Synthesis, crystal and antibacterial studies of diversely functionalized tetrahydropyridin-4-ol

Article abstract of DOI:10.1016/j.bmcl.2010.02.015

In an effort to expand the spectrum of antibacterial activity associated with piperidin-4-one derivatives, we have synthesized two series of 3-carboxyethyl-2,6-diphenyl-4-hydroxy-Δ³-tetrahydropyridine derivatives bearing diversified heterocyclic and aromatic systems at the nitrogen atom through acetyl (6-18) and 2-propanoyl (9-31) linkers. Unlike acetyl derivatives, NMR spectral pattern of the propanoyl counterparts revealed the existence of pair of rotational isomers (syn and anti) in solution at room temperature due to the hindered rotation about N-CO bond. X-ray crystal studies of 9 and 24 clearly pointed out that all the compounds existed in only one form particularly, in stable syn form in solid state. Each of the compounds was screened for their in vitro antibacterial activity against nine human pathogenic Gram-positive strains including multiple drug resistant organisms and seven problematic Gram-negative strains. Among the various heterocycles examined here, imidazole substituted derivatives 12 and 25 exhibited antibacterial activity approaching that of Linezolid and Trovafloxacin drugs particularly against multiple resistant Enterococcus faecium-VanA phenotype strains.

Full text of DOI:10.1016/j.bmcl.2010.02.015

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2242–2249
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, crystal and antibacterial studies of diversely functionalized
tetrahydropyridin-4-ol
*
Gopalakrishnan Aridoss, Shanmugasundaram Amirthaganesan, Yeon Tae Jeong
Division of Image Science and Information Engineering, Pukyong National University, Busan 608-739, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to expand the spectrum of antibacterial activity associated with piperidin-4-one derivatives,
we have synthesized two series of 3-carboxyethyl-2,6-diphenyl-4-hydroxy-D
3-tetrahydropyridine deriv-
Received 12 November 2009
Revised 31 January 2010
Accepted 3 February 2010
Available online 8 February 2010
atives bearing diversified heterocyclic and aromatic systems at the nitrogen atom through acetyl (6–18)
and 2-propanoyl (9–31) linkers. Unlike acetyl derivatives, NMR spectral pattern of the propanoyl coun-
terparts revealed the existence of pair of rotational isomers (syn and anti) in solution at room tempera-
ture due to the hindered rotation about N–CO bond. X-ray crystal studies of 9 and 24 clearly pointed out
that all the compounds existed in only one form particularly, in stable syn form in solid state. Each of the
compounds was screened for their in vitro antibacterial activity against nine human pathogenic Gram-
positive strains including multiple drug resistant organisms and seven problematic Gram-negative
strains. Among the various heterocycles examined here, imidazole substituted derivatives 12 and 25
exhibited antibacterial activity approaching that of Linezolid and Trovafloxacin drugs particularly against
multiple resistant Enterococcus faecium-VanA phenotype strains.
Keywords:
Tetrahydropyridine
Imidazole
Heterocycles
NMR
X-ray structure
Antibacterial activity
Ó 2010 Elsevier Ltd. All rights reserved.
From the beginning of 1960s, a variety of antibacterial agents
have become available which are structurally assorted and proved
to be efficient against a broad range of pathogenic bacteria. Despite
the available agents, parasitic Gram-positive and Gram-negative
bacteria continue to aggravate and kill millions of people in the sub-
tropical regions of the world. Antibacterial resistance is now well
documented for numerous pathogens and the global threat of such
resistance to those established antibiotics is a serious matter under
review by the World Health Organization (WHO) and many
countries around the world. Of particular concern are nosocomial
and community-acquired infections caused by bacterial pathogens
that have become resistant to one or more antibiotics and particu-
larly, the most challenging are methicillin-resistant Staphylococcus
aureus (MRSA), Staphylococcus epidermidis and vancomycin-resis-
tant Enterococci (VRE). Among them, S. aureus, one of the opportu-
nistic human pathogen is liable for series of infections including
skin boils, bacteremia, pneumonia and endocarditis1 while S. epide-
rmidis is recognized as a pathogen affecting immunocompromised
patients or those with indwelling devices.2 Vancomycin is the con-
sistently active agent for the treatment of serious infections due to
methicillin-resistant staphylococci, but an evident unintentional
consequence of its frequent use has been the emergence of high-
level vancomycin-resistant enterococci (VRE) in hospitals world-
wide.3 Since the late 1990s in Republic of Korea, vancomycin-resis-
tant enterococci turned into main pathogens responsible for
endocarditis, bacteremia, and urinary and wound infections in hos-
pital environment and the majority of vancomycin-resistant entero-
coccal isolates have been VanA phenotype-vanA genotype strains.4
The rising incidence of VRE infections has likewise become very
troublesome as they are unresponsive to the first-line antibiotics
and hence is the predominant concern for clinicians and public
health system. Since the approval of Linezolid in 2000, it has become
the promising therapeutic option for the VRE infections5 in patients
either resistant to or unable to receive a penicillin antibiotic. Even if
laboratory investigations indicate the onset of resistant mutants at a
very low frequency in enterococci,6 several instances of emergence
of resistance by the increased Linezolid use have been documented
in clinical isolates of enterococci viz., Enterococcus faecalis and
Enterococcus faecium.7 The frequent raise of these difficult-to-treat,
multidrug-resistant VRE pathogens augment the mortality rates,
as they cause very delicate and sometimes untreatable infections.
