Solvent-free synthesis of bacillamide analogues as novel cytotoxic and anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2016.07.033

Synthesis of fourteen analogues of bacillamide, a bioactive tryptamide alkaloid of marine origin, has been accomplished through a highly efficient convergent route. The present solvent-free protocol involves the formation of thiazole ring in the initial step followed by amide coupling between substituted ethyl 2-alkyl/aryl/heteroaryl/amino/aminoarylthiazole-4-carboxylates and tryptamine in presence of 2-hydroxy-4,6-dimethylpyrimidine, a solid phase catalyst to yield N-[2-(1H-indol-3-yl)ethyl]-2-alkyl/aryl/heteroaryl/amino/aminoarylthiazole-4-carboxamides as bacillamide analogues having structural variation at position-2 of thiazole ring. Bacillamide and its analogues were evaluated for their cytotoxic activity against three cancer cell lines (HCT-116, MDA-MD-231 and JURKAT cell lines) using colorimetric cell proliferation assay. Compounds 17a and 17b exhibited potent anti-cell proliferation activity with IC₅₀values in the range of ～3.0?μM and ～0.1–0.6?μM, respectively against these cell lines. Preliminary mechanism of action studies indicates that these compounds initiate caspase dependent apoptosis. Also, compounds 16d, 16f, 17a and 17d exhibited excellent anti-inflammatory activity comparable to well-known NSAID indomethacin and better to bacillamide, when evaluated using carrageenan induced rat hind paw oedema method.

Full text of DOI:10.1016/j.ejmech.2016.07.033

Accepted Manuscript
Solvent-free synthesis of bacillamide analogues as novel cytotoxic and anti-
inflammatory agents
Sunil Kumar, Ranjana Aggarwal, Virender Kumar, Rachna Sadana, Bhumi Patel,
Pawan Kaushik, Dhirender Kaushik
PII:
S0223-5234(16)30581-5
10.1016/j.ejmech.2016.07.033
EJMECH 8751
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 April 2016
Revised Date: 15 July 2016
Accepted Date: 18 July 2016
Please cite this article as: S. Kumar, R. Aggarwal, V. Kumar, R. Sadana, B. Patel, P. Kaushik, D.
Kaushik, Solvent-free synthesis of bacillamide analogues as novel cytotoxic and anti-inflammatory
agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.033.
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ACCEPTED MANUSCRIPT
Solvent-free synthesis of bacillamide analogues as novel cytotoxic and anti-
inflammatory agents
Sunil Kumar, Ranjana Aggarwal, Virender Kumar, Rachna Sadana, Bhumi Patel, Pawan
Kaushik and Dhirender Kaushik
Abstract
A highly efficient convergent route for the synthesis of N-[2-(1H-indol-3-yl)ethyl]-2-
amino/aminoaryl/alkyl/aryl/heteroarylthiazole-4-carboxamides (bacillamide analogues) was
described via a two-step solvent-free synthesis. Bacillamide analogues showed excellent to
moderate cytotoxic and anti-inflammatory activity.

ACCEPTED MANUSCRIPT
Solvent-free synthesis of bacillamide analogues as novel cytotoxic and anti-
inflammatory agents
Sunil Kumara, Ranjana Aggarwala*, Virender Kumara, Rachna Sadanab, Bhumi Patelb, Pawan
Kaushikc and Dhirender Kaushikc
aDepartment of Chemistry, Kurukshetra University, Kurukshetra-136119, India.
bDepartment of Natural Sciences, UH-Downtown, Houston TX-77002, USA.
cDepartment of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra-136119, India.
*Corresponding Author: Phone: +91(1744)238734, +919896740740; Fax: +91(1744)238277
E-mail address: ranjanaaggarwal67@gmail.com
Abstract
Synthesis of fourteen analogues of bacillamide, a bioactive tryptamide alkaloid of marine
origin, has been accomplished through a highly efficient convergent route. The present solvent-
free protocol involves the formation of thiazole ring in the initial step followed by amide
coupling between substituted ethyl 2-alkyl/aryl/heteroaryl/amino/aminoarylthiazole-4-
carboxylates and tryptamine in presence of 2-hydroxy-4,6-dimethylpyrimidine, a solid phase
catalyst to yield N-[2-(1H-indol-3-yl)ethyl]-2-alkyl/aryl/heteroaryl/amino/aminoarylthiazole-4-
carboxamides as bacillamide analogues having structural variation at position-2 of thiazole ring.
Bacillamide and its analogues were evaluated for their cytotoxic activity against three cancer cell
lines (HCT-116, MDA-MD-231 and JURKAT cell lines) using colorimetric cell proliferation
assay. Compounds 17a and 17b exhibited potent anti-cell proliferation activity with IC50 values
in the range of ~3.0 ꢀM and ~ 0.1-0.6 ꢀM, respectively against these cell lines. Preliminary
mechanism of action studies indicates that these compounds initiate caspase dependent
apoptosis. Also, compounds 16d, 16f, 17a and 17d exhibited excellent anti-inflammatory
activity comparable to well-known NSAID indomethacin and better to bacillamide, when
evaluated using carrageenan induced rat hind paw oedema method.
Keywords: Bacillamide; Tryptamide thiazole; 2-Hydroxy-4,6-dimethylpyrimidine; Solvent-free
synthesis; Cytotoxic activity; Anti-inflammatory activity.
1

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1.
Introduction
Bacillamide (1), a thiazole containing tryptamide alkaloid, was isolated from marine
bacterium, Bacillus species SY-I during termination of blooms of Cochlodinium polykrikoides by
Jeong and coworkers in 2003 [1]. Bacillamide (1), also known as bacillamide A, is the first
compound which showed excellent algicidal activity against Cochlodinium polykrikoides, one of
the most harmful dinoflagellate associated with mass mortality of fishes and great economic loss
of $95.5 million in a single year (1997) in Japanese and Korean waters [2]. Interestingly, it
neither shows algicidal activity against useful microalgae of other phyla such as diatoms, green
algae and cyanobacteria nor causes growth inhibition against bacteria, fungi and yeast. The only
major effect of bacillamide on the ultrastructure of cyanobacteria cells was the reduction and
collapse of gas vesicles and distortion of the cell shape [3]. Due to high selectivity exhibited by
1, it might become a useful algicidal agent for regulating blooms of harmful dinoflagellate
species.
Subsequently, bacillamides B (2) and C (3) were isolated from Bacillus endophyticus by
Socha and coworkers from Bahamian hypersaline microbial mat [4]. N-3′-β-Indolylethyl-2-α-
aminoethylthiazole-4-carboxamide (4), isolated from Thermoactinomyces sp. strain TM-64 in
1976, was named bacillamide D later on due to its similarity in structure with bacillamide (Fig.
1) [5].
Fig. 1. Bacillamide and its natural and synthetic analogues.
Collectively, bacillamide (1) and its closely related analogues 2-4 are referred as
bacillamides as they differ structurally for the substituent at position-2 of thiazole ring. Potential
2

