Biocatalyzed synthesis of both enantiopure fluoromisonidazole antipodes

Article abstract of DOI:10.1016/j.tetlet.2013.07.013

Fluoromisonidazole (FMISO or F-MISO) is a radiotracer for positron emission tomography when ¹⁸F-labeled and is administrated in its racemic form. Herein, a straightforward synthesis of both enantiopure antipodes is proposed through a one-pot two-step microwave protocol to obtain a fluorinated ketone precursor followed by bioreduction using alcohol dehydrogenases from Rhodococcus ruber (ADH-A) or Lactobacillus brevis (LBADH).

Full text of DOI:10.1016/j.tetlet.2013.07.013

Accepted Manuscript
Biocatalyzed synthesis of both enantiopure fluoromisonidazole antipodes
Wioleta Borzęcka, Iván Lavandera, Vicente Gotor
PII:
S0040-4039(13)01152-0
DOI:
http://dx.doi.org/10.1016/j.tetlet.2013.07.013
TETL 43211
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
22 May 2013
26 June 2013
2 July 2013
Please cite this article as: Borzęcka, W., Lavandera, I., Gotor, V., Biocatalyzed synthesis of both enantiopure
fluoromisonidazole antipodes, Tetrahedron Letters (2013), doi: http://dx.doi.org/10.1016/j.tetlet.2013.07.013
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Graphical Abstract
Biocatalyzed synthesis of both enantiopure
fluoromisonidazole antipodes
Leave this area blank for abstract info.
Wioleta Borzęcka, Iván Lavandera and Vicente Gotor
ADH
One-pot two-step
MW
Enantiopure
F-MISO

1
Tetrahedron Letters
journal homepage: www.elsevier.com
Biocatalyzed synthesis of both enantiopure fluoromisonidazole antipodes
Wioleta Borzęcka, Iván Lavandera and Vicente Gotor*
Department of Organic and Inorganic Chemistry, University of Oviedo, Instituto Universitario de Biotecnología, Calle Julián Clavería 8, 33006 Oviedo, Spain
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Fluoromisonidazole (FMISO or F-MISO) is a radiotracer for positron emission tomography
when 18F-labeled and is administrated in its racemic form. Herein, a straightforward synthesis of
both enantiopure antipodes is proposed through a one-pot two-step microwave protocol to obtain
a fluorinated ketone precursor followed by bioreduction using alcohol dehydrogenases from
Rhodococcus ruber (ADH-A) or Lactobacillus brevis (LBADH)
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Fluoromisonidazole
Fluorohydrins
Microwave-assisted synthesis
Biocatalysis
Alcohol dehydrogenases
The relevance of chiral fluorohydrins has been highlighted
with the recent development of novel methodologies based on,
e.g. the use of transition metal catalysis applied to nucleophilic or
electrophilic fluorination,1 through the aperture of epoxides2 or
the reduction of the corresponding ketones.3 These compounds