Therefore, all these resistant strains complicate the treatment, and
extremely affect the recovery of patients.8 Besides these Gram-posi-
tive pathogens, various life-threatening infections caused by some
Gram-negative pathogens such as Escherichia coli, Pseudomonas
aeruginosa and Klebsiella pneumoniae and, few major respiratory
tract community pathogens such as Haemophilus influenzae and
Moraxella catarrhalis are also of serious concern in last few years. A
striking increase in antibiotic resistance chiefly among Gram-posi-
tive bacteria has stimulated the need to screen new compounds
for the development of novel antimicrobials rather than analogs of
the licensed drugs in market.
* Corresponding author. Tel.: +82 51 629 6411; fax: +82 51 629 6408.
E-mail address: ytjeong@pknu.ac.kr (Y.T. Jeong).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.015

G. Aridoss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2242–2249
2243
The importance of heterocyclic compounds, particularly nitro-
gen, oxygen and sulfur containing compounds has long been recog-
nized in the field of medicinal chemistry as they possessed
noticeable biological activities. Our earlier findings have shown
the utility of N-functionalized 2,6-diarylpiperidin-4-one based
chemical libraries of small molecules for the identification of
highly active antimicrobials, antimycobacterial, potent analgesics
and antipyretics.9 In an attempt to uncover interesting antibacte-
rial leads, recently our group has performed screens versus several
clinically challenging Gram-positive and Gram-negative organ-
isms, and noticed N-chloroacetyl-3-carboxyethyl-2,6-diphenyl-4-
COCHBr(CH3) were relatively well deshielded (in comparison with
4). X-ray and NMR studies therefore confirm that isomer 4 is in syn
form (C@O and C-2 carbon are syn with each other, and hence H-2
was well-deshielded) while 5 is in anti form (C@O and C-2 carbon
are anti with each other). The desired target compounds 6–18 and
19–31 (Scheme 1) were achieved by the nucleophilic substitution
of the diversified nucleophiles with the respective intermediates
2 and 4 [by keeping in view of the better yield and antibacterial
activity (Tables 1 and 2) of rotamer 4 than 5, we have chosen 4
for further functionalization].
Since we have employed different heterocycles, optimization of
reaction condition (with suitable base and solvent) is the decisive
factor here. For six-membered cyclic amines, K2CO3/benzene sys-
tem worked well for both the series (6–9/19–22) except in piperi-
din-4-one, HCl, where NEt3/acetonitrile and crushed KOH/DMSO12
were used, respectively, to get compounds 10 and 23. Among the
different bases used for aromatic heterocyclic amines (for com-
pounds 11/24, 12/25, 13/26 and 14/27), crushed KOH in DMSO12
was chosen as an appropriate one as it push the reaction forward
in 2–3 h at room temperature itself. Obviously, K2CO3/benzene
was also a suitable system to achieve compounds 14 and 27 but re-
quires longer reaction time. In the case of 5-phenyltetrazole (ob-
tained through click reaction13 as shown in Scheme 1A in
Supplementary data), K2CO3/acetone was used to get 15 and 28
whereas for benzoxazol-2-thiol (achieved through the condensa-
tion of 2-aminophenol and CS2 as depicted in Scheme 1B in Supple-
mentary data)14 as nucleophile, NaOH/EtOH:H2O (1:1) and K2CO3/
benzene were used, respectively, to obtain compounds 16 and 29.
Nucleophilic substitution reaction to accomplish compounds 17
and 18 was successful only with KOtBu/dry THF while for 30 and
31, such strong base and dry condition were not needed instead
K2CO3/benzene and crushed KOH/DMSO, respectively, gave satis-
factory results.
hydroxy-
In continuation of our earlier findings, we now focused primar-
ily on 3-carboxyethyl-2,6-diphenyl-4-hydroxy-
3-tetrahydropyri-
D
3-tetrahydropyridine as the potent candidate.10
D
dine as our core structure for the generation of library of
compounds through its combination with other nitrogen, sulfur
and oxygen containing heterocycles, and some aromatic ring sys-
tems by means of acetyl and 2-propanoyl linkers. Thus in this com-
munication, we report on the initial structure–activity relationship
(SAR) studies of these diversified molecules that have been inves-
tigated for their antibacterial activity against a series of human
pathogenic Gram-positive and Gram-negative bacteria.
