to W-shaped coupling (see Figure S1
At room temperature, in ethanol or acetonitrile (or in and the COSY spectrum of 10c in the Supporting Infor-
tert-butyl alcohol just above its melting temperature), linear mation).
hydrazone 7c was formed rapidly (10–15 min) and selec-
Reaction of 4-HNE with meta-substituted phenylhydraz-
tively and could be isolated in 65–80% yield. Trace amounts ines 2b and 2d (3-CF3- and 3,5-di-CF3-, respectively) af-
of compound 11c formed by reaction with the solvent were forded similar results. Linear hydrazones 7b and 7d were
observed in ethanol; however, its formation can be com- selectively obtained at room temperature (isolated yields
pletely suppressed by the addition of 1% water to the reac- were 62 and 80%, respectively, when the reaction occurred
tion mixture, or in less nucleophilic solvents. On increasing in EtOH), whereas formation of dihydropyridazines 8b and
the reaction temperature and time, the hydrazone cyclized 8d, together with tetrahydropyridazine adducts 9b and 9d,
to 1,6-dihydropyridazine 8c, as exemplified in Scheme 5. became increasingly relevant at higher temperatures (40–
Evidence for the mechanism to explain the formation of 8c 70 °C). Oxidation of these last two substrates to diazenyl
was obtained by incubating isolated linear hydrazone 7c derivatives 10 was very rapid for the monotrifluoromethyl-
with 0.3 equiv. of TFA at 50 or 70 °C in acetonitrile for 5 h, ated compound (i.e., 9b to 10b), whereas it occurred only
resulting in nearly complete conversion into 8c. Product 9c very slowly (about two weeks at r.t.) for the more electron
resulting from addition of a second arylhydrazine molecule poor disubstituted derivative (i.e., 9d to 10d).
to 8c became dominant at 70 °C, when acetonitrile was the
Interestingly, the scenario was substantially different
solvent or when an excess amount of hydrazine was present when 4-HNE was treated with ortho-CF3-phenylhydrazine
in the reaction mixture. Furthermore, 9c could be isolated 2a, as linear hydrazone 7a was the only reaction product
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that could be isolated from the reaction mixture (in 60–70% cause formation of 9e implies the transient formation of
yield), in different solvents in the temperature range 20– dihydropyridazine 8e (see Scheme 5), this finding suggests
70 °C. Lack of formation of cyclic adducts 8–10 indicates that the nucleophilicity of hydrazone 7e is not completely
that intramolecular nucleophilic attack from the inner ni- abolished by the presence of the ortho-CF3 group, but it is,
trogen atom in 7a to the carbon atom in the 4-position is nonetheless, significantly hampered, as can be deduced
hampered by intramolecular H-bonding (NH···F) to the from comparison with the different behavior recorded with
CF3 substituent, which stabilizes resonance structure 7aЈ.
hydrazines 2b–d.[25] Electron-poor 9e did not spontaneously
oxidize in solution.
An alternative mechanism to justify the formation of 9e
in the absence of 8e can also be hypothesized, such as that
illustrated in Scheme 8.
Support to the occurrence of such an interaction is pro-
vided by the 1H NMR spectrum, which shows a higher field
signal for the (N)H proton in 7a (δ =9.39 ppm) as compared
to meta isomer 7b (δ =10.34 ppm) or para isomer 7c (δ
=10.46 ppm).
To check this hypothesis, we prepared 2,4-di-CF3-phenyl-
hydrazine (2e) from the corresponding aniline through di-
azotization and subsequent SnCl2 reduction according to
Scheme 6, and subjected it to reaction with 4-HNE under
the usual variety of conditions. In no case could dihydro-
pyridazine 8 be observed among the reaction products, even
at temperatures as high as 70 °C. However, by using an ex-
cess amount of the hydrazine, minor amounts (ca. 10%) of
adduct 9e could be isolated, as illustrated in Scheme 7. Be-
Scheme 8. Alternative mechanism to explain the formation of ad-
duct 9e in the absence of 1,6-piridazine 8e (see Scheme 7). In this
scheme, Michael-type addition of a second molecule of arylhydraz-
ine to hydrazone 7e results in hypothetical adduct 13e (not iso-
lated), in which the ring closure upon nucleophilic intermolecular
substitution of the OH group is facilitated by anchimeric assistance
by the newly entered neighboring NH group (structure 14e).
The reactivity of 4-HNE with phenylhydrazines 2a–f is
summarized in Scheme 9. Overall, it was possible to selec-
tively prepare linear hydrazones 7a–f in good yield by treat-
ing 4-HNE with a slight excess amount (1.2 equiv.) of the
corresponding hydrazines at room temperature for a short
time (10–20 min), and the reaction was tolerant to the pres-
ence of some water (1–2% v/v) and air. This reaction ap-
peared particularly suited to be implemented for analytical
purposes. Selective formation of dihydropyridazines 8b–d[26]
was also possible by performing the reaction at higher tem-
perature (50–70 °C) for a longer time (5–7 h) in tert-butyl
alcohol, but a mixture of products was always observed in
other solvents. Because the reaction appeared somewhat
less useful for 4-HNE derivatization, it was not further opti-
mized.
Scheme 6. Synthesis of 2,4-di-CF3-phenylhydrazine (2e).
Scheme 7. Reaction of 4-HNE (1) with 2,4-di-CF3-phenylhydrazine
(2e).
Scheme 9. Summary of the reactivity of fluorinated phenylhydrazines with 4-HNE.[26]
4
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Derivatization of an Elusive Key Biomarker of Lipid Peroxidation
Optimized Formation and Properties of Linear Hydrazones
7a–f
To be useful for analytical derivatization, a reaction is
expected to satisfy some key requisites: (i) The reaction
should be sufficiently simple and not overly sensitive to
water and air. (ii) The reaction should be as close as pos-
sible to being quantitative. (iii) The reaction should be as
fast as possible as to allow the screening of many samples
in a reasonable time. (iv) The reaction should yield products
that are stable under common laboratory conditions and
that are easily detected (e.g., by UV/Vis spectroscopy). Al-
though it is clear that Equation (1) satisfies some of these
requisites, we sought to optimize the reaction under analyti-
cal settings (i.e., at very low concentration of 4-HNE) and
investigated some relevant properties of product hydrazones
7a–f.
Figure 1. Time course of hydrazones 7a–f formation by reaction of
30 μm 4-HNE with 10-fold excess amount of hydrazines 2a–f in the
presence of 0.5 mm TFA at 298 K in MeCN.
(with respect to meta-substituted 2b). On the other hand,
di-CF3-substituted compounds 2d and 2e reacted signifi-
cantly slower, possibly due to higher steric hindrance.
The stabilities of hydrazones 7a–f also depended to some
extent on the pattern of substitution in the aromatic ring,
although not in an easily predictable manner. Any of the
tested hydrazones was perfectly stable in acetonitrile solu-
tion in air for 24 h, indicating low susceptibility to air oxi-
dation. On the other hand, some of them were less stable
in the presence of water and catalytic amounts of TFA. No
significant hydrolysis was recorded for 7c and 7f, whereas
any other hydrazone gave some degradation over 5 h, which
appeared to proceed to equilibrium [Equation (2)] as illus-
trated in Figure 2. The apparent pseudo-first-order rate
constant of decomposition, (kd, at 298 K) in the presence
of 10% water and 1 μm TFA was obtained from traces of
the initial slopes of decay and confirmed by numerical fit-
tings by using Gepasi software.[27–29] Results are collected
in Table 2. The reasons for the recorded differences among
tested compounds are currently not understood. On the
other hand, their different kinetic behavior is clearly of
major relevance in the design of efficient analytical meth-
ods.
When 4-HNE (1) was treated at room temperature in the
presence of a large excess (10-fold or higher) of hydrazines
2a–f, the best results were always obtained in acetonitrile as
the solvent, where the formation of hydrazones 7a–f pro-
ceeded quantitatively, with calculated yields (by HPLC
analysis) in the range 98Ϯ4%. However, when the initial
concentration of 4-HNE was in the range 1–50 μm (i.e., of
the magnitude expected in oxidized food or in biological
tissues), the reaction time to completion was significantly
longer than under preparative settings, which prompted a
preliminary evaluation of the reaction kinetics. When an
excess amount of the arylhydrazine was used, the reaction
proceeded with apparent pseudo-first-order kinetics, as il-
lustrated in Figure 1.
