N-Heteroarylmethyl-5-hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylic acid a novel scaffold for the design of uncompetitive α-glucosidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.07.012

Abstract The enzyme α-glucosidase has attracted interest owing to its involvement in the digestive process of carbohydrate, its role in intracellular glycoprotein trafficking, tumorigenesis and viral infection. In this study, several members of a new family of N-heteroarylmethyl substituted azasugars were synthesized and evaluated as α-glucosidase inhibitors. We systematically investigated the effect of different N-substituents as well as the role of hydroxyl and carboxylate moieties on the piperidine ring. The compounds N-heteroarylmethyl-5-hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylic acid emerged as potent α-glucosidase inhibitors. Unlike Acarbose and other clinically relevant α-glucosidase inhibitors, these compounds act through a reversible uncompetitive mechanism of inhibition which make them attractive candidates for drug development.

Full text of DOI:10.1016/j.bmc.2013.07.012

Accepted Manuscript
N-Heteroarylmethyl-5-hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylic acid
A Novel Scaffold for the Design of Uncompetitive α-Glucosidase Inhibitors
Sha Long, Francesca Romana Stefani, Stefano Biondi, Giancarlo Ghiselli,
Mauro Panunzio
PII:
S0968-0896(13)00623-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.07.012
BMC 10976
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
17 March 2013
7 June 2013
8 July 2013
Please cite this article as: Long, S., Stefani, F.R., Biondi, S., Ghiselli, G., Panunzio, M., N-Heteroarylmethyl-5-
hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylic acid A Novel Scaffold for the Design of Uncompetitive α-
Glucosidase Inhibitors, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.
2013.07.012
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N-Heteroarylmethyl-5-hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylic acid
A Novel Scaffold for the Design of
Uncompetitive -Glucosidase Inhibitors
Sha Long1, Francesca Romana Stefani2, Stefano Biondi3,
Giancarlo Ghiselli2, and Mauro Panunzio1
1 ISOF-CNR Dep “G. Ciamician” Via Selmi 2, 40126 Bologna, Italy;
e-mail: sha.long2@unibo.it; mauro.panunzio@unibo.it
2 Glyconova Srl, Via Ribes 5, 10010 Colleretto Giacosa, Italy;
e-mail: glyconova@gmail.com
3 Innovative Solution, Via Copernico 22, 20016 Pero, Italy
e-mail: stefano.biondi@innovativesolution.it
Corresponding Author:
Dr. Giancarlo Ghiselli
Glyconova Srl
Via Ribes 5
Colleretto Giacosa 10010 TO
Italy
Tel: +39-0125-538823
Fax: +39-0125-538791
e-mail: glyconova@gmail.com
FRS current address is:
Lund University, BMC B10 / Stem Cell Center
Klinikgatan 26, 22184 Lund, Sweden
e-mail: francesca_romana.stefani@med.lu.se
1

Keywords:
-Glucosidase Inhibitors
Iminosugars
Azasugars
Uncompetitive enzymatic inhibition
1,2,5,6-tetrahydropyridine-3-carboxylic acid
2

Abstract:
The enzyme-glucosidase has attracted interest owing to its involvement in the digestive
process of carbohydrate, its role in intracellular glycoprotein trafficking, tumorigenesis
and viral infection. In this study, several members of a new family of N-heteroarylmethyl
substituted azasugars were synthesized and evaluated as -glucosidase inhibitors. We
systematically investigated the effect of different N- substituents as well as the role of
hydroxyl and carboxylate moieties on the piperidine ring. The compounds N-
heteroarylmethyl-5-hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylic acid emerged as
potent -glucosidase inhibitors. Unlike Acarbose and other clinically relevant -
glucosidase inhibitors, these compounds act through a reversible uncompetitive
mechanism of inhibition which make them attractive candidates for drug development.
3

1. Introduction
Restriction of sugar intake through diet is often recommended as a measure to improve
glycemic control in diabetics.1, 2 Compounds affecting carbohydrate breakdown and
absorption in the gut are frequently prescribed to poorly compliant patients, or as
supplements to the dietary regimen. Drugs known as -glucosidase inhibitors belong to
this class. Three -glucosidase inhibitors - Acarbose, Miglitol and Voglibose - are
currently approved for the treatment of type 2 diabetes. Of the three, Acarbose is the most
widely prescribed.3 It is a pseudo-tetrasaccharide of microbial origin (Actinoplanes) that
acts as reversible competitive inhibitor of extracellular -glucosidase of the brush border
of the small intestine in addition to pancreatic alpha-amylase.4
Owing to the success of -glucosidase inhibitors as antidiabetic agents and the postulated
involvement of this enzyme in tumorigenesis 5 and viral infection, 6 the interest in the
discovery and development of novel, structurally different inhibitors is much alive.7-10
Several of the compounds described in the literature are drawn from plants used in the
folk medicine around the world as remedy against diabetes. 11 Fewer -glucosidase
inhibitors have originated through rational drug design and novel chemical synthesis. 12-14
None has supplanted the use of Acarbose or the other clinically tested inhibitors.
Iminosugars are a rich source of glycosidase inhibitors.14-16 Since the discovery of
nojirimicin,17 the first identified natural -glucosidase inhibitor, a number of synthetic
mimetics based on the same scaffold of that of azasugars 18 and aminocyclitols 19, have
been evaluated. As with nojirimycin, the prototypical iminosugar and enzymatic
transition state analogue, a protonated nitrogen in these compounds is responsible for the
formation of a stable electrostatic interaction with the critical carboxylate ion located
within the catalytic pocket of the -glucosidase. It is postulated that the ammonium ion
by mimicking the positively charged oxycarbenium ion in the pyranose ring of the natural
substrate during catalysis, accounts for the competitive mechanism of inhibition of the
compounds.11 Since most glycosidases utilize the carboxylate ion as a catalytic group, the
4

poor specificity of iminosugars and the related scaffolds toward -glucosidase is a
limiting factor for their development as drugs.
Additional experimentation has involved compounds where the protonable nitrogen is
substituted or is replaced with another atom. Of these, Emiglitate, a synthetic analogue
of deoxynojirimycin 4 and Salacinol, a naturally occurring thiasugar 20 display remarkable
potency both in vitro and in vivo. Interestingly in these compounds the protonable center
(either nitrogen or sulphur) carries a substituent harboring an electron rich region. A
structural feature that is uncommon in natural and synthetic iminosugars or in -
glucosidase inhibitors in general. Also there is little information on the effect of replacing
the hydroxyl residues on the iminosugar pyranose ring with different substituents. Such a
substitution may drastically affect the inhibitory activity since key features of the natural
substrate would be missing. However, the recent discovery 21 that 4,5 dihydroxy-1,2
cyclohexanedicarboxylic acids are endowed with -glucosidase inhibitory activity
challenges this view. We were intrigued by the presence of carboxylic acid, a feature that
could lead to new molecules with binding mode that is different from that of the current
drugs. Given these premises, we embarked in a chemical program aimed at synthesizing a
novel series of azasugars embedding in their structure some of the non-canonical features
discussed above. In particular the role of carboxylic acid on the azasugar ring and of a N-
linked heteroaryl substituent bearing hydrogen bond donor/acceptors has been
investigated. Here we report the finding that a series of N-heteroarylmethyl-5-hydroxy-
1,2,5,6-tetrahydropyridine-3-carboxylic acid compounds act as potent -glucosidase
inhibitors. Unlike Acarbose and the other clinically available -glucosidase inhibitors,
the compounds act through a reversible uncompetitive mechanism of inhibition which
make them attractive as candidate for drug development.
2. Results and Discussions
2.1 Chemistry
5

The scaffold 1-heteroarylmethyl-4-hydroxy-piperidine-3-carboxylic acids 7a-d (Fig.1) 22
was prepared with slight modifications of the literature procedures. In detail the
commercially available ethyl 1-benzyl-4-oxo-3-piperidine carboxylate hydrochloride 1
(from Aldrich), after neutralization by TEA (triethylamine) was reduced by sodium
borohydride to give an inseparable mixture of the corresponding alcohol 2 (mixture of
cis- and trans- isomers).23 This mixture was treated with tert-butyldimethylsilyl chloride
(TBSCl) in the presence of imidazole in DMF to give the TBS-ether 5 and the cis-isomer
of alcohol 2b.24 Column chromatography on silica gel gave the pure alcohol 2b. Removal
of benzyl protecting group by means of palladium on carbon (10%) in ethanol, followed
by alkylation of the chlorohydrate, by means of Hüning base in acetonitrile 25 in the
presence of alkylating halides, yielded the targets 7a-7d. Sample 3 plus 4 was prepared
by simple NaOH/MeOH mediated hydrolysis of sample 2. Preparations of samples 10a-
10f were realized as outlined into Fig.2 22 starting from commercially available arecoline
hydrobromide 8. Accordingly demethylation of arecoline was performed by known
procedure.26 Product 15 was obtained from 9 by its hydrolysis and tested as such. The
demethylated product 9, in turn, was alkylated by the suitable alkyl halide in the presence
of bicarbonate 27, followed by hydrolysis with sodium hydroxide, to give the targets 10a-
10d. Targets 10e and 10f were synthesized by adopting the alkylating procedure (DIPEA,
acetonitrile and the corresponding alkylating agent), followed by hydrolysis by
NaOHaq/MeOH.
25 In order to test the potential activity of other double bond isomer,
product 14 was prepared via an alternative demethylation procedure of arecoline with
chloroformiate followed by isomerization of the resulting carbamate 12 (Fig.2) with
LDA, in the presence of hexametapol (HMPA), and SO2Cl2 mediated esterification with
MeOH. The 5-hydroxy guvacine scaffold 20 was obtained by means of oxidation of
product 17 (Fig.2) by meta-chloroperbenzoic acid followed by elimination to give
intermediate 20. After removal of N-Boc protecting group, the corresponding alkylation
25 yielded targets 21a-21e. Hydrolysis of the said products with hydrogen chloride gave
the end products 22a-22e (Fig.2).
2.2 Enzyme inhibition and mechanism
2.2.1 Screening of the glycosidase activity
6

