Synthesis, stereochemical, structural, and biological studies of a series of N′-(2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)pyrazine-2-carbohydrazides

Article abstract of DOI:10.1016/j.molstruc.2016.09.033

A new series of N′-(2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)pyrazine-2-carbohydrazides (8–14) were synthesized by the corresponding 2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-ones (1–7) reaction with pyrazine-2-carbohydrazide. The stereochemistry of

Full text of DOI:10.1016/j.molstruc.2016.09.033

Accepted Manuscript
Synthesis, stereochemical, structural and biological studies of some N′-(2r,4c-diaryl-3-
azabicyclo[3.3.1]nonan-9-ylidene)pyrazine-2-carbohydrazide
M. Mangalam, C. Sebastian Antony Selvan, C. Sankar
PII:
S0022-2860(16)30960-7
10.1016/j.molstruc.2016.09.033
MOLSTR 22945
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 21 April 2016
Revised Date: 20 August 2016
Accepted Date: 12 September 2016
Please cite this article as: M. Mangalam, C.S.A. Selvan, C. Sankar, Synthesis, stereochemical,
structural and biological studies of some N′-(2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)pyrazine-2-
carbohydrazide, Journal of Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.09.033.
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ACCEPTED MANUSCRIPT
Synthesis, Stereochemical, Structural and Biological studies of some
N'-(2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)pyrazine-2-carbohydrazide
a
b*
c
M. Mangalam , C. Sebastian Antony Selvan and C. Sankar
a
Department of Chemistry, Rajah serfoji Govt. College (auto), Thanjavur – 613005, Tamilnadu, India.
b
Department of Chemistry, R.V. Govt. Arts College, Chengalpattu, Chennai, India
c
Department of Chemistry, TRP Engineering College, Tiruchirappalli – 626 126, Tamil Nadu, India
A new series of N′-(2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)pyrazine-2-
carbohydrazides (8-14) have been synthesized. All the compounds have been tested for their
antibacterial activity.
N
O
O
N
H
NH
O H
N
R
R
O
+
N
H
CH COON
H4
3
R
R
*
Corresponding author
E. mail address: sebantonyselvan@gmail.com
Department of Chemistry, R.V. Govt. Arts College, Chengalpattu, Chennai, Tamil Nadu, India
*

ACCEPTED MANUSCRIPT
Synthesis, Stereochemical, Structural and Biological studies of some
N'-(2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)pyrazine-2-carbohydrazide
a
b*
c
M. Mangalam , C. Sebastian Antony Selvan and C. Sankar
a
Department of Chemistry, Rajah serfoji Govt. College (auto), Thanjavur– 613005, Tamilnadu,India.
b
Department of Chemistry, R.V.Govt.Arts College, Chengalpattu, Chennai, India
c
Department of Chemistry, TRP Engineering College, Tiruchirappalli – 626 126, Tamil Nadu, India
ABSTRACT
A new series of N
carbohydrazides (8-14) were
′
-(2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)pyrazine-2-
synthesized by corresponding 2r,4c-diaryl-3-
azabicyclo[3.3.1]nonan-9-ones (1-7) reaction with pyrazine-2-carbohydrazide. The
stereochemistry of the newly synthesized compounds were unambiguously assigned by using
1
13
FT-IR, H, C and 2D (COSY, HSQC, HMBC, ROESY) NMR spectral data. The chemical
shifts suggest that in all these compounds adopts twin-chair conformation with equatorial
orientation of aryl substitutions in solution. Hydrazones were screened for their in vitro
antitubercular activity against Mycobacterium Tuberculosis H Rv and antibacterial activity
3
7
against a set of pathogenic bacteria’s. Most of the halogenated compounds expressed
promising antitubercular and antibacterial activities.
Keywords: Stereochemistry
-
Conformation
-
Anti-TB
–
Hydrazone
-
Azabicyclo[3.3.1]nonan-9-ones.
*
Corresponding author,
E. mail address: sebantonyselvan@gmail.com
*
Department of Chemistry, R.V.Govt. Arts College, Chengalpattu, Chennai, Tamil Nadu, India

ACCEPTED MANUSCRIPT
Introduction
NMR techniques have been extensively applied in deriving stereo-dynamical
information about a wide variety of organic systems. Conformation studies of heterocyclic
compounds have helped in understanding the influence of electronic and conformation effects
on chemical shifts, coupling constant and bioactivity [1-10]. Such, studies have furnished
information about the dependence of NMR spectral parameters such as chemical shifts and
coupling constants on electronegativities of heteroatoms. Proton chemical shifts are
influenced by electronic and magnetic anisotropic effects, whereas carbon chemical shifts are
largely influenced by electronic and steric effects only.
The heterocyclic system of 3-azabicyclononane are distributed in various alkaloids
such as kobusine, hetisine, delcorine, deltaline, elatine, inulin, etc., They are having
interesting chemical and biological actions such as antibacterial antimycobacterial,
antiinflammatory, antiarrhythmic, antifungal, antiallergic, antiprotozoan, antioxidant,
antitumor, anticonvulsant, antiviral, antimalarial and cytotoxic activity [11-17]. In addition a
family of hydrazone / hydrazide derivatives are containing highly reactive azomethine group
(CO-NH-N=CH) and thus useful in new drug development [18]. Also, these are found to
possess antimicrobial [16,19], antimalarial [20], anticonvulsant [21], analgesic [22],
antiinflammatory [23], antiplatelet [24], antitumoral [25] and antiviral [26] activities. The
structural requirement for inhibition of antimycobacterial activity suggests the presence of a
pyrazine ring with an acyl moiety [27].
The main reason for selection of pyrazine-2-carbohydrazide as a lead molecule in this
study that are simplicity of synthesis methodology and search on new compounds related to
pyrazine derivatives which are known for their antitubercular activity [28]. In the present
study, 3-azabicyclo[3.3.1]nonan-9-one carbohydrazide which are active against MTB as
attempt has been made to condense with pyrazine-2-carbohydrazide to explore the