Analysis of the kinetic traces under different initial con-
centrations of the arylhydrazine allowed the apparent sec-
ond-order rate constants of formation (kf) to be estimated.
Data recorded in the presence of 0.5 mm TFA are collected
in Table 2.[27] The recorded rate constants differed by as
much as sevenfold among the tested reactions under iden-
tical settings. The presence of a moderately electron-with-
drawing groups (CF3 or F) in conjugated positions (i.e., in
compounds 2a, 2c, and 2f) slightly accelerated the reaction
Table 2. UV/Vis spectral properties, rate of formation, and stability of hydrazones 7a–f at 298 K.
ε[a] / abscm–1 mol–1
kf[b] / m–1 s–1
kd / 10–5 s–1
[c]
% Degrad. (5 h)[d]
[a]
Compound
λmax / nm
7a
7b
7c
7d
7e
7f
310
305
324
306
312
294
26000
18130
35800
37960
21980
31670
2.8Ϯ0.4
1.7Ϯ0.1
3.0Ϯ0.2
0.6Ϯ0.1
0.5Ϯ0.1
3.5Ϯ0.5
1.4Ϯ0.2
1.7Ϯ0.4
ca. 0
1.9Ϯ0.3
2.8Ϯ0.8
ca. 0
8.7
11.0
ca. 0
10.4
16.9
ca. 0
[a] Determined in MeCN. [b] Apparent rate constant of formation of the hydrazone in MeCN at 298 K in the presence of 0.5 mm TFA.
[c] Apparent pseudo-first-order rate constant of degradation of the hydrazone in MeCN/H2O (9:1) at 298 K in the presence of 1 μm TFA.
[d] Percent degradation at the equilibrium under identical settings as determined from spectrophotometric measurements.
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Experimental Section
General: Reagents and solvents were purchased at the highest com-
mercial quality and used without further purification. Flash col-
umn chromatography was performed by using silica gel (particle
size 40–63 μm, 230–400 mesh) at increased pressure. NMR (1H,
13C) spectra were recorded with Varian Unity INOVA 600 MHz,
Varian Mercury Plus 400 MHz, and Varian Gemini 300 MHz spec-
trometers. The chemical shifts (δ) are referenced to residual undeu-
terated solvent as an internal reference. The 13C NMR spectra of
fluorinated compounds were registered by using relaxation delay (t1
= 8 s) for JC–F evaluation. IR spectra were recorded with a Nicolet
Protégé 460 FTIR spectrometer. Low-resolution mass spectra
(LRMS) were recorded with a Thermo LCQ-Fleet (ESI) or with a
Varian Saturn 2000 (EI) instrument, while high-resolution spectra
(HRMS) were recorded with a Thermo-Finnigan MAT95 XP in-
strument. UV/Vis spectra were recorded in MeCN in a Perkin El-
mer Lambda-20 double-beam spectrometer with bandwidth of
1 nm.
Figure 2. Degradation of hydrazones 7a–f at 298 K in MeCN/H2O
(9:1) and 1 μm TFA.
Indeed, upon consideration of their kinetic behavior, de-
rivatives 7c and 7f are clearly the most interesting for fur-
ther implementation of analytical derivatization methods
for 4-HNE. Not only were they perfectly stable to both hy-
drolysis and air oxidation under common laboratory set-
tings, but they had also the highest rate of formation, being
potentially suitable for the rapid screening of many samples
(by using initial concentration of hydrazines 2c and 2f
around 1 mm the reaction was complete after ca. 10–
15 min). Of interest, hydrazones 7c and 7f also had among
the highest UV extinction coefficients (see Table 2) in a
spectral region (300–320 nm), perfectly suited to spectro-
photometric detection coupled to separation techniques
(e.g., HPLC–UV).
(E)-Methyl 4-Hydroxynon-2-enoate (3) and (E)-Methyl 4-Oxonon-
2-enoate (3a): A round-bottom flask with a condenser was charged
with selenium dioxide (3.26 g, 29.4 mmol, 2.0 equiv.) in dry dioxane
(20 mL) and stirred at room temperature under N2 atmosphere for
15 min before adding (E)-methyl non-2-enoate (2.79 mL,
14.7 mmol, 1.0 equiv.). The reaction mixture was heated at reflux
for 4 h under N2 atmosphere. The obtained yellow solution was
allowed to cool at room temperature, filtered through a pad of
silica and Celite, and concentrated, and the resulting red suspen-
sion was washed with brine. The dried (Na2SO4) organic layer was
concentrated in vacuo. The crude product was purified by flash
chromatography (petroleum ether/EtOAc, 9:1 to 8:2). Alcohol 3
was obtained as the main product as a yellow oil (1.64 g,
1
8.82 mmol, 60%). H NMR (400 MHz, CDCl3): δ = 6.94 (dd, J =
15.6, 5.2 Hz, 1 H), 6.01 (dd, J = 15.2, 1.6 Hz, 1 H), 4.27 (dt, J =
6.4, 1.6 Hz, 1 H), 3.71 (s, 3 H), 2.11 (br. s, 1 H), 1.60–1.50 (m, 2
H), 1.47–1.20 (m, 6 H), 0.86 (t, J = 6.4 Hz, 3 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 167.3, 150.9, 119.8, 71.3, 51.8, 36.8, 31.8,
25.1, 22.7, 14.2 ppm. LRMS (EI): m/z (%) = 186 (0.3) [M]+·, 157
(16), 125 (28), 115 (51), 98 (49), 87 (50), 55 (100). Amounts of
ketone 3a as white solid (0.27 g, 1.47 mmol, 10) and starting mate-
rial (0.35 g, 2.06 mmol) were also recovered (spectroscopic data in
Conclusions
An improved strategy for the gram-scale synthesis of (E)-
4-HNE has allowed detailed investigation of its reactivity
with fluorinated phenylhydrazines as promising novel rea- the Supporting Information).
gents for analytical derivatization of such a relevant, yet
(E)-Methyl 4-(Tetrahydro-2H-pyran-2-yloxy)non-2-enoate (4): A dry
unstable, biomarker of lipid peroxidation. The product dis-
tribution largely depends on the reaction conditions,
strengthening the concept that the reactivity of 4-HNE
might be more complex than often supposed in other works
dealing with its chemical derivatization. Expected linear
hydrazones are formed quantitatively and rapidly at room
temperature (in MeCN) in the presence of catalytic
round-bottomed flask capped with a rubber septum was loaded
under N2 with dry CH2Cl2 (25 mL), alcohol 3 (1.05 g, 5.64 mmol,
1.0 equiv.), and 3,4-dihydro-2H-pyran (2.57 mL, 28.2 mmol,
5.0 equiv.). The solution was stirred at 0 °C before adding a solu-
tion of PPTS (0.140 g, 0.564 mmol, 0.1 equiv.) in CH2Cl2 (1 mL)
by syringe. The resulting mixture was warmed up to room tempera-
ture and stirred for 2 h. The mixture was diluted with CH2Cl2 and
amounts of TFA. At higher temperature, intramolecular cy- washed with an aqueous solution of NaCl (10%) and brine. The
organic layer was dried (Na2SO4) and concentrated. The crude
product was purified by flash chromatography (petroleum ether/
EtOAc, 9.5:0.5 to 9:1) to afford protected alcohol 4 (1:1dr) as a
clization products (1,6-dihydropyridazines and tetra-
hydropyridazine adducts) become prevalent. On the basis
of the relative stability of the adducts, their favorable UV
absorbance, and the kinetics of the reaction, 4-trifluorome-
thylphenylhydrazine and 2,3,4,5,6-pentafluophenylhydraz-
ine are the most promising reactants for derivatization of 4-
HNE under analytical settings. Comparative evaluation of
the reactivity of such derivatizing agents with other relevant
aldehyde products formed during lipid peroxidation will
also be pursued in future work.
