Regio- and stereoselective preparation of ascomycin-d1 and FK 506-d1

Article abstract of DOI:10.1002/jlcr.558

The immunosuppressive macrolides ascomycin 1 and FK 506 2 were stereoselectively deuteriated at C(32) using Curran's radical translocating method. Both AIBN and Et₃B/O₂ were tested as radical initiator for the radical translocation/reduction step with Bu₃SnD as reducing agent. Despite only minor structural differences, ascomycin and FK 506 showed remarkably different behaviour under the radical translocation/reduction conditions. Higher stereoselectivities were observed with Et₃B/O₂ as initiator, presumably due to lower reaction temperatures applied in this case. Copyright

Full text of DOI:10.1002/jlcr.558

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 361–370.
DOI: 10.1002/jlcr.558
Research Article
Regio- and stereoselective preparation of
ascomycin-d1 and FK 506-d1
Murat Acemoglu1*, Hendrik Andres2 and Thomas Moenius2
1 Chemical and Analytical Development, Novartis Pharma AG, CH-4002 Basel,
Switzerland
2 Preclinical Safety, Novartis Pharma AG, CH-4002 Basel, Switzerland
Summary
The immunosuppressive macrolides ascomycin 1 and FK 506 2 were
%
%
stereoselectively deuteriated at C(32) using Curran’s radical translocating
method. Both AIBN and Et3B/O2 were tested as radical initiator for the radical
translocation/reduction step with Bu3SnD as reducing agent. Despite only
minor structural differences, ascomycin and FK 506 showed remarkably
different behaviour under the radical translocation/reduction conditions.
Higher stereoselectivities were observed with Et3B/O2 as initiator, presumably
due to lower reaction temperatures applied in this case. Copyright # 2002
John Wiley & Sons, Ltd.
Key Words: stereoselective deuteriation; radical translocation; ascomycin;
FK506
Introduction
Translocation of radical sites by intramolecular H-atom transfer have
found many applications in organic synthesis, e.g. remote functionalisa-
tion,1 translocation with subsequent addition reaction2 or more
frequently, translocation with subsequent cyclisation.3–7 Protecting/
*Correspondence to: M. Acemoglu, Chemical and Analytical Development, Novartis Pharma AG,
WSJ-145.965, CH-4002, Basel, Switzerland. E-mail: murat.acemoglu@pharma.novartis.com
Copyright # 2002 John Wiley & Sons, Ltd.
Received 11 October 2001
Revised 19 November 2001
Accepted 21 November 2001

362
M. ACEMOGLU ET AL.
radical translocating (PRT) groups, such as (2-bromophenyl)dimethyl-
silyl ether were introduced by Curran et al.,5,6 and were designed to
serve a dual function, as a protecting group and as a group to selectively
activate a remote C–H bond of the molecule for a radical bond forming
reaction. Recently, the 1,5-hydrogen translocation was utilised for
highly enantioselective synthesis of b-substituted b-amino acids by
1,3-asymmetric induction.8
Curran et al.7 demonstrated the suitability of the (2-bromophenyl)di-
methylsilyl derivatives for the regio- and stereoselective [2H]-labelling of
rapamycin at position C(42). Curran’s method was adapted for tritium
labelling of RAD001, a new immunosuppressant.9 In this paper, the
application of the method to the regio- and stereoselective preparation
of [2H]-labelled derivatives of ascomycin and FK 506 is reported.
