An efficient synthesis of [D6]leucocrystal violet

Article abstract of DOI:10.1002/jlcr.1849

This report presents an efficient synthesis of [D6]leucocrystal violet. Esterification of p-toluenesulphonyl chloride with [D4]methanol provided [D ₃]methyl p-toluenesulphonate, which was used to methylate aniline to give [D₆]N, N-di

Full text of DOI:10.1002/jlcr.1849

Research Article
Received 3 September 2010,
Revised 2 October 2010,
Accepted 13 October 2010
Published online 30 November 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1849
An efficient synthesis of [D6]leucocrystal
violet
Ã
Weicheng Yang,a Yong Luo,a Weixia Liu,a Xiaojun Deng,b Xiaoning Du,a
and Meihua Lia
This report presents an efficient synthesis of [D6]leucocrystal violet. Esterification of p-toluenesulphonyl chloride with
[D4]methanol provided [D3]methyl p-toluenesulphonate, which was used to methylate aniline to give [D6]N,N-
dimethylaniline. Condensation of [D6]N,N-dimethylaniline with bis[4-(dimethylamino)phenyl]methanol afforded [D6]leuco-
crystal violet in an overall yield of 30% in three steps.
Keywords: [D6]leucocrystal violet; synthesis; labelled internal standards
(17.46 g, 0.44 mol ) and [D4]methanol (4.52 g, 0.125 mol) in a
two-phase system of water (50 mL) and THF (50 mL) was added
Introduction
Crystal violet (CV) is a well known and inexpensive dye effective
against fungal and parasite infection in fish.1 Similar to malachite,
crystal violet is readily absorbed into fish tissue in an aquatic
environment and is reduced metabolically by fish to its persistent
leuco moiety, leucocrystal violet (LCV).2,3 Owing to its potential
carcinogenic, genotoxic, mutagenic and teratogenic properties in
many animal species and cell lines,4 CV has never been registered
as a veterinary drug in any nation. Numerous methods have been
developed for the determination of CV and its metabolite LCV in
fish.1,5–8 Liquid chromatography isotope diluted tandem mass
spectrometry (LC-IDMS/MS) methods proved sensitive and
reliable among various analytical approaches.9
Reports of the synthesis of labelled leucocrystal violet are very
rare. In this study, we describe an efficient synthesis of
[D6]leucocrystal violet starting from commercially available
[D4]methanol. The target compound, with excellent isotopic
and chemical purities, was synthesized in acceptable yield and
could be used as an internal standard in the determination of
LCV in aquatic products.
dropwise a solution with p-toluenesulphonyl chloride (47.60 g,
0.25 mol) in tetrahydrofuran (80 mL) at ice bath temperature.
The reaction solution was kept at À5 to 01C for another 6.5 h
and then poured into ice-water. The mixture was extracted with
CH2Cl2 (500 ml  3) and the combined organic layers were
washed with water. After drying over MgSO4, the solvent was
evaporated in vacuo to give 23.2 g of pure product as a
colourless oil in 98% yield based on labelled substrate
consumed. LC-MS-ESI1: [M1H]1 = 190.2 (100%).
[D6]N,N-Dimethylaniline 2
To a mixture of [D3]methyl p-toluenesulphonate (22.65 g,
0.12 mol) and aniline (5.18 g, 0.056 mol) was added a solution
of 30% NaOH solution (30 mL), followed by the addition of
phase transfer catalyst, tetrabutylammonium bromide (0.75 g,
2.3 mmol). The reaction proceeded for 4 h at 701C, then the
reaction mixture was cooled to room temperature, the mixture
was extracted with diethyl ether (50 ml  3) and the combined
extracts were dried over MgSO4 and evaporated. The crude
product was purified by chromatography on silica gel column
(n-hexane: ethyl acetate: triethylamine = 25:1:0.05) to give 4.73 g
of yellow oil [D6]N,N-dimethylaniline 2 in 62% yield. LC-MS-ESI1:
[M1H]1 = 128.37(100%).
Experiment
Materials and instruments
[D4]Methanol (99.8 atom% D) was provided by J&K Chemical. All
1
other chemicals were of analytical grade. H-NMR (500.13 MHz)
[D6]Leucocrystal violet 3
and 13C-NMR (125.70 MHz) spectra were recorded on a Bruker
DRX500 spectrometer in CD3Cl (TMS as internal standard). FT-IR
spectra were recorded on a Nicolet FT-IR 6700 spectrometer
using KBr pellets. EI-MS spectra were obtained with TSQ
Quantum Access spectrometer.
[D6]Leucocrystal violet (3) was obtained by the modification of a
known procedure.11 Bis[4-(dimethylamino)phenyl]methanol
(13.52 g, 0.05 mol), [D6]N,N-dimethylaniline (3.18 g, 0.025 mol)
aShanghai Research Institute of Chemical Industry, Shanghai, China
Synthesis
bShanghai Exit and Entry Inspection and Quarantine Bureau, Shanghai, China
[D3]Methyl p-toluenesulphonate 1
*Correspondence to: Yong Luo, Shanghai Research Institute of Chemical
Industry, China.
[D3]methyl p-toluenesulphonate (1) was obtained by the
modification of a previous procedure.10 To a mixture of NaOH E-mail: real-luoyong@hotmail.com
J. Label Compd. Radiopharm 2011, 54 211–213
Copyright r 2010 John Wiley & Sons, Ltd.