1
yellow oil (1.34 g, 4.96 mmol, 88%). H NMR (400 MHz, CDCl3):
δ = 6.96 (dd, J = 16.0, 5.2 Hz, 1 H), 6.78 (dd, J = 16.0, 6.4 Hz, 1
H), 6.06 (dd, J = 15.6, 7.6 Hz, 1 H), 5.94 (d, J = 15.6 Hz, 1 H),
4.70 (t, J = 3.6 Hz, 1 H), 4.56 (t, J = 3.2 Hz, 1 H), 4.32–4.21 (m,
2 H), 3.91–3.76 (m, 2 H), 3.74 (s, 3 H), 3.73 (s, 3 H), 3.54–3.42 (m,
2 H), 1.80–1.55 (m, 2 H), 1.54–1.48 (m, 14 H), 1.45–1.20 (m, 12
H), 0.88 (t, J = 6.8 Hz, 3 H), 0.87 (t, J = 6.8 Hz, 3 H) ppm. 13C
NMR (100 MHz, CDCl3): δ = 167.3, 166.9, 149.7, 148.9, 121.8,
6
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O20346
/KAP1
Date: 30-05-12 16:44:57
Pages: 12
Derivatization of an Elusive Key Biomarker of Lipid Peroxidation
120.1, 97.5, 96.3, 75.3, 74.5, 62.6, 62.5, 51.8, 51.7, 35.4, 34.1, 32.0, by TLC until complete consumption of the starting material (about
31.9, 30.9, 30.8, 25.6, 25.6, 25.2, 24.5, 22.7 (2 C), 19.6, 19.5, 14.2,
14.2 ppm. LRMS (EI): m/z (%) = [M]+· absent, 169 (9), 152 (10),
137 (10), 128 (13), 125 (22), 115 (20), 113 (35), 111 (33), 85 (47),
84 (40), 55 (100).
2 h). The reaction mixture was diluted with CH2Cl2 and washed
with brine. The organic layers were dried with Na2SO4 and concen-
trated under reduced pressure. The residue was purified by flash
chromatography (petroleum ether/EtOAc, 8:2 to 7.5:2.5) to give 1
(0.18 g, 1.15 mmol, 70%) as a colorless oil. 1H NMR (400 MHz,
CDCl3): δ = 9.57 (d, J = 8.0 Hz, 1 H), 6.82 (dd, J = 15.6, 4.8 Hz,
1 H), 6.30 (ddd, J = 16.0, 7.6, 1.6 Hz, 1 H), 4.43 (quint., J = 5.6 Hz,
1 H), 1.88 (d, J = 4.8 Hz, 1 H), 1.68–1.57 (m, 2 H), 1.50–1.40 (m,
6 H), 0.89 (t, J = 5.2 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3):
δ = 193.8, 159.1, 130.8, 71.4, 36.7, 31.8, 25.1, 22.7, 14.2 ppm.
LRMS (EI): m/z (%) = 157 (1) [M]+·, 138 (12), 123 (7), 109 (22),
96 (22), 81 (100), 67 (26), 55 (20), 53 (30).
(E)-4-(Tetrahydro-2H-pyran-2-yloxy)non-2-en-1-ol (5):
A
dry
round-bottomed flask capped with a rubber septum was flushed
with N2 and then dry CH2Cl2 (40 mL) and ester 4 (1.10 g,
4.07 mmol, 1.0 equiv.) were introduced by syringe. The solution
was stirred and cooled down to –10 °C with an ice–NaCl bath, and
DIBAL-H (1.0 m in hexanes, 8.95 mL, 8.95 mmol, 2.2 equiv.) was
added dropwise by glass syringe. The resulting mixture was allowed
to come to room temperature over 1 h. The mixture was quenched
with H2O, stirred, and filtered through a small pad of Celite. The
filtrate was washed with water and brine, dried (Na2SO4), concen-
trated. The crude was purified by flash chromatography (petroleum
ether/EtOAc, 7:3) to afford allylic alcohol 5[20] (1:1dr) as a trans-
parent oil (0.89 g, 3.67 mmol, 90%). 1H NMR (400 MHz, CDCl3):
δ = 5.81 (tdd, J = 15.6, 5.2, 0.8 Hz, 1 H), 5.79 (tdd, J = 15.6, 5.2,
0.8 Hz, 1 H), 5.73 (ddt, J = 15.6, 6.4, 1.2 Hz, 1 H), 5.65 (ddt, J =
15.6, 8.0, 1.6 Hz, 1 H), 4.67 (m, 1 H), 4.64 (t, J = 4.0 Hz, 1 H),
4.13 (t, J = 5.2 Hz, 4 H), 4.12–4.02 (m, 2 H), 3.90–3.82 (m, 2 H),
3.52–3.42 (m, 2 H), 1.84–1.78 (m, 2 H), 1.76–1.60 (m, 4 H), 1.60–
1.40 (m, 10 H), 1.38–1.20 (m, 12 H), 0.87 (t, J = 6.8 Hz, 3 H), 0.87
(t, J = 6.8 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 133.3,
132.3, 131.9, 129.9, 98.0, 95.0, 77.3, 75.5, 63.4, 63.1, 62.9, 62.3,
36.0, 34.9, 32.1, 32.0, 31.1, 30.9, 25.8, 25.6, 25.4, 24.9, 22.8 (2 C),
19.9, 19.7, 14.3, 14.2 ppm. MS (EI): m/z (%) = [M]+· absent, 169
(6), 91 (16), 85 (75), 69 (50), 55 (100).
[2,4-Bis(trifluoromethyl)phenyl]hydrazine (2e): To a fine suspension
of 2,4-bis(trifluoromethyl)aniline (0.60 g, 2.62 mmol, 1 equiv.) in
conc. HCl (4 mL) cooled in an ice–water bath was added dropwise
a solution of sodium nitrite (0.198 g, 2.88 mmol, 1.1 equiv.) in
water (1 mL). After 30 min, a chilled solution of SnCl2·2H2O
(1.29 g, 5.76 mmol, 2.2 equiv.) in conc. HCl (1 mL) was then added
dropwise to the cooled diazonium salt yellow solution, keeping the
reaction below –5 °C. The mixture was stirred an additional 1 h at
0 °C. The pink solid was collected by vacuum filtration, dissolved
in hot water, and filtered. The filtrate was basified with NaOH
(aq. 40%) and a precipitate suddenly formed. The white solid was
recovered by filtration and redissolved in EtOAc. The solution was
then washed with brine, dried with Na2SO4, and evaporated under
reduced pressure to obtain pure hydrazine 2e (0.25 g, 1.02 mmol,
40%) as white needles. M.p. 46–48 °C. 1H NMR (400 MHz,
CDCl3): δ = 7.67 (s, 1 H), 7.65 (d, J = 8.4 Hz, 1 H), 7.46 (d, J =
8.4 Hz, 1 H), 6.10 (br. s, 1 H), 3.70 (br. s, 2 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 150.7, 130.3, 124.4 (q, J = 270 Hz, 2 C),
124.2 (q, J = 3.2 Hz), 119.3 (q, J = 33.0 Hz), 112.3, 111.8 (q, J =
30.6 Hz) ppm. LRMS (EI): m/z (%) = 244 (100) [M]+·, 224 (82),
205 (45), 176 (73), 145 (18). HRMS (EI): calcd. for C8H6F6N2
[M]+· 244.0435; found 244.0439.
Diastereomeric Mixture of (E)-4-(Tetrahydro-2H-pyran-2-yloxy)-
non-2-enal (6): To a solution of oxalyl chloride (2.0 m in CH2Cl2,
3.96 mL, 7.92 mmol, 3.0 equiv.) diluted with CH2Cl2 (15 mL) at
–78 °C under N2 atmosphere was dropwise added a solution of
DMSO (0.94 mL, 13.2 mmol, 5 equiv.) in CH2Cl2 (5.0 mL) in a dry
round-bottomed flask capped with a rubber septum. After 10 min,
a solution of alcohol 5 (0.63 g, 2.64 mmol, 1 equiv.) in CH2Cl2
(10 mL) was added dropwise. After 1 h, Et3N (3.68 mL, 26.4 mmol,
10 equiv.) was added, and stirring was continued at –78 °C for
30 min. The reaction mixture was allowed to reach room tempera-
ture over a period of 1 h. NH4Cl (aq. sat.) was added, and the
mixture was extracted with CH2Cl2. The combined organic layers
were dried with Na2SO4 and concentrated in vacuo, and the re-
sulting residue was purified by flash chromatography (petroleum
ether/EtOAc, 8.5:1.5) to give aldehyde 6[20] (0.54 g, 2.25 mmol, 87%
General Procedure for the Synthesis of Linear Hydrazones 7a–f: A
dry round-bottomed flask capped with a rubber septum was
charged with aldehyde 1 (0.15 mmol, 1 equiv.), flushed with N2,
and then dry EtOH (or tBuOH or MeCN, 2 mL) and solid MgSO4
were added. Neat aromatic hydrazine 2a–f (1.2 equiv.) and a solu-
tion of TFA (5% v/v in the chosen solvent, 0.3 equiv.) were success-
ively added by syringe at room temperature. The reaction was
judged complete within 10–15 min by TLC. The solvent was evapo-
rated under reduced pressure maintaining the rotary evaporator
water bath at room temperature. The obtained crude was purified
directly by flash chromatography (petroleum ether/EtOAc, from 9:1
to 7:3) to afford title compounds 7a–f.
