Convenient synthesis of D-threo-[dichloroacetyl 1-14C] chloramphenicol

Article abstract of DOI:10.1002/jlcr.932

A convenient synthesis of chloramphenicol labelled with carbon-14 in the dichloroacetyl group at the 1 position is described. It was prepared as part of a 4-step sequence from [1-¹⁴C] glycine and the product was purified by preparative HPLC. A radiochemical yield of 47% was obtained based on [1- ¹⁴C] glycine and the product had a specific activity of 0.47 mCi/mmol. The procedure can be employed for the synthesis of high specific activity [¹⁴C] chloramphenicol, labelled at 1, 2 or both the positions of dichloroacetyl group. Copyright

Full text of DOI:10.1002/jlcr.932

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 597–604.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.932
Research Article
Convenient synthesis of d-threo-[dichloroacetyl 1–14C]
chloramphenicol
C.V. Sontakke, S. P. Patil, V. K. P. Unny* and N. Sivaprasad
Labelled Compounds, Immunoassay, Labelled Compounds & Jonaki, Board of Radiation
and Isotope Technology, Vashi Complex, Navi Mumbai 400705, India
Summary
A convenient synthesis of chloramphenicol labelled with carbon-14 in the
dichloroacetyl group at the 1 position is described. It was prepared as part of a 4-
step sequence from [1-14C] glycine and the product was purified by preparative HPLC.
A radiochemical yield of 47% was obtained based on [1-14C] glycine and the product
had a specific activity of 0.47 mCi/mmol. The procedure can be employed for the
synthesis of high specific activity [14C] chloramphenicol, labelled at 1, 2 or both the
positions of dichloroacetyl group. Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: ethyl [1-14C] diazoacetate; ethyl [1-14C] dichloroacetate; d-threo-
[dichloroacetyl 1-14C] chloramphenicol
Introduction
The broad-spectrum antibiotic, chloramphenicol, was isolated from Strepto-
myces venezuelae in 1947.1 It had a wide spectrum of activity only in its d-threo
configuration against gram positive and gram negative bacteria, many anaerobic
bacteria, chlamydia and Rickettsia.2 It is widely used to treat typhoid, dysentery
and bacterial infection of the eye. Although this drug was commonly used
earlier, it is now reserved for microbial infections that are resistant to antibiotics
that have fewer side effects. Chloramphenicol is primarily bacteriostatic. It binds
to the 50S subunit of the 70S ribosomes and inhibits bacterial protein synthesis.
Quantitative measurement of transgenic expression is necessary to study the
mechanism of eucaryotic genetic expression.3,4 One way to study this is by use
of the gene, expressing the enzyme – chloramphenicol acetyl transferase (CAT).
When encoded by a bacterial drug-resistance gene, it inactivates chloramphe-
nicol by acetylating the drug at one or both of its hydroxyl groups. This gene is
*Correspondence to: V. K. P. Unny, LC/ILCJ, Board of Radiation and Isotope Technology, BARC Vashi
Complex, Navi Mumbai 400 705, India. E-mail: vkpunny@rediffmail.com
Copyright # 2005 John Wiley & Sons, Ltd.
Received 28 September 2004
Revised 11 October 2004
Accepted 31 January 2005

