Preparation of tritium-labelled BIIL 260 of high specific radioactivity

Article abstract of DOI:10.1002/jlcr.661

Various approaches to the synthesis of tritium-labelled BIIL 260 with high specific radioactivity were investigated. Attempts to incorporate tritium directly into BIIL 260 were made by solid-phase isotope exchange with tritium gas and by isotope exchange with tritiated water which yielded a final product with specific activities ranging from 2 to 7 Ci/mmol. However, the solid-phase and liquid-phase dehalogenations of an appropriate synthon fragment of BIIL 260 followed by its subsequent conversion to the final product via chemical synthesis yielded the desired tritium-labelled BIIL 260 with specific activities of 25 or 71 Ci/mmol, depending upon the precursors and methods used in the dehalogenation step. Copyright

Full text of DOI:10.1002/jlcr.661

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 221–230.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.661
Research Article
Preparation of tritium-labelled BIIL 260 of
high specific radioactivity
V.P. Shevchenko1, I.Yu. Nagaev1, N.F. Myasoedov1,*, A.B. Susan2,
R. Anderskewitz3, F.W. Birke3 and K-H. Switek3
1 Institute of Molecular Genetics, Russian Academy of Sciences, Kurchatov Sq.,
123182 Moscow, Russia
2 International Isotopes Clearing House, Inc., 9110 Lee Blv., Leawood,
KS 66206, USA
3 Boehringer Ingelheim Pharma KG, Birkendorfer Str. 65, D-88397 Biberach an
der Riss, Germany
Summary
Various approaches to the synthesis of tritium-labelled BIIL 260 with high
specific radioactivity were investigated. Attempts to incorporate tritium
directly into BIIL 260 were made by solid-phase isotope exchange with tritium
gas and by isotope exchange with tritiated water which yielded a final product
with specific activities ranging from 2 to 7 Ci=mmol. However, the solid-phase
and liquid-phase dehalogenations of an appropriate synthon fragment of BIIL
260 followed by its subsequent conversion to the final product via chemical
synthesis yielded the desired tritium-labelled BIIL 260 with specific activities of
25 or 71 Ci=mmol, depending upon the precursors and methods used in the
dehalogenation step. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: tritiation; synthesis of labelled BIIL 260
*Correspondence to: N.F. Myasoedov, Institute of Molecular Genetics, Kurchatov sq., 2, 123182
Moscow, Russia. E-mail: nagaev@img.ras.ru
Copyright # 2002 John Wiley & Sons, Ltd.
Received 7 June 2002
Revised 5 September 2002
Accepted 9 September 2002

222
V.P. SHEVCHENKO ET AL.
Introduction
BIIL 284 BS is a new LTB4 receptor antagonist developed as a novel
anti-inflammatory agent for several chronic inflammatory conditions.
BIIL 284 is a pro-drug which is metabolized to the active metabolite
BIIL 260. BIIL 260 has a high affinity for the LTB4 receptor with a Ki
value of 1:7 nM on human neutrophilic granulocytes.1
For direct characterization of BIIL 260 in its interaction with the
LTB4 receptor, the preparation of tritium-labelled BIIL 260 with a high
molar radioactivity was required. Methods allowing the direct tritium
labelling of the final product226 are always preferable over the more
time-consuming and complex synthetic methods7;8 which need to be
used if the compound is unstable under the given reaction conditions.
The purpose of the present work was the synthesis of tritium-labelled
BIIL 260 with a high specific radioactivity.
Results and discussion
The first attempts to introduce tritium directly into BIIL 260 were made
via the solid-phase isotope exchange method2;3 (reaction times ranging
from 15 to 120 min and temperatures from 1408C to 2008C; palladium
catalysts on various carriers – carbon, barium sulphate, etc.) and via the
isotope exchange method4;5 with tritiated water of high specific
radioactivity (reaction times from 0.5 to 16 h, reaction temperatures
ranging from 238C to 1608C, using various palladium catalysts). We
found out that under these conditions only preparations with specific
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TRITIUM-LABELLED BILL 260
223
radioactivities from 2 to 7 Ci=mmol were obtained, possibly due to the
instability of the BIIL 260 molecule under the given reaction conditions.
