Synthesis, spectral, structural and biological studies of N-cyclohexyl-2-(2,4-diphenyl-3-azabicyclo[3.3.1]nonan-9-ylidene)hydrazinecarbothioamide derivatives

Article abstract of DOI:10.1016/j.molstruc.2015.01.044

Potential antimicrobial activities of azabicyclic skeleton based N-cyclohexyl-2-(2,4-diphenyl-3-azabicyclo[3.3.1]nonan-9-ylidene)hydrazinecarbothioamides (6-10) was synthesized. The newly synthesized compounds were characterized by FT-IR, ¹H NM

Full text of DOI:10.1016/j.molstruc.2015.01.044

Accepted Manuscript
Synthesis, spectral, structural and biological studies of N –cyclohexyl–2–(2,4–
diphenyl–3–azabicyclo[3.3.1]nonan–9–ylidene)hydrazinecarbothioamide de-
rivatives
A. Kamaraj, R. Rajkumar, K. Krishnasamy
PII:
S0022-2860(15)00063-0
DOI:
http://dx.doi.org/10.1016/j.molstruc.2015.01.044
MOLSTR 21289
Reference:
Journal of Molecular Structure
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 September 2014
26 January 2015
26 January 2015
Please cite this article as: A. Kamaraj, R. Rajkumar, K. Krishnasamy, Synthesis, spectral, structural and biological
studies of –cyclohexyl–2–(2,4–diphenyl–3–azabicyclo[3.3.1]nonan–9–ylidene)hydrazinecarbothioamide
N
derivatives, Journal of Molecular Structure (2015), doi: http://dx.doi.org/10.1016/j.molstruc.2015.01.044
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Synthesis, spectral, structural and biological studies of N–cyclohexyl–2–(2,4–
diphenyl–3–azabicyclo[3.3.1]nonan–9–ylidene)hydrazinecarbothioamide
derivatives
A. Kamaraj, R. Rajkumar and K. Krishnasamy*
Department of Chemistry, Annamalai University, Annamalainagar–608 002, Tamilnadu. India.
Corresponding Author– K. Krishnasamy
K. Krishnasamy
Professor
Department of Chemistry
Annamalai University
Annamalainagar
Chidambaram
Mobile no.: 09942547856
Fax: 91 – 4144 – 238080
Email: krishnasamybala56@gmail.com

Synthesis, spectral, structural and biological studies of N–cyclohexyl–2–(2,4–
diphenyl–3–azabicyclo[3.3.1]nonan–9–ylidene)hydrazinecarbothioamide
derivatives
A. Kamaraj, R. Rajkumar and K. Krishnasamy*
Department of Chemistry, Annamalai University, Annamalainagar–608 002, Tamilnadu. India.
ABSTRACT
Potential antimicrobial activities of azabicyclic skeleton based N–cyclohexyl–2–(2,4–diphenyl–
3–azabicyclo[3.3.1]nonan–9–ylidene)hydrazinecarbothioamides (6–10) was synthesized. The
1
newly synthesized compounds were characterized by FT–IR, H NMR, 13C NMR, HRMS and
elemental analysis. The synthesized compound (6) was further compared by 2D NMR
techniques (HOMOCOSY, NOESY and HSQC). The single crystal XRD study revealed that the
compound 6 adopts a twin–chair conformation with equatorial orientation of the aryl groups.
Antimicrobial activities have been carried out for compounds 6-10 against a panel of bacterial
and fungal strains using Streptomycin and Amphotericin B as standards.
Keywords:
3–Azabicyclo[3.3.1]nonan-9–one, 2D NMR, Cyclohexylthiosemicarbazide, Antibacterial
activity, Antifungal activity, Crystal structure
1. Introduction
3–Azabicyclo[3.3.1]nonanes (3–ABN) have attracted the attention of researchers owing
to their extensive scale of microbial potencies. Antifungal and antibacterial studies proved that
3–ABN skeleton seems to be an effective pathogen killer [1, 2]. Further reports revealed that the

analgesic [3], anti–inflammatory [4], antipyretic [5] and antiphologistic [6] capabilities of
azabicyclic ketones. Narcotic antagonism and antitussive and sedative properties of these
heterobicycles were also described [7]. Calcium antagonist, hypotensive, and local anesthetic
activities [8] are measured. A wide variety of naturally abundant diterpenoid/norditerpinoid
alkaloids have been found to possess various pharmacological actions due to the presence of the
3–azabicyclo[3.3.1]nonane pharmocopore. The wide–spread nature and diverse biological
actions of this heterocycle attracted more attention in the recent years and as a consequence,
synthesis of large number of analogous molecules to improve their biological efficacy [9]. The
chemistry of 3–azabicyclo[3.3.1]nonan–9–one has been reviewed [10]. The use of different
substituents particularly aromatic substituents with regard to biological activity has been
demonstrated [11]. The biological importance of bridged bicyclic compounds exhibit twin chair,
chair-boat or twin boat conformations with interesting stereochemistry [12].
The correlation of reactivity and stability of heterocyclic compounds with their
predominant conformation has provided a valuable method of assigning the stereochemical
conformation of the isomers. The multicomponent reactions are importance now-a-days in
synthetic organic chemistry, since they offer one pot combination of more than two components
in one step allowing direct access to target molecule [13].
Thiosemicarbazones were reported to be associated with antimicrobial activities [14–16].
Several semicarbazones, as well as their sulfur analogues and derivatives, have proved their
efficiency and efficacy in combating various diseases [17]. It is great interest because of their
chemistry and beneficial biological activities such as antitumor, antibacterial, antiviral,
antimalarial and antiprotozoal [18–21]. Semicarbazones have documented consistent advances
in the design of novel anticonvulsant agents through the work of Dimmock and others [22–24].

