Mycobacterium tuberculosis lysine-?-aminotransferase a potential target in dormancy: Benzothiazole based inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.03.053

MTB lysine-?-aminotransferase (LAT) was found to play a crucial role in persistence and antibiotic tolerance. LAT serves as a potential target in the management of latent tuberculosis. In present work we attempted to derivatize the benzothiazole lead iden

Full text of DOI:10.1016/j.bmc.2017.03.053

Accepted Manuscript
Mycobacterium tuberculosis lysine-ɛ-aminotransferase a potential target in dor-
mancy: benzthiazole based inhibitors
Rudraraju Srilakshmi Reshma, Variam Ullas Jeankumar, Nidhi Kapoor, Shalini
Saxena, Karyakulam Andrews Bobesh, Astakala Rishi Vachaspathy, Pappachan
E Kolattukudy, Dharmarajan Sriram
PII:
S0968-0896(17)30057-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.053
BMC 13651
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
12 January 2017
24 March 2017
Please cite this article as: Reshma, R.S., Jeankumar, V.U., Kapoor, N., Saxena, S., Bobesh, K.A., Vachaspathy,
A.R., Kolattukudy, P.E., Sriram, D., Mycobacterium tuberculosis lysine-ɛ-aminotransferase a potential target in
dormancy: benzthiazole based inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/
j.bmc.2017.03.053
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Mycobacterium tuberculosis lysine-ɛ-aminotransferase a potential target in dormancy:
benzthiazole based inhibitors
Rudraraju Srilakshmi Reshma a, Variam Ullas Jeankumar a, Nidhi Kapoor b, Shalini Saxena a,
Karyakulam Andrews Bobesh a, Astakala Rishi Vachaspathy a, Pappachan E Kolattukudy b,
Dharmarajan Sriram a, *
a
b
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Hyderabad-500078, India.
Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida,
Orlando, Florida, United States of America.
Corresponding Author*

D. Sriram, Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Jawaharnagar, Hyderabad - 500078. INDIA. Telephone: +91-40663030506; Fax: +91-4066303998;
Email: dsriram@hyderabad.bits-pilani.ac.in
ABSTRACT:
MTB lysine-ɛ-aminotransferase (LAT) was found to play a crucial role in persistence and
antibiotic tolerance. LAT serves as a potential target in the management of latent tuberculosis. In
present work we attempted to derivatize the benzthiazole lead identified through high throughput
virtual screening of Birla Institute of Technology and Science in house database. For Structure
activity relationship purpose 22 derivatives were synthesized and characterized. Among
synthesized compounds, eight compounds were found to be more efficacious in terms of LAT
inhibition when compared to lead compound (IC50 10.38 1.21 µM). Compound 22 exhibits
bactericidal action against nutrient starved Mycobacterium tuberculosis (MTB). It also exhibits
significant activity in nutrient starvation model (2.9 log folds) and biofilm model (2.3 log folds).
Key words: Dormancy, Mycobacterium tuberculosis, Lysine-ɛ amino transferase, 3D granuloma
model, Benzthiazole derivatives.

1. Introduction
Latent tuberculosis (TB) can be defined as a torpid noncontagious form of mycobacterium
residing in host macrophages without symptoms of disease. One third of the global population
1
has latent TB and chances upon reactivation to active TB are 5-10% . The currently available
option of treatment of latent TB is based on the monotherapy with Isoniazid (INH), for 6 to 9
months or 3- or 4-month INH + Rifampicin (Rif) combination therapy. Current treatment
regimens are lengthy due to resistance, persistence of TB and also associated side effects in
therapy is a major drawback.2 Genetic expression profiling helped in identifying new targets
associated with latency one such target is lysine-ɛ amino transferase (LAT) 3. It has a key role in
4
antibacterial resistance and persistence . LAT is a PLP dependent type II aminotransferase
enzyme which catalyzes reversible transamination from lysine to α-keto glutaric acid resulting in
piperidine-6-carboxylic acid and glutamate.4,24 In present work we focused our attention in
expanding new class of benzthiazoles identified through virtual screening from our laboratory.5
A set of twenty-two compounds were synthesized and evaluated for their potency against both
replicative and non-replicating stages of bacteria. By molecular derivatization approach lead
molecule (LAT IC50 10.38 µM) was modified to develop new hit compound 21 (LAT IC50 1.15
µM) with ten folds increases in inhibitory potency.
2. Results and discussion
2.1. Design and Synthesis
Recently we identified nine novel MTB LAT lead compounds by employing e-pharmacophore
approach for crystal structure of LAT from MTB in internal aldimine form with a bound
substrate of 〈-ketoglutarate (PDB code: 2CJH) with 2.00 Å resolution.5 Among the nine initial
leads compound 1 (2-(benzo[d]thiazol-2-yl)-3-(4-hydroxyphenyl)acrylonitrile) was found to

have an IC50 of 10.38 µM. Modelling studies revealed that Compound 1 exhibited good binding
affinity to active site of LAT enzyme. As the findings of the study are fascinating we attempted
some modifications in compound 1 by introducing various changes in aromatic handle and the
benzthiazole scaffold is unaltered. For structure activity relationship purpose various phenyl and
heterocycles were introduced at R position (Fig. 1) and a library of twenty-two compounds were
synthesized by two steps synthetic protocol (Scheme 1).
Figure 1: Design strategy employed for lead derivatization.
Synthesis started with the construction of benzothiazole ring; which was achieved by
condensation of 2 aminothiophenol with malononitrile using ethanol as solvent in presence of
catalytic amount of acetic acid. The second step involves Knoevenagel condensation of
synthesized 2-(benzo[d]thiazol-2-yl) acetonitrile with substituted aryl/heteroaryl aldehydes. In
this step nucleophilic addition of active hydrogen from acetonitrile part with carbonyl group of

substituted aldehydes followed by water elimination to form α-β unsaturated aldehydes
(conjugated enone) as final compounds. In this step weak base piperidine used in catalytic
amount. For SAR purpose Benzoxazole derivatives are prepared using similar protocol
mentioned above. The compounds were characterized by mass, NMR spectroscopic methods and
also by elemental analysis. NMR spectra and mass data were found to be unison with theoretical
values confirming structure of final compounds. The elemental analysis results were within
0.4% of the theoretical values.
Scheme 1: Schematic representation of protocol employed for synthesis of benzthiazole derivatives.
2.2. Biological Evaluation
LAT catalyses reversible transamination from lysine to α-keto glutaric acid resulting in
piperidine-6-carboxylic acid and glutamate. As the end products have absorbance maxima at 465
nm and 280 nm all compounds were evaluated for their MTB LAT inhibitory potency by
spectroscopic method and the results are tabulated in Tab 1. Five compounds exhibited IC50 less
than 5 µM and compound 19 was found to be most potent among all compounds with an IC50
value of 1.15 0.27 µM. Compound 19 showed 10-fold reduction in IC50 when compared to lead
compound 1 (10.38 1.21 µM); eight compounds have more efficacy in terms of LAT inhibitory
potency than lead compound 1. Compounds 9, 17 and 22 also exhibited good inhibitory potency
of 3.08 0.37, 3.74 0.27, 2.62 0.37 µM respectively.

