Enantioselective biocatalytic synthesis of (S)-monastrol

Article abstract of DOI:10.1016/j.bmcl.2010.05.063

The first enantioselective biocatalytic synthesis of (S)-monastrol has been developed via an unexpected and unusual enzymatic pathway as suitable route. Whereas attempts for a direct hydrolysis of racemic monastrol were not successful, formation of racemic O-butanoyl monastrol and subsequent enantioselective hydrolysis furnished O-butanoyl (S)-monastrol with 97% ee. Cleavage of the O-butanoyl moiety then gave the desired (S)-monastrol with 96% ee.

Full text of DOI:10.1016/j.bmcl.2010.05.063

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4679–4682
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Enantioselective biocatalytic synthesis of (S)-monastrol
Maria Alfaro Blasco a, Silvia Thumann a, Jürgen Wittmann a, Athanassios Giannis b, Harald Gröger a,
*
a Department of Chemistry and Pharmacy, University of Erlangen-Nuremberg, Henkestr. 42, 91054 Erlangen, Germany
b Institute of Organic Chemistry, University of Leipzig, Johannisallee 29, 04103 Leipzig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The first enantioselective biocatalytic synthesis of (S)-monastrol has been developed via an unexpected
and unusual enzymatic pathway as suitable route. Whereas attempts for a direct hydrolysis of racemic
monastrol were not successful, formation of racemic O-butanoyl monastrol and subsequent enantioselec-
tive hydrolysis furnished O-butanoyl (S)-monastrol with 97% ee. Cleavage of the O-butanoyl moiety then
gave the desired (S)-monastrol with 96% ee.
Received 22 January 2010
Revised 12 May 2010
Accepted 13 May 2010
Available online 25 May 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Anticancer drugs
Biocatalysis
Biotransformations
Enzymatic resolution
KSP inhibitors
Monastrol
Monastrol, rac-1, is the first small molecule inhibitor of the mi-
totic motor Eg5 (kinesin spindle protein, KSP), and represents a
promising current lead structure in anticancer research.1,2 Several
KSP inhibitors are currently being studied in clinical trials and pro-
vide new opportunities for the development of novel anticancer
drugs alternative from the available microtubule targeting agents.2
Based on monastrol as a lead structure novel derivatives bearing
the 4-aryl-3,4-dihydropyrimidin-2(1H)-thione scaffold of rac-1
have been identified recently by Giannis and co-workers, and these
compounds turned out to act as very potent cell-permeable inhib-
itors of Eg5.3 While both enantiomers of monastrol abolished basal
Eg5 ATPase activity, the (S)-enantiomer shows a 15 times higher
potency compared with the opposite (R)-enantiomer.1b Thus, syn-
thetic efforts based on different approaches have been made for
the stereoselective preparation of the (S)-enantiomer of monastrol
[(S)-monastrol, (S)-1, Fig. 1]. A route to (S)-1 reported by Dondoni
et al. is based on the formation of diastereomeric N-3-ribofurano-
syl amides from racemic monastrol, separation of both diastereo-
mers and subsequent amide hydrolysis of the desired
diastereomer.4 An enantioselective Biginelli reaction using
10 mol % of a bis-phenyl-substituted H8-binaphthol-based phos-
phoric acid as a chiral organocatalyst, leading to TBS-protected
monastrol with 91% ee, was developed by Gong and co-workers.5
Zhu and co-workers reported an enantioselective Biginelli reaction
in the presence of a chiral metal catalyst synthesized from ytter-
bium triflate and a hexadentate ligand. At a catalyst loading of
10 mol % the (R)-enantiomer of monastrol was obtained in 80%
yield and with excellent 99% ee.6 Surprisingly, however, to the best
of our knowledge enzymatic routes have not been used so far in
spite of the known efficiency of biocatalysis for the synthesis of
chiral compounds, and their wide use in industrial drug synthesis.7
In the following we report the first enantioselective biocatalytic
synthesis of (S)-monastrol, (S)-1.
Since the racemic compound monastrol, rac-1, is easily pre-
pared in the Biginelli reaction8 starting from simple starting mate-
rials it appeared attractive to us to use this racemate as a starting
material for an enzymatic resolution process.
