A facile synthesis of α,α′-(EE)-bis(benzylidene)-cycloalkanones and their antitubercular evaluations

Article abstract of DOI:10.1016/j.ejmech.2008.09.026

An economical and facile synthesis of α,α′-(EE)-bis(benzylidene)-cycloalkanones was achieved by the reaction of cycloalkanones with different aromatic aldehydes using ethanolic KOH in good yields. Few of the selected compounds were reduced with NaBH₄ to the respective α,α′-(EE)-bis(benzylidene)-cycloalkanols. All these compounds and our earlier synthesized cyclohexyl phenyl methanols were evaluated for their antitubercular, antifungal and antibacterial activities. Several compounds displayed moderate antitubercular activity with MIC = 12.5-1.56 μg/mL. However, none of the compounds displayed any significant antifungal activity.

Full text of DOI:10.1016/j.ejmech.2008.09.026

European Journal of Medicinal Chemistry 44 (2009) 1705–1709
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A facile synthesis of a,a
0-(EE)-bis(benzylidene)-cycloalkanones and their
antitubercular evaluations
Nimisha Singh a, Jyoti Pandey a, Amit Yadav a, Vinita Chaturvedi b, Shalini Bhatnagar b,
,
Anil N. Gaikwad b, Sudhir Kumar Sinha b, Awaneet Kumar c, P.K. Shukla c, Rama P. Tripathi a
*
a Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow 226001, India
b Division of Drug Target Discovery and Development, Central Drug Research Institute, Lucknow 226001, India
c Fermentation Technology Division, Central Drug Research Institute, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 June 2008
Received in revised form 8 September 2008
Accepted 11 September 2008
Available online 30 September 2008
An economical and facile synthesis of
reaction of cycloalkanones with different aromatic aldehydes using ethanolic KOH in good yields. Few of
the selected compounds were reduced with NaBH4 to the respective
0-(EE)-bis(benzylidene)-cyclo-
alkanols. All these compounds and our earlier synthesized cyclohexyl phenyl methanols were evaluated
for their antitubercular, antifungal and antibacterial activities. Several compounds displayed moderate
a,a
0-(EE)-bis(benzylidene)-cycloalkanones was achieved by the
a,a
antitubercular activity with MIC ¼ 12.5–1.56
m
g/mL. However, none of the compounds displayed any
Keywords:
significant antifungal activity.
Mycobacterium tuberculosis
a,a
0-(EE)-Bis(benzylidene)-cycloalkanones
Ó 2008 Elsevier Masson SAS. All rights reserved.
Antifungal agents
Antitubercular agents
1. Introduction
antitubercular activity both in vitro and in vivo. Moreover, several
acetophenones [12], chalcones [13] and Mannich bases of benzy-
lidene cycloalkanones [14] have also been disclosed to possess
moderate antitubercular activity and their mode of action is also
postulated to be the inhibition of initial steps in fatty acid biosyn-
thesis. Bis-benzylidene cycloalkanones have been reported to
possess drug resistance reversal [15], cytotoxicity [16] and histone
acetyl transferase (HAT) [17] inhibitory activities. N-Acetyl trans-
ferase (NAT), a subtype of HAT plays a very important role in the
initial stages of mycolic acid biosynthesis in mycobacterium and it
is known that human NAT inactivates [18] the antitubercular drug
INH (isoniazid) in humans. The above facts prompted us to
synthesize benzylidene cycloalkanone analogs in an efficient and
cost effective manner, and evaluate them for their antitubercular
and antifungal activities.