6
Further, treatment of BIIL 260 with gaseous tritium via the method
(using palladium catalysts; solvents such as methanol, ethanol,
acetonitrile or their mixtures; and tritium pressure – 400 hPa) led to a
complete destruction of the substance during reaction times ranging
from 10 to 30 min. This was probably due to the previously
described9211 phenomenon of hydrogenolysis of compounds
with the general formula R0O-CH2-C6H4-R with the formation of R0
-OH and R00-C6H4-CH3 moieties. Thus, 2,2-bis-(4-hydroxyphenyl)
propane, m-xylene, and HO-C6H4-CðNH2Þ ¼ NH resulted from the
degradation of the BIIL 260 molecule. Consequently, in order to
achieve our goal to prepare BIIL 260 with a higher specific radio-
activity we devised two labelling pathways described below (Schemes 1
and 2):
1. Preparation of the tritiated 4-hydroxybezonitrile (½3H2ꢀ VIII) intermediate
via the dehalogenation of 4-hydroxy-3,5-dibromobezonitrile (I), with
subsequent condensation of the resulting labelled product (½3H2ꢀ II) with
the chloride (III) and conversion of the resulting nitrile (½3H2ꢀ IV) to ½3H2ꢀ
BIIL 260 as per Scheme 1.
2. Preparation of tritiated 2,2-bis-(4-hydroxyphenyl)propane (½3H4ꢀ VIII) via
dehalogenation of (VII) and subsequent condensation of the labelled
product with nðm-CICH2C6H4CH2OÞC6H4CN (IX) and conversion
of the resulting nilrile (½3H4ꢀ V) to ½3H4ꢀBIIL 260 as per
Scheme 2.
The first reaction pathway yielded tritium-labelled BIIL 260 with a
specific radioactivity of 25 Ci=mmol, while the second reaction pathway
yielded tritium-labelled BIIL 260 with a specific radioactivity of
71 Ci=mmol. The key reactions in the overall process were the
introduction of the label and the reduction of ½3Hꢀamidoximes
(½3H2ꢀ VI and ½3H4ꢀ VI) to ½3HꢀBIIL 260 in the presence of Raney
nickel.
The dehalogenation was carried out via both the solid-phase and the
liquid-phase methods. In the case of the dehalogenation of nitrile (I), the
best results were achieved via the liquid-phase method. Apparently,
considerable loss of substrate occurred via sublimation of the un-
changed reactant (high reaction temperature of above 1008C is needed
for an efficient high-temperature solid-phase dehalogenation) making
this method not practical in this case. In the case of the dehalogenation
Copyright # 2002 John Wiley & Sons, Ltd.
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V.P. SHEVCHENKO ET AL.
Scheme 1. Synthesis of the ½3H2ꢀBIIL 260, labelled in the moiety (II)
Scheme 2. Synthesis of the ½3H4ꢀBIIL 260, labelled in the moiety (VIII)
Copyright # 2002 John Wiley & Sons, Ltd.
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TRITIUM-LABELLED BILL 260
225
of 2,2-bis-(4-hydroxy-3,5-dibromophenyl)propane (VII), the solid-phase
method gave better results (Table 1).
The difficulty of completing the remaining reaction steps resided in
the fact that only milligram quantities of the key starting labelled
product could be prepared via the above reactions. Using the reaction
pathway 1, high yields (up to 90% and above) of the product (½3H2ꢀ IV)
were obtained when using a rather large excess of the chloride (III) by
comparison with the nitrile (½3H2ꢀ II). We also found out that upon
reacting (½3H4ꢀ VIII) with the chloride (IX), an equimolar ratio of these
reagents should be used in order to minimize the formation of a dimeric
condensation by-product. Due to this, the yield of the desired labelled
product (½3H4ꢀ VIII) cannot exceed 50% of the theoretical value.