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of bacillamides as biological agents have not been fully explored due to the low availability of
the compounds from natural resources. Pereira et al. synthesized several 5-substituted
bacillamides (5) which controlled the growth of particular species of cyanobacteria; in particular,
5-iodobacillamide exhibited algistatic activity against eukaryotic algae depending on its
concentration [3].
Indole alkaloids isolated from diverse marine invertebrates including coelenterates,
sponges, algae, tunicates, symbiotic bacteria, and fungi have shown different type of biological
activities such as cytotoxic, antitumor, antiviral, antihypertensive, antibacterial, and anti-
inflammatory activities [6-11]. Also, it has been noticed continuously over the years that
interesting biological activities are associated with thiazole derivatives [12-14]. Specially 2-
aminothiazoles were found in drug development for the treatment of cancer [15-16],
hypertension [17], inflammation [18-19], microbial [20] and HIV infections [21-22], and as new
inhibitors of cyclin-dependent kinase 5/p25 as a potential treatment for Alzheimer’s disease [23].
Bacillamides contain a tryptamide thiazole motif that serves as a building block in some potently
bioactive cyclic peptides, such as the antibiotic zelkovamycin (6) produced by a Streptomyces sp.
and the protein synthesis inhibitors Argyrin A and B (A-21459A and A-21459 B) (7a and 7b)
from an Actinoplanes sp. (Fig. 2) [24-25].
Fig. 2. Bioactive peptides containing a tryptamide-thiazole motif.
In view of the biological activities exhibited by tryptamide thiazole moiety it was
envisaged
in
the
present
study
to
synthesize
N-[2-(1H-indol-3-yl)ethyl]-2-
3

ACCEPTED MANUSCRIPT
alkyl/aryl/heteroaryl/amino/aminoarylthiazole-4-carboxamides (16a-g and 17a-g) which may be
considered as close analogues of bacillamide having thiazole and indole rings linked via a
peptide bond analogous to bacillamide but with a structural variation at position-2 of thiazole
ring in the form of an amine, substituted amine, alkyl, aryl or heteroaryl group through an highly
efficient, concise, solvent-free and ecofriendly route. Bacillamide A (1) and its synthetic
analogues were screened for their cytotoxic and anti-inflammatory activity.
2.
Results and Discussion
2.1
Chemistry
Figueria et al. developed a straight forward total synthesis of bacillamide in six steps
[26]. Their synthesis suffered several drawbacks. Starting from commercially available 4-
methylthiazole, the desired natural product was obtained in only about 14% overall yield.
Formation of bye products in crucial steps namely benzylic bromination and amide coupling
required tedious work up and purification procedure. But the most significant limitation to their
approach was that it does not provide scope to introduce specified structural variation in the
bacillamide structure to obtain better biological active analogues.
Recently, the use of solid phase synthesis, also referred as mechanochemistry, in organic
synthesis has gained importance. The mechanical energy generated by grinding results in the
formation of new surfaces and cracks by breaking the order of crystalline structure and this
results in the formation of products. Solid phase synthesis provides the opportunity to complete
complex reactions within limited time with significant energy saving, reduction or elimination of
by-products, and increased reaction yields, thus complying with the environmental guidelines.
We have recently reported a solid phase synthesis of 4-aryl-2-(3,5-dimethylpyrazol-1-
yl)thiazoles comprising of grinding α-bromoketones with 3,5-dimethylpyrazole-1-
thiocarboxamide in presence of sodium carbonate which not only resulted in a clean reaction
with excellent yield but also avoided the side product formation in this reaction [27]. Prompted
by these observations, it was envisaged to explore a convergent synthesis of bacillamide
analogues involving construction of thiazole ring in the initial step followed by amide bond
formation using ethylpyruvate as an easily accessible precursor (Scheme 1).
4

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Scheme
1.
Solvent-free
synthesis
of
N-[2-(1H-indol-3-yl)ethyl]-2-
alkyl/aryl/heteroaryl/amino/aminoarylthiazole-4-carboxamides (16a-g and 17a-g).
Initially, ethyl α-bromopyruvate (9) obtained by bromination of ethyl pyruvate (8) in
presence of dry CO2 [28], was ground with thioacetamide (10a) in the presence of sodium
carbonate for 5-6 minutes in a pestle mortar. Upon grinding the oily mixture became solid which
after usual work up afforded exclusively ethyl 2-methylthiazole-4-carboxylate (12a) with ~83%
5

ACCEPTED MANUSCRIPT
yield. Encouraging by the success of this reaction, differently substituted aryl thioamides 10b-g
and thioureas 11a-g were condensed with ethyl α–bromopyruvate (9) under identical conditions,
which furnished 12b-g and 13a-g in excellent yield and their formation was confirmed by
comparing their melting points with literature melting points (Table 1) [29].
Table 1 Physical characterization data of ethyl 2-alkyl/aryl/heteroaryl/amino/aminoarylthiazole-
4-carboxylates (12a-12g, 13a-13g).
S. No. Compound
Subtituent (R)
Melting Point (oC)
Obs. Lit. [Ref.]
Yield (%)
1
2
3
4
5
6
7
CH3 -
C6H5-
55-57
48-49
99-101
43-45
77-78
69-70
82-83
57 [29a]
49 [29a]
100 [29a]
44 [29a]
77 [29a]
70 [29b]
83
85
85
83
80
78
78
12a
12b
12c
12d
12e
12f
4-Cl-C6H4-
4-CH3-C6H4-
C6H5-CH2-
2-thienyl-
2-furyl-
(mp not reported)
[29d]
12g
8
H -
172-174
137-138
154-156
120-122
172-174
107-109
165-167
176 [29c]
88
85
85
80
86
80
78
13a
13b
13c
13d
13e
13f
9
C6H5-
139 [29c]
156 [29c]
122 [29c]
176 [29c]
110 [29c]
166 [29c]
10
11
12
13
14
4-Br-C6H4-
4-F-C6H4-
4-Cl-C6H4-
4-CH3-C6H4-
4-NO2-C6H4-
13g
The next challenge was the coupling reaction between ethyl thiazole-4-carboxylate (12
and 13) and tryptamine (15). A perusal of literature survey revealed that several reagents are
reported in literature for the amide bond formation between an ester and an amine e.g., titanium-
tetra-iso-propoxide and hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt),
diethyl aluminium dimethyl amide, 2-pyridone, potassium tert-butoxide, boron tribromide etc.
[30-33]. However, use of all these reagents is associated with disadvantages such as formation of
byproducts, long reaction times, harsh conditions etc. In the present work, 2-hydroxy-4,6-
dimethylpyrimidine (14), prepared by mixing alcoholic solution of acetylacetone and urea, was
6

