Enzymatic multicomponent reaction for simultaneous synthesis of two important scaffolds, pyridin-2-ones and α-alkylated nitriles

Article abstract of DOI:10.1016/j.tet.2014.12.021

An efficient approach for the synthesis of highly substituted pyridin-2-one derivatives and α-alkylated nitriles through enzymatic multicomponent reaction (MCR) was developed. This MCR involved bio-mimetic reduction between 3,4-dihydropyridin-2-one and activated olefin, both of which were in situ generated in the Acylase Amano (AA)-catalyzed domino reaction starting from benzaldehyde, cyanoacetamide and ketone. A wide range of substituted benzaldehydes and ketones were accepted by this reaction. Both final products (pyridin-2-one derivatives and α-alkylated nitriles) were important skeletons and synthetic intermediates. The synthetic application of prepared α-alkylated nitrile was demonstrated by converting it into the corresponding α-alkyl-β²-amino acid with high yield.

Full text of DOI:10.1016/j.tet.2014.12.021

Accepted Manuscript
Enzymatic multicomponent reaction for simultaneous synthesis of two important
scaffolds, pyridin-2-ones and α-alkylated nitriles
Zhi-Qiang Liu , Yu-Jing Hu , Xiao-Yang Chen , Qi Wu , Xian-Fu Lin
PII:
S0040-4020(14)01711-6
10.1016/j.tet.2014.12.021
TET 26242
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 19 July 2014
Revised Date: 2 October 2014
Accepted Date: 4 December 2014
Please cite this article as: Liu Z-Q, Hu Y-J, Chen X-Y, Wu Q, Lin X-F, Enzymatic multicomponent
reaction for simultaneous synthesis of two important scaffolds, pyridin-2-ones and α-alkylated nitriles,
Tetrahedron (2015), doi: 10.1016/j.tet.2014.12.021.
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ACCEPTED MANUSCRIPT
Enzymatic multicomponent reaction for simultaneous synthesis of two important scaffolds,
pyridin-2-ones and α-alkylated nitriles
Zhi-Qiang Liu, Yu-Jing Hu, Xiao-Yang Chen, Qi Wu,* Xian-Fu Lin*
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China,
Fax: + 86 571 87952618; E-mail: llc123@zju.edu.cn
1

ACCEPTED MANUSCRIPT
Enzymatic multicomponent reaction for simultaneous synthesis of two important scaffolds,
pyridin-2-ones and α-alkylated nitriles
Zhi-Qiang Liu, Yu-Jing Hu, Xiao-Yang Chen, Qi Wu,* Xian-Fu Lin*
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China,
Fax: + 86 571 87952618; E-mail: llc123@zju.edu.cn
Abstract
An efficient approach for the synthesis of highly substituted pyridin-2-one derivatives and α-alkylated
nitriles through enzymatic multicomponent reaction (MCR) was developed. This MCR involved
bio-mimetic reduction between 3,4-dihydropyridin-2-one and activated olefin, both of which were in situ
generated in the Acylase Amano (AA)-catalyzed domino reaction starting from benzaldehyde,
cyanoacetamide and ketone. A wide range of substituted benzaldehydes and ketones were accepted by
this reaction. Both final products (pyridin-2-one derivatives and α-alkylated nitriles) were important
skeletons and synthetic intermediates. The synthetic application of prepared α-alkylated nitrile was
2
demonstrated by converting it into the corresponding α-alkyl-β -amino acid with high yield.
Keywords
Enzyme catalysis; Multicomponent reactions; Transfer hydrogenation; Pyridin-2-one; Alkylated nitrile
1
. Introduction
As one representative of substituted six-membered lactams, compounds with pyridine-2-one scaffold
1
2
3
have shown significant pharmaceutical activities such as antifungal , antibacterial and antitumor .
Specifically, amrinone (a) and milrinone (b) in Fig. 1 have been found to display effective activities on
therapy of myocardial infarction, and compound (c) has the activity of antihypertensive. Meanwhile, the
4
pyridin-2-one ring system has successfully been applied in the natural synthesis as a building block .
Thus, development of convenient, efficient and mild strategy to establish these lactam cores is still
interesting in the realm of synthetic organic chemistry. Although several synthetic methodologies have
5
been reported , most of them have their own drawbacks such as harsh reaction conditions, low yields or
complicated multistep routes. Similarly, α-alkylated nitriles are also important building blocks for
6
synthesizing ketones and biologically active agents , the main methods for constructing α-alkylated
nitriles are reactions of nitriles with alkyl halides (or alcohols or carbonyl compounds) using
7
homogeneous inorganic bases such as NaH and NaNH as catalysts. Toxicity of halogenated substrates,
2
the use of homogeneous strong bases and metals are general drawbacks. With ever-increasing
environmental concerns, developing new method to construct pyridine-2-one and α-alkylated nitrile
derivatives still remains a significant challenge.
Fig. 1. Compounds with pyridin-2-one core
2

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Since enzymes were discovered to maintain activity in organic solvents by Klibanov in the early
8
1
980s , enzymes have been proved as efficient and green biocatalysts in organic synthesis due to their
intrinsic advantages of high selectivity, mild reaction conditions, simple operation and environmentally
benign behavior. In the past decade, a growing number of enzymes were found to be capable of
catalyzing not only their ‘‘natural’’ reactions but also one or more alternative reactions, and this feature is
9
called catalytic promiscuity . Our group has found that the Michael and Markovnikov addition could be
1
0
catalyzed by alkaline protease and acylase . And recently we have discovered many enzymatic
11
multicomponent reactions to synthesize heterocyclic compounds . More and more new cases of
1
2
12b
enzymatically promiscuous reactions were reported by other groups , for example, Zhang provided a
new strategy to synthesize spirooxindole derivatives via hydrolase, then Trypsin was founded to catalyze
the formation of 4-thiazolidinones and 3,4-dihydropyrimidin-2-ones through three-component
12e,12f
12a
reaction
, and Guan reported the first enzymatic aza-Diels-Alder Reaction . Though many works
involved single-enzyme catalyzed multistep transformations were found, more explorations concerning
new enzymatic domino reaction are still urgent in terms of the development of enzymology and industrial
importance. Herein, we discovered an interesting reaction to simultaneously synthesize important
pyridine-2-ones and α-alkylated nitriles via enzymatic multicomponent reaction involving in situ
hydrogen transfer.
2
. Results and discussion
Our initial idea was the synthesis of 3,4-dihydropyridin-2-one derivatives (5’) via a multicomponent
reaction starting from benzaldehyde (1), cyanoacetamide (2) and monoketone (3) catalyzed by enzyme
Scheme 1). Surprisingly, compounds 4 and 5 were obtained after flash chromatograph separation, which
(
were very similar to the direct Knoevenagel condensation product (4’) and expected product of three
components (5’). It seemed that hydrogen transfer process happened between compounds 4’ and 5’.
Scheme 1. enzymatic one-pot multicomponent reaction involving in situ hydrogen transfer
Considering the efficiency of this amusing phenomenon, we chose 4-chlorobenzaldehyde (1a),
3