By considering the synthetic and biological significance of 3–azabicyclo[3.3.1]nonan–9–one
thiosemicarbazones, it was thought worthwhile to synthesize them and screen their biological
activities. This study will be much useful for the development of new biologically active
molecules.
A new series of N–cyclohexyl–2–(2,4–diphenyl–3–azabicyclo[3.3.1]nonan–9–ylidene)
hydrazinecarbothioamides (6–10). The synthesized compounds (6-10) were confirmed by
physical constants, elemental analysis and spectral techniques. Single crystal X–ray diffraction
analysis has also been carried out for the representative compound 6. The synthesized
compounds were screened for their antibacterial and antifungal activities and their results were
interpreted in terms of the SAR principles.
2. Experimental
2.1. General
All the chemicals used were of high grade and purchased from sigma Aldrich. Solvents
used were of commercial grade and used after purification. The synthesized compounds melting
points were taken in open capillaries and are uncorrected. FT–IR spectra were recorded in
AVATAR 330 FT–IR Thermo Nicholet spectrophotometer (range 4000–400 cm–1) as KBr
13
pellets as reference standard. NMR spectra (1H and C) were recorded at ambient temperature
1
on a BRUKER AMX 400 and 500 NMR spectrometer operating at 400 and 500 MHz for H and
100 and 125 MHz for 13C. Solutions for the measurement of spectra were prepared by dissolving
10 mg of the sample in 0.5 mL CDCl3 and chemical shifts (δ) are expressed in ppm. High–
resolution mass spectra (HRMS) were performed on VG 7070H mass spectrometer. Elemental
analyses were performed on FINNAGAN EA1112 analyzer, their results were found to be in
good agreement with the calculated values.

2.2. Synthesis of 2,4–diaryl– 3–azabicyclo[3.3.1]nonan–9–ones (1–5)
Substituted 3–azabicyclo[3.3.1]nonan–9–ones were prepared by following the procedure
of Baliah and Jayaraman [25]. Dry ammonium acetate (100 mmol) was dissolved in ethanol and
the
solution was mixed with cyclohexanone (100 mmol) and substituted benzaldehyde
(200 mmol). These reaction mixtures were very gently warmed and stirred until the completion
of the reaction. The crude products formed were filtered and washed with an ethanol–ether (1:5)
mixture to yield the compounds 1–5 and recrystallized from chloroform to obtain the pure
compounds 1–5.
2.3. N–cyclohexyl–2–(2,4–diphenyl–3–azabicyclo[3.3.1]nonan–9–ylidene)hydrazinecarbothio
amides (6–10)
Title compounds (6–10) were synthesized by stirring a mixture of respective
3–azabicyclo[3.3.1]nonan–9–ones (1–5) (25 mmol) and cyclohexylthiosemicarbazide (25 mmol)
in ethanol (10 ml) containing and few drops of acetic acid at room temperature for 5 h. Then the
separated solid was washed with ice–cold water [26]. The crude product was recrystallized from
chloroform. A single-crystal suitable for X–ray structure analysis was obtained for compound 6
by slow evaporation of a solution in a mixture of chloroform and ethanol (1:1 v/v) at room
temperature.
2.4. Analytical data for compounds (6-10)
2.4.1. N–cyclohexyl–2–(2,4–diphenyl–3–azabicyclo[3.3.1]nonan–9–ylidene)hydrazine
carbothioamide (6)
1
White solid, mp=161–163 °C, FT–IR (KBr): 3331, 3303, 1528, 1204 cm–1, H NMR
(400 MHz, CDCl3): 8.51 (s, NH, 1H), 7.51 (d, NH, 1H), 7.29-7.58 (m, Ar–CH, 8H), 4.37

