Practical Manufacture of 4-Alkyl-4-aminocyclohexylalcohols Using Ketoreductases

Article abstract of DOI:10.1021/acs.oprd.7b00048

The diastereoselective preparation of cis- or trans-4-substituted-4-aminocyclohexyl alcohols by conventional chemical processes reported in the literature requires long synthetic sequences and is not amenable to scale-up. In the past decade, ketoreductases (KREDs) have emerged as a powerful approach for the preparation of chiral secondary alcohols from prochiral ketones. This paper describes the diastereoselective preparation at the kilo scale of cis-allyl N-(4-hydroxy-1-methyl-cyclohexyl)carbamate (6a) via ketoreductase transformation. The methodology was also applicable to a set of different analogous cyclic ketones.

Full text of DOI:10.1021/acs.oprd.7b00048

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Communication
Practical manufacture of 4-alkyl-4-
aminocyclohexylalcohols using ketoreductases
Susana Garcia-Cerrada, Laura Redondo-Gallego, Francisco
Jose Martínez-Olid, Juan A. Rincon, and Pablo Garcia-Losada
Org. Process Res. Dev., Just Accepted Manuscript • Publication Date (Web): 20 Apr 2017
Downloaded from http://pubs.acs.org on April 20, 2017
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Practical manufacture of 4-alkyl-4-
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aminocyclohexylalcohols using ketoreductases
Susana GarcíaꢀCerrada,* Laura RedondoꢀGallego,
Francisco MartínezꢀOlid, Juan A. Rincón, Pablo
GarcíaꢀLosada*
Centro de Investigación Lilly S.A., Avda. de la Industria, 30, Alcobendas-Madrid 28108, Spain
AUTHOR EMAIL ADDRESS: garcia_susana_maria@lilly.com; garciaꢀlosada_pablo@lilly.com
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Table of contents
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KEYWORDS: Aminocyclohexylalcohols, stereospecific ketone reduction, biocatalysis,
ketoreductases.
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ABSTRACT: Diastereoselective preparation of cisꢀ or transꢀ 4ꢀsubstitutedꢀ4ꢀaminocyclohexyl
alcohols by conventional chemical processes reported in the literature requires long synthetic
sequences and are not amenable to scaleꢀup. In the last decade, ketoreductases (KREDs) have
emerged as a powerful approach for the preparation of chiral secondary alcohols from prochiral
ketones. This paper describes the diastereoselective preparation at kilo scale of cisꢀallyl Nꢀ(4ꢀ
hydroxyꢀ1ꢀmethylꢀcyclohexyl)carbamate (6a) via ketoreductase transformation. The
methodology was also applicable to a set of different analogous cyclic ketones.
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1.-Introduction
Diastereoselective synthesis of cisꢀ and transꢀ 4ꢀsubstitutedꢀ4ꢀaminocyclohexyl alcohols and
their analogs is in demand due to their application as building blocks in the pharmaceutical
industry for the synthesis of more complex structures.1 Unfortunately, synthetic routes towards
these compounds still remain challenging. Other than chromatographic separation of the cis/trans
isomer mixtures, some interesting examples involving sequential reactions have been developed,
but neither the isolated yields neither the selectivity were completely satisfactory.2 A highly
efficient system based on allyl addition alkyl to aldehydes and ketones catalyzed by rhenium
oxide has also been reported to afford excellent selectivity (98%) for alkyl ketones, but the
selectivity decreased for cyclic ketones.3
Biocatalysis is well matched to chemical synthesis in the pharmaceutical industry.4 A
significant number of pharmaceutical products are optically active, and many compounds have
multiple chiral centers. The efficient generation of chiral centers often requires the use of
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different catalytic systems including enzymes, which possess a unique mechanistic action.
Catalytic ketone reductions using enzymes (KREDs) are a reliable method of accessing optically
enriched alcohols in a highly stereoselective way and often exceed the ability of conventional
chemical catalysts to perform the same transformations.