ACCEPTED MANUSCRIPT
possibilities of their conformation and the MTB activity. Based on the higher bioactivity of
hydrazones, we have synthesized novel hydrazones (8-14) from piperazine-4-carbohydrazide
coupled with 2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-ones (1-7). The assignment of all
proton and carbon resonances was achieved by double resonance experiments. The
antitubercular and antibacterial studies were effectively done for newly synthesized
hydrazones with different concentrations.
Experimental
Purchasing materials and recording spectra
All the solvents and chemicals were purchased from Sigma–Aldrich and were used as
received and the purity of the compounds was checked by TLC. The melting points were
recorded in open capillaries and are uncorrected. FT-IR spectra were recorded in KBr (pellet
forms) on AVATAR-330 FT-IR spectrophotometer (Thermo Nicolet) and noteworthy
1
13
absorption levels (reciprocal centimeters) alone are listed. H and C NMR spectra of 8-14
have been recorded on a BRUKER AMX 400 NMR spectrometer operating at 400.13 MHz
1
13
for H and 100.62 MHz for C, in DMSO-d . The following spectral parameters were used
6
1
H; acquisition time = around 3.0 s, number of scans = around 100 s, number of data points =
13
3
2 K and special with = 5000 Hz. C; acquisition time = around 0.5 s, number of scans =
1
1
around 1000, number of data points = 32 K and special with = 30,000 Hz. H– H COSY,
HSQC, HMBC and ROESY NMR spectra were recorded on a BRUKER DRX 500 MHz
NMR spectrometer using standard parameters. All NMR measurements were made on 5 mm
NMR tubes. The solutions were prepared by dissolving about 10 mg of the compound in 0.5
mL of DMSO-d6.
Synthesis of 2,4-diaryl-3-azabicyclo [3.3.1]nonan-9-ones
The synthetic route of the target compounds 8-14 is illustrated in Scheme 1. The
starting compounds 2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-ones 1-7, were prepared by the
mixture of cyclohexanone, substituted benzaldehyde and ammonium acetate in 1:2:1.5 ratio

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in ethanol was gently warmed and allowed overnight [29]. The crude products formed were
filtered and washed with an ethanol-ether (1:5) mixture. Then the bicyclic ketones were
recrystallized from ethanol-chloroform to obtain the pure compounds.
General procedure for synthesis of
N′-(2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-
ylidene)pyrazine-2-carbohydrazide (8-14)
The quest for synthesis of
N′-(2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-
ylidene)pyrazine-2-carbohydrazide (8-14) was accomplished in two steps as outlined in
Scheme 1. A mixture of 2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-one (1-7) (1 mmol),
commercially available pyrazine-2-carbohydrazide (PZH) (1.5 mmol) in methanol and
chloroform mixture (1:1 v/v), catalytic amount of acetic acid (0.1 ml) was added and refluxed
for 2-3 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 [16].
N′-(2r,4c-diphenyl-3-azabicyclo[3.3.1]nonan-9-ylidene)pyrazine-2-carbohydrazide (8)
–1
M.P 220-221°C. Yield 95%. IR (KBr, νmax cm ): 3322 (N–H stretching), 1650 (C=O
1
stretching), 1546 (C=N stretching). H NMR (δ, DMSO-d -ppm): 11.08 (s, 1H, amide NH),
6
9
.65 (s, 1H, H-3″′), 8.72 (d, 1H, H-5″′), 7.93 (d, 1H, H-6″′), 7.64 (t, 2H, o-H),7.63 (t, 2H, o′-
H), 7.41 (m, 4H, m-H, m′-H), 7.28 (m, 2H, p-H, p′-H), 4.32 (bs, 1H, H-2a,), 4.20 (bs, H, H-
4
3
6
1
1
a), 3.28 (s, 1H, H-5e), 2.73 (m, 1H, H-7a), 2.54 (s, 1H, H-1e), 1.63 (m, 1H, H-8e), 1.46 (m,
1
3
H, H-6a, H-6e, H-8a), 1.26 (m, 1H, H-7e), 2.93 (s, 1H, ring NH). C NMR (δ, DMSO-d ):
6
4.4 (C-2), 62.2 (C-4), 45.9 (C-1), 41.0 (C-5), 28.2 (C-8), 26.9 (C-6), 20.8 (C-7), 170.7 (C-9),
62.5 (NHCO), 149.3 (C-3″′), 121.8 (C-2″′), 141.4 (C-5″′), 150.37 (C-6″′) 142.8 (C-2′, C-4′),
27.0 (o-C, o′-C), 128.2 (m-C), 128.0 (m′-C), 126.8 (p-C, p′-C). Anal. Calculate (Found) for
C H N O (%): C, 72.90 (72.97); H, 6.12 (6.18); N, 17.02 (16.97).
25
25
5

