Synthesis of 1,4- and 1,4,4-substituted piperidines for the inhibition of neuronal T-type Ca2+ channels and mitigation of neuropathic pain in mice

Article abstract of DOI:10.24820/ark.5550190.p010.752

A dysregulation in function of neuronal low-voltage-activated T-channel causes neuropathic pain. A group of ten 1,4-disubstituted and 1,4,4-trisubstituted piperidines were synthesized through short sequences of reactions from 4-cyanopiperidine, which may potentially produce a library of analogs. Inhibitions of T-channel in rat dorsal root ganglion neurons and/or Ca_v3.2 channel in human embryonic kidney-293 cells were screened, and subsequent analgesic effects and inhibition of seizure in pentylenetetrazole-induced seizure mouse model were examined. The two most promising piperidine molecules showed analgesic effects in rat model using a spared nerve injury protocol. One displayed a long withdrawal latency in response to thermal stimulation, and an 80% increase in mechanical threshold assessment studies.

Full text of DOI:10.24820/ark.5550190.p010.752

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Synthesis of 1,4- and 1,4,4-substituted piperidines for the inhibition of neuronal
T-type Ca2+ channels and mitigation of neuropathic pain in mice
Medha J. Gunaratna,a Duy H. Hua,*a Bende Zou,b Conrado Pascual,b William Cao,b Man Zhang,a
Sahani Weerasekara,a Thi D. T. Nguyen,a Kui Xiao,b and Xinmin Simon Xie*b
aDepartment of Chemistry, Kansas State University, Manhattan, KS 66506, U.S.A.
bAfaSci Research Laboratories, Redwood City, CA, U.S.A.
Email: duy@ksu.edu; simonxie@afasci.com
This manuscript is in celebration of Professor George A. Kraus for his dedication to research and education
Received 09-14-2018
Accepted 12-16-2018
Published on line 01-12-2019
Abstract
A dysregulation in function of neuronal low-voltage-activated T-channel causes neuropathic pain. A group of
ten 1,4-disubstituted and 1,4,4-trisubstituted piperidines were synthesized through short sequences of
reactions from 4-cyanopiperidine, which may potentially produce a library of analogs. Inhibitions of T-channel
in rat dorsal root ganglion neurons and/or Cav3.2 channel in human embryonic kidney-293 cells were screened,
and subsequent analgesic effects and inhibition of seizure in pentylenetetrazole-induced seizure mouse model
were examined. The two most promising piperidine molecules showed analgesic effects in rat model using a
spared nerve injury protocol. One displayed a long withdrawal latency in response to thermal stimulation, and
an 80% increase in mechanical threshold assessment studies.
O
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Keywords: Neuropathic pain, patch clamp recording, 1,4-disubstituted piperidines, 1,4,4-trisubstituted
piperidines, T-type calcium channel inhibitors, alkylation of 4-cyanopiperidines
DOI: https://doi.org/10.24820/ark.5550190.p010.752
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Introduction
Intracellular calcium concentrations play a vital role in neuronal functions and signal transduction.1,2 Control of
calcium entry into cells through the cell membrane is partly regulated by a family of transmembrane proteins
called voltage-gated calcium channels.3 Voltage-gated calcium channels modulate the entry of extracellular
calcium ions into cells in response to the changes in electrical potential differences across the cell
membrane.4,5 Depending on the membrane potential required for activation, voltage-gated calcium channels
are divided into two types.5 They are called low-voltage-activated channels (T-type) and high-voltage-activated
channels (L-, N-, P/Q-, and R-types).5-7
Low-voltage-activated channels stimulate by negative membrane depolarization potentials (from about
-40 mV to -70 mV) and have fast voltage-dependent inactivations.3,5,6,8 Therefore, they produce transient
currents.3-6 High-voltage-activated channels are provoked by relatively positive membrane depolarization
potentials (from about -35 mV to +15 mV).3 Voltage-gated calcium channels are divided into three main
channel families called Cav1 (L-type), Cav2 (P/Q, N, and R-types) and Cav3 (T-type).8 Based on the sequence of
their membrane pore-forming α1 subunit, each channel family is further classified into subtypes such as Cav1.1
- Cav1.4, Cav2.1 - Cav2.3, and Cav3.1 - Cav3.3.8 L-type calcium channels are mainly distributed in cardiac, skeletal,
vascular smooth muscles and endocrine cells; P/Q-, N- and R- type channels are expressed in neuronal
presynaptic terminals.8-11 T-type calcium channels Cav3.1 and Cav3.2 are mainly expressed in the brain,
peripheral nervous tissues (dorsal root ganglia and autonomic ganglia) and peripheral tissues such as heart,
smooth muscle, skeletal muscle and endocrine cells,12 while Cav3.3 is mainly expressed in the brain and
peripheral nerve tissues such as dorsal root ganglia and autonomic ganglia.12-14
T-type calcium channels are involved in regulating neuronal excitability, burst firing and transmitter
release.15-19 Therefore, dysregulation in function of neuronal T-type calcium currents has been proposed to be
a link to various types of neurological disorders, including neuropathic pain, hypertension, and absence
seizures.17,18,20,21 Neuropathic pain is a dysfunction of the central and peripheral nervous systems, resulting
from nerve injury or disease conditions such as inflammation, multiple sclerosis, autoimmune diseases,
diabetes or cancer.22,23 Neuropathic pain is often characterized by pain responses such as dysesthesia
(abnormal sense of touch), hyperalgesia (increased sensitivity to painful stimuli) and allodynia (painful
response to normally not painful stimuli).22
Although the molecular mechanisms underlying the development of neuropathic pain are poorly
understood, it is suggested that injured nerve fibers induce neuropathic pain by altering ion channels which
affect the sensory signaling.24 It has been found that T-type calcium currents were up-regulated in sensory
neurons in rat models of neuropathic pain25, and an increase of T-type Ca2+ (mainly Cav3.2) currents promoted
the nociceptor excitability and hyperalgesia in rat dorsal root ganglion (DRG) neurons, subsequently
influencing pain processing.26 Another study has shown that axonal accumulation of Cav3.2 T-type calcium
channels in uninjured rat sural nerves with spared nerve injury (SNI) enhanced sensitization, and subsequently
contributed to neuropathic pain.27
Since T-type calcium channel is involved in the progression of the neuropathic pain, T-type calcium
channel blockers have been investigated intensely for the discovery of neuropathic-pain therapeutics.17,28 It
has been shown that perineural administration of a T-type calcium channel blocker, mibefradil, increased
mechanical thresholds of Aβ-, Aδ- and C-fibers in the sural nerve of neuropathic pain models.17 A number of
studies using the antisense of Cav3.2 T-type channel have shown to reduce nociceptive response in mouse
models of neuropathic pain.26,29,30
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Literature data have shown that T-type channel blockers with a piperidine scaffold have good potencies,
with minimal effects on L-type Ca2+, K+, human ether-a-go-go related gene (hERG) channels and other off-
targets. hERG Is a gene that codes a protein known as Kv11.1, the α subunit of a potassium ion channel in the
heart that coordinates heart beating. Therefore, inhibition of the hERG channel is detrimental.31
Although several T-type calcium channel inhibitors have shown success in relieving neuropathic pain in rat
models, there has been no FDA-approved T-type calcium channel blocker available for clinical use until now.
Therefore, there is a high demand for the discovery of novel, potent T-type-channel-specific inhibitors.
Herein, we report the synthesis of a series of ten 1,4-disubstituted and 1,4,4-trisubstituted piperidines, and
their inhibitory efficacy of neuropathic pain.
Results and Discussion
In search of potent and selective T-channel inhibitors for the treatment of neuropathic pain, we used a
rational drug-design approach. A series of 1,4-di- and 1,4,4-trisubstituted piperidines were synthesized due to
their broad scope of potential therapeutic applications, and their activities against T-type calcium channels
were evaluated for the possible treatment of neuropathic pain.
