Novel pyridinone derivatives as non-nucleoside reverse transcriptase inhibitors (NNRTIs) with high potency against NNRTI-resistant HIV-1 strains

Article abstract of DOI:10.1021/jm400102x

Novel 6-substituted-4-cycloalkyloxy-pyridin-2(1H)-ones were synthesized as non-nucleoside reverse transcriptase inhibitors (NNRTIs), and their biological activity was evaluated. Most of the compounds, especially 26 and 22, bearing a 3-isopropyl and 3-iodine group, respectively, exhibited highly potent activity against wild-type HIV-1 strains and those resistant to reverse transcriptase inhibitors (RTIs). The diastereoisomers of 26-trans and 26-cis were synthesized separately and confirmed with HPLC and NOESY spectra. The 26-trans isomers had an activity about 400-fold more potent than that of 26-cis. The pair of 26-trans enantiomers, one of the most potent inhibitors with EC₅₀ of 4 nM and selectivity index (SI) of 75000, was highly effective against a panel of RTIs-resistant strains with single (Y181C and K103N) or double (A17) mutations in reverse transcriptase. The results suggest that these novel pyridinone derivatives have the potential to be further developed as new antiretroviral drugs with improved antiviral efficacy and drug resistance profile.

Full text of DOI:10.1021/jm400102x

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Article
Novel pyridinone derivatives as non-nucleoside reverse transcriptase
inhibitors (NNRTIs) with high potency against NNRTI-resistant HIV-1 strains
Amin Li, Yabo Ouyang, Ziyun Wang, Yuanyuan Cao, Xiangyi Liu, Li Ran, Chao Li, Li
Li, Liang Zhang, Kang Qiao, Weisi Xu, Yang Huang, Zhili Zhang, Chao Tian, Zhenming
Liu, Shibo Jiang, Yiming Shao, Yansheng Du, Liying Ma, Xiaowei Wang, and Junyi Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400102x • Publication Date (Web): 29 Mar 2013
Downloaded from http://pubs.acs.org on March 30, 2013
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Novel pyridinone derivatives as non-nucleoside reverse transcriptase inhibitors
(NNRTIs) with high potency against NNRTI-resistant HIV-1 strains
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†
,#
§,#
‡
†
†
‡
Amin Li, Yabo Ouyang, Ziyun Wang, Yuanyuan Cao, Xiangyi Liu, Li Ran, Chao
†
†
†
‡
§
§
†
†
Li, Li Li, Liang Zhang, Kang Qiao, Weisi Xu, Yang Huang, Zhili Zhang, Chao Tian,
‡
┴
§
ǂ
,§
Zhenming Liu, Shibo Jiang, Yiming Shao, Yansheng Du, Liying Ma,* Xiaowei
,
†
,†,‡
Wang* and Junyi Liu*
†
‡
Department of Chemical Biology, School of Pharmaceutical Sciences; State Key
§
Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191; State
Key Laboratory for Infection Disease Prevention and Control, National Center for
AIDS/STD Control and Prevention (NCAIDS), Chinese Center for Disease Control and
┴
Prevention, Beijing 102206 and Laboratory of Medical Molecular Virology of
Ministries of Education and Health, Shanghai Medical College and Institute of Medical
ǂ
Microbiology, Fudan University, Shanghai 200032, China. Department of Neurology,
Indiana University School of Medicine, Indianapolis, Indiana 46202, USA
Keywords: HIVꢀ1, nonꢀnucleoside reverse transcriptase inhibitors (NNRTIs), drug
resistance
#
These authors have contributed equally.
*
To whom correspondence should be addressed: Phone/Fax: +86ꢀ10ꢀ82805203, eꢀmail:
xiaoweiwang@bjmu.edu.cn (X.W.); Phone/Fax: +86ꢀ10ꢀ82805203,
eꢀmail: Jyliu@bjmu.edu.cn (J.L.); or Phone: +86ꢀ10ꢀ58900976, Fax: +86ꢀ10ꢀ58900980,
eꢀmail: mal@chinaaids.cn (L.M.).
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Abstract
Novel 6ꢀsubstitutedꢀ4ꢀcycloalkyloxyꢀpyridinꢀ2(1H)ꢀones were synthesized as
nonꢀnucleoside reverse transcriptase inhibitors (NNRTIs), and their biological activity was
evaluated. Most of the compounds, especially 26 and 22, bearing a 3ꢀisopropyl and
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ꢀiodine group, respectively, exhibited highly potent activity against wildꢀtype HIVꢀ1
strains and those resistant to reverse transcriptase inhibitors (RTIs). The diastereoisomers
of 26-trans and 26-cis were synthesized separately, and confirmed with HPLC and
NOESY spectra. The 26-trans isomers had an activity about 400ꢀfold more potent than
that of 26-cis. The pair of 26-trans enantiomers, one of the most potent inhibitors with
EC50 of 4 nM and selectivity index (SI) of 75,000, was highly effective against a panel of
RTIsꢀresistant strains with single (Y181C and K103N) or double (A17) mutations in
reverse transcriptase. The results suggest that these novel pyridinone derivatives have the
potential to be further developed as new antiretroviral drugs with improved antiviral
efficacy and drug resistance profile.
Abbreviations: CC50, concentration for 50% cytotoxicity; EC50, effective concentration
for 50% inhibition; ELISA, enzymeꢀlinked immunosorbent assay; HAART, highly active
antiretroviral therapy; HIVꢀ1, human immunodeficiency virus type 1; NNBP,
nonꢀnucleoside binding pocket; NNRTI, nonꢀnucleoside reverse transcriptase inhibitor;
PDB, protein database; RT, reverse transcriptase; SI, selectivity index (ratio of CC /EC ).
50
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Introduction
The development of potent and effective antiviral drugs for the control of human
immunodeficiency virus type 1 (HIVꢀ1) infection is one of the most pressing goals of
contemporary medicinal chemistry. Reverse transcriptase (RT) is an essential enzyme in
the infectious life cycle of HIV and plays a multifunctional role in viral replication. RT
inhibitors were used as important components of HAART and as a method of preventing
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, 2
sexual transmission or motherꢀtoꢀchild transmission of HIVꢀ1 . Two functionally distinct
classes of HIVꢀ1 RT inhibitors (nucleoside and nonꢀnucleoside) have been discovered and
are being used clinically. Especially, nonꢀnucleoside RT inhibitors (NNRTIs) have gained a
definitive and important place in clinical use based on their favorable pharmacokinetic
3
, 4
properties and wide range of chemically diverse structures. To date, five NNRTIs have
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been approved for clinical use: nevirapine (NVP) , delavirdine (DLV) , efavirenz (EFV) ,
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etravirine (ETR) and rilpivirine (RPV) . However, DLV is rarely used because of its poor
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0ꢀ13
pharmacokinetics
.
Like other types of antiꢀHIV drugs, the therapeutic efficacy of NNRTIs is weakened
by the very rapid development of drugꢀresistant mutations, in vitro and in vivo, both in
1
4ꢀ16
monotherapy and in multidrug trials
. The commonly observed drugꢀresistant virus
contains the single amino acid mutation Tyr181Cys and Lys103Asn, which are resistant to
1
7
NVP, DLV, EFV and other NNRTIs. Therefore, it is important to develop new NNRTIs
that can effectively inhibit commonly observed NNRTIꢀassociated resistant viruses and
have a higher genetic barrier to resistance.
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Crystallographic studies showed that NNRTIs bind in an allosteric pocket located
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about 10 Å from the polymerase active site and cause large conformational changes in
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HIVꢀ1 RT, which could influence the geometry of the polymerase catalytic site
.
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Recently, several compounds based on the 2ꢀpyridinone scaffold, such as 1 (Lꢀ697,661) ,
have been reported as having significant antiviral potency. In our previous study, we
2
2
developed 2 (TNKꢀ651) analogues as antiviral agents with better drugꢀresistance
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profiles . Therefore, we designed novel pyridinone derivatives with respective hybrids of
analogues and 2ꢀpyridinone, in which the central pyrimidinone ring of 2 is replaced with
a pyridinꢀ2ꢀone ring, and the substituents on Cꢀ3, 4 positions are corresponding to the Cꢀ5,
positions of 2 analogues. Subsequently, modifications were performed on positions 3, 4
2
6
and 6 with different substituents (Figure 1).
O
N
Cl
O
H
O
N
HN
HN
N
O
Cl
O
1
(L-697,661)
2 (TNK-651)
O
R2
3
HN
R3
6
4 O
R1
R1 cycloallkyl; R2 -X, -COOEt, -CH(CH3)2, H; R3 Aryl
target compounds
Figure 1. Structures of typical NNRTIs and target compounds
Structure and activity relationship (SAR) studies have revealed that the substituents on
Cꢀ3 and Cꢀ4 of the pyridinone ring were very important for antiviral activity. Based on our
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previous results the optimization of position Cꢀ3 with the isopropyl and halogen
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substituent would have a twist effect on the conformation of position Cꢀ4 . Moreover, the
halogen substituent could act as a vehicle to incorporate diverse functionality at Cꢀ3 of the
2
4
pyridinone ring .
