Highly functionalized tetrahydropyridines are cytotoxic and selective inhibitors of human puromycin sensitive aminopeptidase

Article abstract of DOI:10.1016/j.ejmech.2015.10.026

Efficient one-pot five-component synthetic protocols for highly functionalized tetrahydropyridines (THPs) and their biological evaluation have been illustrated. Synthesis of novel functionalized tetrahydropyridines containing differential substitutions at 2,6-positions has been achieved via a modified MCR. Cytotoxic studies of 23 synthesized compounds have been carried out against three different cell lines, namely A-549, HeLa and HepG2, wherein some compounds have displayed appreciable cytotoxicity. Further, investigation of enzyme inhibition by the synthesized THPs has been carried out against four members of M1 family aminopeptidases. Several compounds have selectively inhibited only one member of this enzyme family i.e., human puromycin sensitive aminopeptidase (hPSA). Among the compounds; 4b, 9b, 9e and 10a demonstrated best inhibition against hPSA.

Full text of DOI:10.1016/j.ejmech.2015.10.026

European Journal of Medicinal Chemistry 106 (2015) 26e33
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Highly functionalized tetrahydropyridines are cytotoxic and selective
inhibitors of human puromycin sensitive aminopeptidase
,
,
Raghunath Aeluri a 1, Roopa Jones Ganji b 1, Anil Kumar Marapaka b,
Vijaykumar Pillalamarri b, Manjula Alla a *, Anthony Addlagatta b
,
, **
a Crop Protection Chemicals Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, 500607, India
b Center for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, 500607, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Efficient one-pot five-component synthetic protocols for highly functionalized tetrahydropyridines
(THPs) and their biological evaluation have been illustrated. Synthesis of novel functionalized tetrahy-
dropyridines containing differential substitutions at 2,6-positions has been achieved via a modified MCR.
Cytotoxic studies of 23 synthesized compounds have been carried out against three different cell lines,
namely A-549, HeLa and HepG2, wherein some compounds have displayed appreciable cytotoxicity.
Further, investigation of enzyme inhibition by the synthesized THPs has been carried out against four
members of M1 family aminopeptidases. Several compounds have selectively inhibited only one member
of this enzyme family i.e., human puromycin sensitive aminopeptidase (hPSA). Among the compounds;
4b, 9b, 9e and 10a demonstrated best inhibition against hPSA.
Received 13 May 2015
Received in revised form
8 October 2015
Accepted 13 October 2015
Available online 19 October 2015
Keywords:
Tetrahydropyridine
Multicomponent reaction
Cytotoxicity
© 2015 Elsevier Masson SAS. All rights reserved.
Puromycin sensitive aminopeptidase and
inhibitors
1. Introduction
piperidines [11]. They exist in numerous natural products such as
(þ)-cannabisativine, (ꢀ)-palustrine, (ꢀ)-sedacrine. 1-Methyl-4-
Heterocyclic nitrogenous compounds and their analogues
represent an important class of bioactive compounds. Many of
these compounds are employed in the treatment of human diseases
due to their specific activity [1]. Among a wide range of heterocyclic
compounds that have been explored for the development of
pharmaceutically important molecules, tetrahydropyridines
constitute an interesting class of pharma actives due to their
versatility and broad-spectrum activities such as anticancer [2],
anti-hypertensive [3], antibacterial [4], anti-malarial [5], anticon-
vulsant [6] and anti-inflammatory [7] activities. Moreover, they are
reported as inhibitors of farnesyl transferase [8], dihydroorotate
dehydrogenase [9] and also involved in the MAO-based mechanism
in Parkinson's disease [10]. In addition, the 2,6-disubstituted tet-
rahydropyridine framework has been regarded as a valuable basic
unit since the possibility of modification and functionalization of
the double bond enables the preparation of polysubstituted
phenyl-1,2,5,6-tetrahydropyridine (MPTP) is a neurotoxin which
induces Parkinson's disease [12]. In addition, this skeleton is an
integral part of many drugs such as xanomeline, sabcomeline,
tazomeline, alvameline, milameline etc., involved in treatment of
cognitive diseases like Alzheimer's and Schizophrenia [13]. Diverse
activity profile has inspired development of many new strategies to
construct
skeleton.
the
2,6-disubstituted
1,2,5,6-tetrahydropyridine
Our interest in developing new chemical entities with anti-
proliferative activity led us to pay much attention towards the
synthesis of polysubstituted tetrahydropyridines. Multicomponent
reactions (MCRs) have become important tools for the rapid gen-
eration of molecular complexity and diversity with predefined
functionality in chemical biology and drug discovery. MCRs have
been extensively used in the construction of target compounds by
the introduction of several diversity elements in a single operation,
resulting minimization of waste, labor, time, and cost. Therefore,
new protocols with operational simplicity, efficiency, high selec-
tivity and minimal environmental hazards are still in demand and
offer a challenging task to chemists. Recently, we have published a
MCR approach [14] based on modified Hantzsch synthesis for
construction of polysubstituted tetrahydropyridines. An improved
* Corresponding author.
** Corresponding author.
E-mail
addresses:
manjula@iict.res.in (M.
Alla),
anthony@iict.res.in
(A. Addlagatta).
1
Indicates equal contribution.
http://dx.doi.org/10.1016/j.ejmech.2015.10.026
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

R. Aeluri et al. / European Journal of Medicinal Chemistry 106 (2015) 26e33
27
version of the reported protocol has been currently extended for
construction of a library of 2,6-differentially substituted THPs for
the first time. Further the THPs have been used as synthons for
construction of piperidine-4-one and piperidine-4-ol frameworks,
to generate an exhaustive library of six membered N-heterocycles.
The library has been evaluated for cytotoxicity and enzyme
inhibition.
carbaldehyde and p-toluidine is expected to take place through
intermolecular Mannich reaction. Later one equivalent of benzal-
dehyde was added to the reaction mixture, which yields iminium
species. An intramolecular Mannich reaction completes the pro-
tocol. After completion of the reaction, solid precipitate was filtered
off and purified by column chromatography to obtain the desired
product i.e., ethyl 6-phenyl-2-(pyridin-3-yl)-1-p-tolyl-4-(p-tolyla-
mino)-1,2,5,6-tetrahydropyridine-3-carboxylate (9a) in 60% yield
(Scheme 2).
