Synthesis of 5α-androstane-3α,17β-diol 17-O-glucuronide histaminyl conjugate for immunoassays

Article abstract of DOI:10.1016/j.steroids.2016.02.011

Simple method of preparation of 5α-androstane-3α,17β-diol 17-O-glucuronide N-histaminyl amide was developed for the construction of immunoanalytical kit. Improved method of glucuronide derivative synthesis was used, followed by hydroxybenzotriazole-dicyclohexylcarbodiimide coupling with histamine.

Full text of DOI:10.1016/j.steroids.2016.02.011

Steroids 109 (2016) 56–59
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of 5a-androstane-3
a
,17b-diol 17-O-glucuronide histaminyl
conjugate for immunoassays
⇑
ˇ
´
Vinš Petr, Cerny Ivan, Mikšátková Petra, Drašar Pavel
Department of Chemistry of Natural Products, University of Chemistry and Technology, Technická 5, CZ 166 28 Praha 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Simple method of preparation of 5a-androstane-3a,17b-diol 17-O-glucuronide N-histaminyl amide was
Received 24 February 2015
Received in revised form 15 January 2016
Accepted 16 February 2016
Available online 18 February 2016
developed for the construction of immunoanalytical kit. Improved method of glucuronide derivative
synthesis was used, followed by hydroxybenzotriazole–dicyclohexylcarbodiimide coupling with
histamine.
Ó 2016 Elsevier Inc. All rights reserved.
Keywords:
Glucuronide histaminyl conjugate
Conjugate for immunoassays
5a-Androstane-3a,17b-diol
Simple efficient synthesis
1. Introduction
2. Experimental
Immunoassays, especially in the field of biological steroids,
retain its importance even in time of progress of sophisticated
instrumental analyses mainly for its simplicity and costs [1].
Syntheses of components for construction of a kit may bring some
challenging tasks due to the structural complexity of products.
Similar studies from our laboratory targeted to preparation of
haptens for endocrinologists and clinical analysts may serve as
an example [2,3].
2.1. General
NMR spectra were measured at Varian Gemini 300 HC instru-
ment (300 MHz, FT mode) in solvents indicated below and were
referenced to solvent signals. Spectra of final compound 4 were
taken on Bruker Avance 600 III (600 MHz; 150 MHz). Chemical
shifts are given in ppm, coupling constants (J) and ranges of mul-
tiplets in Hz. Beside 1H-NMR and 13C-NMR (APT), homonuclear
2D-spectrum (COSY) and heteronuclear 2D-spectra (HSQC, HMBC)
were taken for complete structural assignment of compound 4.
High resolution mass spectra were recorded using LTQ Orbitrap
Velos (Thermo Scientific) instrument with electrospray ionisation
in positive mode. Analytical TLC was performed on Merck TLC
Silica gel 60 F254 plates using visualization by solution of H2SO4
in MeOH (1/1) and consecutive heating. For column chromatogra-
Current project is aimed on the simple synthesis of histaminyl
amide 17-O-glucuronide conjugate of 5
Suitably protected starting compound, acetate 1, was accessible
from commercial 3 -acetoxy-5 -androstan-17-one. For a glycosy-
a-androstane-3a,17b-diol.
a
a
lation into hindered 17b-position of androstane skeleton, the mod-
ified Koenigs–Knorr reaction [4] was selected. The mentioned
paper [4] is aimed on the mass spectrometry and gives only limited
information on the synthesis and its results (esp. yields), so we pre-
sent our procedure which may be considered in part as experimen-
tal specification of the published troublesome general procedure.
Several methods were tested for the deprotection of glucuronide
2, and also for the preparation of N-histaminyl amide 4.
phy, silica gel 60 (100–160
phase flash column chromatography, C-18 modified silica gel
(Sigma-Aldrich) was filled into glass columns. Starting 3 -ace-
toxy-5 -androstan-17-one was purchased from Steraloids, so as
lm, Fluka) was used. For reverse-
a
a
a reference sample of deprotected acid 3. Other chemicals and
solvents were purchased from commercial sources in reagent
grade and were not further purified before use if not stated
otherwise below. The solutions were dried over anhydrous MgSO4
and the solvents were removed on rotary evaporator under
reduced pressure.