In order to study the impact of substituents about N-acyl sys-
tem [N–COCH2–Nu/N–COCH(CH3)-Nu (Nu-nucleophile)] in 1 on
antibacterial activity, we have prepared the respective N-acyl
intermediates 2 and 4/5 by simple coupling of 1 with bromoacetyl
chloride and 2-bromopropionyl bromide, respectively. Unlike
bromoacetylation, 2-bromo propionylation resulted in the forma-
tion of two separable isomers 4 and 5. Of the two, the isomer 4
formed as white crystal suitable for X-ray analysis. The ORTEP11
plot of 4 (Fig. 1) demonstrate that it crystallized with two indepen-
dent molecules per asymmetric unit, where the methyl group of
the ethoxycarbonyl unit is disordered over two positions. More-
over, the tetrahydropyridine ring in one of the independent mole-
cules adopts a half-chair conformation, while the other takes
distorted envelope conformation. Room temperature NMR re-
vealed that H-2 proton in 4 was deshielded significantly than H-
6 (in comparison with 5) whereas in 5, H-6, –COCHBr(CH3) and –
Earlier, the dynamic NMR study had confirmed the existence of
rotational isomers (syn and anti rotamers) at low temperatures in
the case of N-benzimidazolylacetyl-2,6-diarylpiperidin-4-ones
caused by restricted rotation about N–C@O bond.15 However, a sin-
gle set of signals characteristic for the above said compounds were
Figure 1. ORTEP plot of 4.11 (Compound crystallizes with two independent molecules per asymmetric unit and the methyl group of the ethoxycarbonyl unit is disordered
over two positions.

2244
G. Aridoss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2242–2249
O
OH
N
O
COOCH CH
2
3
COOCH CH
2
COOCH CH
3
3
H C
3
O
2
4
5
6
3
2
O
+
Mannich reaction
EtOH
1
N
H
1
BrCH2COCl
NEt3/Benzene
H H
O
NH OAc
4
2
Br
Suitable base
and solvent
R1
BrCH(CH3)COBr
DMAP/Dry DCM
PhCOCl
NEt3/Benzene
Z
H
R2
OH
N
OH
OH
OH
COOCH CH
COOCH CH
2
COOCH CH
2 3
COOCH CH
2
3
3
2
3
Suitable base
and solvent
N
N
N
R1
Br
3
O
Ph
Br
O
O
O
R
Z
H
3
CH
CH
Z = N/ O/ S
3
Z
R2
5 - anti rotamer
R2
4 - syn rotamer
R1
.
O
N
S
O
6-18 (R = H)
N
N
19-31 (R = CH3)
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
R1
R2
Z
H
12/25
11/24
13/26
9/22
10/23
6/19
7/20
8/21
N
HS
O
N
HO
N
HN
N
N
HS
N
H
N
18/31
17/30
14/27
16/29
15/28
Scheme 1. Synthesis of target compounds 6–35.
observed at room temperature (rt) NMR because the said rotamers
undergo faster interconversion on NMR time scale at rt. Likewise,
the compounds 6–18 in the present study (having the same struc-
tural analogy about N–C@O) also furnished only one set of signals
(average NMR signal) in CDCl3 at room temperature NMR. The par-
tial double bond character of the amide bond and unsaturation
about C(3)–C(4) bond due to keto–enol tautomerism in 6–18
provide approximately a non-chair conformation for the tetrahy-
dropyridine moiety where the ring becomes flattened at the nitro-
gen end with coplanarity of amide framework. This is also revealed
from the broadening of benzylic proton (H-2/H-6) signals with
their respective resonances in the most downfield region. As well,
more deshielding of H-2 proton compared to H-6 in 6–18 suggests
that in the preferred conformation of these compounds, H-2 proton
is syn to carbonyl oxygen and H-6 proton is anti to carbonyl group.
In order to confirm all these remarks, single crystal X-ray analysis
was carried out for the representative compound 1-(2-morpholi-
is about 34.5(1)° which suggests that phenyl at C5 is oriented in
equatorial direction while the other at C1 is in axial orientation.
As well, the observed bond angles and bond lengths about amide
group (given with Fig. 2) clearly reveal coplanarity of acetyl func-
tion besides syn orientation of carbonyl group to H-2.