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N′-[2r,4c-bis(p-methylphenyl-3-azabicyclo[3.3.1]nonan-9-ylidene]pyrazine-2-carbohydrazide(9)
–1
M.P 210-212°C. Yield 80%. IR (KBr, νmax cm ): 3303 (N–H stretching), 1654 (C=O
1
stretching), 1512 (C=N stretching). H NMR (δ, DMSO-d -ppm): 11.08 (s, 1H, amide NH),
6
9.65 (s, 1H, H-3″′), 8.73 (d, 1H, H-5″′), 7.94 (d, 1H, H-6″′), 7.49 (m, 4H, o-H, o′-H), 7.20 (t,
4H, m-H, m′-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,
1
3
ring NH), 2.31 (d, 3H, p-CH ), 2.29 (s, 3H, p-CH ). C NMR (δ, DMSO-d ): 64.3 (C-2), 62.1
3
3
6
(
C-4), 46.1(C-1),41.0 (C-5), 28.2(C-8), 26.9(C-6), 20.8(C-7), 20.7 (CH at p), 170.9(C-9),
3
1
62.5(NHCO), 149.3 (C-3″′), 121.8 (C-2″′), 141.4 (C-5″′), 150.37(C-6″′), 126.9 (o-C),
26.7(o′- C), 128.7 (m-C), 128.6 (m′-C), 139.7 (p-C, p′-C). Anal. Calculate (Found) for
1
C H N O (%): C, 73.78 (73.7); H, 6.65 (6.59); N, 15.93 (15.97).
27
29
5
N′-[2r, 4c-bis(p-chlorophenyl-3-azabicyclo[3.3.1]nonan-9-ylidene]pyrazine-2-carbohydrazide (10)
–
1
M.P 245-246°C. Yield 88%. IR (KBr, νmax cm ): 3301(N–H stretching) 1652 (C=O
1
stretching), 1489 (C=N stretching). H NMR (δ, DMSO-d -ppm): 11.09 (s, 1H, amide NH),
6
9
.65 (s, 1H, H-3″′), 8.75 (d, 1H, H-5″′), 7.78 (d, 1H, H-6″′), 8,75 (d, 2H, α-H), 7.76 (d, 2H,
β-H), 7.67 (q, 4H, o-H, o′-H), 7.46 (m, 4H, m-H, m′-H), 4.28 (bs, 2H, H-2a, H-4a), 3.20 (s,
1
6
6
H, 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-
1
3
e, H-8a), 1.24 (m, 1H, H-7e), 3.08 (s, 1H, ring NH). C NMR (δ,DMSO-d ): 63.6 (C-2),
6
1.4 (C4), 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), 149.3 (C-3″′), 121.8 (C-2″′), 141.4 (C-5″′), 150.37(C-6″′), 141.6 (C-2’, C-4’),
28.1 (o-C), 127.9 (o′-C), 128.8 (m-C), 128.6(m′-C), 131.3 (p-C, p′-C). Anal. Calculate
Found) for C H Cl N O (%): C, 62.51 (62.47); H, 4.83 (4.89); N, 14.58 (14.65).
1
(
2
5
23
2
5

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N′-[2r, 4c-bis(p-fluorophenyl-3-azabicyclo[3.3.1]nonan-9-ylidene]pyrazine-2-carbohydrazide (11)
–
1
M.P 235-236 °C. Yield 90%. IR (KBr, νmaxcm ): 3330 (N–H stretching), 1638 (C=O
1
stretching). 1510 (C=N stretching). H NMR (δ, DMSO-d -ppm): 11.08 (s, 1H, amide
6
NH),9.68 (s, 1H, H-3″′), 8.75 (d, 1H, H-5″′), 7.76 (d, 1H, H-6″′), 7.65 (m, 4H, o-H, o′-H),
7
.22 (t, 4H, m-H, m′-H), 4.30 (bs, 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), 1.26
1
3
m, 1H, H-7e), 3.02 (s, 1H, ring NH). C NMR (δ, DMSO-d ): 63.6 (C-2), 61.4 (C-4), 45.7
6
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), 149.3 (C-
3
″′), 121.8 (C-2″′), 141.4 (C-5″′), 150.37(C-6″′), 138.8 (C-2’, C-4’), 128.8 (o-C), 128.6(o′-
C), 114.9 (m-C), 114.7 (m′-C), 162.4 (p-C,p′-C). Anal. Calculate (Found) for C H F N O
2
5
3
2
5
(%): C, 67.10 (67.15); H, 5.18 (5.13); N, 15.36 (15.40).
N′-[2r, 4c-bis(p-methoxyphenyl-3-azabicyclo[3.3.1]nonan-9-ylidene]pyrazine-2-carbohydrazide (12)
–1
M.P 221-223°C. Yield 89%. IR (KBr, νmax cm ): 3324 (N–H stretching), 1653 (C=O
1
stretching), 1510 (C=N ring stretching), H NMR (δ DMSO-d -ppm): 11.06 (s, 1H, amide
6
NH), 9.65 (s, 1H, H-3″′), 8.75 (d, 1H, H-5″′), 7.75 (d, 1H, H-6″′), 7.51 (q, 4H, o-H, o′-H),
6
.96 (m, 4H, m-H, m′-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,
13
1
H, H-7e), 2.75 (s, 1H, ring NH),3.74 (s, 3H, p-OCH ), 3.75 (d, 3H, p-OCH ). C NMR (δ,
3 3
DMSO-d ): 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),
6
5
5.0 (-OCH at p), 170.9(C-9), 162.5 (NHCO), 149.3 (C-3″′), 121.8 (C-2″′), 141.4 (C-5″′),
3
1
50.37(C-6″′), 134.7 (C-2′, C-4′), 128.0 (o-C), 127.7 (o′-C), 113.6 (m-C), 113.4 (m′-C), 158.2
(
p-C, p′-C). Anal. Calculate (Found) for C H N O (%): C, 68.77 (68.70); H, 6.20 (6.28);
27 29 5 3
N, 14.85 (14.90).
N′-[2r, 4c-bis(m-chlorophenyl-3-azabicyclo[3.3.1]nonan-9-ylidene]pyrazine-2-carbohydrazide(13)

