Synthesis and anti-tubercular and antimicrobial activities of some 2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-one N-isonicotinoylhydrazone derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.08.024

In this study, seven 2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-one N-isonicotinoylhydrazones 8-14 were synthesized. The structure and stereochemistry of these compounds were established by IR and NMR spectral data. The purities were checked by elemental analysis. The synthesized compounds adopt twin-chair conformation with equatorial orientations of the aryl groups. The compounds were evaluated for their in vitro anti-tubercular and antimicrobial activities. The initial screen was conducted against Mycobacterium tuberculosis H₃₇Rv (ATTCC 27294) and INH-TB by luciferase reporter phage assay method. All the synthesized compounds showed very good activity against MTB and INH-TB. Though all the compounds showed good antimicrobial activity only 11 (Ar = p-chlorophenyl), 12 (Ar = p-fluorophenyl), 13 (Ar = m-chlorophenyl) and 14 (Ar = m-methoxyphenyl) exhibited activity against all the tested (bacterial and fungal) microorganisms. The results suggest that the formation of hydrogen bonds may play a significant role in drug action.

Full text of DOI:10.1016/j.ejmech.2010.08.024

European Journal of Medicinal Chemistry 45 (2010) 5480e5485
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and anti-tubercular and antimicrobial activities of some
2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-one N-isonicotinoylhydrazone
derivatives
*
C. Sankar, K. Pandiarajan
Department of Chemistry, Annamalai University, Annamalainagar 608002, Tamilnadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, seven 2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-one N-isonicotinoylhydrazones 8e14 were
synthesized. The structure and stereochemistry of these compounds were established by IR and NMR
spectral data. The purities were checked by elemental analysis. The synthesized compounds adopt twin-
chair conformation with equatorial orientations of the aryl groups. The compounds were evaluated for
their in vitro anti-tubercular and antimicrobial activities. The initial screen was conducted against
Mycobacterium tuberculosis H37Rv (ATTCC 27294) and INH-TB by luciferase reporter phage assay method.
All the synthesized compounds showed very good activity against MTB and INH-TB. Though all the
compounds showed good antimicrobial activity only 11 (Ar ¼ p-chlorophenyl), 12 (Ar ¼ p-fluorophenyl),
13 (Ar ¼ m-chlorophenyl) and 14 (Ar ¼ m-methoxyphenyl) exhibited activity against all the tested
(bacterial and fungal) microorganisms. The results suggest that the formation of hydrogen bonds may
play a significant role in drug action.
Received 19 May 2010
Received in revised form
9 August 2010
Accepted 9 August 2010
Available online 14 August 2010
Keywords:
Antituberculosis activity
Isoniazid
Mycobacterium tuberculosis
Antimicrobial activity
3-Azabicyclo[3.3.1]nonane
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
INH and rifampicin, often taking a further two years to treat with
second-line drugs [4,5].
Tuberculosis (TB) is one of the leading infectious disease among
adults and youth, with one-third of the world’s population infected
with Mycobacterium tuberculosis. Two developments make the
resurgence in TB especially alarming. The first is pathogenic
synergy with HIV. The overall incidence of TB in HIV-positive
patients is 50 times that of the rate for HIV-negative individuals [1].
The second is the emergence of drug-resistant and multi-drug
resistant TB (MDR-TB). According to the World Health Organization
(WHO), in 2006 there were 9.2 million new cases and 1.7 million
deaths from TB around the world. The incidence of TB infection has
steadily risen in the last decade [2]. Also WHO has reported that
XDR TB, a virtually untreatable form of the respiratory disease is
found in 45 countries [1,3]. The standard “short” course treatment
for tuberculosis (TB) involves oral administration of isoniazid (INH),
rifampicin, pyrazinamide and ethambutol for two months followed
by INH and rifampicin alone for a further four months. For latent
tuberculosis, the standard treatment involves oral administration
of INH alone for six to nine months. MDR-TB is resistant to at least,
Three reasons are usually given for needing new tuberculosis
drugs: (i) to improve current treatment by shortening the total
duration of treatment and/or by providing for more widely spaced
intermittent treatment, (ii) to improve the treatment of MDR-TB,
and (iii) to provide for more effective treatment of latent tubercu-
losis infection (LTBI) [6].
There are two basic approaches to develop a new drug for TB:
(i) synthesis of analogues by modifications of existing compounds
for shortening and improving TB treatment and, (ii) searching
novel structures which the TB organism has never encountered
with before, for the treatment MDR-TB [7].