All the compounds underwent initial testing at 100 M level. In addition to -
glucosidase, the compounds were also tested on -glucuronidase and hyaluronidase. This
was justified by the presence of a carboxylate moiety on the azasugar ring as in some
analogues of siastatin B reported in the literature 14 displaying activity against -
glucuronidase. None of our compounds showed activity toward these two enzymes
(Table 1). On the other hand, compounds 22b, 22c, 22d, and 22e were identified as
potent α-glucosidase inhibitors. The IC50 of these compounds fell between 2 and 13 M
when evaluated over the 0.17 to 100 M concentration range. In particular compound 22c
displayed the highest inhibitory activity (IC50 2.34 M). An inspection of the structure of
the active compounds highlights key features required for activity. Beside the critical
presence of the 5-hydroxy residue (which cannot be replaced by the same substituent in 4
as in compounds 7a-c) a 3-carboxylate residue on the 1,2,5,6-tetrahydropyridine ring
appears crucial. Specifically its esterification leads to loss of activity as seen in
compounds 21b-e,. Also critical is the presence of an N-substituent carrying an electron-
rich region. Indeed, when the pyridine or the 1,3-diazacyclohexan-2,6-dione rings are
replaced by a benzene ring (as in compound 22a), the activity is lost. The remarkable
inhibitory effect of these compounds in spite of the reduced protonability of the tertiary
amine and as well as other major structural changes such as a reduced hydroxylation of
the tetrahydropyridine ring, leads us to hypothesize alternative mechanism of inhibition
to that of the canonical transition state analogues of the D-glucosyl carbocation. The
modality through which the novel inhibitors affect -glucosidase kinetically, was
therefore investigated.
2.2.2 Modality of enzymatic inhibition
A study of the mechanism of enzymatic inhibition was carried out on the active
compounds. The inhibitory kinetics were investigated in the presence of increasing
concentrations (0.1 to 1.5 mM) of the 4-nitrophenyl-α-glucopyranoside (pNPG) substrate.
For all the compounds, both Km and Vmax, calculated from Michaelis-Menten plots of the
results (see Supp. Fig A), decreased at increasing inhibitor concentrations. In order to
gain insight into the modality of inhibition, the results were analyzed according to Dixon
7

28 (Fig. 3A-D). In all cases, families of 1/V vs [I] regression lines displaying nearly
identical slope were computed strongly suggesting the compounds acted as uncompetitive
inhibitors. This same pattern was seen when data where plotted according to Lineweaver-
Burk (1/V vs 1/[S]) (Supp. Fig A) thus strengthening the previous conclusion. Because of
the identical slopes, Ki values could not be derived from the Dixon plot and were thus
calculated according to Cornish–Bowden 29 (Fig. 3E-H). For the four compounds tested,
the Ki values ranged between 1.2 and 12.5 uM.
To further corroborate our findings, the enzymatic kinetic for compound 22c was re-
examined using maltose as substrate. The results shown in Fig. 4, are in agreement with
the conclusions reached from the experiments using pNPG as substrate. Recently Yang et
al. 30 have proposed a different method for the analysis of the kinetic data that overcomes
possible misinterpretation of the kinetic results.30 In particular this problem applies to
uncompetitive inhibitors that may be erroneously assigned to the mixed inhibitors
category. According to this procedure Km and Vmax are plotted against the inhibitor
concentration and two new kinetic constants, Kik and Kiv, are derived. The found Kik/Kiv
ratios comprised between 1 and 2 at all the inhibitor’s concentrations tested (see Table 2),
fits in well with Yang’s computed parameter for uncompetitive inhibitors.30
Uncompetitive inhibitors of -glucosidase have rarely been reported and the structural
features underlying their mode of action have not been systematically investigated.
Cornish-Bowden 31 has discussed as to why uncompetitive inhibition is rarely seen in
metabolic system by comparing the “catastrophic” response of a metabolic pathway to
the presence of an uncompetitive inhibitor to the much milder response elicited by a
competitive inhibitor. Based on his conclusions, uncompetitive inhibitors are to be
regarded as superior for drug development when compared to competitive and non-
competitive inhibitors and are expected to display better in-vivo efficacy. Westley et al. 32
reached similar conclusions, and further suggested that specific enzymic reaction
products - rather than analogs of substrates, provide a promising basis for the systematic
design of uncompetitive inhibitors. Given the paucity of structure-activity relationship
8

data on uncompetitive inhibitors, the issue of the identity of the interacting site in yeast
-glucosidase involved in binding of the novel inhibitors, remains at present unresolved
and must await further investigation including computer-assisted docking studies.
To gain information on the specificity of action, a survey of the literature was conducted
to identify compounds with structural features that are shared by the active azasugars we
report. A series of 1-benzyl-1,2,5,6-tetrahydropyridine-3-carboxylic acid derivates has
been reported as platelet antiaggregants.33 However in these compounds the
tetrahydropyridine ring is lacking both hydroxyl and N-linked heteroaromatic
substituents. A series of tertiary and quaternary methyl and propargyl N-substituted
1,2,5,6-tetrahydropyridine-3-carboxylic acids derivatives has been reported to be
endowed with muscarinic activity.34 In these compounds the tetrahydropyridine ring is
not hydroxylated and the replacement of the N-alkyl group by larger substituents results
in loss of activity. A number of siastatin B derivatives also bears partial resemblance to
our compounds. 14 While harboring a 3-carboxyl azasugars backbone, their activity
against -glucuronidase, heparanase or neuraminidase 35-37, is critically dependent upon
the presence of a 2-animoacetyl substituent which is absent in our products. None of the
structure surveyed display activity against -glucosidase of the same order as our
compounds. In summary, the structures described in this paper differ from previously
reported compounds in incorporating on the same structure three key features: 1) a
carboxylic acid at position 3 attached to a sp2 carbon atom, 2) an hydroxyl group at
position 5 and, 3) an heteroaryl substituent at position 1 bearing hydrogen bond
donor/acceptors.
3. Conclusions
In the present investigation, a series of N-heteroarylmethyl-5-hydroxy-1,2,5,6
tetrahydropyridine-3-carboxylic acid compounds was found endowed with potent α-
glucosidase inhibitory activity. Unlike Acarbose and the other clinically available
inhibitors, the compounds displayed uncompetitive modality of enzyme inhibition which
makes them good candidates for in vivo testing. The results from the biological
9

evaluation of compounds, whose structure is comprised within a rather limited chemical
space, allowed for the identification of structural features that are critical for the activity
of this novel class of -glucosidase inhibitors. A carboxylate and a hydroxyl residues
properly positioned on the azasugar ring, were found both necessary and sufficient for
activity when coupled with a suitable aglycone attached to the protonable nitrogen. This
simple scaffold and the significant inhibitory activity it affords, are good starting points
for further structural investigations. The data gathered reduce, at least in part, the paucity
of information on the structure-activity relationship of uncompetitive -glucosidase
inhibitors. They should serve as a basis for the design and development of novel
therapeutics for the treatment of diabetes mellitus and other diseases where α-glucosidase
is involved.
4. Experimental
4.1 Chemistry: Material and Methods
All starting materials, unless otherwise stated, were purchased and used without any
further purification. The starting material for the 4-hydroxy-piperidine-3-carboxylic acid
scaffold was the commercially available ethyl 1-benzyl-4-oxopiperidine-3-carboxylate
from Sigma Aldrich. For the compounds reported in Fig.2 the starting material was the
commercially available Arecoline hydrobromide from Qingdao Sicemo, LTD, Qingdao,
China. Solvents were distilled and dried according to standard procedures. Column
chromatography was performed using Merck KGaA Silicagel 60 (230-400 Mesh-ASTM).
Melting points were obtained using a Stuart Scientific SMP3 Melting Point apparatus. IR
spectra were recorded on a Nicolet 380 FT-IR infrared spectrometer. NMR spectroscopy
was performed on a Varian-Mercury 400 spectrometer using the residual signal of the
solvent as the internal standard. The chemical shifts are reported in ppm and coupling
constants (J) in Hz. GC–MS spectra were recorded using Agilent Technologies 6850 and
5975 GC-Mass instrumentation. LC–MS spectra were obtained using an Agilent
Technologies MSD1100 single-quadrupole mass spectrometer. Elemental analyses were
obtained at Laboratorio Scienze Ambientali (Ravenna, Italy) using a Flash 2000, series
CHNS/O Analyzer (Thermo Scientific).
10