ACCEPTED MANUSCRIPT
–1
M.P 239-240°C. Yield 86%. IR (KBr, νmaxcm ): 3321 (N–H stretching, 1653 (C=O
1
stretching), 1532 (C=N stretching). H NMR (δ, DMSO-d -ppm): 11.09 (s, 1H, amide NH),
6
9
.67 (s, 1H, H-3″′), 8.76 (d, 1H, H-5″′), 7.78 (d, 1H, H-6″′), 7.67(m, 2H,o,o-H, o′,o-H), 7.55
d, 2H, o,p-H, o,p′-H)), 7.47 (t, 2H,p,o-H, p,o′-H),7.36 (m, 2H, m, m′), 4.30(d, 2H, H-2a, H-
a), 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-
(
4
8
4
1
1
3
a), 1.27 (m, 1H, H-7e), 3.19 (s, 1H, ring NH). C NMR (δ, DMSO-d ): 63.6 (C-2), 61.4 (C-
6
), 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),
50.0 (C-3″′), 121.9 (C-2″′), 141.4 (C-5″′), 150.37(C-6″′), 133.0 (m-C), 132.8 (m′-C), 130.1
(m,m-C), 130.0 (m,m′-C), 126.9 (p,o-C, p′,o-C) 126.8, 126.6 (o′,p-C, o,p-C), 125.8 (o, o-C),
1
25.6 (o′,o-C). Anal. Calculate (Found) for C H Cl N O (%): C, 62.51 (62.56); H, 4.83
2
5
23
2
5
(4.82); N, 14.58 (14.53).
N′-[2r, 4c-bis(m-methoxyphenyl-3-azabicyclo[3.3.1]nonan-9-ylidene]pyrazine-2-carbohydrazide(14)
–
1
M.P 210-211°C. Yield 73%. IR (KBr, νmaxcm ): 3325 (N–H stretching), 1644 (C=O
1
stretching), 1593 (C=N ring stretching). H NMR (δ, DMSO-d -ppm): 11.07 (s, 1H, amide
6
NH), 9.64 (s, 1H, H-3″′), 8.75 (d, 1H, H-5″′), 7.76 (d, 1H, H-6″′), 7.32 (m, 2H, m-H, m′-H),
7
.20 (m, 2H, o,p-H, o,p′-H), 7.14 (m, 2H, p,o-H, p,o′-H), 6.86 (d, 2H, o,o-H, o,o′-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-
1
2
6
e), 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),
1
3
.88 (s, 1H, ring NH), 3.78 (s, 3H, p-OCH ), 3.77 (s, 3H, p-OCH ). C NMR (δ, DMSO-d ):
3
3
6
4.1 (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), 54.9 (OCH3
at m), 170.5(C-9), 162.5 (NHCO), 149.9 (C-3″′), 121.7 (C-2″′), 141.4 (C-5″′), 150.37(C-6″′),
44.3(C-2’, C-4’), 159.2 (o,m-C, o,m′-C),119.2(o,p-C), 118.9(o′,p-C), 113.2, 112.2 (p,o-C,
p,o′-C), 112.0, 111.5 (o,o-C, o′,o-C). Anal. Calculate (Found) for C H N O (%): C, 68.77
1
2
7
29
5
3
(68.83); H, 6.20 (6.16); N, 14.85 (14.80).

ACCEPTED MANUSCRIPT
Evaluation of antitubercular activity
The in vitro antitubercular activity of the synthesized compounds was evaluated by
the help of Microplate Alamar Blue Assay method (MABA). The resazurin colorimetric MIC
assay method was used to test the MIC of compounds [30]. A color change from blue to pink
was observed when growth occurs. Compounds were initially tested at a single point
concentration of 10 µg/mL against Mycobacterium Tuberculosis (H Rv), obtained from
3
7
Colorado State University. Test compounds were logged into the inventory spreadsheet and
placed in a -20ºC freezer.
H Rv is grown in Middle brook 7H9 broth medium (7H9 medium) supplemented
3
7
with 0.2% (v/v) glycerol, 10% (v/v) ADC (albumin, dextrose, catalase), and 0.05% (v/v)
Tween 80. The bacteria were inoculated in 100 ml of 7H9 medium in 1 liter roller bottles
o
that were placed on a roller bottle apparatus in an ambient 37 C incubator. When the cells
reach an OD600 of 0.150 (equivalent to ~1.5 x 107 CFU/ml), they were diluted 200-fold in
7
H9 medium. The procedure was the same as that used for the MIC procedure described, but
only the first 2 fold dilution was made that reduces the stock solution to 1.6 mg/ml. An
additional 1:10 dilution was made in water, which reduces the stock solution further to 0.16
mg/ml. Addition of 6.25 ꢀl of the 1:10 dilution to the wells in a final volume of 100 ꢀl will
give rise to a concentration equivalent to 10ꢀg/ml.
Antibacterial studies
The in vitro antibacterial activity of the compounds was tested in Nutrient broth (NB;
Hi-media, Mumbai) by two-fold serial dilution method [31]. The test compounds were
-1
dissolved in dimethylsulfoxide (DMSO) to obtain 1mg mL 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±1 °C. The colony forming units (cfu) of the seeded
(
4
5
-1
broth were determined by plating technique and adjusted in the range of 10 -10 cfu mL .