ACCEPTED MANUSCRIPT
tried as a catalyst for amide bond formation. This reagent led to an efficient solvent-free protocol
for
the
amide
coupling
of
tryptamine
(15)
and
ethyl
2-
amino/aminoaryl/alkyl/aryl/heteroarylthiazole-4-carboxylate (12 and 13) to afford desired
bacillamide analogues (16, 17) with promising yields (~72-83%) as compared to Figueria et al’s
mixed anhydride approach (60% yield) [26] (Scheme 1). Structure of all the synthesized
compounds was established by spectral data such as IR, NMR (1H and 13C), mass and elemental
analyses.
The 1H NMR spectra of 16a-g and 17a-g displayed two characteristic signals: a triplet of
two proton intensity at δ 2.9-3.3 ppm attributed to the CH2 linked to indole ring and a double
triplet, partially overlapped, (two proton intensity) at δ 3.6-3.9 ppm attributed to the other
methylene group coupled both with the adjacent CH2 and with the NH group [1]. Compounds
16a-g and 17a-g, also exhibited characteristic singlet at 7.3-8.2 ppm, corresponding to proton at
position-5 of thiazole ring.
2.2
2.2.1 Cytotoxic Activity
Bacillamide (1) and eleven synthesized analogues (16a-g, 17a-c and 17g) were screened
Biological Evaluation
for their cytotoxicity in human colorectal tumor cell lines HCT-116, human breast
adenocarcinoma cell lines MDA-MB-231 and immune system cancer cell line (JURKAT) using
the well established MTT cell proliferation assay. Bacillamide (1) was synthesized by following
the literature procedure given by Figueria et al. [26]. The three cell lines are efficient in vitro
models which have been used in the literature for the evaluation of cytotoxic activity of different
types of chemotherapeutic agents such as DNA minor groove binders [34], protein kinase
inhibitors [35] and caspases inhibitors [36] among others. This assay was chosen as a suitable
model for the assessment of the effect on cell viability.
All the synthesized compounds were initially screened at 10 µM concentration against
these cancer cell lines (Table 2). Compounds 17a and 17b showed cytotoxic effects comparable
to commercial drug doxorubicine against the tested cancer cell lines and were further
investigated for apoptosis production and caspase assay. Compounds 17d, 17e and 17f could not
be screened for their cytotoxic activity as these compounds precipitated out on addition of buffer.
7

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Table 2 Percentage cell survival of bacillamide and its analogues at 10 µM concentration against
three cancer cell lines.
Percent Cell Survival ± S.D.*
Sr. No.
Compound
MDA-MB-231
105.1 ± 6.5
90.4 ± 11.1
88.3 ± 22.6
94.8 ± 7.2
87.3 ± 5.2
89.0 ± 9.9
85.4 ± 4.1
84.4 ± 5.9
24.8 ± 2.7
20.1 ± 5.2
41.5 ± 7.2
92.3 ± 10.2
22.1 ± 1.2
HCT-116
81.1 ± 4.5
97.0 ± 14.3
97.6 ± 8.4
99.2 ± 2.8
108.0 ± 18.3
100.7 ± 2.0
93.9 ± 6.6
96.9 ± 2.9
20.3 ± 8.2
10.3 ± 0.5
55.3 ± 5.2
72.1 ± 4.8
23.1 ± 2.3
JURKATs
85.1 ± 5.5
88.07 ± 1.7
87.3 ± 9.4
84.8 ± 3.3
88.3 ± 7.7
87.3 ± 1.7
87.7 ± 1.1
87.2 ± 1.7
42.3 ± 1.2
40.1 ± 3.2
85.5 ± 1.0
92.5 ± 6.5
18.7 ± 1.6
1
1
2
16a
3
16b
16c
4
5
16d
16e
6
7
16f
8
16g
9
17a
10
11
12
13
17b
17c
17g
Doxorubicin
*The activity data represents mean values
± SD of experiments conducted in triplicates at three independent times.
From the cytotoxic assay results, it is evident that synthetic analogues 17a and 17b,
having amino and aminophenyl group at position-2 of thiazole ring are the most potent amongst
the tested compounds. Incorporation of substituent on the phenyl ring viz. Br and NO2 (17c and
17g) decreases the cytotoxicity against the three cell lines. It is also interesting to note that
replacement of amino functionality by an alkyl and aryl group results in the sharp increase of cell
viability therefore indicating that amino group at position-2 of thiazole ring play crucial role in
imparting cytotoxicity to the molecule. For instance, at 10 ꢀM concentration, compounds 17a (R
= NH2) and 17b (R = NHC6H5) show only 20.3 and 10.3% cell survival against HCT-116 cell
line and 24.8 and 20.1%, respectively against MDA-MB-231 cell lines survival viability,
respectively comparable to commercial drug doxorubicin with percentage cell survival 22.1%.
The marine natural product, bacillamide showed poor cytotoxicity against these cell lines (Table
2).
8