Preparation of ethyl 1-benzyl-4-hydroxypiperidine-3-carboxylate 2
Ethyl 1-benzyl-4-oxo-3-piperidine carboxylate hydrochloride 1 (5 g, 16.8 mmol) was
dissolved into 130 ml methanol. NEt3 (2.3 ml, 16.8 mmol) was dropped into this solution
at 0 oC and stirred for 10 min at this temperature. NaBH4 (2.1 g, 3 eq) was added into the
reaction mixture in portions. The reaction mixture was kept at 0 oC for 2hrs. HClaq (5 M)
was added to adjust the pH =2~3. The solvent was partially removed to small volume
under vacuum and the residue was neutralized by saturated NaHCO3 solution. This
mixture was extracted by CH2Cl2 (30 ml×3). The organic phase was dried (Na2SO4) and
the solvent removed under vacuum to obtain 3.64 g of yellow liquid as a mixture of two
diastereomers which was purified by flash chromatography (AcOEt: cyclohexane =3:2)
to give product 2 (3.64 g, ratio: trans/cis=4/7, yield: 82.7%).
Spectra for (3S*,4S*)-ethyl 1-benzyl-4-hydroxypiperidine-3-carboxylate 2a
IR (film): 1731 cm−1; 1H NMR (400 MHz, CDCl3) δ= 1.18 (t, J=7.2 Hz, 3H), 1.57 (m,
1H), 1.87 (m, 1H), 2.03 (m, 2H), 2.53 (m, 1H), 2.78 (m, 1H), 3.04 (m, 1H), 3.45 (qAB,
J=13.6 Hz, 2H), 3.71 (m, 1H), 4.08 (m, 2H), 7.15-7.27 (m, 5H); 13C NMR (100 MHz,
CDCl3) δ= 14.12, 32.57, 49.64, 51.33, 53.41, 60.88, 62.24, 69.67, 127.8, 128.28, 128.96,
173.38; MS (EI) m/z =263 [M]; Elemental Analysis: Calcd. for C15H21NO3: C, 68.42; H,
8.04; N, 5.32; O, 18.23; Found: C, 68.64; H, 8.07; N, 5.34
Spectra for (3S*,4R*)-ethyl 1-benzyl-4-hydroxypiperidine-3-carboxylate 2b
IR (film): 1731 cm−1; 1H NMR (400 MHz, CDCl3) δ= 1.16 (t, J=7.2 Hz, 3H), 1.71 (m,
1H), 1.80 (m, 1H), 2.41 (m, 2H), 2.58 (m,1H), 2.68 (m, 2H), 3.41 (qAB, J=13.2 Hz, 2H),
4.01 (m, 1H), 4.08 (m, 2H), 7.15-7.26 (m, 5H); 13C NMR (100 MHz, CDCl3) δ= 14.08,
31.51, 46.17, 50.62, 60.76, 62.70, 77.50, 127.19, 128.24, 129.08, 173.70; MS (EI) m/z =
263 [M].
Preparation of sodium 1-benzyl-4-hydroxypiperidine-3-carboxylates 3 and 4
Compound 2 (150 mg, 0.57 mmol) was mixed with NaOHaq (0.32 ml, 2M) and MeOH (3
ml) for 3 hrs at r.t. Once the reaction was complete, the solvent was removed under
vacuum. The residue was treated with anhydrous MeOH and filtered. The filtrate was
11

concentrated under vacuum to obtain the mixture of two diastereomers (3 and 4) (140
mg, ratio: trans/cis= 2:3, yield: 95%)
Spectral data of mixture of compounds 3 and 4)
Sodium (3S*,4S*)-1-benzyl-4-hydroxypiperidine-3-carboxylate 3
IR (film): 1644 cm−1; 1H NMR (400 MHz, CD3OD) δ= 1.57 (dq, J1=4.0 Hz, J2=10.8 Hz,
1H), 1.88 (m, 1H), 2.05 (m, 2H), 2.31 (ddd, J1=4.0 Hz, J2=1.2 Hz, J2=10.0 Hz,1H), 2.88
(m, 1H), 3.15 (m, 1H), 3.54 (qAB, J=12.8 Hz, 2H), 3.61 (dt, J1=4.8 Hz, J2=10.8 Hz, 1H),
7.30 (m, 5H); 13C NMR (100 MHz, CDCl3) δ= 33.94, 49.43, 53.01, 55.88, 63.68, 71.89,
128.32, 129.25, 130.75, 138.61, 181.01; MS (ESI) m/z =234 [M-Na]-; Elemental
Analysis: Calcd. for C13H16NNaO3: C, 60.69; H, 6.27; N, 5.44; Na, 8.94; O, 18.66;
Found:C, 60.85; H, 6.29; N,5.45.
Sodium (3S*,4R*)-1-benzyl-4-hydroxypiperidine-3-carboxylate 4 (for the spectra of
corresponding acid derivative see product 7d)
IR (film): 1644 cm−1; 1H NMR (400 MHz, CD3OD) δ=1.75 (m, 2H), 2.44(m, 1H), 2.52
(m, 3H), 2.86 (m, 1H), 3.54 (qAB, J=12.8 Hz, 2H), 4.11 (m, 1H), 7.30 (m, 5H); 13C NMR
(100 MHz, CDCl3) δ= 32.16, 49.63, 52.57, 55.87, 64.22, 66.87, 128.26, 129.21, 130.65,
138.55, 181.56; MS (ESI) m/z =234 [M-Na]-.
Preparation of (3S*, 4R*)-ethyl 1-benzyl-4-hydroxypiperidine-3-carboxylate 2b and
(3S*, 4S*)-ethyl 1-benzyl-4-(ter-butyldimethylsilyloxy)piperidine-3-carboxylate 5.
The inseparable mixture of two diastereomers 2 (2.5g, 9.5 mmol) was treated with
imidazole (0.36 g, 5.2 mmol) and TBDMSCl (0.72g, 4.75) in anhydrous DMF (16 ml) at
r.t. After stirring for 5hrs the reaction mixture was decomposed with ice/water and
extracted with CH2Cl2 (30 ml x 3). The organic phase was washed by brine, dried by
sodium sulfate and the solvent removed under vacuum to obtain a yellow oil (3.3g).
Purification by flash chromatography (Ether : Cyclohexane=1:1) gave 2b (1.17g) and 5
(1.31 g) in 85% overall yields. Spectral data as follow:
(3S*, 4R*)-ethyl 1-benzyl-4-hydroxypiperidine-3-carboxylate 2b（for spectral data
see compound 2）
(3S*, 4S*)-ethyl 1-benzyl-4-(ter-butyldimethylsilyloxy)piperidine-3-carboxylate 5
12

IR (film): 1732 cm−1; 1H NMR (400 MHz, CDCl3) δ= 0.01 (s, 3H), 0.04 (s, 3H), 0.85 (s,
9H), 1.23 (t, J= 7.2 Hz, 3H), 1.65 (m, 1H), 1.84 (m, 1H), 2.16 (t, J=12.0 Hz, 1H), 2.28 (t,
J=12.0 Hz, 1H), 2.62 (dt, J1=2.8 Hz, J2=12.0 Hz, 1H), 2.80(m, 1H), 2.89 (d, J=10.8 Hz,
1H), 3.52 (s, 2H), 3.90 (ddd, J1=4.0 Hz, J2=4.4 Hz, J3=5.6 Hz, 1H), 4.09 (m, 2H), 7.20-
7.31(m, 5H); 13C NMR (100 MHz, CDCl3) δ= 14.13, 20.26, 32.57, 44.42, 46.92, 60.80,
66.08, 173.82; MS (EI) m/z =377 [M]; Elemental Analysis: Calcd. for C21H35NO3Si: C,
66.80; H, 9.34; N, 3.71; O, 12.71; Si, 7.44; Found: C,66.98; H, 9.37; N, 3,72.
Preparation of (3S*,4R*)-ethyl 4-hydroxypiperidine-3-carboxylate hydrochloride 6
Product 2b (1.03 g, 3.9 mmol), Pd/C 10% (1g) and EtOH (100 ml) were mixed together
and left to react in Parr apparatus for 1.5 hrs under hydrogen pressure (15 psi). The
reaction mixture was filtered through celite. The filtrate was concentrated under vacuum
to obtain compound 6 as a yellow oil (0.65 g, yield: 96%). Spectral data were
superimposable with literature data. 38
Synthesis of (3S*,4R*)-4-hydroxy-1-(heteroarylmethyl)piperidine-3-carboxylic
acids:
Preparation of (3S*,4R*)-4-hydroxy-1-(pyridin-2-yl-methyl)piperidine-3-carboxylic
acid 7a as general procedure.
Compound 6 (87 mg, 0.5 mmol), 2-(bromomethyl)-pyridine hydrobromide (126 mg, 0.5
mmol), N,N-diisopropylethylamine (DIPEA) (0.35 ml, 2.0 mmol) and CH3CN (10 ml)
were mixed together at 0 oC. The reaction was kept at r.t. for 5 hrs. Once the reaction was
completed (t.l.c. test) the solvent was concentrated under vacuum and the residue was
dissolved in 20 ml CH2Cl2. The organic phase was washed by saturated NaHCO3. The
aqueous phase was extracted by CH2Cl2 (15 ml×2). The organic phase was collected,
dried with sodium sulphate and the solvent removed under vacuum to afford a residue
which was purified by flash chromatograph (AcOEt: MeOH=1:1) to obtain pure
compound 7a (100 mg, yield: 75.8%) identified as ethyl ester. Spectral data as follow
Ethylester of (3S*,4R*)-4-hydroxy-1-(pyridin-2-yl-methyl)piperidine-3-carboxylic
acid 7a
13

IR (film): 1728 cm−1; 1H NMR (400 MHz, CDCl3) δ= 1.17 (t, J=6.4 Hz, 3H), 1.81 (m,
2H), 2.51 (m, 2H), 2.71(m, 2H), 3.38 (bs, 1H, OH), 3.64 (qAB, J=14.4 Hz, 2H), 4.11(m,
3H), 7.11(m, 1H), 7.34 (d, J=8.0 Hz, 1H), 7.59 (dt, J1=2.0 Hz, J2=7.6 Hz, 1H), 8.50 (m,
1H); 13C NMR (100 MHz, CDCl3) δ= 14.05, 31.71, 46.30, 47.13, 50.32, 60.61, 64.26,
65.74, 121.98, 122.98, 136.35, 149.10, 158.49, 173.56; MS (EI) m/z =264 [M];
Elemental Analysis: C14H20N2O3 C, 63.62; H, 7.63; N, 10.60; O, 18.16; Found: C, 63.81;
H, 7.65; N, 10.63.
Product 7a was obtained by treatment of the corresponding ester (100 mg, 0.43 mmol)
with NaOHaq (0.32 ml, 2 M) and MeOH (2.5 ml) at r.t. for 5 hrs. The solvent was
removed under vacuum to obtain a crude mixture which was purified by flash
chromatography (AcOEt: acetone: H2O: CH3COOH= 5:3:2:2) to obtain pure 7a (87 mg,
yield: 92%).
IR (film): 1658 cm−1; 1H NMR (400 MHz, CD3OD) δ= 1.79 (m, 2H), 2.48 (m, 1H), 2.55
(m, 2H), 2.70 (bs, 2H), 3.61 (qAB, J=13.6 Hz, 2H), 4.05 (bs, 1H), 7.33 (m, 1H), 7.30 (m,
1H), 7.54 (d, J=7.6 Hz, 1H), 7.81 (dt, J1=1.2 Hz, J2=7.6 Hz, 1H), 8.48 (m, 1H); 13C NMR
(100 MHz, CD3OD) δ= 32.57, 49.63, 49.85, 53.10, 65.09, 66.74, 123.78, 125.20, 138.60,
149.70, 159.44, 181.52; MS (EI) m/z =235 [M-H]-; Elemental Analysis: Calcd. for
C12H16N2O3: C, 61.00; H, 6.83; N, 11.86; O, 20.32; Found: C, 61.15; H, 6.85; N, 11.89.
Preparation of (3S*,4R*)-4-hydroxy-1-(pyridin-3-ylmethyl)piperidine-3-carboxylic
acid 7b.
This product was prepared following the reported procedure for compound 7a. Starting
from compound 6 (87 mg, 0.5 mmol), 3-(bromomethyl)-pyridine hydrobromide (126 mg,
0.5 mmol), the ethyl ester of 7b was obtained after the flash chromatography (AcOEt:
MeOH=1:1) (95 mg, yield: 72%).
IR (film): 1728 m−1;1H NMR (400 MHz, CDCl3) δ= 1.17 (t, J=7.2 Hz, 3H), 1.76 (m, 1H),
1.82 (m, 1H), 2.46 (m, 2H), 2.67 (m, 2H), 3.50 (bs, 1H), 3.50 (qAB,J=13.6 Hz, 2H), 4.10
(m, 3H), 7.21 (dd, J1=4.8 Hz, J2=7.6 Hz,1H), 7.62 (d, J=7.6 Hz, 1H), 8.46 (d, J=14.4 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ= 14.05, 31.76, 46.33, 48.12, 50.93, 59.95, 60.66,
65.94, 123.28, 133.77, 136.59, 148.42, 150.19, 173.41; MS (EI) m/z =264 [M];
14