Table 1: Structure and biological results of studied compounds
R
LAT (IC50) µM
MIC
µM
MTT RAW cell
lines 50 µg/ml
CODE
1
10.38 1.21
4.11 0.78
7.83 0.31
>89.93
89.29
20.29
2.26 0.11
10.56 0.63
34.78 0.82
2
3
23.19 0.89
61.41 1.56
10.61
45.72 0.21
32.45 0.92
4
5
>77.64
64.89 2.31
47.93 1.82
>71.02
67.95
12.59 0.38
22.82 0.21
6
7
65.98 0.63
3.08 0.37
81.69
4.64
45.67 0.91
12.67 0.35
8
9

53.78 0.96
2.32
52.31 1.92
10
16.23 0.26
92.57 1.94
17.05 1.21
49.60
>93.28
>99.60
47.31 1.81
17.34 0.92
56.42 0.97
11
12
13
19.59 0.32
54.76 0.21
37.91 0.48
>84.18
>79.87
>83.06
19.64 0.62
60.31 1.23
45.69 0.92
14
15
16
3.74 0.27
>67.20
3.12 0.67
17
14.06 0.16
1.15 0.27
>67.20
60.09
15.24 0.51
14.38 0.82
18
19
5.73 0.79
64.43
18.94 0.92
20

6.72 0.27
2.62 0.37
2.01
5.64 0.76
21
22
>57.60
32.17 1.56
--
--
0.4
0.5
93.21 1.25
23.48 0.31
INH
RIF
Nutrient starvation model was found to mimic microenvironment of granuloma as MTB exhibits
significantly reduced intracellular ATP levels, a very low but continuous respiration and Loss of
Ziehl-Neelsen staining characteristic 6,7. This model is also characterized by resistance to
8
commonly used antibiotics (INH, Rif, metronidazole, some fluoroquinolines) . After 96hr of
starvation 323 genes involved in amino acid biosynthesis, biosynthesis of cofactors, prosthetic
groups and carriers, DNA replication, repair, recombination and restriction/modification, energy
metabolism, lipid biosynthesis, translation and post-translational modification and virulence
were down regulated. Genes which are found to be upregulated are involved in antibiotic
production and resistance, insertion sequence elements, repeated sequences and phage,
3
nucleotide biosynthesis and metabolism, putative enzymes and regulatory function . LAT
expressed by Rv3290c gene was found to be upregulated 41.86 folds as it involved in antibiotic
resistance.3 All the synthesized compounds were evaluated for their activities against dormant
phase of mycobacterium at 10 µg/ml. Most of the compounds have well correlation between
enzyme inhibitory concentration values and log reduction in this model. Compound 20 and 22
showed a bacterial log reduction of 2.9 fold and were more potent than first line TB drugs INH

(1.2 log fold) and Rif (1.3 log fold) and also Moxi (2.2 log fold). Compounds 9, 14, 19 also
showed 2.8-log fold reduction and were more potent when compared to standard drugs (Fig 2).
Figure 2: Biological activities of the synthesised compounds against M. tuberculosis in the nutrient
starvation model. Bacterial count estimation (Mean
S.D., n = 3) for control and treated groups
conducted by using the MPN (most probable number) assay. most of the compounds gave significant
inhibition of growth of M. tuberculosis in this model as compared to the control (p < 0.0001, two way
ANOVA usingGraphPad Prism Software).
Biofilm formation explains the reasons for the long term persistence of MTB in human host and
longer duration of treatment to completely recover from TB. Biofilm is a structured community
of persistent cells (low nutrients and oxygen) enclosed in a self-produced polymeric matrix and
adherent to an inert or living surface 9,10. In case of MTB bacilli are embedded in a lipid-rich
extracellular matrix containing free methoxy mycolic acids resulting in drug tolerance (50 times
higher than MIC) and persistence 11. Mycobacterial biofilms also called as pellicle or cords

forms a barrier for antibiotic penetration and environmental stress. Biofilm also plays a vital role
in transmission, infection, persistence, chronic nature of disease, virulence, immunomodulation
and relapse of TB. Within the primary granulomas in guinea pig lungs, an acellular rim has been
observed adjacent to the edge of the mineralizing central necrosis containing drug-tolerant
bacteria in micro colonies with features reminiscent of biofilms which are analogous to lesion
environment in granuloma12. Identification of drugs that inhibit biofilm formation could enable
the dramatic shortening of TB treatments using standard antibiotics, with substantial potential
impact on global health and reduction of antibiotic resistance associated with non-compliance.9-10
The effect of compounds 20 and 22 on viability of MTB biofilms was tested at a concentration of
10 µg/ml. Both compounds shown promising results and has 2.1 and 2.3 log reduction in
bacterial count similar to standard drugs INH (1.9 log fold), Rif (2 log fold), Moxi (1.8 log fold)
as shown in Fig 3.
Figure 3: Biological activities of compounds 20 and 22 against biofilm forming M. tuberculosis.
Bacterial count estimation (Mean S.D., n = 4) for control and treated groups conducted by using the
MPN (most probable number) assay. Both compounds gave significant inhibition of growth of M.

tuberculosis in this model as compared to the control (p < 0.0001, two way ANOVA usingGraphPad
Prism Software).
Kill curve experiments give an insight on nature of kill (bactericidal or static) and kinetics of kill
under given set of conditions. MBC is defined as the lowest concentration of compound needed
to kill 3 logs of MTB in 21 days under the given conditions. Compounds that are bactericidal
may also exhibit time dependent killing or concentration-dependent killing. For compounds that
have time-dependent kill, any concentration at or above the MBC will result in a constant rate of
kill of the bacteria. For compounds that are concentration dependent, the rate of kill will increase
as the concentration of compound increases. Knowledge of nature of kill and factors effecting
kill can be related to pharmacokinetic parameters and is very much crucial in deciding
appropriate antibiotic for therapy.13 For further evaluation on its mechanism of action persistors
obtained after 2 weeks’ starvation were evaluated at 0, 7, 14, 21 days after drug treatment at
varying drug concentrations (5, 10, 20 µg/ml). At all concentrations tested there was almost 3 log
fold log reduction from 7th day to 21st day. Based on this observations kinetic profiling of
compound 22 against dormant forms of mycobacteria was neither dependent on concentration
nor time as observed in the Fig 4. It also has bactericidal effect against non-replicative stages of
mycobacterium.

