Remarkable Chichibabin-type cyclotrimerisation of 3-nitrotyrosine, tyrosine and phenylalanine to 3,5-diphenylpyridine derivatives induced by hypochlorous acid

Article abstract of DOI:10.1016/j.tetlet.2005.07.106

Reaction of 3-nitrotyrosine with HOCl in aqueous phosphate buffer (pH 7.4) leads to a mixture of extractable products, including 3,5-di(4-hydroxy-3- nitrophenyl)pyridine (15% isolated yield) and 3,5-di(4-hydroxy-3-nitrophenyl)-2- (4-hydroxy-3-nitrophenylmethyl)pyridine (3%) arising by a Chichibabin-like pyridine synthesis via N-chloroimine intermediates. Under the same conditions, phenylalanine gives 3,5-diphenylpyridine in 9% isolated yield, while tyrosine leads to 3,5-di(4-hydroxyphenyl)pyridine (3%) and 3-(3-chloro-4-hydroxyphenyl)- 5-(4-hydroxyphenyl)pyridine (3%).

Full text of DOI:10.1016/j.tetlet.2005.07.106

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 6457–6460
Remarkable Chichibabin-type cyclotrimerisation of
3-nitrotyrosine, tyrosine and phenylalanine to
3,5-diphenylpyridine derivatives induced by hypochlorous acid
L. Panzella,a P. Di Donato,b S. Comes,b A. Napolitano,a A. Palumbob and M. dÕIschiaa,*
aDepartment of Organic Chemistry and Biochemistry, Complesso Universitario di Monte S. Angelo via Cinthia,
University of Naples Federico II, I 80126 Naples, Italy
bZoological Station Anton Dohm, Villa Comunale, I 80121, Naples, Italy
Received 9 May 2005; accepted 22 July 2005
Abstract—Reaction of 3-nitrotyrosine with HOCl in aqueous phosphate buffer (pH 7.4) leads to a mixture of extractable products,
including 3,5-di(4-hydroxy-3-nitrophenyl)pyridine (15% isolated yield) and 3,5-di(4-hydroxy-3-nitrophenyl)-2-(4-hydroxy-3-nitro-
phenylmethyl)pyridine (3%) arising by a Chichibabin-like pyridine synthesis via N-chloroimine intermediates. Under the same con-
ditions, phenylalanine gives 3,5-diphenylpyridine in 9% isolated yield, while tyrosine leads to 3,5-di(4-hydroxyphenyl)pyridine (3%)
and 3-(3-chloro-4-hydroxyphenyl)-5-(4-hydroxyphenyl)pyridine (3%).
ꢀ 2005 Elsevier Ltd. All rights reserved.
The reactions of a-amino acids with hypochlorous acid
(HOCl) have been extensively investigated over the
past decades for their potential synthetic interest2–4 as
well as for their importance in biology and medicine,5,6
being implicated in the inflammatory response
mediated by activated neutrophils through the myelo-
peroxidase–H2O2–Clꢀ system.7 As a rule, the HOCl-
mediated oxidation of the amino acidic functionality
at neutral pH proceeds with decarboxylation to yield
mainly aldehyde and nitrile products.4–6 In the case
of tyrosine, aromatic ring chlorination may occur as
well, leading, besides 4-hydroxyphenylacetaldehyde, to
3-chlorotyrosine, 3,5-dichlorotyrosine and 3-chloro-4-
hydroxyphenylacetaldehyde.5,8,9 The accepted mecha-
nism of the deamination/decarboxylation of amino
acids by HOCl involves formation of N-chloramines,
unstable intermediates that spontaneously decompose
with loss of NH3, CO2 and Clꢀ to give aldehyde spe-
cies.4,10,11 With excess HOCl, dichloramines may
also be formed, which are responsible for nitrile
formation.12
Recently, we revisited the reaction13 of HOCl (1.5 molar
equiv) with 3-nitrotyrosine (1) (0.02–2 mM), a biochemi-
cal marker of peroxynitrite, at pH 7.4, and noticed the
presence in the ethyl acetate extractable fraction of
two main products whose properties did not match with
those of any of the known oxidation products of amino
acids and that were relatively retained on reverse phase
HPLC (tR 33.4 and 36.2 min) and TLC (Rf 0.23 and
0.33).à The product at Rf 0.23 could be isolated by pre-
cipitation from the organic residue dissolved in ethyl
acetate, while that at Rf 0.33 was isolated by preparative
TLC.
The product at Rf 0.23 displayed in the ESI-/MS
spectrum, a pseudomolecular ion peak at m/z 352 and
Keywords: Aminoacid chlorination; N-Chloroimines; 3,5-Diphenyl-
pyridines; Cyclotrimerisation.
à HPLC analyses were run on octadecylsilane coated columns,
250·4.6 mm, 5 lm particle size, at a 1.0 mL/min flow rate; eluent:
2% acetic acid in water (solvent A) and 2% acetic acid in acetonitrile
(solvent B), gradient from 10% to 60% B (25 min), from 60% to 90%
B (15 min). Analytical and preparative TLC was performed on 0.25
and 0.5 mm silica gel plates, respectively (eluent: cyclohexane–ethyl
acetate 60:40 v/v).
*
Corresponding author. Tel.: +39 081674132; fax: +39 081674393;
e-mail: dischia@unina.it
HOCl has a pKa of 7.46,1 therefore it exists as a mixture of the
protonated and dissociated form (ꢀOCl, hypochlorite) at pH 7.4;
throughout this paper HOCl will be used to designate this mixture
unless otherwise stated.
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.07.106