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C. V. SONTAKKE ET AL.
not found in eucaryotes and therefore eucaryotic cells contain little or no
chloramphenicol acetyl transferase activity. Enzyme activity being low, its assay
requires labelled chloramphenicol tagged with either carbon-14 or tritium.
Chloramphenicol acetyl transferase activity in transfected cells is monitored
by liquid scintillation counting (LSC) or thin-layer chromatography (TLC).5
Both LSC and TLC are performed in parallel from a single cell extract. Cell
3
extracts are incubated in a reaction mixture containing 14C- or H-labelled
chloramphenicol and n-butyryl coenzyme A. Chloramphenicol acetyl transfer-
ase transfers the n-butyryl moiety of the cofactor to chloramphenicol. After
organic extraction, the radiolabelled butyryl chloramphenicol is then detected
with either LSC or TLC followed by radiochromatogram scanning. Hence the
need to synthesize the corresponding carbon-14 version of chloramphenicol
arose. The preparation of chloramphenicol by condensing d-threo-2-amino-1-
p-nitrophenyl-1,3-propanediol (chloramphenicol base) with methyl dichloro-
acetate is one of the several methods described in the literature.6 Since the
synthesis of chloramphenicol base is somewhat complex,7–9 the labelled
analogue prepared by Iliceto resulted in a very low radiochemical yield.10
Carbon-14-labelled chloramphenicol, labelled in the dichloroacetyl portion of
the molecule, was prepared by Ise11 from malonic [2-14C] acid with a
radiochemical yield of 19.5%. Here, we present the preparation of d-threo-
[dichloroacetyl 1-14C] chloramphenicol 5 from [1-14C ] glycine 1 with the label
situated in the dichloroacetyl group at the 1 position. The preparative
procedure can very well be employed for the synthesis of high specific activity
d-threo-[dichloroacetyl 1-14C] chloramphenicol 5, at 1, 2 or both the positions
of the acetyl group. The procedure is efficient, easy to perform and the product
obtained has a comparatively good radiochemical yield. The product was
synthesized as part of a 4-stage sequence (Scheme 1).
O
O
N-
O
N+
NaNO2 / H2SO4
C2H5OH
*
2
*
O
H2N
HH2N
O
*
HCl
OH
(g )
Cl
3
1
Cl2 ( g )
CH2Cl2
OH
OH
OH
OH
O
abs. C2H5OH
reflux
Cl
O
NH
Cl
O-
*
NH2
O-
+
O
N+
*
N+
Cl
O
O
Cl
5
4
denotes position of 14C label
*
Scheme 1.
Copyright # 2005 John Wiley & Sons, Ltd.
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CONVENIENT SYNTHESIS OF D-
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[1-14C] glycine 1 on esterification was converted to [1-14C] glycine ethyl ester
hydrochloride12 2 which on diazotization formed ethyl [1-14C] diazoace-
tate13,14 3. Ethyl [1-14C] diazoacetate 3 on chlorination was converted to ethyl
[1-14C ] dichloroacetate15 4 which on condensation with chloramphenicol base
gave d-threo-[dichloroacetyl 1-14C] chloramphenicol 5. In our cold synthesis
on 2 mM scale, the reaction product was identified as chloramphenicol from its
Rf value by comparison with the authentic sample. The pure product was
obtained by preparative HPLC.16,17 Further confirmation was obtained by
determining the melting point along with the superimposable IR and UV
spectra with an authentic sample of the product.
Experimental
The radioactive starting material, [1-14C] glycine 1 was taken from ex-stock
which was prepared in-house by another group following a published
procedure.18 Chloramphenicol and chloramphenicol base were procured from
M/S Sigma. Other chemicals and solvents were obtained from M/S S.D. Fine
Chemicals, India and were of A.R. grade. Melting point of the pure product
obtained as well as the authentic sample of chloramphenicol was determined
with an Expo capillary melting point apparatus. The radioactivity was
determined using a LSS 4029 liquid scintillation counter supplied by
Electronics Division, BARC, India. The radiolabelled compound was
estimated using a Shimadzu UV-VIS spectrophotometer (Model UV – 265).
TLC analysis was performed on a silica gel 60F (E. Merck) plastic sheet. The
radiochromatograms were scanned with a Bioscan radiochromatogram
scanner (Model AR 2000). HPLC analysis was performed using a Dionex
HPLC system equipped with P680 HPLC pump and UVD 170U UV–detector.
IR–spectra were recorded on a PYE UNICAM infrared spectrophotometer
(Model SP3 – 300).
½1-14Cꢀ glycine ethyl ester hydrochloride 2
[1-14C] glycine 1 (1.0 mCi, 2 mmol, 150 mg) and 10 ml of absolute ethanol
were inserted in a long-necked Pyrex glass tube (15 mm OD and 20 cm long).
Dry hydrogen chloride gas was gently bubbled through the solution
while heating it at 65–708C in an oil-bath. Gas flow was continued for
10 min more after the crystals dissolved in the solution. The solvent was
removed in vacuo to give the title compound as a solid (0.98 mCi, 276 mg).
Radiochemical yield: 98%.
Ethyl [1-14C] diazoacetate 3
Ethyl [1-14C] diazoacetate 3 was prepared by using the specially designed
reactor/extractor apparatus where the temperature was controlled by
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C. V. SONTAKKE ET AL.
circulating cold water maintained at 108C. The apparatus was specially
fabricated so that the diazotization reaction and the subsequent extraction
could be performed in the same apparatus. To a cooled mixture of (0.98 mCi,
1.98 mmol, 276 mg) [1-14C] glycine ethyl ester hydrochloride 2, 260 mg
(3.7 mmol) of sodium nitrite, 8 mg (0.098 mmol) of sodium acetate, 5 ml of
dichloromethane and 10 ml of water, taken in the reactor/extractor, and 100 ml
of 10% aqueous sulphuric acid solution were added. The dichloromethane
layer was thoroughly equilibrated, separated and passed with mild suction
through a column (12 mm OD and 10 cm long) containing potassium
carbonate which was maintained at the same reaction temperature. The
aqueous layer in the reactor/extractor was subjected to diazotization which
was repeated twice using 50 ml of 10% sulphuric acid each time. The total
extract of dichloromethane (15 ml) obtained was used without purification for