Unlike compounds 6–18, 1H and 13C NMR spectra of 19–31 were
quite complex owing to substantial peak doubling. It seemed likely
that rotamers (syn and anti) of 19–31 exist in equilibrium in solu-
tion as a result of hindered or slow rotation of the amide C–N bond,
giving rise to this doubling phenomenon. To clarify these rotational
behaviors and NMR complexity, we have selected 1-[2-(1H-indol-
1-yl)propanoyl]-3-carboxyethyl-2,6-diphenyl-4-hydroxy-D
3-tetra-
hydropyridine (24) as a representative compound from these set of
compounds and studied extensively with the help of different NMR
techniques (dynamic, one and two-dimensional NMR) and single
crystal XRD.
Solution state 1H NMR spectrum of 24 showed two distinguish-
able sets of signals except for aromatic protons (complex multiplet
observed for aromatic protons) and hence could be unambiguously
assigned based on their relative intensity, multiplicity, integral and
chemical shifts values in support of 2D NMR spectral data, and also
by comparing the well assigned chemical shifts of the respective
intermediate rotamers (4 and 5). Thus, methyl protons of –
COOCH2CH3 and N–COCH(CH3) groups appeared as triplet and dou-
blet, respectively, at 0.59/1.09 ppm and 1.69/1.79 ppm while
methylene protons at C-5 appeared as doublet and multiplet at
2.69 and 2.81–2.94 ppm, respectively. In the region 3.12–
4.16 ppm, we have encountered another multiplet and two equal
intense sextets. Here, the multiplet (centered at 4.15 ppm) could
be assigned to –COOCH2CH3 of one of the rotamer whereas for
the counter rotamer, these geminal protons became non-equiva-
lent (diasteriotopic protons: –COOCHaHbCH3) and hence showed
two equal intense sextets centered, respectively, at 3.14 and
3.78 ppm. Similarly, pair of each singlet, triplet and quartet at
noacetyl)-3-carboxyethyl-2,6-diphenyl-4-hydroxy-D
3-tetrahydro-
pyridine 9 [the crystallographic data and refinement parameters of
this compound are furnished in Supplementary data (Table 1)] and
its ORTEP diagram is shown in Figure 2 with important bond
lengths and bond angles. The X-ray data of 9 suggests that sum
of the bond angles at N1 (357.9(6)°) and N2 (329.4(6)°) are in
accordance with sp2 and sp3 hybridization, respectively. The puck-
ering parameters16 and the smallest displacement asymmetry
parameters17 for the morpholine and tetrahydropyridine rings
are q2 = 0.014(3), 0.356(2) Å, q3 = À0.576(3), 0.291(2)
Å QT =
0.576(3), 0.460(2) Å and h = 177.5(3)°, 50.8(2)°, respectively. The
morpholine moiety adopts chair conformation with atoms N2
and O5 deviating by À0.678(1) and 0.648(1) Å, respectively, from
the least square plane defined by rest of the atoms in the ring
and the tetrahydropyridine moiety (atoms N1/C1–C5) adopts dis-
torted half-chair conformation. And, the dihedral angle between
the two phenyl rings attached to the tetrahydropyridine moiety

G. Aridoss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2242–2249
2245
Table 1
In vitro antibacterial activities (MIC90 in
bacterial strains
l
g/mL) of compounds 2–31 and, reference drugs Linezolid and Trovafloxacin against selected sensitive and resistant Gram-positive
Entry
Strains
S.Aa
S.Ab
S.Ec
S.Ed
E.Fe
E.Ff
E.Fg
E.Fh
E.Fi
Minimum inhibitory concentration (MIC90) of compounds in
l
g/mL
2
3
4
5
6
7
8
9
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
128
>128
128
>128
128
128
>128
>128
>128
>128
>128
64
64
128
>128
128
128
>128
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
10
11
12
1310
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
X
32
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
2
128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
64
64
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
2
>128
>128
>128
>128
>128
2
>128
>128
>128
>128
>128
8
Y
<0.0625
2
<0.0625
<0.0625
<0.0625
16
16
128
32
LCB—M/s LegoChem Biosciences, Inc., Daejeon, Republic of Korea (where the activity test was carried out); X—Linezolid (standard drug); Y—Trovafloxacin (standard drug).
a
S. aureus (LCB0001).
Methicillin-resistant S. aureus (LCB0002).
S. epidermidis (LCB0003).
Methicillin-resistant S. epidermidis (LCB0004).
E. faecalis (LCB0005).
Vancomycin-resistant E. faecalis Van A phenotype (LCB0006).
Vancomycin and Linezolid-resistant E. faecalis Van A phenotype (LCB0007).