⇑
Corresponding author.
E-mail address: Pavel.Drasar@vscht.cz (P. Drašar).
http://dx.doi.org/10.1016/j.steroids.2016.02.011
0039-128X/Ó 2016 Elsevier Inc. All rights reserved.

P. Vinš et al. / Steroids 109 (2016) 56–59
57
2.2. Chemical synthesis
3.68–3.63 m, 2 H (H-50, H-17
a
); 3.57–3.43 m, 3 H (2 ꢀ H-700,
1 ꢀ H-40); 3.39–3.36 m, 1 H (H-30); 3.21 t, 1 H, J = 8.8 (H-20); 2.82
br t, 2 H, J = 6.6 (2 ꢀ H-600); 1.99–1.90 m, 2 H (H-12a, H-16a);
1.73–1.66 m, 2 H (H-7a, H-2a); 1.64–1.58 m, 5 H (H-5, H-8,
H-11a, H-15a, H-16b); 1.52 dt, 1 H, J1 = 13.2, J2 = 2.2 (H-4a);
1.48–1.40 m, 2 H (H-1a, H-2b); 1.39–1.35 dd, 1 H, J1 = 13.7,
J2 = 3.3 (H-11b); 1.29–1.21 m, 3 H (H-6a, H-6b, H-15b); 1.15 dt, 1
H, J1 = 12.6, J2 = 3.8 (H-12b); 1.03–0.91 m, 2 H (H-14, H-7b);
0.84 s, 6 H (3 ꢀ H-18, 3 ꢀ H-19); 0.77 dt, 1 H, J1 = 12.1, J2 = 3.8
2.2.1. Methyl 2,3,4-tri-O-acetyl-1-O-(3a-acetyloxy-5a-androstan-
17b-yl)-b-D-glucopyranosiduronate (2)
17b-Hydroxy-5 -androstan-3 -yl acetate [4–6] (1, 100 mg,
1 eq, 0.299 mmol) was dissolved in dry freshly distilled benzene
(8 ml) and treated by methyl 2,3,4-tri-O-acetyl-1 -bromo-1-
a
a
a
deoxy-b-D-glucuronate (6) (300 mg, 2.5 eq, 0.75 mmol) in pres-
ence of silver carbonate (200 mg, 2.4 eq, 0.725 mmol) and ground
molecular sieves (250 mg). Reaction mixture was stirred in dark,
under argon at room temperature for 3 days. Then, the mixture
was diluted by dichloromethane, filtered, and the solvent was
evaporated. The crude product was chromatographed on silica
gel column using gradient of diethyl ether (1–10% v/v) in dichlor-
omethane. Glycoside 2 (36 mg, 18%) was obtained as white amor-
phous solid. 1H NMR (CDCl3, 300 MHz): 5.24–5.15 m, 2 H; 5.01 m, 2
H; 4.58 d, 1 H, J = 7.9; 3.99 d, 1 H, J = 9.7; 3.75 s, 3 H; 3.57 t, 1 H,
J = 8.8; 2.05 s, 3 H; 2.04 s, 3 H; 2.01 m, 6 H; 0.79 s, 3 H; 0.70 s, 3
H. The title compound was previously prepared by another method
[7] and was found to have identical structure.
13
(H-9). C NMR (CD3OD, 150 MHz): 170.49 (C-60); 134.74, 2 C (C-
200, C-400); 117.99 (br.[8], C-500); 103.58 (C-10); 89.22 (C-17); 76.17
(C-30); 74.80 (C-50); 73.50 (C-20); 72.15 (C-40); 65.78 (C-3); 54.62
(C-9); 50.85 (C-14); 42.93 (C-13); 38.93 (C-5); 38.59 (C-700);
37.31 (C-12); 35.81 (C-10); 35.33 (C-4); 35.31 (C-8); 32.12 (C-1);
31.46 (C-7); 28.56 (C-16); 28.28 (C-2); 28.19 (C-600); 28.18 (C-6);
22.97 (C-15); 20.08 (C-11); 10.69 (C-18); 10.30 (C-19). HRMS ESI
+ (m/z): [M+H]+ calcd. for C30H47N3O7: 562.349341. Found:
562.34900.