are very interesting precursors for agrochemicals and
pharmaceuticals,4 and they also present a remarkable role as
liquid crystals,5 and as radiotracers when they are 18F-labeled for
positron emission tomography (PET).6
In particular, fluoromisonidazole (FMISO or F-MISO), 1-(2-
nitroimidazol-1-yl)-3-fluoropropan-2-ol (1, Scheme 1), is a
derivative of the nitroimidazole group of compounds which have
widely been investigated as hypoxic cell sensitizers. 18F-Labeled
derivative of F-MISO has extensively been used to image
hypoxic tissues in vivo with PET technology and also to predict
cancer recurrence in living cells.7
Scheme 1. Previous synthesis of: A) racemic labeled, and B)
enantioenriched unlabeled F-MISO.
Due to the short half-life of 18F-labeled F-MISO, it would be
highly desirable to find a methodology that could easily afford
both stable unlabeled F-MISO antipodes so the different
biological properties of each enantiomer could be more easily
assessed. To achieve this goal, here we propose the synthesis of a
fluorinated ketone precursor (5, Scheme 2) that could be
selectively reduced employing stereocomplementary alcohol
dehydrogenases (ADHs),11 enzymes able to achieve the selective
reduction of carbonyl compounds or the oxidation of alcohol
derivatives under mild hydrogen transfer reaction conditions in
aqueous medium. Starting from this prochiral ketone, this
methodology could afford the final enantiopure fluorohydrin with
a theoretical yield of 100%.
Since this compound presents a chiral center at position 2, it is
administrated in its racemic form although it is well-known that
the biological activity of a racemate can largely differ from each
enantiomer.8 Among the different methodologies described to
obtain this derivative, it can be highlighted the synthesis through
fluoride displacement of a primary tosylated alcohol, and
subsequent deprotection of the tetrahydropyranyl protecting
group from the secondary alcohol in acidic conditions (Scheme
1A).9 Very recently it has been proposed the first asymmetric
synthesis of (S)-1 via enantioselective aperture of an epoxide
precursor with 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), 5-
diazabicyclo[4.3.0]non-5-ene (DBN), benzoyl fluoride, and a
linked (salen)Co catalyst to achieve the kinetic resolution
obtaining it with 93% ee and 40% yield (Scheme 1B).10
Therefore, in order to obtain ketone 5, the straightforward
synthesis shown in Scheme 2 was envisaged. Starting from
commercially available 2-nitroimidazole
2
and 1,3-
———
 Corresponding author. Tel.: +34 985 103452 or +34 985 103448; fax: +34 985 103448; e-mail: lavanderaivan@uniovi.es or vgs@uniovi.es