1
yield) as a colorless oil (1:1dr). H NMR (400 MHz, CDCl3): δ =
9.58 (d, J = 3.6 Hz, 1 H), 9.56 (d, J = 4.0 Hz, 1 H), 6.84 (dd, J =
16.0, 5.6 Hz, 1 H), 6.68 (dd, J = 16.0, 6.4 Hz, 1 H), 6.31 (ddd, J =
15.6, 8.0, 0.8 Hz, 1 H), 6.21 (dd, J = 15.6, 8.0 Hz, 1 H), 4.71 (t, J
= 3.6 Hz, 1 H), 4.56 (t, J = 3.2 Hz, 1 H), 4.43 (q, J = 6.4 Hz, 1 H),
4.36 (q, J = 6.0 Hz, 2 H), 3.92–3.84 (m, 1 H), 3.83–3.76 (m, 1 H),
3.54–3.42 (m, 2 H), 1.90–1.78 (m, 2 H), 1.77–1.45 (m, 14 H), 1.43–
1.20 (m, 12 H), 0.88 (t, 6 H) ppm. 13C NMR (100 MHz, CDCl3):
δ = 194.0, 193.7, 158.6, 157.5, 132.6, 131.3, 98.3, 96.6, 75.7, 74.5,
62.9, 62.7, 35.3, 34.1, 31.9, 31.9, 30.9, 30.8, 25.6, 25.5, 25.2, 24.7,
22.7 (2 C), 19.7, 19.6, 14.2, 14.2 ppm. MS (EI) m/z (%) = [M]+·
absent, 204 (1), 193 (1), 147 (3), 139 (11), 125 (6), 109 (10), 81 (76),
55 (100).
(1E,2E)-1-{2-[2-(Trifluoromethyl)phenyl]hydrazono}non-2-en-4-ol
(7a): In EtOH, 30.2 mg of a yellow oil (0.096 mmol, 64 %); in
CH3CN, 36 mg (0.115 mmol, 76 %) 1H NMR (400 MHz, [D6]-
DMSO): δ = 9.39 (s, 1 H), 8.01 (d, J = 9.6 Hz, 1 H), 7.54 (d, J =
8.8 Hz, 1 H), 7.50–7.43 (m, 2 H), 6.87 (t, J = 7.6 Hz, 1 H), 6.27
(dd, J = 16.0, 9.6 Hz, 1 H), 5.96 (dd, J = 15.6, 5.6 Hz, 1 H), 4.83
(d, J = 4.4 Hz, 1 H) 4.07 (quint., J = 5.6 Hz, 1 H), 1.50–1.37 (m,
2 H), 1.35–1.20 (m, 6 H), 0.89 (t, J = 6.4 Hz, 3 H) ppm. 13C NMR
(100 MHz, [D6]DMSO): δ = 145.5, 143.1, 134.1, 126.7 (q, J =
5.7 Hz, 2 C), 126.6, 125.0 (q, J = 270 Hz), 118.9, 115.2, 111.5 (q,
J = 29.8 Hz), 70.8, 37.6, 32.0, 25.3, 22.8, 14.6. MS (EI) m/z (%) =
[M]+· absent, 296 (17), 239 (24), 225 (51), 212 (100), 145 (34), 114
(29) ppm. LRMS (ESI+) = 315.0 [M + H]+. HRMS (ESI+): calcd.
for C16H22F3N2O [M + H]+ 315.1684; found 315.1689. IR (CDCl3):
(E)-4-Hydroxynon-2-enal (1): To a solution of protected aldehyde
6 (0.40 g, 1.66 mmol, 1 equiv.) in MeOH (5 mL) was added para-
toluenesulfonic acid monohydrate (TsOH, 32 mg, 0.166 mmol,
0.1 equiv.) solubilized in a small amount of MeOH at 0 °C. The
reaction mixture was warmed to room temperature and monitored
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7

Job/Unit: O20346
/KAP1
Date: 30-05-12 16:44:57
Pages: 12
R. Matera, S. Gabbanini, A. Valvassori, M. Triquigneaux, L. Valgimigli
FULL PAPER
ν = 3607, 3383, 1612, 1594, 1524, 1469, 1323, 1277, 1138, 1109, H), 4.81 (d, J = 4.8 Hz, 1 H), 4.04 (quint., J = 5.2 Hz, 1 H), 1.35–
˜
1082 cm–1.
1.27 (m, 2 H), 1.27–1.18 (m, 6 H), 0.84 (t, J = 6.8 Hz, 3 H) ppm.
13C NMR (100 MHz, CDCl3): δ = 145.0, 142.3, 138.6 (dm, J =
247 Hz, 2 C), 138.4 (dm, J = 250 Hz, 2 C), 136.1 (dm, J = 246 Hz),
126.4, 120.5 (tm, J = 10.0 Hz), 72.4, 37.2, 31.9, 25.2, 22.7,
14.2 ppm. HRMS (ESI+): calcd. for C17H21F6N2O [M + H]+
337.1339; found 337.1345.
(1E,2E)-1-{2-[3-(Trifluoromethyl)phenyl]hydrazono}non-2-en-4-ol
(7b): In EtOH, 30 mg of a yellow oil (0.095 mmol, 63%. GC–MS
calculated yield = 83%). 1H NMR (400 MHz, [D6]DMSO): δ =
10.34 (s, 1 H), 7.58 (d, J = 9.6 Hz, 1 H), 7.36 (t, J = 7.6 Hz, 1 H),
7.18 (s, 1 H), 7.12 (d, J = 8.4 Hz, 1 H), 6.98 (d, J = 7.6 Hz, 1 H),
6.28 (dd, J = 15.6, 9.2 Hz, 1 H), 5.97 (dd, J = 15.6, 5.6 Hz, 1 H),
4.80 (d, J = 4.4 Hz, 1 H), 4.05 (quint., J = 5.6 Hz, 1 H), 1.46–1.34
(m, 2 H), 1.32–1.18 (m, 6 H), 0.84 (t, J = 6.8 Hz, 3 H) ppm. 13C
NMR (100 MHz, [D6]DMSO): δ = 146.5, 142.1, 141.5, 130.8, 130.6
(q, J = 33.8 Hz), 126.6, 125.0 (q, J = 270 Hz), 116.1, 115.0 (q, J =
3.5 Hz), 108.1 (q, J = 3.5 Hz), 70.8, 37.7, 32.0, 25.3, 22.8, 14.6.
LRMS (EI): m/z (%) = [M]+· absent, 296 (8), 277 (2), 239 (16), 225
(100), 160 (2), 145 (5) ppm. LRMS (ESI+) = 315 [M + H]+. HRMS
(ESI+): calcd. for C16H22F3N2O [M + H]+ 315.1684; found
315.1678.