To pursue this goal, our research efforts are directed to the
modification of INH, which is a well known anti-tubercular agent
bearing pyridine and hydrazine moieties. Modifying either of these
moieties has been taken up by several research groups [8e12].
On the basis of these observations, we had the impetus to
synthesize a number of 3-azabicyclo[3.3.1]nonan-9-one hydrazones
and subsequently evaluate their in vitro antimicrobial activity.
Molecules with the 3-azabicyclo[3.3.1]nonane nucleus are of great
interest due to their presence in a wide variety of naturally occurring
diterpenoid/norditerpenoid alkaloids and biological activities [13].
The chemistry of 3-azabicyclo[3.3.1]nonan-9-one has been reviewed
* Corresponding author. Tel.: þ91 9994469980.
E-mail address: profkprajan@yahoo.co.in (K. Pandiarajan).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.024

C. Sankar, K. Pandiarajan / European Journal of Medicinal Chemistry 45 (2010) 5480e5485
5481
α
[14]. The use of different substituents particularly aromatic substit-
β
γ
N
uents, with regard to biological activity, has been demonstrated [15].
In this paper, we report the synthesis of seven 2r,4c-diaryl-3-
azabicyclo[3.3.1]nonan-9-one N-isonicotinoylhydrazones 8e14 and
there in vitro anti-tubercular and antimicrobial activities.
O
N
7
H
N
6
5
8
9
2. Chemistry
1
2
4
2'
3
4'
N
H
The synthetic route of the compounds is outlined in Scheme 1.
For the synthesis of the title compounds, 2r,4c-diaryl-3-azabicyclo
[3.3.1]nonan-9-ones 1e7, required as starting materials, were
prepared by the reaction of cyclohexanone, aldehydes and ammo-
nium acetate. The reaction of 1e7 with isonicotinoyl hydrazide, in
the presence of acetic acid, resulted in the formation of the title
compounds 8e14. The structure and stereochemistry of
compounds 8e14 have been established by IR and NMR spectral
data. Their purities were checked by elemental analysis. For 8 all 1H
and 13C signals have been assigned unambiguously using HOMO-
COSY, HSQC and HMBC spectra.
Fig. 1. Numbering of the atoms.
the aryl group at C-2 are denoted as o, m and p- carbons and those
of the aryl group at C-4 are denoted as o0, m0 and p0-carbons. In the
meta-substituted compounds the o-carbon ortho to the substituent
is denoted as o, o-C and that para to the substituent is denoted as o,
p-C. The other aromatic carbons are designated in an analogous
manner. The carbons of the pyridine ring are designated using
Greek letters
example, the benzylic proton at C-2 is denoted as H-2 that at C-
denoted as H- and so on. The methylene protons in the cyclo-
a
,
b
and
g
. The protons are numbered accordingly. For
a
is
3. Pharmacology
a
hexane ring are denoted as axial and equatorial protons assuming
chair conformation for the cyclohexane ring. Thus, the methylene
protons at C-7 are denoted as H-7a and H-7e.
3.1. Anti-tubercular activity
Initial screen was conducted against M. tuberculosis H37Rv (ATCC
27294) and INH-TB by luciferase reporter phage assay method [16].
All the compounds showed very good anti-tubercular activity.
4.2. IR spectra
3.2. Antimicrobial activity
In all cases two separate bands were observed for the two
different NH-stretching vibrations. The piperidine NH-stretching
frequency was in the range 3310e3370 cmꢀ1 and the amide NH-
stretching frequency was in the range 3059e3183 cmꢀ1. The amiꢀd1e
The in vitro activities of the compounds were tested in Nutrient
broth (NB; Hi-media, Mumbai) for bacteria and Sabourauds
dextrose broth (SDB; Hi-media, Mumbai) for fungi by the two-fold
serial dilution method [17,18]. Some compounds showed significant
antibacterial and antifungal activity.
carbonyl stretching frequency was in the range 1635e1640 cm
.
The C]N stretching frequency was in the range 1509e1524 cmꢀ1
.
4.3. NMR spectra
4. Results and discussion
For 8 the 1H and 13C signals were unambiguously assigned based
on the observed correlations in the HOMOCOSY, HSQC and HMBC
spectra. For all the other compounds the 1H and 13C signals were
assigned by comparison with 8. The signals for the aromatic
protons and carbons in 9e14 were assigned based on known
substituent effects [19,20]. In the case of 12 coupling with 19F was
4.1. Designation of atoms
The atoms of the bicycle [3.3.1]nonane part are numbered as
shown in Fig. 1. The ipso carbons of the aryl groups at C-2 and C-4
are designated as C-20 and C-40, respectively. The other carbons of
Scheme 1. Synthesis of 2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-one N-isonicotinoyl hydrazones, Reagents and conditions: (a) CH3COONH4, EtOH, warm; (b) Isoniazid, Methanol:
Chloroform (1:1 v/v), AcOH, reflux.