2. Results and discussion
Later, the scope of the reaction was extended by varying both
aromatic amines and substituted aromatic aldehydes as shown in
Scheme 3. The isolated yields of products obtained by this route are
in the range of 50e65%. To the best of our knowledge this is the first
report on synthesis of 2,6-unsymmetrical THPs using a one pot
protocol.
Additionally, piperidine-4-one-3-carboxylates and piperidine-
4-ol derivatives, required for the comparative study, were obtained
from corresponding polysubstituted 1,2,5,6-tetrahydropyridines.
Piperidine-4-one-3-carboxylates (10a & 10b) were synthesized by
acidic hydrolysis of polysubstituted 1,2,5,6-tetrahydropyridines.
Due to enamine nature of eNH on C-4 carbon of tetrahydropyridine
ring, it could be easily hydrolyzed in acetone at ambient tempera-
tures to obtain piperidine-4-one-3-carboxylates as shown in
Scheme 4. The isolated yields of products were about 60%.
Piperidine-4-ol derivatives (11a & 11b) could be obtained by
2.1. Chemistry
Requisite compounds were synthesized in two phases. In the
first phase, highly substituted tetrahydropyridines containing same
substitutions at 2 and 6 positions (Fig.1) were synthesized adopting
an earlier reported procedure. In this protocol we have demon-
strated a highly efficient one-pot five component MCR protocol
using benign Lewis acid namely, zirconium tetrachloride and a
green solvent, ethanol for construction of THPs. This protocol has
advantage of higher yields in lesser reaction times and occurs at
ambient temperatures. A series of tetrahydropyridines were syn-
thesized employing a variety of aromatic aldehydes, aromatic
amines and methyl/ethyl acetoacetate as shown in Scheme 1.
In the second phase, a series of tetrahydropyridines containing
different substitutions at 2 and 6-positions have been synthesized.
A brief introspection of the mechanism has led to the conclusion
that the reaction proceeds via inter and intramolecular Mannich
reactions to result in the formation of THP. With this understanding
it was envisaged that a one-pot sequential addition protocol would
yield desired molecules. Therefore, a one-pot reaction was carried
out by sequential addition of different aromatic aldehydes (one
equivalent each) for synthesis of THPs with different 2,6-
substitutions. At the outset, a model reaction was carried out by
taking two equivalents of p-toluidine and one equivalent of ethyl
acetoacetate in ethanol at room temperature. After formation of
enamine (confirmed by TLC), one equivalent of pyridine-3-
carbaldehyde was added followed by catalyst (ZrCl4). An in situ
attack of enamine on imine formed between pyridine-3-
simple
basic
hydrolysis
of
corresponding
1,2,5,6-
tetrahydropyridine in presence of lithium hydroxide (LiOH) in
methanol/tetrahydrofuran/water (1:1:1) under reflux conditions as
shown in Scheme 5. The isolated yields were 60 and 58%
respectively.
2.2. Biological assays
2.2.1. Cytotoxic assay
All synthesized compounds were evaluated for their in vitro
cytotoxicity against three different cancer cell lines. Table 1 depicts
the active compounds among those tested. Surprisingly, many
compounds showed high selectivity towards HepG2 cell lines. None
of the compounds were active against the A-549 cell lines. Com-
pounds 9d and 10a were effective against HeLa and HepG2 cell lines
in low micromolar concentrations while 4b had feeble effect on
both these cell lines. Compounds 4a (symmetric substitution of 4-
F-C6H4 at C-2 and C-6 positions) and 9e (unsymmetric substitutions
with 3-pyridinyl and 4-CN-C6H4 at C-2 and C-6 positions, respec-
tively) selectively inhibit the growth of HepG2 cell lines in low
micromolar concentration while 9b and 9f had marginal effect on
HeLa cells. Although aromatic groups at C-2 and C-6 positions
Fig. 1. Pictorial representation of 1,2,5,6-tetrahydropyridine.
Scheme 1. One-pot MCR approach for synthesis of tetrahydropyridines.

28
R. Aeluri et al. / European Journal of Medicinal Chemistry 106 (2015) 26e33
Scheme 2. One-pot sequential addition protocol for synthesis of 9a.
Scheme 3. Protocol for synthesis of differentially substituted THPs.
seemed to be important, exact structure activity relationship could
not be established based on the activity data. Several factors like
solubility and cell entry could be affecting the activity outcome of
these compounds.
2.2.2. Enzymatic assay
We have long term interest in understanding the structure and
function of M1 family aminopeptidases [15e19]. M1 family ami-
nopeptidases are present in all forms of life except for viruses and
play a very important role in the physiology of the organism [20]. In
human nine proteins have been identified so far. Of these puro-
mycin sensitive aminopeptidase from human (hPSA), is a zinc
metallo peptidase that hydrolyzes amino acids from the N-termi-
nus of its substrate [21]. hPSA plays a major role in mitosis and
meiosis [22,23]. This enzyme is known to degrade enkephalins in
Scheme 4. Piperidine-4-one-3-carboxylates synthesis via acidic hydrolysis.

R. Aeluri et al. / European Journal of Medicinal Chemistry 106 (2015) 26e33
29
Table 2
IC50 values were determined in
m
M concentrations.
S. No.
Entry
hPSA pfAPN
ePepN
pmAPN
1
2
3
4
5
6
7
8
4b
4d
4i
9b
9d
9e
9h
9i
1.79 0.37
68.81 3.59
92.51 2.04
8.53 0.46
98.19 2.96
10.18 0.64
80.97 3.91
32.68 0.55
42.96 1.73
8.04 0.21
92.87 3.83
36.27 1.30
36.42 0.76
2.45 0.40
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9
9j
10a
10b
11a
11b
puromycin
10
11
12
13
14
Scheme 5. Synthesis of piperidine-4-ol derivatives via basic hydrolysis.
the brain [24,25], and recent studies show that hPSA degrades
aggregating toxic proteins involved in various neurodegenerative
diseases [26e29]. Studies in mouse suggest that hPSA involved in
proteolytic events that regulate the cell cycle. PfAPN is a malarial
essential enzyme [30]. pmAPN is a porcine enzyme equivalent to
human APN which is a drug target in cancer. ePepN is a bacterial
enzyme (E. coli) usually used as model system to study this family of
enzymes.