Figure 4: Kill kinetic curve of compound 22 at three different concentrations.
Zebra fish has genetic, physiological and immune (adaptive and innate) related similarities with
mammalian host 14-16. When infected with M. marinum it forms granulomas with central necrosis
and hypoxia similar to human 17. Oxidative stress stimulates the expression of bacterial efflux
pumps in active stage of mycobacterium in zebra fish model 14,18. Crucial virulence factors, host
genes and immune cell types implicated in human MTB pathogenesis are retained in zebrafish–
M. marinum model 19. The natural heterogeneity of the zebrafish population proved to be
beneficial as it helps in understanding of genetic differences of different individuals. It also
serves as a link between in vitro and in vivo models as it provides insight into
pharmacodynamic and kinetic parameters 19,20. M. marinum is relatively safe for humans as it
is restricted to topical lesions, has genetic similarity with MTB and also has higher replication
rate than M. tuberculosis. Activity and dosage of antimycobacterial compounds in zebrafish
closely resemble characteristics in humans. Owing to all the mentioned advantages and ease of
handling, cost effectiveness zebra fish model can be used for high throughput screening of
antimycobacterial agents.17,19 Most potent molecule in the series 22 was tested for in vivo

antimycobacterial potency by well validated M. marinum induced zebra fish model. It showed
1.2 log bacterial reduction whereas INH and Moxi showed 2.8 and 2.7 log reduction (Fig 5).
Figure 5: Bacterial count estimation (Mean S.E.M., n = 6) for control and treated groups by zebra fish
model conducted by using MPN (most probable number) assay. The statistical significance (p < 0.001)
with respect to infected control group has been analyzed by Two-way ANOVA using GraphPad Prism
Software.
3D granuloma model is a biomimetic model of human granuloma dormancy as well as
reactivation in favorable conditions such as immunosuppression. The mycobacterium in this
model exhibits characteristics of dormancy such as loss of acid fast staining, accumulation of
fats, Rifampicin tolerance and gene expression changes. The other features of model which
makes it a replica of human granuloma are as follows- granuloma formation, multinucleated
giant cell formation, decrease in CD4 T cell counts, unchanged CD8 T cell values, increase in

+
CD4+ CD25
T cells, decrease in activated macrophage cells, increase in cytokine and
chemokine secretion by host immune cells in response to MTB infection, and resuscitation upon
immunosuppression by treatment with anti-TNFα mAbs. 21 After formation of 3D granuloma it
was treated with compound 22 for 4 days followed by Rifampicin for 3 days. Then the
granuloma was hydrolyzed followed by enumeration of % rifampicin tolerant persisters and
MTB cfu. When the granuloma was treated with compound 22 alone (30.85 X105) cfu were
much higher but when treated in combination with Rif, the cfu counts were less (2.11X 105), as
compared to the cfu counts for MTB from granulomas treated with Rif alone (3.3X105) as shown
in Fig 6 and 7. This indicates that 22, is a more effective killer in the presence of Rif.
40.
30.
20.
10.
0.
Ctrl
Compd 22
COMPOUND
Figure 6: After the formation of granuloma, the granulomas were either left untreated or were treated
with the compound 22 for 4 days and then with Rif for further 3 days. The graph represents the % rif
resistance of Mtb recovered from these granulomas.

40.
30.
20.
10.
0.
Ctrl
Rif
Compd 22
Rif + Compd
22
COMPOUNDS
Figure 7: Data showing cfu counts for Mtb recovered from granulomas treated either with no drugs or
treated with Rif, compd 22 alone or treated with compd 22+ Rif.
When tested against active forms of mycobacterium the MIC were in the range of 2.01 to 99.60
µM. surprisingly compounds 21 and 9 has good potency against both replicating and non-
replicating stages of bacteria as their MIC values are 2.01 and 4.64 µM respectively (Tab 1).
Compound 10 was found to be inactive against LAT enzyme but has low MIC of 2.32 µM
indicating that its mechanism of action might be different than LAT.
Compound 22 ((E)-4-(5-(2-(benzo[d]thiazol-2-yl)-2-cyanovinyl)thiophen-2-yl)isophthalic
acid) on docking gave a glide score of -6.12 kcal/mol. The ligand had four hydrogen bonding
with Arg422, Lys300 and GLn274 with a well fitted pose in the active site. The ligand found
in the hydrophobic pocket within the vicinity of Val30, Leu24, Met28, Leu28, Leu414,
Pro415, Phe167 and Val63. The binding pattern within the active site pocket of the crystal
ligand and reference ligand was quite similar and additionally the solvent exposure area
around the ligand constituted for a stable binding profile of the molecule as shown in the Fig
8. SAR analysis revealed that when compared to substituted phenyl ring, heterocycles are
favoring activity. Heterocycles attached with isophthalic acid were more promising hits than

heterocycles attached to benzoic acid. When attempts were made to modify the benzthiazole
core with benzoxazole ring it resulted in complete loss of activity.
Figure 8: Interactions of most active inhibitor 22 with the active site residues of MTB LAT.
Figure 9: Interactions of reference inhibitor with the active site residues of MTB LAT and
superimposition of docked pose of the reference ligand to the original pose of the ligand.

To know the effect of compound on macrophages where the parasite resides RAW cell lines
were used for MTT studies. All the compounds were tested at a concentration of 50 µg/ml and
the percentage inhibition were found to be in range of 2.26 to 60.31% (Tab 1). Most of the
compounds are devoid of effects on metabolism.
3. Conclusions
In present work an existing lead was modified to develop more active compound against MTB
LAT a crucial enzyme for regulation of amino acid pool which in turn key for antibiotic
tolerance and persistence. In our attempts we also unexpectedly arrived at an interesting
compound 21 ((E)-4-(5-(2-(benzo[d]thiazol-2-yl)-2-cyanovinyl)thiophen-2-yl)benzoic acid)
which even though has moderate activity against persistent phase of mycobacterium it has
significant potency against active phase. In the entire series compound 22 emerged as potent
molecule with LAT IC50 of 2.62 µM. It has a significant log reduction of 2.9 and 2.3 log fold
against nutrient starved and biofilm forming mycobacteria. It was found to be inactive against
active MTB and also in M. marinum induced zebra fish model indicating that it acts through
dormant targets. Compound 22 was also found to possess bactericidal effect which is
independent of concentration and time. It was found to be effective in combination with Rif in
3D granuloma model. All these parameters make it a better candidate for further development.
4. Materials and methods
4.1. Synthesis
Benzthiazole scaffold was prepared by condensation of ortho-aminothiophenol with
malanonitrile. Knoevenagel condensation of active methylene group with corresponding
aldehydes resulted in final compounds as shown in Scheme 1. 22,23
4.1.1. General:

All commercially available chemicals and solvents were used without further purification. TLC
experiments were performed on alumina-backed silica gel 40 F254 plates (Merck, Darmstadt,
Germany). The homogeneity of the compounds was monitored by thin layer chromatography
(TLC) on silica gel 40 F254 coated on aluminum plates, visualized by UV light and KMnO4
treatment. All 1H and 13C NMR spectra were recorded on a Bruker AM-300 (300.12 MHz, 75.12
MHz) NMR spectrometer, BrukerBioSpin Corp, Germany. Molecular weights of the synthesized
compounds were checked by LCMS 6100B series Agilent Technology. Chemical shifts are
reported in ppm (δ) with reference to the internal standard TMS. The signals are designated as
follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet. Elemental analyses
were carried out on an automatic Flash EA 1112 Series, CHN Analyzer (Thermo).
4.1.2.
2-(Benzo[d]thiazol-2-yl)acetonitrile: To solution of 2-aminobenzenethiol (2.5g,
19.97 mmol) in absolute ethanol (25ml), malanonitrile (1.32g, 19.97 mmol) was added,
0
stirred and refluxed at 50 C for 2hrs along with catalytic amount of glacial acetic acid. the
solvent was evaporated under reduced pressure and the residue was extracted with
chloroform and purified by column chromatography using hexane–ethylacetate as eluent and
decolourised with activated carbon to afford 2-(benzo[d]thiazol-2-yl)acetonitrile (2.3g,
66.11%) as yellow solid.
4.1.3. General procedure for the synthesis of benzothiazole acrylonitrile derivatives:
Procedure A: To a warm solution of the corresponding 2-(benzo[d]thiazol-2-yl) acetonitrile (1
mmol) in absolute ethanol (8 mL) was added the corresponding aldehyde (1 mmol) and catalytic
amount of piperidine (0.2 mmol). The reaction mixture was then stirred and heated to 80oC for
0.5-1 h, (as monitored by TLC and LCMS for completion), the precipitate formed was collected

by suction and recrystallised from ethanol to give the desired product in good yield as mentioned
below
Procedure B: To a warm solution of the corresponding 2-(benzo[d]thiazol-2-yl) acetonitrile (1
mmol) in absolute ethanol (8 mL) was added the corresponding aldehyde (1 mmol) and catalytic
amount of 10% methanolic KOH (0.2 mmol). The reaction mixture was then stirred and heated
to 80oC for 0.5-1 h, (as monitored by TLC and LCMS for completion), the precipitate formed
was collected by suction and recrystallised from ethanol to give the desired product in good yield
as mentioned below
4.1.3.1. (E)-2-(Benzo[d]thiazol-2-yl)-3-(4-hydroxyphenyl)acrylonitrile (1): The compound
was synthesized according to the above general procedure A using 2-(benzo[d]thiazol-2-yl)
acetonitrile (0.25g, 1.44mmol), 4-hydroxy benzaldehyde(0.18g, 1.44mmol), piperidine (0.025g,
0.29mmol) to afford 1 (0.32g, 80.2%) as yellow coloured solid. M.p: 218-220oC. . 1H NMR
(DMSO-d6): δH. 8.34 (s, 1H), 8.16 (t, J = 7.4Hz, 1H), 8.05 (t, J = 7.8Hz, 1H), 7.62 – 7.49 (m,
4H), 6.82 (m, 2H), 5.46 (s, 1H). 13C NMR (DMSO-d6): δc. 163.4, 158.2, 152.5(2C), 134.6,
130.3(2C), 128.1, 126.6, 123.4(2C), 122.3, 120.4, 116.3, 114.8, 105.7.ESI-MS m/z 279.3
(M+H)+. Anal Calcd.for C16H10N2OS; C, 69.04; H, 3.62; N, 10.06; Found: C, 69.13; H, 3.61; N,
10.07.
4.1.3.2. (E)-2-(Benzo[d]thiazol-2-yl)-3-(4-fluorophenyl)acrylonitrile (2): The compound was
synthesized according to the above general procedure A using 2-(benzo[d]thiazol-2-
yl)acetonitrile (0.25g, 1.44mmol), 4-fluoro benzaldehyde(0.18g, 1.44mmol), piperidine (0.025g,
0.29mmol) to afford 2 (0.32g, 79.6 %) as red coloured solid. M.p: 174-176oC. 1H NMR (DMSO-
d6): δH. 8.37 (s, 1H), 8.18 (t, J = 8.0Hz, 1H), 8.04 (t, J = 8.2Hz, 1H), 7.84 - 7.53 (m, 4H), 7.25
(m, 2H). 13C NMR (DMSO-d6): δc. 164.1, 162.4, 155.7(2C), 138.7, 131.1(2C), 125.6, 123.6,

122.8(2C), 120.4, 118.7, 115.3(2C), 106.5.ESI-MS m/z 281.4 (M+H)+. Anal Calcd.for
C16H9FN2S; C, 68.55; H, 3.24; N, 9.99; Found: C, 68.38; H, 3.23; N, 10.01.
4.1.3.3. (E)-2-(Benzo[d]thiazol-2-yl)-3-(4-hydroxy-3-methoxyphenyl)acrylonitrile (3): The
compound was synthesized according to the above general procedure A using 2-
(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), 4-hydroxy-3-methoxy benzaldehyde
(0.218g, 1.44mmol), piperidine (0.025g, 0.29mmol) to afford 3 (0.39g, 88.04%) as green
coloured solid. M.p: 175-177oC. 1H NMR (DMSO-d6): δH. 8.31 (s, 1H), 8.16 (t, J = 7.8Hz, 1H),
8.05 (t, J = 7.4Hz, 1H), 7.55 – 7.18 (m, 4H), 7.02 (d, J = 8.2Hz, 1H), 5.41 (s, 1H), 3.84 (s, 3H).
13C NMR (DMSO-d6): δc. 161.2, 153.8(2C), 148.8, 148.3, 137.6, 129.4, 124.2, 123.6(2C),
122.6, 119.6, 118.3, 115.9, 112.1, 107.5, 57.9.ESI-MS m/z 309.4(M+H)+. Anal Calcd.for
C17H12N2O2S; 66.22; H, 3.92; N, 9.08; Found: C, 66.17; H, 3.91 N, 9.10.
4.1.3.4. (E)-2-(Benzo[d]thiazol-2-yl)-3-(2,4-dihydroxyphenyl)acrylonitrile (4):
The
compound was synthesized according to the above general procedure A using 2-
(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), 2,4-dihydroxy benzaldehyde (0.198g,
1.44mmol), piperidine (0.025g, 0.29mmol) to afford 4 (0.35g, 82.9%) as orange coloured solid.
1
M.p: 151-153oC. H NMR (DMSO-d6): δH. 8.43 (s, 1H), 8.15 (t, J = 8.0Hz, 1H), 8.03 (t, J =
7.4Hz, 1H), 7.95 -6.56 (m, 4H), 6.42 (s, 1H), 5.38 (s, 2H).13C NMR (DMSO-d6): δc. 161.1,
160.2, 157.4, 153.9(2C), 137.9, 131.4, 126.9, 124.2(2C), 120.4, 118.7, 110.2, 108.6, 107.8,
103.2.ESI-MS m/z 295.4 (M+H)+. Anal Calcd.for C16H10N2O2S; C, 65.29; H, 3.42; N, 9.52;
Found: C, 65.17; H, 3.43; N, 9.51.
4.1.3.5. (E)-2-(Benzo[d]thiazol-2-yl)-3-(3,4-dimethoxyphenyl)acrylonitrile (5):
The
compound was synthesized according to the above general procedure A using 2-