ACCEPTED MANUSCRIPT
Compounds 17a and 17b with less than 50% cell survival were chosen as lead
compounds and further investigated for their IC50 values (Table 3). Compound 17a was
moderately active against MDA-MB-231 and Jurkat cancer cell line with an IC50 value of 3.0
µM. Compound 17b exhibited excellent cytotoxicity against HCT-116 (IC50 = 0.10 µM), MDA-
MB-231 (IC50 = 0.60 µM), and Jurkat cancer (IC50 = 0.50 µM) cell lines.
Table 3 IC50 values of bacillamide analogues against three cancer cell lines.
Compound
IC50 value (in µM) against cancer cell line
MDA-MB-231
HCT-116
5.40 ± 1.2
0.10 ± 0.02
0.33 ± 0.11
JURKAT
3.00 ± 0.20
0.60 ± 0.03
0.45 ± 0.25
3.00 ± 0.32
0.50 ± 0.02
0.23 ± 0.12
17a
17b
Doxo
Compounds 17a and 17b were selected to study if these compounds caused cell death via
apoptosis. Caspase 3/7 activation is one of the hallmarks of apoptotic cell death. Camptothecin
was used a positive control.
6
5
4
3
2
1
0
Control
17a
17b
Camptothecin
Fig. 3. Caspase activation in JURKAT cells treated with bacillamide analogues (Data represents
mean values ± SD of experiments conducted in duplicates at two independent times).
As shown in Fig. 3, compounds 17a and 17b caused ~4-5 fold increase in fluorescence
respectively indicative of caspase 3/7 activation. This data suggests that these compounds kill
cancer cells via apoptosis induction.
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To further investigate if cells maintained cell membrane integrity upon treatment with
17a and 17b, Lacate dehydrogenase (LDH) assay was performed. As shown in Fig. 4, 17a and
17b did not cause cell lysis so no LDH activity was found. LDH was used a positive control.
Fig. 4. LDH activity in JURKAT cells treated with bacillamide analogues and cells (control cells
without any treatment) using LDH cytotoxicity kit from Cayman chemicals (Data represents
mean values ± SD of experiments conducted in duplicates at two independent times).
In nutshell, bacillamide analogues 17a and 17b having amino and aminophenyl group at
position-2 of thiazole, respectively possess comparable potencies to that of the clinically
available anticancer therapeutic drug, doxorubicine. On the contrary, at the same concentration,
the bacillamide (1) and its synthetic analogues 16a-16g and 17g having substituted
aminophenyl/methyl/aryl/heteroaryl group showed almost nil cytotoxicity against these cell lines
and 17c showed cell survival viability about 41.5% indicating that its ability to generate scarce
toxicity in cancer cells (Table 3).
2.2.2 Anti-inflammatory activity
Bacillamide (1) and its analogues (16a-g and 17a-g) were tested for their anti-
inflammatory activity using carrageenan induced rat hind paw oedema method of Winter et al.
[38] The results of activity are compiled in Table 4.
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Table 4 Percent anti-inflammatory activity of test compounds (Carrageenan-induced paw
oedema test in rats).
S. No. Compound
Mean value of oedema volume (% inhibition)
1 hour
2 hour
3 hour
4 hour
1.
2.
Control
1
0.71 ±0.08
0.30±0.03**
(58)
1.21±0.10
0.53±0.08**
(56)
1.36±0.15
0.74±0.10
(46)
1.25±0.09
0.72±0.8
(42)
3.
4.
0.26±0.14**
(63)
0.32±0.13
0.48±0.20**
(60)
0.46±0.27**
0.76±0.10**
(44)
1.02±0.34
0.62±0.08*
(50)
0.54±0.13**
16a
16b
(55)
(62)
(25)
(57)
5.
6.
7.
8.
0.23±0.14**
(68)
0.13±0.06**
(81)
0.17±0.12**
(76)
0.13±0.06**
(82)
0.45±0.05**
(63)
0.28±0.21**
(77)
0.52±0.11**
(57)
0.29±0.15**
(76)
0.52±0.14*
(62)
0.59±0.23*
(57)
0.75±0.08
(45)
0.57±0.25*
(58)
0.55±0.12**
(56)
0.38±0.15**
(70)
0.72±0.08
(42)
0.47±0.21**
(62)
16c
16d
16e
16f
9.
0.18±0.10**
(75)
0.10±0.02**
0.38±0.12
(69)
0.32±0.07**
0.70±0.23**
(49)
0.63±0.10**
0.68±0.15**
(46)
0.23±0.09**
16g
17a
10.
(86)
(74)
(54)
(82)
11.
12.
13.
0.16±0.04**
(77)
0.55±0.04
(23)
0.12±0.01**
(83)
0.33±0.05**
(73)
0.56±0.29*
(54)
0.49±0.07**
(60)
0.48±0.11**
(65)
0.70±0.11**
(49)
0.56±0.11**
(59)
0.27±0.01**
(78)
0.68±0.16**
(46)
0.72±0.09**
(42)
17b
17c
17d
14.
15.
0.36±0.06**
(49)
0.34±0.06**
0.50±0.10**
(59)
0.52±0.20*
0.68±0.11**
(50)
0.73±0.11**
0.74±0.09**
(41)
0.78±0.13*
17e
17f
(52)
(57)
(46)
(38)
16.
17.
0.24±0.02**
(66)
0.04 ±0.02** 0.19±0.03**
(94) (84)
0.50±0.12**
(59)
0.59±0.10*
(57)
0.26±0.02**
(81)
0.63±0.04**
(50)
0.17±0.03**
(86)
17g
Indomethacin
(Standard)
Significantly different compared to respective control values, **p < 0.01, *p < 0.05.
aAll values are expressed as mean+SEM of five rats in each group analysed by Anova followed by Dunnett’s ‘t’ test.
11

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All the tested compounds exhibited anti-inflammatory activity over a wide range (23-
92%). Among these compounds four compounds 16d (p-tolyl), 16f (2-thienyl), 17a (NH2), and
17d (p-fluoro aminophenyl) were found to exhibit excellent anti-inflammatory activity. Whereas,
bacillamide does not shows any promising activity. The percentage inhibition of 16d, 16f, 17a
and 17d are 81%, 82%, 86% and 83%, respectively in one hour which is close to standard
reference drug i.e. indomethacin (94%). This data promises that these compounds may act as fast
acting anti-inflammatory agents. Compounds 16d, 17a and 17b remain active even after four
hours with percentage inhibition 70%, 82% and 78% respectively. Compounds 16c (68%), 16e
(76%), 16g (75%), 17b (77%), and 17g (66%) also exhibit good anti-inflammatory activity.
3.
Conclusion
In conclusion, we have developed an efficient, concise and ecofriendly route for the
synthesis
of
N-[2-(1H-indol-3-yl)ethyl]-2-aryl/alkyl/aminoaryl/heteroarylthiazole-4-
carboxamides (bacillamide analogues) via a two-step solvent free synthesis. Synthesized
compounds 17a and 17b exhibit excellent cytotoxic activity against three cancer cell lines (HCT-
116, MDA-MD-231 and JURKATs cell lines) as compared to commercial drug doxorubicin.
Four compounds 16d, 16f, 17a, and 17d showed excellent anti-inflammatory activity using
carrageenan induced rat hind paw oedema method.
4.
Experimental Section
4.1
General Chemistry
Thiourea, thioacetamide and ethyl pyruvate were purchased from Spectrochem Pvt. Ltd.
Aryl thioureas and thioamides were prepared by already reported procedures [39-40]. Melting
points were determined in open capillaries in electrical apparatus and are uncorrected. IR spectra
1
13
were recorded on a Buck Scientific IR M500 instrument. The H and C NMR spectra were
recorded on a Bruker instrument at 300 MHz and 75 MHz, respectively. Elemental analysis was
performed at NIPER (Mohali), Chandigarh, India. The compounds gave satisfactory analytical
results (within ± 0.3 of the calculated values). Mass spectra were measured in EI mode on a
Kratos MS-50 spectrometer at MS Facilities at SAIF, Panjab University, Chandigarh, India.
4.2
General procedure for the preparation of ethyl 2-methylthiazole-4-carboxylate (12a)
12

