An efficient synthesis of [13C6]-3,5-dichloroaniline

Article abstract of DOI:10.1002/jlcr.1521

3,5-Dichloroaniline is commonly found in many compounds with pharmacological and other biological activities. [¹³C ₆]-Aniline or its hydrochloride salt was converted in three steps to [¹³C₆]-3,5-dichloroaniline,

Full text of DOI:10.1002/jlcr.1521

Research Article
Received 19 February 2008,
Revised 27 March 2008,
Accepted 7 April 2008
Published online 6 June 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1521
An efficient synthesis of [13C6]-3,5-
dichloroaniline
Ã
Bachir Latli, Matt Hrapchak, Dhileepkumar Krishnamurthy, and Chris H.
Senanayake
3,5-Dichloroaniline is commonly found in many compounds with pharmacological and other biological activities. [13C6]-
Aniline or its hydrochloride salt was converted in three steps to [13C6]-3,5-dichloroaniline, which can be incorporated in
compounds of interest and used as internal standards in drug metabolism and pharmacokinetics (DMPK) studies.
Keywords: 3, 5-dichloroaniline; 1, 3-diamino-5-chlorobenzene; carbon-13; internal standards
2,4-Dichloroacetanilide was also subjected to nitration by
nitric acid:sulfuric acid mixture.10 In our hands the major product
Introduction
3,5-Dichloroaniline (3,5-DCA) is used in the production of a
number of products, including agricultural chemicals, azodyes
and pigments and in pharmaceuticals.
was 2,4-dicholoro-5-nitroacetanilide (78%). The desired 2,4-
dichloro-6-nitroacetanilide was obtained in only 18% yield and
it was difficult to separate it from the above isomer.
For example, dicarboximidic fungicides Vinclozolin and
Iprione, which are used on large scale on grapes, contain the
3,5-DCA moiety.1 Their common metabolite 3,5-DCA has been
used as a biomarker for exposure to these fungicides.2 It has
even been suggested that low-level exposure to 3,5-DCA may
be associated with adverse health effects such as endocrine
disruption and nephrotoxicity.3
In the pharmaceutical industry, the 3,5-DCA moiety is present
in several drug candidates, like the potent LFA-1-mediated cell
adhesion antagonists, which are being developed as potential
drugs to treat autoimmune diseases.4
Herein we report the synthesis of [13C6]-3,5-DCA starting from
the commercially available [13C6]-aniline. [13C6]-3,5-DCA is
incorporated in the synthesis of drug candidates and used as
an internal standard in the drug metabolism and pharmocoki-
netics (DMPK) studies, or it can be used as a biomarker for
biologically active compounds that contain this motif.
Another attempt that failed to give the desired product
included the chlorination of nitrobenzene. We reasoned that the
nitro group would direct the chlorination to the meta positions.
However, several chlorinating reagents and different conditions
failed to give the desired product.
A simple protection of the aniline as the hydrochloride salt is
actually sufficient to obtain 2,4,6-tricholoroaniline in very high
yields and without byproducts.11
Thus, the aniline was protected as the salt by treatment with
concentrated HCl in carbon tetrachloride. The salt can be easily
handled as a white solid in open air. Chlorination in carbon
tetrachloride with chlorine gas at 01C gave 2,4,6-trichloroaniline
in 98% yield. The chlorine should be bubbled slowly to the
suspension of the aniline hydrochloride salt. Deamination as
seen above and amino-dehalogenation gave the desired
3,5-dichloroaniline in quantitative yield (Scheme 1). We found
when excess lithium hexamethyldisilazane (LiHMDS) is used, 1,3-
diamino-5-chlorobenzene is isolated with yields up to 60%. The
triaminobenzene was not produced even with a large excess of
LiHMDS and heating for longer periods of time. Amination of
1,3,5-trichlorobenzene to 3,5-dichloroaniline was reported as
well using copper acetate and ammonia at 1201C in an
autoclave.12
Results and discussion
Direct chlorination of aniline with freshly crystallized N-
chlorosuccinimide (NCS) in either methanol or trifluroroacetic
acid gave 2,4,6-trichloroaniline in yields up to 80% after flash
chromatography purification. In order to improve the isolation,
aniline was protected as the acetanilide and treated with NCS.5
To our surprise and despite the large access of NCS over
extended periods of time up to 2 weeks, the only product
isolated was the 2,4-dichloroacetanilide in 98% yield. Deprotec-
tion of this acetanilide and bromination with freshly crystallized
Department of Chemical Development, Boehringer Ingelheim Pharmaceuticals,
Inc. Research and Development Center, 900 Ridgebury Road, P.O. Box 368,
NBS gave 2-bromo-4,6-dichloroaniline in 99% yield.6 Deamina- Ridgefield, CT 06877-0368, USA
tion via the diazonium salt using either hypophosphorous acid,
*Correspondence to: Bachir Latli, Department of Chemical Development,
or ferrous sulfate in dimethyl formamide gave 1-bromo-3,5-
dichlorobenzene.6,7 Amination according to Hartwig or Buch-
wald procedures gave 3,5-dichloroanilne in 85% yield.8,9
Boehringer Ingelheim Pharmaceuticals, Inc. Research and Development Center,
900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877-0368, USA.
E-mail: blatli@rdg.boehringer-ingelheim.com
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Copyright r 2008 John Wiley & Sons, Ltd.