ACCEPTED MANUSCRIPT
5
-1
The final inoculums size was 10 cfu mL for antibacterial assay. Testing was performed at
pH 7.4 ± 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 simultaneously. The tubes were incubated in BOD incubators at 37 ± 1°C. The
minimum inhibitory concentrations (MICs) were recorded by visual observations after 24 h.
Ciprofloxacin was used as standard drug.
Results and Discussion
Chemistry
In a series of recent reports, methods of synthesis and reactivity of new hydrazone
derivatives bearing pyrazines function were described [21, 26]. N
′-(2r,4c-diaryl-3-azabicyclo
[
3.3.1]nonan-9-ylidene)pyrazine-2-carbohydrazide (8-14) can be smoothly obtained by the
condensation of corresponding 2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-ones (1-7) with
pyrazine-2-carbohydrazide in presence of glacial acetic acid. In all cases expected hydrazones
8
-14 were formed as single compound, and isolated in good to excellent yield.
Numbering and designation of atoms
The numbering of the carbons of the piperidine and cyclohexane rings are shown in
Fig. 1. The ipso carbons of the aryl groups at C-2 and C-4 are designated as C-2′ and C-6′.
The other carbons of 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 o′, m′ and p′-carbons. The carbons of the pyrazine ring are
designated using 2′′, 3′′, 5′′ and 6′′. The protons are denoted accordingly. For example, the
benzylic proton at C-2 is denoted as H-2 and that at C-2′′is denoted as H-2′′ and so on.

ACCEPTED MANUSCRIPT
The methylene protons in the cyclohexane ring at C-6, C-7 and C-8 are denoted as
axial and equatorial protons assuming chair conformation for the cyclohexane ring. Thus, the
methylene protons at C-8 are denoted as H-8a and H-8e.
IR spectral studies
-1
In IR spectra, the presence of C꞊N stretching frequency around 1540 cm confirm the
hydrazone formation. In all cases two separate N-H stretching bands were observed for two
-1
different NH stretching vibrations. In the range 3160–3220 cm are due to N-H of hydrazone
-1
analogues and the piperidine NH stretching frequency are in the range 3230–3310 cm . The
-1
absorption band in the region 3070–2800 cm are ascribed to aromatic and aliphatic C-H
-1
stretching frequencies. The band observed in the region 1645–1660 cm are due to C꞊O
stretching frequency of amide carbonyl group. The important FT-IR stretching frequencies of
8
-14 are given in experimental section.
NMR Spectroscopy
Proton NMR spectral analysis of compound (8)
In order to analysis the spectral assignments of synthesized compounds 8-14, we have
1
13
chosen compound 8 as the representative compound. The H and C signals for the
remaining compounds were assigned by comparison with 8 and using known effects [32] of
1
9
the Cl, CH and OCH substituents in the phenyl rings. In compound 11 coupling with F
3
3
1
was observed for the aromatic carbons except the ipso carbons C-2′ and C-4′ [33]. H NMR
spectrum of 8 there is a sharp singlet at 11.08 ppm, corresponding to one proton. This should
be due to the amide NH proton. There are two doublets at 8.72 and 7.93 ppm, each
corresponding to two protons and a sharp singlet at 9.65 ppm corresponding to one proton.
These signals must be, respectively, due to the H-5″′, H-6″′and H-3″′ protons of pyrazine ring.
There are two doublets at 7.64 and 7.63 ppm, each corresponding to two protons. These are
due to the ortho protons (o-H and o'-H). There is one quartet at 7.41 ppm corresponding to

ACCEPTED MANUSCRIPT
four protons. This signal is an overlap of two triplets and it should be due to the meta protons
(m-H and m'-H). There is one quartet at 7.28 ppm corresponding to two protons. This signal
should be due to the mata protons of the phenyl groups. The ortho protons appear at a higher
frequency than the other aromatic protons due to the deshielding by the nitrogen lone pair.
There are two singlets at 4.32 and 4.29 ppm each corresponding to one proton. These
signals are due to the benzylic protons H-2a and H-4a. There are two broad singlets at 2.54
and 3.28 ppm each corresponding to one proton. These must be due to bridgehead protons H-
1
e and H-5e. Since H-5e should be deshielded by steric interaction with NHCOPZH moiety
the signal at 3.28 ppm should be due to H-5e. It has been shown [16] that in 2r,4c-diaryl-3-
azabicyclo[3.3.1]nonan-9-ones proton H-7a appears as a multiplet around 3.0 ppm. Hence,
the well resolved multiplet at 2.73 ppm, corresponding to one proton, can be assigned to H-
7
a. Proton H-7e appears as a quintet at 1.26 ppm.
There is a broad singlet at 2.93 ppm corresponding to one proton. This signal is due to
the NH proton of the piperidine ring. There are two multiplets at 1.63 and 1.47 ppm. The
multiplet at 1.63 ppm corresponds to one proton. This signal is due to H-8e. The multiplet at
1
.47 ppm corresponds to three protons, ascribed to H-6a, H-6e and H-8a.
1
1
In order to confirm the above assignment H- H COSY and ROESY spectra have
been recorded. The observed correlations of 8 are given in Table 1 and Shown in Fig. 2. In
1
1
the H- H COSY spectrum the bridgehead proton H-1e has weak correlation with the
benzylic proton at 4.32 ppm and has strong correlation with the three protons signal at 1.46
ppm. The signal at 2.73 ppm (H-7a) also has strong correlation with multiplet centered at
1
.46 ppm. These observations suggest that the signal at 1.46 ppm can be unambiguously
assigned to H-6a, H-8a and H-6e. Furthermore, H-7a has strong correlation with the signals at
.63 and 1.26 ppm. Hence, the signal at 1.63 ppm is due to H-8e and that at 1.26 ppm is due
to H-7e. The two benzylic protons at 4.32 and 4.20 ppm have strong correlations with the
1

