Chemoenzymatic Synthesis of δ-Keto β-Hydroxy Esters as Useful Intermediates for Preparing Statins

Article abstract of DOI:10.1002/ejoc.201600268

Enantiopure (R)-3-hydroxy-5-oxohexanoic acid esters have proven to be useful intermediates in the synthesis of the side chain of statins, in view of the recently described preparation of rosuvastatin and other statins through aldol reactions. Herein, an improved synthesis of these intermediates, by combining chemical and enzymatic reactions, is described. In particular, the selective reduction of a δ-ketal β-keto ester was identified as a key step to obtain derivatives with satisfactory optical purities for use in the synthesis of statins.

Full text of DOI:10.1002/ejoc.201600268

DOI: 10.1002/ejoc.201600268
Communication
Statin Synthesis
Chemoenzymatic Synthesis of δ-Keto ꢀ-Hydroxy Esters as Useful
Intermediates for Preparing Statins
Stefano Tartaggia,*[a] Stefano Fogal,[b] Riccardo Motterle,[b] Clark Ferrari,[b] Marta Pontini,[b]
Roberto Aureli,[c] and Ottorino De Lucchi*[a]
Dedicated to Professor Achille Umani-Ronchi on the occasion of his 80th birthday
Abstract: Enantiopure (R)-3-hydroxy-5-oxohexanoic acid esters bining chemical and enzymatic reactions, is described. In partic-
have proven to be useful intermediates in the synthesis of the
side chain of statins, in view of the recently described prepara-
tion of rosuvastatin and other statins through aldol reactions.
Herein, an improved synthesis of these intermediates, by com-
ular, the selective reduction of a δ-ketal ꢀ-keto ester was identi-
fied as a key step to obtain derivatives with satisfactory optical
purities for use in the synthesis of statins.
Introduction
Statins belong to a class of potent inhibitors of 3-hydroxy-3-
methylglutaryl-coenzyme A (HMG-CoA) reductase, which is the
enzyme controlling the rate-determining step of cholesterol
biosynthesis in the liver, and they are widely used for the pre-
vention of several cardiovascular diseases.[1]
The chemical structure of currently approved synthetic sta-
tins, such as atorvastatin (1), fluvastatin (2), pitavastatin (3), and
rosuvastatin (4), includes a common linear side chain having
two syn hydroxy groups and a terminal carboxylate group,
which is usually converted into the calcium salt to provide the
active form of these drugs. The side chain is linked to a different
aromatic unit on each statin through a double bond with (E)
geometry, except for atorvastatin, in which the chain is aliphatic
(Figure 1).
Figure 1. Current approved synthetic statins.
A couple of strategies for the connection of side chain 6
or 9 with the aromatic part of rosuvastatin, pitavastatin, and
fluvastatin, having general structure 5 or 8, have been success-
fully implemented at the industrial level. Specifically, the cou-
pling of phosphorus or sulfur ylides with aldehyde groups
through Wittig, Horner–Emmons, or Julia–Kocienski reactions is
the most frequently used olefination process, as shown in
Scheme 1.[2]
Therefore, several methodologies for the synthesis of enan-
tiopure side-chain intermediates have been developed for ole-
fination by using ylides. In particular, the chemical[3] or bio-
chemical[4] desymmetrization of 3-hydroxyglutaric anhydride is
the most straightforward method to obtain an optical purity
higher than 99 %. More recently, the asymmetric reduction of
3,5-diketo acid derivatives allowed the efficient introduction of
a chiral center on the side chain through careful optimization
of the catalytic hydrogenation of the ꢀ-carbonyl moiety into a
ꢀ-hydroxy group.[5]
[a] Dipartimento di Scienze Molecolari e Nanosistemi, Università Ca' Foscari
Venezia,
Via Torino 155, 30170 Venezia Mestre, Italy
E-mail: stefano.tartaggia@unive.it
delucchi@unive.it
Divergent approaches based on building the side chain step
by step on the aromatic core through an asymmetric aldol[6] or
Claisen[7] reaction are also well known. However, advantages in
terms of convenience are nullified by low yield and optical pu-
rity if applied for the direct connection of a preformed side
chain with the statin core.
http://venus.unive.it/delucchi/index.htm
[b] F.I.S. - Fabbrica Italiana Sintetici S.p.A.,
Viale Milano 26, 36075 Montecchio Maggiore (Vicenza), Italy
[c] ALCHEMY,
Via Saragat 18, 40052 Baricella (Bologna), Italy
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under http://dx.doi.org/10.1002/ejoc.201600268.
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the preparation of statins, there is a need for efficient methods
to prepare the side chain in few steps with high optical purity.