In view of these, it was deemed relevant to evaluate activity of
all compounds against four members of the M1 family aminopep-
tidases. Table 2 summarizes the activities of only those compounds
that show inhibition against at least one enzyme. Surprisingly
except for hPSA none of the M1 family aminopeptidases have been
inhibited. Ethyl-1-phenyl-4-(phenylamino)-2,6-di(pyridin-3-yl)-
1,2,5,6-tetrahydropyridine-3-carboxylate, 4b (symmetric substitu-
tion with 3-pyridinyl at C-2 and C-6 positions) has been found to be
very selective and efficient compared to standard, puromycin
against the hPSA. Compounds 9b (pyridinyl and phenyl), 9e (pyr-
idinyl and 4-CN-C6H4) and 10a (symmetric substitution with 3-
pyridinyl at C-2 and C-6 positions) have also shown inhibition of
Using AutoDock Vina [32], we have docked compound 4b and
puromycin in the active site of the PSA model. The ethyl ester
region of 4b docks nicely against the active site zinc. The entire
molecule binds in the interface of the S1 and S10 sub sites without
experiencing any steric or electrostatic clashes. The central
piperidinyl group forms a good hydrophobic interaction with
Tyr394, one of the critical residues in the enzyme catalysis. The
other substituents spread freely experiencing good hydrophobic
and electrostatic interactions. For example, the 3-pyridinyl sub-
stituents interact with Glu338 and Arg806 (Fig. 2A). When it is a
2-pyridinyl substituent, the nitrogen atom in the aromatic ring,
may not be available for interaction with these residues and hence
the reduced activity is noted. Alignment of the ePepN structure
over the PSA-4b model suggests that active site of the former
could be narrower compared to the PSA (Fig. 2B). However, these
studies are preliminary and need more biochemical and structural
characterization. Puromycin also binds in the S1 and S1’ pockets
making very similar contacts with the enzyme. The affinity value
(docking score) of 4b with hPSA is ꢀ10 kcal/mol while it
is ꢀ9.1 kcal/mol for hPSA-puromycin complex (Fig. 2C).
hPSA with considerable efficiency (~10 mM). On a closer scrutiny of
the active compounds (against hPSA) it can be summarized that 3-
pyridinyl substitutions fared better than the 2-pyridinyl sub-
stitutions with some exceptions.
3. Conclusions
Though good cytotoxic activity was noticed along with hPSA
specific inhibition, a direct correlation if the former effect is due to
the later could not be established. Efforts are underway to syn-
thesize new derivatives of 4b to understand the fine details of SAR
and also to characterize cellular phenotypic behavior.
Multiple routes have been designed for construction of THPs
depending on the ring substitutions required. According to earlier
reported procedure, an efficient one pot five component protocol
consisting aromatic aldehyde, b-keto ester and aromatic amine has
been carried out for construction of 2,6-similarly substituted THPs
using economical, environmentally benign Lewis acid (ZrCl4) as the
catalyst in a green solvent (ethanol) at ambient temperature. A one
pot sequential addition protocol of substrates for construction of
2,6-differential substituted THPs has been developed with simple
modification of earlier mentioned MCR. Besides this, the THPs were
used as synthons for construction of piperidine/piperidone based
six membered heterocycles. Several of the compounds displayed
selective cytotoxicity against the HepG2 cell lines. Furthermore,
among four M1 family aminopeptidases tested, hPSA has been
selectively inhibited by many of the tested compounds, specifically
those containing 3-pyridinyl substitutions.
2.2.3. Molecular modeling studies
The homology model of hPSA was generated using Modeller
9.12 [31] by taking human Aminopeptidase A as template (PDB ID:
4KX7). The model with lowest discrete optimized protein energy
(DOPE) score was selected as the best model and validated it using
Ramachandran parameters (98.4% in acceptable region). Auto-
Dock Tools were used for preparing the model for docking studies.
Table 1
GI50 values were determined in micromolar concentrations.
S. No.
Entry
A-549
HeLa
HepG2
1.
2.
3.
4.
5.
6.
7.
8.
9.