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In our ongoing research focused on the diastereoselective preparation of protected cisꢀ or
transꢀ 4ꢀalkylꢀ4ꢀaminocyclohexylalcohols, we have successfully executed a selective enzymatic
ketone reduction to generate the cis and trans isomers at kilogram scale.
2.-Results and Discussion
The synthesis of the corresponding starting ketones for the alcohol preparation was performed
following the synthetic route described below (Scheme 1).
Scheme 1. General scheme for the preparation of the 4-amino-4-substituted ketones
Stereoselective
Reduction
i)
ii)
iii); iv)
v)
1
2
3a-g
4a-g
5a-g
6a-g
7a-g
Reagents and conditions: (i) ethylene glycol, p-TsOH; (ii)LDA, -78ºC, R-Ior R-Br (60-99%);
(iii) NaOH, EtOH; (iv) Ph3PON3, TEA, CH3CN, allyl alcohol (60-95%); (v) HCl, Acetone (58-93%)
R= Me (a), Et (b), n-Pr (c), Propargyl (d), MOM (e), n-Pentyl (f), i-Bu (g)
Starting from commercially available ethyl 4ꢀoxocyclohexanecarboxylate (1), and after
protection of the ketone group with ethylene glycol,5 the alkylation at the αꢀcarbonyl position
was performed by treatment of the lithium enolate with the corresponding haloꢀalkyl derivative
at low temperature6 to afford 4ꢀsubstituted intermediate compounds 3a-g. Ethyl ester hydrolysis
followed by Curtius rearrangement in the presence of allyl alcohol7 installed the desired
quaternary amine functionality onto the cyclohexyl ring, protected as the allyl carbamate
(compounds 4a-g). Finally, acidꢀcatalyzed ketone deprotection led to the desired 4ꢀsubstituted
cyclohexyl ketones 5a-g with good yields.8
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Our main purpose was the preparation of the cis alcohols 6a-g as pure isomers. However,
synthesis by conventional methods typically afforded a mixture of the two diastereomers that
was difficult to separate by chromatographic methods. In fact, absence of ultraviolet (UV)
chromophore, low molecular weight and poor mass spectroscopy (MS) ionization negatively
impacted on detection.
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Therefore, nuclear magnetic resonance (NMR) was chosen for evaluation of conversion and
selectivity. The comparison of the integrals from the alkyl group resonances of the ketone
starting materials (compounds 5a-g), and the final alcohols (compounds 6a-g and 7a-g), was
taken as reference to measure the conversion. Cis/trans assignation was achieved through
combination of protonꢀproton coupling constants and NOE correlations, as described in the
characterization section. The cis/trans selectivity was determined by integration of the H1 signal
of each isomer in reaction mixtures samples (around 3.63 ppm for the cis isomer and 3.85 ppm
for the trans isomer).
As a first approach, we tested the reduction of the ketones using standard hydride reducing
agents. Substrate 5a was treated with sodium borohydride, sodium triacetoxyborohydride,
lithium borohydride, zinc borohydride and Lꢀselectride as reducing agents (MeOH or THF as
solvent, 0ºC to 22ºC and then 16 h at 22ºC). Under these conditions, a mixture of two
compounds (6a/7a) was obtained in moderate yield but low selectivity, and only freshly
generated zinc borohydride in tetrahydrofuran9 afforded a 3:1 mixture enriched in cis isomer 6a
(72% yield).
Since these conventional methods provided moderate yields and poor stereoselectivity, we
decided to explore biocatalysis for the preparation of our target compounds. Ketoreductases
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(KREDs) are well established as very useful enzymes for the selective preparation of chiral
secondary alcohols from prochiral ketones under mild conditions (Scheme 2).10
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Scheme 2. General mechanism for KREDs.