Elemental Analysis: Calcd. for C14H20N2O3: C, 63.62; H, 7.63; N, 10.60; O, 18.16;
Found: C, 63.80; H, 7.65; N, 10.63.
Following the same procedure for the preparation of compound 7a, the ethyl ester of
compound 7b (95 mg, 0.41 mmol) was treated with NaOHaq (0.31 ml, 2M) and MeOH
(2.5 ml). After chromatography (AcOEt: acetone: H2O: CH3COOH= 5:3:2:2) 7b was
obtained (85 mg, yield: 94.4%).
IR (film): 1654 cm−1; 1H NMR (400 MHz, CD3OD) δ= 1.75 (m, 2H), 2.49 (m, 3H), 2.73
(m, 1H), 3.58 (qAB, J=13.6 Hz, 2H), 4.10 (m, 1H), 7.40 (dd, J1=5.6 Hz, J2=8.0 Hz, 1H),
7.84 (dt, J1=1.6 Hz, J2=8.0 Hz, 1H), 8.43 (dd, J1=1.2 Hz,J2=4.8 Hz, 1H), 8.51 (d, J=1.6
Hz, 1H); 13C NMR (100 MHz, CD3OD) δ= 32.49, 49.43, 49.63, 51.32, 51.32, 61.05,
66.78, 125.03, 125.07, 135.60, 139.35, 148.92, 151.00, 181.46; MS (EI) m/z =235 [M-H]-
; Elemental Analysis: Calcd. for C12H16N2O3: C, 61.00; H, 6.83; N, 11.86; O, 20.32 ;
Found: C, 61.16; H, 6.85; N, 11.89.
Preparation of (3S*,4R*)-4-hydroxy-1-(pyridin-4-ylmethyl)piperidine-3-carboxylic
acid 7c.
This product was prepared following the above reported procedure for compound 7a.
Starting from compound 6 (87 mg, 0.5 mmol), 4-(bromomethyl)-pyridine hydrobromide
(126 mg, 0.5 mmol), ethyl ester of 7c was obtained after the flash chromatography
(AcOEt: MeOH=1:1) (100 mg, yield: 75.8%).
IR (film): 1728 cm−1; 1H NMR (400 MHz, CDCl3) δ= 1.17 (t, J=7.2 Hz, 3H), 1.79 (m,
2H), 2.45 (m, 2H), 2.66 (m, 2H), 3.48 (qAB, J=14.8 Hz, 2H), 3.66 (bs, 1H, OH), 4.08 (q,
J=7.2 Hz, 2H), 4.13(m, 1H), 7.21 (d, J=6.0 Hz, 2H), 8.45 (dd, J1=1.2 Hz, J2=4.4 Hz, 2H);
13C NMR (100 MHz, CDCl3) δ= 14.06, 31.86, 46.41, 48.02, 51.03, 60.63, 61.47, 65.11,
123.67, 147.87, 149.50, 149.54, 173.29; MS (EI) m/z =264 [M]; Elemental Analysis:
Calcd. for C14H20N2O3: C, 63.62; H, 7.63; N, 10.60; O, 18.16; Found: C, 63.79; H, 7.65;
N, 10.63.
Following the same procedure for the preparation of compound 7a, and starting from the
corresponding ethyl ester of 7c (100 mg, 0.43 mmol), NaOHaq (0.33 ml, 2M) and MeOH
15

(2.5 ml), compound 7c was obtained after chromatography (AcOEt: acetone: H2O:
CH3COOH= 5:3:2:2) (80 mg, yield: 84.6%).
IR (film): 1658 cm−1; 1H NMR (200 MHz, CD3OD) δ= 1.77 (m, 2H), 2.58 (m, 4H), 3.57
(s, 2H), 4.06 (s, 1H), 7.40 (m, 2H), 8.40 (m, 2H); 13C NMR (100 MHz, CD3OD) δ=
32.59, 49.45, 49.66, 52.83, 62.58, 66.81, 125.80, 125.84, 149.94, 150.02, 150.42, 181.49;
MS (EI) m/z =235 [M-H]-; Elemental Analysis: Calcd. for C12H16N2O3: C, 61.00; H,
6.83; N, 11.86; O, 20.32 ; Found: C, 61.14; H, 6.85; N, 11.89.
Preparation of (3S*,4R*)-1-benzyl-4-hydroxypiperidine-3-carboxylic acid 7d
Compound 7d was obtained by hydrolysis of compound 2b (100 mg, 0.43 mmol),
NaOHaq (0.33 ml, 2M) and MeOH (2.5 ml), removal of the solvent and flash
chromatography of the residue (AcOEt: acetone: H2O: CH3COOH= 5:3:1:1) (88 mg,
yield: 93.6%).
IR (film): 1644 cm−1; 1H NMR (400 MHz, CD3OD) δ= 1.76 (m, 2H), 2.44 (m, 1H), 2.53
(m, 3H), 2.83 (m, 1H), 3.54 (qAB, J=13.2, 2H), 4.10 (m, 1H), 7.22-7.35 (m, 5H); 13C
NMR (100 MHz, CD3OD) δ= 34.77, 51.86, 52.08, 52.29, 66.90. 69.82, 130.94, 131.89,
133.46, 141.24, 184.22; MS (EI) m/z = 234 [M-H]-; Elemental Analysis: Calcd. for
C13H17NO3:C, 66.36; H, 7.28; N, 5.95; O, 20.40; Found: C, 66.58; H, 7.30; N, 5.97.
Preparation of methyl 1,2,5,6-tetrahydropyridine-3-carboxylate hydrochloride 9
The pH of a solution of Arecoline hydrobromide 8 (Qingdao Sicemo, LTD, Qingdao,
China) (15g) in water (30 ml) was adjusted to pH=13~14 by saturated Na2CO3 solution.
This solution was extracted by ether (50 ml×3). The yellow oil (Arecoline, 9.8g) was
obtained after drying and removing the solvent.
Arecoline (1.55g, 10 mmol) was dissolved into 35 ml CH2Cl2. A mixture of ACE-Cl (1.2
ml, 11 mmol) and ClCH2CH2Cl (2 ml) was dropped into the reaction mixture at 0 °C and
stirred for 15 min. The temperature was increased to 100 °C and the mixture refluxed for
6 hrs (monitor by TLC). The solvent was removed under vacuum. The residue was
dissolved into 25 ml methanol. This reaction mixture was kept refluxing at 110 °C for 6
hrs until the reaction completed (monitor by TLC: AcOEt : Cyclohexane=3:2). Then the
16

solvent was removed to obtain compound 9 (0.825 g, two steps overall yield: 58.5%).
This product is directly used in the next step without further purification.
IR (film): 1710 cm−1; 1H NMR (400 MHz, CDCl3) δ= 1.72 (s, 1H, NH), 2.19 (m, 2H),
2.87 (t, J=5.7 Hz, 2H), 3.49 (m, 2H), 3.68 (s, 3H), 6.99 (m, 1H); 13C NMR (100 MHz,
CDCl3) δ= 26.15, 41.83, 44.02, 51.40, 130.26, 138.22, 166.41; MS (EI) m/z =141 [M-
HCl]; Elemental Analysis: Calcd. for C7H12ClNO2: C, 47.33; H, 6.81; Cl, 19.96; N, 7.89;
O, 18.01; Found: C, 47.48; H, 6.83; N, 7.92.
Synthesis of sodium-1-(heteroarylmethyl)-1,2,5,6-tetrahydropyridine-3-
carboxylates:
Preparation of sodium-1-benzyl-1,2,5,6-tetrahydropyridine-3-carboxylate 10a as
general procedure.
NaHCO3 (179 mg, 2.24 mmol) and EtOH (20 ml) were mixed together and stirred at 0 °C
for 5 min. Product 9 (100 mg, 0.56 mmol) and (bromomethyl)-benzene (96.6 mg, 0.56
mmol) were then added to the mixture at 0° C. The reaction mixture was refluxed at 110
°C for 5 hrs until the reaction was completed (t.l.c. test). The solvent was removed under
vacuum and the residue was dissolved with 20 ml CH2Cl2. The organic phase was
washed by saturated NaHCO3. The aqueous phase was extracted by CH2Cl2 (15 ml×3).
The organic phase was collected, dried with sodium sulfate and concentrated under
vacuum to obtain crude product. This was purified by flash chromatography (AcOEt:
cyclohexane = 1:4) to obtain 10a-methyl ester (50 mg, yield: 55.7%) which was
identified by its spectral data.
IR (KBr): 1722 cm−1; 1H NMR (400 MHz, CDCl3) δ= 2.33 (m, 2H), 2.54 (t, J= 5.6 Hz,
2H), 3.24 (m, 2H), 3.66 (s, 2H), 3.73 (s, 3H), 7.02 (m, 1H), 7.25-7.37 (m, 5H); 13C NMR
(100 MHz, CDCl3) δ= 26.55, 48.30, 51.47, 51.62, 62.44, 127.12, 128.28, 129.05,
138.00, 166.35;MS (EI) m/z =231 [M]; Elemental Analysis: Calcd. for C14H17NO2: C,
72.70; H, 7.41; N, 6.06; O, 13.83; Found: C, 72.90; H, 7.43; N, 6.08.
Methyl ester derivative of 10a (45 mg) was dissolved in 1 ml of MeOH. Then 0.145 ml
of NaOHaq (2 M) was added. This reaction mixture was kept at 50 °C for 2.5 hrs. After
completion, the solvent was removed under vacuum to obtain the crude product which
17