5482
C. Sankar, K. Pandiarajan / European Journal of Medicinal Chemistry 45 (2010) 5480e5485
Table 1
The in vitro activity of compounds against M. tuberculosis H37Rv strain and INH-MTB
(% reduction of RLU).
O
N
C
Compounds
M. tuberculosis
1.00 g/mL
INH-MTB
1.00 g/mL
N
m
2.00
m
g/mL
m
2.00 mg/mL
H
N
8
9
77.76
72.03
73.07
75.61
76.63
77.18
75.42
98.42
87.90
82.04
83.02
85.22
86.12
87.06
85.34
98.42
69.60
62.30
67.07
63.61
67.63
69.18
65.42
89.65
77.52
71.07
74.19
73.28
75.08
78.33
75.83
95.85
10
11
12
13
14
INH
H
H
H
a
H
a
H
a
H
a
H
e
H
Ar
Ar
e
H
e
N
phage assay method against M. tuberculosis H37Rv and INH-resis-
tant M. tuberculosis at two different concentrations level of 1.00 and
2.00 mg/mL and the observed percentage inhibitions are tabulated
H
H
a
Fig. 2. Twin-chair (cc) conformation of compounds.
in Table 1. A compound is considered to be an antimycobacterial
agent if fifty percent reduction in the Relative Light Units (RLU) is
observed when compared to the control using a luminometer. From
Table 1 it is seen that, all the compounds show excellent in vitro
activity against MTB with the percentage of reduction in RLU
ranging 72e80%.
observed for the aromatic carbons except the ipso carbons C-20 and
C-40 [20]. The signal for C-5 merged with the signal of DMSO-d6 in
all cases. This was conformed for 8 from its HSQC spectrum. For 8
the chemical shift of C-5 could be determined from its HSQC
spectrum as 41.0 ppm approximately. The same value is assigned
for all the other compounds.
4.5. Antibacterial activity
In the HOMOCOSY spectrum of 8 the benzylic protons showed
correlation with N(3)-H. Also H-2 showed correlation with H-1 and
H-4 showed correlation with H-5. However, in all cases H-1, H-2, N
(3)-H, H-4 and H-5 gave unresolved signals. The unresolved nature
of the signals for H-1, H-2, H-4 and H-5 suggests that the vicinal
couplings for these protons are very small. However, H-7a showed
three large coupling (one geminal coupling with H-7e and two
diaxial vicinal coupling with H-6a and H-8a) and two small
couplings (one with H-6e and another with H-8e. All these obser-
vations are consistent with twin-chair (CC) conformation for these
compounds (Fig. 2).
The in vitro antibacterial activity of compounds 8e14 was
examined against the bacterial strains viz., Gram positive [Staphy-
lococcus aureus NCIM-2492 and Bacillus subtilis NCIM-2439] and
Gram negative [Escherichia coli NCIM-2345, Klebsiella pneumoniae
(derived from Medical College, Annamalai University) and Pseu-
domonas aeruginosa NCIM-2035] bacteria and are expressed as
minimum inhibitory concentration (MIC, m
g mLꢀ1) Streptomycin
has been taken as the reference drug [22,23] and the observed
minimum inhibitory concentration values are given in Table 2.
Compounds 11, 13 and 14 are active against all the tested
bacterial strains. Compound 10 is not active against P. aeruginosa
and compound 12 is not active against K. pneumoniae. Compound 9
is active against three bacterial strains only, whereas 8 is only active
against S. aureus and P. aeruginosa.
It is interesting to note that 2r,4c-diphenyl-9t-ethynyl-3-azabi-
cyclo[3.3.1]nonan-9t-ol (15) has been to adopt twin-chair confor-
mation, shown in Fig. 3 with equatorial orientations of the phenyl
groups, by X-ray crystallographic study [21].
4.6. Antifungal activity
4.4. Anti-tubercular activity
The in vitro antifungal activity of compounds 8e14 was exam-
ined against the fungal strains viz., Candida albicans-6 (NCIM-C27),
C. albicans (NCIM-C27), Aspergillus niger (NCIM-590), C. albicans-51
(NCIM-C27) and Aspergillus flavus (NCIM-539). Amphotericin B was
used as standard drug, the observed minimum inhibitory concen-
tration values are given in Table 3.