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KRED
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NAD(P)+
NAD(P)H
same KRED
or
spontaneous
hydrolysis
GDH
Gluconic acid
Gluconolactone
Glucose
In an initial set of experiments, a screen of a kit of 24 KREDs was carried out for the selective
reduction of ketone 5a following a similar protocol to the reference procedure reported by the
supplier.11 As result of this screening (Table 1), high selectivity for the cis isomer 6a was
observed with most of the enzymes tested, and five gave the cis isomer with very high
conversion and complete diastereoselectivity (entries 5, 6, 13, 17 and 18). Only one enzyme
showed good selectivity for the trans isomer 7a (entry 22).
Table 1. KRED screening results with compound 5a.
Entry
1
KRED
Cofactor
NADP
NADP
NADP
NADP
NADP
NADP
NADP
NADP
NADP
NADP
NADP
NADP
NADP
NADP
Conversion (%)a 6a (%)a 7a (%)a d.e. (%)b
P1ꢀA04
93
97
99
85
91
98
99
99
99
92
97
95
96
91
83
17
66 (c)
88 (c)
84 (c)
94 (c)
>98 (c)
>98 (c)
94 (c)
90 (c)
96 (c)
72 (c)
50 (c)
40 (c)
>98 (c)
78 (c)
P1ꢀA12
P1ꢀB02
P1ꢀB05
P1-B10
P1-B12
P1ꢀC01
P1ꢀH08
P2ꢀB02
P2ꢀC02
P2ꢀC11
P2ꢀD03
P2-D11
P2ꢀD12
94
6
2
3
92
8
97
3
4
5
>98
>98
97
-
6
-
3
7
95
5
8
97
3
9
86
14
25
30
-
10
11
12
13
14
75
70
>98
89
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P2ꢀG03
P2ꢀH07
P3-B03
P3-G09
P3ꢀH12
101
93
93
82
83
>98
>98
97
ꢀc
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NADP
NADP
NADP
NADP
NADP
NAD
84 (c)
86 (c)
>98 (c)
>98 (c)
96 (c)
ꢀc
93
-
96
-
98
3
<50
<50
73
ꢀc
ꢀc
96
ꢀc
76
119
ꢀc
ꢀc
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NAD
130
4
ꢀc
NAD
92 (t)
ꢀc
NADHꢀ101
NADHꢀ110
<50
90
NAD
24
NAD
52 (t)
a Conversion as percentage, checked by 1HꢀNMR, considered 99% when the starting material was not detected.
b d.e. purity as percentage, checked by 1HꢀNMR, considered >98% when the other isomer was not detected.
(c) for cis isomer, (t) for trans isomer.
c d.e. was not measured due to low conversion observed by TLC.
With these preliminary results, an optimization campaign was initiated to find the best enzyme
for the kilogramꢀscale synthesis of cisꢀ allyl Nꢀ(4ꢀhydroxyꢀ1ꢀmethylꢀcyclohexyl)carbamate (6a).
The five hits identified in the screening were tested at 100 mg substrate scale, under similar
conditions but using 5% w/w enzyme loading.12 All gave complete conversion in 24 h but two of
them, KREDꢀP1ꢀB10 and KREDꢀP2ꢀD11, reached complete conversion with a cleaner impurity
profile. With these two enzymes, a second optimization campaign was carried out. Further
changes in temperature, concentration, and enzyme and NADP+ loading were explored. KREDꢀ
P1ꢀB10 performed as the best enzyme for the reduction of allyl Nꢀ(1ꢀmethylꢀ4ꢀoxoꢀ
cyclohexyl)carbamate (5a) to the corresponding cisꢀ allyl Nꢀ(4ꢀhydroxyꢀ1ꢀmethylꢀ
cyclohexyl)carbamate (6a). A great advantage of this enzyme at scale is that it tolerates
isopropanol (IPA) in high concentration as recycling system (as do all the enzymes 1 to 19 in the
kit), which also function as coꢀsolvent at the same time, simplifying the process and reducing the
cost, since the GDH/glucose recycling system is not required.
T
able 2. KRED-P1-B10 NMR conversion.