Inspired by numerous successful examples of enzymatic resolu-
tions via hydrolysis of esters with a stereogenic center in b-posi-
tion,9 at the start of our experiments we considered this type of
reaction as most promising and straightforward for synthesizing
the (S)-enantiomer of monastrol (according to the synthetic strat-
egy shown in Scheme 1). To our surprise, however, when screening
a set of commercially available hydrolases such as, for example, li-
pases from Aspergillus niger, Candida antarctica B (CAL-B), Candida
OH
OH
O
O
O
NH
O
NH
N
H
S
N
H
S
rac-1
(S)-1
* Corresponding author. Tel.: +49 9131 8522554.
E-mail address: harald.groeger@chemie.uni-erlangen.de (H. Gröger).
Figure 1. Structures of monastrol (rac-1) and (S)-monastrol ((S)-1).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.063

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M. Alfaro Blasco et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4679–4682
enantioselective enzymatic hydrolysis, a two-phase system con-
OH
OH
OH
sisting of water and dichloromethane (80:20 (v/v)) has been used.
When screening12 a set of commercially available lipases we were
pleased to find a promising reaction course in the presence of the
lipase from C. antarctica B (CAL-B). By means of this enzyme a con-
version of 52% was achieved under formation of (R)-1 with 71% ee
(Table 1, entry 1). An enantiomeric excess of 77% ee has been found
for the remaining substrate (S)-3a, which corresponds to an enanti-
oselectivity of E = 13 for this biotransformation. Thus, the desired
(S)-configuration is obtained for the remaining substrate, 3a.
lipase,
H2O
O
O
O
O
NH
HO
NH
O
NH
+
N
H
S
N
H
S
N
H
S
(S)-1
(R)-2
rac-1
Scheme 1. Planned retrosynthetic concept to (S)-1 via enzymatic hydrolysis of rac-1.
OH
OH
OH
commercially
available
O
O
O
hydrolases
O
O
NH
*
O
NH
HO
NH
O
water-organic solvent
(80:20 (v/v)),
N
H
(R)-1
+
S
N
H
S
N
S
O
pH 7, r.t., 14-24 h
lipase
O
H
2
rac-1
<5% conversion
O
NH
water-CH2Cl2
(80:20 (v/v)),
O
N
H
rac-3a
S
pH 7, 25 ºC,25 h
Scheme 2. Enzymatic hydrolysis of monastrol, rac-1 (organic solvents: methylene
chloride, DMSO, hexane, MTBE, i-PrOH).
O
O
NH
N
H
(S)-3a
S
rugosa, Mucor javanicus, and Pseudomonas fluorescens as well as
porcine liver esterase we could not identify a suitable biocatalyst
(Scheme 2). Independent of the type of lipase conversions were be-
low 5% for the studied biocatalysts (data not shown in detail). Due
to the lack of a significant activity of this process, further process
development has not been carried out for this route.
Table 1
Enzymatic hydrolysis of O-acetylated monastrol, rac-3a
As a second option we then focused on the use of racemic O-
acylated monastrol derivatives of type rac-3 as substrates. The es-
ter group in rac-3, which is obtained through derivatization of the
phenolic moiety in rac-1, appeared to us as an interesting func-
tional group for a hydrolytic biotransformation. The retro-synthe-
sis of such an approach to the O-acylated derivative (S)-3 of
(S)-monastrol, (S)-1, is shown in Scheme 3. At the same time, how-
ever, this synthetic route is challenging since in these substrates of
type rac-3 the stereogenic center and the functional group for the
biotransformation are separated from each other by a planar aro-
matic moiety. So far only a few examples are reported for enzy-
matic resolutions of compounds with such a so-called remote
stereogenic center.10
Entrya Lipase Conv.g (%) ee of (R)-1 (%) ee of (S)-3a (%) Eh
1
2
3
4
5
C. antarctica Bb 52
71
66
17
8
77
15
5
8
1
13
6
1
1
1
C. rugosac
A. nigerd
33
35
51
9
P. fluorescense
B. cepaciaf
6
a
b
c
d
e
f
For the experimental protocol, see Ref. 12.
Candida antarctica B (CAL-B), commercial product: lipase Novozym 435.
Candida rugosa, type VII; commercial product from Sigma.
Aspergillus niger, commercial product: lipase AS Amano.
Pseudomonas fluorescens, commercial product: lipase AK Amano 20.
Burkholderia cepacia, commercial product: lipase PS Amano IM.
g
Conversion was determined from the resulting crude product by means of
proton NMR spectroscopy.
The E value (enantioselectivity) has been calculated by means of the measured
ee values of (R)-1 and (S)-3a.