ACCEPTED MANUSCRIPT
An oily mixture of ethyl α-bromopyruvate (9, 2.14 g, 11 mmol) and thioacetamide (10a,
0.75 g, 10 mmol) was ground in presence of Na2CO3 (0.50 g) for 5-6 min. When it becomes
solid, the progress of reaction was monitored with TLC. On completion of the reaction, water (20
ml) was added to the reaction followed by extraction with chloroform (20 ml). The organic layer
thus separated was dried over anhy. Na2SO4. Excess of solvent was removed by distillation.
Filtered the crude product and crystallized from aqueous ethanol (83%) to give pure solid 12a.
Similarly, 12b-12g and 13a-13g were prepared following the above procedure and their
formation was confirmed by comparing their melting points with literature values (Table 1).
o
Ethyl 2-(2-furyl)thiazole-4-carboxylate (12g): mp 82-83 C [29d (mp not reported)]; yield 78%;
1
IR (KBr, cm-1): 1727 (CO); H NMR (300 MHz, CDCl3) δ: 1.47 (t, 3H, J = 7.2 Hz, CH3), 4.42
(q, 2H, J = 7.2 Hz, CH2), 7.15-7.10 (m, 1H, furan 4-H), 7.22 (d, 1H, J = 5.1 Hz, furan 5-H), 7.43
(d, 1H, J = 4.8 Hz, furan 3-H), 8.20 (s, 1H, thiazole 5-H).
4.3
General procedure for the synthesis of N-(2-(1H-indol-3-yl)ethyl)-2-substituted
aminothiazole-4-carboxamides (16 and 17)
Tryptamine (15, 1.76 g, 11 mmol), 2-hydroxy-4,6-dimethylpyrimidine (14, 1.24 g, 10
mmol) and appropriate ethyl 2-alkyl/aryl/heteroaryl/aminoarylthiazole-4-carboxylate (12/13, 10
mmol) were homogenised thoroughly in a pestle mortar. The contents were transferred to a
o
round bottom flask and heated to 100-105 C for 5-6 hours. After completion of the reaction, as
indicated by TLC, water (25 ml) was added. The organic compound was extracted with
chloroform. The organic layer was washed thoroughly with 10% HCl solution (2×25 ml) and
dried over anhy. Na2SO4. The reaction mixture was concentrated and the crude product so
obtained was crystallized from ethanol.
TLC Solvent System: 25% Ethyl acetate in Hexane (for all bacillamide analogues).
o
4.3.1 N-(2-(1H-indol-3-yl)ethyl)-2-methylthiazole-4-carboxamide (16a): mp 124-126 C; IR
1
(KBr, cm-1): 3271 (NH), 1659 (CO); H NMR (300 MHz, DMSO-d6+CDCl3, δ): 2.60 (s, 3H,
CH3), 3.14 (t, yield 78%; 2H, J = 6.9 Hz, CH2-indole), 3.81 (dt, 2H, J = 6.9 Hz, 6.6 Hz, CH2-
NH), 7.09 (s, 1H, indole 2-H), 7.14-7.22 (m, 2H, indole 5,6-H), 7.38 (d, 1H, J = 8.1 Hz, indole 7-
H), 7.51 (s, 1H, NH, exchangeable with D2O); 7.66 (d, 1H, J = 7.5 Hz, indole 4-H), 7.96 (s, 1H,
thiazole 5-H), 8.18 (bs, 1H, NH, exchangeable with D2O); 13C NMR (75 MHz, DMSO-
13

ACCEPTED MANUSCRIPT
d6+CDCl3) δ: 18.93, 25.47, 40.24, 111.34, 112.07, 118.45, 118.72, 121.42, 122.29, 122.45,
127.22, 136.49, 149.73, 160.99, 165.75, Anal. Calcd. for C15H15N3OS: C, 63.13; H, 5.30; N,
14.73; Found: C, 63.31; H, 5.45; N, 14.82. MS (ESI, m/z): 286.1 [M+H]+; Calcd. 285.09.
4.3.2 N-(2-(1H-indol-3-yl)ethyl-2-phenylthiazole-4-carboxamide (16b): mp 144-145 °C; Yield
1
80%. IR (KBr, cm-1): 3333 (NH), 1659 (CONH); H NMR (300 MHz, DMSO-d6+CDCl3) δ:
3.15 (t, 2H, J = 6.9 Hz, CH2-indole), 3.85 (dt, 2H, J = 6.3 Hz, 6.9 Hz, CH2-NH), 7.28-7.13 (m,
4H, indole 2,5,6-H), 7.47-7.41 (m, 4H, phenyl 2,3,5,6-H). 7.73 (d, 1H, J = 8.4 Hz, indole 7-H),
7.64 (s, 1H, NH, exchangeable with D2O), 7.89 (m, 2H, indole 4-H, phenyl 4-H), 8.10 (s, 1H,
thiazole 5-H), 8.13 (bs, 1H, exchangeable with D2O, NH); 13C NMR (75 MHz, DMSO-
d6+CDCl3) δ: 30.20, 43.99, 116.31, 116.80, 123.26, 123.63, 126.22, 127.33, 131.12, 131.35,
133.79, 134.14, 135.35, 137.46, 141.37, 155.70, 165.73, 172.64. Elemental analysis: Calcd. for
C20H17N3OS: C, 69.14; H, 4.93; N, 12.09; Found: C, 69.22; H, 4.98; N, 12.21. MS (ESI, m/z):
348.1 [M+H]+; Calcd. 347.10.
4.3.3 N-(2-(1H-indol-3-yl)ethyl]-2-(4-chlorophenyl)thiazole-4-carboxamide (16c): mp 162-164
1
°C; Yield 76%; IR (KBr, cm-1): 3109 (NH), 1659 (CO); H NMR (300 MHz, CDCl3) δ: 3.14 (t,
2H, J = 6.6 Hz, CH2-indole), 3.85 (dt, 2H, J = 6.3 Hz, 6.6 Hz, CH2-NH), 7.12 (s, 1H, indole 2-
H), 7.28-7.15 (m, 2H, indole 5,6-H), 7.43 (d, 2H, J = 7.5 Hz, Ph 2,6-H), 7.60 (s, 1H, NH,
exchangeable with D2O), 7.72 (d, 1H, J = 7.8 Hz, indole 4-H), 7.80 (d, 2H, J = 7.8 Hz, Ph 3,5-
H), 7.97 (d, 1H, J = 8.4 Hz, indole 7-H), 8.10 (s, 1H, thiazole 5-H), 8.25 (bs, 1H, exchangeable
13
with D2O, NH); C NMR (75 MHz, CDCl3) δ: 25.23, 39.92, 111.25, 113.12, 119.57, 122.11,
122.16, 122.90, 123.26, 127.82, 128.71, 129.24, 129.63, 131.31, 136.59, 151.08, 160.99, 166.64.
Elemental analysis: Calcd. for C20H16ClN3OS: C, 62.90; H, 4.22; N, 11.00; Found: C, 62.98; H,
4.36; N, 11.14. MS (ESI, m/z): 382.1 [M+H]+ ; Calcd. 381.07.
4.3.4 N-(2-(1H-indol-3-yl)ethyl)-2-(4-methylphenyl)thiazole-4-carboxamide (16d): mp 182-183
1
°C; Yield 83%; IR (KBr, cm-1): 3330 (NH), 1659 (CO); H NMR (300 MHz, CDCl3) δ: 2.42 (s,
3H, 4-CH3) 3.15 (t, 2H, J = 6.9 Hz, CH2-indole), 3.84 (dt, 2H, J = 6.9 Hz, 6.6Hz, CH2-NH), 7.14
(s, 1H, indole 2-H), 7.27-7.16 (m, 4H, indole 5,6-H, Ph 2,6-H), 7.43 (d, 1H, J = 8.1 Hz, indole 7-
H), 7.62 (s, 1H, NH, exchangeable with D2O) 7.89-7.78 (m, 3H, indole 4-H, Ph 3,5-H), 8.07 (s,
13
1H, thiazole 5-H), 8.14 (bs, 1H, exchangeable with D2O, NH). C NMR (75 MHz, CDCl3) δ:
14