We designed this series of molecules by changing substituents independently in three regions (A, B and C)
of the 1,4-disubstituted piperidine core 1 (Figure 1). The selection of core 1 was based on reported piperidine
inhibitors such as ML218 and Z944.28,32,33 We hypothesize that the piperidine ring adapts a chair conformation
and, with the N-1 and C4 substituents oriented at the equatorial positions, leads to an extend structure for
hydrophilic (from amide and amine functions) and hydrophobic (aryl or alkyl group) interactions with the
targeted calcium channels. Modification in part A focused on the substitution of different aromatic-ring
equivalents, while modifications in part B focused on the replacement of C4-hydrogen with methyl,
hydroxymethyl or arylcarbonyloxymethyl groups. Modification in part C focused on the replacement of
benzoyl amide with a tertiary-butyl amide. These structural modifications were expected to change the
hydrophilic, hydrophobic, rigidity, planarity, steric hindrance, and number of hydrogen bondings of each
molecule, which may allow future studies for improving the potency and selectivity.
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A
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t-Bu
ML218
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gabapentin
Z944
10
Figure 1. Designed 1,4- and 1,4,4-substituted piperidine compounds as T-type calcium channel inhibitors and
reported inhibitors ML218, Z944, and gabapentin
Synthesis of T-type calcium channel inhibitors
The synthetic routes of compounds 1-10 are shown in Scheme 1 - Scheme 4 starting from commercially
available 4-cyanopiperidine (11). The synthetic methods for the syntheses of these molecules are relatively
short. Hence, in the syntheses of 1 and 3, an initial N-alkylation was carried out by the treatment of 4-
cyanopiperidine with N-phenyl-2-chloroacetamide under basic conditions that yielded compound 13 (Scheme
1). Reduction of the nitrile function of 13 by cobalt(II) chloride•hexahydrate and sodium borohydride yielded
amine 14. Coupling of amine 14 with benzoyl chloride and pyrazinoyl chloride, separately, in the presence of
pyridine, gave compounds 1 and 3, respectively.
Similar amide coupling of compound 14 with p-hydroxybenzoic acid via activation of the acid with 1-ethyl-
3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in the presence of a catalytic amount of N,N´-
dimethylaminopyridine (DMAP), gave a poor yield of amide 2. An improved synthesis was found by using acyl
imidazole 15, derived from 4-(tert-butyldimethylsilyloxy)benzoic acid and 1,1'-carbonyldiimidazole (CDI).
Hence, coupling of 14 and 15 in acetonitrile:DMF (20:1) at 90 oC gave a 51% yield of 2.
Amine 14 was treated with 2-chloroacetylpyrrole (16) in the presence of sodium bicarbonate in
o
acetonitrile:DMF (20:1) at 50 C to yield compound 4 (56% yield). It is important to keep a higher ratio of
amine to alkyl chloride to prevent the formation of N,N-dialkylated product. The desired product 4 and
unreacted amine 14 were separated by silica gel column chromatography.
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Scheme 1. Syntheses of compounds 1 to 4
The introduction of a hydrophilic or hydrophobic group at C-4 of piperidines 1 – 4 may increase binding to
the targeted Ca2+ channel. Hence compounds 5-7, 9, and 10 were synthesized. Deprotonation of 4-
cyanopiperidine (11) using 1 equivalent of lithium diisopropylamide (LDA) followed by methylation with
methyl iodide (1 equivalent) furnished 4-cyano-4-methylpiperidine (17) (Scheme 2).34 The C4-H (adjacent to
CN group) is more acidic (pKa ~27) than N1-H (pKa ~40); hence, methylation took place preferably at the C4-
carbanion instead of N1. However, we cannot rule out that methylation at N-1 (as a minor by-product) did not
occur to yield a minor byproduct since the isolated yield of 17 was not quantitative (74%). Installation of the
usual phenylamidomethyl group to the piperidine nitrogen of 17 with N-phenyl-2-chloroacetamide under
basic conditions gave compound 18. Reduction of the cyano function, followed by amide formation of 18
under a similar reaction sequence as described above, afforded piperidine derivative 5 containing a
hydrophobic methyl group at C4 of the piperidine core (Scheme 2). Proton and carbon-13 NMR spectra of
molecule 5 showed a single isomer in which the larger C4-benzoylaminomethyl group, compared with the
methyl substituent, likely orients in the equatorial position.
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Scheme 2. Synthesis of compound 5
Piperidine derivatives 6, 7, 9 and 10, containing a hydrophilic group, were synthesized via a modification
of Scheme 2 due to the competing reactivity of piperidinyl amine and the newly installed hydroxyl group
(Scheme 3). Hence, 4-cyanopiperidine was first protected using di-tert-butyl dicarbonate (Boc2O) and
triethylamine to give N-Boc protected compound 20. Deprotonation of compound 20, using an equivalent of
LDA followed by paraformaldehyde, and quenching with ammonium hydroxide/ammonium chloride, yielded
the C4-hydroxymethylated molecule 21. Benzoylation of 21 with benzoyl chloride under basic conditions
resulted in benzoate 22. The Boc protecting group of 22 was removed using 10% trifluoroacetic acid (TFA) in
methylene chloride to give the amine intermediate 23. Subsequent alkylation of 23 using N-phenyl-2-
chloroacetamide under basic conditions provided compound 24. Reduction of the cyano moiety of compound
24 was carried out by the treatment with cobalt(II) chloride hexahydrate and sodium borohydride to obtain
the corresponding amine compound 25. Amidation of 25 with benzoyl chloride and 2-fluorobenzoyl chloride,
separately, under basic conditions, resulted in compounds 9 and 10, respectively. Basic hydrolysis of
compounds 9 and 10 with potassium carbonate gave the debenzoylated products 6 and 7, respectively.
Molecules 6, 7, 9, and 10 (vide infra) are mixtures of cis- and trans-isomers based on their NMR spectra.
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Scheme 3. Synthesis of compounds 6, 7, 9, and 10
We considered, and subsequently investigated, whether a replacement of the phenyl ring of the amido
group at C1 of 1 with a t-butyl group, such as molecule 8, would enhance the bioactivity. The synthesis of the
1,4-diamide analog 8 was similar to that of compound 1 except in the initial N-alkylation step of the piperidine
ring (Scheme 4). Treatment of piperidine 11 with N-tert-butyl-2-chloroacetamide under basic conditions
afforded compound 27. The cyano function of 27 was reduced with cobalt(II) chloride•hexahydrate and
sodium borohydride to obtain the corresponding amine molecule 28, which, upon benzoylation with benzoyl
chloride, furnished analog 8.
Scheme 4. Synthesis of compound 8
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Biological evaluation of the synthesized molecules was carried out using patch clamp recordings of either
dissociated mouse dorsal root ganglion (DRG) neurons or Cav3.2 channel expressed in human embryonic kidney
(HEK)-293 cells from a tetracycline-inducible expression system [see Table S1 and supporting information
published in the Supplementary Material (SM) for details]. The tested compounds 4 and 8 have increased
thermal and mechanical thresholds compared to vehicle. The tested compounds produced analgesic effects
on both thermal and mechanical pain thresholds, while the vehicle group continued to produce thermal
hyperalgesia and mechanical allodynia conditions (see SM). Therefore, compounds 4 and 8 have the ability to
mitigate induced-neuropathic pain in rats, and 4 appears to have a greater effect than 8 at the 3-hour time
point (an 80% increase in mechanical threshold for 4 and ~60% for 8) (see Figures S1 and S2 in SM). We have
also examined the prolonged latency to seizures and decreased fatality rate in pentylenetetrazole (PTZ)-
induced mouse model to assess the inhibition of T-channel activity. In agreement with the aforementioned
studies, molecules 4 and 8 exhibited 86 and 89%, respectively, seizure-inhibitory activities.
