Coupling of biologically active steroids to conjugating arms through ether linkages for use in immunochemistry

Article abstract of DOI:10.1016/S0039-128X(01)00134-9

Conjugation of haptens through ether linkages avoids leakage problems in immunoassays, but this procedure is not easily applied to most steroids that bear low reacting hydroxyls. A new technique allowing the ether coupling of biologically active steroids with conjugating arms in mild conditions compatible with thermosensitive protecting groups is presented. In the first step, the solvent (an aromatic hydrocarbon) was dehydrated by azeotropic distillation in a soxhlet apparatus using a cartridge filled with 0.3 nm and 0.4 nm molecular sieves. In this protected medium, a thallium steroid alkoxide was completely formed by reaction of the steroid with thallium ethoxide and by the continuous elimination of ethanol. The halogenated chain was then introduced into the same medium and reacted in the absence of moisture to give the ether. 17β-Hydroxy and 11α-hydroxy derivatives were involved in this reaction. The coupling was effective for all of the compounds tested after 2-36 h of reaction time and at temperatures between 80 and 140°C. The conjugates were at least 95% pure, and yields ranged from 15 to 95%.

Full text of DOI:10.1016/S0039-128X(01)00134-9

Steroids 67 (2002) 71–75
Coupling of biologically active steroids to conjugating arms through
ether linkages for use in immunochemistry
Michel J. Kohl1, Robert G. Lejeune*1
Universite´ de Lie`ge, Institut de Pharmacie, Service de Chimie Analytique, rue Fusch, 3, B-4000 Lie`ge, Belgium
Received 26 June 2000; received in revised form 7 May 2001; accepted 24 May 2001
Abstract
Conjugation of haptens through ether linkages avoids leakage problems in immunoassays, but this procedure is not easily applied to most
steroids that bear low reacting hydroxyls. A new technique allowing the ether coupling of biologically active steroids with conjugating arms
in mild conditions compatible with thermosensitive protecting groups is presented. In the first step, the solvent (an aromatic hydrocarbon)
was dehydrated by azeotropic distillation in a soxhlet apparatus using a cartridge filled with 0.3 nm and 0.4 nm molecular sieves. In this
protected medium, a thallium steroid alkoxide was completely formed by reaction of the steroid with thallium ethoxide and by the
continuous elimination of ethanol. The halogenated chain was then introduced into the same medium and reacted in the absence of moisture
to give the ether. 17␤-Hydroxy and 11␣-hydroxy derivatives were involved in this reaction. The coupling was effective for all of the
compounds tested after 2–36 h of reaction time and at temperatures between 80 and 140°C. The conjugates were at least 95% pure, and
yields ranged from 15 to 95%. © 2002 Elsevier Science Inc. All rights reserved.
Keywords: Ether linkages; Low reacting hydroxyls; Steroid conjugates; Thermosensitive compounds; Synthesis
1. Introduction
in situ by reaction of the steroid and thallium ethoxide [4].
Unfortunately, this method has never been applied to ste-
roids as 4-pregnen-11␣-ol-3,20-dione (11␣-hydroxypro-
gesterone), 4-androsten-17␤-ol-3-one (testosterone), 4-es-
tren-17␤-ol-3-one (nandrolone) and 1,3,5(10)estratrien-
3,17␤-diol (estradiol) are involved in the control of the
fertility and the metabolism. Other well-proven methods for
ether synthesis involving phase-transfer or bis [acetylaceto-
nato]nickel or silver trifluoromethanesulfonate catalysis
have been no more successfully applied to these same ste-
roids [5–7].
In a previous work, conjugates of steroids to penicillins
through ester linkage were prepared for use in homogeneous
immunoassays, but these bonds are very sensitive to chem-
ical or enzymatic hydrolysis [1]. Ether linkages are suited to
considerably reduce these leakage problems in immunoas-
says [2] but attempts at synthesizing ether bonds using the
classic approach failed due to low reactivity and thermo-
sensitivity of the reagents involved in this synthesis proce-
dure. Nevertheless, the advantages resulting from stable
steroid conjugates prompted us to investigate more efficient
synthesis routes.
The aim of this work was to set up a synthesis route that
combined mild reaction conditions to preserve thermosen-
sitive reagents from degradation and a strong reactivity to
allow for the conjugation of low reacting compounds.
Ether formation is usually performed according to Wil-
liamson: an alcohol is reacted with a halogenated derivative
[3]. Thallium I is claimed to accelerate the reaction rates and
is often introduced as a thallium steroid alkoxide prepared
2. Experimental
* Corresponding author. Tel.: ϩ32-4-366-43-26; fax: ϩ32-4-366-43-
17.