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Several studies have pointed out that compound flexibility could act as an important
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parameter for conserving potency against various mutant viral strains . As we know,
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among the pyridinone compounds previously described, only the aromatic and
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heterocyclic substituents are included at position Cꢀ4 of the pyridinone ring. In our report,
the modification on position Cꢀ4 by introducing saturated rings could increase the
molecular flexibility and result in improving the potency against various mutant viral
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7
strains . In addition, a methylꢀbearing moiety on the cycle ring may result in inducing
better contact with the conserved W229, while reducing aromatic stacking interactions
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8
with the highly mutable Y181 side chain .
Although considerable SAR information was available on the Cꢀ3 and Cꢀ4 positions,
only a few studies have reported on the effect of 6ꢀsubstituted pyridinones. The
optimization we carried out was mainly to bind the side chain in a mode similar to the
orientation of the Nꢀ1 side chain of 2, which extends into a tunnel leading toward the
catalytic site comprised of Tyr318 and Pro236, thus providing a favourable interaction
with HIVꢀ1 RT, and then enhancing the potency against wildꢀtype HIVꢀ1 and several
clinically relevant mutant strains with low cytotoxicity.
To verify the relationship between structure and activity of the target compounds, we
synthesized a series of novel pyridinone derivatives (Figure 1), and we detected their
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inhibitory activity on HIVꢀ1 RT. The active compounds were further evaluated in vitro
against a HIVꢀ1 laboratoryꢀadapted strain on both TZMꢀbl and MT4 cell lines, as well as
against principal drugꢀresistant mutant strains, K103N or Y181C and both mutation, which
confer resistance to the NNRTIs currently and commonly used in the clinical genotypic
drugꢀresistant mutation.
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Chemistry
Almost all the target compounds have been synthesized from the common
intermediate 4, which is easily obtained from ethyl 3ꢀoxoꢀ4ꢀphenylbutanoate 3 reacting
with ammonia and diethylmalonate.
The synthetic procedure used for the preparation of 7f-p is illustrated in Scheme 1. For
preparation of 6 analogues, it was first necessary for Oꢀbenzylate to give 5a, which readily
underwent Mitsunobu reaction with the corresponding cycloalkyl alcohol to yield 6f-p.
Catalytic hydrogenation led to the target compounds 7f-p.
a
Scheme 1. Synthesis of compounds 7f-p
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a
3
Reagents and reaction conditions: (a) NH /MeOH, diethyl malonate/EtONa, reflux, 4
d, yields 42~72%; (b) PhCH Br, Ag CO , THF, 60 °C, 8 h, yields 68~88%; (c)
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corresponding cyclohexanol, DIAD, PPh
MeOH, rt, 8 h, yields 75~99%.
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, THF, rt, 20 h, yields 27~94%; (d) H , Pd/C,
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Using a halogen atom replacement on position Cꢀ3, the halogenated compounds were
synthesized as described in Scheme 2. The benzylꢀprotected ester 6e was heated in
aqueous HCl to effect hydrolysisꢀ decarboxylation to afford 8, which reacted with NBS or
NIS to yield 9 and 10, respectively.
a
Scheme 2. Synthesis of compounds 9-10
a
Reagents and reaction conditions: (a) 1N HCl, reflux, 48 h, yield 49%; (b) NIS/NBS,
rt, 8 h, yields 90~99%.
Further modifications were carried out according to Scheme 3, in which 5a-e were
used as the starting materials. The ester intermediates 5a-e were subjected to reductive
methylation, yielding the expected tertiary alcohols 11a-e. Dehydration was performed
with thionylchloride to afford 12a-e. A Mitsunobu reaction with 2’ꢀmethyl cyclohexanol
(mixture) was carried out, followed by hydrogenation to install the desired target
compounds 14a-e with high yields.
a
Scheme 3. Synthesis of derivatives 14a-e
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OBn
OBn
OBn
OH
COOEt
OH
N
a
N
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OH
OH
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a-e
11 a-e
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R : a = H, b = para-F , c = para-Cl,
d = para-MeO, e = para-Me
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OBn
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14 a-e
a
Reagents and reaction conditions: (a) MeLi, THF, ꢀ78 °C, yields 67~97%; (b) SOCl2,
yields 64~90%; (c) 2ꢀmethylcyclohexanol (mixture), DIAD, PPh , THF, rt, 8 h, yields
3
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2~36%; (d) H , Pd/C, MeOH, rt, yields 85~96%.
2
Using the similar strategy described in Schemes 1ꢀ3, we synthesized the derivatives
1
9-26 (Scheme 4). Structure assignments of these compounds were identified by NMR and
mass spectral data.
a
Scheme 4. Synthesis of derivatives 19-26
a
Reagents and reaction conditions were consistent with Scheme 1ꢀ3.
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Results and Discussion
For each rank of our compounds, their inhibition of HIV polymerase activity was
initially assessed using purified recombinant wildꢀtype HIV polymerase and a
poly(rA)/oligo(dT)16 template primer.
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In the first step, the impact of the Cꢀ4 substituent was investigated, including the ring
size and substituent on the cyclohexyl rings. As shown in Table 1, 7f-p, which contained
rings ranging from cyclopentyl to cycloheptyl, were evaluated against HIVꢀ1 RT. The
biological result showed that the substituent with a cyclohexyl ring was superior to both
that of cyclopentyl and cycloheptyl, especially 7j with the 2’ꢀmethylcyclohexyl substituent,
which is a mixture of stereoisomers, had inhibitory activity superior to the other
compounds. When more methyl moieties were introduced to the cyclohexyl ring, the
activity of the compounds disappeared, notably 7m-p. The biological results showed that
the cyclohexyl ring, with its optimized flexibility, could provide the better interaction with
the NNBP.
Structureꢀactivity relationships on position Cꢀ3 of the pyridineꢀ2(1H)ꢀone ring showed
that isopropyl or halo sustitutents could initiate the “trigger action” required for improved
activity against mutant viral strains. The higher potency of 9, 10 and 14a against HIVꢀ1 RT
suggested that the overall steric and lipophilic characteristics were clearly beneficial for
activity, as shown in Table 2.
Completing the modifications on positions Cꢀ3 and Cꢀ4 of the pyridinone ring, we
focused our attention on the modulation of the Cꢀ6 side chain with different length and
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aromatic rings. To verify our design, target compounds (14a-e, 19, 21, 22, 26, 26-cis and
26-trans) were initially tested for their inhibitory activity against HIVꢀ1 RT with
29
nevirapine and 2 as reference compounds . As shown in Table 3, most compounds
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displayed promising antiꢀRT activities.
Recently, the existence of a halogen bond with the Tyr188 carbonyl oxygen was
reported. This was regarded as one of the important interactions leading to overall
2
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proteinꢀligand binding affinity and was credited with improving antiꢀHIVꢀ1 activity
.
31
Upon inspection of the 22-trans/1RT2 complex (Figure 2), we found that the distance
between iodine atom with the carbonyl oxygen of Tyr188 are within the van der Waals
contact, which could result in halogen bond. Moreover, the large iodine atom may also
provide sufficient steric hindrance to modulate the conformation of cyclohexyl ring on the
Cꢀ4 position.
Figure 2. Superimposed stereoview of the docked (±)-22-trans with 1RT2. The hydrogen
bonds are shown as yellow dashed lines, and the halogen bonds are shown as purple
dashed lines.
In addition, the docking results revealed that the Cꢀ6 side chain could be twisted and
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located at the region similar to that of compound 2. Therefore, it was presumed that
changing the chain length could modulate its flexibility and provide better interactions
with Tyr318 and Pro236 (Figure 3), which would generate the compensation of entropic
penalty by these favorable interactions and result in increased antivirus activity.
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Figure 3. Superimposed stereoview of the docked (±)-26-trans and 2 (green) with 1RT2.
The hydrogen bonds are shown as yellow dashed lines.
These target compounds were synthesized as mixtures of diastereomers. From
comparison of the antiviral activity of these compounds, the 26-trans enantiomers were
prepared by using a stereoselective Mitsunobu reaction of cisꢀ2ꢀmethylcyclohexanol,
followed by hydrogenation. The diastereomers of 26-cis and 26-trans were synthesized by
the corresponding transꢀ and cisꢀ 2ꢀmethylcyclohexanol respectively, and confirmed by
1
HighꢀPerformance Liquid Chromatography (HPLC) (Figure 4) and Hꢀnuclear Overhauser
effect (NOE) (Figure 5). Because the 1’ꢀH of the trans isomers were close to the 2’ꢀmethyl,
a spatial correlation would be established, as irradiation of 1’ꢀH resulted in NOE in the
3
2’ꢀCH groups. The NOE spectra proved the conclusion.