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cyanoacetamide (2) and butanone (3a) as the starting materials, catalyst screening was firstly performed
o
in ethylene glycol (EG) at 50 C. Among the selected commercial enzymes, Acylase Amano (AA)
exhibited the best catalytic activity with 50% yield (Table 1, entry 1), 33% yield was obtained by
D-aminoacylase (DA) (Table 1, entry 2). The moderate yields were mainly due to the left substrates.
Butanone could form two pyridine-2-one products due to its different active positions. While other
hydrolases, such as CAL-B (Table 1, entry 3), Lipase from Candida rugosa, Lipozyme from Mucor
miehei and Amano Lipase PS (immobilized on diatomite), showed no activity toward the reaction (data
not shown). Next, we investigated the effect of different solvents on the model reaction, probably
because cyanoacetamide had low solubility in dioxane, toluene and acetonitrile, and no products were
observed in these generally used solvents (data not shown). Fortunately, the reaction in diethylene glycol
o
(
Di-EG) gave the best yield of 71% at 50 C after 48 h (Table 1, entry 9), and 50% yield in EG was
gotten (Table 1, entry 1), while both DMF and DMSO provided the yields less than 20% (Table 1, entries
and 8). Determining Di-EG as the optimum solvent, we performed some control experiments to verify
7
the specific catalytic effect of the enzyme on the initial three component reaction. No product was
detected when the reaction underwent in the absence of enzyme (Table 1, entry 4). In addition, since AA
is a zinc-dependent enzyme, EDTA (ethylene diamine tetraacetic acid) was used to denature the enzyme.
We found that no reaction happened with EDTA-denatured AA or bovine serum albumin (BSA) (Table 1,
entries 6 and 5), ruling out the possibility that amino acid of the protein surface or other impurities in AA
could promote the initial three component reaction. On the other hand, these results also validated that
the specific active site and the tertiary structure of AA was essential for the three component reaction.
It is noteworthy that the ratio of three starting materials has important influence on the output of the
multicomponent reaction. When the ratio of compounds 1a:2:3a was 0.5:0.75:0.5, the total yield reached
to 80%. Further changing the ratio, no improvement of yield was obtained. Thus, we chose the molar
ratio of substrate (1:2:3=0.5:0.75:0.5) for subsequent studies. Actually in our previous report, the
interesting in situ hydrogen transfer process to form α-alkylated nitriles (4) and pyridine-2-ones (5) was
not observed because of the much lower amount of aromatic aldehyde in the optimized feeding ratio of
three starting materials (1:2:3=0.125:1:0.75), thus the hydrogen-accepting Knoevenagel product (4’) was
11b
not enough and only 3,4-dihydropyridin-2-ones (5’) was obtained .
Table 1
a
Optimization of reaction conditions
H
H
N
O
NH2
N
O
O
CHO
H N
2
O
catalyst
solvent
CN
O
CN
CN
CN
Cl
Cl
4a
Cl
Cl
1a
2
3a
5
a
6a
b
b
b
d
Entry
Catalyst
AA
Solent
EG
4a (µmol)
5a (µmol)
6a (µmol)
Total yield(%)
1
2
3
131
84
95
26
50
DA
EG
65
18
33
CAL-B
EG
n.d.
n.d.
n.d.
n.d.
4

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4
5
6
7
8
9
-
Di-EG
Di-EG
Di-EG
DMF
n.d.
n.d.
trace
trace
trace
33
n.d.
Trace
Trace
Trace
5
n.d.
trace
trace
trace
16
BSA
Trace
Trace
Trace
41
c
AA
AA
AA
AA
DMSO
Di-EG
175
124
58
71
a
Reactions were carried out in EG (1 mL) with equimolar substrate (0.5 mmol) in the presence of 20 mg
o
enzyme at 50 C for 48h.
b
Determined by HPLC.
c
o
Denatured by EDTA in water for 12h at 100 C.
d
Total yields of 4+5+6 based on the amount of aromatic aldehyde, calculating equation is Yield =[(Amount
+5+6) / 500] × 100%.
4
n.d.: Not determined.
With the optimal conditions in hand, we further examined the scope and limitation of the new
enzymatic multicomponent reaction. It can be seen from Table 2 that a wide range of substituted
benzaldehydes could successfully participate in the reaction. Generally, the enzyme exhibited better
activity for benzaldehydes with electron-withdrawing substitutions than with electron-donating ones. For
example, 4-(trifluoromethyl)benzaldehyde gave an excellent yield of 94% (Table 2, entry 1), while
4
3
-isopropyl and 2-methoxybenzaldehyde only could get products in 70% yields (Table 2, entries 2 and 4).
-Substituted benzaldehyde could also be accepted in the reaction (Table 2, entry 5).
Table 2
a
Examination of the substituent effect of the aromatic aldehyde
H
N
H
N
O
NH2
CHO H N
O
O
2
O
O
AA
Di-EG
CN
R
CN
CN
CN
R
R
R
1
2
3a
4
5
6
Amount
Amount
Amount
Total
yield(%)
94
Entry
R
Product
Product
Product
b
b
b
c
(µmol)
(µmol)
(µmol)
1
2
3
4
4-CF3
4-iPr
4-F
4b
4c
4d
233
172
212
171
205
5b
5c
5d
5e
5f
156
122
155
135
155
6b
6c
6d
6e
6f
81
56
51
37
51
70
84
2-OCH3 4e
3-Cl
4f
69
5
82
a
Reactions were carried out in Di-EG (1 mL) with 1 (0.5 mmol), 2 (0.75 mmol) and 3 (0.5 mmol) in the
o
presence of 20 mg AA at 50 C for 48h.
b
Determined by HPLC.
c
Total yields of 4+5+6 based on the amount of aromatic aldehyde, calculating equation is Yield =[(Amount
5