Vancomycin-resistant E. faecium Van A phenotype (LCB0008).
b
c
d
e
f
g
h
i
E. faecium (LCB0009).
7.01/5.89, 5.24/5.82 and 5.42/5.73 ppm are, respectively, due to H-
2, H-6 and N–COCH(CH3). In order to confirm the above made
assignments and to differentiate two sets of signals corresponding
to each rotamer, COSY NMR was recorded. COSY spectrum exhib-
ited strong mutual correlations between 1.09/4.16–4.10; 1.79/
5.42; 2.81–2.94/5.24 ppm in one set of signals (for rotamer 1)
and 0.59/3.14, 3.78; 1.69/5.73; 2.69/5.82; 3.14/3.78 ppm in other
set of signals (for rotamer 2). Thus, the noticed correlations al-
lowed for the easy identification and assignment of set of signals
pertaining to each rotamer (refer Supplementary data). Here the
ratio of major (signals with more intensity) to minor (signals with
less intensity) rotamer was evaluated to be 60:40 on the basis of
the integral value of ester methyl protons. Furthermore, exchange
cross-peaks18 observed in NOE (refer Supplementary data) also
confirmed the existence of two exchangeable rotamers in solution.
By taking into account the d values of H-2 (7.01 ppm) proton in
major isomer which experiences strong anisotropic deshielding ef-
fect by C@O group of propanoyl linker, it was inferred that H-2 is
oriented syn (cis) to C@O group and anti (trans) to –CH(CH3) group
whereas that in minor isomer, H-2 is comparatively shielded
(5.89 ppm) and thus directed anti (trans) to C@O group and syn
(cis) to –CH(CH3) group. As well, the observed vicinal coupling con-
stant values (6.85 and 6.43 Hz) for H-6 proton in both the rotamers
suggest that the tetrahydropyridine ring is not in the normal chair
conformation. Furthermore, to check the dynamics of the intercon-
version between the two rotamers, a variable temperature NMR
study was carried out in CDCl3 over the temperature range 293–
333 K (20–60 °C). Figure 3 shows that upon increasing the temper-
ature, line broadening occurs up to 313 K and finally, each couple
of signals corresponding to major and minor rotamers, almost coa-
lesces to one resonance (average NMR) at 333 K. Dynamic NMR
study confirms our hypothesis that at room temperature, slow ex-
change process occurs between the rotamers when compared to
NMR time scale (and thus gave two sets of signals) while at high
temperature, fast exchange process leads to broadening and coa-
lescence of the each set of signals in 24.
Observations of two sets of signals in 13C NMR was also in
agreement with the existence of both syn and anti rotamers in
solution (refer Supplementary data). Once the resonances of the
respective protons were established, analysis of the HSQC spectra
allowed for the distinction between the chemical shift values of
the carbons of each rotamer and their respective assignments.
Unequivocal assignment of important quaternary carbons such as
C-3, amide and ester carbonyl carbon for both the series of signals
was derived from HMBC experiment because it verified the exis-
tence of two and three-bond connectivities of the said carbons

2246
G. Aridoss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2242–2249
Table 2
In vitro antibacterial activities (MIC90 in
l
g/mL) of compounds 2–31 and, reference drugs Linezolid and Trovafloxacin against selected Gram-negative bacterial strains
Entry
Strains
E.Ca
E.Cb
E.Cc
P.Ad
K.Pe
H.If
M.Cg
Minimum inhibitory concentration (MIC90) of compounds in
lg/mL
2
3
4
5
6
7
8
9
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
<0.0625
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
64
>128
>128
64
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
128
>128
>128
>128
>128
32
32
64
64
64
32
64
32
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
10
11
12
1310
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
X
64
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
4
128
64
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
0.5
>128
>128
>128
>128
>128
>128
>128
0.125
Y
<0.0625
<0.0625
<0.0625
<0.0625
LCB—M/s LegoChem Biosciences, Inc., Daejeon, Republic of Korea (where the activity test was carried out); X—Linezolid (standard drug); Y—Trovafloxacin (standard drug).
a
E. coli (LCB0010).
E. coli (LCB0011).
E. coli (LCB0012).
P. aeruginosa (LCB0013).
K. pneumoniae (LCB0014).
H. influenzae (LCB0015).
M. caterrhalis (LCB0016).
b
c
d
e
f
g
with the adjacent protons (refer Supplementary data for HMBCcor-
relations-Table 2).
of 2-propanoyl moiety with C1–N1–C5 plane is noticed from the
observed bond parameters (given with Fig. 4). Moreover, the two
phenyl rings attached to the tetrahydropyridine moiety are nearly
perpendicular to each other (i.e., to relieve A1,3 strain by the coplanar
carbonyl group, phenyl groups are flipped from equatorial to axial
orientation) with the dihedral angle of 88.3(1)°. Thus, according to
the observed NMR and X-ray data, two boat conformers (which
are in equilibrium with each other in solution) are proposed for
syn and anti rotamers of compound 24 as shown in Figure 5.