Entry
time (h)
Conversion (% 6a)
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1.25
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23
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30
51
64
99
Conversion (% 6a)
100
80
60
40
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0
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0
4
8
12
16
20
24
Time (h)
Once KREDꢀP1ꢀB10 had been selected as the best enzyme, a multigramꢀscale proof of concept
run was conducted at 30 g scale under more concentrated conditions, to optimize the
concentration and to characterize the reaction progress (Table 2). The analysis showed that after
just 4 hours of reaction the conversion was 50%, while 24 hours was required for total
conversion, no erosion in diastereoselectivity was observed, giving pure desired compound in up
to 95% isolated yield.
A successful 1 kg scale campaign was finally performed using substrate 5a in a 20 L reactor.13
Compound 6a was isolated in 99% yield and 98% purity (>98% d.e., trans isomer not detected)
by simple direct extraction of the crude mixture.
3.-Extension to analogous alkyl groups
With the excellent results obtained in optimization and scale up in the case of allyl Nꢀ(1ꢀ
methylꢀ4ꢀoxoꢀcyclohexyl)carbamate (5a), the study was extended to other analogues, to better
understand the scope of ketoreductases for the preparation of allyl Nꢀ(4ꢀhydroxyꢀ1ꢀalkylꢀ
cyclohexyl)carbamates.
Scheme 3. General scheme for biocatalytic amino alcohol preparation.
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24 KREDs kit
IPA or GDH/glucose
Buffer/NAD(P)H/MgSO4
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7b-g
5b-g
6b-g
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R= Ethyl (5b);R= n-Propyl (5c); R= Propargyl (5d); R= Methoxymethyl (5e); R= n-Pentyl (5f); R= i-Bu (5g)
In general, most of the enzymes were selective for the cis isomer (6), and only a few of them
for the trans isomer (7) (Table 3).14 Conversion under the screening conditions (10% enzyme
loading w/w, rt, 24 h) was tipically high, especially with the propargyl (d) and methoxymethyl
(e) substituents, but pentyl (f) and isobutyl (g) afforded lower conversion. Steric hindrance
would influence the conversion trends.
Table 3. KRED screening summary with substrates 5a-g.13
Substrate (R)
5a
5b
5c
5d
5e
5f
5g
KRED
P1-B10
P1-B12
P2-B02
P2-D11
P3-H12
130
C a
91%
98%
99%
96%
98%
73%
90%
d.e.b
C a
94%
97%
97%
94%
95%
94%
94%
d.e.b
C a
98%
86%
97%
92%
84%
94%
95%
d.e.b
C a
99%
99%
99%
99%
99%
87%
99%
d.e.b
C a
99%
99%
99%
99%
99%
99%
99%
d.e.b
C a
78%
93%
98%
<50%
<50%
91%
90%
d.e.b
80% (c)
74% (c)
46% (c)
ꢀc
C a
89%
93%
97%
96%
71%
79%
96%
d.e.b
Entry
>98%(c)
>98%(c)
96% (c)
>98%(c)
96% (c)
92% (t)
52% (t)
>98%(c)
>98%(c)
94% (c)
>98%(c)
80% (c)
94% (t)
56% (t)
94% (c)
90% (c)
98% (c)
>98%(c)
31% (c)
48% (t)
75% (t)
98% (c)
>98%(c)
96% (c)
>98%(c)
12% (t)
90% (t)
40% (t)
96% (c)
98% (c)
94% (c)
96% (c)
86% (t)
56% (t)
92% (t)
96% (c)
76% (c)
96% (c)
32% (c)
30% (t)
38% (t)
88% (t)
1
2
3
4
5
6
7
ꢀc
42% (t)
36% (t)
NADH-110
a Conversion as a percentage, checked by 1HꢀNMR, considered 99% when the starting material was not detected.
b d.e. as a percentage, checked by 1HꢀNMR, considered >98% when the other isomer was not detected.
(c) for cis isomer, (t) for trans isomer.
c d.e. was not measured due to low reactivity observed by TLC.