ACCEPTED MANUSCRIPT
4
+5+6) / 500] ×100%
Then reactions of various ketones with different aldehydes were further tested. Both acetone and
3
-pentanone could successfully participate in the reaction to form α-alkylated nitriles and
pyridine-2-ones. But the yield declined sharply with the increase of chain length, from 70% to 32%
Table 3, entries 1 and 2), possibly because the activity of α-hydrogen in carbonyl compound reduced
when increasing the chain length of ketones. Theoretically, 1,3-dicarbonyl compounds like methyl
(
11b
acetoacetate could also give the corresponding products. In our previous report,
the amount of
cyanoacetamide and methyl acetoacetate was excessive compared to aromatic aldehyde, and these
substrates were simultaneously added to the reaction flask, giving the product of
3
,4-dihydropyridin-2-one. Herein we first mixed aromatic aldehyde and cyanoacetamide together, when
they fully changed to the Knoevenagel condensation product, half amount of methyl acetoacetate was
introduced to the system. Finally, the expected α-alkylated nitrile and pyridine-2-one were obtained
through slow transformation after 4 days (Table 3, entry 4).
Table 3
a
Variation of aldehyde and ketone
R2
O
CHO
R3
O
NH2
R2
R3
AA
Di-EG
NH2
NH
NC
R1
R1
CN
O
O
CN
5
R1
1
2
3
4
Amount
Amount Total yield
1
2
3
Entry
R
R
R
Product
Product
b
b
c
(µmol)
(µmol)
176
83
(%)
70
1
2
4-iPr
4-iPr
4-iPr
p-Cl
H
H
4c
4c
4c
4a
175
79
5g
5h
5h
5i
CH3
CH3
H
CH3
CH3
32
d
3
84
89
35
e
4
COOCH3
158
154
62
a
Reactions were carried out in Di-EG (1 mL) with 1 (0.5 mmol), 2 (0.75 mmol) and 3 (0.5 mmol) in the
o
presence of 20 mg AA at 50 C for 48h.
b
Determined by HPLC.
c
Total yields of 4+5+6 based on the amount of aromatic aldehyde, calculating equation is Yield =[(Amount 4+5)
/
500] ×100%.
d
Protected under nitrogen atmosphere.
e
o
After 6h the reaction of 1 (0.5 mmol) and 2 (0.5 mmol) in the presence of 20 mg AA at 50 C, 1 mL Di-EG, 3
(0.25 mmol) was added for 4 days
Basing on the above successful synthesis of two series of important compounds, i.e. pyridine-2-one
derivatives and α-alkylated nitriles, we hope to confirm the existence of hydrogen transfer process.
Firstly, we should determine whether the oxygen acts as oxidant in the system. Therefore, controlled
reaction under the protection of nitrogen was performed, the yield was in accordance with that in air
6

ACCEPTED MANUSCRIPT
(Table 3, entries 2 and 3). This result excluded the role of oxygen as oxidant in the reaction. Secondly,
can the solvent be the hydrogen donor? When we only put Knoevenagel product and the enzyme in the
solvent (Di-EG), no hydrogenation product was observed after two days, which excluded the possibility
that the solvent acted as hydrogen donor. The amounts of pyridine-2-one derivatives should be equal to
that of α-alkylated nitriles if hydrogen transfer process existed, which were confirmed by their respective
amount in Table 1-3. In order to further verify the internal transfer hydrogenation process quantitatively,
we tried to prepare two supposed intermediates, 3,4-dihydropyridin-2-one (Scheme 2, 5i’ and Scheme 3,
5
h’) and Knoevenagel condensation products (4a’). When we mixed 5i’ with 4a’ in Di-EG containing
AA, two compounds (5i and 4a) were successfully detected after chromatography as we expected. To
have a better understanding for the reaction process, we monitored the transforming course of 5i’ and 4a’
quantitatively by HPLC, which was shown in Scheme 2. Gratifyingly, the amount of 5i and 4a increased
as the reaction proceeded. Correspondingly, the substrates (5i’ and 4a’) gradually reduced. Same results
were observed for 5h’ and 4a’, which further confirmed the in situ hydrogen transfer process.
Scheme 2. Quantitatively monitoring the transforming process between two intermediates (5i’ and 4a’)
Another question needs to be considered is whether the in situ hydrogen transfer process happened in
the active site of this enzyme. We mixed 0.2 mmol 5h’ and 4a’ in Di-EG for one day under the
catalyst-free condition, or in the presence of BSA or AA (Scheme 3). No reaction occurred under the
catalyst-free condition, and the catalysis of AA could provide the products with 90% yields (5h and 4a).
Interestingly the control reaction under the catalysis of BSA also gave the products in 70% yields. Then
could amino acid catalyze this process? Aspartate (acidic), lysine (basic) and proline (neutral) as
biocatalyst were investigated respectively, only lysine could promote the transformation with 71% yield,
indicating the in situ hydrogen transfer process was possibly catalyzed by some surface basic amino
acids of the enzyme.
7

ACCEPTED MANUSCRIPT
Scheme 3. Controlled experiments to explore the hydrogen transfer process
Thus, a tentatively proposed mechanism was illustrated on the basis of these data in Scheme 4. First,
AA catalyzed the fast formation of Knoevenagel condensation reaction of aldehyde and cyanoacetamide
to compound 1. Then the zinc ion would coordinate with carbonyl group of cyanoacetamide and ketone
to increase the nucleophilicity, Asp 366 of AA deprived one α-H of ketone, which occurred the Michael
addition with compound 1 to produce intermediate 2. Next, the electron-rich N-H performed the
nucleophilic addition to the carbonyl group of ketone, at the same time, one molecular water was lost
from intermediate 2 to form product 3. Lysine catalyzed the transfer hydrogenation process as a hydrogen
carrier, obtained proton from compound 3 and transferred to compound 1. The final products
pyridin-2-one derivatives 4 and α-alkylated nitriles 5 were obtained.
R2
O
H O
2
O
NH2
CN
H
R3
Zn2+
H
H
O
O
O
R1
O
O
Asp
NH2
O
H
O NH
H
O
Asp
O
R1
1
H CN
O
NC
R1
R2
R3
Zn2+
Asp
O
NC
2
NH
R2
R1
H O
2
R3
O
O
Residue-NH2
NH2
NH2
Residue-NH +
3
CN
CN
R1
O
R1
5
1
NC
NH
R2
R3
R1
4
Scheme 4. The proposed mechanism of this multicomponent reaction.
8