ACCEPTED MANUSCRIPT
21.39, 25.33, 40.05, 111.16, 112.90, 118.74, 119.48, 122.01, 126.56, 127.41 127.83, 129.65,
130.17, 136.30, 140.96, 150.29, 161.65, 168.30. Elemental analysis: Calcd. for C21H19N3OS: C,
69.78; H, 5.30; N, 11.63; Found: C, 69.92; H, 5.44; N, 11.78. MS (ESI, m/z): 362.1 [M+H]+;
Calcd. 361.12.
4.3.5 N-(2-(1H-indol-3-yl)ethyl)-2-benzylthiazole-4-carboxamide (16e): mp 140-141 °C; Yield
83%; IR (KBr, cm-1): 3323 (NH), 1666 (CO); 1H NMR (300 MHz, CDCl3) δ: 3.14 (t, 2H, J = 6.9
Hz, CH2-indole), 3.81 (dt, 2H, J = 6.3 Hz, 6.9 Hz¸ CH2-NH), 4.27 (s, 2H, -CH2-Ph ), 7.09 (s,
1H, indole 2-H), 7.26-7.12 (m, 2H, indole 5,6-H), 7.42-7.30 (m, 6H, Ph 2,3,4,5,6-H, indole 7-H),
7.52 (s, 1H, NH, exchangeable with D2O), 7.70 (d, 1H, J = 7.5 Hz, indole 4-H), 7.99 (s, 1H,
13
thiazole 5-H), 8.11 (s, 1H, exchangeable with D2O, NH); C NMR (75 MHz, CDCl3) δ: 25.48,
39.08, 40.55, 111.37, 112.16, 118.50, 118.81, 121.50, 122.33, 122.90, 127.20, 127.26, 128.77,
128.87, 136.52, 137.15, 149.88, 170.17, 161.03. Elemental analysis: Calcd. for C21H19N3OS: C,
69.78; H, 5.30; N, 11.63; Found: C, 69.88; H, 5.39; N, 11.82. MS (ESI, m/z): 384.1 [M+Na]+;
Calcd. 361.12.
4.3.6 N-(2-(1H-indol-3-yl)ethyl)-2-(2-thienyl)thiazole-4-carboxamide (16f): mp 168-170 °C;
1
Yield 72%; IR (KBr, cm-1): 3337 (NH), 1659 (CO). H NMR (300 MHz, DMSO-d6) δ: 2.91 (t,
2H, J = 6.9 Hz, -CH2-indole), 3.58 (dt, 2H, J = 6.6 Hz, CH2-NH), 6.37 (m, 1H, thiophene 2-H),
6.78 (ds, 1H, J = 3.0 Hz, indole 2-H), 6.99-6.85 (m, 3H, thiophene 3-H, indole 5,6-H), 7.22 (d,
1H, indole 4-H), 7.36 (m, 1H, thiophene 4-H), 7.47-7.44 (m, 2H, indole 7-H and NH,
exchangeable with D2O) 7.85 (s, 1H, thiazole 5-H), 9.67 (bs, 1H, exchangeable with D2O, NH);
13C NMR (75 MHz, DMSO-d6) δ: 25.72, 40.03, 111.85, 112.21, 118.72, 118.87, 121.43, 123.09,
127.43, 127.73, 128.69, 128.95, 130.05, 136.18, 136.78, 150.90, 160.58, 161.54. Elemental
analysis: Calcd. for C18H15N3OS2: C, 61.16; H, 4.28; N, 11.89; Found: C, 61.08; H, 4.20; N,
11.74. MS (ESI, m/z): 354.0 [M+H]+; Calcd. 353.06.
4.3.7 N-(2-(1H-indol-3-yl)ethyl)-2-(2-furyl)thiazole-4-carboxamide (16g): mp 192-193 °C; Yield
76%; IR (KBr, cm-1): 3387 (NH), 1651 (CO); 1H NMR (300 MHz, DMSO-d6) δ: 3.15 (t, 2H, J =
7.2 Hz, -CH2-indole), 3.82 (dt, 2H, J = 6.6, 6.9 Hz, CH2-NH), 6.62 (d, 1H, J = 3.3 Hz, furan 3-
H), 7.10-7.24 (m, 3H, indole 5,6-H, furan 4-H), 7.44 (d, 1H, J = 8.1 Hz, indole 7-H), 7.47 (s, 1H,
NH, exchangeable with D2O); 7.53 (d, 1H, J = 3.3 Hz, furan 5-H), 7.61 (s, 1H, indole 2-H), 7.72
15

