(2-Ethyl-5-oxo-hexyl) Methylthiomethyl Phthalate as By-product of the Swern Oxidation: Improved Synthesis of Ring-deuterated Major Metabolites of Bis(2-ethylhexyl) Phthalate

Article abstract of DOI:10.1007/s00706-003-0044-y

Two ring-deuterated major metabolites of bis(2-ethylhexyl) phthalate were obtained in excellent yields by means of an improved oxidation procedure. The by-product of their interconversion by Swern oxidation is elucidated and a possible mechanism of its formation is proposed.

Full text of DOI:10.1007/s00706-003-0044-y

Monatshefte f€ur Chemie 134, 1207–1213 (2003)
DOI 10.1007/s00706-003-0044-y
(2-Ethyl-5-oxo-hexyl) Methylthiomethyl
Phthalate as By-product of the Swern
Oxidation: Improved Synthesis
of Ring-deuterated Major Metabolites
of Bis(2-ethylhexyl) Phthalate
Hans-Detlev Gilsing1;ꢀ, Ju¨rgen Angerer2, and Dietrich Prescher1
1
€
Institut fur Dunnschichttechnologie und Mikrosensorik e. V., D-14513 Teltow, Germany
Institut und Poliklinik fur Arbeits-, Sozial- und Umweltmedizin der Universitat Erlangen-Nurnberg,
€
2
€
€
€
D-91054 Erlangen, Germany
Received February 27, 2003; accepted March 3, 2003
Published online July 28, 2003 # Springer-Verlag 2003
Summary. Two ring-deuterated major metabolites of bis(2-ethylhexyl) phthalate were obtained in
excellent yields by means of an improved oxidation procedure. The by-product of their interconversion
by Swern oxidation is elucidated and a possible mechanism of its formation is proposed.
Keywords. Bis(2-ethylhexyl) phthalate; Metabolites; Isotopic labelling; Oxidations; Methylthio-
methyl esters.
Introduction
Phthalates are ubiquitous in the environment [1] due to their usage as additives to
poly(vinyl chloride) (PVC) and similar plastics, which serve as basic materials for
many products of everyday life. Recently the omnipresent body burden of the
general population with phthalates has been proven [2]. Persons undergoing hemo-
dialysis [3] and workers in PVC factories [4] are potentially stronger exposed to
phthalates than other parts of the population. Phthalic acid esters undergo biotrans-
formation in living systems [5] such as the majority of drugs do.
In order to reveal toxic pathways of the metabolisms of exogenous compounds
labelling of the metabolites with stable isotopes such as 13C or 2H has been estab-
lished as a versatile tool [6]. Stable isotope labelling also allows the quantification
of the metabolites in body fluids [7] by isotope dilution analysis [8]. The detection
requires sophisticated analytical methologies such as mass spectrometry linked to a
ꢀ
Corresponding author. E-mail: gilsing@idm-teltow.de

1208
H.-D. Gilsing et al.
Scheme 1
gas chromatograph (GC-MS) or to a liquid chromatograph (HPLC-MS) [9]. In
order to establish calibration curves the original compound must be available as
reference material and the stable isotopically labelled analog of the analyt is neces-
sary as internal standard. Recently some of us have developed a new powerful
method [10] of biological monitoring which allows the quantification of the two
major metabolites 1 and 2 of bis(2-ethylhexyl) phthalate (DEHP) in urine samples.
We also have developed a synthetic access to reference material of 1 and 2 [11]. A
little drawback of the procedure was that the conversion of 1 to 2 by Swern
oxidation worked only with moderate yields. We now report an improvement giv-
ing excellent yields of the ring-deuterated major metabolites 3 and 4 of DEHP
(Scheme 1).
Results and Discussion
The isotopically labelled ring positions were introduced by the usage of phthalic an-
hydride-d4 as starting material. The reaction with 5-(tert-butyldiphenylsilyloxy)-2-
ethyl-1-hexanol [11] gave the intermediate 5 wherein the protective group was
cleaved with tetrabutyl ammonium fluoride leading to almost quantitative yields
of the deuterated metabolite 3. From the synthesis of 2 it was known [11] that the
complete conversion of 1 by Swern oxidation demanded a tenfold excess of the
oxidizing agent, which indeed could not avoid the formation of side products.