ACCEPTED MANUSCRIPT
In order to validate the utility of this strategy, firstly, we examined the reaction on tenfold amount of
2
entry 3 in Table 2, total isolated yield was 69%. Then we synthesized the α-substituted β -amino acid
from one product, α-alkylated nitrile via simple conversions. β-amino acids are key building blocks of
1
3
natural products, β-peptides and pharmaceuticals . Compounds with this skeleton usually exhibit
1
4
antivirus and antibacterial activity . So developing new method to construct β-amino acids is still of
urgency. Our protocol is outlined in Scheme 5. Initially, Raney Ni was used as the catalyst for the
hydrogenation of cyano group in the reductive product 4d under hydrogen atmosphere, 4da was obtained
in high yield of 92%. Further simple treatment of 4da with concentrated hydrochloric acid at room
2
temperature for 3 h provided the target α-substituted β -amino acid (4db) in excellent yield of 96%. This
successful demonstration of converting prepared α-alkylated nitrile into the corresponding
2
α-alkyl-β -amino acid with high yield displayed the great potential of this enzymatic multicomponent
reaction in organic synthesis.
o
Scheme 5. Reagents and conditions: (i) Raney Ni (10% w/w), H (1 atm), 2 M NH /MeOH, 50 C, 20 h;
2
3
(ii) aq HCl, rt, 3h
3
. Conclusions
In summary, we have discovered an interesting process involving transfer hydrogenation between
3
,4-dihydropyridin-2-one and activated olefin, both of them were in situ generated in an Acylase Amano
(AA)-catalyzed multicomponent domino reaction starting from benzaldehyde, cyanoacetamide and
ketone. A wide range of substituted benzaldehydes and ketones were accepted by this reaction. The
resultant two final products (pyridin-2-one derivatives and α-alkylated nitriles) were both building blocks
of a variety of biologically active agents. As a demonstration, these α-alkylated nitriles could be
2
transformed to synthetically important α-substituted β -amino acids via simple conversions. The reaction
process was verified in detail by some controlled experiments, separating intermediates and monitoring
the transfer hydrogenation course between 3,4-dihydropyridin-2-one and activated olefin quantitatively.
This novel single enzyme-catalyzed multicomponent reaction is attractive in terms of high atom economy,
easy workup procedure, tolerance of various functional groups and mild reaction conditions, and will
widen the application of enzyme in organic synthesis.
9

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4
. Material and methods
4
.1 General information for reagents and analytical methods
D-aminoacylase from E. coli (10000 U/mg, 1 U is defined as enzyme quantity, which produces 1
mmol of D-Amino acid per 30 min) and Acylase ‘Amano’ (AA) from Aspergillus oryzae (≥ 30000 U/g, 1
U is defined as enzyme quantity, which produces 1 mmol of L-Amino acid per 30 min) were purchased
from Amano Enzyme Inc (Japan). Lipase immobilized on acrylic resin from C. antarctica (10000 U/g,
1
recombinant, expressed in Aspergillus oryzae) was purchased from Sigma (Steinheim, Germany). The H
1
3
and C NMR spectra were recorded with TMS as internal standard using a Bruker AMX-400 or 500
MHz spectrometer. Chemical shifts were expressed in parts per million and coupling constants (J) in
hertz. Analytical HPLC was performed using an Agilent TC-C18 series (250×4.6 mm) and a UV detector
(210 nm). The mobile phase was methanol and water. IR spectra were measured with a Nicolet Nexus
FTIR 670 spectrophotometer. Melting points were determined using XT-4 apparatus and were not
1
corrected. All the known products were characterized by comparing the H NMR data with those
1
13
reported in the literature. The structures of new compounds were confirmed by H NMR, C NMR, and
HRMS.
4
.2 General procedure for the enzymatic multicomponent reaction
A mixture of aromatic aldehyde (0.5 mmol), cyanoacetamide (0.75 mmol), monoketone (0.5 mmol),
o
and 20 mg AA in 1 mL Di-EG was shaken at 50 C and 200 rpm for 48h. After completion of the reaction
as monitored by TLC, the detecting solvent was petroleum ether/ethyl acetate mixture (1/2 v/v), then the
mixture was loaded directly onto a silica gel column using petroleum ether/ethyl acetate mixture (1/1 v/v)
as eluent to afford the desired product of α-alkylated nitriles and pyridin-2-ones, the α-alkylated nitriles
was first obtained, importantly, it had very slight UV absorption at 254 nm, but it can be confirmed in the
iodine jar.
4
.3 Procedure for the scale-up synthesis of α-alkylated nitrile and pyridin-2-one
A mixture of 4-fluorobenzaldehyde (5 mmol), cyanoacetamide (7.5 mmol), butanone (5 mmol), and
o
1
50 mg AA in 10 mL Di-EG was shaken at 50 C and 200 rpm for 72h. The mixture was loaded directly
onto a silica gel column using petroleum ether/ethyl acetate mixture (1/1 v/v) as eluent to afford the
desired product of 4d (337 mg), 5d (350mg) and 6d (61 mg), the total isolated yield was 69 %.
2
4
.4 Procedure for the synthesis of α-alkyl-β -amino acid
0
.3 mmol compound 4d and Raney Ni (10% w/w) were put into 10 mL NH /MeOH solvent (2 M), the
3
o
mixture was stirred at 50 C for 20 h under hydrogen atmosphere. After completion of the reaction,
Raney Ni was filtered and the solvent was removed under reduced pressure, then the residual solid was
washed with 2 mL petroleum ether/ethyl acetate mixture (1/1 v/v) twice. The obtained compound 4da
was put into 10 mL concentrated hydrochloric acid for 3 h at room temperature, the final product was
gotten after removing solvent.
Acknowledgements
The financial support from the National Natural Science Foundation of China (No. 21272208), the
Zhejiang Provincial Natural Science Foundation (No. LY14B020006) and Ph.D. Programs Foundation of
Ministry of Education of China (20110101110008) is gratefully acknowledged.
1
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Chem. 2008, 32, 259-286; (f) Misic-Vukovic, M.; Mijin, D.; Radojkovic-Velickovic, M.; Valentic, N.;
Krstic, V. J. Serb. Chem. Soc. 1998, 63, 585-599.
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(a) Wu, Z. L.; Li, Z. Y. J. Org. Chem. 2003, 68, 2479-2482; (b) Ooi, T.; Takeuchi, M.; Kameda, M.;
Maruoka, K. J. Am. Chem. Soc. 2000, 122, 5228-5229; (c) Kulp, S. S.; Mcgee, M. J. J. Org. Chem.
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983, 48, 4097-4098.
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107-4110; (b) Trost, B. M. Angew. Chem. Int. Ed. Engl. 1995, 34, 259-281; (c) Sheldon, R. A. Pure
4
Appl. Chem. 2000, 72, 1233-1246; (d) Motokura, K.; Fujita, N.; Mori, K.; Mizugaki, T.; Ebitani, K.;
Htsukawa, K.; Kanedar, K. Chem.-Eur. J. 2006, 12, 8228-8239.
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(a) Busto, E.; Gotor-Fernandez, V.; Gotor, V. Chem. Rev. 2011, 111, 3998-4035; (b) Busto, E.;
Gotor-Fernandez, V.; Gotor, V. Chem. Soc. Rev. 2010, 39, 4504-4523; (c) Humble, M. S.; Berglund, P.
Eur. J. Org. Chem. 2011, 3391-3401; (d) Wu, Q.; Liu, B. K.; Lin, X. F. Curr. Org. Chem. 2010, 14,
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966-1988; (e) Groger, H.; Asano, Y.; Bornscheuer, U. T.; Ogawa, J. Chem.- Asian J. 2012, 7,
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Nature 2012, 485, 185-194.
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1
0. (a) Cai, Y.; Sun, X. F.; Wang, N.; Lin, X. F. Synthesis-Stuttgart 2004, 671-674; (b) Wu, W. B.; Wang, N.;
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1
1