ACCEPTED MANUSCRIPT
(d, 1H, J = 7.5 Hz, indole 4-H), 8.2 (s, 1H, thiazole 5-H), 9.9 (bs, 1H, exchangeable with D2O,
13
NH). C NMR (75 MHz, DMSO-d6) δ: 25.72, 40.85, 110.59, 111.85, 112.19, 113.20, 118.70,
118.86, 121.42, 123.09, 123.50, 127.74, 136.77, 145.71, 148.07, 151.40, 157.73, 160.63.
Elemental analysis: Calcd. for C18H15N3O2S: C, 64.08; H, 4.48; N, 12.45; Found: C, 64.12; H,
54.3; N, 12.57. MS (ESI, m/z): 338.3 [M+H]+; Calcd. 337.08.
o
4.3.8 N-(2-(1H-indol-3-yl)ethyl)-2-aminothiazole-4-carboxamide (17a): mp 142-143 C; yield
1
77%; IR (KBr); 3394 (NH), 3317 (NH), 3271 (NH), 1651 (CO) cm-1; H NMR (300 MHz,
CDCl3, δ): 3.34 (t, 2H, J=6.9 Hz, CH2-In), 3.95 (dt, 2H, J=6.9Hz, 6.6 Hz, CH2-NH), 6.98-7.17
(m, 3H, indole 2,5-H and NH (exchangeable with D2O)), 7.41 (m, 1H, indole 6-H), 7.76-7.69
(m, 2H, indole 7-H and thiazole 5-H), 7.83 (d, 1H, J=7.5 Hz, indole 4-H), 8.17 (s, 1H, NH,
exchangeable with D2O), 8.27 (s, 2H, NH, exchangeable with D2O). 13C NMR (75 MHz, CDCl3)
δ: 25.81, 64.70, 111.64, 112.31, 118.64, 118.29, 119.23, 121.38, 121.56, 127.24, 137.64, 146.24,
160.24, 165.42. Anal. Calcd. for C14H14N4OS: C, 58.72; H, 4.93; N, 19.57; Found: C, 58.70; H,
5.01; N, 19.54. MS (ESI, m/z): 287.1 [M+H]+; Calcd. 286.08.
o
4.3.9 N-(2-(1H-indol-3-yl)ethyl)-2-aminophenylthiazole-4-carboxamide (17b): mp 144-145 C;
1
yield 80%; IR (KBr): 3371(NH), 3317 (NH), 3279 (NH), 1612(CO) cm-1; H-NMR (300 MHz,
DMSO-d6, δ): 2.98 (t, 2H, J=6.9 Hz, CH2-In), 3.58 (dt, 2H, J=6.6Hz, 6.9 Hz, CH2-NH), 7.00 (t,
1H, J=7.2 Hz, indole 5-H), 7.11-7.13 (m, 2H, indole 2,6-H), 7.23-7.19 (m, 3H, Ph 2,4,6-H), 7.36
(d, 1H, J =8.1 Hz, indole 7-H), 7.48 (s, 1H, NH, exchangeable with D2O), 7.64 (d, 2H, J=7.8 Hz,
13
Ph 3,5-H), 7.71 (s, 1H, thiazole 5-H), 8.11 (s, 1H, NH, exchangeable with D2O); C NMR (75
MHz, DMSO-d6) δ: 25.74, 65.39, 111.84, 112.14, 115.80, 118.71, 118.89, 119.02, 119.11,
121.46, 123.10, 127.67, 137.72, 137.96, 146.47, 161.00, 163.56. Anal. Calcd. for C20H18N4OS:
C, 66.28; H, 5.01; N, 15.46; Found: C, 66.20; H, 5.07; N, 15.52. MS (ESI, m/z): 363.2 [M+H]+;
Calcd. 362.12.
4.3.10 N-(2-(1H-indol-3-yl)ethyl)-2-(4-bromophenyl)aminothiazole-4-carboxamide (17c): mp
140-141 oC; yield 80%; IR (KBr): 3370(NH), 3317 (NH), 1612 (CO) cm-1; 1H-NMR (300 MHz,
CDCl3, δ): 2.99 (t, 2H, J=6.9 Hz, CH2-In), 3.59 (dt, 2H, J=6.3 Hz, 6.0 Hz, CH2-NH), 7.00-7.09
(m, 3H, indole 5,6-H, NH (exchangeable with D2O)), 7.22 (s, 1H, indole 2-H), 7.32 (d, 2H, J=8.7
Hz, Ph 2,6-H), 7.36 (d, 1H, J=7.8 Hz, indole 4-H), 7.46 (d, 1H, J = 8.7 Hz, indole 7-H), 7.52 (s,
16

ACCEPTED MANUSCRIPT
1H, thiazole 5-H), 7.66 (d, 2H, J=8.4 Hz, Ph 3,5-H), 8.09 (s, 1H, NH, exchangeable with D2O);
13C NMR (75 MHz, CDCl3) δ: 25.40, 65.32, 110.68, 112.43, 119.43, 119.64, 120.09, 121.78,
124.87, 125.38, 128.55, 130.18, 130.92, 138.68, 139.15, 149.22, 163.40, 164.37. Anal. Calcd. for
C20H17BrN4OS: C, 54.43; H, 3.88; N, 12.69; Found: C, 54.45; H, 3.97; N, 12.62. MS (ESI, m/z):
441.1 [M+H]+; Calcd. 440.03.
4.3.11 N-(2-(1H-indol-3-yl)ethyl)-2-(4-fluorophenyl)aminothiazole-4-carboxamide (17d): mp
135-136 oC; yield 76%; IR (KBr): 3371 (NH), 3317 (NH), 1612(CO) cm-1; 1H-NMR (300 MHz,
DMSO-d6, δ): 3.10 (t, 2H, J=6.9 Hz, CH2-In), 3.73 (dt, 2H, J=6.3Hz, 6.0 Hz, CH2-NH), 7.05-
7.11 (m, 3H, indole 5,6-H and NH (exchangeable with D2O)), 7.14 (s, 1H, indole 2-H) 7.38 (d,
2H, J=7.8 Hz, Ph 2,6-H), 7.46-7.53 (m, 3H, indole 4,7-H and NH (exchangeable with D2O)),
7.62 (s, 1H, thiazole 5-H), 7.65 (d, 2H, J=7.8 Hz, Ph 3,5-H), 8.08 (s, 1H, NH, exchangeable with
13
D2O); C NMR (75 MHz, DMSO-d6) δ: 25.14, 65.81, 110.36, 113.77, 115.41, 118.24, 118.46,
119.14, 121.32, 124.67, 128.83, 128.89, 131.54, 136.64, 138.42, 147.92, 162.34, 165.22. Anal.
Calcd. for C20H17FN4OS: C, 63.14; H, 4.50; N, 14.73; Found: C, 63.12; H, 4.46; N, 14.62. MS
(ESI, m/z): 381.1 [M+H]+; Calcd. 380.11.
4.3.12 N-(2-(1H-indol-3-yl)ethyl)-2-(4-chlorophenyl)aminothiazole-4-carboxamide (17e): mp
148-149 oC; yield 80%; IR (KBr): 3371 (NH), 3317 (NH), 1612(CO) cm-1; 1H-NMR (300 MHz,
DMSO-d6+CDCl3, δ): 2.99 (t, 2H, J=7.2 Hz, CH2-In), 3.62 (dt, 2H, J=6.9 Hz, 6.6 Hz, CH2-NH),
6.95-7.10 (m, 2H, indole 5,6-H), 7.13 (s, 1H, indole 2-H), 7.23 (d, 1H, J=6.6 Hz, indole 4-H),
7.37 (m, 3H, indole 7-H, Ph 2,6-H), 7.48 (s, 1H, thiazole 5-H), 7.71-7.61 (m, 4H, Ph 3,5-H and
2NH (exchangeable with D2O)), 8.09 (s, 1H, NH, exchangeable with D2O); 13C NMR (75 MHz,
DMSO-d6+CDCl3) δ: 29.04, 64.03, 115.24, 115.97, 122.32, 122.98, 125.60, 126.33, 127.18,
130.49, 131.22, 132.34, 132.89, 134.54, 136.54, 140.44, 154.03, 165.74. Anal. Calcd. for
C20H17ClN4OS: C, 60.52; H, 4.32; N, 14.12; Found: C, 60.58; H, 4.27; N, 14.08. MS (ESI, m/z):
397.1 [M+H]+; Calcd. 396.08.
4.3.13 N-(2-(1H-indol-3-yl)ethyl)-2-(4-methylphenyl)aminothiazole-4-carboxamide (17f) [41]:
o
1
mp 138-139 C; yield 72%; IR (KBr): 3371 (NH), 3317 (NH), 1612(CO) cm-1 ; H-NMR (300
MHz, DMSO-d6, δ): 2.35 (s, 3H, CH3), 2.95 (t, 2H, J=6.6 Hz, CH2-In), 3.68 (dt, 2H, J=6.3 Hz,
6.0 Hz, CH2-NH), 7.16-7.11 (m, 3H, indole 2,5,6-H), 7.32 (d, 2H, J = 8.7 Hz, Ph 2,6-H), 7.36 (d,
17