General Procedure for the Synthesis of Substituted 1,6-Dihydro-
pyridazines 8b–d and Michael Addition Products 9b–d and 10b–d: A
dry round-bottomed flask was charged with a magnetic stirrer and
capped with a rubber septum. The reaction vessel was charged with
aldehyde 1 (0.25 mmol, 1 equiv.) and flushed with N2 and then an-
hydrous MeCN (8 mL) and solid MgSO4 were added. Neat aro-
matic hydrazine 2b–d (2.0 equiv.) and a solution of TFA (5% in
MeCN, 0.3 equiv.) were successively added by syringe at room tem-
perature. The reaction was heated at 70 °C for 6 h. The solvent was
evaporated under reduced pressure to give a crude that was purified
by flash chromatography (petroleum ether/EtOAc, from 95:5 to
7:3) to afford 1,6-dihydropyridazine 8b–d and Michael addition
products 9b–d. The anti isomers were predominantly formed, as
judged by 1H NMR spectroscopy (Ն8:1dr). The isolated hydrazinyl
products were moderately stable and were rapidly or slowly oxid-
ized by O2/air towards oxidized diazenyl derivatives upon standing
at room temperature. Diazenyl products 10b–d were purified again
by flash column chromatography (petroleum ether/EtOAc, from
85:15 to 75:25). To obtain selectively substituted 1,6-dihydropyrid-
azines 8b–d, the reaction was performed in tBuOH by using a solu-
tion of 5% TFA in tBuOH (0.3 equiv.) and flash chromatography
(petroleum ether/EtOAc, from 95:5 to 85:15).
(1E,2E)-1-{2-[4-(Trifluoromethyl)phenyl]hydrazono}non-2-en-4-ol
(7c): In EtOH, 31 mg of a white solid (0.098 mmol, 65% GC–MS
calculated yield = 87%): in MeCN, 37.7 mg (0.120 mmol, 80%).
1
M.p. 118–120 °C. H NMR (400 MHz, [D6]DMSO): δ = 10.46 (s,
1 H), 7.60 (d, J = 9.6 Hz, 1 H), 7.46 (d, J = 8.8 Hz, 1 H), 7.02 (d,
J = 8.4 Hz, 2 H), 6.27 (dd, J = 16.0, 9.6 Hz, 1 H), 5.99 (dd, J =
15.6, 5.6 Hz, 1 H), 4.81 (d, J = 4.8 Hz, 1 H) 4.06 (quint., J =
5.6 Hz, 1 H), 1.50–1.40 (m, 2 H), 1.40–1.27 (m, 6 H), 0.84 (t, J =
6.8 Hz, 3 H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 148.9,
142.1, 142.1, 127.1 (q, J = 4.0 Hz, 2 C), 126.5, 125.7 (q, J =
269 Hz), 118.7 (q, J = 31.4 Hz), 112.1 (2 C), 70.8, 37.7, 32.0, 25.3,
22.8, 14.6 ppm. HRMS (ESI+): calcd. for C16H22F3N2O
[M + H]+ 315.1684; found 315.1690. IR (CDCl ): ν = 3608, 3343,
˜
3
6-Pentyl-1-[3-(trifluoromethyl)phenyl]-1,6-dihydropyridazine (8b): In
MeCN, 14.8 mg of a yellow oil (0.050 mmol, 20 %); in tBuOH,
1616, 1531, 1467, 1325, 1264, 1165, 1121, 1064 cm–1.
1
44.4 mg (0.15 mmol, 60%). H NMR (400 MHz, CDCl3): δ = 7.54
(1E,2E)-1-{2-[3,5-Bis(trifluoromethyl)phenyl]hydrazono}non-2-en-
4-ol (7d): In EtOH, 46.8 mg of a yellowish solid (0.120 mmol,
80%). M.p. 82–84 °C. 1H NMR (400 MHz, [D6]DMSO): δ = 10.70
(s, 1 H), 7.63 (d, J = 9.2 Hz, 1 H), 7.40 (s, 2 H), 7.25 (s, 1 H), 6.30
(dd, J = 15.6, 9.2 Hz, 1 H), 6.07 (dd, J = 15.6, 5.6 Hz, 1 H), 4.83
(d, J = 4.4 Hz, 1 H) 4.07 (quint., J = 5.6 Hz, 1 H), 1.50–1.40 (m,
2 H), 1.39–1.20 (m, 6 H), 0.84 (t, J = 6.8 Hz, 3 H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 145.6, 141.6, 141.3, 132.7 (q, J = 33.0 Hz,
2 C), 126.9, 123.6 (q, J = 270 Hz, 2 C), 113.0 (q, J = 3.0 Hz),
112.4 (q, J = 3.7 Hz), 72.6, 37.3, 31.9, 25.2, 22.8, 14.2 ppm. HRMS
(ESI+): calcd. for C17H21F6N2O [M + H]+ 383.1558; found
383.1562.
(br. s, 1 H), 7.38 (dd, J = 4.8, 1.5 Hz, 2 H), 7.16 (m, 1 H), 7.06
(dd, J = 3.3, 2.1 Hz, 1 H), 6.05 (ddd, J = 8.7, 6.0, 2.0 Hz, 1 H),
5.96 (dd, J = 9.6, 3.0 Hz, 1 H), 4.78 (m, 1 H), 1.80–1.65 (m, 1 H),
1.58–1.42 (m, 1 H), 1.40–1.20 (m, 6 H), 0.87 (t, J = 6.9 Hz, 3
H) ppm. 13C NMR (75 MHz, CDCl3): δ = 146.5, 136.3, 131.6 (q,
J = 32.2 Hz), 129.7, 127.0, 124.5 (q, J = 271 Hz), 118.1, 116.9 (q,
J = 3.6 Hz, 2 C), 111.2 (q, J = 3.6 Hz), 52.1, 31.9, 31.3, 23.6, 22.7,
14.1 ppm. HRMS (ESI+): calcd. for C16H20F3N2 [M + H]+
297.1579; found 297.1575.
Mixture of 6-Pentyl-1-[3-(trifluoromethyl)phenyl]-5-{2-[3-(trifluoro-
1
methyl)phenyl]hydrazinyl}-1,4,5,6-tetrahydropyridazine (9b): H
NMR (400 MHz, CDCl3, 9b/10b = 1.7:1): δ = 7.80 (s, 0.41) minor,
7.67 (d, J = 8.0 Hz, 0.45 H) minor, 7.64 (d, J = 8.0 Hz, 0.43 H)
minor, 7.50 (t, J = 7.2 Hz, 0.55) minor, 7.44 (m, 1.13 H) overlapped,
7.32 (m, 1.35 H) overlapped, 7.25 (m, 1.78 H) overlapped, 7.15 (t,
J = 8.0 Hz, 1 H) major, 7.08 (d, J = 7.2 Hz, 1 H) major, 7.03 (m,
0.78 H) minor, 6.96–6.89 (m, 2 H) overlapped, 6.81 (m, 1 H) major,
4.55 (br. t, 0.60 H) minor, 4.46 (m, 0.53 H) minor, 4.18 (m, 1 H)
major, 3.41 (m, 1 H) major, 2.74 (d, J = 18.8 Hz, 0.71 H) minor,
2.64 (dd, J = 19.4, 4.8 Hz, 0.65 H) minor, 2.50 (dd, J = 17.6, 4.0 Hz,
1 H) major, 2.20 (d, J = 19.2 Hz, 1 H) major, 1.85 (m, 0.76 H)
minor, 1.90–1.75 (m, 1 H), 1.70–1.60 (m, 1.78 H) overlapped, 1.60–
1.40 (m, 6.06 H) overlapped, 1.42–1.30 (m, 3.15 H) overlapped,
1.28–1.25 (m, 4.23 H) overlapped, 0.92 (t, 1.71 H) minor, 0.86 ppm
(t, J = 6.8 Hz, 3 H) major. 13C NMR spectrum could not be re-
corded because 9b was oxidizing to 10b in the NMR tube the dur-
ing analysis time. LRMS (EI+): m/z (%) = [M]+· absent, 312 (12),
(1E,2E)-1-{2-[2,4-Bis(trifluoromethyl)phenyl]hydrazono}non-2-en-
4-ol (7e): A yellowish solid (35.5 mg, 0.093 mmol, 62%). M.p. 79–
81 °C. 1H NMR (400 MHz, [D6]DMSO): δ = 9.92 (s, 1 H), 8.13 (d,
J = 9.2 Hz, 1 H), 7.79 (d, J = 8.8 Hz, 1 H), 7.72 (s, 1 H), 7.70 (d,
J = 8.8 Hz, 1 H), 6.30 (dd, J = 15.6, 9.6 Hz, 1 H), 6.08 (dd, J =
15.6, 5.6 Hz, 1 H), 4.83 (d, J = 4.8 Hz, 1 H) 4.09 (quint., J =
5.6 Hz, 1 H), 1.50–1.40 (m, 2 H), 1.39–1.17 (m, 6 H), 0.84 (t, J =
6.8 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 144.6, 143.6,
142.4, 130.4 (q, J = 3.0 Hz), 126.5, 124.2 (q, J = 270 Hz), 124.1 (q,
J = 270 Hz), 124.0 (q, J = 3.2 Hz), 121.1 (q, J = 33.8 Hz), 114.8,
111.7 (q, J = 31.4 Hz), 72.3, 37.3, 31.9, 25.2, 22.8, 14.2 ppm.