3. Results and discussion
2.2.2. 3
a-Hydroxy-5a-androstan-17b-yl b-D-glucopyranosiduronic
For the synthesis of complex target amide 4 from commercial
acid (3)
building blocks of reasonable price, pathway starting with 3
a-ace-
Protected glycoside 2 (165 mg, 1 eq, 0.254 mmol) was dissolved
in methanol (2 ml) and the solution was diluted by 10 ml of water.
Solution of lithium hydroxide in methanol (0.5 M, 10 ml) was then
added. Reaction mixture was stirred in dark at room temperature
for 3 days. After dilution by another 10 ml of methanol, pH was
adjusted to neutral by a solution of acetic acid (10% in methanol).
Resulting solution was passed through a column of catex (Amber-
lite, IR-120, H-cycle, 20–50 mesh, 15 ml) using the methanol/water
(2/1) mixture as eluent. After evaporation, the crude product was
purified by column chromatography on reverse phase (C-18 mod-
ified silica gel), using gradient of MeOH in water. Deprotected glu-
curonide 3 (63 mg, 53%) was obtained as amorphous white solid.
This commercial product was described previously [4,7]. 1H NMR
(CD3OD, 300 MHz): 4.36 d, 1 H, J = 7.91 (H-10); 3.95 br t, 1 H
(H-3); 3.77–3.64 m, 2 H (H-50, H-17); 3.50 t, 1 H, J = 9.1 (H-40);
3.34 t, 1 H, J = 9.1 (H-30); 3.20 t, 1 H, J = 7.91 (H-20); 0.82–0.81
2 ꢀ s, 6 H (3 ꢀ H-18, 3 ꢀ H-19). HRMS ESI + (m/z): [M+Na]+ calcd.
for C25H40O8: 491.262087. Found: 491.26158.
toxy-5 -androstan-17-one was selected. Protected androstanolone
a
was reduced to secondary alcohol (Scheme 1), glycosylated by
acetylated halogenose, and after hydrolysis of protecting ester
groups subjected to the conjugation with histamine. Glycosylation
and amide formation with subsequent purification were critical
steps.
Ketone group of starting material was readily reduced to obtain
corresponding alcohol 1 in very good yield by common procedure
using sodium borohydride in mixture of methanol and ethyl acet-
ate. As reported previously [4–6], only 17b isomer was isolated.
The suitable halogenose 6 was prepared (see Scheme 2) with
the method of Bowering and Timell [9] from b-D-glucuronolactone.
From obtained building blocks, glycoside 2 was prepared by
modified Koenigs–Knorr reaction [10], following to method of
Thevis et al. [11] using silver carbonate as catalyst (even though
in stoichiometric excess).
It is noteworthy that this reaction is very sensitive and requires
absolutely dry solvents, giving increasing ratio of corresponding
orthoester 7 (Fig. 1) hand in hand with rising moisture content.
During our early experiments with this method, the orthoester
was even isolated in good yield as main product, using ‘‘dry”
reagent-grade toluene. After such experience, toluene was substi-
tuted by freshly dried and distilled benzene over grinded activated
molecular sieves. Nevertheless, the orthoester remained trouble-
some side product and thorough purification via column chro-
matography was necessary. This caused additional losses and the
overall yield was low (about 20% in total). Orthoester content can
be detected by characteristic signal of its methyl group in 1H
NMR spectrum (1.71 ppm in CDCl3).
2.2.3. 3a-Hydroxy-5a-androstan-17b-yl N-(4-imidazolyl)ethyl-b-D-
glucopyranosiduronamide (4)
To a solution of glucuronide 3 (9 mg, 1 eq, 19.2 nmol) in DMF
(200 ll), hydroxybenzotriazole (hydrate, 90%, 6 mg, 3.5 eq,
6.7 nmol) was added. DCC (14 mg, 3.5 eq, 6.7 nmol) was being
added in 3 portions. 6 mg were added at the beginning of the reac-
tion, another 6 mg were added after 2 days and at last, 2 mg were
added after 2 more days. The reaction was completed after 6 days
of slow stirring at room temperature. The reaction mixture was fil-
tered from crystals of dicyclohexylurea, which were washed by
another 100
6.7 nmol) in DMF (100
l
l of DMF. Solution of histamine (7.5 mg, 3.5 eq,
The deprotection of acetyl groups was achieved by treatment by
solution of LiOH in water/methanol over two days [12]. Despite the
general swiftness and ease of deacetylation reactions, ordinary
protocols using MeONa or NaOH with reaction times of several
hours did not lead to complete deacetylation of the substrate.