The preliminary in vitro antimycobacterial activity of the
synthesized compounds were evaluated by luciferase reporter
CH
HO
C
Table 2
In vitro antibacterial activity of compounds 8e14.
Compounds
Minimum Inhibitory Concentration (MIC) in
mg/mL
Bacillus Klebsiella Escherichia Staphylococcus Pseudomonas
subtilis pneumoniae coli
aureus
aeruginosa
8
9
10
11
12
13
14
e
e
e
100
200
100
50
200
100
e
200
200
100
50
50
100
25
e
e
200
100
e
100
50
100
200
50
100
100
200
100
50
Ph
Ph
50
100
50
12.5
50
200
100
12.5
50
N
H
Penicillin G
Streptomycin 12.5
50
12.5
25
Fig. 3. Twin-chair (cc) conformation of compound 15.
‘e’ No inhibition even at a higher concentration of 200 mg/mL.

C. Sankar, K. Pandiarajan / European Journal of Medicinal Chemistry 45 (2010) 5480e5485
5483
Table 3
The substituents in the aromatic ring do not have significant
effect on the anti-tubercular activity. However, the antibacterial
and antifungal activities are significantly influenced by the
aromatic substituents. Analysis of these results and the observation
earlier made on 4-aryl-5-isopropoxycarbonyl-6-methyl-3,4-dihy-
dropyrimidinones suggest that the formation of hydrogen bond
may play a significant role in drug action.
In vitro antifungal activity of compounds 8e14.
Compounds
Minimum Inhibitory Concentration (MIC) in
m
g/mL
Candida
C. albicans Aspergillus C. albicans-51 Aspergillus
albicans-6
niger
flavus
8
9
e
e
200
100
200
100
100
100
100
50
100
100
100
50
e
200
100
100
50
200
50
100
200
100
50
100
200
25
e
10
11
12
13
14
200
200
100
100
200
50
6. Experimental
50
200
200
25
6.1. Materials and methods
Amphotericin B 25
Isoniazid was purchased from SigmaeAldrich and all other
chemical were used as Analytical grade. Reactions were monitored
by TLC. All the reported melting points were measured in open
capillaries and are uncorrected. FT-IR spectra were recorded as
potassium bromide pellets on AVATAR 330 FT-IR Thermo Nicolet
Spectrometer. 1H & 13C NMR spectra were recorded at ambient
temperature on a Bruker AMX 400 NMR spectrometer operating at
400.13 MHz for 1H and 100.62 MHz for 13C. Solutions for the
measurement of spectra were prepared by dissolving 10 mg of the
‘e’ No inhibition even at a higher concentration of 200 mg/mL.
Compounds 10e14 are active against all the tested fungal
strains. Compound 9 is not active against A. flavus whereas, 8 is
active against only C. albicans-51 and A. niger.
4.7. Influence of aromatic substituents
The substituent in the aryl ring seems to have only little influ-
sample in 0.5 mL DMSO-d6 and chemical shifts (d) are expressed in
ence on the anti-tubercular activity. Compound
8 without
ppm. Elemental analyses were carried out in Heraeus Carlo Erba
1108 CHN analyzer.
a substituent in the aromatic ring and compound with an m-OCH3
substituent in the aromatic ring show greater activities compared
to the other compounds studied. Since the molecular mass of 8 is
6.2. General procedure for synthesis of 2,4-Diaryl-3-azabicyclo
60 units less than that of 13 by 60 units in 1
mg of 13 should contain
[3.3.1]nonan-9-one N-isonicotinoylhydrazone (8e14)
less number of molecules than that 1 g of 8. Hence, 8 can be
m
considered to be the most active compound studied.
However, the aromatic substituents substantially influence the
antimicrobial activities. Thus, in the case of S. aureus the activity
2r,4c-Diaryl-3-azabicyclo[3.3.1]-nonan-9-ones 1e7 were prepared
by the condensation of appropriate ketones, aldehydes and ammo-
nium acetate in 1:2:1 ratio, using literature procedure [24]. A mixture
of 2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-one (1e7) (1 mmol),
isoniazid (1.5 mmol) in methanol and chloroform (1:1 v/v), few drops
of acetic acid was added and refluxed for 2e3 h. On the completion of
reaction a solid mass was formed. After cooling to room temperature
the precipitate was filtered off and washed with cold mixture of
ethanol and water. The crude product was recrystallized from ethanol.
decreases in the order p-Cl > p-F > m-Cl >p-OCH3 > m-OCH3
p-CH3, if the MICs are expressed in mol/mL. However, the same
trend is not observed for the other bacterial strains. Thus, if MICs
are expressed in mol/mL the m-OCH3 compound is the most active
>
m
m
compound against B. subtilis. However, its activity against S. aureus
is only slightly greater than that of the least active compound 9.