Following these conditions the preparation of the deuterated metabolite 4 gave
as crude product the expected compound and a smaller amount of an additional
compound which was isolated by column chromatography using a nonpolar solvent
as eluent. The side product was identified as the ring-deuterated methylthiomethyl
(MTM) phthalate 6. This was derived from the absence of aromatic proton NMR
signals and the occurrence of two singlets at 2.3 and 5.4 ppm, which were in
agreement with the MTM ester group [12] and a singlet at 2.1 ppm indicating
the methyl group adjacent to the keto function. Further evidence for the diester
structure 6 was advanced by the absence of the carboxyl infrared absorption
(Scheme 2).
The repetition of the Swern oxidation of 1 to 2 as described in Ref. [11]
revealed after careful examination of the by-products that the analogous compound
7 was formed also from the unlabelled monophthalate in a comparable portion.
Although oxidations with activated DMSO can be accompanied with the MTM
esterification of a free carboxyl [13] several examples have been described [14]

Metabolites of Bis(2-ethylhexyl) Phthalate
1209
Scheme 2
wherein the acid group remains unaffected. The formation of the by-products 6 and
7 may proceed via the following steps (Scheme 3):
According to the mechanism commonly accepted [15] the Swern oxidation of 1
or 3 involves the formation of the ylide I from which the main products 2 or 4
result by elimination of dimethyl sulfide. To a smaller probability the ylide I
collapses under formation of the alkoxy ion II and the tautomeric methyl methy-
lenesulfonium ion III [16]. The species III is assumed to be an intermediate in the
Pummerer rearrangement [17]. Several mechanisms of the attack of III on the
carboxylate ion are discussed [18]. The alkoxy ion may remove a proton from
the carboxyl group to rebuild the alcohol function which generates the ylide IV
with a second sulfonium ion [19] in the presence of triethylamine. The formation
of 6 or 7 is completed as above.
Although MTM serves as a protective group for the carboxyl function [20]
especially in peptide synthesis [21] and several methods of smooth deprotection
have been developed [22] the selective cleavage of the MTM ester in the target
molecule may be difficult to achieve. We therefore used chromic acid [23] as
oxidizing agent for the conversion of 3 to 4. The reaction proceeded without the
formation of by-products although an excess of the oxidant was still necessary for
the complete conversion of 3. Optimized yields were obtained when 4 was purified
by extraction from acidified aqueous solution.
Experimental
IR spectra were recorded on a Perkin-Elmer 1430 spectrophotometer using the film technique. NMR
spectra were recorded on a Varian Gemini 300 spectrometer operating at 300 MHz for 1H and
75MHz for 13C. High resolution mass spectra were recorded on a VG 7070 double focusing mass
spectrometer using the CI and the EI mode. Elemental analyses were carried out using a CE
Instruments CHNS-Elemental Analyzer EA 1110. All new compounds gave satisfactory data of
elemental analysis or high resolution mass spectrometry. All reactions except the chromic acid
oxidation were carried out under an atmosphere of Ar. Phthalic acid anhydride-d4 (99.4%) was
purchased from Aldrich and used as received. Purifications by column chromatography were per-
formed on silica gel (Merck 63–200 mesh).
(2-Ethyl-5-oxo-hexyl) methylthiomethyl phthalate (7, C18H24O5S)
Compound 7 was prepared by repetition of the procedure described for the preparation of 2 [11] using
exactly the same amounts of each component. After work-up as described 7 was isolated from the

1210
H.-D. Gilsing et al.