(s, H–2, 1H), 4.34 (m, H–1', 1H), 4.23 (s, H–4, 1H), 2.86 (s, H–5e, 1H), 2.82 (m, H–7a, 1H),
2.50 (s, H–1e, 1H), 2.17 (m, H–2'e/H-6'e, 2H), 1.76–1.83 (m, H–6e/H–8e/H–3'e/H–5'e/ NH,
5H), 1.68 (m, H–4'e, 1H), 1.25–1.54 (m, H–4'a/H–2'a/H–6'a/H–7e/H–3'a/H–5'a/H–6'a/H–6a/
H–8a, 9H), 13C NMR (100 MHz, CDCl3), 176.5 (C=S), 159.9 (C=N), 126.9–142.1 (Aromatic
carbons), 65.3 (C–2), 63.7 (C–4), 53.2 (C–1'), 46.2 (C–1), 38.9 (C–5), 32.9 (C–2' and C–6'),
28.6 (C–8), 27.2 (C–6), 25.6 (C–4'), 24.9 (C–3' and C–5'), 21.5 (C–7), Anal. Found (Cal.) for
C27H34N4S (%): C, 72.38 (72.60); H, 7.75 (7.67); N, 12.36 (12.54), HRMS (m/z) = 447.2581
(M + H)+.
2.4.2. 2–(2,4–bis(4–fluorophenyl)–3–azabicyclo[3.3.1]nonan–9–ylidene)–N–cyclohexyl
hydrazinecarbothioamide (7)
1
White solid, mp=196–198 °C, FT–IR (KBr): 3336, 3254, 1533, 1215 cm–1, H NMR
(400 MHz, CDCl3): 8.46 (s, NH, 1H), 7.50 (d, NH, 1H), 7.09–7.57 (m, Ar, 8H), 4.37 (s,
H–2, 1H), 4.32 (m, H–1', 1H), 4.23 (s, H–4, 1H), 2.82 (s, H–5e, 1H), 2.76 (m, H–7a, 1H),
2.48 (s, H–1e, 1H), 2.18 (q, H-2'e/H-6'e, m, 2H), 1.72–1.84 (m, H–6e/H–8e/H–3'e/H–5'e/
NH, 5H), 1.63 (d, H–4'e, 1H), 1.25–1.49 (m, H–4'a/H–2'a/H–6'a/H–7e/H–3'a/H–5'a/H–
13
6'a/H–6a/H–8a, 9H), C NMR (100 MHz, CDCl3), 176.5 (C=S), 159.2 (C=N), 115.2–163.2
(Aromatic carbons), 64.6 (C-2), 63.0 (C-4), 53.3 (C–1'), 46.1 (C–1), 38.8 (C–5), 32.9 (C–2'
and C–6'), 28.4 (C–8), 27.1 (C–6), 25.5 (C–4'), 24.9 (C–3' and C–5'), 21.4 (C–7), Anal.
Found (Cal.) for C27H32F2N4S (%): C, 67.06 (67.19); H, 6.78 (6.68); N, 11.48 (11.61),
HRMS (m/z) = 483.2396 (M + H)+.
2.4.3. 2–(2,4–bis(4–methoxyphenyl)–3–azabicyclo[3.3.1]nonan–9–ylidene)–N–cyclohexyl
hydrazinecarbothioamide (8)
1
White solid, mp=181–183 °C, FT–IR (KBr): 3325, 3265, 1533, 1248 cm–1, H NMR
(400 MHz, CDCl3): 8.46 (s, NH, 1H), 7.77 (d, NH, 1H), 6.94-7.52 (m, Ar–CH, 8H), 4.32–
4.34 (d, H–2 and H–1',2H), 4.19 (s, H–4, 1H), 3.84–3.86 (d, p–OCH3, 3H), 2.80 (s, H–5e,
1H), 2.83 (m, H–7a, 1H), 2.45 (s, H–1e, 1H), 2.18 (q, H–2'e/H–6'e, 2H), 1.79–1.87 (m, H–6e/
H–8e/H–3'e/H–5'e/ NH, 5H), 1.68 (d, H–4'e, 1H), 1.25–1.56 (m, H–4'a/H–2'a/H–6'a/H–7e/
H–3'a/H–5'a/H–6'a/H–6a/H–8a, 9H), 13C NMR (100 MHz, CDCl3), 176.4 (C=S), 160.2
(C=N), 113.7–159.0 (Aromatic carbons), 64.8 (C–2), 63.2 (C–4), 55.3 (p–OCH3), 53.2
(C–1'), 46.3 (C–1), 39.1 (C–5), 32.9 (C–2' and C–6'), 28.5 (C–8), 27.2 (C–6), 25.5 (C–4'),