Conclusions
In summary, a series of ten 1,4-disubstituted and 1,4,4-trisubstituted piperidines were synthesized from 4-
cyanopiperidine through short sequences of reactions.
Molecule
4, containing
a
pyrrolylcarbonylmethylaminomethyl at C4 and N-phenylacetamide at N-1, showed strong inhibition of the
Cav3.2 channel and equal potency of analgesic effect as two known potent T-channel inhibitors, ML218 and
Z944. Importantly, 4 revealed a much stronger inhibition of seizure in PTZ-induced fatality than the two
known inhibitors, suggesting that 4 can be a lead compound for discovery of novel analgesics in the future.
Experimental Section
General. Chemicals were purchased from Fisher Scientific, VWR international LLC and Chem-Impex
International, Inc. All solvents were dried over appropriate drying agents such as CaH2 (for DMF,
dichloromethane and acetonitrile) or Na/benzophenone (for THF and diethyl ether) followed by distillation.
1
13
Column chromatography was carried out on silica gel (200-400 mesh). H NMR spectra (400 MHz) and C
NMR spectra (100 MHz) were recorded on a Varian Unity plus 400 MHz Spectrometer in CDCl3 or DMSO-d6
relative to TMS (δ = 0 ppm) or CHCl3 (δ = 7.26 ppm) (1H NMR) or CDCl3 (δ = 77.0 ppm) or DMSO (δ = 39.5 ppm)
(13C NMR). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, dd =
doublet of doublets, td = triplet of doublets, ddd = doublet of doublet of doublets, m = multiplet, br = broad),
coupling constant (Hz). Mass spectra were obtained from an API 2000-triple quadrupole ESI-MS/MS mass
spectrometer (Applied Biosystems).
Synthesis of T-type calcium channel inhibitors
N-({1-[2-Oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl)benzamide (1). A mixture of 4-cyanopiperidine
(11) (1.0 g, 9.1 mmol), N-phenyl-2-chloroacetamide (12) (1.54 g, 9.1 mmol), and anhydrous potassium
o
carbonate (2.5 g, 18.2 mmol) in acetonitrile (30 mL) was heated to reflux at 90 C under argon for 8 hours. The
resulting mixture was cooled and filtered to remove inorganic salts. The filtrate was concentrated, and
column chromatographed on silica gel using a mixture of dichloromethane and methanol (30:1) as an eluent
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1
to give 2-(4-cyanopiperidin-1-yl)-N-phenylacetamide (13) (2.2 g, 99.7% yield) as a white solid. H NMR (CDCl3):
δH 8.94 - 8.90 (1H, br. s., NH), 7.52 (2H, d, J 7.8 Hz, CH x 2 o-aromatic), 7.29 (2H, t, J 8.0 Hz, CH x 2 m-Aromatic),
7.08 (1H, t, J 7.4 Hz, CH p-aromatic), 3.08 (2H, s, NCH2CO), 2.72 - 2.79 (2H, m, CH2N), 2.63 (1H, td, J 7.7, 3.8 Hz,
13
CHCN), 2.44 (2H, t, J 8.4 Hz, CH2N), 1.84 - 2.00 (4H, m, 2 CH2); C NMR (CDCl3): δC 167.8 (C=O), 137.4 (C
aromatic), 128.9 (CH x 2 aromatic), 124.2 (CH aromatic), 121.2 (CN), 119.4 (CH x 2 aromatic), 62.1 (CH2N), 51.7
(2 CH2N), 28.8 (2 CH2), 25.4 (CHCN); MS (ESI): m/z 244.5 (M+H)+ (75%), 123.1 (100%).
o
To a cold (0 C) solution of 13 (0.80 g, 3.29 mmol) in MeOH (25 mL), cobalt(II) chloride•hexahydrate (0.39 g,
1.65 mmol) was added followed by the addition of sodium borohydride (0.62 g, 16.5 mmol). After stirring for
15 hours at 25 °C, the reaction solution was diluted with 25 mL of 5% aqueous ammonium hydroxide and
extracted three times with dichloromethane. The combined organic layer was washed with brine, dried over
anhydrous Na2SO4, concentrated, and column chromatographed on silica gel using a mixture of dichloro-
methane and methanol (2:1) as an eluent to give 2-[4-(aminomethyl)piperidin-1-yl]-N-phenylacetamide (14)
1
(0.81 g, 80% yield) as a colorless oil. H NMR (CDCl3): δH 9.18 (1H, br. s., NH), 7.56 (2H, d, J 8.2 Hz, CH x 2
o-aromatic), 7.32 (2H, t, J 7.8 Hz, CH x 2 m-aromatic), 7.09 (1H, t, J 7.4 Hz, CH p-aromatic), 3.09 (2H, s, CH2N),
2.92 (2H, d, J 11.3 Hz, CH2N), 2.63 (1H, d, J 5.5 Hz, CH), 2.18 - 2.30 (2H, m, CH2N), 1.78 (2H, d, J 10.5 Hz, CH2N),
1.21 - 1.37 (4H, m, 2 CH2); 13C NMR (CDCl3): δC 168.9 (C=O), 137.8 (C aromatic), 129.1 (CH x 2 aromatic), 124.2
(p-CH aromatic), 119.5 (CH x 2 aromatic), 62.5 (CH2N), 54.2 (3 CH2N), 48.0 (CH), 30.4 (2 CH2); MS (ESI): m/z
270.2 (M+Na)+, 248.2 (M+H)+ (100%), 231.3.
To a cold (0 °C) solution of 0.13 g (0.61 mmol) of 14 in 3 mL of freshly distilled dichloromethane under argon,
pyridine 78 µL (0.915 mmol) was added followed by the addition of 70 µL (0.61 mmol) of benzoyl chloride.
The reaction was stirred at 0 °C for 30 min. and 25 °C for 3 hours. The reaction solution was diluted with 20 mL
of aqueous sodium bicarbonate and extracted three times with dichloromethane. The combined organic layer
was washed with brine, dried (anhydrous Na2SO4), concentrated, and column chromatographed on silica gel
using a mixture of dichloromethane and methanol (10:1) as an eluent to give N-({1-[2-oxo-2-
1
(phenylamino)ethyl]piperidin-4-yl}methyl)benzamide (1) (0.114 g, 53% yield) as a white solid. H NMR (CDCl3):
δH 9.85 (1H, br. s., NH), 9.59 (1H, br. s., NH), 8.09 (2H, d, J 7.4 Hz, CH 2 x aromatic), 7.79 (1H, d, J 7.4 Hz, CH
aromatic), 7.54 - 7.58 (2H, m, CH x 2 aromatic), 7.40 - 7.43 (2H, m, CH x 2 aromatic), 7.30 (1H, t, J 7.8 Hz, CH
aromatic), 7.09 (1H, t, J 7.4 Hz, CH aromatic), 6.65 (1H, t, J 5.9 Hz, CH aromatic), 3.40 (2H, t, J 6.4 Hz, CH2N),
3.31 (2H, s, O=CCH2N), 3.08 (2H, d, J 11.7 Hz, CH2N), 2.34 - 2.45 (2H, m, CH2N), 1.82 (2H, d, J 12.5 Hz, CH2),
1.66-1.77 (1H, m, CH), 1.44-1.57 (2H, m, CH2); 13C NMR (CDCl3): δC 168.3 (C=O), 167.6 (C=O), 137.8 (C
aromatic), 134.6 (C aromatic), 131.8 (CH aromatic), 129.2 (CH x 2 aromatic), 128.6 (CH x 2 aromatic), 127.2 (CH
x 2 aromatic), 124.6 (CH aromatic), 119.9 (CH x 2 aromatic), 62.0 (CH2N), 53.7 (2 CH2N), 45.3 (CH2N), 35.5 (CH),
29.7 (2 CH2); MS (ESI): m/z 374.2 (M+Na)+, 352.1 (M+H)+ (100%).