Results and discussion
Synthesis of the (o-bromophenyl)dimethylsilylether derivatives
of ascomycin and FK506
For the regioselective introduction of the radical translocating (2-
bromophenyl)dimethylsilylether group, ascomycin ð1Þ was silylated with
TBDMS-Cl at positions O–C(24) and O–C(32), to give the bis-
%
(TBDMS)-ether 3 (Scheme 1). The TBDMS-ether at O–C(32) was
%
cleaved regioselectively with 40% aqueous HF in CH3CN10 to obtain
the corresponding mono-(TBDMS)-ether 4 in 93% yield. The reaction
%
of compound 4 with (2-bromophenyl)dimethylchlorosilane ð5Þ pro-
ceeded smoothly with imidazole as base in dimethylformamide and gave
%
%
the corresponding silyl ether 6 in 92.8% yield. An excess (2.0 moleq.) of
%
the silylating agent 5 was necessary for complete conversion. The
%
silylating agent, (2-bromophenyl)dimethylchlorosilane ð5Þ was prepared
%
from 1,2-dibromobenzene and dimethyldichlorosilane by a slight
modification of a known procedure.5,11 The process was modified to
minimise side reactions (formation of benzyne and multiple metallation/
polymerization processes) as well as to protect the highly sensitive
product against hydrolysis. For this purpose, BuLi was added dropwise
to a solution of 1,2-dibromobenzene at ꢀ1108C and the formed, mono-
lithiated intermediate was allowed to react immediately by parallel slow
addition of a solution of dimethyldichlorosilane. To avoid hydrolysis by
uptake of water during work-up and to ensure dryness of the crude
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REGIO- AND STEREOSELECTIVE PREPARATION
363
Scheme 1. Preparation of the (o-bromophenyl)dimethylsilylether derivatives of
ascomycin and FK 506
product, a solvent exchange was performed by diluting the reaction
mixture with cyclohexane, distillation of the ether solvents, filtration of
the formed suspension and subsequent evaporation to dryness. The
crude product was distilled in Kugelrohr to obtain compound 5 in 65%
%
yield. If purified by Kugelrohr distillation, compound 5 was stable for
%
several months in DMF-solution at 0–48C.
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364
M. ACEMOGLU ET AL.
Starting from FK 506 ð2Þ; the corresponding (o-bromophenyl)di-
methylsilyl derivative 9 was prepared in comparable yields using the
%
%
same procedures as described for the preparation of 6 (Scheme 1).
%
Synthesis of 24-O-TBDMS-32-[2H]-ascomycin
When the ascomycin derivative 6 was exposed to the classical radical
%
translocating/reducing conditions with AIBN and Bu3SnD in refluxing
benzene, slow decomposition of the starting material was observed
without the formation of significant amounts of the desired product.
However, with Et3B/O2 as radical initiator and THF as solvent at room
temperature, 76% conversion to deuteriated products was achieved
2
(Scheme 2). The H-NMR spectrum of the crude product mixture
revealed that ca 30% of the deuterium label was located at the aromatic
ring (multiplet at 7.5–7.8 ppm) and ca 70% of it was on the ascomycin
residue (broad multiplets at 3.2–3.8 and 1.0–1.5 ppm). It is worth
mentioning that only one deuterium atom can be transferred to each
2
molecule by this method. From the H-NMR data given above it
follows that ca 70% of the initially formed aryl radicals lead to
translocation and in ca 30% of the occurrences, the aryl radicals were
directly reduced by Bu3SnD. Based on model studies with silylated
cyclohexane-1,2-diols,7 the hydrogen abstraction from position C(32) of
ascomycin (1,5-hydrogen shift) was supposed to dominate over
abstraction from positions C(31) and C(33) (1,6-hydrogen shift).