was dissolved into anhydrous MeOH and filtered. The filtrate was concentrated under
vacuum to obtain the pure 10a (40 mg, yield: 85%).
IR (film): 1700 cm−1; 1H NMR (400 MHz, CD3OD) δ= 2.63 (m, 2H), 3.31 (m, 4H), 4.44
(s, 2H), 7.21 (m, 1H), 7.44-7.59 (m, 5H); 13C NMR (100 MHz, CD3OD) δ= 23.91, 60.79,
125.41, 130.26, 130.39, 131.29, 132.41, 138.05, 138.08, 166.65; MS (ESI) m/z = 216
[M-Na]- ; Elemental Analysis: Calcd. for C13H14NNaO2: C, 65.26; H, 5.90; N, 5.85; Na,
9.61; O, 13.37; Found: C, 65.48; H, 5.92; N, 5.87.
Preparation of sodium 1-(pyridin-2-ylmethyl)-1,2,5,6-tetrahydropyridine-3-
carboxylate 10b
Following the same procedure for the preparation of compound 10a, starting from
NaHCO3 (239 mg, 2.84 mmol), EtOH (20 ml), product 9 (126 mg, 0.71 mmol) and 2-
(bromomethyl)pyridine hydrobromide (179 mg, 0.71 mmol), compound 10b-methyl
ester was obtained after flash chromatography (AcOEt : MeOH =9 :1) (127 mg, yield:
77.0%)
IR (film): 1722 cm−1;1H NMR (400 MHz, CDCl3) δ= 2.36 (m, 2H), 2.61 (t, J= 5.6 Hz,
2H), 3.28 (qAB, J= 2.8 Hz, 2H), 3.71 (s, 3H), 3.80 (s, 1H), 7.02 (m, 1H), 7.17 (m, 1H),
7.42 (d, J= 8.0 Hz, 1H), 8.44 (dt, J1= 1.2 Hz, J2= 7.6 Hz, 1H), 8.57 (d, J= 4.8 Hz , 1H);
13C NMR (100 MHz, CDCl3) δ= 26.46, 48.73, 51.62, 63.96, 122.07, 122.99, 128.93,
136.45, 137.94, 149.25, 158.48, 166.79; MS (EI) m/z =232[M]; Elemental Analysis:
Calcd. for C13H16N2O2: C, 67.22; H, 6.94; N, 12.06; O, 13.78; Found: C, 67.46; H, 6.96;
N, 12.10.
Following the same procedure for the preparation of compound 10a, starting from the
corresponding ester of 10b (87 mg, 0.38 mmol), and 0.28 ml of NaOHaq (2M), compound
10b was obtained (72 mg, yield: 80%).
IR (film): 1662 cm−1; 1H NMR (400 MHz, CD3OD) δ= 2.29 (m, 2H), 2.57 (t, J=5.6 Hz,
2H), 3.27 (dd, J1=2.8 Hz, J2=4.8 Hz,2H), 3.79 (s, 2H), 6.69 (m, 1H), 7.32 (dddd, J1=1.2
Hz, J2=4.8 Hz,J3=7.6 Hz,1H), 7.56 (dt, J1=7.6 Hz, J2=1.2 Hz,1H), 7.83 (dt, J1= 2.0 Hz,
J2=7.6 Hz, 1H), 8.50 (m, 1H); 13C NMR (100 MHz, CD3OD) δ= 26.85, 50.30, 54.39,
64.61, 123.92, 125.24, 132.14, 135.78, 138.70, 149.65, 159.21, 174.70; MS (ESI) m/z =
18

217 [M-Na]-; Elemental Analysis: Calcd. for C12H13N2NaO2: C, 60.00; H, 5.45; N,
11.66; Na, 9.57; O, 13.32; Found: C, 60.17; H, 5.47; N, 11.69.
Preparation of sodium 1-(pyridin-3-ylmethyl)-1,2,5,6-tetrahydropyridine-3-
carboxylate 10c
Following the same procedure for the preparation of compound 10a, starting from
NaHCO3 (239 mg, 2.84 mmol), EtOH (20 ml), product 9 (126 mg, 0.71 mmol) and 3-
(bromomethyl)pyridine hydrobromide (179 mg, 0.71 mmol), compound (10c-methyl
ester) was obtained after flash chromatography (AcOEt : MeOH =98:2) (70 mg, yield:
42.4%).
IR (film): 1712 cm−1; 1H NMR (400 MHz, CDCl3) δ= 2.31 (m, 2H), 2.53 (t, J= 5.6 Hz,
2H), 3.20 (m, 2H), 3.63 (s, 2H), 3.70 (s, 3H), 7.00 (m, 1H), 7.24 (m, 1H), 7.67 (d, J= 8.0
Hz, 1H), 8.50 (d, J= 4.4 Hz, 1H), 8.55 (s, 1H); 13C NMR (100 MHz, CDCl3) δ= 26.44,
48.35, 51.53, 51.56, 59.58, 123.36, 128.79, 133.52, 136.56, 137.90, 148.68, 150.30,
166.17; MS (EI) m/z =232 [M]; Elemental Analysis: Calcd. for C13H16N2O2: C, 67.22; H,
6.94; N, 12.06; O, 13.78; Found: C,67.38; H, 6.96; N,12.09.
Following the same procedure for the preparation of compound 10a, starting from the 10c
-methyl ester (70 mg, 0.30 mmol), and 0.23 ml of NaOHaq (2 M), compound 10c was
obtained (60 mg, yield: 83%)
IR (film): 1663 cm−1; 1H NMR (400 MHz, CD3OD) δ= 2.30 (m, 2H), 2.60 (t, J=5.6 Hz,
2H), 3.21 (dd, J1=2.4 Hz, J2=4.4 Hz, 2H), 3.70 (s, 2H), 6.69 (m, 1H), 7.41 (dddd, J1=0.8
Hz,J2=5.2 Hz, J3=7.6 Hz,1H), 7.89 (dt, J1=1.6 Hz, J2=8.0 Hz, 1H), 8.45 (dd, J1=1.6 Hz,
J2=4.8 Hz, 1H), 8.53 (m, 1H); 13C NMR (100 MHz, CD3OD) δ= 26.80, 50.11, 54.11,
60.57, 125.12, 125.14, 132.04, 132.16, 135.36, 135.61, 139.51, 148.99, 151.12, 174.59;
MS (ESI) m/z =217 [M-Na]-; Elemental Analysis: Calcd. for C12H13N2NaO2: C, 60.00;
H, 5.45; N, 11.66; Na, 9.57; O, 13.32; Found: C, 60.19; H, 5.47; N, 11.70.
Preparation of sodium 1-(pyridin-4-ylmethyl)-1,2,5,6-tetrahydropyridine-3-
carboxylate 10d
Following the same procedure for the preparation of compound 10a, starting from
NaHCO3 (239 mg, 2.84 mmol), EtOH (20 ml), product 9 (126 mg, 0.71 mmol) and 4-
19

(bromomethyl)pyridine hydrobromide (179 mg, 0.71 mmol), compound 10d-methyl
ester was obtained after flash chromatography (AcOEt : MeOH =8:1) (80 mg, yield:
48.5%)
IR (film): 1710 cm−1; 1H NMR (400 MHz, CDCl3) δ= 2.35 (m, 2H), 2.52 (m, 2H), 3.22
(m, 2H), 3.64 (s, 2H), 3.72 (s, 3H), 7.02 (m, 1H), 7.28 (d, J= 6.0 Hz, 2H), 8.54 (m, 2H);
13C NMR (100 MHz, CDCl3) δ= 26.44, 48.59, 51.51, 51.65, 61.12, 123.67, 128.81,
137.88, 147.50, 149.83, 165.15; MS (EI) m/z =232 [M]; Elemental Analysis: Calcd. for
C13H16N2O2: C, 67.22; H, 6.94; N, 12.06; O, 13.78; Found: C, 67.41; H, 6.96; N, 12.09.
Following the same procedure for the preparation of compound 10a, starting from 10d -
methyl ester (150 mg, 0.56 mmol), and 0.70 ml of NaOHaq (2 M), compound 10d was
obtained (114 mg, yield: 85%)
IR (film): 1666 cm−1; 1H NMR (400 MHz, CD3OD) δ= 2.31 (m, 2H), 2.55 (t, J=6.0 Hz,
2H), 3.22 (dd, J1=2.4 Hz,J2=4.8 Hz, 2H), 3.70 (s, 2H), 6.69 (m, 1H), 7.46 (d, J=7.2 Hz,
2H), 8.46 (d, J=7.2 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ= 26.84, 50.31, 54.30,
62.24, 125.93, 125.96, 132.03, 132.14, 135.68, 149.99, 150.07, 174.61; MS (EI) m/z
=217 [M-Na]- ; Elemental Analysis: Calcd. for C12H13N2NaO2: C, 60.00; H, 5.45; N,
11.66; Na, 9.57; O, 13.32; Found: C, 60.18; H, 5.47; N,11.69.
Preparation of sodium 1-((2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-yl)methyl)-
1,2,5,6-tetrahydropyridine-3-carboxylate 10e
Product 9 (150 mg, 0.85 mmol), 6-(chloromethyl)uracil (137 mg, 0.85 mmol) and
diisopropyl amine (0.739 ml, 4.25 mmol) were mixed in 15 ml of CH3CN. This reaction
mixture was kept at r.t. for 3 hrs. After the reaction completed (t.l.c testing), the solvent
was removed under vacuum to obtain the crude product. This crude product was purified
after flash chromatography (AcOEt : MeOH =3:1) to obtain 10e-methyl ester (150 mg,
yield: 66.7%).
IR (film): 1733, 1716, 1700, 1653 cm−1; 1H NMR (400 MHz, CD3OD) δ= 2.40 (m, 2H),
2.58 (t, J=6.0 Hz, 2H), 3.23 (qAB, J= 2.8 Hz, 2H), 3.42 (d, J= 1.2 Hz , 2H), 3.73 (s, 3H),
4.35 (s, 1H), 7.03 (m, 1H); 13C NMR (100 MHz, CD3OD) δ= 27.19, 41.27, 48.37, 52.21,
58.66, 100.40, 129.61, 139.33, 155.35, 167.64; MS (EI) m/z = 266 [M+1]; Elemental
20