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(±)ꢀ26-trans
(±)ꢀ26-cis
2
6 (mixture) + (±)ꢀ26-trans
26 (mixture) + (±)ꢀ26-cis
Figure 4. The HPLC results of 26 diastereomers.
(
±)ꢀ26-trans
(±)ꢀ26-cis
(±)ꢀ26-trans
(±)ꢀ26-cis
Figure 5. NOE spectra of (±)ꢀ26-trans and (±)ꢀ26-cis.
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It can be observed that 26-trans enantiomers with the 2’ꢀmethyl substituent in the
trans position had an antiviral potency (0.003 ꢀM) which is 1483ꢀ and 29ꢀfold more potent
than Nevirapine and 2, respectively (Table 3). Thus, the configuration of substituted
cyclohexyl would bring a significant impact on the antivirus activity, especially for the
mutant strains.
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Subsequently, the antiꢀHIVꢀ1 activity and cytotoxicity of target compounds, except
1
4a and 19, were determined using Nevirapine and 2 as reference compounds. As shown
in Table 4, all these compounds were capable of inhibiting wildꢀtype virus infection by
HIVꢀ1SF33 in TZMꢀbl and MT4 cells with EC values at 0.003 ꢀ 0.136ꢁM (Table 4).
50
In addition, 26-trans and 22, which had an excellent preliminary antiviral profile and
higher selectivity index (SISF33/MT4 75000 and 17647, repectively), were selected for
further profiling. Tests with a panel of NNRTIꢀresistant mutant viruses revealed that
2
6-trans and 22 strongly inhibit replication of the prevalent single mutations of K103N,
Y181C, and double mutant A17 with EC nanomolar values, which compared to that of 2
5
0
(Table 5). Therefore, these compounds could be further developed as nextꢀgeneration
NNRTIs with improved antiviral efficacy and drug resistance profile.
Moreover, docking studies displayed that the isopropyl or iodine group on Cꢀ3 located
in a hydrophobic pocket, which could provide a favorable interaction with the NNBP.
Meanwhile, they could regulate the conformation of the Cꢀ4 cyclohexyl, which is located
in a binding pocket lined by the aromatic residues Tyr188, Phe227 and Trp229 (Figure 3).
With the presence of 2’ꢀmethyl, the cyclohexyl ring could be close to Trp229, which
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would be conducive to forming πꢀσ interaction with the highly conserved amino residue
Trp229, instead of the readily mutated residue Tyr181. Especially, the 26-trans
enantiomers displayed the best profile against the mutant strains.
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Therefore, we docked 26-trans enantiomers into the clinically important K103N (PDB.
1
FKP) mutant RT (Figure 6), as well as the Y181C (PDB. 1JLA) (Figure 7) respectively,
to evaluate whether conformational flexibility and configuration would allow adaptation of
the inhibitor to the mutant binding site.
Figure 6. Docking results of (±)ꢀ26-trans into the nonꢀnucleoside bindingꢀpocket
1FKP Lys103Asn. Hydrogen bonds are shown as dashed lines.
Figure 7 Docking results of (±)ꢀ26-trans into the nonꢀnucleoside bindingꢀpocket
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JLA Tyr181Cys. Hydrogen bonds are shown as dashed lines.
The docking results revealed that some changes took place with 26-trans/mutated RT
complex. First, with RTꢀdirected mutagenesis (1FKP and 1JLA), the binding mode toward
NNBP produced a little change compared to the wildꢀtype (Figure 3), especially
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2
6-trans/1FKP complex (Figure 6). Second, for the 26-trans/1JLA complex (Figure 7), the
mainly hydrogen bond was preserved with Lys101, while the hydrogen bond with Lys103
was disappeared. However, binding with 1FKP (Figure 6), the hydrogen bonds formed
with not only Lys101, but also Asn103. Third, the cyclohexyl ring was buried deep in the
hydrophobic pocket composed of Trp229 and Tyr188 for both of 1FKP and 1JLA, which
increased πꢀσ interaction with the amino acid residues. Fourth, the Cꢀ6 side chain of
2
6-trans/1JLA complex could extend into a tunnel which would provide πꢀπ and πꢀσ
interactions with Tyr318 and Pro236. However, the substituent on Cꢀ6 of 26-trans/1FKP
complex closed to Val179.
Because the shape of NNBP changed as the K103N and Y181C mutated, some of the
original interaction forces disappeared. Therefore, the inhibitory activity of 26-trans and
2
2 against the mutated strains was less than the wild strain. However, the affinity involved
in these networks of van der Waals contacts stilled remained, which would be important
for stabilizing both wildꢀtype and mutant complexes. These results appear significant for
understanding the resistance profiles of the target compounds.
In addition, the docking results displayed similar binding modes of the enantiomers of
2
6-trans. Thus, we assume that the enantiomers may have the same contribution to the
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antiꢀHIVꢀ1 activity.
Conclusion
In summary, we have identified a new series of 3ꢀisopropylꢀ4ꢀcyclohexyloxy
pyridinꢀ2(1H)ꢀones and 3ꢀiodoꢀ4ꢀcyclohexyloxypyridinꢀ2(1H)ꢀones derivatives as
effective antiꢀHIV agents in comparison with nevirapine and 2. The biological results
confirmed our hypothesis that the introduction of an isopropyl or iodine group on Cꢀ3
displayed a “trigger” action on the conformation of the Cꢀ4 cyclohexyl and that the Cꢀ6
side chain with two carbon linker could extend into the tunnel forming the πꢀπ interaction
with the Tyr318 and Pro236 residues. Additionally, the configuration of 2’ꢀmethyl on the
cyclohexyl ring would significantly improve the potency against a panel of single and
double mutant strains often observed in patients treated with NNRTIs. In
particular, 26-trans and 22 were superior to 2 in that they displayed the best profile against
the wildꢀtype HIV, K103N, Y181C, and double mutant strains, making them potential lead
compounds with improved resistance profiles and low toxicity. Further investigation is
ongoing to characterize the pharmacokinetic profiles.
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Experimental Section
Chemistry The structural characterization was performed with an NMR spectrometer and
a high resolution mass spectrometer (HRMS). The purity of tested compounds was
determined by HPLC (Dionex Ultimate 3000 HPLC system). All the assayed compounds
displayed a purity of ≥95% (Table S1, Supporting Information). Melting points were
determined on a WBSꢀ1B type digital meltingꢀpoint apparatus and are uncorrected. NMR
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spectra were recorded on a Bruker Avance 300 or Avance 500 with tetramethylsilane (TMS)
as an internal standard, and chemical shifts are reported in δ (ppm). All the reactions were
routinely monitored by TLC, which was performed on aluminumꢀbacked silica gel plates
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(
60 F254) with spots visualized by UV light. Silica gel (0.040 ꢀ 0.064 mm) was used for
column chromatography and analytical silica gel for TLC plates. Unless otherwise stated,
all reagents were purchased from commercial sources. When necessary, they were purified
and dried by standard methods. Organic solutions were dried over anhydrous sodium
sulfate. The cisꢀ and transꢀ 2ꢀmethyl cyclohexanols were used as materials for synthesizing
the compounds 26-trans and 26-cis, respectively. Other target compounds were prepared
with the mixture of 2ꢀmethylcyclohexanol.
Ethyl 6-aryl-4-alkyl-2-oxo-1,2-dihydropyridine-3-carboxylate (7f-p).
General Procedure:
Aryl acetyl chloride (37.81 mmol) was added dropwise to a solution of Meldrum’s
acid (5.45 g, 37.81 mmol) and pyridine (6.2 mL) in CH Cl (70 mL) at 0 °C. The solution
2
2
was stirred for 30 min at 0 °C and then allowed to warm slowly to ambient temperature for
h. The reaction mixture was washed with 10% aqueous HCl (2 × 50 mL) and H O (50
8
2
mL) separately. The organic layer was dried with MgSO , filtered and concentrated under
4
reduced pressure. The crude residue was dissolved in EtOH (100 mL) and heated to reflux
for 4 h. The mixture was allowed to cool to room temperature and then concentrated under
reduced pressure. The dark oily residue was purified by column to give required
compounds 3a-e.
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A mixture of 3a-e (2.04 mmol) and 2 M NH
3
in MeOH (100 mL) was heated to reflux
for 8 h in a sealed tube. After cooling, the mixture was concentrated to dryness to provide
3
ꢀaminoꢀ4ꢀarylbutꢀ2ꢀenoic acid ethyl ester which was used without further purification.