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L. Panzella et al. / Tetrahedron Letters 46 (2005) 6457–6460
exhibited a chromophore characterised by maxima at
260 and 368 nm. The H NMR spectrum (DMSO-d6)
Other reaction products that were isolated from the
ethyl acetate fraction after removal of the solvent at rt
were 4-hydroxy-3-nitrophenylacetaldehyde 4 (20% iso-
1
showed as salient features: (a) the lack of aliphatic pro-
ton signals; (b) the typical spin system of the 4-hydroxy-
3-nitrophenyl moiety and (c) a highly deshielded doublet
at d 8.84 (J = 2.0 Hz) coupled to a multiplet at d 8.31
partially overlapped to one of the aromatic proton reso-
nances. Scrutiny of the spectrum run in different solvents
revealed that the integrated area of the signal at d 8.31
was invariably half that of the other resonances. Based
lated yield), 4-hydroxy-3-nitrophenylacetonitrile
5
(20% isolated yield), the N-chloroimines 6 (inseparable
mixture of E/Z isomers, 17% isolated yield)§ and little
4-hydroxy-3-nitrobenzaldehyde.
Formation of products 2 and 3 is reminiscent of the
Chichibabin pyridine synthesis via condensation of car-
bonyl compounds with ammonia or amines in boiling
ethanol or in autoclave under pressure.14–17 Whereas
3 is evidently the result of a cyclotrimerisation process
involving 4 and an imine intermediate, the pyridine 2
would arise by a more complex mechanism in which
a 4-hydroxy-3-nitrophenylmethyl moiety is lost. In
the classical Chichibabin condensation, phenylacetalde-
hyde reacts with ammonia to give 3,5-diphenylpyridine
with loss of toluene,16 whereas in the reaction of 1 with
HOCl no detectable formation of 4-hydroxy-3-nitrotol-
uene was observed after the usual work-up, suggesting
that a different mechanism was operative involving
direct loss of 4-hydroxy-3-nitrobenzaldehyde. Since in
separate experiments it was found that 4-hydroxy-3-
nitrobenzaldehyde does not arise by oxidation of 4-
hydroxy-3-nitrotoluene under the reaction conditions,
a series of experiments were performed to gain a deeper
mechanistic insight. When the pure N-chloroimines 6
were heated in ethyl acetate at 40 ꢁC for 30 min, the
nitrile 5 was formed as the main product, along with
the pyridines 2 and 3, the aldehyde 4 and 4-hydroxy-
3-nitrobenzaldehyde, whereas at rt the trimer 3 was
the prevalent species along with 5. Addition of the
aldehyde 4 to the solution of 6 in ethyl acetate at
40 ꢁC led to an increase in the yield of formation of
2 and 3. Addition of formaldehyde16 to the reaction
mixture of 1 with HOCl did not affect formation of 2
or 3, thus ruling out its involvement in the build-up
of the pyridine ring. On this basis, it is argued that for-
mation of pyridine derivatives depends on the genera-