E-mail address: rlejeune@ulg.ac.be (R.G. Lejeune).
1 Present address: Universite´ de Lie`ge, Institut de Pharmacie, Service
de Chimie Analytique, CHU, Tour 4, Niveau ϩ2 (Baˆtiment B 36), avenue
de l’Hoˆpital 1, Sart-Tilman, B-4000 Lie`ge, Belgique.
All reagents were of analytical or biochemical grade
products purchased from Aldrich Chemical Company (Mil-
waukee, Wisconsin, USA), from Steraloids (Wilton, NH,
USA), or from Merck (E. Merck, Darmstadt, Germany).
Columns filled with Silica gel 60 (230–400 mesh
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(01)00134-9

72
M.J. Kolh, R.G. Lejeune / Steroids 67 (2002) 71–75
ASTM) from Merck (E. Merck, Darmstadt, Germany) or
plates Alugram௡ Sil6/UV254 from Macherey-Nagel (Mach-
erey-Nagel, Du¨ren, Germany) were used for preparative and
analytical chromatography.
Purity was estimated by thin layer chromatography.
Equal volumes of the sample, twenty-fold and hundred-fold
dilutions of the sample were deposited on the plate. After
migration, the spots of the compound and the impurities
were visually observed under a U-V lamp at 254 nm. The
intensity of impurity spots in the sample were compared to
the intensity of the spots of the main compound after twen-
ty-fold and hundred-fold dilutions.
Mass spectra (MS) were obtained by electrospray ion-
ization (ES) on a VG platform Fisons mass spectrometer
[solvent:acetonitrile/water (50:50 v/v)] (Danvers, MA,
USA). Infrared (IR) spectra of solutions (0.1% in tetrachlor-
ethylene - pathlength: 500 ␮m) or pellets (0.4% dispersion
in KBr) were recorded on a Perkin Elmer spectrum 2000
FTIR spectrophotometer (Beaconsfield, Bucks, England).
Melting points were obtained on a Bu¨chi B 530 apparatus
(Flawil, Switzerland), and analytical data on a Carlo-Erba
EA 1108 analyser (Milano, Italy).
2.1.3. t-Butyl 3-[4-(2-bromoethoxy)-3-chlorophenyl]-5-
methylisoxazole-4-carboxylate [3]
Refrigerated solutions (Ϫ 5°C) of 7 g (26.6 mmol) of [2]
in acetonitrile (100 ml) and 5.89 g (30 mmol) of t-butyl
acetoacetate potassium enolate in acetonitrile (60 ml) were
slowly mixed and incubated overnight at the same temper-
ature. The mixture was acidified with 0.01 N HCl to pH 6
and then extracted with chloroform. The product was puri-
fied by column chromatography using a petroleum ether
(b.p.: 100–140°C)/ethyl-acetate mixture (75:25 v/v) as the
mobile phase and crystallized from a mixture of chloroform/
petroleum ether (b.p.: 40°C) (10:90 v/v) with a 76% yield.
M.p.: 93°C. IR (cmϪ1) (KBr): 1720 (␯ C ϭ O). (ESϩ) m/z:
418.6 (MϩH)ϩ. Anal. Calcd for C17H19O4NBrCl: C, 49.01;
H, 4.59; N, 3.36. Found: C, 49.28; H, 4.95; N, 3.58.
2.1.4. 3-[4-(2-Bromoethoxy)-3-chlorophenyl]-5-
methylisoxazole-4-carboxylic acid [4]
Samples of 4 g (10.46 mmol) of [3] and of 2.08 g (12
mmol) of p-toluenesulfonic acid were stirred in 70 ml of a
mixture of toluene/nitromethane (65: 35 v/v) at 25°C for
6 h. The suspension was then diluted with chloroform and
washed with water. After evaporation, the residue was crys-
tallized from a mixture of toluene/petroleum ether (b.p.:
100–140°C) (10: 90 v/v) with a 81% yield. M.p.: 163°C. IR
(cmϪ1) (KBr): 1689 (␯ C ϭ O). (ESϪ) m/z: 360.3 (M-H)Ϫ.
Anal. Calcd for C13H11O4NBrCl: C, 43.30; H, 3.07; N,
3.88. Found: C, 43.54; H, 3.41; N, 3.99.
Unless otherwise specified, solvent evaporations were
carried out under reduced pressure (15 mmHg). Organic
extracts were dried over anhydrous Na2SO4 and products
over P2O5 under reduced pressure (1 mmHg).