2
Tetrahedron Letters
dichloroacetone 3, the chlorinated ketone
4
could be
24 h was achieved using a 1:1 ratio between 2 and 3 (entry 11),
synthesized through nucleophilic substitution, which would
afford desired ketone 5 using a fluoride source.
the employment of lower temperatures and a higher amount of
1,3-dichloroacetone afforded very low yields of undesired ketone
6, although conversions of chlorinated derivative 4 were also low
(up to 31%, entries 13 and 14).
To achieve a faster and more efficient synthesis, the reaction
was carried out under microwave conditions,12 studying again
different parameters in order to optimize this transformation
(Table 2). Thus, when this process was performed in the absence
of a base (entries 1 to 3), even at temperatures of 175ºC the
conversion of ketone 4 was extremely low. Therefore, several
basic compounds were added into the reaction medium to obtain
higher conversions with good selectivities. While the proportion
between 2 and 3 was kept 1:3 to minimize the formation of 6, a
substoichiometric amount of the base (0.5 equivalents) was
selected to compare their efficiency as catalysts, and among
them, triethylamine (entry 7), 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU, entry 11), and potassium carbonate (entry 14) afforded the
best results (43-75%) after 10 min at 80ºC, forming disubstituted
ketone 6 in very low quantities (<10%). A lower amount of the
base (entry 6) or the addition of 1,3-dichloroacetone 3 in portions
(entry 13) did not improve the previous results, although leaving
the reaction for a longer time (entry 12), slightly enhanced the
formation of derivative 4. Then, the transformations were carried
out with these three bases in stoichiometric quantity during 30
min (entries 15 to 17), and it was clearly observed that K2CO3
appeared as the best one affording a high conversion of the
desired compound (86%) avoiding the formation of ketone 6.
Gladly, under these conditions and just after 2 min of reaction,
the same results were observed (entry 19), and the employment
of higher temperatures (entry 20), did not improve the conversion
obtained, so conditions shown in entry 19 were selected as the
most appropriate to synthesize derivative 4.
Scheme 2. Proposed retrosynthesis to obtain fluorinated
ketone 5.
In a first set of experiments, different ratios of compounds 2
and 3 were treated with several solvents and bases under various
temperatures to synthesize ketone 4, although it was noticed that
in many cases disubstituted derivative 6 was also obtained as by-
product (Table 1). Thus, using an equimolar ratio between
compounds
2 and 3 to minimize the formation of 6,
dichloromethane (entry 1), acetone (entries 2 and 3), and
acetonitrile (entries 4 and 5) were employed at different
temperatures in the presence of sodium carbonate as base,
observing that while for the first solvent no reaction occurred,
probably due to the low solubility of the base, better results were
achieved with the other two solvents, although undesired ketone
6 (14-30%) was obtained as by-product. Since the use of
acetonitrile allows higher reaction temperatures, we further
studied more conditions with this organic solvent. When the
process was performed under reflux (entries 6 and 7), the
employment of 3 equivalents of the base helped to achieve a
higher conversion of ketone 4, obtaining 57% of conversion after
2 h. Potassium carbonate was also used (entry 8) under these
conditions, affording 64% of conversion of ketone 4 after 2 h of
reaction. In another set of experiments, triethylamine was studied
as base to get access to the desired derivative (entries 9 to 14).
While at room temperature a maximum of 57% of conversion at
Table 1. Synthesis of α-chloro ketone 4 by reaction of nitroimidazole 2 and 1,3-dichloroacetone 3 in the presence of different
bases.
Entry
1
Ratio 2:3
1:1
Conditions
t (h)
24
48
24
48
24
2
2 (%)a
>97
28
4 (%)a
<3
51
6 (%)a
<3
21
28
14
30
57
40
10
5
CH2Cl2, 41ºC, inert atmosphere, Na2CO3 (1 equiv.)
acetone, rt, Na2CO3 (0.5 equiv.)
acetone, 56ºC, Na2CO3 (0.5 equiv.)
CH3CN, rt, Na2CO3 (0.5 equiv.)
CH3CN, 56˚C, Na2CO3 (0.5 equiv.)
CH3CN, 82ºC, Na2CO3 (0.5 equiv.)
CH3CN, 82ºC, Na2CO3 (3 equiv.)
CH3CN, 82ºC, K2CO3 (3 equiv.)
CH3CN, rt, Et3N (1 equiv.)
2
1:1
3
1:1
15
57
4
1:1
40
46
5
1:1
12
58
6
1:1
<3
<3
26
43
7
1:1
2
57
8
1:1
2
64
9
1:1
1
67
28
10
11
12
13
14
1:1
CH3CN, rt, Et3N (1 equiv.)
2
57
36
7
1:1
CH3CN, rt, Et3N (1 equiv.)
24
48
1
22
57
21
22
<3
4
1:1
CH3CN, rt, Et3N (1 equiv.)
22
56
1:2
CH3CN, 0˚C, Et3N (1 equiv.)
CH3CN, 0˚C, Et3N (1 equiv.)
80
20
1:2
2
65
31
a Measured by 1H-NMR.