24.9 (C-3' and C–5'), 21.5 (C–7), Anal. Found (Cal.) for C29H38N4O2S (%): C, 68.85 (68.74);
H, 7.48 (7.56); N, 11.16 (11.06), HRMS (m/z) = 507.2794 (M + H)+.
2.4.4. 2– (2,4–bis(3–methoxyphenyl) –3–azabicyclo[3.3.1]nonan–9–ylidene)–N–cyclohexyl
hydrazinecarbothioamide (9)
1
White solid, mp=187–189 °C, FT–IR (KBr): 3353, 3216, 1534, 1265 cm–1, H NMR (400
MHz, CDCl3): 8.45 (s, NH, 1H), 7.52 (d, NH, 1H), 6.85-7.53 (m, Ar–CH, 8H), 4.35 (s, H–2,
1H), 4.33 (m, H–1', 1H), 4.21 (s, H–4, 1H), 3.87 (d, m–OCH3, 3H), 2.86 (s, H–5e, 1H), 2.81
(m, H–7a, 1H), 2.51 (s, H–1e, 1H), 2.19 (q, H–2'e/H–6'e, 2H), 1.78–1.88 (m, H–6e/H–8e/
H–3'e/H–5'e/NH, 5H), 1.70 (m, H–4'e, 1H), 1.23–1.56 (m, H–4'a/H–2'a/H–6'a/H–7e/H–3'a/
H–5'a/H–6'a/H–6a/H–8a, 9H), 13C NMR (100 MHz, CDCl3), 176.6 (C=S), 159.9 (C=N),
112.1–159.8 (Aromatic carbons), 65.1 (C–2), 63.5 (C–4), 55.3 (m–OCH3), 53.2 (C–1'), 46.2
(C–1), 38.9 (C–5), 32.8 (C–2' and C–6'), 28.7 (C–8), 27.3 (C–6), 25.5 (C–4'), 24.9 (C–3' and
C–5'), 21.4 (C–7), Anal. Found (Cal.) for C29H38N4O2S (%): C, 68.85 (68.74); H, 7.48
(7.56); N, 11.21 (11.06), HRMS (m/z) = 507.2795 (M + H)+.
2.4.5. 2–(2,4–bis(4–chlorophenyl)–3–azabicyclo[3.3.1]nonan–9–ylidene)–N–cyclohexyl
hydrazinecarbothioamide (10)
1
White solid, mp=183–185 °C, FT–IR (KBr): 3347, 2931, 1523, 1210 cm–1, H NMR
(400 MHz, CDCl3): 8.51 (s, NH, 1H), 7.52 (d, NH, 1H), 7.29-8.03 (m, Ar–CH, 8H), 4.77
(s, H–2, 1H), 4.35 (m, H–1', 1H), 4.66 (s, H–4, 1H), 3.21 (s, H–1e, 1H), 2.79 (m, H–7a, 1H),
2.86 (s, H–5e, 1H), 2.16 (q, H–2'e/H–6'e, 2H), 1.74–1.79 (m, H–6e/H–8e/H–3'e/H–5'e/ NH,
5H), 1.66 (m, H–4'e, 1H), 1.27–1.56 (m, H–4'a/H–2'a/H–6'a/H–7e/H–3'a/H–5'a/H–6'a/H–
6a/H–8a, 9H), 13C NMR (100 MHz, CDCl3), 176.6 (C=S), 158.3 (C=N), 126.8–138.8
(Aromatic carbons), 62.0 (C–2), 60.3 (C–4), 53.2 (C–1'), 41.9 (C–1), 34.6 (C–5), 32.8 (C–2'
and C–6'), 28.5 (C–8), 27.1 (C–6), 25.5(C–4'), 24.9 (C–3' and C–5'), 21.0 (C–7), Anal. Found
(Cal.) for C27H32Cl2N4S (%): C, 62.76 (62.90); H, 6.35 (6.26); N, 10.65 (10.87), HRMS
(m/z) = 515.5487 (M + H)+.
2.5. X–ray data collection and structure refinement
A colorless crystal of the compound 6 was mounted on a glass fiber with epoxy cement
for the X–ray crystallographic study. The selected crystal data and data collection parameters of

the compound 6 were collected at 293 K. Bruker area data detector diffract meter was used for
the measurement of data. The collected data were reduced using the SAINT program and
structural refinement was carried out by Full–matrix least–squares on F2
(SHELXL–97).
2.6. In vitro antibacterial and antifungal activity
The in vitro activities of the compounds were tested in Sabourauds dextrose broth (SDB;
Hi–media, Mumbai) for bacteria by the 2–fold serial dilution method. The test compounds were
dissolved in dimethylsulfoxide (DMSO) to obtain 1 mg/ml stock solutions. Seeded broth (broth
containing microbial spores) was prepared in NB (Nutrient Broth) from 24–h–old bacterial
cultures were suspended in SDB. The colony forming units (cfu) of the seeded broth were
determined by plating technique and adjusted in the range of 104–105 cfu/ml. The final inoculum
size was 105 cfu/ml for antibacterial assay and 1.1–1.5 x102 cfu/ml for antifungal assay and
testing was performed at pH 7.4 ± 0.2. Exactly 0.2 ml of the solution of each test compound was
added to 1.8 ml of seeded broth to form the first dilution. One milliliter of this was diluted with
a further 1 ml of the seeded broth to give the second dilution and so on till six such dilutions
were obtained. A set of assay tubes containing only seeded broth was kept as control and
likewise solvent controls were also run simultaneously. The tubes were incubated in BOD
incubators at 37 ± 1°C for bacteria and 28 ± 1°C for fungi. The minimum inhibitory
concentrations (MICs) were recorded by visual observations after 24 h (for bacteria) and 72–96 h
(for fungi) of incubation. Streptomycin and Amphotericin B were used as standards for
antibacterial and antifungal activity evaluation respectively.