tion and reactivity of the N-chloroimines 6. These
can undergo condensation with two molecules of
the aldehyde 4 to give eventually 3 (Scheme 1). Alter-
natively, further chlorination of 6 by HOCl or by
interaction with another molecule of 6 may give the
a-chloroderivatives, which after condensation with 4
would eliminate 4-hydroxy-3-nitrobenzaldehyde.
1
on H, 13C heteronuclear multiple quantum coherence
and heteronuclear multiple bond correlation experi-
ments, the compound was identified as 3,5-di(4-hydro-
xy-3-nitrophenyl)pyridine (2).
The product at Rf 0.33 displayed in the ESI-/MS spec-
1
trum a pseudomolecular ion peak at m/z 503. The H
NMR spectrum was characterised by the presence of
spin systems for three different 4-hydroxy-3-nitrophenyl
moieties. Moreover, the low field region showed a doub-
let (J = 2.4 Hz) at d 8.84, coupled to a doublet at d
1
7.68. Finally, the aliphatic region of the H NMR spec-
trum was characterised by the presence of a 2H singlet at
d 4.17. On this basis, the product was identified as 3,5-
di(4-hydroxy-3-nitrophenyl)-2-(4-hydroxy-3-nitrophenyl-
methyl)pyridine (3).
NMR resonances of compounds 2 and 3 and assign-
ments based on 2D correlation experiments are listed
in Table 1.
Isolated yields of 2 and 3 were 15% and 3%, respectively,
based on reacted 1.
Table 1. NMR spectral data for 2 and 3
2 (DMSO-d6)
1H (J, Hz)
3(CDCl3)
1H (J, Hz)
13C
13C
1a
2
3
4
—
—
146.1
133.9
131.4 7.68 (d, 2.4)
—
—
—
—
—
8.84 (d, 2.0)
—
8.31 (m)
157.2
136.0
135.8
133.0
147.1
130.5
125.3
135.0
155.8
121.2
137.9
131.5
123.1
134.5
156.0
120.7
135.8
40.1
5
6
8.84 (d, 2.4)
—
10
—
126.8
123.7 8.00 (d, 2.0)
138.0
153.1
120.4 7.30 (d, 8.4)
20
8.33 (d, 2.4)
—
—
7.19 (d, 8.8)
8.00 (dd, 8.8, 2.4) 133.5 7.48 (d, 8.4, 2.0)
30
—
—
40
500
Under similar conditions, tyrosine reacted with HOCl
to give 3,5-di(4-hydroxyphenyl)pyridine (7) and 3-(3-
chloro-4-hydroxyphenyl)-5-(4-hydroxyphenyl)pyridine
600
100
200
3
—
8.34 (d, 2.4)
—
—
7.28 (d, 8.8)
7.84 (d, 8.8, 2.4)
4.17 (s)
400
500
600
CH2a
1000
2000
3000
4000
5000
6000
§ 1H NMR (CDCl3) d (ppm) (major/minor 1:0.4): 3.69 (2H, d,
J = 5.6 Hz), 3.89 (0.8H, d, J = 4.8 Hz), 7.14 (1H, d, J = 8.4 Hz),
7.16 (0.4H, d, J = 8.4 Hz), 7.43 (1H, dd, J = 8.4, 2.0 Hz), 7.46 (0.4H,
dd, J = 8.4, 2.0 Hz), 7.95 (1H, d, J = 2.0 Hz), 7.99 (0.4H, d,
J = 2.0 Hz), 8.06 (0.4H, t, J = 4.8 Hz), 8.25 (1H, t, J = 5.6 Hz),
10.50 (1.4H, br s); 13C NMR (CDCl3) d (ppm) major/minor: 40.1/
38.3 (CH2), 120.7/120.9 (CH), 125.1/124.9 (CH), 126.1/126.3 (C),
133.4 (C), 138.2/137.9 (CH), 154.4 (C), 173.8/171.5 (CH); ESI-/MS:
m/z 215 ([M+2ꢀH]ꢀ, 33), 213 ([MꢀH]ꢀ, 100), 177 ([MꢀHClꢀH]ꢀ,
38).
—
132.0
124.5
134.0
154.5
120.1
7.76 (d, 2.4)
—
—
7.04 (d, 8.4)
7.39 (dd, 8.4, 2.4) 138.2
a Numbering as shown in structural formulas 2 and 3.