3
Table 2. Synthesis of α-chloro ketone 4 by reaction of nitroimidazole 2 and 1,3-dichloroacetone 3 (molar proportion 1:3) under
microwave conditions.
Entry
1
Conditions
MW setup
t (min)
10
2 (%)a
>97
96
4 (%)a
<3
4
6 (%)a
<3
<3
<3
<3
<3
<3
3
CH3CN
150˚C, 300 W, 250 psi
175˚C, 300 W, 250 psi
150˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
80˚C, 200 W, 250 psi
120˚C, 200 W, 250 psi
2
CH3CN
30
3
CH3CN/DMSO (4:1)
10
>97
92
<3
8
4
CH3CN, NH3 (0.5 equiv.)
CH3CN, Py (0.5 equiv.)
CH3CN, Et3N (0.2 equiv.)
CH3CN, Et3N (0.5 equiv.)
CH3CN, DMAP (0.5 equiv.)
CH3CN, DABCO (0.5 equiv.)
CH3CN, piperidine (0.5 equiv.)
CH3CN, DBU (0.5 equiv.)
CH3CN, DBU (0.5 equiv.)
CH3CN, DBU (0.5 equiv.)
CH3CN, K2CO3 (0.5 equiv.)
CH3CN, Et3N (1 equiv.)
CH3CN, DBU (1 equiv.)
CH3CN, K2CO3 (1 equiv.)
CH3CN, K2CO3 (1 equiv.)
CH3CN, K2CO3 (1 equiv.)
CH3CN, K2CO3 (1 equiv.)
10
5
10
>97
79
<3
20
43
16
13
20
48
53
43
75
48
62
86
83
86
84
6
10
7
10
54
8
10
82
<3
5
9
10
82
10
11
12
13b
14
15
16
17
18
19
20
10
79
<3
4
10
48
30
43
4
10+10+10 43
14
10
6
10
30
30
30
10
2
15
46
34
14
17
14
16
4
<3
<3
<3
<3
2
a Measured by 1H-NMR. b 1,3-Dichloroacetone was added in three portions every 10 min of microwave reaction.
This transformation was achieved at 2 mmol scale obtaining,
after 2 min of MW reaction and purification, compound 4 with
78% isolated yield. As the next step, fluorination of this ketone to
obtain 5 (Scheme 2), was tried under several conditions. Hence,
the use of KF and ZnF2 in CH3CN at 120˚C for 24 h;13 PhCOF,
HFIP and DBU in TBME at 50˚C for 24 h;10 or CsF in CH3CN
under reflux for 24 h in the presence or in the absence of 18-
crown-6, did not afford any conversion recovering the starting
chlorinated ketone. Due to this, the corresponding chlorohydrin 7
was synthesized by reduction of 4 using NaBH4 in MeOH at 0ºC
in 97% yield, and then the nucleophilic fluorination over this
compound was also tried. In this case it was observed the
formation of a new derivative when employing KF and ZnF2 or
CsF with 18-crown-6 in CH3CN, although unfortunately it was
not the desired racemic F-MISO but the epoxide 8 (Scheme 3).
Due to the fact that this strategy seemed to be not appropriate
to obtain the fluorinated ketone 5, precursor of enantioenriched
F-MISO antipodes, the synthetic route was modified. Since 1-
chloro-3-fluoropropan-2-ol (9) is commercially available, it was
envisaged the oxidation of this derivative into 1-chloro-3-
fluoroacetone (10), which under the previous optimized MW
reaction conditions could directly afford the final derivative 5.
Due to the high difficulty to oxidize halohydrins,14 it was
envisioned the employment of a potent oxidant such as Jones’
reagent. Thus, following a typical procedure,15 using a mixture of
acetone/water (40:1 v v-1) at 0ºC afforded, by TLC, the desired
compound, but when the solvent was evaporated under vacuum
to obtain 10, the high volatility of this ketone avoided its
adequate isolation. In order to circumvent this fact, the use of
microwave conditions in the absence of an organic solvent
employing just alcohol 9 with the Jones’ reagent under different
setups was done (see Supplementary Data). Hence, a temperature
of 80ºC was too high since several by-products were observed in
the reaction crude, therefore 60ºC was fixed as the most
adequate. Maintaining a pressure of 250 psi, several powers
(from 10 to 200 W) and reaction times (from 5 to 15 min) were
studied, but in some cases the formation of by-products was still
detected or incomplete transformations were achieved, so finally
10 W during 15 min were chosen as the most appropriate
Scheme 3. Fluorination conditions over chlorohydrin 7
afforded epoxide 8.