the next reaction.
Ethyl [1-14C] dichloroacetate 4
The combined dichloromethane extract of ethyl [1-14C] diazoacetate 3 taken in
a 100 ml R.B. flask was cooled in an ice-bath. Dry chlorine gas was gently
bubbled through this solution until the yellow colour disappeared. Dichlor-
omethane was removed by distillation and the product was dissolved in 5 ml
absolute ethanol and assayed for radioactivity. 0.82 mCi (30.34 MBq) of the
product obtained was used without purification for the next reaction.
d-threo [dichloroacetyl 1–14C]– chloramphenicol 5
A mixture of chloramphenicol base (424 mg, 2 mmol) and ethyl [1-14C]
dichloroacetate 4 (0.82 mCi) in absolute ethanol (6.5 ml) was refluxed for 6 h.
The solution was allowed to cool to room temperature and ethanol was
removed in vacuo. The solid residue was dissolved in ethyl acetate (30 ml) and
washed with 1 N HCl (10 ml) followed by water (2 ꢁ 15 ml). The ethyl acetate
layer was again washed with 5% NaHCO3 (10 ml) followed by water
(2 ꢁ 15 ml) and then dried over anhydrous sodium sulphate. On concentrating
under reduced pressure, an oily residue resulted which was triturated with
diethyl ether to give the title compound. The compound was purified by
preparative HPLC using C-18 reverse-phase preparative column
(25 cm ꢁ 2.5 cm ) and methanol:water (37:63, v/v) as mobile phase at a flow
rate of 4 ml/min. UV-detector was set at 278 nm (retention time for
chloramphenicol=69.43 min). The compound was then analyzed by silica
gel TLC using the solvent system – chloroform:methanol (95:5). A single peak
in the TLC radiochromatogram scan was obtained corresponding to the
authentic sample. Radiochemical purity of the d-threo-[dichloroacetyl 1–14C]–
chloramphenicol 5 was estimated to be greater than 99%. The radiolabelled
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CONVENIENT SYNTHESIS OF D-
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compound was estimated spectrophotometrically at 278 nm and radioactivity
measured by liquid scintillation counter. The title compound (0.47 mCi,
0.99 mmol, 320 mg) was obtained with a radiochemical yield of 57.31%.
Specific activity:0.47 mCi/mmol. The overall radiochemical yield of the title
product based on [1-14C] glycine 1 was 47%.
Results and discussion
The main problem we faced was the low yield of ethyl diazoacetate which
subsequently led to the low yield of chloramphenicol. We used diethyl
ether for the extraction of ethyl diazoacetate after the diazotization of glycine
ethyl ester hydrochloride. Also the diazotization and extraction were
carried out in two different apparatus. Ethyl diazoacetate is reported to
decompose rapidly in the presence of acid. It is explosive and hence distillation
even under reduced pressure is risky and dangerous. Therefore, the product
should be used as soon as possible. Dichloromethane instead of diethyl
ether protects the ester from the excess acid used in the diazotization
reaction,19 so we decided to use dichloromethane instead of diethyl ether for
the extraction of ethyl diazoacetate and also for the next conversion to ethyl
dichloroacetate. The apparatus was so designed that the temperature could be
controlled by circulating cold water maintained at 108C. It was fabricated
so that the diazotization reaction and the subsequent extraction could be
performed in the same apparatus. With the above modifications, we could
successfully prepare labelled ethyl diazoacetate and subsequently chloram-
phenicol in good yield.
The prepared carbon–14-labelled chloramphenicol on analysis by silica gel
TLC using the solvent system – chloroform:methanol (95:5) followed by
autoradiography, showed three radioactive impurities along with radioactive
chloramphenicol. Purification of this crude radiolabelled chloramphenicol was
first tried by silica-gel column chromatography using the mobile phase
containing chloroform:methanol (90:10) but separation was not achieved.
Preparative HPLC purification was then tried following the analytical
procedure as reported16 using mobile phase 0.1 N sodium acetate and
acetonitrile (85:15) in which the impurities merged with the product and no
clear-cut separation was obtained as shown in Figure 1, whereas on using
methanol:water (37:63) as mobile phase at a flow rate of 4 ml/min and UV-
detector set at 278 nm and as followed by Zeegers and co-workers,17 we
successfully separated all the impurities and the radiochemically pure carbon–
14-labelled chloramphenicol was obtained. It was observed that in the first
HPLC purification process, the retention time for the impurities and
chloramphenicol was found to be more or less close by whereas in the latter
case good resolution was obtained as revealed by the vast difference in the
retention time of the impurities and that of chloramphenicol which was found
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C. V. SONTAKKE ET AL.
Figure 1. Preparative RP-HPLC chromatogram of the labelled chloramphenicol
using solvent system: 0.1 N sodium acetate/acetonitrile (85:15)
Figure 2. Preparative RP-HPLC chromatogram of the labelled chloramphenicol
using solvent system: methanol/water (37:63)
to have got shifted to 69.43 min from 31 min as shown in Figure 2. Figure 3
shows RP-HPLC chromatogram of pure labelled chloramphenicol obtained
on an analytical column.
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CONVENIENT SYNTHESIS OF D-
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Figure 3. Analytical RP-HPLC chromatogram of the labelled chloramphenicol
using solvent system: methanol/water (37:63)
Acknowledgements
The authors wish to thank Mr J. K. Ghosh, Chief Executive, BRIT for his
support and encouragement in the work. Thanks are also due to our
colleagues, Mr N. Jayachandran for his valuable assistance in the HPLC
analysis and Mr J. M. Gawas for fabricating the special apparatus for the
preparation and extraction of labelled ethyl diazoacetate.
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