To a solution of 1.2 g (48.0 mmol) of sodium in 60 mL of ethanol was added a
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solution of 7.4 mL (48.0 mmol) of diethyl malonate in 10 mL of ethanol. The resulting
yellow mixture was stirred for 1 h, and another solution of 5 g (24.0 mmol) of
3
ꢀaminoꢀ4ꢀarylbutꢀ2ꢀenoic acid ethyl ester in 24 mL of ethanol was added. The mixture
was refluxed for 4 d, and a white precipitate was formed. After removing the solvent, the
remainder was acidified with conc. HCl. The precipitate was filtered, washed with water
and dried to yield 4a-e as a white, crystalline solid.
Amounts of 4a-e (5.05 mmol), 0.77 g (2.78 mmol) of silver carbonate and 0.66 mL
(
5.56 mmol) of benzyl bromide were placed in a 20 mL THF and heated to reflux for 8 h.
After cooling to room temperature, the mixture was filtered through a filter agent pad
Celite 521, Aldrich), and the solvent was evaporated. The crude product was purified by
(
column chromatography to give 5a-e as a white solid.
Diisopropyl azodicarboxylate (DIAD) (0.26 g, 1.27 mmol) was added dropwise at
room temperature to a solution of 5a (0.23 g, 0.63 mmol), triphenylphosphine (PPh ) (0.33
3
g, 1.27 mmol) and the corresponding cyclohexanol (1.27 mmol) in 20mL of THF. After
stirring overnight, the solvent was evaporated, and the crude product was purified by
column chromatography to give 6f-p as a liquid.
To a pressure reaction bottle were added 6f-p (0.23 mmol) and 0.02 g of Pd/C 10% in
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0 mL of methanol. The reaction was under three air/hydrogen exchange cycles and stirred
at 15 psi for 8 h. After evaporating the solvent, the residue was purified by column
chromatography to give 7 f-p as a white solid.
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Ethyl 3ꢁoxoꢁ4ꢁphenylbutyrate (3a). pale yellow liquid, yield 57%; H NMR (300
MHz, CDCl
3
): δ 7.20ꢀ7.37 (m, 5H, PhꢀH), 4.16 (q, 2H, J = 7.2 Hz, OCH
2
CH
3
), 3.83 (s, 2H,
+
CH ꢀPh), 3.51 (s, 2H, CH
2
2
), 1.26 (t, 3H, J = 7.2 Hz, OCH CH
2
3
); m/z (EI): 206 [M + H] .
33
1
Ethyl 4ꢁ(4ꢁfluorophenyl)ꢁ3ꢁoxobutanoate (3b). pale yellow liquid, yield 78%; H
NMR (300 MHz, CDCl ): δ 6.98ꢀ7.25 (m, 4H, PhꢀH), 4.18 (q, 2H, J = 7.2 Hz, OCH CH ),
.81 (s, 2H, CH Ar), 3.46 (s, 2H, CH ), 1.26 (t, 3H, J = 7.2 Hz, OCH CH ).
3
2
3
3
2
2
2
3
32
1
Ethyl 4ꢁ(4ꢁchlorophenyl)ꢁ3ꢁoxobutanoate (3c). pale yellow liquid, yield 77%; H
NMR (300 MHz, CDCl ): δ 7.13ꢀ7.30 (m, 4H, PhꢀH), 4.17 (q, 2H, J = 7.2 Hz, OCH CH ),
.80 (s, 2H, CH Ar), 3.45 (s, 2H, CH ), 1.26 (t, 3H, J = 7.2 Hz, OCH CH ).
3
2
3
3
2
2
2
3
3
2
1
Ethyl 4ꢁ(4ꢁmethoxylphenyl)ꢁ3ꢁoxobutanoate (3d). pale yellow liquid, yield 84%; H
NMR (400 MHz, CDCl ): δ 6.86ꢀ7.27 (m, 4H, PhꢀH), 4.16 (q, 2H, J = 7.2 Hz, OCH CH ),
3
2
3
3
.79 (s, 3H, CH
3
OꢀPh), 3.75 (s, 2H, CH
2
Ar), 3.43 (s, 2H, CH
2
), 1.25 (t, 3H, J = 7.2 Hz,
OCH CH ).
2
3
3
4
1
Ethyl 4ꢁ(4ꢁmethylphenyl)ꢁ3ꢁoxobutanoate (3e). pale yellow liquid, yield 89%; H
NMR (400 MHz, CDCl
3
): δ 7.10ꢀ7.14 (m, 4H, PhꢀH), 4.62 (s, 1H, CH
2
), 4.10 (q, 2H, J =
7
.2 Hz, OCH CH ), 3.41 (s, 3H, CH ), 2.33 (s, 3H, CH ꢀPh), 1.25 (t, 3H, J = 7.2 Hz,
2 3 2 3
2 3
OCH CH ).
3
5
1
Ethyl 3ꢁoxoꢁ5ꢁphenylpentanoate (15) pale yellow liquid, yield 95%; H NMR (400
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MHz, CDCl
3
2 3
): δ 7.17ꢀ7.29 (m, 5H, PhꢀH), 4.16 (q, 2H, J = 7.2 Hz, OCH CH ), 3.41 (s, 2H,
CH ), 2.85ꢀ2.92 (m, 4H, CH CH Ph), 1.25 (t, 3H, J = 7.2 Hz, OCH CH ).
2
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3
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Ethyl 6ꢁbenzylꢁ4ꢁhydroxyꢁ2ꢁoxoꢁ1,2ꢁdihydropyridineꢁ3ꢁcarboxylate (4a). white solid,
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yield 46%; m.p. 208ꢀ209 °C; H NMR (400 MHz, DMSOꢀd ): δ 12.43 (s, 1H, OH), 11.57
6
(
br s, 1H, NH), 7.24ꢀ7.36 (m, 5H, PhꢀH), 5.72 (s, 1H, C
OCH CH ), 3.77 (s, 2H, CH Ar), 1.25 (t, 3H, J = 7.2 Hz, OCH
DMSOꢀd ): δ 172.22 (COOEt), 170.12 (Cꢀ4), 161.14 (Cꢀ2), 154.35 (Cꢀ6), 137.21, 129.39,
5
ꢀH), 4.25 (q, 2H, J = 7.2 Hz,
13
2
3
2
2 3
CH ); C NMR (101 MHz,
6
1
2 3 2
29.05, 127.37 (aryl), 99.12 (Cꢀ3), 97.84 (Cꢀ5), 61.06 (OCH CH ), 38.37 (CH Ar), 14.56
+
(
OCH CH ); m/z (EI): 274.2 [M + H] .
2
3
Ethyl 6ꢁ(4ꢁfluorobenzyl)ꢁ4ꢁhydroxyꢁ2ꢁoxoꢁ1,2ꢁdihydropyridineꢁ3ꢁcarboxylate (4b).
1
white solid, yield 42%; m.p. 221ꢀ222 °C; H NMR (400 MHz, DMSOꢀd
6
): δ 12.43 (s, 1H,
OH), 11.57 (br s, 1H, NH), 7.15ꢀ7.40 (m, 4H, aryl), 5.72 (s, 1H, C ꢀH), 4.24 (q, 2H, J =
5
7
2 3 2 2 3
.2 Hz, OCH CH ), 3.76 (s, 2H, CH Ar), 1.25 (t, 3H, J = 7.2 Hz, OCH CH ); m/z (EI):
+
2
92.4[M + H] .
Ethyl 6ꢁ(4ꢁchlorobenzyl)ꢁ4ꢁhydroxyꢁ2ꢁoxoꢁ1,2ꢁdihydropyridineꢁ3ꢁcarboxylate (4c).
1
white solid, yield 54%; m.p. 227ꢀ228 °C; H NMR (400 MHz, DMSOꢀd
6
): δ 12.40 (s, 1H,
OH), 11.57 (br s, 1H, NH), 7.35 (m, 4H, aryl), 5.71 (s, 1H, C ꢀH), 4.21 (q, 2H, J = 7.2 Hz,
5
13
OCH
2 3 2 2 3
CH ), 3.75 (s, 2H, CH Ar), 1.22 (t, 3H, J = 7.2 Hz, OCH CH ); C NMR (101 MHz,
DMSOꢀd ): δ 172.02 (COOEt), 169.96 (Cꢀ4), 161.14 (Cꢀ2), 153.75 (Cꢀ6), 136.23, 132.14,
6
1
2 3 2
31.28, 128.99 (aryl), 99.32 (Cꢀ3), 97.89 (Cꢀ5), 61.07 (OCH CH ), 37.57 (CH ꢀPh), 14.56
+
(OCH
2
CH
3
); m/z (EI): 307.9[M + H] .