Analysis: Calcd. for C12H15N3O4: C, 54.33; H, 5.70; N, 15.84; O, 24.13; Found: C,
54.43; H, 5.71; N, 15.87.
Compound 10e-methyl ester (50 mg, 0.18 mmol) was treated with NaOHaq (2 M) (0.43
ml) at 50°C for 3 hrs. After the hydrolysis was complete, the solvent was removed under
vacuum. The residue was dissolved in anhydrous MeOH (5 ml). The mixture was filtered.
The solution was concentrated under vacuum to give 10e (25 mg, yield: 48 %)
IR (film): 1715, 1670 cm−1; 1H NMR (400 MHz, D2O) δ= 2.50 (m, 2H), 3.03 (m, 2H),
3.59 (m, 2H), 3.85 (m, 2H), 5.92 (m, 1H), 6.77 (m, 1H); 13C NMR (100 MHz, D2O) δ=
23.61, 48.36, 51.35, 56.09, 102.44, 130.12, 133.00, 149.98, 152.81, 166.53, 172.85; MS
(ESI) m/z =274 [M+H]; Elemental Analysis: Calcd. for C11H12N3NaO4: C, 48.36; H,
4.43; N, 15.38; Na, 8.41; O, 23.42; Found: C, 48.46; H, 4.44; N, 15.41.
Preparation of sodium 1-((1H-benzo[d]imidazol-2-yl)methyl)-1,2,5,6-
tetrahydropyridine-3-carboxylate 10f
Following the same procedure for the preparation of 10e, starting from 9 (150 mg, 0.85
mmol), 2-(chloromethyl)-benzimidazole (142 mg, 0.85 mmol) and diisopropyl amine
(0.443 ml, 2.55 mmol), compound 10f-methyl ester (50 mg, yield: 21.7%) was obtained
after flash chromatography (AcOEt : cyclohexane = 3:1)
IR (film): 1709 cm−1; 1H NMR (400 MHz, CDCl3) δ= 2.33 (m, 2H), 2.64 (t, J=5.2 Hz,
2H), 3.31 (qAB, J=2.8 Hz, 2H), 3.70 (s, 3H), 3.96 (s, 2H), 7.01 (m, 1H), 7.22 (m, 2H),
7.57(m, 2H), 10.42 (bs, 1H, NH); 13C NMR (100 MHz, CDCl3) δ= 26.30, 48.90, 51.63,
51.72, 55.85, 12.38, 137.82, 152.09, 166.05; MS (EI) m/z = 240 [M-OCH3]; Elemental
Analysis: Calcd. for C15H17N3O2: C, 66.40; H, 6.32; N, 15.49; O, 11.79; Found: C,
66.57; H, 6.34; N, 15.53.
Following the same procedure for the preparation of compound 10e, starting from 10f-
methyl ester (50 mg, 0.18 mmol), and 0.14 ml of NaOHaq (2 M), compound 10f was
obtained (30 mg, yield: 60%)
IR (film): 1663 cm−1; 1H NMR (400 MHz, D2O) δ= 2.42 (m, 2H), 2.96 (m, 2H), 3.55 (m,
2H), 4.22 (m, 2H), 6.70 (m, 1H), 7.32 (m, 2H), 7.61 (m, 2H); 13C NMR (100 MHz, D2O)
δ= 23.73, 47.94, 51.06, 52.63, 115.02, 123.30, 130.54, 132.58, 147.06, 173.10; MS
21

(ESI) m/z =280 [M+H]; Elemental Analysis: Calcd. for C14H14N3NaO2: C, 60.21; H,
5.05; N, 15.05; Na, 8.23; O, 11.46; Found: C, 60.43; H, 5.07; N,15.10.
Preparation of 1-ethyl 3-methyl 5,6-dihydropyridine-1,3(2H)-dicarboxylate 11
The pH of a solution of Arecoline hydrobromide 8 (Qingdao Sicemo, LTD, Qingdao,
China) (15g) in water (30 ml) was adjusted to 13~14 by saturated Na2CO3 solution. This
solution was extracted by ether (50 ml×3). The yellow oil (Arecoline, 9.8g) was obtained
after drying and removing the solvent.
Arecoline (6.59g, 42.5 mmol) was dissolved into 80 ml toluene at 0 oC. A mixture of
ClCOOEt (12.24 ml, 11 mmol) and toluene (20 ml) was dropped into the reaction
mixture at 0 oC in 1 hour and kept stirring for 15 min at this temperature. Then the
temperature was increased to 100 oC and the mixture refluxed for 2.5 hrs. This reaction
was decomposed with ice water and the pH adjusted to 2-3 with HClaq (1 M). The
mixture was extracted by AcOEt (20 ml×3). The organic phase was washed by NaHCO3
and brine, dried and the solvent removed under vacuum to obtain 11 as yellow oil (5.8g,
yield: 64%)
IR (film): 1704 cm−1; 1H NMR (400 MHz, CDCl3) δ= 1.26 (t, J=7.1 Hz, 3H), 2.31(m,
2H), 3.52 (t, J=5.6 Hz, 2H), 3.75(s, 3H), 4.16 (m, 4H), 7.07 (m, 1H); 13C NMR
(100 MHz, CDCl3) δ= 14.65, 25.37, 39.19, 42.49, 51.69, 61.44, 128.96, 137.78, 155.54,
165.64; MS (EI) m/z =213 [M]; Elemental Analysis: Calcd. for C10H15NO4: C, 56.33; H,
7.09; N, 6.57; O, 30.01; Found: C, 56.54; H, 7.12; N,6.59.
Preparation of 1-(ethoxycarbonyl)-1,2,5,6-tetrahydropyridine-3-carboxylic acid 12
Product 11 (462 mg, 2.0 mmol) was dissolved into 10 ml toluene at 0 °C. A solution of
NaOHaq (0.6 ml, 5 M) was dropped into the reaction mixture at 0 °C. Then the
temperature was increased to 50 °C for 5.5 hrs. The reaction mixture was decomposed
with ice water, adjusted the pH to 2-3 with HClaq (1 M) and extracted with AcOEt (25
ml×3). The organic phase was collected, dried with Na2SO4 and the solvent removed
under vacuum to obtain compound 12 (390 mg, yield: 98%). This product was directly
used for the next step without any further purification.
22

IR (film): 1705, 1687 cm−1; 1H NMR (400 MHz, CDCl3) δ= 1.28 (t, J=8.8 Hz, 3H), 2.32
(m, 2H), 3.53 (t, J=5.6 Hz, 2H), 3.76 (m, 4H), 7.08(s, 1H); 13C NMR (100 MHz, CDCl3)
δ= 14.60, 25.51, 42.24, 48.11, 61.74, 127.87, 140.23, 157.89, 169.91; MS (EI) m/z =199
[M]; Elemental Analysis: Calcd. for C9H13NO4: C, 54.26; H, 6.58; N, 7.03; O, 32.13;
Found: C, 54.44; H, 6.60; N,7.05.
Preparation of 1-ethyl 3-methyl 5,6-dihydropyridine-1,3(4H)-dicarboxylate 14
BuLi (2.2 ml, 2.5M in THF, 5.5 mmol) was added into a solution of diisopropyl amine
(0.70 ml, 5.0 mmol) in THF (20 ml) at -78 °C. The temperature was increased to -10 °C
and 4 ml of HMPA were added at this temperature. This reaction mixture was kept at this
temperature for 30 min and then cooled down to -78 °C again. A solution of compound
12 (500 mg, 2.5 mmol) in THF (5 ml) was dropped into the base solution at -78 °C. This
reaction mixture was kept at -78 °C for 3 hrs, then poured into a cold saturated NH4Claq
(20 ml). The pH was adjusted to pH=2-3 and the mixture was extracted with AcOEt (30
ml×3). The organic phase was washed with HCl (1M) and brine, dried with Na2SO4 and
evaporated to obtain crude product which was purified by flash chromatography (AcOEt :
Cyclohexane =3:1) to obtain a mixture of ene-isomers 13. This mixture was processed in
the next step without any purification.
The mixture of isomers 13 (360 mg, 1.81 mmol) was directly dissolved into 1 ml of
MeOH. This solution was added into a solution of SO2Cl2 (2.88 ml) in MeOH (20 ml) at
0 oC. This reaction mixture was kept at 0 oC for 1hr. After the reaction completed, the
solvent was removed to obtain the crude product which was purified by column
chromatography (AcOEt : cyclohexane= 1:4) to give pure compound 14.
IR (film): 1724, 1700 cm−1; 1H NMR (400 MHz, CDCl3) δ= 1.34 (t, J=6.8 Hz, 3H), 1.85
(m, 2H), 2.32 (dt, J=1.2 Hz, J=6.0 Hz, 2H), 3.61 (m, 2H), 3.74(s, 3H), 4.27 (q, J=6.8 Hz,
1H), 8.03 (s, 1H); 13C NMR (100 MHz, CDCl3) δ= 14.45, 20.63, 20.89, 42.16, 51.32,
62.76, 102.12, 135.47, 153.36, 167.96; MS (EI) m/z =213 [M]; Elemental Analysis:
Calcd. for C10H15NO4: C, 56.33; H, 7.09; N, 6.57; O, 30.01; Found: C, 56.50; H, 7.11; N,
6.59.
Preparation of 1,4,5,6-tetrahydropyridine-3-carboxylic acid hydrochloride 15
23