The removal of protective groups and the transformation of the
tritium-labelled nitriles (V) to the labelled amidoximes (VI) did not
cause problems since these reactions could be carried out with a large
excess of non-labelled reagents. For reasons of safety, all reactions
involving labelled intermediates were carried out in sealed ampoules.
The stirring of the reaction mixtures was achieved by rotation of the
ampoule in a rotary evaporator. A water bath provided the necessary
temperature control.
The most complex reaction step was the reduction of the tritium-
labelled amidoximes (VI) by Raney nickel. This reaction was carried out
with microgram amounts of the labelled precursor (VI). The reaction
yield was strongly influenced by the conditions used to prepare the
Raney nickel. Thus, we found that the reduction yield of amidoximes
(VI) was raised by up to 40% by treating the Raney nickel with acetic
acid while also using a substance-to-catalyst ratio of 1:40 (by weight). In
Table 1. Dehalogenation of 2,2-bis-(4-hydroxy-3,5-dibromophenyl)propane
(VII); tritium pressure 400 hPa, 5% Pd/C catalyst
Temp.
ð8CÞ
Reaction
time
(min)
Solvent
Yield
(%)
Specific activity
(Ci/mmol)
Method
140
160
160
180
200
20
15
15
30
15
15
80
}
}
}
}
}
68
54
50
23
17
73
54
69
71
83
88
63
Solid-phase
Solid-phase
Solid-phase
Solid-phase
Solid-phase
Liquid-phase
Ethylacetate-
triethylamine (20:1)
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226
V.P. SHEVCHENKO ET AL.
addition, the yield of the final labelled product was strongly dependent
on the reaction time (Table 2).
Experimental
The solvents, catalysts and other chemicals were commercial reagents.
Initial preparations and standard compounds (including BIIL 296 – a
hydrolysis product of BIIL 260) were commercial reagents or were
prepared by Boehringer Ingelheim Pharma KG, Germany.
The analysis and/or purification of various intermediates and final
products was carried out by high-performance liquid chromatography
(HPLC) and by thin layer chromatography (TLC).
TLC analysis was carried out using silica gel plates and the following
solvent system: ethylacetate–methylene chloride–acetic acid–water
(40:20:20:5:5). The Rf values were (0.64) for BIIL 260, and (0.88) for
BIIL 296.
Analytical HPLC was performed on a 4 ꢁ 150 mm column packed
with Separon SGX C18, 7 mm, at a flow rate of 0:8 ml=min, using the
following systems:
(A) methanol–water–trifluoroacetic acid (78:22:0.1);
(B) acetonitrile–50 mM potassium phosphate buffer, pH 3, (4:6);
(C) acetonitrile–50 mM potassium phosphate buffer, pH 3, (1:1);
(D) acetonitrile–50 mM potassium phosphate buffer, pH 3, (6:4);
(E) methanol–50 mM potassium phosphate buffer, pH 3, (7:3);
(F) methanol–50 mM potassium phosphate buffer, pH 3, (3:1).
Table 2. Kinetics of the conversion of ½3Hꢀ amidoximes (VI) to tritium-labelled
BIIL 260
Reaction time
(min)
Composition of the reaction mixture
Amount of BIIL 260 in
½3Hꢀamidoximes (%) ½3HꢀBIIL 260 (%) the standard sample ðmgÞ
30
55
60
75
80
90
135
180
300
81
69
66
39
30
16
11
8
11
15
20
52
70
74
62
17
8
0.2
0.3
0.5
1.1
2.2
1.9
1.2
0.3
0.1
3
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TRITIUM-LABELLED BILL 260
227
Preparative HPLC purifications were carried out on a 10 ꢁ 250 mm
column packed by
Silasorb C18, 13 mm, at a flow rate of 2:0 ml=min, using the following
mobile phases:
(G) acetonitrile–50 mM potassium phosphate buffer, pH 3, (6:4);
(H) methanol–50 mM potassium phosphate buffer, pH 3, (4:1);
(I) methanol–water–trifluoroacetic acid (850:150:1).