Tuberculosis has re-emerged as a growing public health threat,
killing young and middle-aged people faster than any other
diseases. Infection of Mycobacterium tuberculosis remains a leading
cause of death [1–3]. It is estimated that nearly 9 ꢀ 106 new cases of
active TB disease occur every year [4]. A vast majority of the world’s
burden of tuberculosis (TB) is in developing countries, and it is
estimated that only 23% of the prevalent active cases receive an
appropriate antitubercular treatment [5]. An appropriate treatment
of tuberculosis has led to the development of several bottlenecks in
chemotherapy of tuberculosis. Emergence of MDR (multi-drug
resistance) and XDR (extremely drug resistance) tuberculosis and
its synergy with HIV have further aggravated the problem of
chemotherapy in tuberculosis. Although, a number of new chemical
entities have been discovered recently as potent antituberculars,
yet no new drug has entered clinics since 1965 [6–9]. Therefore,
there is an urgent need to develop new drugs that act through
a novel mode of action for the chemotherapy of TB. Recently, we
have designed and developed novel aryloxy cyclopropyl phenyl
methanones [10] as possible inhibitors of FAS-II enzyme which is
required in chain elongation of mycolic acids keeping in mind the
structure of triclosan [11] and the compounds displayed potent
2. Chemistry
a,a
0-(EE)-Bis(benzylidene)-cycloalkanones (1–15) were prepared
by reacting 2 equivalents of aromatic aldehydes with 1 equivalent of
cycloalkanones in the presence of solid KOH (5 mol%) in ethanol
(Scheme 1). The selected aldehydes comprise furfuraldehyde, benz-
aldehyde, 4-bromobenzaldehyde, 4-chlorobenzaldehyde, 4-fluo-
robenzaldehyde, 4-nitrobenzaldehyde, 4-methoxybenzaldehyde,
3,4-dimethoxybenzaldehyde and 3,4,5-trimethoxybenzaldehyde.
* Corresponding author. Tel.: þ91 522 2612412; fax: þ91 522 2623405.
E-mail address: rpt.cdri@gmail.com (R.P. Tripathi).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.026

1706
N. Singh et al. / European Journal of Medicinal Chemistry 44 (2009) 1705–1709
O
O
O
Ethanol, Solid KOH
0-30°C, 6-12 h
H
+
Ar
Ar
n
n
R
1-15
OH
NaBH4/EtOH/0-30°C
2 and 5
R
R
16, R=Br; 17, R=NO2
Scheme 1. Synthesis of bis-benzylidene cycloalkanones and cycloalkanols.
The selected cycloalkanones were cyclopentanone, cyclohexanone
and cycloheptanone.
(trichloro-1,3,5-triazine). We have used aqueous KOH/NaOH, TCT
and many other catalysts for the condensation of cycloalkanones
Two of the most active compounds 2 and 5 were reduced with
and aldehydes to get the desired a,a
0-(EE)-bis(benzylidene)-
NaBH4 in ethanol to give the respective
a
,
a
0-bis-(benzylidene)-
cycloalkanones. In our work, most of the catalysts resulted in poor
yields of the products as several byproducts were formed and it
was difficult to isolate the desired compounds in pure form from
the reaction mixture. Application of solid KOH (5 mol%) as a cata-
lyst for the condensation of different aldehydes with cyclo-
alkanones in minimum amount of ethanol resulted in the desired
cycloalkanols 16 and 17 in good yields.
Cyclohexylphenyl methanols 18, 19, 20 and 21 were prepared by
D-glucosamine catalyzed aldol reaction of cyclohexanone with 4-
nitro-, 3-nitro-, 2-nitro- and 4-bromobenzaldehydes as reported by
us [19] (Scheme 2).
a,a
0-(EE)-bis(benzylidene)-cycloalkanones (1–15) in good yields
(Scheme 1 and Table 1) and proved to be the most convenient
method. This method is economical and eco-friendly as neither
any byproduct was formed nor any toxic material was used during
the synthesis and the reactions were carried out at ambient
temperature. Work up of the reaction mixture was very simple
involving just filtration of the product and washing with cold
water followed by drying and crystallization. The structures of bis-
benzylidene cycloalkanones (1–15) were in accordance with their
spectroscopic data and microanalyses. The IR spectrum of the
compounds in general exhibited the absorption band at around
1731–1593 cmꢁ1 indicating that the carbonyl group and olefinic
3. Biology
All of the synthesized compounds were evaluated for their
antitubercular activity against M. tuberculosis H37Ra by MABA
(Microplate Alamar Blue Assay) method [21], while agar micro-
dilution method [22] was used against M. tuberculosis H37Rv. INH
and ethambutol were used as standard drugs. All of the above
compounds were also screened against different strains of bacteria
and fungi viz. Escherichia coli (ATCC 9637), Pseudomonasaeruginosa
(ACTT BAA-427), Staphylococcus aureus (ACTT-25923), Klebsiella
pneumoniae (ACTT-27736), Candida albicans (ATCC-1405B),
Cryptococcus neoformans, Sporothrix schenckii, Trichophyton men-
tagrophytes, Aspergillus fumigatus and Candida parapsilosis (ATCC-
22019). Fungi were tested by NCCLS (National Committee for
Clinical Laboratory Standards) method in RPMI 1640 medium and
bacteria in Mueller Hinton Broth [34–37].