Product 9 (500 mg, 2.82 mmol) was dissolved into 10 ml toluene at 0 °C. A solution of
NaOHaq (8.6 ml, 2 M) was dropped into the reaction mixture at 0 °C. Then the
temperature was brought to 110 °C and kept for 6 hrs (monitoring by tlc: AcOEt/
acetone/ water/ acetic acid=5:3:2:2). The reaction mixture was decomposed with ice
water and the pH adjusted to 2-3 with HClaq (3 M). The solvent was removed under
vacuum. The residue was treated with ethereal hydrogen chloride and filtered to obtain
pure 15 38, 39 (330 mg, yield: 92%).
Mp: 305 °C (Decomp). IR (film): 1665 cm−1; 1H NMR (400 MHz, CD3OD) δ= 2.27 (m,
2H), 2.92 (t, J=6.0 Hz, 2H), 3.56 (m, 2H), 6.75 (m, 1H); 13C NMR (100 MHz, CD3OD)
δ= 24.32, 41.00, 44.13, 130.86, 134.50, 173.12.
Preparation of 1-tert-butyl 3-methyl 5,6-dihydropyridine-1,3(2H)-dicarboxylate 16
Compound 9 (5.49 g, 31.0 mmol) was mixed with triethylamine (5.65 ml, 40.3 mmol)
and di-tert-butyl dicarbonate (10.14g, 46.5 mmol) in 60 ml of anhydrous methanol at r.t.
The reaction mixture was kept overnight. After removing the solvent under vacuum, 60
ml of water were added to the residue. The pH of the mixture was adjusted to 4-5 and
extracted with ethyl acetate (80 ml×3). The collected organic phase were dried and
evaporated to obtain compound 16 (11.08 g, yield: 79%). Spectral data were
superimposable with literature data.40
Preparation of 1-tert-butyl 3-methyl 2,3-dihydropyridine-1,3(6H)-dicarboxylate 17
BuLi (8.96 ml, 2.5 M in THF) was added into a solution of diisopropyl amine (3.15 ml,
22.41 mmol) in THF (35 ml) at -78°C. Then the temperature was increased to -10 °C for
30 min and then cooled down to -78°C. A solution of compound 16 (3g, 12.45 mmol) in
THF (10 ml) was dropped into the base solution and kept for 3 min. The reaction mixture
was poured into a cold NH4Cl solution (1M, 80 ml) and extracted with ether (60 mlx3).
The organic phase was collected, dried and evaporated to obtain a crude product which
was purified by flash chromatography (AcOEt : Cyclohexane =1:4) to give pure
compound 17 (1.48 g. yield 49.3%). Spectral data were superimposable with literature
data.40
24

Preparation of (1S*,5S*,6R*)-3-tert-butyl 5-methyl 7-oxa-3-
azabicyclo[4.1.0]heptane-3,5-dicarboxylate 18 and (1R*,5S*,6S*)-3-tert-butyl 5-
methyl 7-oxa-3-azabicyclo[4.1.0]heptane-3,5-dicarboxylate 19
Product 17 (250 mg, 1.03 mmol) was dissolved into anhydrous CH2Cl2 (20 ml). 3-
Chloroperbenzoic acid (MCPA, 357 mg, 2.06 mmol) was added. The reaction mixture
was kept at r.t for 3 hrs, then an extra 1 eq. of MCPA (179 mg, 1.03 mmol) was added
and the mixture left overnight. The reaction was decomposed with Na2S2O5 and extracted
with AcOEt (15 ml × 3). The organic phase was collected, washed with Na2S2O5, brine,
dried with Na2SO4 and evaporated to obtain a crude mixture of 18 and 19 which was
purified by flash chromatography (AcOEt : cyclohexane =1:4) (175 mg, overall yield:
66%).
An aliquot of the mixture was further chromatographed to obtain pure isolated 18 and 19.
Spectral data as follow
18 White solid. Mp: 70-74 oC; IR (film): 1738, 1698 cm−1; 1H NMR (400 MHz, CDCl3)
δ= 1.45 (s, 9H), 3.00 (m, 2H), 3.34 (m, 1H), 3.42 (d, J= 15.2 Hz 1H), 3.61 (d, J= 4.0 Hz,
1H), 3.76 (s, 3H), 3.84-4.20 (m, 2H); 13C NMR (100 MHz,CDCl3) δ= 28.33, 38.10,
38.92, 41.28, 50.89, 51.22, 52.17, 80.30, 154.32, 170.88. MS (EI) m/z =242 [M-CH3];
Elemental Analysis: Calcd. for C12H19NO5: C, 56.02; H, 7.44; N, 5.44; O, 31.09; Found:
C, 56.15; H, 7.46; N, 5.45.
19 Oil. IR (film): 1738, 1698 cm−1; 1H NMR (400 MHz, CD3OD) δ=1.41 (s, 9H), 3.03 (d,
J= 19.2 Hz 1H), 3.23 (d, J= 20.8 Hz 1H), 3.44 (m, 1H), 3.48 (dd, J1= 4.0 Hz, J2= 1.2 Hz,
1H), 3.57 (m, 1H), 3.71(s, 3H), 3.78 (m, 1H); 13C NMR (100 MHz,CDCl3) δ= 28.26,
39.80, 40.35, 42.03, 50.00, 50.90, 52.14, 80.13,154.75, 171.48. MS (EI) m/z =242 [M-
CH3]; Elemental Analysis: Calcd. for C12H19NO5: C, 56.02; H, 7.44; N, 5.44; O, 31.09;
Found: C, 56.17; H, 7.46; N, 5.46.
Preparation of 1-tert-butyl 3-methyl 5-hydroxy-5,6-dihydropyridine-1,3(2H)-
dicarboxylate 20.
A mixture of compounds 18 and 19 (500 mg, 2.02 mmol) and KOH solid (226 mg, 4.04
mmol) were dissolved in 20 ml of MeOH. The reaction mixture was kept at r.t for 30
25

min. The pH of the reaction was adjusted to 6-7 by HCl aq (2 M). The solvent was
removed under vacuum. The residue was mixed with 20 ml of water and extracted with
AcOEt (15 ml × 3). The organic phase was collected, dried with Na2SO4 and evaporated.
The crude product was purified by flash chromatography (AcOEt : cyclohexane =2:3) to
obtain pure compound 20 (350 mg, yield: 67%).
White solid. Mp: 78-85 oC; IR (film): 1699 cm−1; 1H NMR (400 MHz, CD3OD) δ=1.47
(s, 9H), 3.49-3.51(m, 1H), 3.65-3.73 (m, 1H), 3.78 (s, 3H), 3.97 (dt, J1= 18.4 Hz, J2= 2.4
Hz, 1H), 4.18 (dt, J1= 18.4 Hz, J2= 2.0 Hz, 1H), 4.26 (m, 1H), 6.92 (m, 1H); 13C NMR
(100 MHz,CDCl3) δ= 28.43, 42.78, 46.16, 52.05, 63.46, 80.72, 129.34, 139.79, 154.95,
165.66. MS (EI) m/z =257 [M]; Elemental Analysis: Calcd. for C12H19NO5: C, 56.02; H,
7.44; N, 5.44; O, 31.09; Found: C, 56.17; H, 7.46; N, 5.46.
Synthesis of methyl 1-(heteroarylmethyl)-5-hydroxy-1,2,5,6-tetrahydropyridine-3-
carboxylates:
Preparation of methyl 1-benzyl-5-hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylate
21a and 1-benzyl-5-hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylic acid
hydrochloride 22a (as general procedure).
Compound 20 (100 mg, 0.39 mmol) was dissolved in 3 ml of CH2Cl2. TFA (3 ml) was
added into this solution at 0 oC. The reaction mixture was kept at 0 oC for 20 min. The
solvent was removed under vacuum. The crude residue was dissolved in 10 ml of
CH3CN. (Bromomethyl)benzene (67 mg, 0.389 mmol) and diisopropyl amine (0.27 ml,
1.56 mmol) were added into the solution at 0 oC. This reaction mixture was kept at r.t. for
3hrs. After the reaction completed, the solvent was removed under vacuum. The residue
was dissolved with CH2Cl2 and washed with brine. The organic phase was separated,
dried and the solvent was removed. This crude product was purified by flash
chromatography (AcOEt : cyclohexane = 2:3) to obtain pure compound 21a (20 mg,
yield: 21%).
IR (film): 1718 cm−1; 1H NMR (400 MHz, CDCl3) δ= 2.36 (bs, 1H, OH), 2.53 (dd, J1=
11.6 Hz, J2= 3.2 Hz, 1H), 2.75 (dd, J1= 11.6 Hz, J2= 3.6 Hz, 1H), 2.99 (d, J= 17.2 Hz,
1H), 3.43 (d, J= 16.8 Hz, 1H), 3.68 (qAB, J= 12.8 Hz, 2H), 3.75 (s, 3H), 4.21 (m, 1H),
7.31 (m, 5H); 13C NMR (100 MHz, CDCl3) δ= 51.69, 51.85, 56.39, 62.00, 63.97, 127.51,
26