4a
4b
9b
9d
9e
9f
9g
10a
Doxo
>100
>100
>100
>100
>100
>100
>100
>100
>100
14.89 0.42
81.53 0.33
>100
11.88 0.05
13.73 0.97
>100
>100
11.21 0.16
4.45 0.08
87.93 1.47
90.73 6.56
9.95 1.18
>100
88.16 5.5
10.55 0.31
9.32 1.05
7.51 0.12
4. Experimental protocols
4.1. Synthesis
All the chemicals used for the synthesis are of the synthetic
grade procured from Spectrochem, Aldrich, SR Laboratories and SD-
fine chemicals. Completion of the reaction was monitored by thin
8.14 0.15

30
R. Aeluri et al. / European Journal of Medicinal Chemistry 106 (2015) 26e33
Fig. 2. A) The ethyl ester region makes of 4b makes strong interaction with active site zinc ion. In addition, Glu338, Tyr394, Phe802 and Arg806 form the basis for the hydrophobic
and electrostatic interactions. B) Alignment of the PSA-4b model (colored surface) and ePepN structure (yellow sticks). From this it is understood that some of the residues in the
ePepN structures could have a steric clash with the compound 4b there by not allowing it to bind in the active site. C) Comparison of binding modes of 4b and puromycin in the
active site of hPSA model indicates that they bind similarly making analogous interactions. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
layer chromatography (TLC) using E-Merck 0.25 mm silica gel
plates using ethyl acetate/hexane (2:8) as eluting medium. Visu-
alization was accomplished by using UV light (256 nm) and iodine
chamber. Melting points were determined in open capillaries and
are uncorrected. 1H-NMR (300 MHz and 500 MHz) and 13C-NMR
(75 MHz) spectra were recorded on Avance 300 MHz, Avance
500 MHz and JCAMP DX-50 in CDCl3 and DMSO-d6 unless other-
60%; M. P. 170e172 ꢁC; 1H-NMR (CDCl3, 300 MHz)
d ppm 1.44 (t,
J ¼ 6.98 Hz, 3H), 2.12 (s, 3H), 2.26 (s, 3H), 2.76(dd, J ¼ 2.26, 15.10 Hz,
1H), 3.50(dd, J ¼ 6.04, 15.10 Hz, 1H), 4.24e4.51 (m, 2H), 5.19 (brs,
1H), 6.22 (d, J ¼ 8.12 Hz, 2H), 6.37 (d, J ¼ 9.44 Hz, 1H), 6.43 (d,
J ¼ 8.68 Hz, 2H), 6.83 (d, J ¼ 8.30 Hz, 1H), 6.90(quin, J ¼ 3.96,
8.30 Hz, 3H), 7.04e7.39 (m, 6H), 7.47e7.64 (m, 2H), 8.44e8.55 (m,
1H), 10.24 (s, 1H). 13C-NMR (CDCl3, 125 MHz)
d ppm 14.8, 20.0, 20.8,
wise mentioned. Chemical shifts are reported in
from internal standard Tetramethylsilane (TMS) and coupling
d
(ppm) downfield
32.6, 57.6, 59.5, 59.8, 95.6, 112.5, 121.3, 121.6, 121.8, 125.2, 125.3,
129.4, 129.5, 135.2, 135.4, 135.9, 136.6, 144.8, 149.2, 149.4, 157.1,
162.9, 163.9, 168.1. IR (KBr, cmꢀ1) 3241, 3001, 2921, 1647, 1588, 1514,
1253, 1077, 780. ESI-MS m/z 504 [M þ H]þ; HRMS m/z calculated for
constants (J) values are in Hz. IR spectra were recorded on a Thermo
Nicolet NEXUS 670 spectrometer in KBr with absorption in cmꢀ1
.
Electronic Spin ionization Mass spectra were recorded on Thermo
Finnigan ESI ion trap mass spectrometer and HRMS done on high
resolution QSTAR XL Hybrid/MS system. The compounds 4a-i were
synthesized according to our previous reported procedure [14]. The
spectral data of unreported compounds are reported here.
C
33H34O2N3 ¼ 504.26381, found ¼ 504.26205.
4.1.1.3. Ethyl 6-(4-bromophenyl)-2-(pyridin-3-yl)-1-p-tolyl-4-(p-tol-
ylamino)-1,2,5,6-tetrahydropyridine-3-carboxylate (9c). White solid,
Yield: 59%; M. P. 216e219 ꢁC; 1H-NMR (CDCl3, 300 MHz)
d ppm 1.44
(t, J ¼ 7.17 Hz, 3H), 2.17 (s, 3H), 2.29 (s, 3H), 2.66e2.80 (m, 2H),
4.23e4.49 (m, 2H), 5.05 (brs, 1H), 6.30 (d, J ¼ 8.12 Hz, 2H), 6.37 (d,
J ¼ 6.79 Hz, 3H), 6.89 (d, J ¼ 8.49 Hz, 2H), 6.96 (d, J ¼ 7.55 Hz, 2H),
7.01 (d, J ¼ 8.30 Hz, 2H), 7.15e7.22 (m, 2H), 7.39 (d, J ¼ 8.49 Hz, 2H),
7.54e7.619 (m, 1H), 8.44e8.49 (m, 1H), 8.62 (d, J ¼ 2.07 Hz, 1H),
4.1.1. General procedure for synthesis of 1,2,5,6-tetrahydropyridine-
3-carboxylates (9a-j)
To the solution of aromatic amine (2 mmol) in ethanol 8e10 mL,
b-keto ester (1 mmol) was added. After formation of enamine (as
monitored by TLC), aromatic aldehyde (1 mmol) followed by ZrCl4
(15 mol%) were added. The reaction mixture was allowed to stir at
room temperature for about 4 h. Then another aromatic aldehyde
(1 mmol) was added with constant stirring. After completion of the
reaction solid obtained was filtered off and washed with ethanol.
The crude product was purified by column chromatography to get
the desired product. The yields of the products were ranging from
50 to 63%.
10.25 (s, 1H). 13C-NMR (CDCl3, 75 MHz)
d ppm 14.7, 20.1, 20.9, 33.6,
53.7, 57.2, 59.8, 96.5, 113.2, 120.8, 123.0, 125.8, 126.1, 128.2, 129.6,
131.7, 134.2, 134.8, 135.9, 137.5, 139.6, 141.6, 144.1, 147.7, 148.7, 156.2,
167.8. IR (KBr, cmꢀ1) 3228, 2984, 2920, 1649, 1595, 1515, 1252, 1071,
790. ESI-MS m/z 582 [M þ H]þ; HRMS m/z calculated for
C33H33O2N3Br ¼ 582.17507, found ¼ 582.17409.