Several methods of amide formation were tried out for the final
step. First, T3PÒ [13] (propylphosphonic anhydride, 50% solution in
EtOAc) was employed in DMF/pyridine. Even with an excess (4–
6 mol. eq.) of the reagent, only starting steroid 3 was detected in
reaction mixture. Then, experiments with DEPC (diethyl phospho-
ryl cyanide) were made [14]. This reagent readily promoted amide
group formation in DMF with DIPEA, but only phosphate of target
structure 4 was retrieved (single product was obtained, showing
MW = 698 in MS spectrum and two excessive ethyl groups in 1H
l
l) and DIPEA (20 l) were added. After stir-
l
ring in dark at room temperature for 3 days, the reaction was com-
pleted. Reaction mixture was evaporated to dryness and residue
was submitted to the column chromatography on reverse phase,
using gradient of MeOH in water as eluent. Fractions containing
target compound were combined and evaporated, yielding 7 mg
of compound 4 which was further purified by column chromatog-
raphy on silica gel (CH2Cl2/MeOH). Target compound 4 (3.7 mg,
34%) was obtained as amorphous white solid. Prior to measure-
ments of NMR spectra, the compound was further purified from
trace impurities by semi-preparative HPLC (gradient of MeOH in
1
water). H NMR (CD3OD, 600 MHz): 7.60 s, 1 H (H-200); 6.89 br s,
1 H (H-500); 4.38 d, 1 H, J = 7.7 (H-10); 3.97 br s, 1 H (H-3b);

58
P. Vinš et al. / Steroids 109 (2016) 56–59
AcO
O
OAc
AcO
O
OH
O
O
O
a
b
O
O
AcO
HO
AcO
O
H
H
AcO
Br
AcO
H
1
AcO
OAc
2
OH
HO
OH
HO
O
HO
O
O
O
O
OH
O
NH
c
d
H
H
H
N
HO
HO
HN
H
H
3
4
Scheme 1. Proposed synthetic route towards amide 4. (a) NaBH4, MeOH, EtOAc; (b) Ag2CO3, 6, benzene, mol. sieves; (c) LiOH, MeOH, H2O; (d) 1. HOBT, DCC, DMF; 2.
Histamine, DIPEA, DMF.
O
O
HO
O
O
O
a
b
O
O
O
OH
AcO
OAc
OAc
AcO
Br
O
OH
AcO
AcO
OAc
5
6
Scheme 2. Preparation of bromosugar for glycosylation reaction. (a) 1. NaOH, MeOH; 2. Ac2O, Py; (b) HBr, AcOH.
developed. It consists of conventional reactions, which were veri-
OAc
O
O
AcO
fied for given reagents, affording new reproducible synthetic route.
Very low overall yield of 3–4% offers possibilities for further opti-
mization, however it is strongly dependent on desired purity of
target product. Small quantity of conjugate prepared was enough
to be successfully used in immunoanalytical sets both in perfor-
mance and stability by the industrial partner.
O
O
O
O
Acknowledgements
AcO
H
The authors are indebted to the project of the Czech Ministry of
Interior No. VG20112015045. They also express their gratitude to
Fig. 1. Structure of orthoester impurity detected after glycosylation reaction.
ˇ
Dr. Hana Dvoráková for measurement of NMR spectra of com-
NMR). Finally, method using hydroxybenzotriazole with dicyclo-
hexylcarbodiimide [15] and consecutive active-ester addition to
histamine in DMF/DIPEA solution was employed to obtain amide
4. Followed by reverse-phase and subsequent normal-phase chro-
matography, this method provided desired conjugate in sufficient
purity.
pound 4.