(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), 3,4-dimethoxy benzaldehyde (0.239g,
1.44mmol), piperidine (0.025g, 0.29mmol) to afford 5 (0.27g, 58.32%) as orange coloured solid.
1
M.p: 147-1490C. H NMR (DMSO-d6): δH. 8.36 (s, 1H), 8.16 (t, J = 8.8Hz, 1H), 8.06 (t, J =
13
7.2Hz, 1H), 7.81 – 7.47 (m, 4H), 7.19 (d, J = 8.4Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H). C NMR
(DMSO-d6): δc. 163.3, 153.5, 152.8, 149.3, 146.8, 134.7, 126.8, 126.6, 125.7, 125.4, 123.3,
121.6, 117.1, 111.3, 111.1, 102.3, 56.1(2C).ESI-MS m/z 323.4(M+H)+. Anal Calcd.for
C18H14N2O2S; C, 67.06; H, 4.38; N, 8.69; Found: C, 66.92; H, 4.39 N, 8.71.
4.1.3.6. (E)-2-(Benzo[d]thiazol-2-yl)-3-(3,4,5-trimethoxyphenyl)acrylonitrile (6):
The
compound was synthesized according to the above general procedure A using 2-
(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), 3,4,5-trimethoxy benzaldehyde (0.283g,
1.44mmol), piperidine (0.025g, 0.29mmol) to afford 6 (0.31g, 61.27%) as yellow coloured solid.
M.p 262-2640C. 1H NMR (DMSO-d6): δH.. 8.16 (s, 1H), 8.07 (t, J = 7.2Hz, 1H), 8.05 (t, J =
7.6Hz, 1H), 7.57 – 7.32 (m, 4H), 3.95 (s, 3H), 3.94 (s, 6H). 13C NMR (DMSO-d6): δc. 163,
153.7(2C), 153.5(2C), 135.0, 127.6, 127.1, 126.1, 123.6(2C), 121.8, 117.0, 107.9, 104.1(2C),
61.2, 56.4(2C).ESI-MS m/z 353.42 (M+H)+. Anal Calcd.for C19H16N2O3S; C, 64.76; H, 4.58; N,
7.95; Found: C, 64.63; H, 4.57; N, 7.97.
4.1.3.7. (E)-2-(Benzo[d]thiazol-2-yl)-3-(4-benzyloxyphenyl)acrylonitrile (7): The compound
was synthesized according to the above general procedure A using 2-(benzo[d]thiazol-2-
yl)acetonitrile (0.25g, 1.44mmol), benzyloxybenzaldehyde (0.31g, 1.44mmol), piperidine
(0.025g, 0.29mmol) to afford 7 (0.37g, 69.94%) as brown coloured solid. M.p 217-219 0C. 1H
NMR (DMSO-d6): δH.. 8.28 (s, 1H), 8.16 (t, J = 8.2Hz, 1H), 8.06 (t, J = 7.8Hz, 1H), 7.74 (d, J =
13
7.8Hz, 2H), 7.51 – 7.37 (m, 7H), 7.04 (d, J = 7.8Hz, 2H), 5.28 (s, 2H). C NMR (DMSO-d6):
δc. 162.3, 159.8, 157.4, 153.8(2C), 136.8, 135.9, 129.8(2C), 128.6(2C), 127.5(2C), 127.4, 126.3,

124.2(2C), 120.1, 118.8, 114.3(2C), 107.8, 69.7.ESI-MS m/z 369.45(M+H)+. Anal Calcd.for
C23H16N2OS; C, 74.98; H, 4.38; N, 7.60; Found: C, 75.08; H, 4.39; N, 7.62.
4.1.3.8. (E)-3-(Benzo[d][1,3]dioxol-5-yl)-2-(benzo[d]thiazol-2-yl)acrylonitrile (8): The
compound was synthesized according to the above general procedure B using 2-
(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), piperonal (0.22g, 1.44mmol), alc. KOH
(0.02g, 0.29mmol) to afford 8 (0.22 g, 50.11%) as red coloured solid. M.p219-221 0C.1H NMR
(DMSO-d6): δH. 8.30 (s, 1H), 8.15 (t, J = 7.8Hz, 1H), 8.07 (t, J = 7.2Hz, 1H), 7.83 (d, J = 7.4Hz,
1H), 7.60 (m, 2H), 7.37 (s, 1H), 7.02 (d, J = 7.6Hz, 1H), 6.12 (s, 2H). 13C NMR (DMSO-d6): δc.
158.4, 154.4(2C), 149.1, 147.8, 137.6, 129.1, 126.7, 123.3(2C), 122.3, 119.2, 118.4, 112.1,
108.4, 107.3, 100.8.ESI-MS m/z 307.32 (M+H)+. Anal.Calcd.for C17H10N2O2S; C, 66.65; H,
3.29; N, 9.14; Found: C, 66.59; H, 3.28; N, 9.12.
4.1.3.9. (E)-2-(-4-(2-(Benzo[d]thiazol-2-yl)-2-cyanovinyl)phenoxy)acetic acid (9):
The
compound was synthesized according to the above general procedure A using 2-
(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), 4-formyl-phenoxy acetic acid(0.26 g,
1.44mmol), piperidine (0.025g, 0.29mmol) to afford 9 (0.27g, 55.9%) as green coloured solid.
1
M.p 231-2330C. H NMR (DMSO-d6): δH. 12.68 (s, 1H), 8.38 (s, 1H), 8.15 (t, J = 7.6Hz, 1H),
13
8.07 (d, J = 7.8Hz, 1H), 7.65 – 7.56 (m, 4H), 7.06 (d, J = 7.4Hz, 2H), 4.44 (s, 2H). C NMR
(DMSO-d6): δc. 169.7, 162.8, 157.6, 153.8(2C), 137.6, 130.4(2C), 127.4, 126.9, 123.1(2C),
122.8, 118.7, 114.5(2C), 107.9, 63.9.ESI-MS m/z 335.36 (M-H)+. Anal Calcd.for C18H12N2O3S;
C, 64.27; H, 3.60; N, 8.33; Found: C, 64.32; H, 3.61; N, 8.31.
4.1.3.10. (E)-2-(-2-(2-(Benzo[d]thiazol-2-yl)-2-cyanovinyl)phenoxy)acetic acid (10): The
compound was synthesized according to the above general procedure A using 2-
(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), 2-formyl-phenoxy acetic acid(0.26g,