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Mei, Y. J.; Du, K.; Shi, Q. Y.; Zhang, P. F. Catal. Lett. 2013, 143, 298-301; (f) Xie, Z. B.; Wang, N.; Wu,
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Gossett, L. S.; Gruber, J. M.; Grossman, C. S.; Andis, S. L.; Schultz, R. M.; Worzalla, J. F.; Corbett, T.
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Cebula, R.; Wattanasin, S. Biorg. Med. Chem. Lett. 2008, 18, 1840-1844.
1
2

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Table 1
a
Optimization of reaction conditions
H
N
H
N
O
NH2
O
O
CHO
H N
2
O
catalyst
solvent
CN
O
CN
CN
CN
Cl
Cl
4a
Cl
Cl
1
a
2
3a
5
a
6a
b
b
b
d
Entry
Catalyst
AA
Solent
EG
4a (µmol)
5a (µmol)
6a (µmol)
26
Total yield(%)
1
2
3
4
5
6
7
8
131
84
95
50
DA
EG
65
18
33
CAL-B
-
EG
n.d.
n.d.
Trace
Trace
Trace
41
n.d.
n.d.
trace
trace
trace
33
n.d.
n.d.
n.d.
trace
trace
trace
16
Di-EG
Di-EG
Di-EG
DMF
n.d.
BSA
Trace
Trace
Trace
5
c
AA
AA
AA
AA
DMSO
Di-EG
9
175
124
58
71
a
Reactions were carried out in EG (1 mL) with equimolar substrate (0.5 mmol) in the presence of 20
o
mg enzyme at 50 C for 48h.
b
Determined by HPLC.
c
o
Denatured by EDTA in water for 12h at 100 C.
d
Total yields of 4+5+6 based on the amount of aromatic aldehyde, calculating equation is Yield
=
[(Amount 4+5+6) / 500] × 100%.
n.d.: Not determined.

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Table 2
a
Examination of the substituent effect of the aromatic aldehyde
H
H
N
O
NH2
CHO H N
N
O
O
2
O
O
AA
Di-EG
CN
R
CN
CN
CN
R
R
R
1
2
3a
4
5
6
Amount
Amount
Amount
Total
yield(%)
94
Entry
R
Product
Product
Product
b
b
b
c
(µmol)
(µmol)
(µmol)
1
2
3
4
5
4-CF3
4-iPr
4-F
4b
4c
4d
4e
4f
233
172
212
171
205
5b
5c
5d
5e
5f
156
122
155
135
155
6b
6c
6d
6e
6f
81
56
51
37
51
70
84
2-OCH
3
69
3-Cl
82
a
Reactions were carried out in Di-EG (1 mL) with 1 (0.5 mmol), 2 (0.75 mmol) and 3 (0.5 mmol) in the
o
presence of 20 mg AA at 50 C for 48h.
b
Determined by HPLC.
c
Total yields of 4+5+6 based on the amount of aromatic aldehyde, calculating equation is Yield =[(Amount 4+5+6)
/
500] ×100%

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Table 3
a
Variation of aldehyde and ketone
R2
O
CHO
R3
O
NH
2
AA
Di-EG
NH
2
NH
NC
R2
R3
R1
CN
O
R1
O
CN
5
R1
1
2
3
4
Amount
Amount Total yield
1
2
3
Entry
R
R
R
Product
Product
b
b
c
(µmol)
(µmol)
176
83
(%)
70
1
2
4-iPr
4-iPr
4-iPr
p-Cl
H
H
4c
4c
4c
4a
175
79
5g
5h
5h
5i
CH3
CH3
H
CH3
CH3
32
d
3
84
89
35
e
4
COOCH
3
158
154
62
a
Reactions were carried out in Di-EG (1 mL) with 1 (0.5 mmol), 2 (0.75 mmol) and 3 (0.5 mmol) in the
o
presence of 20 mg AA at 50 C for 48h.
b
Determined by HPLC.
c
Total yields of 4+5+6 based on the amount of aromatic aldehyde, calculating equation is Yield
=
[(Amount 4+5) / 500] ×100%.
d
Protected under nitrogen atmosphere.
e
o
After 6h the reaction of 1 (0.5 mmol) and 2 (0.5 mmol) in the presence of 20 mg AA at 50 C, 1 mL Di-EG,
(0.25 mmol) was added for 4 days
3

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Enzymatic multicomponent reaction for simultaneous synthesis of two
important scaffolds, pyridin-2-ones and α-alkylated nitriles
Zhi-Qiang Liu, Yu-Jing Hu, Xiao-Yang Chen, Qi Wu,* Xian-Fu Lin*
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China
Supplementary Data
Table of Content
Characterizations for Compounds………………………………… S-1
1
13
Copy of H NRM, C NRM and HRMS spectra…………………..S-8
[
键入文字]