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Ethyl 6ꢁ(4ꢁmethoxybenzyl)ꢁ4ꢁhydroxyꢁ2ꢁoxoꢁ1,2ꢁdihydropyridineꢁ3ꢁcarboxylate (4d).
1
white solid, yield 46%; m.p. 209ꢀ210 °C; H NMR (400 MHz, CDCl ): δ 13.32 (s, 1H,
3
5
OH), 12.51 (br s, 1H, NH), 6.83ꢀ7.30 (m, 4H, aryl), 5.78 (s, 1H, C ꢀH), 4.40 (q, 2H, J =
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7
.2 Hz, OCH CH ), 3.81 (s, 2H, CH Ar), 3.78 (s, 3H, OCH ꢀPh), 1.37 (t, 3H, J = 7.2 Hz,
2
3
2
3
13
OCH
2
CH
3
); C NMR (101 MHz, CDCl
3
): δ 176.11 (COOEt), 172.00 (Cꢀ4), 163.50 (Cꢀ2),
1
58.95 (Cꢀ6), 155.18, 130.43, 127.47, 114.25 (aryl), 99.28 (Cꢀ3), 96.84 (Cꢀ5), 61.60
+
(OCH CH ), 55.26 (OCH ), 38.46 (CH Ar), 14.24 (OCH CH ); m/z (EI): 304.2[M + H] .
2 3 3 2 2 3
Ethyl 6ꢁ(4ꢁmethylbenzyl)ꢁ4ꢁhydroxyꢁ2ꢁoxoꢁ1,2ꢁdihydropyridineꢁ3ꢁcarboxylate (4e).
1
white solid, yield 72%; m.p. 205ꢀ206 °C; H NMR (400 MHz, CDCl ): δ 13.32 (s, 1H,
3
OH), 12.33 (br s, 1H, NH), 7.11ꢀ7.26 (m, 4H, aryl), 5.79 (s, 1H, C
.2 Hz, OCH CH ), 3.83 (s, 2H, CH Ar), 2.32 (s, 3H, CH ꢀPh), 1.37 (t, 3H, J = 7.2 Hz,
OCH CH ); C NMR (101 MHz, CDCl ): δ 176.12 (COOEt), 172.01 (Cꢀ4), 163.37 (Cꢀ2),
5
ꢀH), 4.40 (q, 2H, J =
7
2
3
2
3
13
2
3
3
1
54.89 (Cꢀ6), 137.16, 132.32, 129.56, 129.23 (aryl), 99.38 (Cꢀ3), 96.87 (Cꢀ5), 61.60
+
(OCH CH ), 38.91 (CH ꢀPh), 21.05 (CH ), 14.24 (OCH CH ); m/z (EI): 287.7[M + H] .
2
3
2
3
2
3
Ethyl 4ꢁhydroxyꢁ2ꢁoxoꢁ6ꢁphenethylꢁ1,2ꢁdihydropyridineꢁ3ꢁcarboxylate (16). white
1
solid, yield 33%; m.p. 192ꢀ193 °C; H NMR (400 MHz, DMSOꢀd
6
): δ 12.58 (s, 1H, OH),
1
1.51 (br s, 1H, NH), 7.18ꢀ7.31 (m, 5H, aryl), 5.81 (s, 1H, C ꢀH), 4.26 (q, 2H, J = 7.2 Hz,
5
OCH
2
CH
3
), 2.86ꢀ2.90 (m, 2H, CH
2
CH
2
2 2
ꢀPh), 2.69ꢀ2.73 (m, 2H, CH CH ꢀPh), 1.26 (t, 3H, J
13
=
7.2 Hz, OCH CH ); C NMR (101 MHz, DMSOꢀd ): δ 172.82 (COOEt), 170.57 (Cꢀ4),
2 3 6
1
61.14 (Cꢀ2), 154.97 (Cꢀ6), 140.68, 128.78, 126.61 (aryl), 98.53 (Cꢀ3), 97.52 (Cꢀ5), 61.09
(OCH
2
CH
3
2 2 2 2 2 3
), 34.54 (CH CH ꢀPh), 34.09 (CH CH ꢀPh), 14.58 (OCH CH ); m/z (EI):
+
2
88.5[M + H] .
2
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Ethyl 6ꢁbenzylꢁ2ꢁ(benzyloxy)ꢁ4ꢁhydroxynicotinate (5a). white solid, yield 80%; m.p.
1
67ꢀ68 °C; H NMR (400 MHz, CDCl ): δ 12.25 (s, 1H, OH), 7.20ꢀ7.47 (m, 10H, aryl),
3
6
.35 (s, 1H, C
5 2 2 3
ꢀH), 5.44 (s, 2H, OCH Ph), 4.36 (q, 2H, J = 7.2 Hz, OCH CH ), 3.93 (s,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
2
H, CH Ph), 1.33 (t, 3H, J = 7.2 Hz, OCH CH ); C NMR (101 MHz, CDCl ): δ 171.56
2
2
3
3
(
COOEt), 170.99 (Cꢀ4), 163.63 (Cꢀ2), 162.51 (Cꢀ6), 138.46, 137.41, 129.30, 128.50,
1
2
28.20, 127.62, 127.54, 126.52 (aryl), 106.23 (Cꢀ3), 95.35 (Cꢀ5), 68.20 (OCH Ph), 61.74
+
(
(OCH CH ), 44.55 (CH Ph), 14.06 (OCH CH ); m/z (EI): 364.2 [M + H] .
2
3
2
2
3
Ethyl 6ꢁ(4ꢁfluorobenzyl)ꢁ2ꢁ(benzyloxy)ꢁ4ꢁhydroxynicotinate (5b). white solid, yield
1
7
8%; m.p. 60ꢀ61 °C; H NMR (400 MHz, CDCl ): δ 12.29 (s, 1H, OH), 6.94ꢀ7.47 (m, 9H,
3
Ar), 6.33 (s, 1H, C
H, CH Ar), 1.34 (t, 3H, J = 7.2 Hz, OCH
COOEt), 170.97 (Cꢀ4), 163.31 (Cꢀ2), 162.55 (Cꢀ6), 160.49, 137.35, 134.15, 130.75,
30.67, 128.23, 127.58, 127.54, 115.36, 115.15 (aryl), 106.15 (Cꢀ3), 95.41 (Cꢀ5), 68.18
OCH Ph), 61.81 (OCH CH ), 43.63 (CH Ar), 14.07 (OCH CH ); m/z (EI): 382.9 [M +
5
ꢀH), 5.43 (s, 2H, OCH
2
Ph), 4.36 (q, 2H, J = 7.2 Hz, OCH
2
CH
3
), 3.88 (s,
1
3
2
2
2
CH
3
); C NMR (101 MHz, CDCl ): δ 171.60
3
(
1
(
2
2
3
2
2
3
+
H] .
Ethyl 6ꢁ(4ꢁchlorobenzyl)ꢁ2ꢁ(benzyloxy)ꢁ4ꢁhydroxynicotinate (5c). white solid, yield
1
7
4%; m.p. 81ꢀ82 °C; H NMR (400 MHz, CDCl ): δ 12.30 (s, 1H, OH), 7.16ꢀ7.45 (m, 9H,
3
aryl), 6.33 (s, 1H, C
5
2 2 3
ꢀH), 5.41 (s, 2H, OCH Ph), 4.36 (q, 2H, J = 7.2 Hz, OCH CH ), 3.87
13
(
s, 2H, CH Ar), 1.34 (t, 3H, J = 7.2 Hz, OCH CH ); C NMR (101 MHz, CDCl ): δ
2
2
3
3
1
1
71.63 (COOEt), 170.95 (Cꢀ4), 162.89 (Cꢀ2), 162.58 (Cꢀ6), 160.49, 137.34, 136.98,
30.65, 130.62, 128.63, 128.59, 128.54, 128.43, 128.24, 127.56 (aryl), 106.23 (Cꢀ3), 95.49
(
Cꢀ5), 68.60 (OCH Ph), 61.82 (OCH CH ), 43.87 (CH Ar), 14.08 (OCH CH ); m/z (EI):
2 2 3 2 2 3
2
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+
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98.9[M + H] .