1.44mmol), piperidine (0.025g, 0.29mmol) to afford 10 (0.21g, 43.48%) as green coloured solid.
0
M.p 257-259 C. 1H NMR (DMSO-d6): δH. 12.63 (s, 1H), 8.72 (s, 1H), 8.17 (t, J = 7.2Hz, 2H),
8.09 (d, J = 8.0Hz, 1H), 7.59 (t, J = 7.8Hz, 1H), 7.52 (t, J = 8.2Hz, 2H), 7.09 (t, J = 8.2Hz, 1H),
7.01 (d, J = 8.2Hz, 1H), 4.42 (s, 2H).13C NMR (DMSO-d6): δc.170.7, 163.4, 158.8, 152.9(2C),
143.3, 134.0, 127.7, 127.0, 126.1, 123.0, 122.4, 120.8, 120.0, 116.3, 113.6, 104.1, 68.5. ESI-MS
m/z 335.36(M-H)+. Anal Calcd.for C18H12N2O3S; C, 64.27; H, 3.60; N, 8.33; Found: C, 64.22; H,
3.59; N, 8.34.
4.1.3.11. (E)-2-(Benzo[d]thiazol-2-yl)-3-(furan-2-yl)acrylonitrile (11): The compound was
synthesized according to the above general procedure B using 2-(benzo[d]thiazol-2-
yl)acetonitrile (0.25g, 1.44mmol), furaldehyde (0.14g, 1.44mmol), alc. KOH (0.02g, 0.29mmol)
1
to afford 11 (0.28 g, 77.78%) as yellow coloured solid. M.p: 137-139oC. H NMR (DMSO-d6):
δH. 8.22 (s, 1H), 8.14 (t, J = 7.8Hz, 1H), 8.07 (t, J = 8.0Hz, 1H), 7.82 (d, J = 7.2Hz, 1H), 7.52 (m,
13
2H), 7.03 (d, J = 8.0Hz, 1H), 6.67 (t, J = 8.2Hz, 1H). C NMR (DMSO-d6): δc. 162.4, 152.2,
150.6, 144.8, 143.6, 135.3, 124.5, 123.4(2C), 120.1, 118.9, 113.8, 112.9, 109.4. ESI-MS m/z
253.3(M+H)+. Anal Calcd.for C14H8N2OS; C, 66.65; H, 3.20; N, 11.10; Found: C, 66.58; H,
3.21; N, 11.12.
4.1.3.12. (E)-2-(Benzo[d]thiazol-2-yl)-3-(thiophen-2-yl)acrylonitrile (12): The compound was
synthesized according to the above general procedure B using 2-(benzo[d]thiazol-2-
yl)acetonitrile (0.25g, 1.44mmol), 2-thiophenecarboxaldehyde (0.16g, 1.44mmol), alc. KOH
(0.02g, 0.29mmol) to afford 12 (0.13 g, 33.77%) as yellow coloured solid. M.p 149-151 0C. 1H
NMR (DMSO-d6): δH. 8.26 (s, 1H), 8.13 (t, J = 8.2Hz, 1H), 8.05 (t, J = 7.8Hz, 1H), 7.73 (d, J =
13
8.2Hz, 1H), 7.59 (m, 2H), 7.26 (t, J = 8.8Hz, 1H), 7.11 (d, J = 7.6Hz, 1H). C NMR (DMSO-
d6): δc. 162.1, 154.7, 142.3, 137.6, 135.3, 133.2, 129.8, 128.6, 124.7, 123.2(2C), 120.2, 118.3,

113.8. ESI-MS m/z 269.41 (M+H)+. Anal.Calcd.for C14H8N2S2; C, 62.66; H, 3.00; N, 10.44;
Found: C, 62.75; H, 2.99; N, 10.42.
4.1.3.13. (E)-2-(Benzo[d]thiazol-2-yl)-3-(1H-pyrrol -2-yl)acrylonitrile (13): The compound
was synthesized according to the above general procedure B using 2-(benzo[d]thiazol-2-
yl)acetonitrile (0.25g, 1.44mmol), pyrrole-2-carboxaldehyde(0.14g, 1.44mmol), alc. KOH
(0.02g, 0.29mmol) to afford 13 (0.19g, 52.63%) as brown coloured solid. M.p: 176-178oC. 1H
NMR (DMSO-d6): δH. 9.12 (s, 1H), 8.21 (s, 1H), 8.12 (t, J = 7.2Hz, 1H), 8.05 (t, J = 7.8Hz, 1H),
7.57 (m, 2H), 7.03 (d, J = 8.0Hz, 1H), 6.67 (d, J = 8.2Hz, 1H), 6.26 (t, J = 7.4Hz, 1H). 13C NMR
(DMSO-d6): δc. 162.6, 154.1, 145.4, 137.0, 126.6, 125.4(2C), 122.8, 120.5, 118.7, 118.0, 113.9,
112.0, 107.9. ESI-MS m/z 252.4 (M+H)+. Anal Calcd.for C14H9N3S; C,66.91; H, 3.61; N, 16.72;
Found: C, 66.87; H, 3.62; N, 16.74.
4.1.3.14. (E)-2-(Benzo[d]thiazol-2-yl)-3-(5-nitrofuran-2-yl)acrylonitrile (14): The compound
was synthesized according to the above general procedure B using 2-(benzo[d]thiazol-2-
yl)acetonitrile (0.25g, 1.44mmol), 5-nitrofurfural (0.20g, 1.44mmol), alc. KOH (0.02g,
0.29mmol) to afford 14 (0.28g, 65.57%) as orange coloured solid. M.p 245-247 0C.1H NMR
(DMSO-d6): δH. 8.26 (s, 1H), 8.15 (t, J = 7.2Hz, 1H), 8.04 (t, J = 7.8Hz, 1H), 7.64 – 7.58 (m,
3H), 6.97 (d, J = 8.0Hz, 1H). 13C NMR (DMSO-d6): δc. 161.3, 155.9, 154.2, 153.9, 144.8,
137.6, 124.6, 123.7(2C), 122.8, 118.1, 116.5, 114.8, 113.6. ESI-MS m/z 298.3 (M+H)+.
Anal.Calcd.for C14H7N3O3S; C, 56.56; H, 2.37; N, 14.13; Found: C, 56.48; H, 2.36; N, 14.12.
4.1.3.15. (E)-2-(Benzo[d]thiazol-2-yl)-3-(5-nitrothiophen-2-yl)acrylonitrile (15): The
compound was synthesized according to the above general procedure B using 2-
(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), 5-nitrothiophene-2-carboxaldehyde (0.23g,
1.44mmol), alc. KOH (0.02g, 0.29mmol) to afford 15 (0.32g, 71.27%) as green coloured solid.