4-Hydroxy-N-((1-(2-oxo-2-(phenylamino)ethyl)piperidin-4-yl)methyl)benzamide (2). To a solution of 0.10 g
(0.28 mmol) of 14 in a freshly distilled 20:1 mixture of acetonitrile:DMF under argon, 0.86 g (0.28 mmol) of (4-
(tert-butyldimethylsilyloxy)phenyl)(1H-imidazol-1-yl)methanone (15) was added. The reaction mixture was
heated to reflux at 90 °C for 24 hours, cooled to room temperature, diluted with 20 mL of aqueous sodium
bicarbonate, and extracted three times with dichloromethane. The combined organic layer was washed with
brine, dried (anhydrous Na2SO4), concentrated, and column chromatographed on silica gel using a mixture of
1
dichloromethane and methanol (15:1) as an eluent to give 2 (52 mg, 51% yield) as a white solid. H NMR
(DMSO-d6): δH 9.16 (1H, br. s., OH), 8.25 (1H, s, NH), 7.68 (2H, d, J 8.6 Hz, CH 2 x aromatic), 7.52 - 7.61 (2H, m,
CH x 2 aromatic), 7.35 (2H, d, J 7.4 Hz, CH x 2 aromatic), 7.07 - 7.18 (1H, m, CH aromatic), 6.87 (2H, d, J 8.6 Hz,
CH x 2 aromatic), 3.12 (2H, t, J 6.4 Hz, CH2N), 3.04 - 3.15 (2H, s, CH2N), 2.93 (2H, d, J 11.7 Hz, CH2N), 2.25 (2H, t,
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J 10.7 Hz, CH2N), 1.81 (2H, d, J 10.5 Hz, CH2), 1.60-1.58 (1H, m, CH), 1.37 - 1.46 (2H, m, CH2); 13C NMR (DMSO-
d6): δC 168.6 (C=O), 166.0 (C=O), 160.0 (C aromatic), 138.6 (C aromatic), 129.1 (CH x 2 aromatic), 128.7 (CH x 2
aromatic), 125.4 (C aromatic), 123.4 (C aromatic), 119.5 (CH x 2 aromatic), 114.8 (CH x 2 aromatic), 62.3
(CH2N), 53.3 (2 CH2N), 44.7 (CH2N), 35.4 (CH), 29.8 (2 CH2); MS (ESI): m/z 389.9 (M+Na)+, 368.4 (M+H)+ (100%).
o
N-({1-[2-Oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl)pyrazine-2-carboxamide (3). To a cold (0 C)
solution of 14 (80 mg, 0.32 mmol) in 3 mL of freshly distilled dichloromethane under argon, pyridine 40 µL
(0.48 mmol) was added followed by the addition of 52 mg (0.36 mmol) of pyrazinoyl chloride. The reaction
was stirred at 0 oC for 30 min. and 25 oC for 3 hours. The reaction solution was diluted with 20 mL of aqueous
sodium bicarbonate and extracted three times with dichloromethane. The combined organic layer was
washed with brine, dried (anhydrous Na2SO4), concentrated, and column chromatographed on silica gel using
a mixture of dichloromethane and methanol (10:1) as an eluent to give 3 (62 mg, 54% yield) as a white solid.
1H NMR (CDCl3): δH 9.42 (1H, s, CH pyrazine), 9.14 (1H, br. s., NH), 8.77 (1H, d, J 2.3 Hz, CH pyrazine), 8.53 (1H,
s, CH pyrazine), 7.86 - 7.99 (1H, m, NH), 7.56 (2H, d, J 8.2 Hz, CH x 2 o-aromatic), 7.33 (2H, t, J 7.8 Hz, CH x 2 m-
aromatic), 7.11 (1H, t, J 7.4 Hz, CH p-aromatic), 3.44 (1H, t, J 6.6 Hz, CH or CH2N), 3.11 (2H, s, CH2N), 2.95 (2H,
d, J 11.7 Hz, CH2N), 2.26 (2H, t, J 10.7 Hz, CH2N), 1.84 (2H, d, J 12.1 Hz, CH2), 1.69 (1H, ddd, J 3.9, 7.3, 11.0 Hz,
13
CH), 1.39 - 1.49 (2H, m, CH2); C NMR (CDCl3): δC 168.6 (C=O), 163.3 (C=O), 147.5 (CH pyrazine), 144.6 (CH
pyrazine), 144.5 (C pyrazine), 142.6 (CH pyrazine), 137.7 (C aromatic), 129.2 (CH x 2 aromatic), 124.3 (CH
aromatic), 119.6 (CH x 2 aromatic), 62.5 (CH2N), 55.0 (2 CH2N), 44.9 (CH2N), 35.9 (CH), 30.5 (2 CH2); MS (ESI):
m/z 376.1 (M+Na)+, 354.3 (M+H)+(100%), 233.4 (75%).
2-(4-{[2-Oxo-2-(1H-pyrrol-2-yl)ethylamino]methyl}piperidin-1-yl)-N-phenylacetamide (4). A mixture of 14
(0.10 g, 0.40 mmol), 2-chloroacetyl pyrrole (16) (0.04 g, 0.28 mmol), and sodium bicarbonate (0.11 g, 0.40
mmol) in a mixture of 20:1 distilled acetonitrile:DMF (5 mL) was heated at 50oC under argon for 8 hours. The
resulting mixture was cooled to room temperature and filtered to remove insoluble inorganic salt. The filtrate
was concentrated and column chromatographed on silica gel using a mixture of dichloromethane and
1
methanol (30:1) as an eluent to give 4 (56 mg, 56% yield) as a red solid. H NMR (CDCl3): δH 9.96 (1H, br. s.,
NH), 9.19 (1H, br. s., NH), 7.53 (2H, d, J 1.0 Hz, CH 2 x o-aromatic), 7.33 (2H, t, J 1.0 Hz, CH x 2 m-aromatic),
7.10 (1H, t, J 7.4 Hz, CH p-aromatic), 7.06 (1H, br. s., CH imidazole), 6.93 (1H, d, J 3.5 Hz, CH imidazole), 6.27
(1H, dd, J 1.0 Hz, CH imidazole), 3.89 (2H, s, CH2N), 3.18 (2H, s, CH2N), 2.90 (2H, d, J 11.3 Hz, CH2N), 2.64 (2H, d,
J 6.6 Hz, CH2N), 2.38 (1H, s, NH), 2.23 (2H, t, J 10.7 Hz, CH2N), 1.84 (2H, d, J 11.7 Hz, CH2), 1.62-1.70 (1H, m,
13
CH), 1.13 - 1.47 (2H, m, CH2); C NMR (CDCl3): δC 188.6 (C=O), 169.1 (C=O), 137.9 (C aromatic), 130.6 (C
imidazole), 129.2 (CH x 2 aromatic), 124.9 (CH imidazole), 124.3 (CH aromatic), 119.6 (CH x 2 aromatic), 116.0
(CH imidazole), 111.0 (CH imidazole), 62.6 (CH2N), 55.9 (CH2N), 55.0 (CH2N), 54.3 (2 CH2N), 36.0 (CH), 31.0 (2
CH2); MS (ESI): m/z 355.4 (M+H)+ (30%), 377.5 (M+Na)+, 231.3 (100%).
o
N-({4-Methyl-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl)benzamide (5). To a cold (-78 C)
solution of 11 (0.5 g, 4.54 mmol) in 25 mL of dry THF under argon was added a solution of lithium
diisopropylamide (LDA) (0.23 M in 20 mL of THF, 4.54 mmol) slowly over 30 min. A pale brown solution
o
resulted. Stirring was continued for 2 hours at -78 C and iodomethane (0.64 g, 4.54 mmol) was added
o
dropwise into the reaction solution while maintaining the temperature at –78 C. The reaction solution was
o
o
stirred at –78 C for 2 hours, diluted with 5 mL of saturated aqueous NH4Cl solution at –78 C, and then
warmed to room temperature. The organic layer was separated, and the aqueous layer was extracted three
times (100 mL each) with dichloromethane. The combined organic layers were washed with brine (100 mL),
dried (anhydrous Na2SO4), concentrated, and column chromatographed on silica gel using a gradient mixture
of CH2Cl2 and MeOH as eluent to give 4-methylpiperidine-4-carbonitrile (17)34 (0.42 g, 74% yield) as a white
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1
solid. H NMR (CDCl3): δH 4.82 (1H, br. s., NH), 3.23 (2H, d, J 11.4 Hz, CH2N), 2.98 (2H, t, J 12.6 Hz, CH2N), 1.97
(2H, d, J 13.5 Hz, CH2), 1.72-1.63 (2H, m, CH2), 1.41 (3H, s, CH3); MS (ESI): m/z 125.1 (M+H)+ (100%).