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Supporting Information
Characterizations for Compounds
4
a: 3-(4-chlorophenyl)-2-cyanopropanamide
O
NH2
CN
o
Cl
1
m.p. 180-181 C; IR (neat): 3423, 3315, 3206, 2252, 1667, 1611;
H NMR (400 MHz, DMSO-d ) δ 7.78 (s, 1H), 7.49 (s, 1H), 7.41-7.31 (m, 4H), 3.96-3.93
6
13
(
m, 1H), 3.14 (dd, J = 13.6, 6.8 Hz, 1H), 3.16 (dd, J = 13.6, 8.8 Hz, 1H); C NMR (125
MHz, DMSO-d ) δ 171.3, 141.1, 137.0, 136.2, 136.2, 133.6, 133.6, 123.5, 39.6; HRMS (EI)
6
+
calcd. for C H N OCl [M ]: 208.0403, found: 208.0403
1
0
9
2
6
a: 4-(4-chlorophenyl)-6-ethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile
NH
O
CN
o
1
Cl
m.p. 260-261 C; IR (neat): 2220, 1645, 1614; H NMR (400 MHz,
DMSO-d ) δ 12.65 (s, 1H), 7.66-7.60 (m, 4H), 6.34 (s, 1H), 2.58 (q, J = 7.6 Hz, 2H), 1.19 (t,
6
1
3
J = 7.4 Hz, 3H); C NMR (125 MHz, DMSO-d ) δ 161.4, 159.1, 157.5, 135.2, 134.9, 129.9,
6
+
1
28.9, 116.4, 104.9, 97.7, 26.1, 12.7; HRMS (EI) calcd. for C H N OCl [M ]: 258.0560,
1
4
11
2
found: 258.0562
5
a: 4-(4-chlorophenyl)-5,6-dimethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile
NH
O
CN
o
1
Cl
m.p. 278-279 C; IR (neat): 2223, 1652, 1604; H NMR (400 MHz,
13
DMSO-d ) δ 12.64 (s, 1H), 7.60-7.33 (m, 4H), 2.30 (s, 3H), 1.68 (s, 3H); C NMR (125
6
MHz, DMSO-d ) δ 159.9, 150.7, 150.7, 135.1, 133.9, 129.7, 128.8, 116.2, 111.3, 99.7, 18.0,
6
+
1
3.9; HRMS (EI) calcd. for C H N OCl [M ]: 258.0560, found: 258.0561
1
4
11
2
4
b: 2-cyano-3-(4-(trifluoromethyl)phenyl)propanamide
O
NH2
CN
o
1
F3C
m.p. 148-149 C; IR (neat): 3361, 3193, 2248, 1677, 1627; H
S-1

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NMR (400 MHz, DMSO-d ) δ 7.79 (s, 1H), 7.71-7.51 (m, 5H), 4.01 (t, J = 7.6 Hz, 1H),
6
1
3
3
.25 (dd, J = 13.8, 6.6 Hz, 1H), 3.16 (dd, J = 13.6, 8.4 Hz, 1H); C NMR (125 MHz,
DMSO-d ) δ 171.2, 147.0, 135.2, 135.1, 133.1, 132.9, 130.6, 130.5, 128.4, 123.4, 40.0;
6
+
HRMS (EI) calcd. for C H N OF [M ]: 242.0667, found: 242.0667
11
9
2
3
6
b: 6-ethyl-2-oxo-4-(4-(trifluoromethyl)phenyl)-1,2-dihydropyridine-3-carbonitrile
NH
O
CN
o
1
3
F C
m.p. 200-201 C; IR (neat): 2272, 1694, 1617; H NMR (400 MHz,
DMSO-d ) δ 12.74 (s, 1H), 7.92-7.81 (m, 4H), 6.37 (s, 1H), 2.58 (q, J = 7.6 Hz, 2H), 1.18 (t,
6
1
3
J = 7.6 Hz, 3H); C NMR (125 MHz, DMSO-d ) δ 161.3, 158.9, 157.9, 140.2, 130.4, 130.2,
6
1
29.0, 126.5, 125.7, 125.7, 125.0, 122.8, 116.2, 105.0, 98.1, 26.1, 12.6; HRMS (EI) calcd.
+
for C H N OF [M ]: 292.0823, found: 292.0827
1
5
11
2
3
5
5
b:
,6-dimethyl-2-oxo-4-(4-(trifluoromethyl)phenyl)-1,2-dihydropyridine-3-carbonitrile
NH
O
CN
o
1
3
F C
m.p. 264-265 C; IR (neat): 2227, 1652, 1617; H NMR (400 MHz,
13
DMSO-d ) δ 12.73 (s, 1H), 7.92-7.56 (m, 4H), 2.33 (s, 3H), 1.68 (s, 3H); C NMR (125
6
MHz, DMSO-d ) δ 160.2, 159.8, 151.1, 140.4, 129.5, 129.3, 128.7, 125.7, 125.7, 125.0,
6
+
1
22.9, 116.1, 111.1, 99.5, 18.0, 13.9; HRMS (EI) calcd. for C H N OF [M ]: 292.0823,
1
5
11
2
3
found: 292.0826
4
c: 2-cyano-3-(4-isopropylphenyl)propanamide
O
NH2
CN
o
m.p. 150-151 C; IR (neat): 3433, 3367, 3194, 2253, 1659, 1630;
1
H NMR (400 MHz, DMSO-d ) δ 7.78 (s, 1H), 7,48 (s, 1H), 7.20 (s, 4H), 3.93-3.89 (m, 1H),
6
3
.11 (dd, J = 13.8, 6.6 Hz, 1H), 2.99 (dd, J = 13.6, 8.8 Hz, 1H), 2.89-2.83 (m, 1H), 1.19 (d,
1
3
J = 6.8 Hz, 6H); C NMR (125 MHz, DMSO-d ) δ 171.6, 152.3, 139.4, 134.1, 131.6, 123.7,
6
+
4
0.1, 38.3, 29.1; HRMS (EI) calcd. for C H N O [M ]: 216.1263, found: 216.1266
1
3
16
2
6
c: 6-ethyl-4-(4-isopropylphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile
S-2