bonds of a,a
0-(EE)-bis(benzylidene)-cycloalkanones are in conju-
gation. The ESMS (mass spectra) of the compounds showed their
respective [M þ H]þ peaks. In the 1H NMR spectrum of the
compounds the vinylic protons either occur as singlet at around
d
7.50–7.30 ppm or occur merged with the multiplet of aromatic
4. Results and discussion
Table 1
Synthesis of bis-benzylidene cycloalkanones and cycloalkanols (1–17).
4.1. Chemistry
Compound
no.
n
Ar/R
m.p. (ꢂC) Known m.p
(ꢂC) [Ref]
% Yield
69
Several reports exist for the synthesis [38–44] of benzylidene
cycloalkanones involving the use of organic and inorganic bases,
metal catalyst, different types of Friedel Craft catalysts and TCT
1
2
3
4
5
6
7
8
2
2
2
2
2
3
2
2
2
2
3
3
3
1
2
2
2
Furyl
131–133 140–142 [20]
163–164 165–168 [26,27] 86
141–143 138–140 [23]
154–156 156–158 [28]
154–157 161–162 [43]
129–131 137 [24]
4-Bromophenyl
3,4-Dimethoxyphenyl
4-Fluorophenyl
4-Nitrophenyl
4-Bromophenyl
4-Benzyloxyphenyl
92
85
87
83
86
88
91
85
85
87
90
84
89
75
65
O
OH
CHO
O
199–201 190–191 [25]
3,4,5-Trimethoxyphenyl 218–219 208 [23]
Phenyl
9
117–119
117–118 [43]
D-glucosamine(10mol%)
H2O, 30°C
10
11
12
13
14
15
16
17
4-Chlorophenyl
4-Chlorophenyl
4-Methoxyphenyl
3,4-Dimethoxyphenyl
3,4-Dimethoxyphenyl
2-Naphthyl
142–145 147–148 [43]
118–120 Not reported
+
95–97
126–127 [39]
R
R
155–157 164–165 [43]
190–192 186–188 [23]
199–202 212–213 [29]
131–132 Not reported
143–145 Not reported
18,R = 4-nitro; 19, R = 3-nitro
20,R = 2-nitro; 21, R = 4-bromo
4-Bromophenyl
4-Nitrophenyl
Scheme 2. Synthesis of hydroxy-(phenyl)-methyl cycloalkanones.

N. Singh et al. / European Journal of Medicinal Chemistry 44 (2009) 1705–1709
1707
Table 2
protons ranging from
protons of C-3 appeared as multiplet at around
and the methylene protons of C-4 was observed as multiplet at
around 1.97–1.67 ppm.
d
8.25 to 6.65 ppm, while the methylene
In vitro antitubercular activity of bis-benzylidene cyclohexanones (1–15), bis-ben-
zylidene cyclohexanols (16 and 17) and cyclohexyl phenyl methanols (18–21).
d
2.96–2.36 ppm
Compound no.