4.1.1.4. Ethyl 6-(4-chlorophenyl)-1-phenyl-4-(phenylamino)-2-(pyr-
idin-3-yl)-1,2,5,6-tetrahydropyridine-3-carboxylate (9d). White
solid, Yield: 61%; M. P. 186e189 ꢁC; 1H-NMR (CDCl3, 300 MHz)
4.1.1.1. Ethyl 6-phenyl-2-(pyridin-3-yl)-1-p-tolyl-4-(p-tolylamino)-
1,2,5,6-tetrahydropyridine-3-carboxylate (9a). White solid, Yield:
d
ppm 1.46 (t, J ¼ 7.17 Hz, 3H), 2.74e2.83 (m, 2H), 4.25e4.53 (m, 2H)
63%; M. P. 179e181 ꢁC; 1H-NMR (CDCl3, 300 MHz)
d
ppm 1.45 (t,
5.12 (brs, 1H), 6.36e6.53 (m, 5H), 6.66 (t, J ¼ 6.79 Hz, 1H), 7.03e7.25
(m, 9H), 7.59 (d, J ¼ 7.55 Hz, 1H), 8.47 (d, J ¼ 3.77 Hz, 1H), 8.62e8.67
J ¼ 6.79 Hz, 3H), 2.16 (s, 3H), 2.27 (s, 3H), 2.68(dd, J ¼ 5.28, 15.1 Hz,
1H), 2.78(dd, J ¼ 2.26,15.1 Hz,1H), 4.24e4.50 (m, 2H), 5.10 (brs,1H),
6.19 (d, J ¼ 8.30 Hz, 2H), 6.42 (d, J ¼ 8.30 Hz, 3H), 6.89 (t, J ¼ 8.30 Hz,
4H), 7.12e7.36 (m, 6H), 7.58 (d, J ¼ 8.30 Hz, 1H), 8.41e8.52 (m, 1H),
8.65 (d, J ¼ 2.26 Hz, 1H), 10.25 (s, 1H). 13C-NMR (CDCl3, 75 MHz)
(m, 1H), 10.34 (s, 1H). 13C-NMR (CDCl3, 75 MHz)
d ppm 14.7, 33.7,
53.4, 57.1, 59.9, 97.1, 112.9, 116.9, 123.0, 125.6, 125.9, 127.7, 128.7,
128.9, 129.1, 132.9, 134.1, 137.5, 139.3, 140.7, 146.2, 147.7, 148.6, 155.8,
167.8. IR (KBr, cmꢀ1) 3241, 2976, 2925, 1655, 1596, 1501, 1247, 1194,
1067, 746, 694. ESI-MS m/z 510 [M þ H]þ; HRMS m/z calculated for
d
ppm 14.7, 20.1, 20.8, 33.6, 53.6, 57.8, 59.7, 96.5, 113.1, 122.9, 125.7,
125.9, 126.3, 127.1, 128.6, 129.4, 129.5, 134.2, 134.9, 135.8, 139.7,
142.5, 144.3, 147.6, 148.8, 156.5, 167.9. IR (KBr, cmꢀ1) 3238, 2986,
2914, 1650, 1593, 1514, 1252, 1073, 790, 704. ESI-MS m/z 504
[M þ H]þ; HRMS m/z calculated for C33H34O2N3 ¼ 504.26383,
found ¼ 504.26212.
C
31H29O2N3Cl ¼ 510.19428, found ¼ 510.19140.
4.1.1.5. Ethyl 6-(4-cyanophenyl)-1-phenyl-4-(phenylamino)-2-(pyr-
idin-3-yl)-1,2,5,6-tetrahydropyridine-3-carboxylate (9e). White
solid, Yield: 59%; M. P. 228e231 ꢁC; 1H-NMR (CDCl3, 300 MHz)
d
ppm 1.46 (t, J ¼ 6.98 Hz, 3H), 2.78e2.88 (m, 2H), 4.24e4.54 (m,
4.1.1.2. Ethyl 6-phenyl-2-(pyridin-2-yl)-1-p-tolyl-4-(p-tolylamino)-
1,2,5,6-tetrahydropyridine-3-carboxylate (9b). White solid, Yield:
2H), 5.20 (brs, 1H), 6.36e6.51 (m, 5H), 6.68 (t, J ¼ 6.79 Hz, 1H),
7.03e7.31 (m, 8H), 7.38e7.68 (m, 3H), 8.38e8.69 (m, 2H), 10.32 (s,

R. Aeluri et al. / European Journal of Medicinal Chemistry 106 (2015) 26e33
31
1H). 13C-NMR (CDCl3, 75 MHz)
111.1, 112.9, 117.3, 118.7, 123.1, 125.4, 126.1, 127.1, 129.1, 129.2, 132.4,
134.1, 137.3, 139.0, 145.9, 147.9, 148.5, 155.2, 167.7. IR (KBr, cmꢀ1
d
ppm 14.7, 33.5, 53.4, 57.5, 60.1, 97.1,
1H), 2.80(dd, J ¼ 2.45, 14.91 Hz, 1H), 3.70 (s, 6H), 3.85 (s, 3H),
4.24e4.29 (m, 2H), 5.05 (brs, 1H), 6.30e6.48 (m, 4H), 6.54 (d,
J ¼ 8.30 Hz, 2H), 6.67 (t, J ¼ 7.17 Hz, 1H), 7.04e7.24 (m, 5H), 7.57 (d,
J ¼ 7.93 Hz, 1H), 8.47 (d, J ¼ 3.96 Hz, 1H), 8.62 (s, 1H), 10.34 (s, 1H).
)
3235, 3044, 2978, 2223, 1652, 1596, 1500, 1369, 1256, 1068, 747,
696. ESI-MS m/z 501 [M þ H]þ; HRMS m/z calculated for
13C-NMR (CDCl3, 75 MHz)
d ppm 14.7, 33.7, 53.4, 55.7, 57.9, 59.7,
C
32H29O2N4 ¼ 501.22850, found ¼ 501.22668.
60.7, 96.6, 102.8, 112.8, 116.7, 122.9, 125.4, 126.0, 128.7, 128.9, 133.9,
136.7, 137.4, 137.9, 139.1, 146.4, 147.6, 148.5, 153.2, 156.7, 167.6. IR
(KBr, cmꢀ1) 3147, 2933, 1648, 1594, 1500, 1325, 1256, 1125, 1066,
751, 690. ESI-MS m/z 566 [M þ H]þ; HRMS m/z calculated for
4.1.1.6. Ethyl 6-(4-fluorophenyl)-2-(4-nitrophenyl)-1-phenyl-4-(phe-
nylamino)-1,2,5,6-tetrahydropyridine-3-carboxylate (9f). White
solid, Yield: 52%; M. P. 210e212 ꢁC; 1H-NMR (CDCl3, 500 MHz)
C34H36O5N3 ¼ 566.26495, found ¼ 566.26316.