The enzymes that tolerate IPA as recycling system (from 1 to 19 in the Codexis® screening kit)
were the most reactive and almost always showed selectivity for the cis isomer 6. On the other
hand, the enzymes that required GDH/glucose as recycling system (from 20 to 24 in the
Codexis®screening kit) were less reactive, and were selective for the trans isomer 7 in almost all
the examples. Similar behavior was observed for some KREDs across the different substrates.
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Enzymes KREDꢀP1ꢀB10, KREDꢀP1ꢀB12 and KREDꢀP2ꢀD11 were completely selective for the
cis isomer 6 in substrates 5a, 5b and 5d (R: methyl, ethyl and propargyl, entries 1,2,4), and
showed very high cis selectivity (>90%) in substrates 5c (R: propyl, in this case KREDꢀP2ꢀD11
was completely selective) and 5e (R: methoxymethyl). Alkyl size negatively impacted
selectivity, being slightly or significantly reduced for 5f (R: pentyl group), in which the d.e. was
80%, 74% and 46% with enzymes KREDꢀP1ꢀB10, KREDꢀP1ꢀB12, KREDꢀP2ꢀB02 (entries
1,2,3) and 5g (R: isobutyl group; d.e. 76% and 32% with enzymes KREDꢀP1ꢀB12 and KREDꢀ
P2ꢀD11 (entries 2,4). Substrate 5f (R: pentyl) was the only example in which the selectivity for
the cis isomer was lower than 96% with all the enzymes tested.
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Regarding the trans isomer, enzyme KREDꢀ130 showed very good selectivity for the trans
isomer in the case of substrates 5a, 5b, and 5d (entry 6), and KREDꢀNADHꢀ110 was the most
selective in the case of substrates 5c, 5e, and 5g (entry 7). The pentyl substrate 5f was the
example in which lowest trans selectivity was observed.
Interesting results were obtained with ketoreductase KREDꢀP3ꢀH12, wich showed dramatic
changes in selectivity as a function of the substituent, from the cis isomer (96% d.e. in the case
of methyl group 6a) to the trans isomer (86% d.e. in the case of the methoxymethyl group 7e).
To further demonstrate the synthetic applicability and scalability of these procedures, we also
ran the KRED reduction of substrate 5b on larger scale. After some optimization efforts,
including, as previously, variations of enzyme and NADP+ loading, temperature and
concentration conditions, the reaction was performed at 50 g scale of ketone starting material
5b,15 obtaining cisꢀisomer (6b) in 97% isolated yield, with d.e. >98% (the trans isomer was not
detected).
4.-CIS/TRANS isomer assignation by NMR
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The cis/trans assignment of the 4ꢀhydroxycyclohexylcarbamates 6 and 7 was performed
(NMR) through protonꢀproton coupling constants (3JHH) and confirmed by NOE experiments.
The isomer assignment is described in detail for isomer pairs 6a and 7a, the approach being
similar for the rest of the compounds in this communication.
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The assignment of the cis/trans isomers was achieved through the combination of NOEs and
protonꢀproton coupling constants. In both derivatives, the NH of the carbamate substituent shows
NOEs to H2ax and H3eq, whereas the methyl protons show NOEs to H3eq but not to H2ax,
indicating that both isomers adopt a chair conformation in which the carbamate is in an axial and
the methyl group is in an equatorial position (Figure 1). The position of the hydroxyl group was
determined based on the NOEs observed for H1. In 7a, H1 shows NOEs with H2eq and H2ax
whereas in 6a this proton shows NOEs to H2eq and H3ax, indicating that H1 occupies an
equatorial position in 7a and an axial position in 6a and, therefore, the hydroxyl is axial in 7a
(trans) and equatorial in 6a (cis).
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Figure 1. 2DNOESY expansion where the most important correlations are showed for 6a.