n
c Log Pa
MIC (H37Ra)
MIC (H37Rv)
d
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
2
2
2
2
2
3
2
2
2
2
3
3
3
1
2
2
2
2
2
2
2
–
–
3.68
6.46
4.64
5.18
7.29
6.78
7.59
4.43
4.64
5.76
6.17
>12.5
1.56
>12.5
12.5
>12.5
12.5
>12.5
>12.5
12.5
>12.5
>12.5
>12.5
6.25
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
12.5
The EE geometry of the double bonds in the above compounds
(1–15) was based on earlier literature reports [30–33]. It is reported
that the vinylic protons are in close proximity to the carbonyl group
which exerted an anisotropic effect, resulting in the downfield
shifting and overlapping of the vinylic protons with the aromatic
3.12
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
6.25
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
–
protons, and appearance of vinylic protons in the region of
7.95 ppm is an indication of such compounds with E configuration
and in the region of 6.8 ppm indicates Z configuration [30,31], as
d 7.15–
d
for example, the olefinic protons of Z-2-phenyl methyl-
enecyclohexanones and Z-2-phenyl methylene-6,6-diphenyl
4.8
4.55
3.72
7.67
6.13
3.32
3.43
3.53
3.55
3.07
ꢁ0.668
0.1188
cyclohexanones are generally observed at
[32,33]. In the 1H NMR spectra of compounds 1–15 the vinylic
protons appear either as singlet at around 7.50–7.30 ppm or
observed along with the multiplets of aromatic protons ranging
from 8.25 to 6.65 ppm. The EE geometry of the two double bonds
was further substantiated by NOE experiment on a prototype
compound (10). The vinylic proton at 7.7 ppm showed NOE with
the aromatic ortho protons and at the same time it did not show any
NOE with the methylene protons at C-3 ( 2.8 ppm) (Fig. 1). The
d 6.27 and 6.22 ppm
d
17
18
19
20
>12.5
12.5
12.5
>12.5
12.5
0.75
d
d
21
Isoniazid
Ethambutol
d
–
3.25
a
latter clearly shows the EE geometry of the olefinic bond.
c Log P was determined by OSIRIS Property Explorer Programme which is
The IR spectrum of the bis-benzylidene cycloalkanols (16 and
17) exhibited the absorption band at around 3300 and 1480 cmꢁ1
indicating the presence of hydroxyl group and olefinic bonds. In the
1H NMR spectrum of the compound the vinylic protons are
observed to be merged with aromatic protons and appeared in the
available at http://www.organic-chemistry.org/prog/peo/.
cycloalkanones, compounds 1, 3, 6, 7, 8, 10, 11, 13, 14, 15, 17 and 20
do not exhibit antitubercular activity against either of the strains M.
tuberculosis H37Ra or M. tuberculosis H37Rv as they have
range of
d
8.21–6.54 ppm and hydroxyl proton observed around
2.79 ppm and the
MIC > 12.5 mg/mL. Further, it is also evident that the reduction of
d
4.50 ppm while the proton of C-1 occurred at
d
carbonyl group in the most potent compounds (2 and 5) of the
series to their respective bis-benzylidene cycloalkanols (16 and 17),
results in the loss of antitubercular activity indicating that the
carbonyl group is essential for their antitubercular activity. It is also
interesting to note that among the bis-benzylidene cycloalkanones,
only bis-benzylidene cyclohexanones display antitubercular
activity while their counter parts with cyclopentanone and cyclo-
methylene protons of C-3 and C-4 appeared as such.
4.2. Biology
Out of all the screened compounds 2, 4, 5, 9, 12, 16, 18, 19 and 21
displayed antitubercular activity with MIC ranging from 12.5 to
1.56 mg/mL against either the avirulent strain M. tuberculosis
heptanone moieties are inactive as their MIC values were >12.5 mg/
H37Ra or the virulent strain M. tuberculosis H37Rv. As evident
from Table 2, compounds 2, 4, 5 and 12 display antitubercular
activity against the avirulent strain M. tuberculosis H37Ra,
mL. It is also clear that the substitution on aromatic ring at the 4th
position generally leads to compounds having better activity in
comparison to other compounds either with unsubstituted
aromatic ring or having substitution on other aromatic positions.