HRMS (ESI+): calcd. for C17H21F6N2O [M + H]+ 383.1558; found
383.1553.
(1E,2E)-1-[2-(Perfluorophenyl)hydrazono]non-2-en-4-ol (7f): A white
solid (42.8 mg, 0.128 mmol, 85 %). M.p. 110–112 °C. 1H NMR
(400 MHz, [D6]DMSO): δ = 9.91 (s, 1 H), 7.76 (d, J = 9.6 Hz, 1 241 (100), 213 (8), 145 (11). LRMS (ESI+) = 473 [M + H]+. HRMS
H), 6.18 (dd, J = 16.0, 9.6 Hz, 1 H), 5.98 (dd, J = 15.6, 5.6 Hz, 1
(ESI+): calcd. for C23H27F6N4 [M + H]+ 473.2140; found 473.2131.
8
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Eur. J. Org. Chem. 0000, 0–0

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Date: 30-05-12 16:44:57
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Derivatization of an Elusive Key Biomarker of Lipid Peroxidation
(E)-6-Pentyl-1-[3-(trifluoromethyl)phenyl]-5-{[3-(trifluoromethyl)-
phenyl]diazenyl}-1,4,5,6-tetrahydropyridazine (10b): In MeCN,
37.6 mg of a yellow oil (0.080 mmol, 32%). 1H NMR (400 MHz,
(ddd, J = 9.2, 6.0, 1.6 Hz, 1 H), 5.99 (dd, J = 9.6, 3.2 Hz, 1 H),
4.80 (m, 1 H), 1.80–1.70 (m, 1 H), 1.55–1.45 (m, 1 H), 1.40–1.25
(m, 6 H), 0.87 (t, J = 6.8 Hz, 3 H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 7.80 (s, 1 H), 7.67 (d, J = 7.6 Hz, 1 H), 7.64 (d, J = CDCl3): δ = 147.1, 137.3, 132.4 (q, J = 32.2 Hz, 2 C), 128.1, 123.7
7.2 Hz, 1 H), 7.50 (t, J = 7.2 Hz, 1 H), 7.44 (s, 1 H), 7.31 (t, J =
7.6 Hz, 1 H), 7.25 (m overlapped, 1 H), 7.05 (d, J = 7.2 Hz, 1 H),
6.90 (br. s, 1 H), 4.55 (br. t, 1 H), 4.46 (m, 1 H), 2.75 (d, J =
18.8 Hz, 1 H), 2.64 (dd, J = 19.4, 4.8 Hz, 1 H), 1.90–1.75 (m, 1 H),
1.70–1.60 (m, 1 H), 1.60–1.45 (m, 2 H), 1.42–1.30 (m, 4 H), 0.90
(t, J = 6.8 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ = 151.7,
147.4, 134.9, 131.8 (q, J = 33.0 Hz), 131.5 (q, J = 33.0 Hz), 129.8,
129.6, 127.4 (q, J = 3.2 Hz), 125.3, 124.6 (q, J = 270 Hz), 123.8 (q,
J = 270 Hz), 119.9 (q, J = 4.0 Hz), 116.4, 116.1 (q, J = 3.2 Hz),
110.7 (q, J = 4.0 Hz), 67.5, 54.9, 31.8, 29.6, 25.6, 23.4, 22.7,
14.1 ppm. LRMS (EI): m/z (%) = 470 (20) [M]+·, 451 (6), 297 (55),
225 (60), 213 (40), 145 (100). LRMS (ESI–) = 469.13 [M – H]–.
HRMS (EI): calcd. for C23H24F6N4 [M]+· 470.1905; found
470.1910.
(q, J = 271 Hz, 2 C), 118.0, 113.5 (q, J = 3.5 Hz, 2 C), 113.2 (q, J
= 4.1 Hz), 52.1, 31.8, 31.4, 23.6, 22.7, 14.1 ppm. HRMS (ESI+):
calcd. for C17H19F6N2 [M + H]+ 365.1452; found 365.1461.
1-[3,5-Bis(trifluoromethyl)phenyl]-5-{2-[3,5-bis(trifluoromethyl)-
phenyl]hydrazinyl}-6-pentyl-1,4,5,6-tetrahydropyridazine (9d): In
MeCN, 78 mg of a yellow oil (0.128 mmol, 51 %). 1H NMR
(400 MHz, CDCl3): δ = 7.45 (s, 2 H), 7.25 (s, 1 H), 7.13 (s, 2 H),
7.11 (s, 1 H), 6.88 (s, 1 H), 5.49 (s, 1 H), 4.18 (br. t, 1 H), 3.61 (br.
s, 1 H), 3.44 (m, 1 H), 2.57 (dd, J = 19.2, 5.2 Hz, 1 H), 2.16 (dd, J
= 19.6 Hz, 1 H), 1.70–1.60 (m, 1 H), 1.55–1.40 (m, 1 H), 1.40–1.20
(m, 6 H), 0.85 (t, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ =
150.2, 147.6, 135.8, 132.5 (q, J = 33.0 Hz, 2 C), 132.4 (q, J =
33.0 Hz, 2 C), 123.7 (q, J = 271 Hz, 2 C), 123.4 (q, J = 271 Hz, 2
C), 112.4 (m, 4 C), 112.1 (m, 2 C), 52.6, 52.1, 31.8, 29.3, 25.7, 24.7,
22.6, 14.0 ppm. HRMS (ESI+): calcd. for C25H25F12N4 [M + H]+
609.1888; found 609.1893.
6-Pentyl-1-[4-(trifluoromethyl)phenyl]-1,6-dihydropyridazine (8c): In
tBuOH, 47.4 mg of a yellow oil (0.160 mmol, 64 %). 1H NMR
(400 MHz, CDCl3): δ = 7.52 (d, J = 8.4 Hz, 2 H), 7.30 (d, J =
8.8 Hz, 2 H), 7.07 (m, 1 H), 6.07 (td, J = 6.4, 1.6 Hz, 1 H), 5.96
(dd, J = 9.2, 3.2 Hz, 1 H), 4.79 (m, 1 H), 1.82–1.72 (m, 1 H), 1.54–
1.45 (m, 1 H), 1.38–1.22 (m, 6 H), 0.87 (t, J = 6.8 Hz, 3 H) ppm.
13C NMR (100 MHz, CDCl3): δ = 148.5, 136.7, 127.5, 126.5 (q, J
= 4.1 Hz, 2 C), 126.4 (q, J = 269 Hz), 122.0 (q, J = 32.2 Hz), 118.0,
113.7 (2 C), 52.0, 31.9, 31.5, 23.6, 22.7, 14.1 ppm. LRMS (EI+):
m/z (%) = 296 (0.5) [M]+·, 277 (5), 251 (2), 225 (100). LRMS (ESI+)
= 297.05 [M + H]+. HRMS (EI): calcd. for C16H19F3N2 296.1502;
found 296.1510.
(E)-1-[3,5-Bis(trifluoromethyl)phenyl]-5-{[3,5-bis(trifluoromethyl)-
phenyl]diazenyl}-6-pentyl-1,4,5,6-tetrahydropyridazine (10d): A yel-
low oil (18 mg, 0.029 mmol) 1H NMR (400 MHz, CDCl3): δ = 7.96
(s, 2 H), 7.91 (s, 1 H), 7.54 (s, 2 H), 7.28 (s, 1 H), 6.98 (m, 1 H),
4.65 (br. t, J = 6.8 Hz, 1 H), 4.57 (m, 1 H), 2.83 (dd, J = 19.2,
4.0 Hz, 1 H), 2.70 (ddd, J = 19.2, 6.0, 1.6 Hz, 1 H), 1.90–1.75 (m,
1 H), 1.75–1.62 (m, 1 H), 1.60–1.48 (m, 2 H), 1.45–1.30 (m, 4 H),
0.91 (t, J = 6.4 Hz, 3 H) ppm. 13C NMR (100 MHz, CDCl3): δ =
151.8, 147.8, 136.4, 132.9 (q, J = 33.1 Hz, 2 C), 132.5 (q, J =
33.1 Hz, 2 C), 124.3, 123.4 (q, J = 270 Hz, 2 C), 123.0 (q, J =
270 Hz, 2 C), 122.8 (2 C), 113.0 (2 C), 112.7, 67.7, 54.6, 31.7, 29.6,
25.6, 23.2, 22.7, 14.0 ppm. LRMS (ESI–): m/z = 605 [M – H]–.