ACCEPTED MANUSCRIPT
signal at 2.93 ppm. These correlations suggest that the signal 2.93 ppm is due to piperidine
NH proton.
In the ROESY spectrum the doublet at 7.64 ppm has strong nOes with H-4a at 4.29
ppm and H-7a at 2.73 ppm. Hence, the doublet at 7.64 ppm must be due to the ortho protons
(o'-H) of the phenyl ring at C-4. Likewise, the doublet at 7.63 ppm has strong nOes with H-2a
at 4.32 ppm and H-1e at 2.54 ppm. Hence, the doublet at 7.63 ppm must be due to the ortho
protons (o-H) of the phenyl ring at C-2.
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
positions, respectively. There is significant nOe between the hydrazone NH proton at 11.08
ppm and bridgehead proton at 3.28 ppm. This shows that the signal at 3.28 ppm is due to H-
1
5
e. All these observations are consistent with the assignments of H signals and conformation
8
CC assigned to 8 (Fig. 3).
In addition, the NOE between NH proton in hydrazone analogue and H-5e bridgehead
proton clearly reveals that the H-5e proton is syn to the hydrazone analogue.
In 9-14 individual assignments of the protons were made based on their positions,
multiplicities, integral values of the signals and by comparison with 8. Normally, the ortho,
meta and para protons of piperidine ring give separate signals. Ortho protons are deshielded
by the lone pair of electron on the nitrogen while the meta and para protons are shielded and
resonate in the upfield region.
13
Analysis of C NMR spectra
1
3
In order to assign the C signals unambiguously HSQC and HMBC spectra have
been recorded. The observed correlations in the HSQC and HMBC spectra are given in Table
2
. There is a weak signal at 170.7 ppm. This signal has no correlation in the HSQC spectrum.
In the HMBC spectrum, this signal shows correlation with NH proton at 11.08 ppm. Hence,

ACCEPTED MANUSCRIPT
this signal must be due to C-9. There are four other weak signals at 162.5, 142.8 and 141.4
ppm. These signals have no correlation in the HSQC spectrum. The signal at 162.5 ppm
shows correlation with amide NH proton at 11.08 ppm in the HMBC spectrum. Hence, this
signal should be due to the carbonyl carbon (C=O). The signals at 142.8 ppm show
correlation with the meta protons at 7.41 ppm, can be assigned to the ipso-carbons C-2' and
C-4'.
In the HMBC (Fig. 4) spectrum the signal at 141.4 ppm has correlation with H-5″′ at
8
.72 ppm. Hence, this signal should be due to C-5″′. The other signals are assigned based on
1
3
the observed correlations in the HSQC spectrum. The C chemical shifts are given
experimental section.
The proton signal at 3.28 ppm shows correlation with the signal of DMSO-d . From
6
this correlation, it is obvious that the signal for C-5 is mixed with the solvent signal. By
careful inspection the chemical shift of C-5 was determined as 41.0 ppm.
Among the bridgehead carbons C-5 is shielded by about 5 ppm relative to C-1. This is
due to the interaction between H-5e and nitrogen in hydrazone unit (Fig. 5). This interaction
induces a polarity in the C-5 - H-5e bond so that the proton H-5e gets a partial positive charge
and C-5 gets a partial negative charge. Consequently, the proton is deshielded and the carbon
is shielded. Such an effect has been observed in related compounds and has been termed as γ-
syn effect [8].
Interestingly, among the three methylene carbons of the cyclohexane ring, C-7 is
shielded by about 7 ppm than C-6 and C-8. This is due to the steric interaction between H-7a
and lone pair on the nitrogen atom of the piperidine ring. Owing to this interaction, C-7 - H7a
bond is polarized, H-7a acquires a partial positive charge and C-7 acquires a partial negative
charge. Therefore, C-7 is shielded and H-7a is deshielded. The partial negative charge on C-7
shields H-7e. Hence, H-7e appears at a considerably lower frequency (δ = 1.26 ppm).

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Antitubercular evaluation
The primary antimycobacterial screening was performed in accordance with the protocol
of the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) Southern
Research institute [30]. All compounds were screened for their in vitro antimycobacterial
activity against M. tuberculosis H Rv (ATCC2729) using isoniazid and rifampicin as the
37
standards. Compounds demonstrating a percent inhibition of bacterial growth greater than or
equal to 90% in the primary screening was re-tested against M. tuberculosis H Rv, to
37
determine the actual minimum inhibition concentration (MIC) in the MABA. The MIC was
defined as the minimum concentration of the compound required to inhibit 90% of the
bacterial growth and reported in Table 3 along with the standard drugs for comparison. This
values was determined from the dose-response curve as the IC90 using a curve fitting
program. Any IC value of ≤10 µg/mL was considered “Active” for the antitubercular
9
0
activity. Compounds active in the initial screen were tested for cytotoxicity (IC ) in VERO
5
0
cells. Cytotoxicity was determined from the does-response curve as the IC using a curve
5
0
fitting programs. Concurrent with the determination of MICs, compounds were tested for
cytotoxicity in VERO cells at concentration 10×MIC for the M. tuberculosis H Rv (Table
3
7
3
). The selectivity index (SI) was defined as the ratio of the measured IC in VERO cells to
50
the MIC.
The percentage inhibition was found to lie in a large range of 60-70%. The resazurin
MIC assay is a colorimetric assay used to test compounds for antimycobacterial activity. A
color change from blue to pink is observed when growth occurs. All of these compounds
displayed good in vitro activity against M .tuberculosis H Rv strain. The MIC values are
3
7
within a range of 0.312 µg/mL to 0.625 µg/mL. By comparing their MIC values with INH,
the compounds under study were good active against M. tuberculosis H Rv. Among the
3
7