Compounds 16, 18, 19 and 21 are moderate antitubercular agents as
a
surrogate of M. tuberculosis H37Rv. On the other hand
compounds 2, 5, 9, 16, 18, 19 and 21 show activity against M.
tuberculosis H37Rv. Therefore, two of the above compounds 2 and
5 are active against both the avirulent and virulent strains of M.
tuberculosis.
they have MICs values < 12.5
and 12 are good antituberculars as they have MICs in the range of
1.56–6.25 g/mL. Among the hydroxy-(phenyl)-methyl cyclo-
mg/mL. However, compounds 2, 5, 9
m
Among all the compounds screened against different strains of
bacteria and fungi only one compound (compound 2) showed
activity against the two fungi, S. schenckii and T. mentagrophytes
alkanones 18–21, except compound 20 with nitro substituent at 2-
position of the aromatic ring, other compounds of the series 18, 19
and 21 with substituents (–NO2 or –Br) either at 3- or 4-positions
on the aromatic rings display moderate antitubercular activity
with MIC ¼ 25 and 12.5
displayed MIC ꢃ 50 g/mL against all the fungi which were tested
for these compounds.
mg/mL, respectively. Other compounds
m
against M. tuberculosis H37Rv with MIC ¼ 12.5
m
g/mL while they
g/mL).
were inactive against the avirulent strains (MIC > 12.5
m
A closer look into the SAR (structure activity relationship) of
these compounds reveals that among all the benzylidene
Further as the compounds did not display any significant activity
against fungi or other bacteria, they may be specific to
mycobacterium.
NOE
NOE
O
H
H
H
H
5. Conclusion
1
4
No
NOE
No
2
3
NOE
In conclusion, we have developed a simple, economical and
efficient method for the synthesis of bis-benzylidene cyclo-
alkanones and evaluated them for their antitubercular activity.
Three of the compounds displayed MIC in the range of
H
H
H
H
Cl
Cl
NOE
EE geometry
Fig. 1. NOE in EE geometry.
NOE
6.25–1.56
mg/mL, a criterion for further optimization of the series
for new antitubercular agents.

1708
N. Singh et al. / European Journal of Medicinal Chemistry 44 (2009) 1705–1709
6. Experimental
(m, 6H, ArH), 3.89 (s, 12H, OCH3), 2.71 (m, 4H, CH2), 2.00 (m, 4H,
CH2). 13C NMR (50 MHz, CDCl3):
198.1, 150.6, 149.2, 140.0, 135.9,
d
6.1. Chemistry
129.4, 122.9, 113.4, 111.4, 56.0, 29.1 and 28.6. Anal. Calcd for
C25H28O5: C, 73.51; H, 6.91%. Found: C, 73.49; H, 6.93%.
Commercially available reagent grade chemicals were used as
received. All reactions were followed by TLC on E. Merck Kieselgel
60 F254, with detection by UV light and/or by spraying a 20% KMnO4
aq. solution. Column chromatography was performed on silica gel
(60–120 mesh, E. Merck). IR spectra were recorded as thin films or
in chloroform solution with a Perkin–Elmer Spectrum RX-1 (4000–
450 cmꢁ1) spectrophotometer. 1H and 13C NMR spectra were
recorded on a Brucker DRX-300 in CDCl3. Chemical shift values are
reported in ppm relative to SiMe4 as internal reference, unless
otherwise stated; s (singlet), d (doublet), t (triplet), m (multiplet); J
in hertz. FAB mass spectra were performed using a mass spec-
trometer Jeol SX-102 and ESI mass spectra with Quattro II (Micro-
mass). Elemental analyses were performed on a Perkin–Elmer 2400
II elemental analyzer.
6.1.1.6. 2,5-(EE)-Bis-(3,4-dimethoxy-benzylidene)-cyclopentanone
(14). IR (KBr): nmax cmꢁ1 3020, 1730, 1595; MS (FAB): 367 [M þ H]þ.
1H NMR (200 MHz, CDCl3):
d 7.50 (s, 2H, olefinic protons), 7.18–6.89
(m, 6H, ArH), 3.92 (s, 12H, OCH3), 3.09 (m, 4H, CH2). 13C NMR
(50 MHz, CDCl3): 196.0, 150.6, 149.3, 135.6, 134.0, 129.4, 124.9,
d
113.9, 111.5, 56.2, and 26.8. Anal. Calcd for C25H28O5: C, 76.61; H,
6.36%. Found: C, 76.61; H, 6.39%.