In our initial screening for a suitable biocatalyst O-acetylated
monastrol, rac-3a,11 served as a substrate. This compound was eas-
ily prepared via acetylation of monastrol, rac-1, using acetic anhy-
dride in the presence of DMAP.11 As a reaction media for the
h
With other enzymes the resolutions based on hydrolysis of rac-3a
proceeded less successfully (Table 1, entries 2–5). A low conversion
of 9% was obtained when using a lipase from Burkholderia cepacia
(entry 5). The use of a lipase from C. rugosa led to an improved con-
version of 33%, but a non-satisfying enantioselectivity with an E va-
lue of 6 was found in this experiment (entry 2). Good to high
conversions of 35% and 51% were observed with lipases from A. ni-
ger and P. fluorescens, respectively. However, both reactions pro-
ceeded with very low enantioselectivities, which are indicated by
the E value of only 1 for both reactions (entries 3,4).
After having identified a suitable biocatalyst (with CAL-B), we
focused on the study of the impact of the acyl moiety on conver-
sion and enantioselectivity in order to optimize the ‘leaving group’
in the hydrolytic process. When investigating the influence of sev-
eral aliphatic acyl moieties, O-butanoylated monastrol, rac-3b,13
turned out as the most suitable substrate. In the enzymatic hydro-
lysis of this substrate rac-3b an improved enantioselectivity with
an E value of 20 was obtained (Scheme 4). When this resolution
was stopped at a conversion of 59%, subsequent work-up led to
OH
rac-1
O-acylation
O
O
O
NH
O
R
N
H
(R)-1
S
O
lipase,
H2O
O
O
NH
+
O
R
N
H
S
O
rac-3
(3a: R = Me
O
NH
3b: R = n-Pr)
N
H
(S)-3
S
Scheme 3. Retrosynthetic route to (S)-3 via enzymatic hydrolysis of rac-3.

M. Alfaro Blasco et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4679–4682
4681
In summary, we reported the first enantioselective biocatalytic
synthesis of (S)-monastrol, (S)-1, which has been realized by means
of an unexpected and unusual enzymatic resolution of a substrate
with a remote stereogenic center as the preferred route. Notably,
an easily available commercial biocatalyst can be used for this res-
olution as a key step. Currently extension of this new methodology
towards a technology platform for the enantioselective synthesis of
a broad range of derivatives of (S)-monastrol is in progress. In addi-
tion, based on this methodology enzymatic resolutions of other
types of racemic phenol esters bearing a remote stereogenic center
are also planned.
OH
O
O
NH
N
H
(R)-1
S
O
n-Pr
lipase from
O
C. antarctica B
O
48% yield, 66% ee
(CAL-B)
+
O
NH
water-CH2Cl2
(80:20 (v/v)),
O
n-Pr
N
H
S
pH 7, 25 °C, 25 h,
O
O
59% conversion
rac-3b
Acknowledgments
O
NH
The authors thank Jasmin Düring, Katharina Ritter and Eva
Zolnhofer for experimental assistance, and Evonik Degussa GmbH
and Amano Enzymes Inc. for generous supply with chemicals. A
scholarship for M.A.B. by the German Academic Exchange Service
(Deutscher Akademischer Austausch Dienst, DAAD) is gratefully
acknowledged.
N
H
S
(S)-3b
31% yield, 97% ee
Scheme 4. Enzymatic hydrolysis of O-butanoyl monastrol, rac-3b.
References and notes
the hydrolyzed product (R)-1 in 48% yield and with 66% ee. The
remaining desired (S)-enantiomer, (S)-3b, was isolated after col-
umn chromatography in 31% yield and with a high enantiomeric
excess of 97% ee (Scheme 4).14 When starting from substrates of
type 3 bearing acyl moieties such as propanoyl (R = Et) and hexa-
noyl (R = n-pentyl) reactions also proceeded, but gave less satisfac-
1. (a) Mayer, T. U.; Kapoor, T. M.; Haggarty, S. J.; King, R. W.; Schreiber, S. L.;
Mitchison, T. J. Science 1999, 289, 971; (b) Maliga, Z.; Kapoor, T. M.; Mitchison,
T. J. Chem. Biol. 2002, 9, 989.
2. Review: Sarli, V.; Giannis, A. Clin. Cancer Res. 2008, 14, 7583.
3. Gartner, M.; Sunder-Plassmann, N.; Seiler, J.; Utz, M.; Vernos, I.; Surrey, T.;
Giannis, A. ChemBioChem 2005, 6, 1173: with dimethylenastron
compound has been identified which is even more than 100-times more potent
than monastrol.