To a solution of 17 (0.15 g, 1.21 mmol) in dry CH3CN (8 mL) under argon, N-(chloromethyl)benzamide (0.21 g,
o
1.21 mmol) and anhydrous K2CO3 (0.33 g, 2.42 mmol) were added, and the mixture was stirred at 85 C for 8
o
hours. The reaction mixture was cooled to 25 C, filtered, concentrated, and column chromatographed on
silica gel using a mixture of ethyl acetate and hexane (5:1) as an eluent to give 2-(4-cyano-4-methylpiperidin-1-
1
yl)-N-phenylacetamide (18) (0.30 g, 97% yield). H NMR (CDCl3): δH 8.94 (1H, br. s., NH), 7.57 - 7.52 (2H, m, CH
x 2 o-aromatic), 7.35 - 7.29 (2H, m, CH x 2 m-aromatic), 7.18 - 7.06 (1H, m, CH p-aromatic), 3.15 (2H, s, CH2N),
2.91 - 2.84 (2H, m, CH2N), 2.58 (2H, dt, J 2.3, 12.3 Hz, CH2N), 2.00 - 1.94 (2H, m, CH2), 1.69 - 1.58 (2H, m, CH2),
1.42 (3H, s, CH3); MS (ESI): m/z 258.6 (M+H)+, 280.1 (M+Na)+ (100%).
To a solution of 18 (200 mg, 0.78 mmol) in 8 mL of methanol, CoCl2•6H2O (94 mg, 0.39 mmol) was added, and
o
the solution was stirred for 5 min. The reaction solution was cooled to 0 C and sodium borohydride (132 mg,
3.51 mmol) was added portion-wise over 30 min. The resulting solution was stirred for 18 hours at room
temperature, diluted with 15 mL of cold saturated NH4Cl, 15 mL of water and extracted three times with ethyl
acetate. The combined organic layers were washed with brine (10 mL), dried (anhydrous Na2SO4), filtered,
and concentrated to yield 165 mg (80% yield) of 2-[4-(aminomethyl)-4-methylpiperidin-1-yl]-N-
phenylacetamide (19), as a light yellow oil. MS (ESI): m/z 284.3 (M+Na)+, 262.3 (M+H)+ (70%), 245.5 (100%).
This compound was used in the following reaction without further purification.
To a solution of compound 19 (150 mg, 0.57 mmol) in distilled dichloromethane (7.5 mL) under argon, dry
pyridine (74 μL, 0.57 mmol) was added and stirred at 0oC. To it, benzoyl chloride (80 mg, 0.57 mmol) was
added and stirred for 3 hours at room temperature. The reaction mixture was partitioned between
dichloromethane and water and basified to pH 8 using aqueous NaHCO3. The organic layer was separated,
and the aqueous layer was extracted three times with dichloromethane. The combined organic layers were
washed with brine, dried (anhydrous Na2SO4), filtered, concentrated, and purified by column chromatography
on silica gel using a gradient mixture of CH2Cl2 and MeOH as eluent to obtained 115 mg (55% yield) of N-({4-
1
methyl-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl)benzamide (5) as a white solid. H NMR (CDCl3):
δH 9.39 (1H, br. s., NH), 7.82 - 7.77 (2H, m, CH x 2 o-aromatic), 7.57 (2H, m, CH x 2 aromatic), 7.52 - 7.49 (1H,
m, CH aromatic), 7.46 - 7.42 (2H, m, CH x 2 aromatic), 7.35 - 7.29 (2H, m, CH x 2 aromatic), 7.14 - 7.06 (1H, m,
CH aromatic), 6.41 (1H, t, J 5.9 Hz, NH), 3.45 - 3.37 (2H, m, CH2N), 3.23 (2H, s, CH2N), 2.85 - 2.75 (2H, m, CH2N),
2.66 - 2.56 (2H, m, CH2N), 1.70 (2H, ddd, J 3.7, 9.5, 13.4 Hz, CH2), 1.56 - 1.46 (2H, m, CH2), 1.05 (3H, s, CH3); 13C
NMR (CDCl3): δC 168.0 (C=O), 167.7 (C=O), 137.5 (C aromatic), 134.5 (C aromatic), 131.4 (CH aromatic), 128.9
(CH x 2 aromatic), 128.5 (CH x 2 aromatic), 126.9 (CH x 2 aromatic), 124.2 (CH aromatic), 119.5 (CH x 2
aromatic), 61.8 (CH2N), 53.6 (CH2N), 49.6 (2 CH2N), 35.4 (C), 34.4 (2 CH2), 32.7 (CH3); MS (ESI): m/z = 366.1
(M+H)+ (100%), 245.5.
{4-(Benzamidomethyl)-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl benzoate (9). To a solution of
4-cyanopiperidine (11) (0.5 g, 4.54 mmol) in 25 mL of distilled dichloromethane under argon, dried
triethylamine (1.3 mL, 9.08 mmol) was added. While stirring, the solution was cooled to 0 °C and di-tert-butyl
dicarbonate (1.19 g, 5.4 mmol) was added in several portions over 15 min (bubbling observed). The resulting
mixture was stirred at 25 oC for 12 hours, then the solvent was evaporated and the residue purified by column
chromatography on silica gel using a mixture of dichloromethane and methanol (30:1) as an eluent to give
tert-butyl 4-cyanopiperidine-1-carboxylate (20), 0.95 g (100% yield) as a colorless viscous oil, that solidifies on
1
standing. H NMR (CDCl3): δH 3.64 (2H, ddd, J 3.91, 7.03, 13.67 Hz, CH2N), 3.31 (2H, ddd, J 3.7, 7.8, 13.9 Hz,
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13
CH2N), 2.73 - 2.83 (1H, m, CHCN), 1.82 - 1.92 (2H, m, CH2), 1.72 - 1.82 (2H, m, CH2), 1.44 (9 H, s, 3 CH3); C
NMR (CDCl3): δC 153.6 (C=O), 120.5 (CN), 79.0 (C-O), 40.8 (2 CH2N), 27.8 (CH), 27.7 (3 CH3), 25.5 (2 CH2).