Scheme 3
crude product mixture by column chromatography using ethyl acetate:heptane ¼ 1:5. Further elution
with ethyl acetate gave 0.26 g (52%) of 2. Colourless viscous oil; 0.15g (25%); 1H NMR (300 MHz,
DMSO-d6): ꢀ ¼ 0.87 (t, J ¼ 7.5 Hz, CH2CH3), 1.28–1.66 (m, CH2CHCH2), 2.06 (s, COCH3), 2.25 (s,
SCH3), 2.45 (m, CH2CO), 4.15 (d, J ¼ 5.4 Hz, CO2CH2), 5.38 (s, CO2CH2SCH3), 7.72 (m, ArH) ppm;
13C NMR (75 MHz, DMSO-d6): ꢀ ¼ 11.5, 15.5, 23.8, 24.7, 30.4, 38.3, 40.6, 67.9, 70.0, 129.4, 129.6,
131.5, 132.3, 132.6, 132.7, 167.1, 167.7, 209.0 ppm; IR (film): ꢁꢀ ¼ 2962, 2927, 2877, 1715, 1250,
1111, 1059, 921, 741 cmꢁ 1; HRMS (EI) [M]þ : calcd.: 352.1344, found: 352.1331.

Metabolites of Bis(2-ethylhexyl) Phthalate
1211
Mono[2-ethyl-5-(tert-butyldiphenylsilyloxy)hexyl] (3,4,5,6-2H4)-phthalate
(5, C32H36D4O5Si)
Phthalic acid anhydride-d4 (2.00 g, 13.1mmol) was added to a solution of 3.00g of 5-(tert-butyldi-
phenylsilyloxy)-2-ethyl-1-hexanol [11] (7.7 mmol) in 180 cm3 of anhydrous pyridine. The mixture was
stirred at room temperature for 72 h, and the solvent was evaporated. The residual oil was diluted with
diethyl ether, washed with aqueous HCl and H2O, and dried. After removal of the solvent the residue
was diluted with heptane and the solution was filtered. The solvent was evaporated and 5 was purified
by column chromatography (elution with ethyl acetate:heptane ¼ 5:1). Colourless viscous oil; 3.80g
1
(92%); H NMR (300MHz, DMSO-d6): ꢀ ¼ 0.80 (m, CH2CH3), 0.96 (s, C4H9), 1.01 (d, J ¼ 6.0 Hz,
CH3CHOSi), 1.20–1.58 (m, CH2CH2CHCH2), 3.80 (m, CHOSi), 4.04 (d, J ¼ 5.7 Hz, OCH2CH), 7.41–
7.61 (m, 10H, SiPh2), 13.10 (br s, CO2H) ppm; 13C NMR (75 MHz, CDCl3): ꢀ ¼ 10.8, 19.2, 23.1, 23.6,
26.0, 27.0, 36.3, 38.7, 68.2, 69.7, 127.4, 127.5, 129.4, 129.5, 130.2, 133.0, 134.4, 134.8, 135.8, 168.1,
172.0ppm; IR (film): ꢁꢀ ¼ 3071, 2962, 2931, 2858, 2595, 1729, 1699, 1428, 1240, 1110, 1075, 740,
701 cmꢁ 1; HRMS (NH4–NCl) [M–H]ꢁ : calcd.: 535.2819, found: 535.2807.