B. Latli et al.
Scheme 1. Synthesis of [13C6]-3,5-DCA from [13C6]-aniline.
127.60(dt, J = 8.01, 66.64 Hz, C3), 121.81(dt, J = 5.15, 66.64 Hz, C4),
119.64 (dt, J = 4.08, 67.20 Hz, C2). LCMS: ESI: MH1 = 207 (100%).
This product can be also prepared directly from aniline. Thus,
a solution of the aniline-13C6 (2.0 g, 20.2 mmol) was dissolved in
CH3OH. Freshly crystallized NCS (12 g, 9.0 mmol) was added to
one portion at room temperature. The resulting dark mixture
was stirred in the dark at room temperature until no starting
material was detected (20 h). The dark mixture was concentrated
under reduced pressure and the residue was dissolved in
chloroform (50 ml) and washed with saturated NaHCO3 (200 ml),
dried over MgSO4, filtered and concentrated in vacuo to give
6.7 g of a brown solid. Purification by flash chromatography
Experimental procedures
Materials and methods
LC-MS spectra were acquired by a Hewlett-Packard auto sampler
Series 1100, connected to a Micromass Platform LCZ in the
electron spray positive ion mode using 95% to 5% water/
acetonitrile gradient (0.1% formic acid) for 3 min, and a
SunFireTM C18, 3.5 mm (4.6 Â 30 mm) column. NMR spectra were
recorded with a Bruker-Biospin DPX-400 spectrometer operating
at 400.13 MHz 1H frequency and at 100.66 MHz for 13C using
deuterated chloroform as a solvent and tetramethyl silane as the
internal standard unless stated otherwise. Silica gel TLC was
performed on pre-coated aluminum sheets with fluorescent
indicator (EM Separating, Gibbstown, NJ). All reagents were
obtained from Aldrich Chemical Co. (Milwaukee, WI).
Aniline-13C6 99 atom % 13C was purchased from Isotec
(Miamisburg, OH). Solvents were all of HPLC grade.
1
using CH2Cl2 gave 3.3 g of a solid material in 80% yield. HNMR
was identical to the product isolated from chlorine gas.
[13C6]-1,3,5-Tricholorobenzene
The above compound (1.6 g, 7.9 mmol) was dissolved slowly in a
solution of 70% H2SO4 (8 ml). The solution was then cooled in an ice-
bath to about À51C and a solution of NaNO2 (1.2 g, 17.4 mmol) in
water (2 ml) was added slowly over 30 min. After stirring at À5 to
01C for 30 min, a solution of H3PO2 (30% in water, 12 ml) was added
dropwise in 20 min period and the resulting mixture was warmed
slowly to room temperature and stirred for 12 h. The dark mixture
was poured into ice-cold water and the resulting solid was filtered to
give 1.4 g of a pink solid. Purification by flash chromatography using
Synthesis
[13C6]-2,4,6-Trichloroaniline
A solution of aniline-13C6 (1.0 g, 10.1 mmol) in CCl4 (5 ml) was
cooled to 01C in an ice-bath. Concentrated HCl (12 N, 1.5 ml) was
added dropwise to give a white solid. After stirring for one hour
at 01C, the solid was filtered, washed with CCl4 (2 Â 5 ml) and
dried under reduced pressure to give 1.42 g of a white solid. This
solid was suspended in carbon tetrachloride (12 ml) and
absolute ethanol (0.3 ml). Chlorine gas was bubbled slowly
through the suspension at room temperature to give a fluffy
yellow solid. After 30 min no starting material was detected by
1
CH2Cl2 as eluant gave 1.3 g of a white fluffy solid in 88% yield. H
NMR (CDCl3): d7.27 (dm, J1H-13C = 172.01 Hz, 3H). 13C NMR (CDCl3):
d135.54 (dt, J= 6.65, 67.83 Hz, C1), 127.18 (dt, J= 8.71, 65.01 Hz, C2).
LCMS: ESI: MH1 = 189 (100%).
[13C6]-3,5-Dichloroaniline
LC. Water (25 ml) was added to the mixture and stirred for A mixture of the above trichlorobenzene (0.19 g, 1.01 mmol)
15 min. The solid was filtered, washed with water (25 ml), and Pd2(dba)3 (27 mg, 0.03 mmol), P(t-Bu)3 (10 ml, 0.033 mmol, 90%
dried under reduced pressure for 12 h to give 1.98 g of an off- tech.) in toluene (5 ml) was degassed and nitrogen was
white solid with 98% purity as judged from LC-MS and in 98% introduced. A solution of LiHMDS (1.0 M in THF, 1.1 ml) was
1
yield. H NMR (CDCl3): d7.12 (dm, J1H-13C = 172.22 Hz, 2H), 4.43 added over 10 min and the mixture was heated to 851C and
(brs, NH2). 13C NMR (CDCl3): d139.02(dt, J = 3.3, 69.96 Hz, C1), stirred for 12 h. After cooling to room temperature, aqueous HCl
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J. Label Compd. Radiopharm 2008, 51 283–285