Indeed, the integral of the signal at 1.0–1.5 ppm, corresponding to
deuterium at C(31), showed 55% deuteriation at this C-atom. Due to
overlapping broad signals, the exact ratio of deuteriation at C(33) could
2
not be determined from the H-NMR spectrum. However, although a
higher ratio of deuteriation at C(33) by comparison to C(31) may be
assumed as a consequence of oxy substitution at C(33), the order of
magnitude of deuteriation must be similar in both cases, since both
products are formed via a 7-ring transition state. Thus, minor
deuteriation at C(33) and high regioselectivity of the desired radical
translocation and deuteriation at C(32) can be concluded. After the
radical translocation/reduction step, the phenyl(dimethyl)silyl ether was
cleaved selectively (Scheme 2) by hydrolysis of the product mixture with
acetic acid/H2O in THF. As expected, a mixture of 24-O-TBDMS-32-
[2H]-ascomycin ð32-½2Hꢁ-4Þ and unlabelled 24-O-TBDMS-ascomycin ð4Þ
%
%
was obtained. 32-½2Hꢁ-4 was formed from the hydrolysis of compound
%
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REGIO- AND STEREOSELECTIVE PREPARATION
365
Scheme 2. Deuterium labelling of ascomycin by radical translocation/reduction
10 whilst the hydrolysis of 11 and of the starting material 6 led to the
%
formation of unlabelled 4:
%
1
2
The product mixture was analysed by MS, H-NMR, H-NMR, and
by HPLC. MS analysis revealed a ratio of 53:47 labelled to unlabelled
2
product. The H-NMR spectrum confirmed the high regioselectivity of
the radical translocation/reduction step, showing a broad multiplet at
2
1
3.2–3.7 ppm for H–C(32) as the only significant signal. The H-NMR
spectrum of the mixture was almost identical to that of unlabelled 24-O-
TBDMS-ascomycin ð4Þ, exhibiting minor differences in the shape of the
%
signal of H–C(33) at ca 3.05 ppm. The hydrogen abstraction and
2
subsequent H-incorporation at C(32) was also stereoselective, giving
rise to minor epimerization at this carbon atom. In the HPLC, 24-O-
TBDMS-32-[2H]-epi-ascomycin ðepi-32-½2Hꢁ-4Þ appeared at DRt=ca
%
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M. ACEMOGLU ET AL.
2.0 min (4.7 area%) compared to 24-O-TBDMS-32-[2H]-ascomycin
ð32-½2Hꢁ-4Þ (95.3 area%) and was identified by LC–MS and by co-
chromatography with an authentic sample. The epimerization must
%
have occurred during the reduction of the intermediate radical at C(32)
with Bu3SnD and therefore, each epimer molecule must be deuteriated
at position C(32), whilst the total ratio of deuteriated products including
the epimer was 53 mol% according to MS.
From the data given above, a ratio of ca 9% can be assumed for
epimerization at C(32) and 91% of the radicals formed at C(32) must
have been reduced stereoselectively to afford 32-½2Hꢁ-4:
%
Synthesis of 24-O-TBDMS-32-[2H]-FK 506
Under the radical translocation/reduction conditions, the FK 506
derivative 9 reacted differently to the ascomycin derivative 6 : Both
%
%
Et3B/O2 and AIBN could be used as initiators in this case (Scheme 3).
With Et3B/O2 as initiator, the conversion was 63%. According to the
2H-NMR spectrum, ca 65% of the deuterium label was located on the
FK 506 moiety of the molecule and the remainder on the aromatic ring.
Regioselective desilylation of the mixture at position O–C(32) with
AcOH/H2O gave a mixture of the corresponding 24-O-TBDMS-
derivatives 8 and 32-½2Hꢁ-8 (Scheme 3). MS analysis of the product
%
%
revealed a ratio of ca 41:59 for labelled:unlabelled product. The
2H-NMR spectrum showed a significantly higher deuteriation (ca 7%)
at C(31) and C(33) by comparison to the ascomycin series, where the
deuteriations at these carbons were negligible. Finally, the ratio of
epimerization at C(32) was determined to be ca 3% by HPLC analysis.
AIBN, which was rather ineffective as initiator in the ascomycin
series, could be used in the case of FK 506 derivative 9 to initiate the
%
radical translocation/reduction in refluxing benzene. The crude product
mixture was then selectively desilylated at position O–C(32) to obtain a
mixture of labelled/unlabelled 24-O-TBDMS-FK 506. MS analysis
revealed a ratio of 68:32 for 32-½2Hꢁ-8 : 8: The epimerization at C(32)
%
%
was higher in this case (ca 10%) compared to Et3B/O2 as initiator,
presumably due to the difference in the reaction temperature.