2.1. Preparation of halogenated derivatives
2.1.1. 4-(2-Bromoethoxy)benzaldoxime [1]
Samples of 10 g (43.6 mmol) of 4-(2-bromoethoxy)ben-
zaldehyde and of 6.06 g (87.2 mmol) of hydroxylamine
hydrochloride were stirred for 2 h at 45°C in 200 ml of a
mixture of methanol/water (50:50 v/v) adjusted to pH 4.5
with 4% aqueous NaOH. After removal of the methanol, the
crude product was extracted with chloroform. After evapo-
ration to dryness, the crystallized product was obtained from
a mixture of chloroform/petroleum ether (b.p.: 40°C) (10:90
v/v) with a 92% yield. M.p.: 94°C. IR (cmϪ1) (C2Cl4): 3595
(␯ N-OH). (MS): (ESϩ) m/z: 245.1 (MϩH)ϩ. Anal. Calcd
for C9H10O2NBr: C, 44.29; H, 4.13; N, 5.73. Found: C,
44.58; H, 4.45; N, 5.75.
2.1.5. Phenacyl 3-[4-(2-bromoethoxy)-3-chlorophenyl]-5-
methylisoxazole-4-carboxylate [5]
Samples of 364 mg of potassium fluoride (6.27 mmol),
of 1.24 g of ␣-bromoacetophenone (6.27 mmol), and of
1.9 g (5.2 mmol) of [4] were stirred at 25°C in 55 ml of
N,N-dimethylformamide for 3 h. After concentration under
reduced pressure (1 mmHg), the residue was diluted with
chloroform and washed with water. The product was chro-
matographed as described for [3] and crystallized from a
mixture of toluene/petroleum ether (b.p.: 40°C) (10:90 v/v)
with a 76% yield. M.p.: 119°C. IR (cmϪ1) (KBr): 1725 (␯
C ϭ O); 1692 (␯ C ϭ O). (ESϩ) m/z: 481.3 (MϩH)ϩ. Anal.
Calcd for C21H17O5NBrCl: C, 52.69; H, 3.58; N, 2.92.
Found: C, 52.82; H, 3.72; N, 2.99.
2.1.2. 4-(2-Bromoethoxy)-3-chloro-benzohydroxamoyl
chloride [2]
A solution of 4 g (60 mmol) of chlorine in chloroform
(150 ml) was poured into a refrigerated solution (Ϫ10°C) of
8 g (34.9 mmol) of [1] in 85 ml of an anhydrous mixture of
1,4-dioxane/chloroform (75:25 v/v). The mixture was left
overnight at room temperature. After removal of the sol-
vents, the residual oil was crystallized from a mixture of
chloroform/petroleum ether (b.p.: 40°C) (5:95 v/v) in 78%
yield. IR (cmϪ1) (C2Cl4): 3570 (␯ N-OH). (ESϪ) m/z: 312.2
(MϪH)Ϫ. Anal. Calcd for C9H8O2NBrCl2: C, 34.54; H,
2.57; N, 4.48. Found: unstable.
2.1.6. t-Butyl N-(3-chloropropyl) carbamate [6]
Samples of 2.31 g (22.91 mmol) of chloropropylamine
hydrochloride and of 5 g (22.91 mmol) of ditert-butyl di-
carbonate, dissolved in a mixture of tetrahydrofuran/water
(50:50 v/v), adjusted at pH 8.5 with 4% aqueous NaOH
were stirred for 4 h at room temperature. The solution was
acidified to pH 2 using 0.1 N HCl and then extracted with
chloroform. After evaporation of the solvent, the product
was crystallized from a mixture of chloroform/petroleum

M.J. Kolh, R.G. Lejeune / Steroids 67 (2002) 71–75
73
Table 1
Reacting compounds, reaction conditions, and main analytical characteristics of final products
Steroid
Halogenated
derivative
Chromatography
M.S.
IR cmϪ1
Reaction
time
Analytical
calculations (%)
Melting Yield
point
%
(Solvent)
(°C)
Thin layer (Rf)
Col.
Theor.