3. Results and discussion
3.1. Designation of atoms
The carbon atoms of the bicyclic[3.3.1]nonane part are numbered as shown in Fig. 1.
The carbons of the aryl group at C–2 are denoted as 1'', 2'', 3'', 4'', 5'' and 6''– carbons and those
of the aryl group at C–4 are denoted as 1''', 2''', 3''', 4''', 5'''and 6'''–carbons. The carbons of the
cyclohexyl ring at C–1, C–2, C–3, C–4, C–5 and C–6 are designated as C–1', C–2', C–3', C–4',
C–5' and C–6', respectively and the protons are numbered accordingly. For example, the
benzylic proton at C–2 is denoted as H–2 and so on. The methylene protons in the cyclohexane
ring are denoted as axial and equatorial protons assuming chair conformation for the
cyclohexane ring. The methylene protons at C–6, C–7 and C–8 are denoted as H–6a / H–6e, H–
7a / H–7e and H–8a / H–8e.
Fig. 1. Numbering of the atoms in the target compound

3.2. IR Spectral analysis of compounds 6–10
The synthesized compounds, the NH stretching frequency was in the range
of 3216–3353 cm–1. The characteristic vibrational band in the region 1725-1675 cm-1 was
observed for the carbonyl functionality [27]. For all the parent piperidones, the sharp band in the
region 1720-1700 cm-1 of C=O group 1-5. The synthesized compounds 6–10 showed the
absence of C=O stretching frequency around 1720–1700 cm–1 and the presence of C=N and C=S
stretching frequencies around 1528–1599 cm–1 and 1205–1248 cm–1 confirming the condensation
of 2,4–diaryl–3–aza–bicyclo[3.3.1]nonan–9–ones with cyclohexylthiosemicarbazide. Literature
report [28] shows that NH, C=N and C=S stretching frequency in the region of 3357-3171cm-1,
1533-1516 and 1247-1205 cm-1. The intense aliphatic and aromatic stretching frequencies
appeared are 2969–2843 cm–1 due to the presence of the phenyl and cyclohexyl ring
(Supplementary Material).
3.3. 1H NMR spectral analysis of compound 6
The schematic diagram of compounds 6–10 is shown in Scheme 1. All the synthesized
13
compounds were characterized by 1H and C NMR spectral studies. The signals were assigned
1
13
with the help of earlier reports [28, 29]. The H and C NMR spectral analysis of compound 6
is discussed as follows.
Table 1 and 2 respectively.
The observed 1H and 13C chemical shifts are given in
We have chosen compound 6 as the representative system. 1H NMR spectrum of
1
compound 6 is given in Fig. 2. The H NMR spectrum showed two broad signals in the
downfield region at 8.51 ppm (singlet) with one proton integral, and a doublet at 7.51 ppm with
one proton integral are due to NH protons of thiosemicarbazone moiety (=N-NH-CS and
cyclohexyl-NH-CS). The benzylic protons (H–2 and H–4) appear separately at 4.37 and 4.23

ppm, respectively. Similarly, the bridgehead protons resonate at 2.50 ppm and 2.86 ppm
(singlet) respectively. The shielded signal appeared at 2.50 ppm is attributed to H–1e proton
whereas the deshielded signal observed at 2.86 ppm (singlet) is assigned to H–5e proton. The
observed desheilding experienced by H–1e proton is attributed to the presence of electronegative
nitrogen atom in the adjacent position.
The multiplet observed at 2.82 ppm is assigned for H-7a methylene proton and another
multiplet observed at 4.34 ppm is assigned for H–1'a proton. Further, the multiplet observed at
2.17 ppm with two protons integral is assigned for H–2'e and H–6'e protons. The shielded region
appeared at 1.68 ppm is attributed to the H–4'e proton. Furthermore, the multiplet observed in
the region of 1.76–1.83 ppm is due to H–8e, H–6e, H–5'e and H–3'e and piperidone ring NH
proton whereas another multiplet observed in the region of 1.25–1.54 ppm with eight protons
integral is assigned for H–5'a, H–3'a, H–7e, H–8a, H–6a, H–2'a, H–6'a and H–4'a protons. The
aromatic protons resonate in the region of 7.29–7.58 ppm due to anisotropic effect of the
aromatic ring.
3.4. HOMOCOSY and NOESY spectral analysis
HOMOCOSY and NOESY spectrum of compound 6 shows Fig 3 and 4. The selected
HOMOCOSY and NOESY correlations shown in Fig 5 and 6 whereas Table 3 and 4 represents
the correlations chemical shifts values of compound 6. The weak HOMOCOSY correlation
between the signals at 2.86 and 2.50 ppm indicated that the correlation is due to the long-range
coupling between the bridgehead protons (H-5e and H-1e) and this is possible only when both
the protons are in equatorial orientation in chair conformation. This observation clearly confirms
that the singlet at 2.86 ppm is due to H-5e proton. Another bridgehead proton (H-1e) appears at
2.50 ppm (singlet) has strong correlation with the benzylic proton at 4.23 ppm (singlet). Besides