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NH
O
CN
o
1
m.p. 280-281 C; IR (neat): 2219, 1652, 1605; H NMR (400 MHz,
DMSO-d ) δ 12.54 (s, 1H), 7.57-7.41 (m, 4H), 6.33 (s, 1H), 3.00-2.93 (m, 1H), 2.58 (q, J =
6
1
3
7
.4 Hz, 2H), 1.25-1.17 (m, 9H); C NMR (125 MHz, DMSO-d ) δ 161.6, 160.2, 157.0,
6
1
51.0, 133.6, 128.1, 126.7, 116.7, 105.0, 97.2, 33.3, 26.1, 23.6, 12.6; HRMS (EI) calcd. for
+
C H N O [M ]: 266.1419, found: 266.1417
17
18
2
5
c: 4-(4-isopropylphenyl)-5,6-dimethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile
NH
O
CN
o
1
m.p. 290-291 C; IR (neat): 2219, 1648, 1605; H NMR (400 MHz,
DMSO-d ) δ 12.56 (s, 1H), 7.40-7.20 (m, 4H), 2.99-2.92 (m, 1H), 2.30 (s, 3H), 1.70 (s, 3H),
6
1
3
1
1
.24 (d, J = 6.8 Hz, 6H); C NMR (125 MHz, DMSO-d ) δ 161.8, 160.1, 150.3, 149.2,
6
33.7, 127.7, 126.5, 116.4, 111.4, 99.7, 33.2, 23.7, 18.0, 14.0; HRMS (EI) calcd. for
+
C H N O [M ]: 266.1419, found: 266.1424
17
18
2
4
d: 2-cyano-3-(4-fluorophenyl)propanamide
O
NH2
CN
o
1
F
m.p. 149-150 C; IR (neat): 3468, 3323, 3201, 2250, 1676, 1614; H
NMR (400 MHz, DMSO-d ) δ 7.77 (s, 1H), 7.49 (s, 1H), 7.35-7.14 (m, 4H), 3.95-3.91 (m,
6
1
3
1
H), 3.14 (dd, J = 13.8, 7.0 Hz, 1H), 3.05 (dd, J = 13.8, 8.6 Hz, 1H); C NMR (125 MHz,
DMSO-d ) δ 166.7, 162.8, 160.9, 133.6, 133.6, 131.5, 131.4, 118.8, 115.8, 115.6, 34.8;
6
+
HRMS (EI) calcd. for C H N OF [M ]: 192.0699, found: 192.0703
1
0
9
2
6
d: 6-ethyl-4-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile
NH
O
CN
o
1
F
m.p. 128-129 C; IR (neat): 2272, 1695, 1618; H NMR (400 MHz,
DMSO-d ) δ 12.58 (s, 1H), 7.68-7.33 (m, 4H), 6.30 (s, 1H), 2.55 (q, J = 7.4 Hz, 2H), 1.16 (t,
6
S-3

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1
3
J = 7.4 Hz, 3H); C NMR (125 MHz, DMSO-d ) δ 164.2, 162.2, 161.4, 159.3, 157.3, 132.5,
6
1
32.5, 130.6, 130.5, 116.5, 115.9, 115.7, 105.0, 97.7, 26.1, 12.6; HRMS (EI) calcd. for
+
C H N OF [M ]: 242.0855, found: 242.0856
14
11
2
5
d: 4-(4-fluorophenyl)-5,6-dimethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile
NH
O
CN
o
1
F
m.p. 253-254 C; IR (neat): 2224, 1652, 1603; H NMR (400 MHz,
13
DMSO-d ) δ 12.59 (s, 1H), 7.37-7.33 (m, 4H), 2.30 (s, 3H), 1.69 (s, 3H); C NMR (125
6
MHz, DMSO-d ) δ 164.0, 162.0, 161.6, 160.7, 151.3, 133.3, 133.0, 130.8, 130.7, 117.0,
6
+
1
16.5, 116.3, 112.2, 100.6, 18.7, 14.6; HRMS (EI) calcd. for C H N OF [M ]: 242.0855,
1
4
11
2
found: 242.0856
4
e: 2-cyano-3-(2-methoxyphenyl)propanamide
O
NH2
CN
o
1
OCH
3
m.p. 116-117 C; IR (neat): 3334, 3199, 2245, 1683, 1602; H NMR
(
3
400 MHz, DMSO-d ) δ 7.79 (s, 1H), 7.47 (s, 1H), 7.29-6.88 (m, 4H), 3.92-3.88 (m, 1H),
.08 (dd, J = 13.6, 6.8 Hz, 1H), 3.02 (dd, J = 13.6, 9.2 Hz, 1H); C NMR (125 MHz,
6
1
3
DMSO-d ) δ 166.3, 157.2, 130.4, 128.7, 124.5, 120.3, 118.5, 110.8, 55.4, 37.3, 30.2; HRMS
6
+
(
EI) calcd. for C H N O [M ]: 204.0899, found: 204.0901
11
12
2
2
6
e: 6-ethyl-4-(2-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile
NH
O
CN
o
1
OCH
3
m.p. 249-250 C; IR (neat): 2223, 1644, 1617; H NMR (400 MHz,
DMSO-d ) δ 12.50 (s, 1H), 7.48-7.03 (m, 4H), 6.18 (s, 1H), 3.78 (s, 3H), 2.55 (q, J = 7.4 Hz,
6
1
3
2
1
H), 1.16 (t, J = 7.6 Hz, 3H); C NMR (125 MHz, DMSO-d ) δ 161.7, 159.1, 157.0, 156.2,
6
31.9, 129.8, 125.8, 121.1, 116.7, 112.4, 106.6, 100.6, 56.1, 26.5, 13.1; HRMS (EI) calcd.
+
for C H N O [M ]: 254.1055, found: 254.1057
1
5
14
2
2
5
e: 4-(2-methoxyphenyl)-5,6-dimethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile
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NH
O
CN
OCH3
o
1
m.p. 262-263 C; IR (neat): 2223, 1652, 1605; H NMR (400 MHz,
1
3
DMSO-d ) δ 12.55 (s, 1H), 7.49-7.06 (m, 4H), 3.76 (s, 3H), 2.30 (s, 3H), 1.63 (s, 3H); C
6
NMR (125 MHz, DMSO-d ) δ 160.1, 159.7, 155.2, 149.7, 130.8, 128.8, 124.9, 120.7, 116.3,
6
+
1
12.2, 111.7, 100.4, 55.6, 17.9, 13.5; HRMS (EI) calcd. for C H N O [M ]: 254.1055,
1
5
14
2
2
found: 254.1052
4
f: 3-(3-chlorophenyl)-2-cyanopropanamide
O
NH2
CN
o
1
Cl
m.p. 115-116 C; IR (neat): 3366, 3200, 2250, 1675, 1624; H NMR
(
3
400 MHz, DMSO-d ) δ 7.78 (s, 1H), 7.51 (s, 1H), 7.39-7.25 (m, 4H), 4.00-3.96 (m, 1H),
.16 (dd, J = 13.6, 6.8 Hz, 1H), 3.07 (dd, J = 14.0, 8.8 Hz, 1H); C NMR (125 MHz,
6
1
3
DMSO-d ) δ 166.0, 139.4, 132.9, 130.2, 128.8, 127.8, 127.1, 118.2, 34.6; HRMS (EI) calcd.
6
+
for C H N OCl [M ]: 208.0403, found: 208.0402
1
0
9
2
6
f: 4-(3-chlorophenyl)-6-ethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile
NH
O
CN
o
1
Cl
m.p. 253-254 C; IR (neat): 2220, 1640, 1616; H NMR (400 MHz,
DMSO-d ) δ 12.68 (s, 1H), 7.68-7.58 (m, 4H), 6.37 (s, 1H), 2.58 (q, J = 7.6 Hz, 2H), 1.19 (t,
6
1
3
J = 7.6 Hz, 3H); C NMR (125 MHz, DMSO-d ) δ 161.3, 158.7, 157.6, 138.1, 133.4, 130.7,
6
1
30.1, 127.7, 126.8, 116.3, 105.0, 97.9, 26.1, 12.7; HRMS (EI) calcd. for C H N OCl
14 11 2
+
[
M ]: 258.0560, found: 258.0563
5
f: 4-(3-chlorophenyl)-5,6-dimethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile
NH
O
CN
o
1
Cl
m.p. 249-250 C; IR (neat): 2223, 1652, 1591; H NMR (400 MHz,
S-5