On the same grounds the m-Cl compound 14 is the most active
compound against C. albicans-6, whereas the p-F compound 12 is
the most active compound against C. albicans and the p-Cl
compound 11 is the most active compound against C. albicans-51.
However, in the case of 4-aryl-5-isopropoxycarbonyl-6-methyl-
3,4-dihydropyrimidinones [15], among the p-F, p-Cl, p-CH3 and p-
OCH3 substituted compounds, the p-fluoro compound was found as
the most active against all the tested organisms.
If the antimycobacterial and antimicrobial activities are due to
the release of INH molecule by hydrolysis, the substituents in the
aromatic ring cannot cause such a marked variation in the activi-
ties. The rate of hydrolysis cannot be influenced significantly by the
aromatic substituent.
6.3. Analytical data for compounds (8e14)
6.3.1. 2r,4c-Diphenyl-3-azabicyclo[3.3.1]-nonan-9-one
N-isonicotinoylhydrazone (8)
Yield: 85%, m.p. 216e218 ꢁC. IR (KBr, nmax cmꢀ1): 3312 (NeH
stretching), 1637 (C]O stretching), 1524 (C]N stretching). 1H NMR
(d, DMSO-d6, ppm): 11.09 (s, 1H, amide NH), 8.75 (d, 2H, a-H), 7.77
(d, 2H, b
-H), 7.63 (t, 2H, o-H), 7.62 (t, 2H, o0-H), 7.41 (m, 4H, m-H, m0-
H), 7.28 (m, 2H, p-H, p0-H), 4.31(bs, 1H, H-2a,), 4.29 (bs, H, H-4a),
3.28 (s, 1H, H-5e), 2.74 (m, 1H, H-7a), 2.54 (s, 1H, H-1e), 1.63 (m, 1H,
H-8e), 1.47 (m, 3H, H-6a, H-6e, H-8a), 1.26 (m, 1H, H-7e), 2.93 (s, 1H,
ring NH). 13C NMR (
d, DMSO-d6, ppm): 64.2 (C-2), 62.2 (C-4), 45.9
However, in all cases the most active compound contains
a strong hydrogen-bonding atom O, F or Cl. Thus, it seems that
formation of hydrogen bonds may play a significant role in drug
action.
(C-1), 41.0 (C-5), 28.2 (C-8), 26.9 (C-6), 20.8 (C-7), 170.7 (C-9), 162.5
(NHCO), 150.0 (C-a), 121.8 (C-b), 141.4 (C-g
), 142.8 (C-20, C-40), 127.0
(o-C, o0-C), 128.2 (m-C), 128.0 (m0-C), 126.8 (p-C, p0-C). Anal. Found
(Cal.) for C26H26N4O (%): C, 75.89 (76.00); H, 6.23 (6.23); N, 13.42
(13.47).
5. Conclusions
6.3.2. 2r,4c-Bis(p-methylphenyl)-3-azabicyclo[3.3.1]nonan-9-one
N-isonicotinoylhydrazone (9)
2r,4c-Diaryl-3-azabicyclo[3.3.1]-nonan-9-one N-isonicotinoyl-
hydrazones 8e14 adopt twin-chair conformation with equatorial
orientations of the aryl groups. All the newly synthesized
compounds were screened for their preliminary antituberculosis,
antibacterial and antifungal activities. All these compounds show
good anti-tubercular activity against M. tuberculosis H37Rv and INH-
TB. Most of the compounds show good antibacterial and antifungal
activities.
Yield: 80%, m.p. 210e212 ꢁC. IR (KBr, nmax cmꢀ1): 3303 (NeH
stretching), 1654 (C]O stretching), 1512 (C]N stretching). 1H NMR
(
d
, DMSO-d6, ppm): 11.08 (s, 1H, amide NH), 8.75 (d, 2H,
a-H), 7.75
(d, 2H,
b
-H), 7.49 (m, 4H, o-H, o0-H), 7.20 (t, 4H, m-H, m0-H), 4.25 (bs,
1H, H-2a), 4.33(bs, 1H, H-4a), 3.16 (s, 1H, H-5e), 2.72 (m, 1H, H-7a),
1.65 (m, 1H, H-8e), 1.44 (m, 3H, H-6a, H-6e, H-8a), 1.25 (m, 1H,
H-7e), 2.77 (s, 1H, ring NH), 2.31 (d, 3H, p-CH3), 2.29 (s, 3H, p-CH3).