A solution of tert-butyl 4-cyanopiperidine-1-carboxylate (20) (0.95 g, 4.54 mmol) was dissolved in 25 mL of dry
THF under argon and cooled to –78 °C. Lithium diisopropylamide (0.23M in 20 mL, 4.54 mmol) was slowly
added to the reaction solution over 30 min at –78 °C. A pale brown solution was obtained. Stirring was
continued for 2 hours at –78 °C, and a solution of paraformaldehyde (0.16 g, 5.4 mmol) in 25 mL of freshly
distilled THF was added slowly into the reaction solution while keeping the temperature at –78 °C. The
reaction mixture was allowed to reach room temperature while being stirred for 15 hours under argon. It was
diluted with 100 mL of water and 50 mL of saturated NaCl, and the resulting solution was extracted with
dichloromethane three times (250 mL each). The combined organic layers were washed with brine (100 mL),
dried (anhydrous Na2SO4), concentrated, and column chromatographed on silica gel using a gradient mixture
of hexane and ethyl acetate as eluent to give tert-butyl 4-cyano-4-(hydroxymethyl)piperidine-1-carboxylate
1
(21), 0.78 g (72% yield) as a white solid. H NMR (CDCl3): δH 4.10 - 4.24 (2H, m, CH2O), 3.67 (2H, d, J 6.3 Hz,
CH2N), 2.03 - 2.11 (2H, m, CH2N), 1.91 - 2.00 (2H, m, CH2), 1.95 – 1.85 (2H, m, CH2), 1.46 (9H, s, 3 CH3); 13C
NMR (CDCl3): δC 154.4 (C=O), 121.4 (CN), 80.1 (C-O), 67.4 (CH2O), 40.6 (2 CH2N), 40.0 (2 CH2), 30.8 (C), 28.1 (3
CH3); MS (ESI): m/z 263.5 (M+Na)+ (100%).
To a solution of tert-butyl 4-cyano-4-(hydroxymethyl)piperidine-1-carboxylate (21) (0.49 g, 2.0 mmol) and
pyridine (0.24 mL, 3.03 mmol) in dichloromethane (8 mL) under argon at 0 °C, was added benzoyl chloride
(0.25 mL, 2.0 mmol), and the reaction mixture was stirred for 8 hours at room temperature. The reaction
mixture was partitioned between dichloromethane and water and adjusted the pH to 8 using aqueous
NaHCO3. The organic layer was separated, and the aqueous layer was extracted three times with
dichloromethane. The combined organic layers were washed with brine (10 mL), dried (anhydrous Na2SO4),
filtered, concentrated, and purified by column chromatography on silica gel using a mixture of
dichloromethane and methanol (30:1) to obtain 0.21 g (86% yield) of tert-butyl 4-[(benzoyloxy)methyl]-4-
cyanopiperidine-l-carboxylate (22), as a yellow solid. 1H NMR (CDCl3): δH 7.88 - 8.04 (2H, m, CH x 2 o-
aromatic), 6.81 - 6.95 (2H, m, CH x 2 m-aromatic), 6.41 (1H, s, CH p-aromatic), 4.33 (2H, s, CH2O), 4.3 - 4.17
(2H, m, CH2N), 3.10 – 3.0 (2H, m, CH2N), 2.05 - 2.01 (2H, m, CH2), 1.53 - 1.61 (2H, m, CH2), 1.47 (9H, s, 3 CH3);
13C NMR (CDCl3): δC 165.6 (C=O), 161.0 (C=O), 154.7 (C aromatic), 132.3 (CH x 2 aromatic), 121.3 (CN), 120.5
(CH aromatic), 115.6 (CH x 2 aromatic), 80.7 (C-O), 68.1 (CH2O), 42.0-39.0 (m, 2 CH2N) 38.8 (CH2), 31.8 (CH2),
28.5 (3 CH3); MS (ESI): m/z 367.0 (M+Na)+ (100%), 245.4.
A solution of 4-[(benzoyloxy)methyl]-4-cyanopiperidine-l-carboxylate (22) (1.12 mmol) in 2 mL dichloro-
methane containing 10% trifluoracetic acid (TFA) was stirred at room temperature for 1 hour. The solvent was
removed and the remaining solid was dried under high vacuum to yield 0.27 g (95% yield) of (4-
1
cyanopiperidin-4-yl)methyl benzoate (23) as a yellow solid. H NMR (DMSO-d6): δH 8.8-8.6 (1H, br. s., NH), 8.02
(2H, d, J 7.42 Hz, CH x 2 o-aromatic), 7.71 (1H, t, J 7.42 Hz, CH p-aromatic), 7.57 (2H, t, J 7.62 Hz, CH x 2 m-
aromatic), 4.49 (2H, s, CH2O), 3.46 - 3.48 (2H, m, CH2N), 3.02 (2H, t, J 12.1 Hz, CH2N), 2.28 (2H, d, J 14.5 Hz,
CH2), 1.92 - 2.02 (2H, m, CH2); 13C NMR (DMSO-d6): δC 165.1 (C=O), 134.0 (C aromatic), 129.5 (CH x 2 aromatic),
129.0 (CH x 2 aromatic), 128.9 (CH aromatic), 120.3 (CN), 67.2 (CH2O), 40.3 (2 CH2N), 39.0 (C, overlap with
DMSO-d6), 36.7 (CH2), 27.8 (CH2). This molecule was used in the following reaction without purification.
To a solution of (4-cyanopiperidin-4-yl)methyl benzoate (23) (0.10 g, 0.41 mmol) in dry CH3CN under argon, N-
(chloromethyl)benzamide (12) (57 mg, 0.41 mmol) and anhydrous K2CO3 (0.17 g, 1.23 mmol) were added and
o
the mixture was refluxed at 85 C for 12 hours. The reaction mixture was cooled, filtered through Celite,
concentrated, and purified by column chromatography on silica gel using a mixture of dichloromethane and
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methanol (30:1) as an eluent to give {4-cyano-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl benzoate
1
(24), 0.15 g (97% yield). H NMR (CDCl3): δH 8.9 - 8.8 (1H, br. s., NH), 8.04 - 8.12 (2H, m, CH x 2 aromatic)), 7.58
- 7.64 (1H, m, CH aromatic), 7.52 - 7.57 (2H, m, 2H, CH x 2 aromatic), 7.44 - 7.51 (2H, m, CH x 2 aromatic), 7.28
- 7.36 (2H, m, 2H, CH x 2 aromatic), 7.08 - 7.14 (1H, m, CH aromatic), 4.40 (2H, s, CH2O), 3.21 (2H, s, CH2N),
2.98 (2H, d, J 12.5 Hz, CH2N), 2.69 (2H, dt, J 2.3, 12.3 Hz, CH2N), 2.13 (2H, d, J 13.3 Hz, CH2), 1.77 - 1.86 (2H, m,
CH2); 13C NMR (CDCl3): δC 167.8 (C=O), 165.9 (C=O), 137.4 (C aromatic), 133.8 (CH aromatic), 129.9 (CH x 2
aromatic), 129.1 (CH x 2 aromatic), 128.7 (CH x 2 aromatic), 124.5 (CH aromatic), 124.4 (C aromatic) 120.5
(C≡N), 119.6 (CH x 2 aromatic), 68.3 (CH2O), 62.0 (CH2N), 50.3 (2 CH2N), 37.7 (C), 32.2 (CH2), 27.1 (CH2); MS
(ESI): m/z 378.5 (M+H)+ (100%), 257.3.
To a solution of {4-cyano-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl benzoate (24) (0.16 g, 0.42
mmol) in 5 mL of methanol under argon, CoCl2•6H2O (51 mg 0.21 mmol) was added and stirred for 5 min. The
o
reaction mixture was cooled to 0 C and sodium borohydride (86 mg, 2.27 mmol) was added in several
o
portions over 30 min. The resulting solution was stirred for 18 hours at 25 C, diluted with 15 mL of cold
saturated aqueous NH4Cl solution and 15 mL of water, and extracted three times with ethyl acetate. The
combined organic layers were washed with brine (10 mL), dried (anhydrous Na2SO4), filtered, and
concentrated to yield 0.13 g (78% yield) of {4-(aminomethyl)-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-
yl}methyl benzoate (25), as a white solid. MS (ESI) m/z = 382.2 (M+H)+ (100%). This material was used in the
following reaction without purification.