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The use of the dihedral angle dependence of vicinal proton coupling constants (3JHH) for
structural studies of organic compounds is widely accepted, and was used to confirm the
cis/trans assignation. The magnitude of the coupling constant between vicinal protons is related
to the dihedral angle between them via the Karplus equation.16
The position of the hydroxy group was confirmed based on the coupling constants between H1
and the vicinal H2 protons. In the isomer 6a, H1 appears as a triplet of triplets with a large
coupling constant of 10 Hz and a small coupling constant of 4.5 Hz, corresponding to two axialꢀ
axial couplings and to two axialꢀequatorial couplings, respectively (Figure 2). Therefore, these
coupling constants reveal that H1 is axial and the hydroxy group equatorial, as expected for the
cis isomer. In contrast, in the isomer 7a, H1 appears as a broad signal involving small equatorialꢀ
equatorial and equatorialꢀaxial coupling constants, indicating that the hydroxy group occupies an
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axial position, as expected for the trans isomer. The same conclusions can be reached through
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the analysis of the multiplicity of H2ax as depicted in Figure 2.
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Figure 2. Proton NMR spectra for isomers TRANS (a) and CIS (b) from the methyl hydroxycyclohexylcarbamate
compounds, and proton-proton coupling constants obtained for protons H2 and H3ax (m = multiplet, tt = triplet of
triplets, cd = quadruplet of doublets, ax = axial disposition, eq = equatorial disposition).
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5.-Conclusions
In summary, we have developed an efficient sequence for the stereoselective reduction of a
number of 4ꢀsubstitutedꢀ4ꢀaminocyclohexanones, utilizing KRED reduction to set the absolute
cis or trans configuration of these key intermediates. Initial batches of the intermediates were
produced using a commercial KRED and IPA for cofactor regeneration to give nearꢀquantitative
yields of the alcohols with 98% d.e. The process was intensified to a point that showed sufficient
robustness, efficiency regardless scale, and was easily reproduced at kilogram scale for the
preparation of cisꢀ 4ꢀsubstitutedꢀ4ꢀaminocyclohexyl alcohols. This is the first example of this
type of substrates being prepared in a totally selective manner.
6.-Experimental Section
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Materials and Methods
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All solvents were purchased from SigmaꢀAldrich (HyꢀDry anhydrous solvents), and
commercially available reagents were used as received. Codexis® Ketoreductase Screening Kit
(N14002), NADP (Nicotinamide adenine dinucleotide phosphate, oxidized form, monosodium
salt, NADPꢀ004669) and KREDꢀP1ꢀB10 (D13083) were purchased from Codexis®. Ketones 5a-
g and alcohols 6a-g (as mixture of diastereomers, for reference) were synthesized and
characterized following standard procedures described in reference 1. All reactions were
followed by TLC analysis (TLC plates GF254, Merck) or liquid chromatography mass
spectrometry (LC/MS) using an Agilent 1100 equipped with a solvent degasser, binary pump,
autosampler, thermostatted column compartment with 2ꢀposition/10ꢀport valve, and a diode array
detector (Agilent Technologies, Waldbronn, Germany). The UV wavelength was set at 214 nm.
Electrospray mass spectrometry measurements were performed on a MSD quadrupole mass
spectrometer (Agilent Technologies, Palo Alto, CA, USA) interfaced to the above HPLC system.
MS measurements were acquired in positive ionization mode over the mass range of 100−700.
Data acquisition and integration for LC/UV and LC/MS detection was performed using
Chemstation software (Agilent Technologies). GC/MS measurements were acquired using an
Agilent 7890A with HPꢀChiral 30m, 0.25 mm, 0.25 mm (βꢀcyclodextrin in (35% phenyl)ꢀ
methylpolixiloxane) column, and mass measurements were performed in EI ionization mode
over the mass range of 50−500. NMR spectra were recorded at ambient temperature using
standard pulse methods on any of the following spectrometers and signal frequencies: Bruker
Avance DPX 300 MHz (1H = 300 MHz, 13C =75 MHz), Bruker Avance III HD 400 MHz (1H =
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400 MHz, C = 100MHz), Bruker Avance III HD 500 MHz (1H = 500 MHz, C = 125 MHz).