Retention times are given in Table 3.
Bromination of phenolic precursors
In a typical run, 1:26 mmol of phenolic precursor dissolved in 3 ml of
methanol and 2 ml of concentrated aqueous ammonia was stirred for
70 min with a solution of 2 ml of methanol containing 300 ml of
bromine. Following this, the reaction mixture was evaporated to
dryness, dissolved in 2 ml of methanol and acidified to pH 2 with a 1 N
solution of HCl. The resulting solution was evaporated once
more, dissolved in 5 ml of ethylacetate and washed with water
ð3 ꢁ 1 mlÞ. The organic solvent was removed by evaporation and the
resulting residue was dissolved in 4 ml of methanol, heated to 708C after
which 2 ml of water was added with stirring. The desired brominated
derivative was formed upon slow cooling and the isolated yield
was 60–65%.
Table 3. Retention times of BIIL 260 and its intermediates in various chromato-
graphic systems
Compound
Retention time (min)
A
B
C
D
E
F
G
H
I
I
II
III
IV
V
VI
VII
VIII
IX
BIIL 260
BIIL 296
}
}
}
8.52
}
6.02
6.20
2.65
3.75
9.46
}
}
}
}
}
}
}
}
}
}
}
}
}
}
}
}
}
}
}
}
}
}
}
}
}
3.21
2.21
}
}
}
}
}
}
}
}
}
12.11
}
14.16
}
}
}
}
}
}
}
}
}
}
}
}
}
2.32
}
}
}
}
}
}
}
}
}
}
}
9.76
7.12
}
2.68
}
}
10.94
}
}
8.24
10.15
}
4.31
5.53
}
13.36
}
8.86
}
}
}
}
}
10.79
}
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228
V.P. SHEVCHENKO ET AL.
Dehalogenation of compound (I)
1. A batch of 13 mg of the substance (I), 20 ml of triethylamine in 500 ml of
dioxane, and 40 mg of 5% Pd=BaSO4 were placed in the reaction ampoule
and the mixture was frozen in liquid nitrogen. After evacuation, the
ampoule was filled with gaseous tritium up to a pressure of 400 hPa. The
reaction mixture was stirred for 2:5 h at the given reaction temperature
followed by freezing with liquid nitrogen and evacuation of the unreacted
tritium gas. The catalyst was removed by filtration and washed with
methanol ð3 ꢁ 2 mlÞ, and the filtrates were combined and evaporated 3 times
with methanol ð3 ꢁ 2 mlÞ to remove the labile tritium. The residue left,
representing the crude reaction product, was purified by preparative HPLC.
The yield of (½3H2ꢀ II) was 80–85%, with a specific radioactivity of
25 Ci=mmol.
2. In a typical run, 10 mg of the substance (I), coated on 100 mg of 5% Pd=
BaSO4 was placed in the reaction ampoule which was evacuated and filled
with gaseous tritium at a partial pressure of 400 hPa. The reaction was
carried out for 15 min at various temperatures such as 1008C; 1208C; 1408
C; 1608C and the crude reaction mixture was processed as described above.
The product resulting from the reaction carried out at 1008C contained 46%
of (½3H2ꢀ II), 26% of mono-bromoderivative and 28% starting material (I)
while at 1608C, the reaction mixture consisted of 54% (½3H2ꢀ II), 14% mono-
bromoderivative, and 32% starting material. The specific radioactivity of the
resulting products were in the range 25–30 Ci=mmol.