In order to examine the conformational preference in solid
state, crystal structure was studied for 1-[2-(1H-indol-1-yl)propa-
noyl]-3-carboxyethyl-2,6-diphenyl-4-hydroxy-D
3-tetrahydropyri-
dine (24), which fortunately obtained as a single crystal by slow
evaporation of a solution in ethanol. The crystallographic data
and refinement parameters of 24 are furnished in Supplementary
data (Table 3). ORTEP plot of asymmetric unit of 24 shown in
Figure 4 (with important bond lengths and bond angles) obviously
displays that the crystal structure of 24 is in stable syn form (C@O
syn to H-2). The sum of the bond angles at N1 [359.4(3)°] (revealed
from the shortening of C(6)–N(1) bond length to 1.355 Å by the
partial double bond character of N1–C6@O1) and N2 [359.8(3)°]
are in accordance with sp2 hybridization. The torsion angles C11–
C10–C15–N2 [À179.9(2)] and C9–C10–C15–C14 [179.3(2)] suggest
that the indole moiety is planar. The tetrahydropyridine ring
(atoms N1/C1–C5) in the present structure adopts boat conforma-
tion with atoms C2 and C5 deviating by 0.495(1) Å and 0.500(1) Å,
respectively, from the least square plane defined by rest of the
atoms in the ring. The puckering parameters16 and the smallest
displacement asymmetry parameters17 for tetrahydropyridine ring
In view of the similarity of the 1H and 13C NMR spectra for the
tetrahydropyridine moiety in amide 24 with other derivatives (19–
23 and 25–31), the complete assignment of the individual protons
and carbons for both the rotamers has been made. Finally, NMR
(dynamic, one and two-dimensional NMR) studies concluded the
existence of inseparable rotamers in equilibrium in solution slowly
interconverting to each other on NMR time scale at room temper-
ature, conversely, the single crystal X-ray analysis confirmed the
existence of only one form (i.e., stable syn form) in solid state.
In vitro antibacterial testing of the series of derivatives was car-
ried out by using National Committee for Clinical Laboratory Stan-
dards (NCCLS)19 assay against a panel of both susceptible and
resistant Gram-positive bacterial strains viz. methicillin-sensitive
S. aureus/S. epidermidis, methicillin-resistant S. aureus/S. epidermidis,
E. faecalis, E. faecium, vancomycin-resistant E. faecalis/E. faecium
and vancomycin and Linezolid-resistant E. faecalis. The observed
MIC90 (MIC90 is the minimum concentration of an antibacterial
are q2 = 0.575(2) Å, q3 = À0.001(2) Å, QT = 0.575(2)
Å and
h = 90.1(2)°. And, unsaturation about C(3)–C(4) bond is established
from the decrease in its bond length (1.34 Å) compared to other
single bonds (ꢀ1.5 Å) in the ring system whereas the coplanarity

G. Aridoss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2242–2249
2247
dard drugs Linezolid and Trovafloxacin. Test compounds were pre-
pared at an initial concentration of 128 g/mL in DMSO and
l
serially diluted resulting in twofold dilution. Here DMSO and
growth medium served as growth control and sterility control,
respectively.
A preliminary assay data (Table 1) showed that the compounds
bearing six-membered saturated cyclic amines were not efficient
in inhibiting the growth of all the tested strains except 6 and 7,
which exhibited inhibition at 128 lg/mL against E. faecium.
Replacement of the said amines by aromatic amines has shown
moderate improvement in their activity. Though compound 11
bearing indole moiety is inactive against all the strains under
study, its replacement by imidazole gave compound 12 that
showed better inhibitory potency at a MIC of 64 lg/mL against
methicillin-resistant S. aureus, vancomycin-resistant E. faecium-
VanA phenotype, and vancomycin and Linezolid-resistant E. faecal-
is-VanA phenotype. Here, the effectiveness of compound 12 against
vancomycin-resistant E. faecium-VanA was found to be doubled to
that of Trovafloxacin drug while against vancomycin and Linezo-
lid-resistant E. faecalis-VanA, an equal potency was noted to that
of Linezolid. Similarly, the corresponding benzotriazole derivative
14 showed same potency to that of Travofloxacin against vanco-
mycin-resistant E. faecium-VanA while against E. faecium and
methicillin-resistant S. epidermidis, it has registered MIC at 64
and 128 lg/mL, respectively. On the other hand, the derivatives
15–18 were almost inactive towards all the pathogens with the
exception of E. facium against which compounds 15, 17 and 18
bearing, respectively, 5-phenyltetrazole, 2-naphthalenethiol and
2-naphthol showed inhibition at a minimum concentration of
Figure 2. ORTEP plot of 9. The important bond lengths (Å): C(1)–N(1) = 1.470(3);
C(5)–N(1) = 1.476(3); C(21)–N(1) = 1.356(3); C(21)–O(4) = 1.218(3); C(21)–C(22) =
1.520(3); C(2)–C(3) = 1.484(3); C(3)–C(4) = 1.346(3); C(4)–C(5) = 1.507(3). The
important bond angles (°): N(2)–C(22)–C(21) = 114.4(19); O(4)–C(21)–N(1) =
121.4(2); N(1)–C(21)–C(22) = 120.2(2); N(1)–C(5)–C(6) = 113.3(18); C(1)–N(1)–
C(5) = 116.8(17); C(21)–N(1)–C(5) = 117.1(19); C(21)–N(1)–C(1) = 123.9(18).