ACCEPTED MANUSCRIPT
newly synthesized compounds, two compounds 10 and 14 exhibited excellent
antimycobacterial activity against M. tuberculosis H Rv at MIC 0.312 µg/mL. The obtained
3
7
results revealed that the nature of substituent’s and substitution pattern on the aryl ring may
have a considerable impact on the antimycobacterial activities of the target hydrazones. By
closer look into structure–activity relationship of the compounds, it is evident that
substitution in aromatic ring by any of the group like methyl, methoxy, fluoro and chloro
affects the activity profile. Compounds with aromatic ring having electron donating and
electron withdrawing groups are results in loss of activity with compare unsubstituent.
Compounds with aromatic ring having fluoro group are more active than the compounds with
chloro, methoxy and methyl groups. With respect to structure-MTB activity relationship, the
results demonstrated that the antimycobacterial activity was in the order, 4-fluoro > 3-
methoxy > 4-chloro >phenyl > 4-methyl as evident from the compounds 8 – 14.
Beside on our data it is clear that the antimycobacterial activities of the all compounds
are comparable to the standard drug isoniazid. Among the seven synthesized compounds two
compounds (11 and 14) were found to be most active compounds with high percentage
growth inhibition at 0.62 µg/mL was found. Compound with electron releasing meta
substituted phenyl group showed more activity, when compared to methoxy substituent at
para position. The mycobacterial activity profile of 8, 11, 12 and 14 compounds suggests that
each compound has a bactericidal effect as there has been no growth in treated control.
Antibacterial evaluation
The newly synthesized compounds were assayed for their antibacterial activity against
selected some Gram positive such as Staphylococcus Aureus and Bacillus Subtilis and Gram
negative bacteria Escheichia Coli, Enterobacter Aerogenes. The result of antibacterial
screening is shown in Table 4. In table 4 clearly shows that the compound 11 fluoro
substituent in the phenyl ring exhibited good activity against all the bacterial strains.

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Compounds 10 and 13 had an almost similar range of activity, they differ only by one or two
fold dilution, in spite of having a chloro substituent at different positions. The replacement of
the halogen substituent by a methoxy or methyl group in phenyl ring as in 9, 12 and 14
respectively, caused a reduction of activity. From the antibacterial screening, it can be
concluded that the compounds show better activity toward gram positive bacteria than gram
negative bacteria. Compound 11 shows superior activity against B. Subtilis.
Conclusion
In summary seven N′-(2r,4c-diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)pyrazine-2-
1
13
carbohydrazide (8-14) were synthesized in good yield and characterized by FT-IR , H, C
NMR spectral data and elemental analyses. Based on the observed chemical shifts and 2D
correlations the hydrazones are twin-chair conformation with equatorial orientations of the
aryl groups. All the newly synthesized compounds were screened for their anti-
mycobacterium tuberculosis activity. Among the synthesized hydrazones, 10 and 14 showed
excellent antituberculosis activity against M. tuberculosis H Rv at the concentration of 0.312
3
7
µg /mL. In antibacterial activity that the compound 11 exhibited good activity against all the
bacterial strain.
Acknowledgements
The authors are thankful to SIF, Indian Institute of Science, Bangalore and to SAIF IIT-
Madras for recording NMR spectra. We thank to Mr. Robert C. Goldman, Tuberculosis
Antimicrobial Acquisition and Coordinating Facility (TAACF), Southern Research institute,
USA.
Conflict of Interest
The authors have declared no conflict of interest.

ACCEPTED MANUSCRIPT
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[
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FIGURE CAPTIONS
Scheme 1. Schematic diagram showing the synthesis of title compounds 8-14.
Figure 1.The numbering of the carbons of the piperidine and cyclohexane ring of
compounds.
Figure 2. COSY and (dash line) ROESY correlations of compound 8.
Figure 3. Twin chair conformation of compound 8.
Figure 4. HMBC Correlation of compound 8.
Figure 5. The non-bond interaction between H-5e and Nitrogen in hydrazone unit.
Table 1. Correlations in the COSY and ROESY spectra of compound 8.
Table 2. Correlations in the HSQC and HMBC spectra of compound 8.
Table 3. In vitro antimycobacterial screening data of the compounds 8-14.
Table 4. In vitro antibacterial activity of the compounds 8-14.

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1
1
Table 1: Correlations in the H- H COSY and ROESY spectra of 8
Correlations in the ROESY
Correlations in the COSY
Protons
spectrum
spectrum
7.93 (H-6″′), 3.28 (H-5e)
-
1
1.09 (amide NH)
Nil
8
.72 (H-5″′)
.93 (H-6″′)
7.93 (H-6″′)
8.72 (H-5″′)
7
11.09 (amide NH)
2
.73 (H-7a), 2.93 (NH),
.28 (H-5e), 4.29 (H-4a)
.54 (H-1e), 2.73 (H-7a),
.93 (NH), 4.32 (H-2a)
7
.64 (o'-H)
7.41 (m-H, m'-H)
7.41(m-H m'-H)
3
2
7
.63 (o-H)
2
7
.41 (m-H, m'-H)
.28 (p-H, p'-H)
7.62 (o-H), 7.64 (o'-H)
7.41(m-H, m'-H)
7.63 (o-H), 7.64 (o'-H)
7.63 (o-H), 7.64 (o'-H)
7
2.54 (H-1e)
1.47, 4.31(H-2a)
4.32 (H-2a) 7.64 (o'-H), 7.63 (o-H)
2
.54 (H-1e), 2.93 (NH),
.63 (o-H)
.93 (NH), 3.28 (H-5e),
.64 (o'-H)
4
.32 (H-2a)
2.54 (H-1e), 2.93 (NH)
7
2
4
.29 (H-4a)
.28 (H-5e)
2.93 (NH), 3.28 (H-5e)
1.47, 4.29 (H-4a)
7
3
1.47, 4.29 (H-4a), 7.64 (o'-H)
1
.47 (H-6a, H-6e
H-8a)
2.73 (H-7a)
1.63 (H-8e), 7.64 (o'-H)
&
2
.73 (H-7a)
1.47 (H-6a, H-6e & H-8a)
.47 (H-6a, H-6e & H-8a),
1.26 (H-7e), 1.47, 7.63, 7.64
2.73 (H-7a)
1
1.26 (H-7e)
2.73 (H-7a)
1
.47 (H-6a, H-6e & H-8a),
2.54 (H-1e), 1.47 (H-6a, H-6e
1
.63 (H-8e)
2.73 (H-7a)
&
H-8a)
.28 (H-5e), 4.29 (H-4a),
.32 (H-2a), 7.63 (o-H), 7.64 (o'-H)
3
2
.93 (piperidine NH)
4.29 (H-4a), 4.32 (H-2a)
4