H-1e proton also shows HOMOCOSY correlations with H-8a and H-8e protons. The strong
correlation observed between the H-7a proton at 2.82 ppm and the H-7e proton at 1.39 ppm
infers that both signals are due to the C-7 methylene protons (H-7a and H-7e). Furthermore,
H-7a proton has correlations with H-6a, H-6e, H-8a and H-8e protons.
The signal due to cyclohexane ring proton (H-7a) appears in the higher frequency region
than the equatorial proton (H-7e). The deshielding effect of H-7a proton signal is mainly due to
the steric interaction between H-7a proton and the lone pair electrons on nitrogen atom. Due to
this, C-H(7a) bond becomes polarized and as a consequence, carbon and its attached proton
acquires negative and positive charges, respectively. Hence, the H-7a proton signal is highly
deshielded than the H-7e. Further, H-6a and H-8a protons appear in the shielded region than the
corresponding equatorial protons in accordance with C–C single bond anisotropy effect. In
HOMOCOSY spectrum, the benzylic protons show correlation with that of N(3)-H proton.
Based on the observed chemical shift values and the earlier report [30] it has been concluded that
the compound 6 exists in the twin-chair (Fig. 7) conformation.
The strong nOes between H-2a and H-1e and also between H-4a and H-5e clearly
confirm that the benzylic and bridgehead protons are occupying axial and equatorial dispositions
respectively. Besides, the nOe between the NH proton in hydrazone analogue and H-5e
bridgehead proton suggests that the H-5e proton is syn to the hydrazone group.
The syn α–bridgehead proton (H-5e) is observed in the highly deshielded region than anti
α–bridgehead proton (H-1e). This is due to the non–bonded (spatial) interaction between (H-5e)
proton with nitrogen atom in the thiosemicarbazone moiety (Fig. 7). Owing to this interaction,
syn α–carbon get partial negative charge and the attached proton acquires a slight positive charge

[30]. Based on this report, the shielded signal is assigned to (H-1e) proton whereas the
deshielded signal is assigned to (H-5e) proton.
3.5. 13C NMR spectral analysis of compound 6
In 13C NMR spectrum of compound 6, the thiosemicarbazone C=S and C=N carbons are
resonated at 176.5 and 159.9 ppm respectively. In the aliphatic region the signals observed at
53.2, 25.6 and 32.8 are assigned to C–1' and C–4' carbons. This signal at 32.9 and 24.9 ppm are
assigned to C–2'/C–6' and C–5'/C–3' carbons respectively. Further, two signals appeared at 65.3
and 63.7 ppm are conveniently assigned to C–2 and C–4 carbons whereas the bridged carbons
C–1 and C–5 carbons resonated at 46.2 and 38.9 ppm respectively based on their HSQC
correlations. The cyclohexyl ring methylene carbons C–6 and C–8 are appeared at 27.2 and 28.6
ppm whereas C–7 carbon is resonated at 21.5 ppm. The aromatic carbons are distinguished from
other carbons by their characteristic absorption in the region of 126.9–128.6 ppm. Ipso carbons
of phenyl groups resonated at 141.5–142.1 ppm are assigned based on their intension and
chemical shift values.
3.6. HSQC spectral analysis of compound 6
HSQC spectrum of compound 6 is recorded to confirm the 1D NMR spectral
assignments. In the HSQC NMR spectrum the benzylic protons observed at 4.37 and 4.23 ppm
(H–2 and H–4) have cross peaks with the signals at 65.3 and 63.7 ppm, confirmed the C–2 and
C–4 carbons. The bridgehead methine proton signals at 2.50 (H‒1e) and 2.86 ppm (H‒5e) have
correlations with the carbon signals at 46.2 and 38.9 ppm. This inferred that the signals appeared
at 46.2 and 38.9 ppm is due to C–1 and C–5 carbons. The HSQC spectrum of compound 6
shown in Fig 8 and the chemical shifts are reproduced in Table 5.
3.7. Single crystal XRD study of compound 6

Single crystal XRD analysis was carried out for the representative compound
N–cyclohexyl–2–(2,4–diphenyl–3–azabicyclo[3.3.1]nonan–9–ylidene)hydrazinecarbothioamide
6 and its ORTEP was shown in Fig. 9 with selected bond length and bond angles. The bond
lengths and bond angles are in good agreement with the literature values [28]. The
thiosemicarbazone (6) crystallizes in triclinic system with P-1 space group as a chloroform
solvate. The details of crystal data and refinement are given in Table 6.
From the single crystal XRD analysis, it is interesting to note that N–cyclohexyl–2–(2,4–
diphenyl–3–azabicyclo[3.3.1]nonan–9–ylidene)hydrazinecarbothioamide (6) has adopted a
twin–chair conformation with equatorial orientations of the phenyl groups shown in Fig. 10.
The condensation of ketone with thiosemicabazide resulted in a new C=N bond whose
bond length is 1.265(7) Å [C3–N2]. The configuration about the C=N bond is E. The observed
dihedral angle between S1 and N2 is 175.4(5)° [S1–C21–N3–N2]. The delocalization of π
electron density over thiosemicarbazone moiety resulted in the variations of C=N, N-N and C=S
bond distance from the normal value [31]. The torsion angles of phenyl rings C3–C4–C5–C15
and C3–C2–C1–C9 are –177.0 (6)° and –178.7(7)°, respectively. Thus, all parameters clearly
indicate the twin–chair conformation [32] of the bicycle with an equatorial orientation of the
phenyl groups (Fig 10). The cyclohexyl ring in the thiosemicarbazone fragment also adopts
chair conformation as expected and all other bond parameters are normal. The packing of the
molecule is given in Fig. 11.
3.8. Biological activity
The bacterial and fungal minimum inhibitory concentrations (MIC) values of the test
compounds and the standard are furnished in Table 7.