5484
C. Sankar, K. Pandiarajan / European Journal of Medicinal Chemistry 45 (2010) 5480e5485
13C NMR (
d
, DMSO-d6, ppm): 64.3 (C-2), 62.1 (C-4), 46.1(C-1), 41.0
54.9 (OCH3 at m), 170.5(C-9), 162.5 (NHCO), 149.9 (a-C), 121.7 (b-C),
(C-5), 28.2 (C-8), 26.9(C-6), 20.8(C-7), 20.7 (CH3 at p), 170.9(C-9),
141.3 (g
-C), 144.3 (C-20, C-4’), 159.2 (o,m-C, o,m0-C),119.2(o, p-C),
162.5(NHCO), 150.0(
a
-C), 121.8 (
b
-C), 141.4 (
g
-C), 135.8 (C-20, C-40),
118.9(o0,p-C), 113.2, 112.2 (p,o-C, p,o0-C), 112.0, 111.5 (o,o-C, o0,o-C).
126.9 (o-C), 126.7(o0-C), 128.7 (m-C), 128.6 (m0-C), 139.7 (p-C, p0-C).
Anal. Found (cal.) for C28H30N4O (%): C, 76.52 (76.61); H, 5.40 (5.47);
N, 12.67 (12.77).
6.3.7. 2r,4c-Bis(m-chlorophenyl)-3-azabicyclo[3.3.1]nonan-9-one
N-isonicotinoylhydrazone (14)
Yield: 82%, m.p. 225e227 ꢁC. IR (KBr, nmax cmꢀ1): 3311 (NeH
6.3.3. 2r,4c-Bis(p-methoxyphenyl)-3-azabicyclo[3.3.1]nonan-9-one
N-isonicotinoylhydrazone (10)
stretching, 1650 (C]O stretching), 1522 (C]N stretching). 1H NMR
(d, DMSO-d6, ppm): 11.09 (s, 1H, amide NH), 8,76 (d, 2H, a-H), 7.78
Yield: 82%, m.p. 22e223 ꢁC. IR (KBr, nmax cmꢀ1): 3304 (NeH
(d, 2H, b
-H), 7.67(m, 2H, o,o-H, o0,o-H), 7.55 (d, 2H, o,p-H, o,p0-H)),
stretching), 1653 (C]O stretching), 1510 (C]N ring stretching), 1H
7.47 (t, 2H, p,o-H, p,o0-H),7.36 (m, 2H, m, m0), 4.30 (d, 2H, H-2a, H-
4a), 3.27 (s, 1H, H-5e), 2.64 (m, 1H, H-7a), 1.61 (m, 1H, H-8e), 1.48
(m, 3H, H-6a, H-6e, H-8a), 1.27 (m, 1H, H-7e), 3.19 (s, 1H, ring NH).
NMR (d, DMSO-d6, ppm): 11.06 (s, 1H, amide NH), 8,75 (d, 2H,
a-H),
7.75 (d, 2H,
b
-H), 7.51 (q, 4H, o-H, o0-H), 6.96 (m, 4H, m-H, m0-H),
4.24 (bs, 1H, H-2a), 4.22 (bs, 1H, H-4a), 3.13 (bs, 1H, H-5e), 2.71 (m,
1H, H-7a), 1.67 (m, 1H, H-8e), 1.544 (m, 1H, H-6a), 1.44 (m, 2H, H-6e,
H-8a), 1.24 (m, 1H, H-7e), 2.75 (s, 1H, ring NH),3.74 (s, 3H, p-OCH3),
13C NMR (
d, DMSO-d6, ppm): 63.6 (C-2), 61.4 (C-4), 45.5 (C-1), 41.0
(C-5), 28.2 (C-8), 26.9 (C-6), 20.7 (C-7), 168.7 (C-9), 162.7 (NHCO),
150.0(a-C), 121.9 (b-C), 141.4 (g
-C), 133.0 (m-C), 132.8 (m0-C), 130.1
3.75 (d, 3H, p-OCH3). 13C NMR (
d
, DMSO-d6, ppm): 64.0 (C-2), 61.8
(C-4), 46.1(C-1), 41.0 (C-5), 28.2 (C-8), 26.9 (C-6), 20.8 (C-7), 55.0
(eOCH3 at p), 170.9(C-9), 162.5 (NHCO), 150.0 ( -C), 121.8 ( -C),
(m,m-C), 130.0 (m,m0-C), 126.9 (p,o-C, p0,o-C) 126.8, 126.6 (o0,p-C,
o,p-C), 125.8 (o, o-C), 125.6 (o0,o-C).