W. Yang et al.
Scheme 1. Synthesis of [D6]leucocrystal violet.
and p-toluenesulphonic acid (0.75 g, 4.35 mmol) were heated [D4]methanol with p-toluenesulphonyl chloride in which the
under reflux in toluene (60 mL) for 12 h. After washing with yield of [D3]methyl p-toluenesulphonate (1) was 98% based on
water and removal of toluene in vacuo, the residue was treated [D4]methanol consumed. The alkylation reactions of amines
with acetone to give 4.754 g of colourless crystals in 50% yield. were often carried out under high temperature in the previous
The product obtained showed 99.4% chemically pure and more literature.12–14 We found that [D6]N,N-dimethylaniline (2) could
than 99% isotopically enrichment on the basis of HPLC ananlysis be synthesized in a satisfying 62% yield under mild reaction
and mass spectrometry, respectively. 1H NMR (CDCl3)d: 2.89 conditions by using phase transfer catalysis. The as-synthesized
(s,6H); 5.28 (s,1H); 6.64–7.23 (m,12H); 13C NMR (CDCl3)d: [D6]N,N-dimethylaniline (2) could then be converted to [D6]leu-
40.1(s, 6C); 54.1(s, 1C); 112.6(s, 4C); 112.5(s, 2C); 129.9(m, 6C); cocrystal violet (3) with bis[4-(dimethylamino)- phenyl]methanol
133.8 (s, 3C); 148.8(s, 3C); FT-IR (KBr) cmÀ1: 2879.8, 2792.6, 1612.8, in the presence of p-toluenesulphonic acid in an overall yield of
1563.5, 1479.6, 1442.5, 809.9; LC-MS-ESI1:[M1H]1 = 380.3 (100%). 30% in three steps. The above syntheses could be easily
modified for the preparation of labelled triphenylmethane dyes,
Results and discussion
e.g. [D6]crystal violet in which [D6]N,N-dimethylaniline reacts
with bis(4-dimethylamino)benzophenone.15
Although the manufacture of leucocrystal violet is fairly
common on an industrial scale, reports of the preparation of
isotopically labelled leucocrystal violet and its analogues have
not been found in the literature. Herein, we report a new
synthetic method with satisfying labelling yields, starting from
[D4]methanol.
N,N-dimethylaniline is a versatile and important building
block for many triphenylmethane dyes. The synthesis of labelled
N,N-dimethylaniline is of importance for corresponding labelled
analogues. Our approach to the synthesis of [D6]leucocrystal
violet (3) is summarized in Scheme 1. (2), as the key intermediate
in this synthesis, was obtained in two steps. The isotopic label
was introduced at the first stage by the treatment of
Acknowledgement
This research was supported by the research grant (2009IK134)
from General Administration of Quality Supervision, Inspection
and Quarantine, People’s Republic of China.
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