L. Panzella et al. / Tetrahedron Letters 46 (2005) 6457–6460
6459
O
O
O
O2N
HO
O2N
HO
O N
HOCl
-H2O
HOCl
-H2O
2
OH
OH
OH
NH2
NH
NCl
Cl
-CO2
-HCl
Cl
HO
1
-CO2
-HCl
H2O
-NH3
O2N
HO
N
Cl
-HCl
O2N
HO
CN
O2N
O
6
5
4
HO
HO
HOCl
-H2O
NO2
Cl
O
O2N
HO
N
Cl
NHCl
H2O
4
-HCl
O2N
OH
NO2
O2N
N
Cl
HO
HO
-HCl
O
NO2
HO
-HCN
-HCl
HO
O
Cl
O
NHCl
O2N
HO
H
O2N
NH
O
HO
O2N
O2N
+H2O
-HCl
H
HO
4
HO
O
-H2O
NO2
NO2
NO2
HO
HO
OH
O
N
NH
4
-H2O
NO2
NO2
O2N
HO
OH
O
-H2O
HO
NO2
NO2
3''
3'
OH
HO
2'
1'
N
2''
1''
4
-H2O
NO2
3
NO2
α
3'
2
1'''
N
1
OH
HO
2'
1'
2'''
3'''
3
3
O2N
N
1
2
OH
Scheme 1.
(8) isolated in 3% yield each,– in addition to 3-chloro-
tyrosine, 4-hydroxyphenylacetaldehyde, 3-chloro-4-
hydroxyphenylacetaldehyde and 4-hydroxyphenylaceto-
nitrile as the main species, consistent with previous
reports.5,8,9 Extensive chlorination of the reactive phenol
ring accounts in part for the lower yields of pyridine
products. Phenylalanine also gave a 9% yield of 3,5-
diphenylpyridine (9),k previously prepared by reaction
of phenylacetaldehyde with methyleniminium chlo-
ride,18 while representative aliphatic amino acids, such
as serine, leucine and aspartic acid, failed to give pyri-
dine derivatives with HOCl under a variety of condi-
tions. It may be relevant to notice that Chichibabin
reactions depend on the degree of enolisation of the
aldehyde intermediates,16 a factor that may be impli-
cated in the lack of pyridine formation from aliphatic
amino acids.
– Preparative TLC fractionation (eluent chloroform–methanol 95:5
v/v). 7 (Rf 0.25): 1H NMR ((CD3)2CO) d (ppm): 7.01 (4H, d,
J = 8.4 Hz), 7.66 (4H, d, J = 8.4 Hz), 8.10 (1H, t, J = 2.0 Hz), 8.73
(2H, d, J = 2.0 Hz); 13C NMR (CD3OD) d (ppm): 117.1 (4·CH),
129.4 (4·CH), 131.4 (2·C), 131.9 (2·C), 134.0 (CH), 144.6 (2·CH),
159.4 (2·C); ESI+/MS: m/z 264 ([M+H]+, 100). 8 (Rf 0.33): 1H NMR
((CD3)2CO) d (ppm): 7.01 (2H, d, J = 8.8 Hz), 7.18 (1H, d,
J = 8.4 Hz), 7.63 (1H, dd, J = 8.4, 2.0 Hz), 7.68 (2H, d,
J = 8.8 Hz), 7.83 (1H, d, J = 2.0 Hz), 8.16 (1H, t, J = 2.0 Hz), 8.76
(1H, d, J = 2.0 Hz), 8.77 (1H, d, J = 2.0 Hz); 13C NMR (CD3OD) d
(ppm): 117.1 (2·CH), 118.2 (CH), 127.8 (CH), 129.4 (2·CH), 129.6
(CH), 130.9 (C), 131.2 (C), 131.4 (C), 131.9 (C), 133.4 (CH), 145.5
(CH), 146.1 (CH), 154.9 (C), 159.4 (C); ESI+/MS: m/z 300
([M+2+H]+, 33), 298 ([M+H]+, 100).
k Preparative TLC fractionation (eluent: cyclohexane–ethyl acetate
80:20 v/v). 9 (Rf 0.25): 1H NMR ((CD3)2CO) d (ppm): 7.48 (2H, t,
J = 7.2 Hz), 7.56 (4H, t, J = 7.2 Hz), 7.84 (4H, d, J = 7.2 Hz), 8.28
(1H, m), 8.89 (2H, m); 13C NMR ((CD3)2CO) d (ppm): 128.1 (4·CH),
129.3 (2·CH), 130.0 (4·CH), 134.4 (CH), 137.9 (2·C), 139.2 (2·C),
146.0 (2·CH); ESI+/MS: m/z 232 ([M+H]+, 100).

6460
L. Panzella et al. / Tetrahedron Letters 46 (2005) 6457–6460
trometric facilities and to Mrs. Silvana Corsani for tech-
nical assistance.
OH
HO
HO
N
7
References and notes
Cl
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OH
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8
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To our knowledge, this is the first example of one-pot
formation of 3,5-diarylpyridine derivatives by mild oxi-
dation of an a-amino acid with HOCl. Although the
yield of 2 is low, the reaction is nonetheless of prepara-
tive value because of the simple procedure and the ease
of isolation of the product in pure form (>98% NMR
analysis) without chromatographic steps. Pyridine 2
bears two nitro groups that may be conveniently modi-
fied thus offering a valuable option for the preparation
of structural variants of potential synthetic and pharma-
cological interest, for example, as inhibitory agents of
interleukin-6.19
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Commun. 1999, 258, 168–172.
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1953, 75, 4291–4296.
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Acknowledgements
This study was carried out in the frame of the MIUR
projects (ÔSostanze naturali ed analoghi sintetici con atti-
vita antitumoraleÕ, PRIN 2003). Thanks are also due to
the Centro Interdipartimentale di Metodologie Chimico
`
Fisiche of Naples University for NMR and mass spec-