a
b
141.4 (
g
-C), 134.7 (C-20, C-40), 128.0 (o-C), 127.7 (o0-C), 113.6 (m-C),
6.4. Pharmacology
113.4 (m0-C), 158.2 (p-C, p0-C). Anal. Found (cal.) for C28H30N4O3 (%):
C, 71.35 (71.40); H, 6.28 (6.38); N, 11.82 (11.90).
6.4.1. Anti-tubercular activity
Primary in vitro antimycobacterial activities of the synthesized
compounds were evaluated by luciferase reporter phage assay
method [16] against M. tuberculosis H37Rv and INH-resistant
6.3.4. 2r,4c-Bis(p-chlorophenyl)-3-azabicyclo[3.3.1]nonan-9-one
N-isonicotinoylhydrazone (11)
Yield: 87%, m.p. 230e232 ꢁC. IR (KBr, nmax cmꢀ1): 3301 (NeH
M. tuberculosis at two concentrations (1.00 and 2.00
micro liter bacterial suspension equivalent to MacFarlands No.2
standard was added to 400 L of G7H9 with and without the test
mg/mL). Fifty-
stretching) 1652 (C]O stretching), 1489 (C]N stretching). 1H NMR
(
d
, DMSO-d6, ppm): 11.09 (s, 1H, amide NH), 8.75 (d, 2H,
a-H), 7.76
m
(d, 2H,
b
-H), 7.67 (q, 4H, o-H, o0-H), 7.46 (m, 4H, m-H, m0-H), 4.28
compound. For each sample, two drug-free controls and two drug
concentrations were prepared and this setup was incubated for
(bs, 2H, H-2a, H-4a), 3.20 (s, 1H, H-5e), 2.65 (m, 1H, H-7a), 2.53 (s,
1H, H-1e), 1.60 (m, 1H, H-8e), 1.46 (m, 3H, H-6a, H-6e, H-8a), 1.24
72 h at 37 ꢁC. After incubation 50
reporter phage (PhAE129) and 40 m
m
L of the high titer Luciferase
L of 0.1 M CaCl2 were added to
all the vials and this setup was incubated at 37 ꢁC for 4 h. After
incubation 100 L of the mixture was taken from each tube into
a luminometer cuvette and equal amount of working -luciferin
(m, 1H, H-7e), 3.08 (s, 1H, ring NH). 13C NMR (
d
, DMSO-d6, ppm):
63.6 (C-2), 61.4 (C-4), 45.6(C-1), 41.0 (C-5), 28.1(C-8), 26.8 (C-6),
20.7 (C-7), 169.7 (C-9), 162.6 (NHCO), 150.0 ( -C), 121.8 ( -C), 141.4
a
b
m
(g
-C), 141.6 (C-20, C-40), 128.1 (o-C), 127.9 (o0-C), 128.8 (m-C), 128.6
D
(m0-C), 131.3 (p-C, p0-C). Anal. Found (cal.) for C26H24Cl2N4O (%): C,
(0.3 mM in 0.05 M sodium citrate buffer, pH 4.5) solution was
added. The RLU was measured after 10 s of integration in the
Luminometer (Monolight 2010). Duplicate readings were recorded
for each sample and the mean was calculated. The percentage
reduction in the RLU was calculated for each test sample and
compared with control. The experiment was repeated when the
mean RLU of the control was less than 1000.
65.13 (65.08); H, 5.22 (5.01); N, 11.75 (11.68).