a related
tory results with
E values of 8
and 6, respectively.15 The
conversions of these reactions were 30% and 11%. Notably, a dra-
matic drop of reactivity was observed when the substituent at
the acyl moiety was an isopropyl group (R = i-Pr) as a representa-
4. Dondoni, A.; Massi, A.; Sabbatini, S. Tetrahedron Lett. 2002, 43, 5913.
5. Chen, X.-H.; Xu, X.-Y.; Liu, H.; Cun, L.-F.; Gong, L.-Z. J. Am. Chem. Soc. 2006, 128,
14802.
6. Huang, Y.; Yang, F.; Zhu, C. J. Am. Chem. Soc. 2005, 127, 16386.
7. Biocatalysis in the Pharmaceutical and Biotechnology Industries; Patel, R. N., Ed.;
CRC Press: New York, 2006.
8. Synthesis of rac-1: A solution of 3-hydroxybenzaldehyde (0.15 mol, 18.32 g),
thiourea (0.15 mol, 11.42 g), ethyl acetoacetate (0.23 mol, 29.28 g, 28.46 mL)
and concd HCl (2 mL) in ethanol (85 mL) was heated under reflux for 6 h. The
reaction mixture was filtered and the solid residue was recrystallized from
ethanol twice to afford rac-1 in 69% yield (30.21 g). The synthesis was carried
out according to a protocol reported in: Lewandowski, K. J. Combinat. Chem.
1999, 1, 105.
tive for an a
-branched substituent (<5% conversion).15
Based on the encouraging result in the enzymatic resolution of
rac-3b we then focused on the subsequent cleavage of the
O-butanoyl moiety in (S)-3b in order to obtain (S)-monastrol,
(S)-1, as the desired final product. Although for such a non-enan-
tioselective hydrolysis ‘standard’ chemical hydrolytic methods
might be also conceivable, the presence of the second ester moiety
(ethyl ester) in the molecule (S)-3b, which should not undergo
hydrolysis, makes this step challenging. After a preliminary screen-
ing of base-catalyzed methods (using aluminum oxide or NaOH as
a base) was not successful (data not shown) we identified a non-
selective biocatalytic hydrolysis as the method of choice. This
method is based on the use of a lipase from C. rugosa, which
showed a high activity but low enantioselectivity (E value of 7)
for the resolution of rac-3b. At the same time, this enzyme does
not possess a (significant) activity for the hydrolysis of the ethyl es-
ter moiety of rac-1 and rac-3 (data not shown), thus making it
attractive for the desired non-enantioselective but chemoselective
hydrolysis of (S)-3b under formation of (S)-1. We were pleased to
find that, as expected, in the presence of the lipase from C. rugosa
the desired (S)-enantiomer of monastrol, (S)-1, was then formed
with a high conversion of >95%. After subsequent work-up (S)-
monastrol, (S)-1, was obtained in 98% yield and with a high enan-
tiomeric excess of 96% ee (Scheme 5).16
9. Reviews: (a) Gais, H.-J.; Theil, F. In Enzymes in Organic Synthesis, 2nd ed.; Drauz,
K., Waldmann, H., Eds.; Wiley-VCH: Weinheim, 2002; Vol. 2,
p 335; (b)
Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic Synthesis—Regio- and
Stereoselective Biotransformations, 2nd ed.; Wiley-VCH: Weinheim, 2005; (c)
Liljeblad, A.; Kanerva, L. T. Tetrahedron 2006, 62, 5831.
10. For remote stereogenic center recognition in enzymatic resolutions, see: (a)
Mizuguchi, E.; Takemoto, M.; Achiwa, K. Tetrahedron: Asymmetry 1993, 4, 1961;
(b) Mizuguchi, E.; Achiwa, K. Tetrahedron: Asymmetry 1993, 4, 2303; (c) Yang,
X.; Reinhold, A. R.; Rosati, R. L.; Liu, K. K.-C. Org. Lett. 2000, 2, 4025; (d)
Hedenström, E.; Nguyen, B.-V.; Silks, L. A., III Tetrahedron: Asymmetry 2002, 13,
835; (e) Colombo, G.; Riva, S.; Danieli, B. Tetrahedron 2004, 60, 741; (f) Hu, S.;
Kelly, S.; Lee, S.; Tao, J.; Flahive, E. Org. Lett. 2006, 8, 1653.