Found
toluene. 1
ethyl acetate 1
HOAc 0.01
(0.81)
toluene. 1
ethyl acetate 2
HOAc 0.01
(0.78)
toluene. 1
ethyl acetate 2
HOAc 0.01
(0.77)
toluene. 1
ethyl acetate 2
HOAc 0.01
(0.61)
721.4
v CO: 1725
v O–Si: 1257
24 H
C: 68.24
H: 7.77
N: 1.94
C: 68.31
H: 7.86
N: 2.00
ϩ
(MϪH)Ϫ
649.4
143
155
127
124
22
72
15
74
(Benzene)
12 H
445.6
v NH: 3459
v CO: 1729
v CO: 1681
C: 72.61
H: 9.93
N: 3.13
C: 72.92
H: 10.21
N: 3.25
ϩ
ϩ
(MϪH)ϩ
355.4
(Benzene)
36 H
431.6
v NH: 3460
v CO: 1729
v CO: 1679
C: 72.36
H: 9.57
N: 3.24
C: 72.51
H: 9.89
N: 3.19
(MϪH)ϩ
(Toluene)
12 H
488.3
v NH: 3454
v CO: 1726
v CO: 1711
v CO: 1683
C: 71.43
H: 9.22
N: 2.87
C: 71.58
H: 9.35
N: 3.24
(MϪH)ϩ
(Benzene)
toluene. 2
ethyl acetate 1
HOAc 0.01
(0.88)
343.4
401.3
v CO: 1726
v CO: 1708
v CO: 1691
v CO: 1673
14 H
C: 69.27
H: 6.3
N: 1.92
C: 69.65
H: 6.71
N: 2.13
ϩ
148
55
(Xylene)
ether (b.p.: 40°C) (20:80 v/v) with a 92% yield. M.p.: 32°C.
IR (cmϪ1) (C2Cl4): 1723 (␯ C ϭ O); 3464 (␯ N-H); (ESϩ)
m/z: 193.8 (MϩH)ϩ. Anal. Calcd for C8H16O2 NCl: C,
49.61; H, 8.32; N, 7.23. Found: C, 49.84; H, 8.56; N, 7.46.
3. Results
All of the synthesized conjugating arms and all of the
ether-linked steroid conjugates prepared by this technique
demonstrated the expected characteristics in either elemen-
tal analysis, mass spectrometry, or Fourier transform infra-
red spectrometry (Table 1).
2.2. General procedure for coupling steroids to unstable
alkylhalides (Table 1)
High yields, ranging from 76 to 92%, were obtained for
each step of the synthesis procedure of conjugating arms
(overall yields: 33–92%), while steroid conjugates were
prepared in yields ranging from 15 to 75%, depending on
temperature, reaction time, and steroid reactivity (Table 1).
Each conjugate was purified until any impurity spot did not
show intensity greater than that of the hundred-fold dilution
of the tested compound. In that way, the overall purity of the
conjugate could be estimated to be better than 95%.
Bromoethoxyphenylisoxazolecarboxylic derivatives were
prepared following the procedure developed and discussed
previously for carboxyphenylisoxazolecarboxylic derivatives
[1]. However, during the preparation of 4-(2-bromoethoxy)-
benzohydroxamoyl chloride, the phenyl ring was substituted at
the 3-position by a chlorine atom. This substitution, detected
by mass spectrometry, was confirmed and positioned by an
X-ray diffraction study of t-butyl 3-[4-(2-bromoethoxy)-3-
chlorophenyl]-5-methylisoxazole-4-carboxylate [3] [8]. The
derivative resulting from that reaction was the unstable 4-(2-
bromoethoxy)-3-chlorobenzohydroxamoyl chloride [2].
Only protected estradiol reacted with t-butyl 3-[4-(2-
bromoethoxy)-3-chlorophenyl]-5-methylisoxazole-4-
A solution of steroid (1 mmol) in 100 ml of an aromatic
hydrocarbon (see Table 1) was heated under reflux in a
soxhlet apparatus well protected from light by aluminium
foil applied to the balloon flask. The cartridge of the appa-
ratus was filled with a mixture of 3 Å and 4 Å molecular
sieves in equal proportions. After 60 min, 1.2 mmol of
thallium ethoxide was added to the boiling solution, and the
reaction was continued for an additional 2 h period. After
cooling to room temperature, a solution of 1.0 mmol of
halogenated derivative in 5 ml of the same solvent was
added, and the mixture was boiled again. When no further
evolution of the reaction seemed to occur, as determined
by thin layer chromatography, the solvent was evapo-
rated, and the residue was dissolved in 50 ml of chloro-
form. The organic phase was washed with water, dried,
and evaporated. The oily residue was crystallized from a
mixture of toluene/petroleum ether (b.p.: 40°C) (5:90
v/v) after an optional purification (marked ‘ϩ’ in Table
1) by column chromatography using the mobile phase
defined in Table 1.