Results and Discussion
The synthesis of methyl (R)-3-hydroxy-5-oxohexanoate (17)
from the selective reduction of intermediate 16 was primarily
chosen because of the low cost of dehydroacetic acid (15) as
the starting material. Unfortunately, we were unable to obtain
a robust protocol to prepare desired intermediate 17, as asym-
metric reduction experiments performed on 16 did not show a
satisfactory enantiomeric excess (ee), in accordance with the
previously reported data (Scheme 3).[5f]
Scheme 3. Synthesis of 17 by catalytic asymmetric hydrogenation of diketo
ester 16.[5f]
Transesterification of 16 with enantiopure borneol or menth-
ol in quest for double asymmetric induction did not improve
the ee of the final product.
Scheme 1. General strategies based on connecting the side chain to aromatic
unit 5 or 8 for preparing statins through reaction with phosphorus or sulfur
ylides; TBDMS = tert-butyldimethylsilyl.
The fair selectivity observed in the selective reduction of the
inner carbonyl group of 16 was related to overreduction with
hydrogenation occurring at both carbonyl groups. Instead, pro-
tecting the terminal methyl ketone as an acetal allowed effi-
cient optimization of the desired reduction by catalytic
hydrogenation with high yield and enantioselectivity, as re-
cently demonstrated by Zhang.[5c]
Recently, we outlined an efficient aldol condensation proto-
col between aldehydes 5a and 5b with ketone intermediate 13
to prepare rosuvastatin (14a) and pitavastatin (14b) intermedi-
ates in high yields (Scheme 2).[8]
However, having found the direct acetalization of 16 with
diols to be highly unselective,[9] the preparation of desired
product 23 was consequently approached by using a couple of
reported procedures modified by us (Scheme 4).
Scheme 2. Preparation of ketone 13 and aldol condensation with rosuvastatin
and pitavastatin aldehydes 5a and 5b; CDI = 1,1′-carbonyldiimidazole.
Beneficial implications exploited by the aldol approach, with
respect to the use of ylides, were the clear absence of stoichio-
metric amounts of phosphine oxide or sulfoxide byproducts
and the full selectivity toward the (E) isomer.
Scheme 4. Synthesis of δ-ketal-ꢀ-keto ester 23 by using Meldrum's acid (20)
or malonate 22; DCC = dicyclohexylcarbodiimide, DMAP = 4-(dimethyl-
amino)pyridine, DCM = dichloromethane.
The first synthesis of ketone 13 was based on derivatization
of 3-hydroxyglutaric acid intermediate 11, currently used to
Glycol-protected ethyl acetoacetate 18 was therefore chosen
prepare phosphorus ylide derivatives, through the preparation as the starting material, and it was treated with potassium ma-
of corresponding Weinreb amide 12 followed by the addition lonate monomethyl ester (22)[5c] or, more conveniently, Mel-
of MeMgCl (Scheme 2).
A study of an improved synthesis of ketone 13 is described
herein. As the aldol approach for the synthesis of statins might
drum's acid (20)[10] to obtain δ-ketal ꢀ-keto ester 23 in good
yield.
Although the asymmetric hydrogenation of 23 with a Ru/
effectively provide a feasible alternative to the use of ylides in SunPhos catalyst was reported to provide the desired product
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in good yield with 99 % enantioselectivity,[5c] we examined a
different approach to avoid the use of metal catalysts and chiral
ligands, which are often very expensive or even unavailable,
especially in view of a large-scale production.
Table 1. Enzymatic reduction of 23 to 24.[a]
The enantioselective reduction of 23 was therefore per-
formed by using simple chiral boron hydride 25, prepared
in situ from (+)-tartaric acid and sodium boron hydride, which
allowed the use of a very accessible reagent and avoided the
presence of residual amounts of metal on the final product.
Indeed, the reaction of 23 with 25 under an optimized set up
afforded (R)-δ-ketal ꢀ-hydroxy ester 24 in 71 % yield with
80 % ee at 0 °C in THF as the solvent (Scheme 5).
Entry
Enzyme
Conversion [%][b]
ee [%][b]
1
2
3
4
5
6
7
KRED-P1-A04
KRED-P1-B02
KRED-P1-C01
KRED-P2-D11
KRED-P1-H10
KRED-119
2.9
5.7
9.1
4.5
>99
32.7
25.1
22.6
>99
34.5
>99
3
86.3
KRED-130
100 (86)[c]
[a] Reaction conditions: 23 (40 μmol) in 250 m
(2 mL, pH 7) with enzyme (2 mg), NADPNa (1.1 m
dehydrogenase GDH-105 (10 U mL–1), and MgSO4 (2 m
M
sodium phosphate buffer
M
), glucose (80 mM), glucose
M) at 30 °C for 24 h.
[b] Determined by HPLC on a chiral stationary phase. [c] Yield of isolated
product on 32 mmol scale.
Scheme 5. Enantioselective reduction of 23 by using chiral borane 25 as a
reducing agent.