All the compounds were screened for their antibacterial activity against Staphylococcus
aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella typhi
and antifungal activity against, Candida albicans, Aspergillus flavus, Rhizopus Mucor and
Aspergillus niger. The antimicrobial studies of compounds 6–10 with various substituents in the
aromatic ring will be useful to understand the influence of steric and electronic effects on the
biological activities. Also it reveals the effect of replacement of oxygen by thiosemicarbazone
moiety on the biological activity.
3.9. Antibacterial activity of compounds (6-10)
For evaluating antibacterial activity, streptomycin was used as the standard drug. In
general all the synthesized compounds exert a wide range of modest antibacterial activity in vitro
against the tested organisms. Compound 6 without any substituent in the aryl moiety exhibited
antibacterial activity in vitro at 100 µg ml–1 against Escherichia coli and Pseudomonas aeruginosa
whereas it showed antibacterial activity for the other tested organisms only at 200 µg ml–1.
Fluoro and chloro substituted compounds (7 and 10) demonstrated very good
antibacterial activity against all the bacterial strains used in this study. Further when compared
with standard drug streptomycin, compound 7 has two fold increased activity against
Staphylococcus aureus and Klebsiella pneumoniae. The antibacterial activity of compound 10
has been compared with the antibacterial activity of the standard drug streptomycin and it
indicated that compound 10 has the same efficiency against Staphylococcus aureus, Klebsiella
pneumoniae and Salmonella typhi. Further compounds 8 and 9 exhibited good antibacterial
activity against all the bacterial strains at 50–100 µg ml–1.
3.10. Antifungal activity of compounds (6-10)

For evaluating antifungal activity, Amphotericin B was used as the standard drug. The
results of the present study also provide evidence for the antifungal effects of compounds 6−10
with MIC values ranging from 6.25 to 200 µg ml–1. Compound 9, without any substituents at the
aryl groups, showed mild and comparatively less antimicrobial activity against Candida albicans
(100 µg ml–1), Aspergillus flavus (200 µg ml–1), Rhizopus (100 µg ml–1), Mucor (100 µg ml–1)
and Aspergillus niger (50 µg ml–1) compared to Amphotericin B, a known antifungal agent used
as positive control. Compound 7 exerted 3–fold increased antifungal activity against C. albicans
and A. niger at MIC values of 6.25 µg ml–1 and 2–fold increased activity against Aspergillus
flavus, Rhizopus and Mucor at a MIC value of 12. 5 µg ml–1 when compared to the standard.
Furthermore, phenyl rings with electron–donating methoxy group at the para position of the
phenyl rings (compounds 8 and 9) hinder the growth of all fungal strains at MIC values ranging
from 50 to 100 µg ml–1. Compound 10 exhibited 3–fold increased antifungal activity against
Aspergillus flavus, Mucor and Aspergillus niger and 2–fold enhanced antifungal activity against
Candida albicans and Rhizopus when compared with Amphotericin B. The results of the present
study demonstrate that electron withdrawing groups at the para position of the aromatic ring in
the azabicyclononan–9–one moiety exert superior inhibitory effect against various tested
microbes compared to the other tested compounds and standard drug.
The observed results indicated that the type of functional groups and the pattern of
substitution on the azabicyclononan–9–one moiety could be responsible for the substantial
influence on the antimicrobial activity of the hydrazone derivatives. The antifungal and
antibacterial activities of compounds 6−10 create promising leads for the development of potent
antimicrobial agents.