Mono(2-ethyl-5-hydroxyhexyl) (3,4,5,6-2H4)-phthalate (3, C16H18D4O5)
Tetrabutylammonium fluoride (33.5 cm3 of an 1M solution in THF, 33.5mmol) was added to a solution
of 3.60g of 5 (6.7mmol) in 160 cm3 of THF. The mixture was stirred at room temperature for 72 h and
then diluted with 650 cm3 of H2O containing 0.6 cm3 of concentrated aqueous HCl (7.2mmol). Product
3 was extracted with diethyl ether and dried over MgSO4. The solvent was evaporated and the residual
oil was diluted with 15 cm3 of methanol and then poured into 190 cm3 of an ice-cold aqueous solution
of 0.27 g of NaOH (6.7mmol). After stirring for 1 min the solution was extracted with 2 ꢂ 10cm3 of
diethyl ether, then acidified with 2.0 cm3 of concentrated aqueous HCl (24.0 mmol) and extracted with
4 ꢂ 10cm3 of diethyl ether. The combined extracts of the acidified solution were dried and 3 was
1
isolated by evaporation of the solvent. Colourless viscous oil; 1.90 g (95%); H NMR (300 MHz,
DMSO-d6): ꢀ ¼ 0.87 (t, J ¼ 7.5 Hz, CH2CH3), 1.03 (d, J ¼ 6.0 Hz, CH3CHOH), 1.20–1.48 (m,
CH2CH2CHCH2), 1.62 (m, CH2CH2CHCH2), 3.58 (m, CHOH), 4.12 (m, CO2CH2), 4.38 (br s,
OH), 13.10 (br s, CO2H) ppm; 13C NMR (75 MHz, DMSO-d6): ꢀ ¼ 10.8, 23.2, 23.7, 26.3, 36.0,
38.2, 66.0, 67.2, 132.2, 132.3, 167.6, 168.1ppm; IR (film): ꢁꢀ ¼ 3454, 2965, 2933, 2877, 2598,
1703, 1461, 1419, 1235, 1076, 931,768 cmꢁ 1; HRMS (NH4–Cl) [Mþ H]þ : calcd.: 299.1797, found:
299.1788.
(2-Ethyl-5-oxo-hexyl) methylthiomethyl (3,4,5,6-2H4)-phthalate (6, C18H20D4O5S)
A solution of 0.97cm3 of oxalyl chloride (11.2 mmol) in 25cm3 of CH2Cl2 was added dropwise to a
solution of 1.83 cm3 of DMSO (25.8 mmol) in 15 cm3 of CH2Cl2 at ꢁ65 ꢃC. After 5 min of stirring a
solution of 0.77 g of 3 (2.6mmol) in 15 cm3 of CH2Cl2 was added dropwise at the same temperature.
After stirring for 30min a solution of 4.29cm3 NEt3 (31.0mmol) was added dropwise at ꢁ65 ꢃC.
After additional stirring for 5 min the mixture was allowed to reach room temperature. The mixture
was poured into 600 cm3 of H2O containing 3.8 cm3 of concentrated aqueous HCl (45.6 mmol) and
the organic products were extracted with diethyl ether and dried over MgSO4. After evaporation of
the solvent 7 was isolated from the residue by column chromatography using ethyl acetate:heptane¼
1:5. Further elution with ethyl acetate gave 0.39 g (51%) of 4. Colourless viscous oil; 0.24g (26%);
1H NMR (300MHz, DMSO-d6): ꢀ ¼ 0.87 (t, J ¼ 7.2 Hz, CH2CH3), 1.34 (m, CH2CHCH2), 1.52 (m,
CH2CHCH2), 1.62 (m, CH2CHCH2), 2.07 (s, COCH3), 2.26 (s, CO2CH2SCH3), 2.45 (m, CH2CO),
4.16 (d, J ¼ 5.4 Hz, CO2CH2), 5.38 (s, CO2CH2S) ppm; 13C NMR (75 MHz, DMSO-d6): ꢀ ¼ 10.7,
14.7, 23.0, 23.9, 29.6, 37.6, 39.8, 67.1, 69.2, 127.4, 128.5, 129.1, 130.7, 131.8, 134.4, 166.4, 166.9,
208.2ppm; IR (film): ꢁꢀ ¼ 2961, 2927, 2877, 1716, 1216, 1057, 920, 756 cmꢁ 1; HRMS (NH4–Cl)
[Mþ H]þ : calcd.: 357.1674, found: 357.1665.

1212
H.-D. Gilsing et al.
Mono(2-ethyl-5-oxohexyl) (3,4,5,6-2H4)-phthalate (4, C16H16D4O5)
A solution of 0.65g of 3 (2.2mmol) in 100 cm3 of acetone was added over a period of 1 h to a solution
of 3.84g of K2Cr2O7 (13.1 mmol) and 2.32 cm3 of concentrated H2SO4 in 100 cm3 of H2O (43.4 mmol) at
0 ꢃC. After the addition was complete the mixture was allowed to reach room temperature and stirring
was continued for 4 h. The mixture was poured into 500 cm3 of H2O and 4 was extracted with diethyl
ether and dried over MgSO4. The solvent was evaporated and the residual oil was diluted with 2.0 cm3
of methanol and poured into 100 cm3 of an ice-cold aqueous solution of 0.09 g of NaOH (2.2mmol).