Sheldon and Huisman, who applied a two-step enzymatic ap-
proach for the enantioselective reduction of ethyl 4-chloroace-
toacetate to develop a green-by-design biocatalytic process for
the synthesis of the side chain of atorvastatin.[12]
Under this arrangement, the expensive NADPH cofactor
could be loaded in catalytic amounts, as its regeneration was
associated with the oxidation of glucose to gluconolactone and
gluconic acid.[13]
Amongst several commercially available ketoreductases, the
mutant-KRED series KRED-P1 and KRED-P2 provided conver-
sions that were, in general, lower than 10 %, whereas selectivity
reached the excellent value of 99 % ee with KRED-P1-A04 and
KRED-P1-H10 (Table 1, entries 1 and 5). On the contrary, wild-
type biocatalysts KRED-119 and KRED-130 increased the conver-
sion values up to 86 and 100 %, respectively, and matched our
purpose of obtaining product 24 in good yield with 99 % enan-
tioselectivity in the case of KRED-130 (Table 1, entry 7). Interest-
ingly, we did not find appreciable critical problems associated
with the stability of KRED enzymes during our tests.
After scaling up the procedure to 32 mmol, enantiopure 24
was isolated in 86 % yield by using simple solvent extraction
and without using any further purification steps, such as chro-
matography.
Unfortunately, all our efforts to improve the optical purity of
product 24 by fractional crystallization techniques were fruit-
less, as 24 was always recovered in the form of an oily material,
even after further functionalization with chiral auxiliaries, such
as transesterification with menthol or borneol.
Therefore, the limitation on selectivity attained by this con-
ventional asymmetric chemical reduction strategy prompted us
to search for a different method that could improve the enan-
tioselective reduction of 23 and that, at the same time, could be
compatible and applicable to a possible large-scale production.
Under these circumstances, biochemical transformations
drew our attention.[11] In comparison with most chiral metal
complexes used for asymmetric catalysis, a vast array of natural
and artificial enzymes are now available at relatively low cost
and have the benefit of being easy to handle and renewable,
and they also enable environmentally friendly processes.
In particular, enzymes are active in aqueous buffer solutions
at neutral pH and do not usually require the use of the high
pressure, high temperature, or oxygen-free conditions typically
found in conventional metal-catalyzed reactions. Moreover, all
components used in enzymatic processes are biodegradable,
relatively nontoxic, and avoid contamination problems in the
final product.
Finally, product 13, which is the key intermediate for the
synthesis of statins through the aldol reaction with statin alde-
hydes 5a and 5b, was easily obtained in high yield from 24
through TBDMS protection and glycol acetal cleavage in se-
quence (Scheme 6).
Enzymatic reduction to improve the desired reduction of 23
at the maximum enantioselectivity was finally investigated. Sev-
eral NADPH-dependent ketoreductases (KRED) for the enantio-
selective reduction of the carbonyl function of 23 were there-
fore screened to provide enantiopure product 24 in high yield
(Table 1).
The enzymatic reduction was performed in a phosphate
buffer (pH 7) at 30 °C by using a KRED–glucose dehydrogenase
(GDH) coupled two-enzyme system, as shown in the scheme of
Table 1. A similar configuration was also recently reported by
Scheme 6. Preparation of 13 from 24. Reaction conditions: (a) TBDMSCl, imid-
azole, 98 % yield; (b) 4-toluenesulfonic acid, acetone, 87 % yield.
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Conclusions
The synthesis of ketone 13, an important intermediate used
to prepare statins, was approached through a chemoenzymatic
synthetic sequence as a possible alternative to the use of
hydroxyglutaric acid for the synthesis of the side chain of
statins. Selective reduction of ketal 23 was a crucial step for the
preparation of desired product 13. Careful study of the chemi-
cal reduction by catalytic hydrogenation or chiral borane rea-
gents demonstrated a limitation of such chemical methods, as
the observed ee was limited to 80 %, which is not suitable for
the preparation of statins. Enzymatic reduction was finally dem-
onstrated to be a powerful tool to prepare the desired interme-
diate with good efficacy in terms of yield, enantioselectivity,
and ease of isolation.
Supporting Information (see footnote on the first page of this
article): Experimental procedures and spectroscopic data for all re-
ported compounds.
Acknowledgments
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The authors gratefully acknowledge the Fabbrica Italiana Sintet-
ici S.p.A. (F.I.S.) for financial support.
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Keywords: Asymmetric synthesis · Ketones · Reduction ·
Enzymes · Inhibitors · Statins
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Received: March 7, 2016
Published Online: ■
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Communication
Statin Synthesis
S. Tartaggia,* S. Fogal, R. Motterle,
C. Ferrari, M. Pontini, R. Aureli,
O. De Lucchi* ...................................... 1–5
Chemoenzymatic Synthesis of δ-
Keto ꢀ-Hydroxy Esters as Useful In-
termediates for Preparing Statins
An improved synthesis of an enantio- zymes (e.g., KRED-130) successfully
pure (R)-3-hydroxy-5-oxohexanoic acid provides an intermediate for the syn-
ester is described. The selective reduc- thesis of statins in high yields with ex-
tion of a δ-ketal ꢀ-keto hexanoate cellent optical purities; TBDMS = tert-
methyl ester with ketoreductase en- butyldimethylsilyl.
DOI: 10.1002/ejoc.201600268
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