4
Tetrahedron
conditions to obtain ketone 10 after extraction with diethyl
was an excellent substrate for ADH-A and LBADH, affording
ether in excellent yield (>95%).
both chlorohydrin enantiomers in enantiomerically pure form.
Acknowledgments
W.B. thanks the Ministerio de Educación, Cultura y Deporte
for her predoctoral fellowship (FPU Program). I.L. thanks the
Spanish Ministerio de Ciencia e Innovación (MICINN) for
personal funding (Ramón y Cajal Program). Financial support of
this work by the Spanish MICINN (Project MICINN-12-
CTQ2011-24237) is gratefully acknowledged.
Scheme 4. One-pot two-step synthesis of ketone 5 under MW
conditions.
The subsequent nucleophilic substitution of ketone 10 with 2-
References and notes
nitroimidazole
2 was carried out following the reaction
conditions shown in entry 19 from Table 2 obtaining, by NMR, a
conversion of 63% of fluorinated ketone 5. A longer time (30
min) and a higher number of K2CO3 equivalents (7), improved
the conversion until 70%. Due to the fact that both processes
followed similar MW conditions and since, at first, no reagent
incompatibilities were envisaged, the one-pot two-step procedure
at 1 mmol scale was performed avoiding the isolation of volatile
ketone 10, thus synthesizing derivative 5 with 60% of isolated
yield (Scheme 4). As can be noted, the reaction times were longer
to achieve similar conversions than at smaller scale.
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Having in hand α-fluoro ketone 5, precursor of both F-MISO
enantiomers, and α-chloro ketone analogue 9, we decided to use
different alcohol dehydrogenases with opposite stereopreference
to synthesize both alcohol antipodes. Thus, among the different
ADHs we have in our laboratory, alcohol dehydrogenase from
Rhodococcus ruber ADH-A overexpressed in E. coli (E.
coli/ADH-A),16 and commercially available ADH from
Lactobacillus brevis (LBADH)17 were used. The transformations
were performed in Tris.HCl buffer 50 mM pH 7.5 1 mM
NAD(P)H using 2-propanol (5%
v
v-1) to recycle the
nicotinamide cofactor in a ‘coupled-substrate’ approach11 and to
solubilize the hydrophobic substrate in the reaction medium.
Thus, ketone 5 was reduced at 30 mM concentration by E.
coli/ADH-A and LBADH (Scheme 5) affording, respectively,
enantiopure (R)- and (S)-1 with >99% and 92% of conversion
after 24 h. The bioreduction was especially fast with ADH-A,
achieving 86% of conversion after 1 h (see Supplementary Data).
On the other hand, chlorinated substrate 4 was reduced by both
biocatalysts giving rise to enantiopure (R)- and (S)-7 with
quantitative conversion. These transformations were performed at
higher substrate concentration (100 mM) allowing the synthesis
of both antipodes of F-MISO with high to excellent conversions
(75% for LBADH and >99% for E. coli/ADH-A) while the
chlorinated analogue showed also high conversions (75% for
LBADH and 86% for E. coli/ADH-A) after 24 h.
9. Kämäräinen, E.-L.; Kyllönen, T.; Nihtilä, O.; Björk, H.; Solin, O.
J. Label. Compd. Radiopharm. 2004, 47, 37–45.
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Chem. 2010, 131, 340–344.
Scheme 5. ADH-catalyzed synthesis of both enantiopure
antipodes of alcohols 1 and 7.
14. Bisogno, F. R.; García-Urdiales, E.; Valdés, H.; Lavandera, I.;
Kroutil, W.; Suárez, D.; Gotor, V. Chem. Eur. J. 2010, 16, 11012–
11019.
15. Bisogno, F. R.; Cuetos, A.; Orden, A. A.; Kurina-Sanz, M.;
Lavandera, I.; Gotor, V. Adv. Synth. Catal. 2010, 352, 1657–1661.
16. This (S)-selective enzyme has a strong preference for
NADH/NAD+. See, for instance: Edegger, K.; Gruber, C. C.;
Poessl, T. M.; Wallner, S. R.; Lavandera, I.; Faber, K.; Niehaus,
F.; Eck, J.; Oehrlein, R.; Hafner, A.; Kroutil, W. Chem. Commun.
2006, 2402–2404.
17. This (R)-selective enzyme shows a preference for
NADPH/NADP+. See: Wolberg, M.; Hummel, W.; Wandrey, C.;
Müller, M. Angew. Chem. Int. Ed. 2000, 39, 4306–4308.
Herein we have described an efficient protocol to synthesize
both enantiopure fluoromisonidazole antipodes via one-pot two-
step microwave protocol synthesis of ketone 5 plus bioreduction
catalyzed by two stereocomplementary ADHs under the
‘coupled-substrate’ approach using 2-propanol as hydrogen
donor. Moreover, after extensive optimization, the chlorinated
analogue 4 could also be synthesized through MW reaction and

5
Supplementary Material
Supplementary data (general details, procedures for the
preparation of intermediates and final compounds, enzymatic
1
protocols, analytics and H-, 13C-, and 19F-NMR spectral data)
associated with this article can be found, in the online version, at
http://xxxx.