ACCEPTED MANUSCRIPT
13
DMSO-d ) δ 12.65 (s, 1H), 7.56-7.27 (m, 4H), 2.32 (s, 3H), 1.70 (s, 3H); C NMR (125
6
MHz, DMSO-d ) δ 160.1, 159.9, 150.8, 138.3, 133.3, 130.7, 129.0, 127.4, 126.4, 116.1,
6
+
1
11.3, 99.7, 18.0, 13.9; HRMS (EI) calcd. for C H N OCl [M ]: 258.0560, found:
14
11
2
2
58.0564
5
g: 4-(4-isopropylphenyl)-6-methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile
NH
O
CN
o
1
m.p. 265-266 C; IR (neat): 2224, 1652, 1615; H NMR (400 MHz,
DMSO-d ) δ 12.59 (s, 1H), 7.55-7.40 (m, 4H), 6.33 (s, 1H), 2.99-2.92 (m, 1H), 2.29 (s, 3H),
6
1
3
1
1
.22 (d, J = 6.8 Hz, 6H); C NMR (125 MHz, DMSO-d ) δ 161.6, 160.0, 151.9, 151.0,
6
33.5, 128.0, 126.7, 116.8, 106.5, 96.9, 33.3, 23.6, 19.1; HRMS (EI) calcd. for C H N O
1
6
16
2
+
[
M ]: 252.1263, found: 252.1263
5
h: 6-ethyl-4-(4-isopropylphenyl)-5-methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile
NH
O
CN
o
1
m.p. 254-255 C; IR (neat): 2223, 1652, 1598; H NMR (400 MHz,
DMSO-d ) δ 12.53 (s, 1H), 7.40-7.22 (m, 4H), 2.99-2.92 (m, 1H), 2.60 (q, J = 7.6 Hz, 2H),
6
1
3
1
.74 (s, 3H), 1.24 (d, J = 6.8 Hz, 6H), 1.14 (t, J = 7.2 Hz, 3H); C NMR (125 MHz,
DMSO-d ) δ 162.5, 160.2, 154.6, 149.1, 133.7, 127.7, 126.5, 116.3, 110.5, 100.3, 33.2, 24.6,
6
+
2
3.7, 13.5, 12.7; HRMS (EI) calcd. for C H N O [M ]: 280.1576, found: 280.1579
1
8
20
2
5
6
e
h’:
-ethyl-4-(4-isopropylphenyl)-5-methyl-2-oxo-1,2,3,4-tetrahydropyridine-3-carbonitril
NH
O
CN
o
1
m.p. 184-185 C; IR (neat): 2253, 1699, 1682; H NMR (400 MHz,
CDCl ) δ 8.21, 8.13 (s, 1H), 7.27-7.08 (m, 4H), 4.14-3.72 (m, 1H), 3.60-3.56 (m, 1H),
3
2
.93-2.86 (m, 1H), 2.33-2.21 (m, 2H), 1.70 (s, 3H), 1.30-1.23 (m, 6H), 1.18-1.11 (m, 3H);
1
3
C NMR (125 MHz, CDCl ) δ 162.6, 162.0, 149.0, 148.9, 134.6, 133.1, 132.7, 132.6, 128.1,
3
S-6

ACCEPTED MANUSCRIPT
1
2
2
27.4, 127.4, 127.1, 116.5, 115.1, 110.4, 108.4, 48.0, 46.7, 41.6, 40.9, 33.8, 23.9, 23.9, 23.9,
+
3.4, 23.2, 16.2, 16.0, 12.3, 12.2; HRMS (EI) calcd. for C H N O [M ]: 282.1732, found:
1
8
22
2
82.1734
5
i: Methyl 4-(4-chlorophenyl)-5-cyano-2-methyl-6-oxo-1,6-dihydropyridine-3-
carboxylate
O
O
NH
O
CN
o
1
Cl
m.p. 265-266 C; IR (neat): 2231, 1734, 1651; H NMR (400 MHz,
1
3
CDCl ) δ 13.01 (bs, 1H), 7.58-7.34 (m, 4H), 3.40 (s, 3H), 2.40 (s, 3H); C NMR (125 MHz,
3
CDCl ) δ 165.2, 159.7, 158.3, 153.6, 134.6, 134.3, 129.1, 128.6, 115.3, 111.6, 100.7, 52.1,
3
+
1
8.4; HRMS (EI) calcd. for C H N O Cl [M ]: 302.0458, found: 302.0459
1
5
11
2
3
4
da: 3-amino-2-(4-fluorobenzyl)propanamide
O
NH2
o
1
F
NH2
m.p. 121-122 C; IR (neat): 3355, 3298, 1670, 1606, 1510; H
NMR (400 MHz, DMSO-d ) δ 7.31 (s, 1H), 7.21-7.07 (m, 4H), 6.75 (s, 1H), 2.74-2.42 (m,
6
1
3
5
1
H); C NMR (100 MHz, DMSO-d ) δ175.6, 161.8, 159.4, 136.4, 130.5, 130.4, 114.7,
14.6, 50.7, 43.8, 34.5; HRMS (EI) calcd. for C H FN O [M ]: 196.1012, found:
6
+
10
13
2
1
96.1012
4
db: 3-amino-2-(4-fluorobenzyl)propanoic acid
O
OH
o
1
F
NH2
m.p. 242-243 C; IR (neat): 3362, 3184, 1665, 1637, 1511; H NMR
(
400 MHz, DMSO-d ) δ 8.10 (s, 2H), 7.75 (s, 1H), 7.38-7.10 (m, 4H), 2.91-2.67 (m, 5H);
6
1
3
C NMR (100 MHz, DMSO-d ) δ173.4, 162.1, 159.7, 134.6, 134.6, 130.9, 130.8, 115.0,
6
+
1
14.8, 44.4, 34.8; HRMS (EI) calcd. for C H FNO [M ]: 197.0852, found: 197.0849
1
0
12
2
S-7