128 lg/mL. To investigate the effect of C-methyl group in the
N-acetyl functionality on the antibacterial activity, we have syn-
thesized the other series of compounds (19–31) and screened
against the same panel of Gram-positive strains. However, this sec-
ond series of compounds also displayed the same trend in the anti-
bacterial profile. The saturated cyclic amine derivatives 19–23
were inactive up to a maximum concentration of 128
19 and 22, for which growth inhibition was noticed at a minimum
concentration of 128 g/mL against E. facium. Switching over from
lg/mL except
agent required to inhibit the growth of 90% of organisms) values of
the test compounds are reproduced in Table 1 along with the stan-
l
Figure 3. Temperature dependant NMR spectra of 24.

2248
G. Aridoss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2242–2249
Figure 4. ORTEP plot of 24. The important bond lengths (Å): C(1)–N(1) = 1.479(19); C(5)–N(1) = 1.490(2); C(6)–N(1) = 1.355(2); C(6)–O(1) = 1.219(19); C(6)–C(7) = 1.536(2);
C(2)–C(3) = 1.478(3); C(3)–C(4) = 1.336(2); C(7)–C(28) = 1.521(2); The important bond angles (°): N(2)–C(7)–C(6) = 110.3(13); O(1)–C(6)–N(1) = 122.3(15); N(1)–C(6)–
C(7) = 119.4(14); C(6)–N(1)–C(1) = 122.9(13); C(6)–N(1)–C(5) = 116.5(13); C(1)–N(1)–C(5) = 119.9(13).
CH
3
H
O
H
O
H
CH
O
4
H
H
3
CH
H
O
3
5
4
3
N
N
Ph
Ph
O
Hb
O
Ha
N
N
2
O
1
6
H
H
1
H
CH
O
H
3
H
3
6
2
5
No A1,3 strain between phenyl and
carbonyl group
Ph
Ph
H
H-H COSY
HMBC
anti rotamer
syn rotamer
Figure 5. syn and anti forms of compound 24 in equilibrium in solution. Selected H–H COSY and HMBC correlations are shown (both syn and anti rotamers exhibited the same
kind of correlations while the additional COSY correlations observed in anti rotamer are shown separately). Bonds in red color highlight the coplanarity of 2-propanoyl system
with C2–N1–C6 plane.
saturated cyclic amines to aromatic amines, imidazole (25) and
benzimidazole (26) only exhibited marked activity against some
of the sensitive and resistant Gram-positive pathogens while their
replacement by either of the other heterocyclic amines or naphtha-
lene derivatives led to complete loss of antibacterial potency. Com-
pared to 12 (N-acetyl derivative), Compound 25 [N-2-(propanoyl)
derivative with imidazole nucleus] exhibited twofold increased
inhibitory potency against S. aureus and onefold decreased potency
against vancomycin-resistant E. faecium. Nonetheless, potency of
25 against Vancomycin-resistant E. faecium is equivalent to that
of Trovafloxacin. Substitution of benzimidazole in place of imidaz-
compounds 3, 6, 12, 23–24 registered an attractive and comparable
inhibitory power to Linezolid drug against E. coli LCB0012. N-Ben-
zoyl derivative 3 registered MIC at 64 lg/mL while the incorpora-
tion of piperidinoacetyl (compound 6) and imidazolylacetyl
(compound 12) in lieu of benzoyl group retained the same potency.