6.1.2. General experimental procedure for the preparation of
bis(benzylidene)-cycloalkanols
a, -
a0
A
solution of a,a
0-(EE)-bis(substituted-benzylidene)-cyclo-
alkanones (1 mmol) and ethanol (5 mL) was stirred at 0 ꢂC for
10 min. NaBH4 (1 equivalent) was slowly added and stirred at
30 ꢂC till the disappearance of starting material (TLC). The reaction
mixture was brought to 0 ꢂC and excess of NaBH4 was quenched by
saturated aq. solution of NH4Cl and solid so-obtained was filtered.
The solid cake was washed with ethanol and the combined filtrate
was evaporated under reduced pressure to give a crude mass. The
latter was dissolved in ethylacetate, organic layer was washed
with water, dried (anhy. Na2SO4) and concentrated in vacuum to
give a gummy mass. The latter was chromatographed over SiO2
using a gradient of hexane:EtOAc as eluent to afford the pure
products.
6.1.1. General experimental procedure for the preparation of a, -
a0
(EE)-bis(substituted-benzylidene)-cycloalkanones
To a stirring solution of the cycloalkanone (1 mmol) and
aromatic aldehyde (2 mmol) in minimum amount of ethanol, solid
KOH (5 mol%) was added. The reaction mixture was stirred at
ambient temperature till the disappearance of the starting mate-
rials (TLC). The solid separated was filtered and washed with water
and dried. The product, so-obtained, was crystallized with ethanol
to give the desired compounds in good yields.
6.1.1.1. 2,6-(EE)-Bis-furan-2-yl-methylene-cyclohexanone
(1). IR
6.1.2.1. 2,6-Bis-(4-bromobenzylidene)-cyclohexanol (16). IR (KBr):
nmax cmꢁ1 3021, 1724, 1596; MS (FAB): 417 [M ꢁ OH]þ. 1H NMR
(KBr): nmax cmꢁ1 3452, 1777, 1593; MS (FAB): 255 [M þ H]þ. 1H NMR
(300 MHz, CDCl3):
d
7.56–7.53 (d, J ¼ 7.1 Hz, 4H, olefinic protons and
(200 MHz, CDCl3): d 7.74–6.54 (m, 10H, olefinic protons and ArH),
ArH), 6.66 (d, J ¼ 3.18 Hz, 2H, ArH), 6.51 (dd, J ¼ 1.68 and 1.56 Hz,
4.64 (s, 1H, CHOH), 2.79–2.73 (m, 1H, CH), 2.40–2.33 (m, 4H, CH2),
1.61 (m, 2H, CH2). Anal. Calcd for C20H18O1Br2: C, 55.33; H, 4.18%.
Found: C, 55.30; H, 4.20%.
2H, ArH), 3.05–2.93 (m, 4H, CH2), 1.93–1.88 (m, 2H, CH2). 13C NMR
(50 MHz, CDCl3): d 188.4, 153.2, 144.5, 133.2, 123.6, 116.1, 112.5, 31.0,
30.1, 28.4 and 22.1. Anal. Calcd for C16H14O3: C, 75.57; H, 5.55%.
Found: C, 75.54; H, 5.58%.
6.1.2.2. 2,6-Bis-(4-nitro-benzylidene)-cyclohexanol (17). IR (KBr):
nmax cmꢁ1 3213, 1656 and 1513; MS (FAB): 366.9 [M þ H]þ. 1H NMR
6.1.1.2. 2,7-(EE)-Bis-(4-bromobenzylidene)-cycloheptanone (6). IR
(KBr): nmax cmꢁ1 3449,1673,1606; MS (FAB): 447 [M þ H]þ. 1H NMR
(200 MHz, CDCl3): d 8.21–8.16 (m, 4H, ArH), 7.37–7.33 (m, 6H,
olefinic protons and ArH), 4.25 (s, 1H, CHOH), 2.79–2.72 (m, 1H,
CH), 2.07–1.99 (m, 4H, CH2), 1.67–1.58 (m, 2H, CH2). Anal. Calcd for
C20H18N2O: C, 65.57; H, 4.95%. Found: C, 65.53; H, 4.99%.