Ethyl 6ꢁ(4ꢁmethoxybenzyl)ꢁ2ꢁ(benzyloxy)ꢁ4ꢁhydroxynicotinate (5d). white solid, yield
1
68%; m.p. 54ꢀ55 °C; H NMR (400 MHz, CDCl
3
): δ 12.24 (s, 1H, OH), 76.81ꢀ7.48 (m,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9
H, aryl), 6.32 (s, 1H, C ꢀH), 5.44 (s, 2H, OCH Ph), 4.35 (q, 2H, J = 7.2 Hz, OCH CH ),
5
2
2
3
13
3
.86 (s, 2H, CH
2
3 2 3
Ar), 3.77(s, 3H, CH OAr), 1.33 (t, 3H, J = 7.2 Hz, OCH CH ); C NMR
(101 MHz, CDCl
3
): δ 171.56 (COOEt), 171.00 (Cꢀ4), 164.11 (Cꢀ2), 162.53 (Cꢀ6), 158.37,
1
6
37.48, 130.58, 130.25, 128.20, 127.60, 127.34, 114.01 (aryl), 106.05 (Cꢀ3), 95.29 (Cꢀ5),
8.16 (OCH
2
2 3 3 2 2 3
Ph), 61.89 (OCH CH ), 55.25 (CH OAr), 43.68 (CH Ar), 14.06 (OCH CH );
+
m/z (EI): 394.5 [M + H] .
Ethyl 6ꢁ(4ꢁmethylbenzyl)ꢁ2ꢁ(benzyloxy)ꢁ4ꢁhydroxynicotinate (5e). white solid, yield
1
8
8%; m.p. 54ꢀ55 °C; H NMR (400 MHz, CDCl
3
): δ 12.23 (s, 1H, OH), 7.02ꢀ7.48 (m, 9H,
aryl), 6.34 (s, 1H, C ꢀH), 5.44 (s, 2H, OCH Ph), 4.36 (q, 2H, J = 7.2 Hz, OCH CH ), 3.89
5
2
2
3
13
(s, 2H, CH
2
Ar), 2.32 (s, 3H, CH
3
2 3
Ar), 1.33 (t, 3H, J = 7.2 Hz, OCH CH ); C NMR (101
MHz, CDCl ): δ 171.55 (COOEt), 170.99 (Cꢀ4), 163.95 (Cꢀ2), 162.51 (Cꢀ6), 137.46,
3
1
3
36.03, 135.40 (CH Ph), 129.18, 128.18, 127.62, 127.52 (aryl), 106.13 (Cꢀ3), 95.30 (Cꢀ5),
6
8.18 (OCH
2
2 3 2 3 2 3
Ph), 61.68 (OCH CH ), 44.15 (CH Ar), 21.03 (CH Ar), 14.05 (OCH CH );
+
m/z (EI): 377.7[M + H] .
Ethyl 2ꢁ(benzyloxy)ꢁ4ꢁhydroxyꢁ6ꢁphenethylnicotinate (17). white solid, yield 82%; m.p.
1
5
2ꢀ53 °C; H NMR (400 MHz, CDCl ): δ 12.27 (s, 1H, OH), 7.15ꢀ7.52 (m, 10H, aryl),
3
6
.35 (s, 1H, C
5
ꢀH), 5.44 (s, 2H, OCH
2
2 3
Ph), 4.37 (q, 2H, J = 7.2 Hz, OCH CH ), 3.02 (t, 2H,
J = 6.8 Hz, CH
2
CH ꢀPh), 2.91 (t, 2H, J=6.8 Hz, CH
2
2
CH ꢀPh), 1.35 (t, 3H, J = 7.2 Hz,
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5
5
5
5
5
5
6
1
3
OCH
2 3 3
CH ); C NMR (101 MHz, CDCl ): δ 171.38 (COOEt), 171.09 (Cꢀ4), 164.03 (Cꢀ2),
162.50 (Cꢀ6), 141.44, 137.55, 128.45, 128.40, 128.26, 127.57, 127.50, 126.00 (aryl),
1
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2
ꢀPh), 61.78 (OCH
2
CH
3
2 2
), 39.71 (CH CH ꢀPh),
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0
1
2
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6
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8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
3
4.56 (CH CH ꢀPh), 14.06 (OCH CH ); m/z (EI): 378.2 [M + H] .
2
2
2
3
Ethyl 6ꢁbenzylꢁ2ꢁ(benzyloxy)ꢁ4ꢁ(cyclopentyloxy)nicotinate (6f). pale yellow liquid,
1
yield 71%; H NMR (400 MHz, CDCl
3
): δ 7.18ꢀ7.38 (m, 10H, aryl), 6.30 (s, 1H, C
5
ꢀH),
1
’
5
2
.42 (s, 2H, OCH Ph), 4.70ꢀ4.72 (m, 1H, H ),4.32 (q, 2H, J = 7.2 Hz, OCH CH ), 3.96 (s,
2 2 3
13
H, CH
2
Ph), 1.54ꢀ1.80 (m, 8H, cyclopentyl), 1.29 (t, 3H, J = 7.2 Hz, OCH
2
CH
3
);
C
NMR (101 MHz, CDCl ): δ 165.54 (COOEt), 163.93 (Cꢀ4), 160.86 (Cꢀ2), 160.68 (Cꢀ6),
3
1
1
39.14, 137.73, 129.10, 128.43, 128.21, 127.59, 127.39, 126.37 (aryl), 104.40 (Cꢀ3),
1
’
02.46 (Cꢀ5), 80.59 (C ), 67.52 (OCH
2
Ph), 61.08 (OCH
2
3
CH ), 44.67, 32.75, 23.84
+
(
Ccyclopentyl, CH Ph), 14.25 (OCH CH ); m/z (EI): 432.3 [M + H] .
2
2
3
Ethyl 6ꢁbenzylꢁ2ꢁ(benzyloxy)ꢁ4ꢁ(cyclohexyloxy)nicotinate (6g). pale yellow liquid,
1
yield 77%; H NMR (400 MHz, CDCl ): δ 7.20ꢀ7.38 (m, 10H, aryl), 6.30 (s, 1H, C ꢀH),
3
5
1
’
5
.42 (s, 2H, OCH
2
Ph), 4.28ꢀ4.36 (m, 3H, H , OCH
2
CH
); C NMR (101 MHz, CDCl
63.76 (Cꢀ4), 160.93 (Cꢀ2), 160.63 (Cꢀ6), 139.17, 137.73, 129.08, 128.42, 128.21, 127.61,
3
), 3.95 (s, 2H, CH
2
Ph, 1.29ꢀ1.82
13
(
m, 13H, Cyclohexyl, OCH
2
CH
3
3
): δ 165.62 (COOEt),
1
1
1
’
27.39, 126.35 (aryl), 106.68 (Cꢀ3), 102.26 (Cꢀ5), 75.71 (C ), 67.52 (OCH
2
Ph), 61.13
(
OCH CH ), 44.63, 31.17, 25.44, 22.91 (C
, CH Ph), 14.24 (OCH CH ); m/z (EI):
2
3
cyclohexyl
2
2
3
+
4
46.9 [M + H] .
Ethyl 6ꢁbenzylꢁ2ꢁ(benzyloxy)ꢁ4ꢁ(cycloheptyloxy)nicotinate (6h). pale yellow liquid,
2
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yield 75%; H NMR (400 MHz, CDCl
3 5
): δ 7.20ꢀ7.38 (m, 10H, aryl), 6.24 (s, 1H, C ꢀH),
1
’
5.42 (s, 2H, OCH Ph), 4.37ꢀ4.43 (m, 1H, H ), 4.33 (q, 2H, J = 7.2 Hz, OCH CH ), 3.95 (s,
2
2
3
1
3
2
H, CH
2
Ph), 1.39ꢀ1.88 (m, 12H, cycloheptyl), 1.30 (t, 3H, J = 7.2 Hz, OCH
2
CH
3
);
C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
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33
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36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NMR (101 MHz, CDCl ): δ 165.63 (COOEt), 163.79 (Cꢀ4), 160.93 (Cꢀ2), 160.70 (Cꢀ6),
3
1
1
39.14, 137.74, 129.12, 128.43, 128.21, 127.59, 127.38, 126.37 (aryl), 104.61 (Cꢀ3),
1
’
02.32 (Cꢀ5), 78.65 (C ), 67.50 (OCH
2
Ph), 61.11 (OCH
2
CH
3
), 44.66, 39.40, 28.26, 22.67
+
(
Ccycloheptyl, CH Ph), 14.28 (OCH CH ); m/z (EI): 459.8 [M + H] .
2
2
3
Ethyl 6ꢁbenzylꢁ2ꢁ(benzyloxy)ꢁ4ꢁ(2ꢁchlorocyclohexyloxy)nicotinate (6i). pale yellow
1
liquid, yield 81%; H NMR (400 MHz, CDCl ): δ 7.20ꢀ7.38 (m, 10H, aryl), 6.30 (s, 1H,
3
1
’
C
5
ꢀH), 5.42 (s, 2H, OCH
.95 (s, 2H, CH Ph), 1.25ꢀ1.31 (m, 9H, cyclohexyl); C NMR (101 MHz, CDCl
65.57 (COOEt), 163.90 (Cꢀ4), 160.92 (Cꢀ2), 160.78 (Cꢀ6), 139.14, 137.71, 129.09,
2
Ph), 4.47ꢀ4.53 (m, 1H, H ), 4.33 (q, 2H, J = 7.2 Hz, OCH
2
CH
3
),
13
3
1
1
6
1
2
3
): δ
1
’
28.45, 128.22, 127.60, 127.41, 126.39 (aryl), 104.62 (Cꢀ3), 102.16 (Cꢀ5), 71.29 (C ),
7.53 (OCH Ph), 61.10 (OCH CH ), 44.68, 33.71, 28.83, 23.23, 21.86 (C
, CH Ph),
cyclohexyl 2
2
2
3
+
4.24 (OCH
2
CH
3
); m/z (EI): 480.7 [M + H] .