After stirring for 1 min the solution was extracted with diethyl ether, then acidified with 0.70cm3 of
concentrated aqueous HCl (8.4mmol), and extracted with diethyl ether. The combined extracts of the
acidified solution were dried and 4 was isolated by evaporation of the solvent. Colourless viscous
1
oil; 0.58g (89%); H NMR (300MHz, DMSO-d6): ꢀ ¼ 0.87 (t, J ¼ 7.5 Hz, CH2CH3), 1.20–1.68 (m,
CH2CHCH2), 2.06 (s, COCH3), 2.48 (t, J ¼ 6.6 Hz, CH2CO), 4.12 (d, J ¼ 5.4 Hz, CO2CH2), 13.20 (br
s, CO2H) ppm; 13C NMR (75 MHz, DMSO-d6): ꢀ ¼ 10.9, 23.2, 24.1, 29.7, 37.7, 40.0, 66.9, 132.1,
132.3, 167.7, 168.1, 208.4ppm; IR (film): ꢁꢀ ¼ 2963, 2934, 2878, 2597, 1712, 1421, 1236, 1074, 1019,
767 cmꢁ 1; HRMS (NH4–Cl) [Mþ H]þ : calcd.: 297.1640, found: 297.1637.
Acknowledgements
The authors thank Dr. M. Bartoszek, Dr. D. M€uller, H. Gehrmann, and G. Seidlitzki, ACA Berlin, for
recording the HRMS and NMR spectra and performing the elemental analyses and E. Tews for
conducting the preparative work. Financial support by the Deutsche Forschungsgemeinschaft under
AN107=16-1 is gratefully acknowledged.
References
[1] a) Tomita I, Nakamura Y, Yagi Y (1977) Ecotoxicol Environ Saf 1: 275; b) Griffiths WC,
Camara P, Lerner KS (1985) Ann Clin Lab Sci 15: 140; c) Wams TJ (1987) Sci Total Environ 66:
1; d) Allen B (1999) Green Chem 1: G102; e) Petersen JH, Breindahl T (2000) Food Addit
Contam 17: 133; f) Butte W, Hoffmann W, Hostrup O, Schmidt A, Walker G (2001) Gefahrst
Reinh Luft 61: 19
[2] a) Blount BC, Silva MJ, Claudill SP, Needham LL, Pirkle JL, Sampson EJ, Lucier GW, Jackson
RJ, Brock JW (2000) Environ Health Perspect 108: 979; b) Koch HM, Drexler H, Angerer J
(2003) Int J Hyg Environ Health 206: 77
[3] a) Faouzi MA, Dine T, Gressier B, Kambia K, Luyckx M, Pagniez D, Brunet C, Cazin M,
Belabed A, Cazin JC (1999) Int J Pharm 180: 113; b) Tickner JA, Schettler T, Guidotti T,
McCally M, Rossi M (2001) Am J Ind Med 39: 100; c) Kambia K, Dine T, Azar R, Gressier B,
Luyckx M, Brunet C (2001) Int J Pharm 229: 139
[4] Dirven HAAM, van den Broek PHH, Arends AMM, Nordkamp HH, de Lepper AJGM,
Henderson PT, Jongeneelen FJ (1993) Int Arch Occup Environ Health 64: 549
[5] a) Woodward KN, Smith AM, Mariscotti SP, Tomlinson NJ (1986) Review of the Toxicity of the
Esters of o-Phthalic Acid (Phthalate Esters). HMSO, London, p 159; b) Woodward KN (1988)
Phthalate Esters: Toxicity and Metabolism, vol 1, CRC Press, Boca Raton, p 47; c) Woodward
KN (1990) Hum Exp Toxicol 9: 397
[6] a) Albro PW, Hass JR, Peck CC, Jordan ST, Corbett JT, Schroeder J (1982) J Environ Sci Health