(300 MHz, CDCl3):
d 7.61–7.11 (m, 10H, olefinic protons and ArH),
2.73–2.67 (m, 4H, CH2), 1.98–1.81 (m, 4H, CH2). 13C NMR (50 MHz,
CDCl3): 204.0, 142.0, 134.6, 134.3, 131.7, 131.6, 130.9, 43.3, 31.3,
d
29.9, 28.7, 28.0, 27.7 and 25.4. Anal. Calcd for C21H18O1Br2: C, 56.53;
H, 4.07%. Found: C, 56.54; H, 4.09%.
7. Biological activity
6.1.1.3. 2,7-(EE)-Bis-(4-chlorobenzylidene)-cycloheptanone (11). IR
(KBr): nmax cmꢁ1 3021, 1731, 1603; MS (FAB): 343 [M þ H]þ. 1H NMR
7.1. Activity against M. tuberculosis H37Ra strain
(200 MHz, CDCl3):
d 7.40–7.19 (m, 10H, olefinic protons and ArH),
2.65 (m, 4H, CH2),1.97–1.95 (m, 4H, CH2). 13C NMR (50 MHz, CDCl3):
198.1, 142.1, 134.9, 134.6, 131.0, 129.1, 129.0, 43.6, 31.7, 29.1, 28.8
All of the synthesized compounds were evaluated for their
efficacy against M. tuberculosis H37Ra at active concentration
d
and 25.7. Anal. Calcd for C21H18O1Cl2: C, 70.60; H, 5.08%. Found: C,
70.59; H, 5.10%.
ranging from 50 mg/mL to MIC using two-fold dilutions in the initial
screen. Log phase culture of M. tuberculosis H37Ra is diluted so as to
give final OD550 nm of 0.05 in Sauton’s medium. In 96-well white
6.1.1.4. 2,7-(EE)-Bis-(4-methoxybenzylidene)-cycloheptanone
plates 190
sulfoxide (DMSO) solution of test compounds is dispensed into 96-
well plates so as to make final test concentration of 25 g/mL (5
test compound is dispensed into 10 L of DMSO). Then the plate is
incubated at 37 ꢂC/5% CO2 for 5 days. On 5th day 15
L Alamar Blue
mL of culture is dispensed in each well. A dimethyl
(12). IR (KBr): nmax cmꢁ1 3018, 1731, 1603; MS (FAB): 335
[M þ H]þ. 1H NMR (200 MHz, CDCl3):
d
7.45–7.26 (m, 6H, olefinic
m
mg
protons and ArH), 6.93–6.87 (m, 4H, ArH), 3.81 (s, 6H, OCH3), 2.70
m
(m, 4H, CH2), 1.97 (m, 4H, CH2). 13C NMR (50 MHz, CDCl3):
d
199.7,
m
160.0, 140.0, 135.8, 131.6, 129.0, 114.3, 55.5, 30.2, 28.8, 28.4 and
25.7. Anal. Calcd for C23H24O3: C, 82.63; H, 7.18%. Found: C, 82.68;
H, 7.09%.
solution is added to each well of the plate. The plate is again
incubated overnight at 37 ꢂC/5% CO2 incubator. The fluorescence is
read on BMG polar star with excitation frequency at 544 nm and
emission frequency at 590 nm. The compounds, which were found
to be active (>90% inhibition as compared with control) at this
concentration are then tested at 6 serial dilutions starting from 50
6.1.1.5. 2,7-(EE)-Bis-(3,4-dimethoxybenzylidene)-cycloheptanone
(13). IR (KBr): nmax cmꢁ1 3010, 1695, 1596; MS (FAB): 395 [M þ H]þ.
1H NMR (200 MHz, CDCl3):
d
7.29 (s, 2H, olefinic protons), 7.05–6.83
to 1.56 mg/mL.

N. Singh et al. / European Journal of Medicinal Chemistry 44 (2009) 1705–1709
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Acknowledgements
This paper bears CDRI communication no. 7369. Authors thank
SAIF Division for spectroscopic data. Financial assistance from
DRDO, New Delhi and DBT, New Delhi in the form of sponsored
projects is gratefully acknowledged.
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