Ethyl 6ꢁbenzylꢁ2ꢁ(benzyloxy)ꢁ4ꢁ(2ꢁmethylcyclohexyloxy)nicotinate (6j). pale yellow
1
liquid, yield 80%; H NMR (400 MHz, CDCl ): δ 7.20ꢀ7.38 (m, 10H, aryl), 6.29 (s, 1H,
3
C
5
ꢀH), 5.41 (s, 2H, OCH
2
Ph), 4.33 (q, 2H, J = 7.2 Hz, OCH
2
CH
3
2
), 3.95 (s, 2H, CH Ph),
1
’
13
3
.72ꢀ3.78 (m, 1H, H ), 0.90ꢀ2.02 (m, 15H, OCH CH , 2ꢀmethylCyclohexyl); C NMR
2
3
3
(101 MHz, CDCl ): δ 165.64 (COOEt), 164.39 (Cꢀ4), 160.96 (Cꢀ2), 160.58 (Cꢀ6), 139.17,
1
8
37.74, 129.11, 128.41, 128.22, 127.64, 127.40, 126.36 (aryl), 104.49 (Cꢀ3), 102.26 (Cꢀ5),
1’
2.84 (C ), 67.52 (OCH Ph), 61.10 (OCH CH ), 44.62, 37.68, 33.20, 30.79, 25.01, 24.35,
2
2
3
2
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5
5
5
5
6
+
1
2 2 3
8.44 (2ꢀmethylcyclohexyl, CH Ph), 14.27 (OCH CH ); m/z (EI): 460.8 [M + H] .
Ethyl 6ꢁbenzylꢁ2ꢁ(benzyloxy)ꢁ4ꢁ(3ꢁmethylcyclohexyloxy)nicotinate (6k). pale yellow
1
liquid, yield 97%; H NMR (400 MHz, CDCl
3
): δ 7.20ꢀ7.39 (m, 10H, PhꢀH), 6.31 (s, 1H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
’
C ꢀH), 5.41 (s, 2H, OCH Ph), 4.32 (q, 2H, J = 7.2 Hz, OCH CH ), 4.11ꢀ4.16 (m, 1H, H ),
5
2
2
3
13
3
.95 (s, 2H, CH
2
2 3
Ph), 0.82ꢀ2.01 (m, 15H, OCH CH , 3ꢀmethylCyclohexyl); C NMR (101
MHz, CDCl
3
): δ 165.58 (COOEt), 163.92 (Cꢀ4), 160.93 (Cꢀ2), 160.63 (Cꢀ6), 139.21,
1
7
2
37.73, 129.11, 128.43, 128.23, 127.62, 127.41, 126.38 (aryl), 104.69 (Cꢀ3), 102.40 (Cꢀ5),
1
’
3.38 (C ), 67.52 (OCH
2
Ph), 61.10 (OCH
2
CH
3
), 44.61, 40.45, 38.7, 33.98, 31.30, 23.81,
+
2.28 (3ꢀmethylcyclohexyl, CH Ph), 14.29 (OCH CH ); m/z (EI): 459.7 [M + H] .
2
2
3
Ethyl 6ꢁbenzylꢁ2ꢁ(benzyloxy)ꢁ4ꢁ(4ꢁmethylcyclohexyloxy)nicotinate (6l). pale yellow
1
liquid, yield 94%; H NMR (400 MHz, CDCl
3
): δ 7.19ꢀ7.37 (m, 10H, aryl), 6.31 (s, 1H,
1
’
C ꢀH), 5.41 (s, 2H, OCH Ph), 4.32 (q, 2H, J = 7.2 Hz, OCH CH ), 4.09ꢀ4.14 (m, 1H, H ),
5
2
2
3
13
3
.95 (s, 2H, CH
2
2 3
Ph), 0.88ꢀ2.01 (m, 15H, OCH CH , 4ꢀmethylCyclohexyl); C NMR (101
MHz, CDCl ): δ 165.58 (COOEt), 163.97 (Cꢀ4), 160.94 (Cꢀ2), 160.62 (Cꢀ6), 139.18,
3
1
7
37.72, 129.08, 128.43, 128.22, 127.61, 127.40, 126.37 (aryl), 104.69 (Cꢀ3), 102.34 (Cꢀ5),
1
’
3.68 (C ), 67.53 (OCH
2
Ph), 61.10 (OCH
2
CH
3
), 44.62, 37.75, 31.53, 28.99, 21.75
+
(4ꢀmethylcyclohexyl, CH Ph), 14.28 (OCH CH ); m/z (EI): 459.5 [M + H] .
2
2
3
Ethyl 6ꢁbenzylꢁ2ꢁ(benzyloxy)ꢁ4ꢁ(2,6ꢁdimethylcyclohexyloxy)nicotinate (6m). pale
1
yellow liquid, yield 54%; H NMR (400 MHz, CDCl ): δ 7.21ꢀ7.40 (m, 10H, PhꢀH), 6.36
3
1
’
(s, 1H, C
5
ꢀH), 5.41 (s, 2H, OCH
2
Ph), 4.30ꢀ4.36 (m, 3H, OCH
2 3
CH , H ), 3.95 (s, 2H,
13
CH Ph), 0.80ꢀ1.72 (m, 17H, OCH
2
2
CH , 2, 6ꢀdimethylcyclohexyl); C NMR (101 MHz,
3
2
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CDCl
3
): δ 166.38 (COOEt), 165.75 (Cꢀ4), 160.88 (Cꢀ2), 160.55 (Cꢀ6), 139.13, 137.74,
129.16, 128.42, 128.20, 127.63, 127.38, 126.35 (aryl), 104.20 (Cꢀ3), 101.93 (Cꢀ5), 82.06
1
’
(
C ), 67.54 (OCH
2
Ph), 61.14 (OCH
2
CH
3
), 44.70, 37.39, 28.19, 25.74, 18.71 (2,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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36
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45
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48
49
50
51
52
53
54
55
56
57
58
59
60
+
6
ꢀdimethylcyclohexyl, CH Ph), 14.28 (OCH CH ); m/z (EI): 488.8 [M + H] .
2
2
3
Ethyl 6ꢁbenzylꢁ2ꢁ(benzyloxy)ꢁ4ꢁ(3,5ꢁdimethylcyclohexyloxy)nicotinate (6o). pale
1
yellow liquid, yield 57%; H NMR (400 MHz, CDCl
3
): δ 7.20ꢀ7.38 (m, 10H, aryl), 6.30 (s,
1
H, C ꢀH), 5.41 (s, 2H, OCH Ph), 4.32 (q, 2H, J = 7.2 Hz, OCH CH ), 4.12ꢀ4.21 (m, 1H,
5 2 2 3
1
’
2 2 3
H ), 3.96 (s, 2H, CH Ph), 1.29 (t, 3H, J = 7.2 Hz, OCH CH ), 0.52ꢀ2.00 (m, 14H, 3,
13
5
1
1
4
ꢀdimethylcyclohexyl); C NMR (101 MHz, CDCl ): δ 165.53 (COOEt), 163.92 (Cꢀ4),
3
60.93 (Cꢀ2), 160.58 (Cꢀ6), 139.18, 137.69, 129.09, 128.39, 128.19, 127.59, 127.37,
1
’
26.34 (aryl), 104.67 (Cꢀ3), 102.42 (Cꢀ5), 80.79 (C ), 67.53 (OCH
2 2 3
Ph), 61.08 (OCH CH ),
4.56, 42.97, 39.96, 30.54, 22.10 (3, 5ꢀdimethylcyclohexyl, CH Ph), 14.26 (OCH CH );
2
2
3
+
m/z (EI): 474.6 [M + H] .