To a solution of {4-(aminomethyl)-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl benzoate (25) (50 mg,
0.13 mmol) in dichloromethane (5 mL) under argon, dry pyridine (19 μL, 0.24 mmol) was added and the
mixture was stirred at 0 °C. To it, benzoyl chloride (19 μL, 016 mmol) was added and the mixture was stirred
for 3 hours at room temperature, partitioned between dichloromethane and water, and basified to pH 8 using
aqueous NaHCO3. The organic layer was separated, and the aqueous layer was extracted three times with
dichloromethane. The combined organic layers were washed with brine (10 mL), dried (anhydrous Na2SO4),
filtered, concentrated, and purified by column chromatography on silica gel using a mixture of dichloro-
methane and methanol (30:1) as an eluent to obtained 48 mg (75% yield) of {4-(benzamidomethyl)-1-[2-oxo-2-
(phenylamino)ethyl]piperidin-4-yl}methyl benzoate (9), as a white solid (as a mixture of cis- and trans-
isomers). 1H NMR (CDCl3): δH 9.35 - 9.27 (1H, br. s., NH), 8.70 - 8.65 (1H, br. s., NH), 8.11 (2H, d, J 8.2 Hz, CH x 2
aromatic), 8.07 (2H, d, J 8.2 Hz, CH x 2 aromatic), 7.87 (2H, d, J 8.2 Hz, CH x 2 aromatic), 7.57 - 7.60 (2H, m, CH
x 2 aromatic), 7.44 - 7.49 (4H, m, CH x 4 aromatic), 7.33 (2H, t, J 7.62 Hz, CH x 2 aromatic), 7.08 - 7.14 (1H, m,
CH aromatic), 4.40 (2H, s, CH2O), 3.53 (2H, d, J 5.86 Hz, CH2N), 3.26 (2H, s, CH2N), 2.84 (2H, dd, J 4.3, 5.9 Hz,
13
CH2N), 2.71 - 2.79 (2H, m, CH2N), 1.67 - 1.85 (4H, m, 2 CH2); C NMR (CDCl3): δC 168.1 (C=O), 167.6 (C=O),
167.4 (C=O), 137.5 (C aromatic), 133.7 (CH aromatic), 133.2 (C aromatic), 131.6 (C aromatic), 130.0 (CH
aromatic), 129.8 (CH x 2 aromatic), 129.0 (CH x 2 aromatic), 128.7 (CH x 2 aromatic), 128.4 (CH x 2 aromatic),
127.0 (CH x 2 aromatic), 124.2 (CH aromatic), 119.6 (CH x 2 aromatic), 68.4 (CH2O), 62.1 (CH2N), 49.5 (CH2N),
42.1 (C), 37.0 (2 CH2N), 30.6 (2 CH2); MS (ESI) m/z = 486.2 (M+H)+ (100%), 365.4.
N-({4-(Hydroxymethyl)-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl)benzamide (6). To a solution of
(4-(benzamidomethyl)-1-(2-oxo-2-(phenylamino)ethyl)piperidin-4-yl)methyl benzoate (9) (45 mg, 0.09 mmol)
in methanol, anhydrous K2CO3 (130 mg, 0.93 mmol) was added and the mixture was stirred for 2 hours at
25 oC. The reaction mixture was filtered and purified by column chromatography on silica gel using
dichloromethane:methanol (30:1) as an eluent to give N-({4-(hydroxymethyl)-1-[2-oxo-2-(phenylamino)-
ethyl]piperidin-4-yl}methyl)benzamide (6), 25 mg (71% yield) as a white solid (as a mixture of trans- and cis-
1
isomers). H NMR (CDCl3): δH 9.0 - 8.96 (1H, br. s., NH, major isomer), 8.4 – 8.35 (1H, br. s., NH, minor isomer),
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8.15 – 8.10 (2H, m, CH x 2 o-aromatic, minor isomer), 7.76 - 7.85 (3H, m, CH x 3 aromatic, minor isomer), 7.51 -
7.62 (3H, m, CH x 3 aromatic, major isomer), 7.42 - 7.50 (2H, m, CH x 2 aromatic, major isomer), 7.34 (3H, t, J
8.01 Hz, CH x 3 aromatic, major isomer), 7.13 (2H, t, J 7.42 Hz, CH x 2 aromatic major isomer), 3.72 (2H, s,
CH2O), 3.67 (2H, s, O=CCH2N), 3.22 (2H, s, CH2NC=O), 3.0 – 2.8 (1H, m, CHN) 2.63 (2H, dt, J 2.0, 12.3 Hz, CH2N),
13
2.34 (1H, t, J 7.4 Hz, CHN), 2.06 (2H, d, J 12.9 Hz, CHN + OH), 1.63 - 1.73 (4H, m, 2 CH2); C NMR (CDCl3): δC
168.1 (C=O), 168.0 (C=O), 133.2 (C aromatic), 129.8 (CH x 2 aromatic), 129.3 (CH x 2 aromatic), 129.0 (C
aromatic) 128.6 (CH aromatic), 127.3 (CH aromatic), 124.7 (CH x 2 aromatic), 119.8 (CH x 2 aromatic), 68.3
(CH2O), 62.1 (CH2N), 53.0 (CH2N), 50.8 (CH2N), 40.0 (CH2N), 33.8 (C), 31.7 (CH2), 28.6 (CH2); MS (ESI): m/z 382.2
(M+H)+ (100%).
{4-[(2-Fluorobenzamido)methyl]-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl benzoate (10). To a
solution of {4-(aminomethyl)-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl benzoate (25) (50 mg, 0.13
mmol) in dried dichloromethane (5 mL) under argon was added pyridine (19 μL, 0.17 mmol) and stirred at 0 oC
for 10 min. To the reaction solution, 2-fluorobenzoyl chloride (27 mg, 017 mmol) was added and stirred for 3
hours at room temperature. The reaction mixture was partitioned between dichloromethane and water and
basified to pH 8 using aqueous NaHCO3. The organic layer was separated, and the aqueous layer was
extracted three times with dichloromethane. The combined organic layers were washed with brine (10 mL),
dried (anhydrous Na2SO4), filtered, concentrated, and purified by column chromatography on silica gel using a
mixture of dichloromethane and methanol (30:1) as an eluent to obtain 48 mg (69% yield) of 4-
1
(benzamidomethyl)-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-yl)methyl benzoate (10), as a white solid. H
NMR (CDCl3): δH 9.68-9.55 (1H, br. s., NH), 7.97 - 8.04 (2H, m, CH x 2 aromatic), 7.83 (2H, d, J 7.42 Hz, CH x 2
aromatic), 7.60 (2H, d, J 7.8 Hz, CH x 2 aromatic), 7.52 (2H, d, J 6.6 Hz, CH x 2 aromatic), 7.41 - 7.47 (2H, m, CH
x 2 aromatic), 7.29 - 7.36 (2H, m, CH x 2 aromatic), 7.12 - 7.17 (2H, m, CH x 2 aromatic), 6.10 (1H, br. s., NH),
4.41 (2H, s, CH2O), 3.62 (2H, d, J 5.9 Hz, CH2N), 3.48 (2H, s, CH2N), 2.92 - 3.08 (4H, m, 2 CH2N), 1.85 (4H, m, 2
13
CH2); C NMR (CDCl3): δC 167.9 (C=O), 167.8 (C=O), 166.6 (C=O), 164.4 (d, JC-F 160 Hz, CF aromatic), 137.6 (C
aromatic), 135.2 (C aromatic), 134.6 (C aromatic), 132.6 (CH aromatic), 132.3 (CH aromatic), 131.7 (CH
aromatic), 128.9 (CH x 2 aromatic), 128.6 (CH x 2 aromatic), 127.0 (CH x 2 aromatic), 124.4 (CH aromatic),
124.0 (CH aromatic), 119.7 (CH x 2 aromatic), 117.0 (CH aromatic), 69.2 (CH2O), 61.5 (CH2N), 49.2 (CH2N), 42.3
(C), 36.5 (2 CH2N), 29.7 (2 CH2); MS (ESI): m/z 504.2 (M+H)+ (100%).