Complete desilylations of the mono- or bis-silylether derivatives of
ascomycin and FK 506 were performed with 40% aqueous HF in
CH3CN at room temperature for 2.5 h, to obtain ascomycin or FK 506
in ca 70% yield.
Copyright # 2002 John Wiley & Sons, Ltd.
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REGIO- AND STEREOSELECTIVE PREPARATION
367
Scheme 3. Deuterium labelling of FK 506 by radical translocation/reduction
Conclusions
The protecting/radical translocating (2-bromophenyl)dimethylsilyl
group can be used for regio- and stereoselective [2H]-labelling of
ascomycin and FK 506. Higher regio- and stereoselectivities can be
achieved by performing the radical translocation/reduction step at lower
temperatures. Despite only minor structural differences, the correspond-
ing ascomycin and FK 506 derivatives behave significantly differently
during the radical translocation/reduction step regarding reactivity and
regio-, as well as stereoselectivity. These results suggest different
conformations for the two macrocyclic substances.
Copyright # 2002 John Wiley & Sons, Ltd.
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368
M. ACEMOGLU ET AL.
Experimental
Chemicals were purchased from commercial suppliers. Intermediates
and precursors were identified by either chromatographic and/or
spectroscopic methods (NMR, MS). Mass spectra were measured on
a VG 70-SE mass spectrometer in a nitrobenzylalcohol/lithium iodide
matrix (FAB, Xe: 8 keV) or on a FINNIGAN TSQ 7000 spectrometer
in acetonitrile/water (ESI +QlMS). NMR spectra were measured on a
BRUKER AMX 400 spectrometer.
HPLC: HP Series 1050 with UV–VIS detector; stationary phase:
Nucleosil 120/Cl8, 5 mm, 250 ꢂ 4.6 mm; mobile phase: gradient of A
and B, consisting of H2O/CH3CN/t-butyl methyl ether /H3PO4,
A=(650:240:60:0.2) v/v and B=(200:660:60:0.2) v/v. B:A=5 min
(70:30); 8–22 min (100:0); 27–32 min (70:30).
Modified synthesis of (2-Bromophenyl)dimethylchlorosilane ð5Þ
%
BuLi (1.55 M solution in hexane, 56.46 g, 125 mmo1) was added
dropwise to a solution of 1,2-dibromobenzene (30.4 g, 125 mmol) in
1000 ml of THF/diethyl ether (1:1) at ꢀ1108C and the formed, mono-
lithiated intermediate was allowed to react immediately by parallel slow
addition of a solution of dimethyldichlorosilane (24.30 g, 187 mmol) in
THF/diethyl ether (1:1). When the addition was completed, the reaction
mixture was stirred for 1 h at ꢀ1108C and allowed to warm up to room
temperature. Then, a solvent exchange was carried out by diluting with
cyclohexane and distilling off the ether/THF mixture. The formed
precipitate was removed by filtration and the solvent was evaporated.
The resulting crude liquid was purified by Kugelrohr-distillation at
80–1008C and 0.20–0.15 mbar. Yield: 19.5 g (65%) of (2-bromophe-
nyl)dimethylchlorosilane ð5Þ as colourless liquid.