Ethyl 6ꢁbenzylꢁ2ꢁ(benzyloxy)ꢁ4ꢁ(3,3,5ꢁtrimethylcyclohexyloxy)nicotinate (6p). pale
1
yellow liquid, yield: 27%; H NMR (400 MHz, CDCl
3
): δ 7.20ꢀ7.38 (m, 10H, aryl), 6.29
Ph), 4.61 (s, 1H, H ), 4.29 (q, 2H, J = 7.2 Hz, OCH CH ),
.95 (s, 2H, CH Ph), 0.82ꢀ1.91 (m, 19H, OCH CH , 3,3,5ꢀtrimethylcyclohexyl); C NMR
1
’
(s, 1H, C
5
ꢀH), 5.42 (s, 2H, OCH
2
2
3
13
3
2
2
3
(101 MHz, CDCl
3
): δ 165.73 (COOEt), 163.56 (Cꢀ4), 160.87 (Cꢀ2), 160.66 (Cꢀ6), 139.18,
1
7
2
1
37.75, 129.07, 128.40, 128.16, 127.55, 127.35, 126.34 (aryl), 104.44 (Cꢀ3), 104.51 (Cꢀ5),
1’
4.53 (C ), 67.45 (OCH
2
Ph), 61.22 (OCH
2
CH
3
), 48.15, 44.71, 41.40, 34.1, 30.61, 27.09,
CH ); m/z (EI): 487.8 [M +
3.01, 22.46 (3,3,5ꢀtrimethylcyclohexyl, CH
2
Ph), 14.14 (OCH
2
3
+
] .
2
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6
Ethyl 2ꢁ(benzyloxy)ꢁ4ꢁ(2ꢁmethylcyclohexyloxy)ꢁ6ꢁphenethylnicotinate (18). pale
1
yellow liquid, yield 76%; H NMR (400 MHz, CDCl ): δ 7.12ꢀ7.45 (m, 10H, aryl), 6.14 (s,
3
1
5 2 2 3
H, C ꢀH), 5.45 (s, 2H, OCH Ph), 4.34 (q, 2H, J = 7.2 Hz, OCH CH ), 3.69ꢀ3.70 (m, 1H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
’
H ), 2.91ꢀ3.00 (m, 4H, CH CH ꢀPh), 0.94ꢀ1.92 (m, 15H, OCH CH , 2ꢀmethylcyclohexyl);
2
2
2
3
1
3
3
C NMR (101 MHz, CDCl ): δ 165.70 (COOEt), 164.12 (Cꢀ4), 160.95 (Cꢀ2), 160.88
(
Cꢀ6), 141.51, 137.86, 128.55, 128.31, 128.22, 127.48, 127.38, 125.88 (aryl), 104.40 (Cꢀ3),
1
’
1
3
4
01.99 (Cꢀ5), 82.83 (C ), 67.48 (OCH Ph), 61.06 (OCH CH ), 39.99, 37.70, 35.13, 33.28,
2
2
3
0.75, 25.06, 24.44 18.44, (2ꢀmethylcyclohexyl, CH
2
CH
2
2 3
Ph), 14.26 (OCH CH ); m/z (EI):
+
74.5 [M + H] .
Ethyl 6ꢁbenzylꢁ4ꢁ(cyclopentyloxy)ꢁ2ꢁoxoꢁ1,2ꢁdihydropyridineꢁ3ꢁcarboxylate (7f):
1
white solid, yield 81%, m.p. 176ꢀ177 °C; H NMR (400 MHz, CDCl
3
): δ 7.22ꢀ7.32 (m, 5H,
1
’
aryl), 5.76 (s, 1H, C ꢀH), 4.71 (s, 1H, H ), 4.33 (q, 2H, J = 7.2 Hz, OCH CH ), 3.90 (s, 2H,
5
2
3
1
3
CH
2
ꢀPh), 1.57ꢀ1.78 (m, 8H, cyclohexyl ), 1.32 (t, 3H, J = 7.2 Hz, OCH
2 3
CH ); C NMR
(
101 MHz, CDCl ): δ 166.11 (COOEt), 16528 (Cꢀ4), 163.77 (Cꢀ2), 151.97 (Cꢀ6), 136.16,
3
1
’
1
29.34, 128.77, 127.17 (aryl), 106.76 (Cꢀ3), 95.62 (Cꢀ5), 81.21 (C ), 60.85 (OCH
2
3
CH ),
3
9.50, 32.95, 23.79 (cyclohexyl, CH ꢀPh), 14.29 (OCH CH ); HRMS (m/z): calcd for
2 2 3
+
C H NO [M + H] 342.16998, found 342.16983.
20
24
4
Ethyl 6ꢁbenzylꢁ4ꢁ(cyclohexyloxy)ꢁ2ꢁoxoꢁ1,2ꢁdihydropyridineꢁ3ꢁcarboxylate (7g). white
1
solid, yield 88%, m.p. 203ꢀ204 °C; H NMR (400 MHz, CDCl ): δ 7.24ꢀ7.32 (m, 5H, aryl),
3
1’
5
.75 (s, 1H, C
CH ꢀPh), 1.26ꢀ1.79 (m, 13H, OCH
65.90 (COOEt), 165.34 (Cꢀ4), 163.80 (Cꢀ2), 151.83 (Cꢀ6), 136.16, 129.31, 128.77,
5
ꢀH), 4.32ꢀ4.37 (q, 2H, OCH
2
CH
3
), 4.25ꢀ4.29 (m, 1H, H ), 3.89 (s, 2H,
13
2
2
CH
3 3
, cyclohexyl ); C NMR (101 MHz, CDCl ): δ
1
2
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27.18 (aryl), 107.17 (Cꢀ3), 95.38 (Cꢀ5), 76.29 (C ), 60.92 (OCH
25.30, 27.74 (cyclohexyl, CH ꢀPh), 14.31 (OCH CH ); HRMS (m/z): calcd for C H NO
4
2 3
CH ), 39.50, 31.34,
2
2
3
21 26
+
[
M + H] 356.18503, found 356.18555.
10
11
12
13
14
15
16
17
18
19
20
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27
28
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60
Ethyl 6ꢁbenzylꢁ4ꢁ(cycloheptyloxy)ꢁ2ꢁoxoꢁ1,2ꢁdihydropyridineꢁ3ꢁcarboxylate (7h).
1
white solid, yield 88%, m.p. 206ꢀ207 °C; H NMR (400 MHz, CDCl
3
): δ 7.23ꢀ7.34 (m, 5H,
, H ), 3.89 (s, 2H, CH ꢀPh),
.30ꢀ1.89 (m, 15H, OCH CH , cycloheptyl ); C NMR (101 MHz, CDCl ): δ 165.92
1
’
aryl), 5.69 (s, 1H, C
5
ꢀH), 4.31ꢀ4.39 (m, 3H, OCH
2
CH
3
2
13
1
2
3
3
(
COOEt), 165.37 (Cꢀ4), 163.75 (Cꢀ2), 151.88 (Cꢀ6), 136.07, 129.36, 128.80, 127.21 (aryl),
1
’
1
07.10 (Cꢀ3), 95.50 (Cꢀ5), 79.31 (C ), 60.90 (OCH CH ), 39.50, 33.63, 28.15, 22.55
2 3
+
(
cycloheptyl, CH
2
2 3 4
ꢀPh), 14.32 (OCH CH ); HRMS (m/z): calcd for C22H28NO [M + H]
3
70.20128, found 370.20125.
Ethyl
6ꢁbenzylꢁ4ꢁ(2ꢁchlorocyclohexyloxy)ꢁ2ꢁoxoꢁ1,2ꢁdihydropyridineꢁ3ꢁcarboxylate
1
(
7i). white solid, yield 75%, m.p. 185ꢀ186 °C; H NMR (400 MHz, CDCl
3
): δ 7.23ꢀ7.31
1
’
(
m, 5H, aryl), 5.76 (s, 1H, C ꢀH), 4.47ꢀ4.48 (m, 1H, H ), 4.38 (q, 2H, J = 7.2 Hz,
5
2
’
OCH
2
CH
3
), 4.11ꢀ4.13 (m, 1H, H ), 3.89 (s, 2H, CH
2
ꢀPh), 1.33ꢀ2.05 (m, 11H, OCH
): δ 165.48 (COOEt), 165.02 (Cꢀ4),
63.81 (Cꢀ2), 151.15 (Cꢀ6), 136.01, 129.35, 128.81, 127.23 (aryl), 107.82 (Cꢀ3), 95.30
2 3
CH ,
1
3
2
1
ꢀchlorocyclohexyl ); C NMR (101 MHz, CDCl
3
1
’
(
(
[
Cꢀ5), 61.13 (C ), 60.31 (OCH
2ꢀchlorocyclohexyl, CH ꢀPh), 14.34 (OCH CH ); HRMS (m/z): calcd for C H ClNO
4
2 3
CH ), 39.48, 31.51, 28.66, 23.33, 20.21
2
2
3
22 25
+
M + H] 390.14666, found 390.14648.
Ethyl6ꢁbenzylꢁ4ꢁ(2ꢁmethylcyclohexyloxy)ꢁ2ꢁoxoꢁ1,2ꢁdihydropyridineꢁ3ꢁcarboxylate
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