B. Latli et al.
(1.0 N, 1.1 ml) was added slowly and the mixture was stirred for a introduced in the synthesis of drug candidates and used as an
few minutes. The mixture was then extracted with ether and the internal standard in DMPK studies or as a biomarker for
combined extracts were washed with aqueous NaOH (1.0 M, compounds containing this motif.
50 ml), dried over MgSO4, filtered and concentrated in vacuo to
give 0.24 g of a dark solid. Purification by flash chromatography
using 1–10% CH3OH/CH2Cl2 gave 150 mg of an off-white solid in
87% yield. For larger quantities the product can be purified by
sublimation. 1H NMR (CDCl3): d6.72 (dm, J1H-13C = 172.87 Hz,
1H), 6.54 (dm, J1H-13C = 172.87 Hz, 2H), 3.78 (brs, NH2). 13C NMR
(CDCl3): d148.19 (dt, J = 4.10, 62.60 Hz, C1), 135.40 (dt, J = 4.10,
66.27 Hz, C3), 118.26 (dt, J = 4.26, 66.27 Hz, C4), 113.20 (dt,
J = 4.26, 62.60 Hz, C2). LCMS: ESI: MH1 = 168.35 and 209.42
(MeCN adduct, 100%).
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[13C6]-1,3,5-Trichlorobenzene (195 mg, 1.04 mmol), Pd2(dba)3
(55 mg, 0.06 mmol), P(t-Bu)3 (20 ml, 0.066 mmol, 90% tech.) in
toluene (5 ml) was degassed before nitrogen was introduced. A
solution of LiHMDS (1.0 M in THF, 2.2 ml) was added over 20 min
and the mixture was heated to 851C and stirred for 14 h. Work
up as seen before and purification by flash chromatography
using 1–10% CH3OH/CH2Cl2 gave 90 mg of a yellow solid in 60%
yield of the desired product. 1H NMR (CDCl3): d6.09 (dm,
J1H-13C = 167.87 Hz, 2H), 5.86 (dm, J1H-13C = 154.40 Hz, 1H), 3.61
(brs, 4H, 2NH2). 13C NMR (CDCl3): d148.32 (dt, J = 3.85, 66.12 Hz,
C1 and C3), 135.50 (dt, J = 5.16, 69.60 Hz, C5), 105.85
(t, J = 68.00 Hz, C4 and C6), 99.64 (t, J = 66.00 Hz, C2). LCMS: ESI:
MH1 = 149.46 (100%).
Conclusion
[13C6]-3,5-Dichloroaniline is prepared from the commercially
available [13C6]-aniline or its hydrochloride salt in a three step
synthesis and in 75% overall yield. This moiety can be [12] J. Ulrich, S. Horst, DE 2720316 A1, BASF, 1977.
J. Label Compd. Radiopharm 2008, 51 283–285
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