d
ppm 1.47 (t, J ¼ 7.01 Hz, 3H), 2.76e2.84 (m, 1H), 2.93(dd, J ¼ 5.79,
15.25 Hz, 1H), 4.29e4.50 (m, 2H), 5.13 (brs, 1H), 6.40e6.47 (m, 5H),
6.64e6.70 (m, 1H), 7.00 (d, J ¼ 8.39 Hz, 1H), 7.06e7.22 (m, 7H), 7.28
(d, J ¼ 8.69 Hz, 1H), 7.39e7.43 (m, 1H), 7.50 (d, J ¼ 8.54 Hz, 1H),
4.1.2. Procedure for synthesis of ethyl 4-oxo-1-phenyl-2,6-
di(pyridin-3-yl)piperidine-3-carboxylate (10a)
To the solution of ethyl 1'-phenyl-4'-(phenylamino)-10,20,50,6'-
tetrahydro-[3,2':60,300-terpyridine]-3'-carboxylate (4b) (1 mmol) in
acetone (5 mL), 2 equivalents of Con. HCl was added. The reaction
mixture was stirred at room temperature. After completion of the
reaction (as monitored by TLC), acetone was evaporated under the
vacuum and reaction mixture was diluted with water and extracted
with ethyl acetate (3 x 10 mL). Combined organic layers were dried
over anhydrous Na2SO4 and the solvent was evaporated. Then the
pure product was crystallized from hexane. White solid, Yield: 62%;
8.10e8.16 (m, 2H), 10.28 (s, 1H). 13C-NMR (CDCl3, 75 MHz)
d ppm
14.8, 33.5, 55.2, 59.5, 97.7, 112.9, 124.9, 125.9, 126.1, 126.4, 126.6,
126.9, 128.1, 128.5, 129.4, 129.4, 135.2, 135.5, 143.0, 144.3, 144.8,
156.4,168.2. IR (KBr, cmꢀ1) 3243, 2977,1656,1594,1502,1345,1249,
1068, 749, 696. ESI-MS m/z 538 [M þ H]þ; HRMS m/z calculated for
C
32H29FO4N3 ¼ 538.21346, found ¼ 538.21196.
4.1.1.7. Ethyl
(phenylamino)-1,2,5,6-tetrahydropyridine-3-carboxylate
White solid, Yield: 55%; M. P. 215e218 ꢁC; 1H-NMR (CDCl3,
300 MHz)
6-(4-bromophenyl)-2-(4-nitrophenyl)-1-phenyl-4-
(9g).
M. P. 71e73 ꢁC; 1H-NMR (CDCl3, 500 MHz)
d ppm 1.09e1.18 (m, 3H),
2.27 (d, J ¼ 6.56 Hz, 2H), 4.04e4.17 (m, 2H), 4.28e4.34 (m, 1H),
4.39e4.47 (m, 1H), 5.29e5.37 (m, 1H), 6.64e6.78 (m, 4H), 7.10e7.25
(m, 4H), 7.31e7.60 (m, 3H), 7.68e7.75 (m, 1H), 8.49e8.63 (m, 1H).
d
ppm 1.46 (t, J ¼ 7.01 Hz, 3H), 2.82(dd, J ¼ 2.59, 15.25 Hz,
1H), 2.93(dd, J ¼ 5.79, 15.25 Hz, 1H), 4.29e4.50 (m, 2H), 5.20 (brs,
1H), 6.37 (s, 1H), 6.39e6.46 (m, 4H), 6.68 (t, J ¼ 7.17 Hz, 1H),
7.05e7.22 (m, 7H), 7.28 (d, J ¼ 8.69 Hz, 2H), 7.42 (d, J ¼ 8.39 Hz, 2H),
8.12 (d, J ¼ 8.69 Hz, 2H), 10.28 (s, 1H). 13C-NMR (CDCl3, 75 MHz)
13C-NMR (CDCl3, 75 MHz)
d ppm. 14.8, 49.2, 52.0, 55.1, 64.3, 65.5,
113.4, 113.6, 118.1, 124.1, 125.9, 128.0,128.6, 129.3, 130.3, 131.8, 133.6,
148.6, 158.6, 158.6, 168.1, 202.5. IR (KBr, cmꢀ1) 3041, 3030, 2956,
1755, 1719, 1597, 1493, 1206, 1128, 790, 758, 693. ESI-MS m/z 402
[M þ H]þ; HRMS m/z calculated for C24H24O3N3 ¼ 402.18113,
found ¼ 402.17951.
d
ppm 14.8, 33.4, 54.7, 57.4, 60.0, 97.8, 112.8, 117.2, 120.3, 123.8,
125.3,126.0,127.3, 128.3, 129.1, 129.2,131.4, 137.4, 142.6, 146.1, 147.2,
150.1, 155.0,167.8. IR (KBr, cmꢀ1) 3235, 2980,1652, 1598, 1501, 1350,
150, 1068, 749, 698. ESI-MS m/z 599 [M þ 2]þ; HRMS m/z calculated
for C32H30O4N3Br ¼ 599.12817, found ¼ 599.12635.
4.1.3. Procedure for synthesis of ethyl 4-oxo-1-phenyl-2,6-
di(pyridin-2-yl)piperidine-3-carboxylate (10b)
4.1.1.8. Ethyl 6-(4-bromophenyl)-1-phenyl-4-(phenylamino)-2-(thio-
To the solution of ethyl 1'-phenyl-4'-(phenylamino)-10,20,50,6'-
tetrahydro-[2,2':60,200-terpyridine]-3'-carboxylate (4i) (1 mmol) in
acetone (5 mL), 2 equivalents of Con. HCl was added. The reaction
mixture was stirred at room temperature. After completion of the
reaction (as monitored by TLC), acetone was evaporated under the
vacuum and reaction mixture was diluted with water and extracted
with ethyl acetate (3 x 10 mL). Combined organic layers were dried
over anhydrous Na2SO4 and the solvent was evaporated. Then the
pure product was crystallized from hexane. White solid, Yield: 60%;
phen-2-yl)-1,2,5,6-tetrahydropyridine-3-carboxylate
White solid, Yield: 50%; M. P. 205e207 ꢁC; 1H-NMR (CDCl3,
300 MHz)
(9h).