ACCEPTED MANUSCRIPT
Table 2: Correlations in the HSQC and HMBC spectra of 8
1
3
C Chemical shifts
δ, ppm)
Correlations in the
Correlations in the
(
HSQC spectrum
HMBC spectrum
170.7
162.5
150.0
121.8
-
-
11.09 (amide NH)
11.09, 9.65 (H-3′′′)
7.93 (H-6′′′)
-
8.72 (H-5′′′)
9.65 (H-3′′′)
142.8, 142.7
-
7.41(m-H, m'-H)
1
41.4
27.0
8.72 (H-5′′′)
7.63 (o-H), 7.64 (o'-H)
7.41 (m-H, m'-H)
7.28 (p-H, p'-H)
4.32 (H-2a)
7.93 (H-6′′′)
1
4.32 (H-2a), 4.29 (H-4a)
1
28.2, 128.0
26.8
-
1
-
6
4.4
2.2
5.9
1.0
8.2
6.9
0.8
7.63 (o-H)
6
4.29 (H-4a)
7.64 (o'-H)
4
4
2
2
2
2.54 (H-1e)
-
-
-
-
-
3.28 (H-5e)
1.63 (H-8e), 1.47 (H-8a)
1.47 (H-6a, H-6e)
2.73 (H-7a), 1.26 (H-7e)

ACCEPTED MANUSCRIPT
Table 3. In vitro antimycobacterial activity of the compounds 8-14
a
Comp.
R
MIC
µg/mL)
IC50 IC90
Growth
Inhibition (%)
62.66
log P
SI
(
8
9
H
0.625
0.29 0.32
0.57 0.64
0.57 0.64
3.58
4.50
4.77
3.68
3.41
4.77
3.41
-0.58
-2.38
0.93
0.91
0.91
0.64
062
0.91
0.93
-
p-CH
p-Cl
p-F
0.625
62.30
63.61
67.63
67.07
65.42
59.18
-
3
10
11
12
13
14
0.625
0.312
0.4
0.59
p-OCH
0.625
0.39 0.63
0.57 0.65
0.29 0.32
3
m-Cl
0.625
m-OCH
0.312
3
INH
RIF
-
-
0.125 - 0.0625
0.006 - 0.003
-
-
-
-
-
-

ACCEPTED MANUSCRIPT
Table 4. In vitro antibacterial activity of the compounds 8-14
Gram positive
S. Aureus B. Subtilis
AI ZI AI
8 0.27
Gram negative
E. Coli
E. Aerogenes
Compds. R
ZI
-
ZI
-
AI
-
ZI
-
AI
8
9
H
-
p-CH
8 0.25
-
-
-
-
9
0.29
3
1
1
1
1
1
0 p-Cl
1 p-F
18 0.56
20 0.63
12 0.38
15 0.47
10 0.31
32
20 0.67
25 0.83
14 0.47
17 0.57
7 0.23
30
12 0.43
15 0.54
10 0.36
8 0.29
18 0.64
22 0.79
11 0.39
13 0.46
2 p-OCH
3 m-Cl
3
4 m-OCH
-
-
8
0.29
3
Ciprofloxacin
28
28
Z.I.–Zone of Growth Inhibition (mm); A.I.–Activity Index (Inhibition Zone of Compound /
Inhibition Zone of the Standard Drug).

ACCEPTED MANUSCRIPT
N
N
O
N
NH
H
O
O
O
a
b
2
N
H
N
H
R
+
R
R
R
R
CH COONH
3
4
1-7
8-14
Comp. R
1
2
3
4
5
6
7
, 8
, 9
H
p-CH3
,10 p-Cl
, 11 p-F
, 12 p-OCH3
, 13 m-Cl
, 14 m-OCH3
Scheme 1. Reagents and conditions: (a) EtOH, warm, allow overnight; (b) PZH, 0.2 mL
AcOH, Methanol and Chloroform (1:1 v/v), reflux 2-3 h.

ACCEPTED MANUSCRIPT
N
5''
3''
4
''
2''
6''
O
1''
C
N
N
7
H
N
6
8
9
1
5
4
2
3
4'
2'
N
H
Figure 1. The numbering of the carbons of the piperidine and cyclohexane ring of
compounds 8-14

ACCEPTED MANUSCRIPT
H
H
N
H
O
N
C
N
H
N
9
He
H
5
1
Ha
Ha
He
Ha
2
H
8
4
6
H
He
He
3
H
N
H
He
7
H
Ha
Figure 2. COSY and (dash line) ROESY correlations of compound 8.

ACCEPTED MANUSCRIPT
H
H
N
N
H
O
C
N
H
N
N
He
N
O
H
C
N
Ha
N
H
Ha
Ha
Ha
H
He
He
H
He
H
N
N
H
H
Ha
H
8
CC
8
Figure 3. Twin chair conformation of compound 8.

ACCEPTED MANUSCRIPT
H
H
N
H
O
N
C
N
H
N
9
He
H
5
1
Ha
Ha
Ha
Ha
H
8
4
2
6
H
He
He
3
H
N
H
He
7
H
Ha
Figure 4. HMBC Correlation of compound 8.