11. Synthesis of rac-3a: monastrol rac-1 (10 mmol, 2.92 g), acetic anhydride
(10 mmol, 1.02 g, 0.95 mL) and DMAP (0.20 mmol, 25 mg) were dissolved in
dried pyridine (5 mL). The reaction mixture was stirred for 3 h under reflux.
After cooling down the reaction mixture to room temperature and adding ice-
water, the mixture was stirred for 1 h at room temperature. Then diluted HCl
(0.1 M) was added until a pH of 2 was achieved. The mixture was agitated
overnight at room temperature and the resulting precipitate was filtered,
washed with water and recrystallized from ethanol to furnish rac-3a in 72%
yield (2.42 g).
12. General protocol for the screening of lipases for the hydrolysis of rac-3a: The
screening was carried out at 25 °C and using a Methrom titrino apparatus (with
an automatic titration unit). After dissolving rac-3a (1 mmol, 334 mg) in
CH2Cl2 (20 mL) and addition of water (80 mL) under formation of a two-phase
system, the corresponding lipase (100 mg) was added. The pH was kept
constant at 7 (by dosage of a solution of NaOH (0.21 M)). After a reaction time
of 25 h the reaction mixture was filtered and then extracted with CH2Cl2
(3 Â 60 mL). The combined organic layers were dried over MgSO4 and the
solvent was evaporated. The resulting crude product was used for the
determination of the conversion (via 1H NMR spectroscopy) and
enantiomeric excess (via chiral HPLC chromatography).
O
n-Pr
OH
O
lipase from
O
O
C. rugosa
O
NH
O
NH
buffer-CH2Cl2
(79:21 (v/v)),
N
H
S
N
H
S
pH 7, 40 °C, 49 h,
>95% conversion
(S)-1
(S)-3b
13. Synthesis of rac-3b has been carried out in analogy to the synthesis of rac-3a
(see Ref. 11) using butyric anhydride (15 mmol, 2.37 g), a modified amount of
DMAP (0.21 mmol, 26 mg) and dried pyridine (8 mL). After recrystallization
from ethanol rac-3b was obtained in 42% yield (1.53 g).
98% yield, 96% ee
97% ee
Scheme 5. Synthesis of (S)-monastrol, (S)-1, via enzymatic hydrolysis of (S)-3b.

4682
M. Alfaro Blasco et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4679–4682
14. Synthesis of (S)-3b: In a Methrom titrino reaction apparatus rac-3b (1 mmol,
0.369 g) was dissolved in CH2Cl2 (20 mL). After subsequent addition of water
(80 mL), which results in the formation of a two-phase system, lipase from
Candida antarctica B (Novozym 435, 200 mg) was added. Then the reaction
mixture was stirred for 25 h at 25 °C, and the pH was kept constant at a pH of 7
(by dosage of a solution of NaOH (0.21 M)). Subsequently, the reaction mixture
was filtered and washed with water (5 mL) and CH2Cl2 (10 mL). The filtrate
was extracted with CH2Cl2 (3 Â 60 mL), and the combined organic layers were
dried over MgSO4. Evaporation of the solvent in vacuo furnished a mixture of
(S)-3b and (R)-1 as a crude product, which was then separated and purified by
column chromatography [CH2Cl2/ethyl acetate, 5:1 (v/v)] to give (S)-3b (31%
yield, 97% ee) and (R)-1 (48% yield, 66% ee) in isolated form.
15. These reactions were carried out in analogy to the synthesis described in Ref.
12 at a substrate loading of 0.83 mmol and with an amount of the lipase from
Candida antarctica B (Novozym 435) of 83 mg.
16. Synthesis of (S)-monastrol, (S)-1: After dissolving (S)-3b (0.284 mmol, 0.103 g,
97% ee; for preparation, see Ref. 14 and Scheme 4) in CH2Cl2 (4 mL), phosphate
buffer (15 mL, pH 7, 50 mM) and lipase from Candida rugosa (type VII,
commercial product from Sigma; 150 mg) were added and the reaction
mixture was stirred for 49 h at 40 °C. Subsequently, the reaction mixture was
filtered and the filtrate was extracted with CH2Cl2 (4 Â 30 mL). The combined
organic layers were dried over MgSO4 and the solvent was evaporated in vacuo.
The desired (S)-monastrol, (S)-1, was formed with >95% conversion and obtained
in 98% yield (0.277 mmol, 0.081 g) and with an enantiomeric excess of 96% ee.