B 17: 701; b) Pohl LR, Gillette JR (1985) Drug Metab Rev 15: 1335; c) Cronholm T (1989)
Stable Isotopes as Metabolic Tracers. In: Baillie TA, Jones JR (eds) Synthesis and Applications
of Isotopically Labelled Compounds 1988. Elsevier, Amsterdam, p 1
[7] a) Pyron KH, Kracko DA, Strunk MR, Bechtold WE, Dahl AR, Lewis JL (1997) J Anal Toxicol
21: 363; b) Blount BC, Milgram KE, Silva MJ, Malek NA, Reidy JA, Needham LL, Brock JW
(2000) Anal Chem 72: 4127

Metabolites of Bis(2-ethylhexyl) Phthalate
1213
€
[8] a) Tolgyessy J, Braun T, Kyrs M (1972) Isotope Dilution Analysis. Pergamon Press, Oxford,
p 155; b) Yoshioka H (1995) Isotope Dilution Analysis. In: Townshend A (ed) Encyclopedia of
Analytical Science, vol 4, Academic Press, New York, p 2399
[9] Siekmann L (1989) Reference Methods. In: Lawson AM (ed) Mass Spectrometry. de Gruyter,
Berlin, p 648
[10] Koch HM, Gonzalez-Reche LM, Angerer J (2003) J Chromatogr B 784: 169
[11] Gilsing H-D, Angerer J, Prescher D (2002) Monatsh Chem 133: 1147
[12] Kukla MJ (1982) Tetrahedron Lett 23: 4539
[13] Herdewijn P, Claes PJ, Vanderhaeghe H (1982) Can J Chem 60: 2903
[14] a)Roberts SM, Woolley GT, Newton RF (1981) JChem SocPerkin Trans I 1729; b) Meth-Cohn O,
Reason AJ, Roberts SM (1982) J Chem Soc Chem Commun 90; c) Lowe G, Swain S (1983)
J Chem Soc Chem Commun 1279
[15] a) Omura K, Swern D (1978) Tetrahedron 34: 1651; b) Mancuso AJ, Swern D (1981) Synthesis
165; c) Tidwell TT (1990) Synthesis 857
[16] a) Marx M, Tidwell TT (1984) J Org Chem 49: 788; b) Tidwell TT (1990) Org React 39: 297
[17] a) Parham WE, Bhavsar MD (1963) J Org Chem 28: 2686; b) Parham WE, Groen SH (1965)
J Org Chem 30: 728
[18] a) Johnson CR, Phillips WG (1969) JAm Chem Soc 91: 682; b) Lerch U, Moffat JG (1971) J Org
Chem 36: 3391; c) Itoh O, Numata T, Yoshimura T, Oae S (1983) Bull Chem Soc Jpn 56: 266;
d) Oae S, Itoh O, Numata T, Yoshimura T (1983) Bull Chem Soc Jpn 56: 270
[19] Mancuso AJ, Huang S-L, Swern D (1978) J Org Chem 43: 2480
[20] a) Ho T-L, Wong CM (1973) J Chem Soc Chem Commun 224; b) Wade LG, Gerdes JM, Wirth
RP (1978) Tetrahedron Lett 731; c) Ho T-L (1979) Synth Commun 9: 267; d) Dossena A,
Marchelli R, Casnati G (1979) J Chem Soc Chem Commun 370; e) Dossena A, Marchelli R,
Casnati G (1981) J Chem Soc Perkin Trans I, 2737; f) Liu J-S, Tao Y (1992) Tetrahedron 48:
6793
[21] a) Dossena A, Palla G, Marchelli R, Lodi T (1984) Int J Peptide Protein Res 23: 198; b) Mora N,
Lacombe JM, Pavia AA (1993) Tetrahedron Lett 34: 2461
[22] a) Gerdes JM, Wade LG (1979) Tetrahedron Lett 689; b) Kim S, Park YH, Kee IS (1991)
Tetrahedron Lett 32: 3099
[23] Hussey AS, Baker RH (1960) J Org Chem 25: 1434