M.p 260-262 0C.1H NMR (DMSO-d6): δH. 8.20 (s, 1H), 8.18 (d, J = 8.0Hz, 1H), 8.14 (t, J =
13
7.2Hz, 1H), 8.06 (t, J = 7.8Hz, 1H), 7.50 (m, 2H), 7.41 (d, J = 8.0Hz, 1H). C NMR (DMSO-
d6): δc.162.2, 156.3, 154.7, 147.2, 141.8, 138.2, 135.3, 129.8, 126.8, 123.6(2C), 119.7, 116.5,
111.2. ESI-MS m/z 314.37 (M+H)+. Anal.Calcd.for C14H7N3O2S2; C, 53.66; H, 2.25; N, 13.41;
Found: C, 53.62; H, 2.24; N, 13.39..
4.1.3.16. (E)-2-(Benzo[d]thiazol-2-yl)-3-(1H-indol-3-yl)acrylonitrile (16): The compound was
synthesized according to the above general procedure B using 2-(benzo[d]thiazol-2-
yl)acetonitrile (0.25g, 1.44mmol), indole-3-carboxaldehyde(0.21g, 1.44mmol), alc. KOH (0.02g,
0.29mmol) to afford 16 (0.22g, 50.81%) as green coloured solid. M.p: 289-291oC . 1H NMR
(DMSO-d6): δH. 11.52 (s, 1H), 8.31 (s, 1H), 8.10 (t, J = 7.8Hz, 1H), 8.02 (t, J = 7.2Hz, 1H), 7.82
13
(s, 1H), 7.73 – 7.62 (m, 3H), 7.12 – 7.05 (m, 3H). C NMR (DMSO-d6): δc. 161.2, 154.1(2C),
137.3, 136.1, 129.4, 126.9, 125.0, 124.7(2C), 123.2, 121.8, 119.6, 118.8, 117.9, 113.8, 111.3,
110.7. ESI-MS m/z 302.37 (M+H)+. Anal Calcd.for C18H11N3S; C,71.74; H, 3.68; N, 13.94;
Found: C, 71.65; H, 3.67; N, 13.96.
4.1.3.17. (E)-3-(5-(2-(Benzo[d]thiazol-2-yl)-2-cyanovinyl)furan-2-yl)benzoic acid (17): The
compound was synthesized according to the above general procedure A using 2-
(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), 3-(5-formyl furan-2-yl) benzoic acid
(0.31g, 1.44mmol), piperidine (0.025g, 0.29mmol) to afford 17 (0.31g, 58.05%) as orange
coloured solid. M.p:279-281oC. 1H NMR (DMSO-d6): δH. 13.12 (s, 1H), 8.65 (s, 1H), 8.53 (d, J
= 7.2Hz, 1H), 8.21 (s, 1H), 8.23 – 8.13 (m, 2H), 8.06 (t, J = 7.2Hz, 1H), 7.84 (t, J = 8.2Hz,1H),
7.51 (m, 2H), 7.14 (d, J = 8.2Hz, 1H), 6.94 (d, J = 7.8Hz, 1H). 13C NMR (DMSO-d6): δc. 171.3,
162.3, 156.3, 152.7, 150.8, 144.8, 137.3, 131.1, 130.9, 129.9, 129.8(2C), 126.9, 124.2,

123.1(2C), 122.1, 118.6, 114.7, 110.2, 106.1. ESI-MS m/z 371.05(M-H)+. Anal Calcd.for
C21H12N2O3S; C,67.73; H, 3.25; N, 7.52; Found: C, 67.69; H, 3.24; N, 7.50.
4.1.3.18. (E)-4-(5-(2-(Benzo[d]thiazol-2-yl)-2-cyanovinyl)furan-2-yl)benzoic acid (18): The
compound was synthesized according to the above general procedure A 2-(benzo[d]thiazol-2-
yl)acetonitrile (0.25g, 1.44mmol), 4-(5-formyl furan-2-yl) benzoic acid(0.31g, 1.44mmol),
piperidine (0.025g, 0.29mmol) to afford 18 (0.35g, 65.54%) as yellow coloured solid. M.p:109-
111oC . 1H NMR (DMSO-d6): δH. 13.07 (s, 1H), 8.27 (s, 1H), 8.19 – 8.07 (m, 6H), 7.51 (m, 2H),
7.16 (d, J = 8.0Hz, 1H), 6.96 (d, J = 8.2Hz, 1H). 13C NMR (DMSO-d6): δc. 170.6, 161.0, 155.7,
153.3, 151.2, 143.2, 136.8, 134.3, 130.8, 126.9(2C), 125.1, 124.4(2C), 123.1(2C), 120.5, 117.7,
114.7, 110.8, 107.1. ESI-MS m/z 371.05 (M-H)+. Anal Calcd.for C21H12N2O3S; C,67.73; H, 3.25;
N, 7.52; Found: C, 67.81; H, 3.24; N, 7.50.
4.1.3.19. (E)-5-(5-(2-(Benzo[d]thiazol-2-yl)-2-cyanovinyl)furan-2-yl)isophthalic acid (19):
The compound was synthesized according to the above general procedure A using 2-
(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), 5-(5-formylfuran-2-yl)isophthalic acid
(0.38 g, 1.44 mmol),piperidine (0.025 g, 0.29 mmol) to afford 19 (0.38 g, 61.87%) as yellow
coloured solid. M.p: 269-271 0C. 1H NMR (DMSO-d6): δH. 12.96 (s, 1H), 12.87 (s, 1H), 9.27 (s,
2H), 8.67 (s, 1H), 8.31 (s, 1H), 8.15 (t, J = 7.2Hz, 1H), 8.03 (t, J = 7.0Hz,1H), 7.48 (m, 2H), 7.12
(d, J = 8.2Hz, 1H), 6.96 (d, J = 8.4Hz, 1H). 13C NMR (DMSO-d6): δc. 170.9(2C), 161.8, 155.8,
154.6, 150.3, 144.8, 136.9, 135.9(2C), 130.8(3C), 130.6, 126.2, 125.1(2C), 120.4, 117.7, 114.9,
110.3, 106.6. ESI-MS m/z 415.41 (M-H)+. Anal.Calcd.for C22H12N2O5S; C, 63.46; H, 2.90; N,
6.73; Found: C, 63.39; H, 2.89; N, 6.75.
4.1.3.20. (E)-3-(5-(2-(Benzo[d]thiazol-2-yl)-2-cyanovinyl)thiophen-2-yl)benzoic acid (20):
The compound was synthesized according to the above general procedure A using 2-