Chemical shifts are reported in ppm and are referenced to the following solvent peaks:
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Chloroform (1H = 7.26 ppm, C = 77.16 ppm), Acetone (1H = 2.05 ppm, C = 29.84 ppm),
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Methanol (1H = 3.31 ppm, C = 49.00 ppm), and DMSO (1H = 2.50 ppm, C = 39.52 ppm).
Coupling constants are quoted to the nearest 0.1 Hz, and multiplicities are given by the following
abbreviations and combinations thereof: s (singlet), d (doublet), t (triplet), q (quartet), m
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(multiplet), br (broad). The purity of all compounds tested was determined by LC/MS and H
NMR to be >95%.
General protocol for the screening with KRED Codexis® Kit
KREDs (2.5 mg) were weighted in 24 separate vials. To vials 1ꢀ19 (corresponding to enzymes
P1ꢀA04 to P3ꢀH12) was added KRED Recycle Mix P (300 µL, 125mM potassium phosphate,
1.25 mM magnesium sulfate, 1.0 NADP+, pH 7.0). KRED Recycle Mix N (300 µL, 250 mM
potassium phosphate, 2mM magnesium sulfate, 1.1 NADP+, 1.1 NAD+, 80 mM Dꢀglucose, 10
U/mL glucose dehydrogenase, pH 7.0) was added to vials 20ꢀ24 (enzymes 101 to NADHꢀ110).
The corresponding ketone 5a-g (10 mg) was added to each vial as a solution in isopropanol (30
µL). The reactions were stirred for 24 hours at room temperature. Then, 1 mL of ethyl acetate
was added to each vial and shaken for 3 minutes. The organic phase was separated and dried. An
initial check of conversion was made using TLC (hexane/MTBE 1:1). For reactions showing an
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approximate conversion higher than 50%, precise conversion was determined by H NMR
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(a,b,c,f,g), or GC/MS (d,e). Diastereoselectivity was checked by H NMR (a-g), compared to a
previously prepared diastereomeric mixture of alcohols. The results from the screenings can be
found in the supporting information.
Scale-up of the enzymatic reduction reactions
Ketone 5a (1 Kg, 4.7 mol) was dissolved in isopropanol (3 L, 40.8 mol). Phosphate buffer (9
L, C=0.1M, pH 7.0), magnesium sulfate (3.43 g, 28 mmol), NADP+ (5 g, 5 mg/g) and KREDꢀ
P1ꢀB10 (10 g, 10 mg/g, 1% w/w) were added to the solution. The reaction mixture was stirred at
1
35°C for 24 hours. Conversion and diastereomeric excess were checked by TLC and H NMR,
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compared to the ketone and a cis/trans mixture of alcohols respectively. Then, ethyl acetate (3.5
L) was added to the mixture and stirred for 3 minutes. Once the layers were clearly defined the
phases were separated. This procedure was repeated a second time with 3.5 L EtOAc and a third
with 2 L. The organic layers were combined, dried over anhydrous MgSO4, filtered and
concentrated. Isolated yield 99%, diastereomeric excess >98% for pure cis isomer 6a.
A similar procedure was applied for the preparation at scale of substrate 6b. 17
Supporting Information Available
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Detailed H NMR characterization and enzyme screening results for compounds 5a-g, 6a-g
and 7a-g.
Acknowledgements
The authors want to thank Dr. Graham R. Cumming, Dr. Javier Mendiola, and Dr. Joshua R.
Clayton for their assessment and helpful discussion. Laura RedondoꢀGallego thanks the
Fundación UniversidadꢀEmpresa (FUE) for a fellowship.