6.3.5. 2r,4c-Bis(p-fluorophenyl)-3-azabicyclo[3.3.1]nonan-9-one N-
isonicotinoylhydrazone (12)
Yield: 90%, m.p. 225e227 ꢁC. IR (KBr, nmax cmꢀ1): 3331 (NeH
stretching), 1638 (C]O stretching). 1509 (C]N stretching). 1H NMR
(
d
, DMSO-d6, ppm): 11.08 (s, 1H, amide NH), 8.75 (d, 2H,
a-H), 7.76
(d, 2H,
b
-H), 7.65 (m, 4H, o-H, o0-H), 7.22 (t, 4H, m-H, m0-H), 4.30 (bs,
6.4.2. Antimicrobial studies
1H, H-2a), 4.29 (bs, 1H, H-4a), 3.18 (s, 1H, H-5e), 2.67 (m, 1H, H-7a),
2.52 (s, 1H, H-1e), 1.61 (m, 1H, H-8e), 1.46 (m, 3H, H-6a, H-6e, H-8a),
The in vitro activities of the compounds were tested in Sabo-
urauds dextrose broth (SDB; Hi-media, Mumbai) for fungi and
Nutrient broth (NB; Hi-media, Mumbai) for bacteria by the two-fold
serial dilution method [17,18]. The test compounds were dissolved
in dimethylsulfoxide (DMSO) to obtain 1 mg mLꢀ1 stock solutions.
Seeded broth (broth containing microbial spores) was prepared in
NB from 24-h-old bacterial cultures on nutrient agar (Hi-media,
Mumbai) at 37 ꢂ 1 ꢁC while fungal spores from 24 h to 7-day-old
Sabourauds agar slant 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 104e105 cfu mLꢀ1. The final
inoculums size was 105 cfu mLꢀ1 for antibacterial assay and
1.1e1.5 ꢃ 102 cfu mLꢀ1 for antifungal assay. Testing was performed
at pH 7.4 ꢂ 0.2. Exactly 0.2 mL of the solution of 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 simul-
taneously. The tubes were incubated in BOD incubators at 37 ꢂ 1 ꢁC
for bacteria and 28 ꢂ 1 ꢁC for fungi. The minimum inhibitory
1.26 (m, 1H, H-7e), 3.02 (s, 1H, ring NH). 13C NMR (
d
, DMSO-d6,
ppm): 63.6 (C-2), 61.4 (C-4), 45.7 (C-1), 41.0 (C-5), 28.1(C-8), 26.8
(C-6), 20.7 (C-7), 170.0 (C-9), 159.9 (NHCO), 150.0 ( -C), 121.8 ( -C),
a
b
141.4 (g
-C), 138.8 (C-20, C-4’), 128.8 (o-C), 128.6 (o0-C), 114.9 (m-C),
114.7 (m0-C), 162.4 (p-C, p0-C). Anal. Found (cal.) for C26H24F2N4O
(%): C, 69.79 (69.89); H, 5.25 (5.37); N, 12.45 (12.54).
6.3.6. 2r,4c-Bis(m-methoxyphenyl)-3-azabicyclo[3.3.1]nonan-9-
one N-isonicotinoylhydrazone (13)
Yield: 77%, m.p. 209e211 ꢁC. IR (KBr, nmax cmꢀ1): 3311 (NeH
stretching), 1630 (C]O stretching), 1593 (C]N ring stretching). 1H
NMR (
7.76 (d, 2H,
d
, DMSO-d6, ppm): 11.07 (s, 1H, amide NH), 8,75 (d, 2H,
a-H),
b
-H), 7.32 (m, 2H, m-H, m0-H), 7.20 (m, 2H, o,p-H, o,p0-
H), 7.14 (m, 2H, p,o-H, p,o0-H), 6.86 (d, 2H, o,o-H, o,o0-H), 4.27 (bs,
1H, H-2a),4.26 (bs, 1H, H-4a), 3.19 (s, 1H, H-5e), 2.68 (m, 1H, H-7a),
2.56 (s, 1H, H-1e), 1.66 (m, 1H, H-8e), 1.54 (m, 1H, H-6a), 1.46 (m, 2H,
H-6e, H-8a), 1.26 (m, 1H, H-7e), 2.88 (s, 1H, ring NH), 3.78 (s, 3H, p-
OCH3), 3.77 (s, 3H, p-OCH3). 13C NMR (
d, DMSO-d6, ppm): 64.1 (C-2),
62.0 (C-4), 45.8 (C-1), 41.0 (C-5), 28.2 (C-8), 26.9 (C-6), 20.7 (C-7),

C. Sankar, K. Pandiarajan / European Journal of Medicinal Chemistry 45 (2010) 5480e5485
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Acknowledgements
Authors would like to thank the NMR research centre, IISc-
Bangalore for recording the NMR spectrum. We are thankful to
CECRI e Karaikudi for providing analytical data. We are thankful to
Tuberculosis Research Centre, Chennai, India, for the in vitro eval-
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RMMCH, Annamalai University for the antimicrobial studies. One of
the author C. Sankar is thankful to UGC, India for the award of
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