4. Conclusion
All the target compounds are synthesized successfully and were characterized by FT-IR,
1H NMR, 13C NMR, HRMS and elemental analysis. The piperidone ring adopts a twin–chair
conformation with equatorial orientations of the aryl groups was confirmed by single crystal
XRD (compound 6). The cyclohexyl fragment present in thiosemicarbazone moiety also
exhibits a normal chair conformation. From the close assessment of the in vitro antibacterial and
antifungal results against a panel of microbial organisms revealed that the compounds with
fluorine or chlorine substituents were found to be excellent antimicrobial activities against all the
tested organisms.
Acknowledgments
One of the author K. Krishnasamy thank the University Grant Commission for the
financial support (major research project, F. No.42–342/2013) of this research work. We are
grateful to the UGC Networking Resource Centre, University of Hyderabad, for providing
characterization facilities and Dr. R. Nagarajan, School of Chemistry, University of Hyderabad
for providing laboratory facilities.
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Figure captions
Table 1
Table 2
Table 3
Table 4
Table 5
Table 6
Table 7
1H Chemical shift values of compounds 6-10
13C Chemical shift values of compounds 6-10
Correlations in the HOMOCOSY spectrum of compound 6
Correlations in the NOESY spectrum of compound 6
Correlations in the HSQC spectrum of compound 6
Crystal data and structure refinement parameters of compound 6
In vitro antibacterial and antifungal activity of compounds 6-10
Fig. 1
Fig. 2
Fig. 3
Fig.4
Numbering of the atoms in the target compound
1H NMR spectrum of compound 6
HOMOCOSY spectrum of compound 6
NOESY spectrum of compound 6
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig.10
HOMOCOSY- Selected correlations of compound 6
NOESY- Selected correlations of compound 6
Non-bonded interaction between CH(5e) and NH group.
HSQC spectrum of compound 6
ORTEP view of compound 6
Conformation of compound 6
Fig.11
Packing diagram of compound 6
IR Spectrum of Compounds 6-10
Fig. S1-S5
Fig. S6-S14 1H-13C NMR Spectrum of compounds 6-10
Fig. S6-S14 High–resolution mass spectrum of compounds 6-9

Fig. 2. 1H NMR spectrum of compound 6

Fig. 3. HOMOCOSY spectrum of compound 6

Fig. 4. NOESY spectrum of compound 6

Fig. 5. HOMOCOSY- Selected correlations of compound 6
Fig.6. NOESY- Selected correlations of compound 6

Fig. 7. Non-bonded interactions between CH(5e) and NH group.
Fig. 8. HSQC spectrum of compound 6

Fig. 9. ORTEP view of compound 6
The important bond lengths (Å): N2-C3=1.265(7) ; N2-N3=1.38(1); N3-C21=1.350(7); N4-C21=1.29(1); C1-
N1=1.44(1); N1-C5=1.45(1); C5-C4=1.52(1); C3-C8=2.429(9); C3-C6=2.42(1); C6-C7=1.53(1); C7-C8=1.51(1);
C6-C2=1.51(1); C2-C3=1.48(1); C3-C4=1.49(1); C2-C1=1.53(1); C25-H26B=2.02(1); H24A-C23=2.058(8);C27-
H22=2.0(1); H26B-C27=2.064(8); C27-C22=1.50(2); C26-C27=1.53(1).
The important bond angles (°): C3-N2-N3=118.1(7);C3-C2-C1=107.8(7);C3-C4-C5=109.3(7);C2-H6B-C7=77.0(3);
C7-C8-C4=113.1(7); C21=117.5(7);N2-N3-C21=117.5(7); N3-C21-S1=117.9(6); N1-C5-C15=110.5(7); N1-C1-
C9=111.9(7).
Fig. 10. Conformation of compound 6

Fig. 11. Packing diagram of compound 6

Scheme 1. Schematic diagram showing the synthesis of compounds 6–10

Table 1
1H Chemical shift (ppm) values of compounds 6-10
Compound
Proton
6
7
8
9
10
NHCS
CSNH-Cy
H-2a
8.51(s)
7.51(d)
4.37(s)
4.23(s)
4.34(m)
2.86(s)
2.82(m)
2.50(s)
1.68(m)
2.17(m)
8.46(s)
7.50(d)
4.37(s)
4.23(s)
4.32(m)
2.82(s)
2.76(m)
2.48(s)
1.63(d)
2.18(q)
8.46(s)
7.77(d)
4.34(s)
4.19(s)
4.36(m)
2.80(s)
2.83(m)
2.45(s)
1.68(q)
2.18(dd)
8.45(s)
7.52(d)
4.35(s)
4.21(s)
4.33 (m)
2.86(s)
2.81(m)
2.51(s)
1.70(m)
2.19(q)
8.51(s)
7.52(d)
4.77(s)
4.66(s)
4.35(m)
2.86(s)
2.79(m)
3.21(s)
1.66(m)
2.16(q)
H-4a
H-1'a
H-5e
H-7a
H-1e
H-4'e
H-2'e, H-6'e
H-6e, H-8e,
H-3'e, H-5'e,
NH
1.76-1.83
(m)
1.72-1.84
(m)
1.79-1.87
(m)
1.78-1.88
(m)
1.74-1.79
(m)
H-3'a, H-5'a,
H-7e, H-6a,
H-8a, H-2'a,
H-6'a, H-4'a
O(CH3)
1.25-1.54
(m)
1.25-1.49
(m)
1.25-1.56
(m)
1.23-1.56
(m)
1.27-1.56
(m)
-
-
3.85(d)
3.87(d)
-
Aromatic
protons
7.29-7.58
(m)
7.09-7.57
(m)
6.94-7.52
(m)
6.85-7.53
(m)
7.29-8.03
(m)