REFERENCES
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F.; Masse, C.; Preparation of substituted 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidines and analogs as
IRAK inhibitors, WO 2012097013 ; Madar, D. J.; Djuric, S. W.; Michmerhuizen, M. J.; Kopecka, H. A.; Li, X.;
Longenecker, K. L.; Pei, Z.; Pireh, D.; Sham, H. L.; Stewart, K. D.; Szczepankiewicz, B. G.; Wiedeman, P. E.;
Yong, H.; Preparation of cyanopyrrolidine derivatives and pharmaceutical compositions thereof as inhibitors of
dipeptidyl peptidase-iv (DPP-IV); US 20050215784.
2 Zhang, X. Org. Lett. 2015, 17(23), 5828; Kapat, A. J. Am. Chem. Soc. 2009, 131(49), 17746; Ellman, J. Org. Lett.
2004, 6(10), 1645; Panchaud, P. J. Org. Chem. 2004, 69(8), 2755.
3 Pramanik, S. Chem. Comm. 2012, 48(12), 1820.
4 a) Alcantara, A. R. Curr. Green Chem. 2014, 1(2), 155; b) Reetz, M. J. Am. Chem. Soc. 2013, 135, 12480. c) U. T.
Bornscheuer, U.T. Nature 2012, 485(7397), 185.
5 Kitbunnadaj, R. Bioor. Med. Chem. 2005, 13(23), 6309.
6 Zheng, S. J. Med. Chem. 2012, 55(10), 4837
7 Ley S. Org. Biomol. Chem. 2008, 6, 1577 ; Aicher, T. D.; Chicarelli, M. J.; Hinklin, R. J.; Tian, H.; Wallace, O. B.;
Chen, Z.; Mabry, T. E.; Mccowan, J. R.; Snyder, N.J.; Winnerosky, L. Allen, J.G. Preparation of cycloalkyl lactam
derivatives, particularly N-substituted pyrrolidin-2-ones, as inhibitors of 11beta-hydroxysteroid dehydrogenase 1,
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Schunk, S,; Zemolka, S.; Saunders, D.; Gruss, M.; Graubaum, H. Preparation of spirocyclic indoles as ORL1
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Truppo M. Comprehensive Organic Synthesis (2nd Ed), 2014, 8, 317; Huisman, G. W.; Liang, J.; Krebber, A.
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Current Opinion in Chemical Biology 2010, 14, 122; Kambourakis, S.; Rozzell, J. D. PharmaChem 2006, 5(9), 2ꢀ5.
Potassium phosphate buffer 0.1 M (pH 7), catalytic MgSO4, using NADP+ as cofactor and isopropanol as
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recycling system for the enzymes in entries 1 to 19, and NADP+ or NAD+ as cofactor and GDH/glucose as recycling
system for the enzymes in entries 20 to 24. The enzyme loading was 10% w/w, the temperature was 25ºC and the
reaction time was 24 hours. (http://www.codexis.com/productꢀprotocolsꢀandꢀfaqs)
12 100 mg scale conditions for 5a: 5% w/w enzyme loading, 1% NADP+, 15 mL/g of phosphate buffer pH 7, 8.6 eq.
of isopropanol (3.3 mL/g), 35ºC, 24 h.
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13 1 Kg scale conditions for 5a: 9 L of phosphate buffer pH 7, 3.1 L of isopropanol, 1% w/w enzyme loading, 0.5%
w/w NADP+, 35ºC (inner temperature), 24 h.
14 The complete results from the screenings can be found in the Supporting Information.
15 50 g scale conditions for 5b: 3% w/w enzyme loading, 0.5% w/w NADP+, 9 vol. of buffer phosphate pH 7, 8.6 eq
IPA, 35ºC, 24 h.
16 Buys, H. R. Recl. Trav. Chim. Pays-Bas, 1969, 88, 1003; Karplus, M. J. Am. Chem. Soc., 1963, 85, 2870; Karplus,
M. J. Chem. Phys. 1959, 30, 11; Minch, M. J., Concepts Magnetic Res., 1994, 6, 41.
17 In the case of substrate 6b, the enzyme loading was 3% w/w.
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