But the other kind of heterocycle modification leads to the loss of
activity. Moreover, compounds 10 and 11, which were inactive
against E. coli LCB0012, became active with MIC 128 lg/mL
through the introduction of methyl group in the N-acetyl moiety
(i.e., compounds 23 and 24, respectively, with N-2-propanoyl func-
tionality) but the same kind of modification in 12 (compound 25)
did not enhance the potency. A striking observation from the re-
sults in Table 2 is that compounds 23/24 and 3/6/12/25 demon-
strated, respectively, equivalent and onefold improved inhibitory
power than Linezolid. Apart from 4/25, 25, 4 against P. aeruginosa,
ole in 25 (i.e., compound 26) showed inhibitions at 64 and 128 lg/
mL against S. aureus/MR S. aureus and MR S. epidermidis, respec-
tively, while against rest of the strains, it was almost inactive.
Besides these antibacterial screening against a panel of Gram-
positive organisms, they were also examined towards a group of
pathogenic Gram-negative bacteria such as three different E. coli
strains (LCB0010–LCB0012), P. aeruginosa, K. pneumoniae, H. influen-
zae and M. catarrhalis. The observed MIC90’s are reproduced in
Table 2.
K. pneumoniae and H. influenzae (MIC at 128
all other derivatives were inactive up to a concentration of
128 g/mL. Against M. caterrhalis, most of the N-acetyl derivatives
(6–13 with MIC 32–64 g/mL) seems better than the correspond-
ing N-(2-propanoyl) derivatives (19, 20, 25 with MIC 64 g/mL).
lg/mL) respectively,
l
l
l
It is interesting to note that even if substitution of various het-
erocycles or homocycles results in the devoid of antibacterial activ-
ity against two types of E. coli strains LCB0010 and LCB0011,
Thus substitution of either piperidine (6) or N-methylpiperazine
(7) system in place of bromine in 2 recorded about twofold im-
proved antibacterial activity (32 lg/mL) while their replacement

G. Aridoss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2242–2249
2249
by either thiomorpholine (8) or morpholine (9) or piperidin-4-one
(10) resulted in onefold (64 g/mL) enhancement. Similarly, intro-
duction of aromatic heterocycles such as indole (11) and imidazole
(12) also recorded MIC, respectively, at 32 and 64 g/mL.
References and notes
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l
In conclusion, we have achieved heterocyclic and homocyclic
substituted acetyl (6–18) and propanoyl (19–31) derivatives of
tetrahydropyridin-4-ol in moderate to good yields and studied
extensively through various NMR techniques. The cumulative
NMR and X-ray analysis concluded that all the compounds 6–
31 were in stable syn form in solid state whereas propanoyl
derivatives 19–31 exist in two inseparable rotameric conforma-
tions (syn and anti) in solution. Antibacterial screening study
against clinically relevant Gram-positive and Gram-negative
pathogens revealed that acetyl derivatives were comparatively
better than 2-propanoyl analogues. Generally amide functional
group has different steric, electronic and lipophilic characteristics
depending upon the substituent in it. Moreover, the possible
conformation of the acetyl derivatives may allow for the facile
penetration of the cell wall of the bacteria while for the corre-
sponding 2-propanoyl derivatives, such physical process may
be lacking owing to their assumed conformation. As well, among
the various heterocycles tested here, imidazole derivatives (12
and 25) are emerged as potent compounds as they showed
promising and comparable activity to that of the standard Lin-
ezolid and Trovafloxacin drugs particularly against multiple
resistant enterococci VanA phenotype strains, thereby considered
for further structural optimization.
Acknowledgments
This research work was supported by the grant from second
stage of BK21 program. We are also grateful to M/s LegoChem Bio-
sciences, Inc., Daejeon, Republic of Korea for their help in conduct-
ing antibacterial screening tests and Dr. D. Gayathri, Institute of
Structural Biology and Biophysics-2, Germany for her useful dis-
cussions about crystal structure of the compounds.
11. Aridoss, G.; Gayathri, D.; Velmurugan, D.; Kim, M. S.; Jeong, Y. T. Acta
Crystallogr. Sect. E 2009, 65, o1708.
12. Wilson, Z. E.; Heapy, A. M.; Brimble, M. A. Tetrahedron 2007, 63, 5379.
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Supplementary data
Detailed experimental procedure and characterization data for
all the compounds along with 1H/13C NMR, COSY, NOESY, HSQC
and HMBC spectra for the representative compound 24. The
crystallographic data of 9 and 24 have been deposited at Cam-
bridge Crystallography Data Center (CCDC Nos. 685679 and
748022, respectively). Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.bmcl.2010.02.015.
17. Nardelli, M. Acta Crystallogr. Sect. C 1983, 39, 1141.
18. Marakos, P.; Pouli, N.; Garoufalias, S. P.; Mikros, E. J. Mol. Struct. 2003, 650, 213.
19. National Committee for Clinical Laboratory Standards. Methods for dilution
antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed.
Approved Standard. NCCLS Document M7-A5, National Committee for Clinical
Laboratory Standards, Villanova, PA, 2000.