HRMS (EI): calcd. for C25H22F12N4 [M]+· 606.1653; found
606.1559.
6-Pentyl-1-[4-(trifluoromethyl)phenyl]-5-{2-[4-(trifluoromethyl)-
phenyl]hydrazinyl}-1,4,5,6-tetrahydropyridazine (9c): In MeCN,
65 mg of a yellow oil (0.138 mmol, 55%). 1H NMR (400 MHz,
CDCl3): δ = 7.64 (d, J = 8.4 Hz, 2 H), 7.58 (d, J = 8.4 Hz, 2 H),
7.46 (d, J = 9.2 Hz, 2 H), 7.19 (d, J = 8.8 Hz, 2 H), 6.92 (m, 1 H),
4.57 (br. t, 1 H), 4.45 (m, 1 H), 2.74 (d, J = 19.2 Hz, 1 H), 2.65
(dd, J = 18.4, 5.2 Hz, 1 H), 1.90–1.74 (m, 1 H), 1.73–1.64 (m, 1
H), 1.52–1.45 (m, 2 H), 1.42–1.30 (m, 4 H), 0.91 (t, J = 6.8 Hz, 3
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 153.7, 150.0, 135.4,
130.0, 126.5 (m, 4 C), 123.6 (2 C), 112.9, 112.7 (2 C), 111.9 (2 C),
53.4, 51.6, 31.9, 29.4, 25.7, 24.5, 22.7, 14.2 ppm. LRMS (ESI+):
m/z = 473 [M + H]+. HRMS (ESI+): calcd. for C23H27F6N4 [M +
H]+ 473.2140; found 473.2144.
(E)-1-[(E)-4-Ethoxynon-2-enylidene]-2-[4-(trifluoromethyl)phenyl]-
hydrazine (11c): The reaction was run according to the general pro-
cedure in EtOH (2.0 mL) with aldehyde 1 (0.020 g, 0.128 mmol,
1 equiv.), hydrazine 2c (1.1 equiv.), and a solution of TFA (5% in
EtOH, 0.088 mL, 0.3 equiv.) at 50 °C for 5 h. The crude residue
was purified by flash chromatography (petroleum ether/EtOAc, 9:1
to 8:2) to afford ethyl ether 11c (40%) and 1,6-dihydropyridazine
8c (30%).
11c: Yellow oil (17 mg, 0.078 mmol, 40%). 1H NMR (400 MHz,
[D6]DMSO): δ = 10.5 (s, 1 H), 7.61 (d, J = 9.2 Hz, 1 H), 7.47 (d,
J = 8.4 Hz, 2 H), 7.03 (d, J = 8.4 Hz, 2 H), 6.31 (dd, J = 15.6,
9.6 Hz, 1 H), 5.85 (dd, J = 15.6, 7.2 Hz, 1 H), 3.79 (q, J = 6.4 Hz,
1 H), 3.44 (m, 1 H), 3.30 (m overlapped, 1 H), 1.60–1.45 (m, 2 H),
1.44–1.25 (m, 6 H), 1.08 (t, J = 7.2 Hz, 3 H), 0.84 (t, J = 6.8 Hz,
3 H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 148.8, 141.5,
138.9, 129.3, 127.1 (q, J = 4.0 Hz, 2C), 125.6 (q, J = 270 Hz), 118.9
(q, J = 31.4 Hz), 112.2 (2C), 79.6, 63.8, 35.7, 31.9, 25.1, 22.7, 16.0,
14.6 ppm. LRMS (EI): m/z (%) = 342 (2) [M]+·, 323 (3), 296 (10),
239 (8), 212 (56), 185 (10), 145 (10), 85 (18), 57 (100). HRMS (EI):
calcd. for C1.8H25F3N2O [M]+· 342.1919; found 342.1923.
(E)-6-Pentyl-1-[4-(trifluoromethyl)phenyl]-5-{[4-(trifluoromethyl)-
phenyl]diazenyl}-1,4,5,6-tetrahydropyridazine (10c): In MeCN,
41 mg of a yellow oil (0.087 mmol, 35%). 1H NMR (400 MHz,
CDCl3): δ = 7.64 (d, J = 8.4 Hz, 2 H), 7.58 (d, J = 8.4 Hz, 2 H),
7.46 (d, J = 9.2 Hz, 2 H), 7.19 (d, J = 8.8 Hz, 2 H), 6.92 (m, 1 H),
4.57 (br. t, 1 H), 4.45 (m, 1 H), 2.74 (d, J = 19.2 Hz, 1 H), 2.65
(dd, J = 18.4, 5.2 Hz, 1 H), 1.90–1.74 (m, 1 H), 1.73–1.64 (m, 1
H), 1.52–1.45 (m, 2 H), 1.42–1.30 (m, 4 H), 0.91 (t, J = 6.8 Hz, 3
H) ppm. 13C NMR (100 MHz, CDCl3): δ = 153.6, 149.3, 135.3,
132.6 (q, J = 32.3 Hz), 126.5 (m, 4 C), 123.9 (q, J = 276 Hz), 125.0
(q, J = 276 Hz), 122.7 (2 C), 121.2 (q, J = 33.0 Hz), 112.9 (2 C),
67.5, 54.7, 31.9, 29.7, 25.6, 23.5, 22.7, 14.2 ppm. LRMS (EI+): m/z
(%) = 470 (3) [M]+·, 451 (15), 397 (26), 297 (100), 295 (25), 225
(25), 213 (15), 145 (36). HRMS (EI): calcd. for C23H24F6N4 [M]+·
470.1905; found 470.1909.
Reactions of (E)-4-Hydroxynon-2-enal (1) with [2,4-Bis(trifluoro-
methyl)phenyl]hydrazine (2e): Reaction followed the general pro-
cedure with aldehyde 1 (0.020 g, 0.128 mmol, 1 equiv.) hydrazine 2e
1-[3,5-Bis(trifluoromethyl)phenyl]-6-pentyl-1,6-dihydropyridazine (2.0 equiv.) in CH3CN (3 mL) with TFA (5% in CH3CN, 0.156 mL,
(8d): In MeCN, 13 mg of a yellow oil (0.036 mmol, 14 %); in
tBuOH, 56 mg (0.15 mmol, 61%). H NMR (400 MHz, CDCl3): δ
0.3 equiv.) and solid MgSO4. The reaction was heated at 70 °C for
5 h. The crude was purified by flash chromatography (petroleum
1
= 7.64 (s, 2 H), 7.36 (s, 1 H), 7.10 (dd, J = 2.8, 1.6 Hz, 1 H), 6.11 ether/EtOAc, 95:5) to afford 1,6-disubstituted product 12e. Further
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Job/Unit: O20346
/KAP1
Date: 30-05-12 16:44:57
Pages: 12
R. Matera, S. Gabbanini, A. Valvassori, M. Triquigneaux, L. Valgimigli
FULL PAPER
elution (petroleum ether/EtOAc, 85:15) furnished linear hydrazone
7e and addition product 9e.
Acknowledgments
Authors are grateful to BeC s.r.l (Forlì, Italy), the University of
Bologna (Italy) and Ministero dell’Università e della Ricerca
(MIUR) (Rome, Italy; project PRIN2008: Processi Radicalici e di
Trasferimento Elettronico in Chimica e Biologia: Applicazioni nella
Sintesi, nella Catalisi e nelle Scienze dell’Ambiente e dei Materiali)
for funding, and to the University of Orléans (France) for a grant
to M. T. We thank Sébastien Guesné (Queen Mary, University of
London) for helpful discussion on cyclization products formation.