(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), 3-(5-formyl thiophen-2-yl) benzoic acid
(0.33g, 1.44mmol), piperidine (0.025g, 0.29mmol) to afford 20 (0.36g, 64.6%) as brown
coloured solid. M.p:283-285oC . 1H NMR (DMSO-d6): δH.. 13.32 (s, 1H), 8.63 (s, 1H), 8.25 (s,
1H), 8.16 (t, J = 8.0Hz, 1H), 8.12 - 7.98 (m, 4H), 7.86 (d, J = 4.0Hz, 1H), 7.64 – 7.50 (m, 3H).
13C NMR (DMSO-d6): δc. 171.2, 162.4, 155.3, 144.5, 142.7, 139.7, 135.4, 133.5, 132.3,
130.2(2C), 130.1, 129.9, 127.6, 125.6, 124.3, 122.8(2C), 120.1, 118.2, 113.5. ESI-MS m/z
387.1(M-H)+. Anal Calcd.for C21H12N2O2S2; C, 64.93; H, 3.11; N, 7.21; Found: C, 64.98; H,
3.12; N, 7.23.
4.1.3.21. (E)-4-(5-(2-(Benzo[d]thiazol-2-yl)-2-cyanovinyl)thiophen-2-yl)benzoic acid (21):
The compound was synthesized according to the above general procedure A using 2-
(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), 4-(5-formyl thiophen-2-yl) benzoic acid
(0.33g, 1.44mmol), piperidine (0.025g, 0.29mmol) to afford 21 (0.29g, 52.07%) as brown
coloured solid. M.p:289-291oC. 1H NMR (DMSO-d6): δH. 13.01 (s, 1H), 8.34 (s, 1H), 8.20 (t, J =
7.2Hz, 1H), 8.16 (t, J = 8.0Hz, 1H), 8.11 – 7.87 (m, 4H), 7.67 (m, 2H), 7.28 (d, J = 8.0 Hz, 2H).
13C NMR (DMSO-d6): δc. 170.1, 161.7, 152.4, 143.9, 141.5, 139.8, 138.4, 135.1, 131.0, 130.9,
128.4, 127.9, 126.8(2C), 125.1, 124.9(2C), 123.7, 120.4, 117.4, 113.2.ESI-MS m/z 387.51 (M-
H)+. Anal Calcd for C21H12N2O2S2; C, 64.93; H, 3.11; N, 7.21; Found: C, 64.86; H, 3.12; N,
7.23.
4.1.3.22. (E)-4-(5-(2-(Benzo[d]thiazol-2-yl)-2-cyanovinyl)thiophen-2-yl)isophthalicacid (22):
The compound was synthesized according to the above general procedure A using 2-
(benzo[d]thiazol-2-yl)acetonitrile (0.25g, 1.44mmol), 4-(5-formyl thiophen-2-yl) isophthalic acid
(0.39g, 1.44mmol), piperidine (0.025g, 0.29mmol) to afford 22 (0.38g, 61.19%) as yellow solid.
M.p:281-283oC . 1H NMR (DMSO-d6): δH. 13.01 (s, 1H), 12.96 (s, 1H), 8.52 – 8.48 (m, 2H),

8.27 (s, 1H), 8.18 (m, 1H), 8.14 (t, J = 7.4Hz, 1H), 8.03 (d, J = 7.2Hz, 1H), 7.92 (d, J = 7.6Hz,
1H), 7.58 (m, 2H), 7.43 (d, J = 8.0Hz, 1H). 13C NMR (DMSO-d6): δc. 170.4, 169.1, 158.3,
152.4, 144.2, 141.5, 139.2, 138.5, 135.4, 132.8, 131.3, 129.7(2C), 128.4, 128.1, 126.8, 125.1,
124.2(2C), 120.4, 117.4, 113.0. ESI-MS m/z 431.44(M-H)+. Anal Calcd for C22H12N2O4S2; C,
61.10; H, 2.80; N, 6.48; Found: C, 61.14; H, 2.79; N, 6.50.
4.2. Enzyme inhibitory assay
LAT enzyme has been cloned and purified as per procedure reported in literature. MTB LAT
enzymatic assay was performed in 100 µl volume containing 200 mM phosphate buffer (pH 7.2),
1.5 mM L-lysine, 1.5 mM 〈-ketoglutarate, 15 M pyridoxal 5-phosphate with MTB LAT for 1hr
at 37°C. The compounds were added to the plates with different concentrations from 50 M to 1
M. Reactions were terminated by adding 10% trichloroacetic acid in ethanol. The MTB LAT
activity was monitored and the end product piperidene 6-carboxylate and glutamate was
measured due to its colour intensity of its adduct with o-aminobenzaldehyde at 465 nm and 280
5,24
nm
.
Reactions were carried out in a heat-controlled Perkin Elmer Victor X3
spectrophotometer. 5
4.3. MIC determination
All compounds were screened for their activity against replicative stage of MTB by MABA
method in duplicates. The bacterial inoculums of H37Rv (active TB) was prepared from fresh LJ
medium and re-suspended in Middlebrook 7H9 broth supplemented with OADC growth
supplement. This culture was grown at 37°C until the turbidity matched with McFarland no.1
turbidity standard. The culture was diluted to 1:25 with media and 100 µL was used as an
inoculum. Each drug stock solution was diluted four-fold highest concentration tested. Sterile
medium with and without bacteria as control maintained on each plate. After 7 days of

incubation, 50 µL of 1:1 mixture of alamar blue solution and sterile tween 80 were added to each
well and the plate was re-incubated overnight. 25
4.4. MTT assay
The synthesized compounds were further examined for its effects on metabolism in mouse
macrophage cell line (RAW 264.7) at 50µg/ml concentration. After 48 h of exposure, viability
was assessed on the basis of cellular conversion of MTT into a formazan product using the
Promega Cell Titer 96 non-radioactive cell proliferation assay. The compounds are tested at 50
µg/ml concentration for 48 hrs. 26
4.5. Nutrient starvation model
A culture of M. tuberculosis H37Rv (O.D. of 0.8 - 1.0) grown in Middlebrook 7H9 medium
supplemented with OADC was pelleted and washed twice with PBS. The pellet was resuspended
in PBS in sealed bottles and incubated at 37 °C for 6 weeks. Aliquots of these cultures were then
treated with standard drugs like INH, Rif and Moxifloxacin (Mox) and the lead compounds for 7
days at a concentration of 10 µg/ml. The frequency of persistors was enumerated by MPN
assay.3
4.6. Biofilm formation and determination of frequency of persistors
4.5 ml of Sautons media inoculated with MTB culture with an O.D. of 0.7 - 1.0 (1:100 dilution)
0
was added in each well and sealed with parafilm then incubated at 37 C for 5 weeks. To the
matured biofilm, compound to be evaluated was injected at desired concentration into media and
swirled (n=4). The plates were incubated in incubator for 7 days after sealing with parafilm. At
the end of the incubation, Tween-80 (0.1% volume/volume) was added, swirled and incubated at
room temperature for 15 minutes. The content of each well was mixed with pipette several times