Synthesis of ½3H2ꢀ V
1. 12:8 Mg of (III), K2CO3 ð4 mgÞ, KI ð1:5 mgÞ; 2:6 mg of (½3H2ꢀ II), and
0:2 ml of acetonitrile were placed in an ampoule. The latter was sealed and
the reaction was kept at 858C for 2 h while stirring. The reaction mixture
was diluted with 1 ml of acetonitrile, the solution was filtered off and the
residue was washed with acetonitrile ð3 ꢁ 1 mlÞ. The combined filtrates were
evaporated to dryness and dissolved in 1:5 ml of ethylacetate–methanol (2:1)
mixture.
2. 1:5 ml of the reactant solution was placed in the reaction ampoule, and
10 mg of toluenesulphonic acid was added. After 2 h the solvents were
evaporated and the residue was dissolved in 6 ml of ethylacetate. The
toluenesulphonic acid was extracted with 3 ml of 1 N sodium bicarbonate
and twice with 3 ml of water. The resulting organic phase was evaporated to
dryness and the remaining crude product was purified by HPLC. The
isolated yield of (½3H2ꢀ V) was 80–85%, while the specific radioactivity
remained the same.
Synthesis of ½3H4ꢀ VIII
16 mg of (VII) coated on 160 mg 5% Pd/C was placed in a reaction
ampoule, evacuated, filled with gaseous tritium at a partial pressure of
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TRITIUM-LABELLED BILL 260
229
400 hPa and the reaction carried out at 1608C for 30 min after which the
reaction mixture was processed as described above. The yield of (½3H4ꢀ
VIII) was 50%, the specific radioactivity 71 Ci=mmol and a radio-
chemical purity greater than 98%.
Synthesis of ½3H4ꢀ V
A suspension of 2 mg of (½3H4ꢀ VIII), 3 mg of (IX), 3:52 mg of K2CO3
and 2:8 mg of KI in 0:1 ml of acetonitrile was heated with stirring for
90 min at 808C in a sealed ampoule. The reaction mixture was diluted
with 1 ml of acetonitrile, filtered off, and the solid residue washed with
acetonitrile ð3 ꢁ 1 mlÞ. The combined filtrates were evaporated to
dryness and the rest was dissolved in 1:5 ml of ethylacetate–methanol
(2:1) mixture. After careful HPLC purification the yield of the desired
compound (½3H4ꢀ V) was about 40%, the specific radioactivity remained
unchanged, and a radiochemical purity of 95–97% was achieved.
Synthesis of tritium-labelled amidoximes (VI)
A solution of 1:9 mg of tritium-labelled (V) in 1 ml of methanol was
treated with 0:4 ml of an aqueous solution of sodium carbonate ð70 mgÞ
and hydroxylamine hydrochloride ð62 mgÞ in a sealed ampoule at 1008C
over 2 h. Following this, the ampoule was cooled to room temperature,
opened, and its contents evaporated to dryness. The resulting product
was extracted by shaking with 1:2 ml of a (1:1) mixture of ethylacetate
and water followed by additional extraction of the aqueous layer with
ethylacetate ð3 ꢁ 0:6 mlÞ. The resulting organic extracts were evapo-
rated to dryness and dissolved in 1 ml of methanol containing 40 ml of
acetic acid. The yield at this stage after chromatographic purification
was 80–85% and the radiochemical purity 95–97%.
Synthesis of tritium-labelled BIIL 260
A suspension of 20 mg of fresh Raney nickel in 1 ml of methanol was
treated with 0:1 ml of acetic acid for 1 h, after which the above
methanolic solution of the labelled precursor containing ð0:5 mgÞ of
(VI) was added and the reaction left for 80 min. The yield of ½3Hꢀ BIIL
260 ranged from 10% to 40%, and the specific radioactivity of the final
preparation was equal to that of the labelled precursor.
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V.P. SHEVCHENKO ET AL.
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