d
ppm 1.45 (t, J ¼ 6.98 Hz, 3H), 2.73(dd, J ¼ 2.26, 15.10 Hz,
1H), 2.83(dd, J ¼ 5.28, 15.10 Hz, 1H), 4.24e4.51 (m, 2H), 5.07 (brs,
1H), 6.34 (s, 1H), 6.37e6.71 (m, 4H), 7.0 (d, J ¼ 8.30 Hz, 2H),
7.04e7.23 (m, 6H), 7.35e7.45 (m, 4H),10.28 (s, 1H). 13C-NMR (CDCl3,
75 MHz)
d ppm 14.7, 33.5, 54.7, 57.3, 59.8, 97.6, 112.8, 113.1, 116.7,
120.8,125.6,125.9,128.1,128.3,128.9,128.9,131.3,131.6,137.5,141.4,
142.9, 146.4, 155.7, 167.9. IR (KBr, cmꢀ1) 3229, 3032, 2980, 1651,
1595, 1499, 1253, 1067, 754, 692. ESI-MS m/z 559 [M þ H]þ; HRMS
m/z calculated for C30H28O2N2BrS ¼ 559.10471, found ¼ 559.10569.
M. P. 127e129 ꢁC; 1H-NMR (CDCl3, 500 MHz)
d ppm 1.08e1.17 (m,
3H), 2.44 (s, 1H), 2.52 (s, 1H), 4.03e4.16 (m, 2H), 4.28e4.56 (m, 2H),
5.29e5.37 (m, 1H), 6.64e6.79 (m, 4H), 7.10e7.25 (m, 4H), 7.31e7.44
(m, 2H), 7.51e7.59 (m, 1H), 7.69e7.75 (m, 1H), 8.48e8.63 (m, 1H).
4.1.1.9. Ethyl 6-(4-cyanophenyl)-1-phenyl-4-(phenylamino)-1,2,5,6-
13C-NMR (CDCl3 þ DMSO-d6, 75 MHz)
d ppm 13.5, 57.2, 57.8, 60.7,
tetrahydro-[2,2'-bipyridine]-3-carboxylate (9i). White solid, Yield:
61.8, 63.3, 113.1, 114.2, 117.2, 117.6, 121.7, 123.5, 124.1, 125.5, 128.5,
128.6, 148.4, 148.5, 148.8, 158.9, 167.9, 200.9. IR (KBr, cmꢀ1) 3267,
2986, 2928, 1712, 1596, 1286, 1161, 748. ESI-MS m/z 402 [M þ H]þ;
62%; M. P. 120e123 ꢁC; 1H-NMR (CDCl3, 500 MHz)
d ppm 1.46 (t,
J ¼ 7.17 Hz, 3H), 2.79e2.87 (m, 2H), 4.30e4.50 (m, 2H), 5.18 (brs,
1H), 6.40e6.46 (m, 5H), 6.68 (t, J ¼ 7.17 Hz, 1H), 7.06e7.25 (m, 8H),
7.54e7.62 (m, 3H), 8.47e8.50 (m,1H), 8.62e8.64 (m, 1H), 10.32 (s,
HRMS
m/z
calculated
for
C
24H24O3N3
¼
402.18114,
found ¼ 402.17925.
1H). 13C-NMR (CDCl3, 75 MHz)
d ppm 14.7, 33.4, 55.2, 58.3, 59.9,
98.8, 108.4, 114.5, 117.8, 119.1, 126.2, 126.4, 126.5, 127.2, 127.4, 128.4,
128.8, 131.4, 131.6, 131.9, 136.9, 142.1, 143.1, 145.9, 155.2, 168.1. IR
(KBr, cmꢀ1) 3266, 3026, 2980, 2223, 1652, 1597, 1497, 1369, 1288,
1164, 1023, 750, 692. ESI-MS m/z 501 [M þ H]þ; HRMS m/z calcu-
lated for C32H29O2N4 ¼ 501.22842, found ¼ 501.22658.
4.1.4. Procedure for synthesis of 1-phenyl-2,6-di(pyridin-3-yl)-
1,2,3,6-tetrahydropyridin-4-ol (11a)
To the solution of tetrahydropyridine (4b) (1 mmol) in MeOH/
THF/H2O (3:2:1), lithium hydroxide monohydrate (LiOH.H2O)
(1.2 mmol) was added at 0 ꢁC. After addition the temperature of
reaction mixture was raised to room temperature and subjected to
reflux (at 65 ꢁC) for about 5 h. After completion of the reaction (as
monitored by TLC), the reaction mixture was removed and
concentrated under reduced pressure and extracted with DCM
(3 x 10 mL). The combined organic layers were dried over
4.1.1.10. Ethyl 1,2-diphenyl-4-(phenylamino)-6-(3,4,5-
trimethoxyphenyl)-1,2,5,6-tetrahydropyridine-3-carboxylate
White solid, Yield: 60%; M. P. 207e209 ꢁC; 1H-NMR (CDCl3,
300 MHz)
ppm 1.43 (t, J ¼ 7.17 Hz, 3H), 2.68(dd, J ¼ 5.28, 15.10 Hz,
(9j).
d

32
R. Aeluri et al. / European Journal of Medicinal Chemistry 106 (2015) 26e33
anhydrous Na2SO4 and the product was purified by column chro-
matography. White solid, Yield: 60%; M. P. 142e144 ꢁC; 1H-NMR
4.2.2. Kinetic assays on M1 class aminopeptidases
An investigation of redesigned molecules was carried out on
hPSA and other members of the M1 family. Recombinant hPSA,
LePepN, pfAPN and ePepN proteins were purified in the lab using
affinity chromatography and as reported earlier [15,19]. pmAPN was
obtained from SigmaeAldrich (St. Louis, MO, USA). All chemicals
used for the activity were also obtained from SigmaeAldrich.