References
[1] B. Carlson, Seizing immunoassay opportunities, Genet. Eng. Biotechnol. News
34 (4) (2014) 12–13.
[2] V. Pouzar, I. Cerny´, On steroids. 378. 17-(O(2-Carboxyethyl)oxime) Derivatives
ˇ
It should be stated that all efforts to get the compounds pre-
pared in decent crystalline form were not successful.
of Androstanes of 3-Hydroxy-5-alpha-5-ene, 3-Hydroxy-5-beta-5-ene, 3-
Hydroxy-5-ene and 3-Oxo-4-ene Series, Collect. Czech. Chem. Commun. 60
(1) (1995) 137–149.
ˇ
[3] O. Longin, I. Cerny´, P. Drašar, Novel approach to the preparation of
hemisuccinates of steroids bearing tertiary alcohol group, Steroids 97 (1)
(2015) 67–71.
4. Conclusion
[4] M. Thevis, G. Opfermann, H. Schmickler, W. Schänzer, Mass spectrometry of
steroid glucuronide conjugates. II-Electron impact fragmentation of 3-keto-4-
en- and 3-keto-5alpha-steroid-17-O-beta glucuronides and 5alpha-steroid-
Synthetic pathway towards target androstanyl glucuronate
marked by histamine starting from basic building blocks was

P. Vinš et al. / Steroids 109 (2016) 56–59
59
3alpha,17beta-diol 3- and 17-glucuronides, J. Mass Spectrom. 36 (9) (2001)
998–1012.
[5] T. Ohta, H. Zhang, Y. Torihara, T. Hida, I. Furukawa, Practical synthesis of
androgen: the efficient transformation of 17-oxo group to 17 alpha-hydroxy
group, Steroids 63 (12) (1998) 630–632.
5beta-androstan-3alpha-ol-17-one glucuronides, 5alpha-estran-3alpha-ol-17-
one glucuronide and deuterium-labelled analogues, J. Mass Spectrom. 36 (2)
(2001) 159–168.
[12] T. Iida, S. Tazawa, Y. Ohshima, T. Niwa, J. Goto, T. Nambara, Analysis of
conjugated bile-acids in human biological-fluids
–
synthesis of
[6] P. Purushottamachar, V.C. Njar, A new simple and high-yield synthesis of
hyodeoxycholic acid 3-glycoside and 6-glycoside and related-compounds,
Chem. Pharm. Bull. 42 (7) (1994) 1479–1484.
[13] A.L.L. García, T3P: a convenient and useful reagent in organic synthesis, Synlett
8 (2007) 1328–1329.
5alpha-dihydrotestosterone (DHT),
Steroids 77 (14) (2012) 1530–1534.
a potent androgen receptor agonist,
[7] P.N. Rao, K.M. Damodaran, Synthesis of a precursor for the preparation of 9
alpha,11 alpha-tritiated alpha-androstane-3 alpha,17 beta-diol 17-
5
[14] T. Shioiri, N. Irako, An efficient synthesis of the piperazinone fragment of
glucuronide, Steroids 43 (3) (1984) 343–350.
pseudotheonamide A1 via a stereoselective intramolecular Michael ring
[8] Signal very broadened due to very low product concentration, Corresponding
proton signal in HNMR was found broad as well.
closure, Chem. Lett. 31 (2) (2002) 130–131.
[15] C. Bertolla, S. Rolin, B. Evrard, L. Pocheta, B. Masereela, Synthesis and
[9] W.D.S. Bowering, T.E. Timell, Synthesis and characterization of 2-O-(beta-D-
glucopyranosyluronic acid)-D-xylopyranose, J. Am. Chem. Soc. 82 (11) (1960)
2827–2830.
pharmacological evaluation of a new targeted drug carrier system: b-
cyclodextrin coupled to oxytocin, Bioorg. Med. Chem. Lett. 14 (18) (2008)
1855–1858.
[10] P.N. Rao, A.M. Rodriguez, D.W. Miller, J. Steroid Biochem. 25 (3) (1986) 417.
[11] M. Thevis, G. Opfermann, H. Schmickler, W. Schänzer, Mass spectrometry of
steroid glucuronide conjugates. I. Electron impact fragmentation of 5alpha-/