74
M.J. Kolh, R.G. Lejeune / Steroids 67 (2002) 71–75
carboxylate [3] while 11␣-hydroxyprogesterone that did not
react with [3] gave a conjugate to phenacyl 3-[4-(2-
bromoethoxy)-3-chlorophenyl]-5-methylisoxazole-4-
carboxylate [5]. 11␣-Hydroxyprogesterone also reacted
with t-butyl N-(3-chloropropyl) carbamate [6] in higher
yield and this last reagent was also conjugated to testoster-
one and nandrolone. In all of the reactions considered, the
yield reached a maximum after a reaction time which varied
from one compound to another. As monitored by thin layer
chromatography, the different reagents were progressively
consumed and the final product itself was altered when the
reaction was continued beyond the time recommended.
4. Discussion
In a previous work, steroids were coupled to carbenicillin
and oxacillin side chains through ester linkages [1]. The aim
was to prepare strong ␤-lactamase inhibitors whose effect
could be suppressed after antibody–steroid conjugate interac-
tion. From this point of view, the best compounds were those
having the shortest possible conjugating arm (Steroid-O-
CO-Oxacillin). In order to preserve the conjugates from se-
rum esterase activity, ether linkages are a possible alternative.
t-Butyl 3-[4-(2-bromoethoxy)-3-chlorophenyl]-5-methylisox-
azole-4-carboxylate [3] was first selected for its close similarity
to the ester linked compounds previously described [1] and the
easy removal of the t-butyl group. Unfortunately, [3] failed to
react with most of the investigated compounds at tempera-
tures compatible with the t-butyl group (solvents: benzene or
toluene). Phenacyl 3-[4-(2-bromoethoxy)-3-chlorophenyl]-5-
methylisoxazole-4-carboxylate [5] was prepared with the aim
of increasing stability against decarboxylation, thus allowing
conjugation at higher temperatures (solvent: xylene). t-Butyl
N-(3-chloropropyl) carbamate [6] was selected for the high
reactivity of its halogenate although the length of the coupling
arm should be significantly increased after subsequent reaction
of the amine to the carboxyoxacillin.
For the steroid coupling, the optimal reaction conditions
were set out in the silylation of estradiol on its phenol. A
compound identical to the product described by Top et al.
[9] was obtained with a very good yield (95%). This oper-
ating mode was then applied to the O - alkylation of four
steroids: silylated estradiol, nandrolone, testosterone, and
11␣-hydroxyprogesterone. Silylated estradiol reacted with
[3] in benzene, and the yield reached a maximum of 22%
after 24 h reaction time, but no reaction occurred between
[3] and the other steroids in this solvent. In xylene, trace
amounts of progesterone conjugate were formed in the early
phase of the reaction, but the conjugate was destroyed as
soon as it was formed. Using the less thermosensitive com-
pound [5], the corresponding conjugate was obtained with a
55% yield after 14 h, but neither testosterone nor nan-
drolone did react with [5]. A more reactive compound [6],
although potentially much less interesting in immunochem-
istry, was tested on 11␣-hydroxyprogesterone (benzene,
Fig. 1. Synthesis routes for the preparation of conjugating arms for cou-
pling to steroids. Preparation of halogenated derivatives.
12 h, 74% yield). The yield increase obtained at lower
temperature seemed to be due to the greater reactivity of [6]
more probably than to the better formation of the thallium
steroid alkoxide, and prompted us to apply this reaction for
coupling to testosterone (benzene, 12 h, 72% yield), and
nandrolone (toluene, 36 h, 15% yield).
The halogenated derivatives included in the study, except
for [5], were unstable by decarboxylation, and the steroids
selected, as secondary alcohols bearing long alkyl chains,
were poor nucleophiles. Despite these unfavorable proper-
ties of the reagents involved in the conjugation, a coupling
was obtained with all of the steroids tested. The continuous
elimination of moisture traces by distillation over the mo-
lecular sieves certainly contributed to maintain the best condi-
tions during the rather long reaction time. The maximum yield
varied inversely as a function of the reaction time and resulted
from a competition between the kinetics of the conjugation and
the degradation rate of the final products.
In conclusion, the proposed method is an efficient ap-
proach to the conjugation of steroid by ether formation with
halogenated derivatives protected by thermosensitive
groups (Fig. 1).

M.J. Kolh, R.G. Lejeune / Steroids 67 (2002) 71–75
75
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transfer catalysis. Synthesis 1980:921–4.
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