128.45, 129.09, 131.29, 137.50, 166.03. MS (EI) m/z =247 [M]; Elemental Analysis:
Calcd. for C14H17NO3: C, 68.00; H, 6.93; N, 5.66; O, 19.41; Found: C, 68.18; H, 6.95; N,
5.67.
Preparation of 22a
Compound 21a (20 mg) was dissolved into 2 ml of HClaq (1 M). After refluxing for 4.5
hrs at 110 oC, the solvent was removed under vacuum to obtain compound 22a (20 mg,
yield: 91% ).
IR (film): 1708 cm−1; 1H NMR (400 MHz, D2O) δ= 3.31-3.62 (m, 2H), 3.76-4.25 (m,
2H), 4.50 (m, 2H), 4.67 (m, 1H), 7.11(m, 1H), 7.55 (m, 5H) ; 13C NMR (100 MHz, D2O)
δ= 48.91, 54.14, 59.63, 60.08, 126.53, 127.88, 129.33, 129.49, 130.36, 131,13, 135.87,
166.76. MS (ESI) m/z = 269 [M]; Elemental Analysis: Calcd. for C13H16ClNO3: C,
57.89; H, 5.98; Cl, 13.14; N, 5.19; O, 17.80; Found: C, 58.07; H, 6.00; N, 5.21.
Synthesis of methyl 5-hydroxy-1-(pyridin-2-ylmethyl)-1,2,5,6-tetrahydropyridine-3-
carboxylate 21b and the corresponding acid 5-hydroxy-1-(pyridin-2-ylmethyl)-
1,2,5,6-tetrahydropyridine-3-carboxylic acid hydrochloride 22b
Following the procedure of 21a and starting from 20 (90 mg, 0.35 mmol), 2-
(bromomethyl)-pyridine hydrobromide (87 mg, 0.35 mmol) and diisopropyl amine (0.24
ml, 1.4 mmol) compound 21b was obtained (36 mg, yield: 41%) after flash
chromatography (AcOEt :MeOH=5:1).
IR (film): 1710 cm−1; 1H NMR (400 MHz, CDCl3) δ= 2.56 (d, J= 11.6 Hz, 1H), 2.71 (dd,
J1= 11.6 Hz, J2= 3.6 Hz, 1H), 2.99 (d, J= 16.8 Hz, 1H), 3.38 (d, J= 16.8 Hz, 1H), 3.66 (s,
3H), 3.74 (m, 2H), 4.20 (m,2H), 4.70 (s, 1H, OH), 6.90 (m, 1H), 7.12 (m, 1H), 7.30 (d,
J= 4.8 Hz, 1H), 7.60 (m, 1H), 8.45 (d, J= 4.4 Hz, 1H); 13C NMR (100 MHz, (CD3)2CO)
δ= 51.00, 51.53, 56.57, 62.87, 63.93, 122.19, 122.86, 131.15, 136.71, 139.82, 149.11,
158.41, 165.51; MS (ESI) m/z =249 [M+1]+; Elemental Analysis: Calcd. for C13H16N2O3:
C, 62.89; H, 6.50; N, 11.28; O, 19.33; Found: C, 63.09; H, 6.52; N, 11.32.
Preparation of 22b
27

Compound 21b (15 mg) was dissolved into 2 ml of HClaq (1 M). The reaction was
refluxed for 3 hrs at 110 oC. The solvent was removed under vacuum to obtain compound
22b (15 mg, yield: 81% ).
IR (film): 1708 cm−1; 1H NMR (400 MHz, D2O) δ= 3.52 (m, 2H), 4.00 (m, 2H), 4.74 (m,
2H), 7.13 (m, 1H), 8.00 (m, 2H), 8.45 (m, 1H), 8.83 (d, J=5.6 Hz, 1H); 13C NMR
(100 MHz, D2O) δ= 49.50, 54.54, 57.75, 60.47, 126.37, 126.72, 127.74, 136.92, 143.61,
146.11, 146.14, 166.92; MS (EI) m/z = 269 [M-HCl-H]-; Elemental Analysis: Calcd. for
C12H16Cl2N2O3: C, 46.92; H, 5.25; Cl, 23.08; N, 9.12; O, 15.63; Found: C, 47.04; H,
5.26; N, 9.14.
Synthesis of methyl 5-hydroxy-1-(pyridin-3-ylmethyl)-1,2,5,6-tetrahydropyridine-3-
carboxylate 21c and the corresponding acid 5-hydroxy-1-(pyridin-3-ylmethyl)-
1,2,5,6-tetrahydropyridine-3-carboxylic acid hydrochloride 22c
Following the procedure of 21a and starting from 20 (90 mg, 0.35 mmol), 3-
(bromomethyl)-pyridine hydrobromide (87 mg, 0.35 mmol) and diisopropylamine (0.24
ml, 1.4 mmol), compound 21c was obtained after flash chromatography (AcOEt
:MeOH=5:1)(75 mg, yield: 87%).
IR (film): 1685 cm−1; 1H NMR (400 MHz, CDCl3) δ= 2.56 (dd, J1= 11.2 Hz, J2= 2.4 Hz,
1H), 2.76(dd, J1= 3.6 Hz, J2= 11.2 Hz, 1H), 2.99 (d, J= 16.4 Hz, 1H), 3.42 (d, J= 16.8
Hz, 1H), 3.70 (qAB, J= 13.6 Hz, 2H), 3.76 (s, 3H), 4.24 (m, 1H), 4.76(s, 1H, OH), 6.98
(m, 1H), 7.72 (dd, J1=11.2 Hz, J2= 3.6 Hz, 1H), 8.57 (m, 2H), 8.60 (s, 1H); 13C NMR
(100 MHz, (CD3)2CO) δ= 51.93, 52.07, 57.36, 59.62, 65.13, 124.19, 130.58, 134.65,
137.32, 140.87, 149.40, 151.10, 166.49; MS (ESI) m/z =249 [M+1]; Elemental Analysis:
Calcd. for C13H16N2O3: C, 62.89; H, 6.50; N, 11.28; O, 19.33; Found: C,63.02; H,6.51;
N,11.30.
Preparation of 22c
Compound 21c (20 mg) was dissolved into 2 ml of HClaq (1 M). This reaction was
refluxed for 5 hrs at 110 oC. The solvent was removed under vacuum to obtain compound
22c (23 mg, yield:92% ).
28

IR (film): 1707 cm−1; 1H NMR (400 MHz, D2O) δ= 3.55 (dd, J1=4.0Hz, J2=12.4Hz, 1H),
3.62 (dd, J1=4.4Hz, J2=12.8Hz, 1H), 4.02 (dt, J1=2.4Hz, J2=16.8Hz, 1H), 4.17 (d,
J=15.6Hz, 1H), 4.86 (m, 2H), 7.14 (m, 1H), 8.25 (dd, J1=5.6Hz, J2=8.0Hz, 1H), 8.88 (dt,
J1=1.2Hz, J2=8.4Hz, 1H), 8.99 (d, J=5.6Hz, 1H), 9.12 (s, 1H); 13C NMR (100 MHz,
D2O) δ= 49.21, 54.19, 55.67, 60.07, 126.02, 128.04, 128.78, 136.55, 143.22, 143.79,
149.33, 166.91; MS (EI) m/z =269 [M-HCl-H]-; Elemental Analysis: Calcd. for
C12H16Cl2N2O3:C, 46.92; H, 5.25; Cl, 23.08; N, 9.12; O, 15.63; Found: C, 47.03; H,
5.26; N, 9.14.
Synthesis of methyl 5-hydroxy-1-(pyridin-4-ylmethyl)-1,2,5,6-tetrahydropyridine-3-
carboxylate 21d and the corresponding acid 5-hydroxy-1-(pyridin-4-ylmethyl)-
1,2,5,6-tetrahydropyridine-3-carboxylic acid hydrochloride 22d
Following the procedure of 21a and starting from 20 (100 mg, 0.37 mmol), 4-
(bromomethyl)-pyridine hydrobromide (94 mg, 0.37 mmol) and diisopropyl amine (0.27
ml, 1.56 mmol), compound 21d was obtained after flash chromatography (AcOEt
:MeOH=5:1) (70 mg, yield: 78%).
IR (film): 1685 cm−1; 1H NMR (400 MHz, CDCl3) δ= 2.57 (dd, J1= 12.0 Hz, J2= 2.8 Hz,
1H), 2.74 (dd, J1= 4.0 Hz, J2= 12.0 Hz, 1H), 3.01 (d, J= 16.4 Hz, 1H), 3.42 (d, J= 16.8
Hz, 1H), 3.68 (qAB, J= 14.0 Hz, 2H), 3.76 (s, 3H), 4.25 (m, 1H), 4.76 (bs, 1H, OH), 6.98
(m, 1H), 7.32 (m, 2H), 8.59 (m, 2H); 13C NMR (100 MHz, (CD3)2CO) δ= 51.93, 52.19,
57.50, 61.06, 65.18, 124.48, 130.64, 140.77, 148.59, 150.56, 166.47; MS (ESI) m/z =249
[M+1]; Elemental Analysis: Calcd. for C13H16N2O3: C, 62.89; H, 6.50; N, 11.28; O,
19.33; Found: C, 63.10; H, 6.52; N, 11.32.
Preparation of 22d
Compound 21d (20 mg) was dissolved into 2 ml of HClaq (1 M). This reaction was
refluxed for 5 hrs at 110 oC. The solvent was removed under vacuum to obtain compound
22d (17 mg, yield: 68% ).
IR (film): 1707 cm−1; 1H NMR (400 MHz, D2O) δ= 3.51 (dd, J1=4.0Hz, J2=12.8Hz, 1H),
3.63 (dd, J1=4.0Hz, J2=12.8Hz, 1H), 4.05 (dt, J1=1.2Hz, J2=16.4Hz, 1H), 4.02 (d,
J=16.4 Hz, 1H), 4.91 (m, 2H), 7.17 (m, 1H), 8.33 (d, J=6.4 Hz,1H), 8.98 (d, J=6.4Hz,
29