M. Velayutham Pillai et al. / Journal of Molecular Structure 1076 (2014) 174–182
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wide range of biological activities such as antibacterial and tuber-
culostatic properties. Moreover, hydrazine and acyl hydrazine moi-
eties [16] are also found to be the most important pharmacophore
in many anti-inflammatory, anti-nociceptive and anti-platelet drug
molecules. Cyanoacetyl hydrazones have been reported to exhibit
antitumor activity [17–19] against several cell lines. The molecules
containing piperidine as well as nitrile moieties [20] such as Peri-
cyazine, Alogliptin, Levocabastine, Piritramide, Diphenozylate are
known for their excellent biological potencies.
As the reactivity as well the pharmacological properties of the
drugs depend mostly on their stereochemistry, trials are being con-
tinued to develop new synthetic procedures that lead to stereose-
lectivity [21,22]. Among the several spectral techniques available,
NMR spectroscopy is a well established method for configurational
and conformational studies since the stereoisomers differ consid-
erably in their NMR spectra [23]. Hence, in the present work the
stereoselective synthesis of such hybrid molecules viz. cyanoacetyl
hydrazone derivatives of piperidin-4-ones, NMR spectral studies
along with others to establish the stereochemistry and evaluation
of their antibacterial and antifungal potencies against some
selected micro-organisms have been focused.
Using a sterile inoculation loop, 20 pure colonies of the test
organism were transferred to 5 ml of sterile nutrient broth and
incubated at 37 °C for 18 h. The modified agar well diffusion
method of Perez et al. [27] was employed. Each selective med-
ium was inoculated with the microorganism suspended in ster-
ile water. Once the agar was solidified, it was punched with
6 mm diametre wells and filled with 25 lL of the sample and
blank (ethanol and antibiotic). The test was carried out by trip-
licate. The plates were incubated at 35 2 °C for 24 h. The anti-
microbial activity was calculated by applying the expression in
mm.
Results and discussion
A series of new cyanoacetyl hydrazones of 3-alkyl-2,6-diarylpi-
peridin-4-ones were prepared (Scheme 1) in good to excellent
yields by the condensation of cyanoacetic hydrazide with piperi-
din-4-ones.
A higher degree of diastereoselectivity (selective
formation of E isomer) was achieved owing to the presence of alkyl
substituent adjacent to the carbonyl group which decides the ori-
entation of the hydrazone moiety (ANHAN@) as anti with respect
to it. Therefore the stereoselectivity of the reaction is ascertained
as substrate controlled. The numbering of carbon atoms in the
compounds is shown in Fig. 1 and the protons are numbered
accordingly. The newly synthesized hydrazones were character-
ized by IR, Mass, 1H NMR, 13C NMR, 1H–1H COSY and 1H–13C COSY
spectral studies. The signals in the 1H NMR spectra were assigned
based on their chemical shift values, splitting pattern, coupling
constants and correlations in the 2-D NMR spectra for compound
26 and for other compounds the signals were assigned by compar-
ing chemical shift values of them with the former. The 1H–1H COSY
and 1H–13C COSY unraveled the problems during assignment of the
signals in 1H and 13C NMR spectra.
Experimental
Materials and methods
All the solvents used for recrystallization and thin layer chro-
matography were of analytical grade and used without further
purification. The progress as well as the completion of reactions
was monitored by thin layer chromatography on silica gel
precoated aluminum sheets (Type 60 GF254, Merck). All the final
compounds were purified by repeated crystallization from suitable
solvents. IR spectra were recorded on an AVATAR-330 FT-IR
spectrometer (Thermo Nicolet) using KBr (pellet form). The mass
spectra of the synthesized compounds were recorded on a Varian
Saturn 2000 GC–MS/MS spectrometer using electron impact tech-
nique. 1H and 13C NMR spectra for all the compounds were
recorded at 400 MHz and 100 MHz, in a Bruker instrument, using
CDCl3 as solvent. Tetramethylsilane (TMS) was used as an internal
reference for all NMR spectra, with chemical shifts reported in d
units (parts per million) relative to the standard. 1H NMR splitting
patterns are designated as singlet (s), broad singlet (bs), doublet
(d), doublet of doublets (dd), triplet (t) and multiplet (m). Coupling
constants are expressed in Hertz (Hz).
Analysis of IR and mass spectra
Infrared spectra of the synthesized compounds exhibited signif-
icant absorptions pertaining to the functional groups present such
as imino(C@N), carbonyl(C@O),cyano(CN) and the absorption
frequencies (in cmꢁ1) are presented in Table 1. Mass spectra of
recorded compounds have shown (M + H)+ values and the values
are included in Table 1.
Analysis of 1H NMR spectra
The 1H chemical shifts of all the synthesized compounds are
tabulated in Table 2. The 1H NMR spectrum of compound 26 is
shown in Fig. 2. In the 1H NMR spectrum of compound 26, a sharp
singlet found in the downfield region at 9.21 ppm corresponding to
one proton is ascribable to the ACONHA hydrogen of the hydra-
zone moiety. All the protons of the phenyl rings at C2 and C6 car-
bons resonate from 7.47 ppm to 7.29 ppm as multiplets
corresponding to 10 hydrogens. The methyl protons at nitrogen
(N1) and C3 are observed as sharp singlet and doublet respectively
in the upfield region at 1.73 ppm and 0.81 ppm corresponding to
three protons each.
Two closely spaced doublets forming an AB quartet around
3.78 ppm correspond to two protons are assigned as the methylene
protons of C10 based on their spin–spin coupling constant values.
The actual chemical shift values of these diastereotopic geminal
protons exhibiting AB spin system of coupling was found out using
second order spectral analysis [24]. Further, a multiplet centered at
2.69 ppm with one proton integral is assignable to H3 proton. In
addition, a doublet at 3.22 ppm with a large vicinal coupling con-
stant 3J = 11.2 Hz showing cross peak in the 1H–1H COSY spectrum
Procedure for the synthesis of compounds 15-28
The parent compounds (1–14) were synthesized by Mannich
condensation of aromatic aldehydes, ketones and ammonium ace-
tate in ethanol. Compounds 1–3 were methylated by methyl iodide
in the presence of K2CO3 and acetone at refluxing conditions. The
title compounds 15–28 were prepared as follows: A mixture of
3-alkyl-2,6-diphenylpiperidin-4-ones (2 mmol) and cyanoacetic
hydrazide (2 mmol) in ethanol (10 mL) was refluxed for 3–6 h.
The progress of the reaction was monitored by TLC. After comple-
tion of the reaction, the solid product separated on cooling was
collected by filtration and washed well with water. Pure sample
was obtained by recrystallization from 1:1 mixture of ethanol
and ethyl acetate.
Procedure for antimicrobial assay
The antimicrobial activities of the synthesized compounds
against different pathogens were determined by Agar Well diffu-
sion method or cork borer method.
Velayutham Pillai