1-[2,4-Bis(trifluoromethyl)phenyl]-5-{2-[2,4-bis(trifluoromethyl)-
phenyl]hydrazinyl}-6-pentyl-1,4,5,6-tetrahydropyridazine (9e): Yel-
low oil (15 mg, 0.025 mmol, 13%). 1H NMR (400 MHz, CDCl3):
δ = 7.94 (s, 1 H), 7.72 (dd, J = 8.8, 1.6 Hz, 1 H), 7.66 (s, 1 H), 7.59
(d, J = 9.2 Hz, 2 H), 7.56 (d, J = 9.2 Hz, 2 H), 7.39 (d, J = 8.4 Hz,
1 H), 6.94 (m, 1 H), 6.30 (s, 1 H), 4.01 (d, J = 1.8 Hz, 1 H), 3.88
(br. t, 1 H), 3.30 (br. t, J = 1.5 Hz, 1 H), 2.47 (dd, J = 19.6, 8.4 Hz,
1 H), 2.24 (dm, J = 19.6 Hz, 1 H), 1.55–1.40 (m, 1 H), 1.30–1.20
(m, 6 H), 0.80 (t, J = 6.8 Hz, 3 H) ppm. 13C NMR (100 MHz,
CDCl3): δ = 150.1, 149.6, 139.0, 130.2, 129.4, 126.7, 126.3 (q, J =
33.1 Hz), 125.8 (q, J = 7.2 Hz), 124.4 (q, J = 271 Hz), 124.3 (q, J
= 271 Hz), 124.1 (m), 123.8 (q, J = 271 Hz), 123.7 (q, J = 271 Hz),
122.8 (q, J = 31.4 Hz), 119.7 (q, J = 33.0 Hz), 111.7 (q, J =
31.4 Hz), 58.6, 52.9, 31.6, 29.9, 25.5, 24.2, 22.5, 14.0 ppm. LRMS
(ESI+): m/z = 609.1 [M + H]+. HRMS (ESI+): calcd. for
C25H25F12N4 [M + H]+ 609.1888; found 609.1883.
[1] L. Valgimigli, D. A. Pratt in Encyclopedia of Radicals in Chem-
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1
yellow oil (7 mg, 0.078 mmol, 8%). H NMR (400 MHz, CDCl3):
δ = 7.99 (s, 1 H), 7.75–7.71 (m, 2 H), 7.68–7.61 (m, 2 H), 7.61–7.56
(m, 2 H), 7.51 (d, J = 9.2 Hz, 1 H), 6.39 (dd, J = 15.6, 9.2 Hz, 1
H), 5.97 (br. s, 1 H), 5.78 (dd, J = 15.6, 8.8 Hz, 1 H), 3.53 (br. s, 1
H), 3.39 (q, J = 7.2 Hz, 1 H), 1.66–1.58 (m, 1 H), 1.55–1.45 (m, 1
H), 1.45–1.20 (m, 6 H), 0.89 (t, 3 H) ppm. HRMS (ESI+): calcd.
for C25H25F12N4 [M + H]+ 609.1888; found 609.1896.
HPLC Analysis: The reaction mixture during the synthesis or de-
gradation of hydrazones 7a–f was analyzed by HPLC-DAD (injec-
[10] T. Nystrçm, EMBO J. 2005, 24, 1311–1317.
[11] R. M. LoPachin, D. S. Barber, T. Gavin, Toxicol. Sci. 2008,
tion volume: 10 μL) by using
a
C18 stationary phase
104, 235–249.
(150 mmϫ4.6 mmϫ5 μm particle size), eluting at 500 μL/min with
a gradient mixture of A (0.5% formic acid in MeCN) and B (0.5%
formic acid in water) with the following programming: t = 0 min,
A/B 60:40; t = 10 min, A/B 80:20; t = 15 min A/B 80:20; t = 20 min
A/B 60:40, detecting in the wavelength range 230–450 nm. Cali-
bration curves (8 levels) were built for each analyte at the band
maxima by using authentic standards prepared as described above.
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forth, B. V. Burger, A. M. Crouch, P. Sandra, J. Chromatogr. B
2006, 834, 134–140.
Kinetics of Formation and Degradation of Hydrazones 7a–f: To
study the kinetics of formation of the hydrazones, a solution of 4-
HNE in MeCN (1–50 μm) was incubated with 10-fold or higher
excess of the hydrazines 2a–f in the presence of an excess amount
of trifluoroacetic acid (TFA, 0.01–1 mm) at 25 °C. The progress of
the reaction was monitored at time intervals by HPLC analysis of
the reaction mixture as indicated above. The growth of the concen-
tration of hydrazones 7a–f vs. time was first analyzed against first-
order curves. The pseudo-first-order rate constant was then plotted
vs. the initial concentration of the hydrazine (4 data points or more
for each hydrazine) at fixed concentration of TFA to obtain the
apparent second-order rate constant under the fixed experimental
conditions as reported in Table 2. To study the decomposition of
hydrazones 7a–f solutions in MeCN, either in the presence or ab-
sence of water and TFA, were incubated at 25 °C in air and were
monitored with time, both continuously by spectrophotometry at
the λmax reported in Table 2 and at time intervals by HPLC analysis
(vide supra). Decay plots were first analyzed against first-order de-
cay curves and the initial first-order decomposition rate constants
kd were then used as inputs for numerical fittings using Gepasi
3.30 software[30] and a reversible hydrolysis model to obtain the
optimized kd values collected in Table 2.
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782.
[17] Low recoveries are typically overcome in analytical chemistry
by using isotopically labeled analytes as internal standards;[18]
however, this approach is unpractical in the case of highly
unstable 4-HNE due to the difficult accessibility of suitably
labeled standards in pure form.
[18] X. P. Luo, M. Yazdanpanah, N. Bhooi, D. C. Lehotay, Anal.
Biochem. 1995, 228, 294.
[19] Solutions need to be stored at –160 to –80 °C, and as much as
25% degradation is observed in 1 h at 4 °C.[15a]
[20] I. Jouanin, V. Sreevani, E. Rathahao, F. Guéraud, A. Paris, J.
Labelled Compd. Radiopharm. 2008, 51, 87–92.
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29–98.
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4983–4986.
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ter, L. J. Marnett, Chem. Res. Toxicol. 2008, 21, 432–444.
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2007, 150, 239–243.
[25] The 1H NMR signal for the hydrazone (N)H in 7e was
9.93 ppm as compared to the value of 10.70 ppm for disubsti-
tuted 7d.
[26] The reaction of 2f with 4-HNE at higher temperature to pro-
vide the corresponding dihydropyridazine was not investigated.
[27] The apparent value of the rate constants varied with the con-
centration of the acid catalyst, in line with expectations for
Supporting Information (see footnote on the first page of this arti-
cle): Additional spectroscopic data and copies of the 1H NMR and
13C NMR spectra.
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Date: 30-05-12 16:44:57
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Derivatization of an Elusive Key Biomarker of Lipid Peroxidation
a general acid catalysis mechanism. A more detailed kinetic
investigation was outside the scope of this work, and kinetic
data have been determined for comparative evaluation only at
fixed TFA concentrations.
Cavaleiro, R. Amorati, M. G. Fumo, G. F. Pedulli, L. Valgimi-
gli, Chem. Eur. J. 2006, 12, 4646–4653.
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Received: March 20, 2012
Published Online: ᭿
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Job/Unit: O20346
/KAP1
Date: 30-05-12 16:44:57
Pages: 12
R. Matera, S. Gabbanini, A. Valvassori, M. Triquigneaux, L. Valgimigli
FULL PAPER
Lipid Peroxidation
R. Matera, S. Gabbanini, A. Valvassori,
M. Triquigneaux, L. Valgimigli* ..... 1–12
Reactivity of (E)-4-Hydroxy-2-nonenal
with
Fluorinated
Phenylhydrazines:
4-Hydroxynonenal reacts rapidly at room
temperature with 2-, 3-, or 4-CF3-phenyl-
hydrazine, or with the 3,5-di-CF3, 2,4-di-
CF3, or pentafluoro analogues, to form hy-
drazones, which may undergo cyclization
to 1,6-dihydropyridazines and other ad-
dition/oxidation products. The product dis-
tribution can be controlled by solvent and
temperature to develop rapid derivatization
assays.
Towards the Efficient Derivatization of an
Elusive Key Biomarker of Lipid Peroxid-
ation
Keywords: Synthetic methods / Hydraz-
ones / Cyclization / Lipids / Kinetics
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