2-Fluoro-N-{[4-(hydroxymethyl)-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4yl}methyl)benzamide (7). To a
solution of {4-(benzamidomethyl)-1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl benzoate (10) (30 mg,
0.06 mmol) in methanol under argon, anhydrous K2CO3 (83 mg, 0.6 mmol) was added and the mixture was
o
stirred for 2 hours at 25 C. The reaction mixture was filtered and purified by column chromatography on
silica gel using a mixture of dichloromethane and methanol (30:1) as an eluent to give N-({4-(hydroxymethyl)-
1
1-[2-oxo-2-(phenylamino)ethyl]piperidin-4-yl}methyl)benzamide (7), 13 mg (55% yield) as a white solid. H
NMR (CDCl3): δH 9.20 - 9.07 (1H, br. s., NH), 7.78 (1H, d, J 7.81 Hz, CH aromatic), 7.56 (2H, d, J 7.81 Hz, CH x 2
aromatic), 7.41 - 7.48 (2H, m, CH x 2 aromatic), 7.33 (2H, t, J 7.81 Hz, CH x 2 aromatic), 7.08 - 7.22 (1H, m, CH
aromatic), 6.78 (1H, d, J 5.47 Hz, CH aromatic), 4.1 – 3.7 (1H, br. s., OH), 3.49 (2H, d, J 6.3 Hz, CH2O), 3.46 (2H,
s, CH2N), 3.14 (2H, s, CH2N), 2.61 (4H, dd, J 7.0, 11.3 Hz, 2 CH2N), 1.60-1.68 (4H, m, 2 CH2); 13C NMR (CDCl3): δC
169.1 (C=O), 168.5 (C=O), 135.6 (d, JC-F 380 Hz, CF aromatic), 131.9 (C aromatic), 131.8 (CH aromatic), 129.0
(CH x 2 aromatic), 128.7 (CH x 2 aromatic), 127.0 (CH x 2 aromatic), 124.3 (C aromatic), 119.5 (CH x 2
aromatic), 65.8 (CH2O), 62.3 (CH2N), 49.7 (2 CH2N), 37.0 (CH2N), 30.9 (CH2); MS (ESI) m/z = 400.0 (M+H)+
(100%).
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N-({1-[2-(tert-Butylamino)-2-oxoethyl]piperidin-4-yl}methyl)benzamide (8). A mixture of 4-cyanopiperidine
(11) (1.0 g, 9.1 mmol), N-tert-butyl-2-chloroacetamide (26) (1.35 g, 9.1 mmol), and anhydrous potassium
carbonate (2.5 g, 18.2 mmol) in acetonitrile (30 mL) was heated to reflux under argon for 9 hours. The
resulting mixture was cooled to room temperature and filtered to remove inorganic salts. The filtrate was
concentrated and crystallized from diethyl ether to give N-tert-butyl-2-(4-cyanopiperidin-l-yl)acetamide (27)
1
(1.82 g, 90% yield) as a white solid. H NMR (CDCl3): δH 2.89 (2H, s, CH2N), 2.68 - 2.78 (2H, m, CH2N), 2.67 –
2.55 (1H, m, CH), 2.31 - 2.44 (2H, m, CH2N), 1.93-2.03 (2H, m, CH2), 1.81-1.93 (2H, m, CH2), 1.35 (9H, s, 3 CH3);
13C NMR (CDCl3): δC 168.9 (C=O), 121.5 (CN), 62.5 (CH2N), 51.8 (C-N), 50.6 (2 CH2N), 29.1 (2 CH2), 28.9 (3 CH3),
25.7 (CH); MS (ESI): m/z 262.4 (M+K)+ (100%), 246.3 (M+Na)+ (85%), 224.2 (M+H)+.
To a cold (0 °C) solution of N-tert-butyl-2-(4-cyanopiperidin-lyl)acetamide (27) (0.7 g, 3.1 mmol) in methanol
(20 mL) under argon, cobalt (II) chloride hexahydrate (0.37 g, 1.6 mmol) was added followed by the addition of
sodium borohydride (0.53 g, 14.1 mmol). After stirring for 15 hours at 25 °C, the reaction solution was diluted
with 25 mL of 5% aqueous ammonium hydroxide and extracted three times with dichloromethane. The
combined organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated to give 2-[(4-
(aminomethyl)piperidin-l-yl]-N-tert-butylacetamide (28) (0.65 g, 90% yield) as a crude product, which was
used in the next step without further purification. 1H NMR (CDCl3): δH 7.12 - 7.05 (1H, br. s., NHC=O), 2.86 (2H,
s, O=CCH2N), 2.81 (2H, br. s., CH2N), 2.61 (2H, d, J 6.3 Hz, CH2N), 2.12 (2H, t, J 11.1 Hz, CH2N), 1.74 (2H, d, J
11.7 Hz, CH2), 1.35 (9H, s, 3 CH3), 1.17 - 1.25 (3H, m, CH + CH2) ; MS (ESI): m/z 228.3 (M+H)+ (100%).
o
To a cold (0 C) solution of 0.20 g (0.88 mmol) of 2-(4-(aminomethyl)piperidin-l-yl)-N-(tert-butyl)acetamide
(28) in freshly distilled dichloromethane (2 mL) under argon, pyridine (1.3 mmol) was added followed by the
o
addition of 0.12 g (0.88 mmol) of benzoyl chloride. The reaction was stirred at 0 C for 30 min. and 25 oC for 2
hours. It was diluted with 20 mL of aqueous sodium bicarbonate and extracted three times with
dichloromethane. The combined organic layer was washed with brine, dried (anhydrous Na2SO4),
concentrated, and column chromatographed on silica gel using a mixture of dichloromethane and methanol
(10:1) as an eluent to give N-({1-[2-(tert-butylamino)-2-oxoethyl)]piperidin-4-yl}methyl)benzamide (8) (0.15 g,
1
51% yield) as a white solid. H NMR (CDCl3): δH 7.77 (2H, d, J 7.4 Hz, CH x 2 o-aromatic), 7.49 (1H, t, J 7 Hz, CH
p-aromatic), 7.45 (2H, t, J 7 Hz, CH x 2 m-aromatic), 7.1 (1H, s, NH), 6.38 (1H, br. s., NH), 3.37 (2H, t, J 6.4 Hz,
CH2N), 2.79 - 2.90 (6H, m, 3 CH2N), 2.12 (2H, t, J 11.3 Hz, CH2), 1.77 (2H, d, J 12.5 Hz, CH2), 1.55 - 1.67 (1H, m,
CH), 1.36 (9H, s, 3 CH3); 13C NMR (CDCl3): δC 169.5 (C=O), 167.6 (C=O), 134.6 (C aromatic), 131.4 (CH aromatic),
128. 5 (CH x 2 aromatic), 126.8 (CH x 2 aromatic), 62.5 (CH2N), 53.7 (CH2N), 50.4 (C-N), 45.3 (2 CH2N), 35.9
(CH), 30.3 (2 CH2), 28.7 (3 CH3); MS (ESI): m/z 354 (M+Na)+ (100%), 331.9 (M+H)+ (70%).
Acknowledgements
Research reported in this publication was, in part, supported by the National Institute of Neurological
Disorders and Stroke of the National Institutes of Health under award number R44NS086343 (to XSX). The
content is solely the responsibility of the authors and does not necessarily represent the official views of the
National Institutes of Health. DHH acknowledges financial support from the Johnson Cancer Center for
Research and Travel Awards.
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Supplementary Material
Biological evaluation of the synthesized molecules is reported, and proton and carbon-13 NMR and mass
spectra of compounds 1 – 10 and their synthetic intermediates are presented as supporting information in
Supplementary Material. Readers will be able to access this supporting information using the link
"Supplementary Material" in the journal issue contents page.
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