The reaction buffer used for the hPSA, pfAPN, ePepN assay was
10 mM Tris pH 7.5, 100 mM NaCl. For assays of LePepN 25 mM
phosphate buffer at pH 6.5 was used. Leucine para-nitroanilide
(Leu-pNA) was used as the substrate for hPSA, pfAPN, ePepN, and
glutamic acid para-nitroanilide (Glu-pNA) for LePepN. All enzy-
matic reactions were carried out on a Tecan multimode plate reader
and were performed in triplicate. Various concentrations (1 nM,
(CDCl3, 500 MHz)
d
ppm 2.99(dd, J ¼ 3.35, 17.24 Hz, 1H), 3.19(dd,
J ¼ 6.25, 17.24 Hz, 1H), 3.61(q, J ¼ 7.17, 14.19 Hz, 1H), 5.42 (brs, 1H),
6.50 (d, J ¼ 8.08 Hz, 1H), 6.67e6.93 (m, 3H), 7.09e7.18 (m, 3H),
7.24e7.40 (m, 3H), 7.53e7.57 (m, 1H), 8.50e8.55 (m, 3H). 13C-NMR
(CDCl3, 75 MHz)
d ppm 29.5, 47.9, 52.1, 113.7, 113.9, 115.0, 118.1,
123.8, 126.7, 127.3, 129.1, 129.6, 130.3, 134.5, 139.9, 146.2, 149.8,
151.0, 187.6. IR (KBr, cmꢀ1) 3447, 2922, 2852, 1724, 1635, 1462, 1023,
758. ESI-MS m/z 330 [M þ H]þ; HRMS m/z calculated for
C
21H20ON3 ¼ 330.16011, found ¼ 330.15836.
4.1.5. Procedure for synthesis of 1-phenyl-2,6-di(pyridin-2-yl)-
1,2,3,6-tetrahydropyridin-4-ol (11b)
10 nM, 100 nM, 1 mM, 10 mM, 100 mM) of these compounds were
To the solution of tetrahydropyridine (4i) (1 mmol) in MeOH/
THF/H2O (3:2:1), lithium hydroxide monohydrate (LiOH.H2O)
(1.2 mmol) was added at 0 ꢁC. After addition the temperature of
reaction mixture was raised to room temperature and subjected to
reflux (at 65 ꢁC) for about 5 h. After completion of the reaction (as
monitored by TLC), the reaction mixture was removed and
concentrated under reduced pressure and extracted with DCM
(3 x 10 mL). The combined organic layers were dried over anhy-
drous Na2SO4 and the product was purified by column chroma-
tography. White solid, Yield: 58%; M. P. 107e109 ꢁC; 1H-NMR
incubated with each of the enzymes in triplicate for 30 min at 37 ꢁC
in a 96-well plate. After addition of the substrate, immediate in-
crease in absorption was measured at 405 nm. IC50 values were
computed using Sigma Plot. The results were summarized in the
Table 2.
4.2.3. Molecular modeling and docking
The template for building hPSA model was searched against PDB
database using BLASTp [33]. The human Amino peptidase A (PDB
ID: 4KX7, 2.15 Å resolution) was selected as template (98% query
coverage and 34% identity). The 3D model was generated using
Modeller 9.12 [31]. Best model was selected based on the lowest
DOPE score. The model was assessed with Ramachandran plot at
RAMPAGE server [34].
(CDCl3, 500 MHz)
d
ppm 1.26 (t, J ¼ 7.17 Hz, 2H), 2.05 (s, 2H), 4.12(q,
J ¼ 7.17, 14.34 Hz, 1H), 7.15 (d, J ¼ 16.02 Hz, 2H), 7.27 (s, 1H),
7.36e7.40 (m, 2H), 7.75 (d, J ¼ 16.02 Hz, 2H), 7.92e7.96 (m, 2H),
8.63e8.66 (m, 2H), 8.83e8.87 (m, 2H). 13C-NMR (CDCl3, 75 MHz)
d
ppm 29.5, 47.9, 52.1, 113.7, 113.9, 115.0, 118.4, 123.8, 126.7, 127.3,
The generated hPSA model was used for molecular docking
studies. The coordinates of the compound 4b and puromycin were
drawn using Chemdraw 12 and performed MM2 energy minimi-
zation. The input files for AutoDock Vina [32] were prepared using
AutoDock Tools [35]. The preparation includes addition of polar
hydrogens and gasteiger charges. The grid box size was set to 20,
24, and 24 for X, Y, Z respectively. Of the 20 generated models, the
best model was assessed with negative binding energy. The ligand
with best binding affinity is assessed and extracted using AutoDock
Tools. PyMOL [36] was used to generate the final docked images.
129.1, 129.6, 130.3, 134.5, 139.9, 146.2, 149.8, 151.0, 187.6. IR (KBr,
cmꢀ1) 3450, 2928, 2855, 1725, 1632, 1461, 1030, 760. ESI-MS m/z
330 [M þ H]þ; HRMS m/z calculated for C21H20ON3 ¼ 330.15918,
found ¼ 330.15828.
4.2. Biological assay
4.2.1. Cytotoxicity assay
Three cell lines used for testing in vitro cytotoxicity were A-549
(human lung carcinoma), HeLa (human cervical cancer) and HepG2
(human hepato carcinoma). These cell lines were obtained from
American Type Culture Collection, Manassas, VA, USA. All cell lines
were maintained in a modified Eagle's medium (SigmaeAldrich,
USA) supplemented with 10% fetal bovine serum (Sigma), along
Acknowledgments
ARN, RJG, AKM and VP thank the Council of Scientific and In-
dustrial Research (CSIR) and University Grants Commission (UGC),
New Delhi, India for research fellowships. AA acknowledges the
DBT, New Delhi (BT/PR4965/MED/30/749/2012) and CSIR, New
Delhi (SMiLE) for research grants.
with 1% nonessential amino acids without
L -glutamine (Sigma),
0.2% sodium bicarbonate, 1% sodium pyruvate (Sigma), and 1% of
antibiotic mixture (10,000 U penicillin and 10 mg streptomycin per
mL, Sigma). Cellular viability in the presence of test compounds
was determined by MTT-microcultured tetrazolium assay.
Appendix A. Supplementary data
The cells were seeded to flat bottomed 96 (10,000 cells/100 UL)
well plates and cultured in the media containing 10% FBS and
allowed to attach and recover for 24 h in a humid chamber con-
taining 5% CO2. MTT (3-(4,5-dimethylthiazol-2yl)-2,5diphenyl
tetrazolium bromide; sigma catalog no M2128) was dissolved in
PBS at 5 mg/mL and filtered to sterilize. Different concentrations of
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.10.026.
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