%
Synthesis of 24-O-TBDMS-32-O-(2-bromophenyl)dimethylsilylether
derivatives of ascomycin and FK 506 (compounds 6 and 9)
%
%
A solution of 4 or 810ð3:17l mmolÞ and imidazole (0.876 g, 12.74 mmol)
%
%
in DMF (100 ml) was cooled down to ꢀ98C to ꢀ138C and was treated
dropwise with a solution of (2-bromophenyl)dimethylsilyl-chloride ð5Þ
in DMF (4.6 g of a 34.5% w/w solution, 6.35 mmol) while maintaining
%
the temperature at ꢀ98C to ꢀ138C. Stirring was continued at this
temperature for 90 min, until a TLC (silica gel, hexane/2-propanol 95:5
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REGIO- AND STEREOSELECTIVE PREPARATION
369
as eluent) indicated the completion of the conversion. The reaction
mixture was then poured onto PBS-buffer, pH 7 (600 ml) and diethyl
ether (200 ml) was added. The mixture was stirred for 45 min at room
temperature, the phases were separated and the H2O-phase was
extracted with diethyl ether (2 ꢂ 150 ml). The organic phase was washed
with H2O (150 ml), dried over anhydrous Na2SO4 and the solvent was
evaporated. The crude product was purified by flash chromatography
on silica gel with hexane/2-propanol/N-ethyl-diisopropylamine (95:5:0.1)
as mobile phase, to obtain the pure compounds 6 (93% yield) or 9 (91%
%
%
yield) as white solids. 1H-NMR and MS spectra of the products were in
accordance with the proposed structures.
Procedure for [2H]-labelling by radical translocation/reduction
with Et3B/O2 as initiator
O2-gas (5 ml, 0.22 mmol) was bubbled through a solution of 6 or 9
%
%
(0.22 mmol), Bu3SnD (0.132 g, 0.442 mmol) and Et3B (0.22 ml of a 1 M
THF solution, 0.22 mmol) in dry THF (9 ml) during 3 min at room
temperature and the mixture was stirred for an additional 6 h. For
work-up, the reaction mixture was diluted with diethyl ether (10 ml) and
DBU (0.04 g, 0.257 mmol) was added. After addition of sufficient
amounts of iodine (until coloured solution), the solution was filtered
over silica gel and evaporated to obtain a crude product mixture
including starting material. The crude product mixture was directly
hydrolysed as follows: a solution of the product mixture (1 g) in THF
(60 ml) was treated atꢀ58C with H2O (22.5 ml) and subsequently with
acetic acid (22.5 ml), while maintaining this temperature. The mixture
was stirred for an additional 1 h atꢀ58C to complete the reaction.
For work-up, the reaction mixture was diluted with diethyl ether
(150 ml) and the solution was extracted with H2O (2 ꢂ 300 ml).
The H2O-phases were combined and were extracted with diethyl
ether (2 ꢂ 150 ml). The combined organic phases were washed with
brine (250 ml), dried on Na2SO4 and the solvent was evaporated to
obtain the crude product, which was purified by column chromato-
graphy on silica gel with hexane/2-propanol/N-ethyl-diisopropylamine
(85:15:0.1) as mobile phase. Starting from the ascomycin derivative 6; a
%
47:53 mixture of 4=32-½2Hꢁ-4 was obtained as product in 51%
%
%
overall yield. A 59:41 mixture of 8=32-½2Hꢁ-8; was obtained in 62%
overall yield, when the FK 506 derivative 9 was used as starting
%
%
%
material.
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M. ACEMOGLU ET AL.
Procedure for radical translocation/reduction with AIBN as initiator
Essentially, the procedure of Curran et al.7 was applied: a solution of
24-O-TBDMS-32-O-(o-bromophenyl)dimethylsilyl-FK 506 ð9Þ (0.102 g,
0.09 mmol), AIBN (0.003 g, 0.018 mmol) and Bu3SnD (0.0325 g,
%
0.108 mmol) in benzene (3 ml) was heated to reflux for 2.5 h. The
reaction mixture was then cooled down to room temperature, the
solution was filtered over silica gel and the silica gel was washed using
THF (40 ml) for complete elution of the product/educt mixture. The
mixture was hydrolysed with acetic acid/H2O in THF as described
above and the crude product was purified by column chromatography
on silica gel with hexane/2-propanol/N-ethyl-diisopropylamine
(85:15:0.1) as mobile phase to obtain a 32:68 mixture of 8=32-½2Hꢁ-8
%
%
in 45% overall yield.
Acknowledgements
.
We thank Mr R. Schreiber and Miss N. Brandlin for technical
assistance, Mr J. France for the NMR-spectra and Dr C. Guenat for the
MS spectra and MS calculations.
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Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 361–370