ACCEPTED MANUSCRIPT
1H, J = 8.7 Hz, indole 4-H ), 7.44-7.40 (m, 2H, indole 7-H, Ph 3,5-H, 2NH (exchangeable with
D2O)), 7.50 (s, 1H, thiazole 5-H), 8.14 (s, 1H, NH, exchangeable with D2O); 13C NMR (75 MHz,
DMSO-d6) δ: 21.45, 25.71, 39.41, 111.85, 112.20, 118.72, 118.84, 121.43, 123.10, 126.84,
127.74, 128.94, 130.23, 130.34, 136.73, 141.15, 151.17, 160.82, 167.67. Anal. Calcd. for
C21H20N4OS: C, 67.00; H, 5.35; N, 14.88; Found: C, 66.92; H, 5.37; N, 14.76. MS (ESI, m/z):
377.0 [M+H]+; Calcd. 376.13.
4.3.14 N-(2-(1H-indol-3-yl)ethyl)-2-(4-nitrophenyl)aminothiazole-4-carboxamide (17g): mp 157-
o
1
158 C; yield 75%; IR (KBr): 3385 (NH), 3322 (NH), 1612 (CO) cm-1 ; H-NMR (300 MHz,
DMSO-d6, δ): 2.94 (t, 2H, J=6.6 Hz, CH2-In), 3.76 (dt, 2H, J=6.3 Hz, 6.0 Hz, CH2-NH), 7.16-
7.07 (m, 3H, indole 2,5,6-H), 7.81-7.64 (m, 4H, indole 4,7-H and 2 NH (exchangeable with
D2O)), 8.19 (d, 2H, J=8.4 Hz, Ph 2,6-H), 8.28-8.25 (m, 3H, Ph 3,5-H, 1 NH, exchangeable with
D2O), 8.31 (s, 1H, thiazole 5-H); 13C NMR (75 MHz, DMSO-d6) δ: 25.86, 63.34, 113.80,
113.89, 118.87, 119.15, 124.58, 125.25, 125.60, 125.65, 127.46, 129.22, 140.11, 140.32, 141.90,
146.34, 161.04, 163.23. Anal. Calcd. for C20H17N5O3S: C, 58.96; H, 4.21; N, 17.19; Found: C,
59.04; H, 4.27; N, 17.20. MS (ESI, m/z): 408.2 [M+H]+; Calcd. 407.10.
4.4
Biological Assays
4.4.1 Cytotoxic activity
4.4.1.1 Cell Culture
HCT-116 (colon cancer cell line) and MDA-MB-231 (breast cancer cell line) were
cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal serum
albumin and 50 µg/mL of penicillin and streptomycin. JURKATs (leukemia cell line) were
cultured in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% fetal
serum albumin and 50 µg/mL of penicillin and streptomycin. All cell lines were maintained in an
incubator containing 5% CO2 at 37 °C.
4.4.1.2 Cell Viability Assay
Cells were seeded in a 96-well plate at a density of 75000 per mL and grown overnight.
Cells were treated with various compounds at a final concentration of 10 µM and incubated for
48 hours. Cell viability assay was performed using a MTT cell proliferation kit from ATCC
(American Type Culture Collection) (#30-1010K). In summary, 10 µL MTT reagent was added
18

ACCEPTED MANUSCRIPT
to each well, and cells were placed back in incubator for 4 hours. 100 µL of detergent (from kit)
was added and absorbance data was collected at 570 nm using Biotek synergy 2
spectrophotometer,
Data was calculated as percentage of cell survival using the following formula:
% Cell survival = (100/At * As)
Where At and As are the absorbance of wells treated with test compounds and solvent control
respectively.
4.4.1.3 Apoptotis Induction and Caspase Assay
100,000 JURKAT cells were plated in a 24 well plate and treated with 1µM of different
compounds for 48hours. 100 µL sample was mixed with equal amount of substrate reagent and
analyzed as per kit (Promega G7790). Fluorescence for the samples was measured at 0 min and
180 minutes as apoptosis induction. Camptothecin was used as a positive control for inducing
apoptotic cell death.
4.4.2 Anti-inflammatory activity
Bacillamide (1) and its analogues (16a-g and 17a-g) were tested for their anti-
inflammatory activity using carrageenan induced rat hind paw oedema method of Winter et al.
[38]. Compounds were administered orally at the dose of 50 mg/kg body weight to the test
animals 30 min prior to induction of inflammation by carrageenan injection. Control animals
were treated with 5% (v/v) aqueous Tween-80 solution. Same Tween-80 solution was used as
vehicle for standard and test drugs. The oedema in the hind paw was induced by injection of 0.1
mL of 1% carrageenan solution into sub-planter region of left hind paw. The volume of the paw
was measured plethysmographically immediately and 1 h, 2 h, 3 h and 4 h after the injection of
the irritant. The difference in volume gave the amount of oedema developed. Percent inhibition
of the oedema between control group and the compound treated group was calculated and
compared with the group receiving standard drug. Protocol of animal experiments has been
approved by the Institutional Animal Ethics Committee (IAEC). The results of activity are
compiled in Table 4.
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Statistical analysis: In this study, data is expressed as Mean+SEM. Differences between vehicle
control and treatment groups were tested using one-way ANOVA followed by Dunnet’s ‘t’ test.
A probability value less than 0.01 was considered as statistically significant.
Acknowledgements
Authors are thankful to UGC, New Delhi, India for providing financial assistance in the
form of UGC Major Research Project (F.No. 33-300/2007). Thanks are due to National Institute
of Pharmaceutical Education and Research, Mohali, Chandigarh and Sophisticated Analytical
Instrumentation Facility, Panjab University, Chandigarh, India for providing elemental analyses
and mass spectra, respectively. Thanks are also due to Linus Klingler, Department of Natural
Sciences, UH-Downtown, Houston TX-77002, USA for carrying out LDH activity. We would like to
thank University of Houston-Downtown for providing organized research and creative activities
(ORCA) funds to Dr. Rachna Sadana.
Appendix A. Supplementary data
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Research Highlights
•
•
•
•
Bacillamide analogues (14 examples) were synthesized via two-step solvent-free method.
2-Hydroxy-4,6-dimethylpyrimidine was used for amide bond formation.
Compounds 17a and 17b exhibited excellent cytotoxic activity.
Compounds 16d, 16f, 17a and 17d exhibited excellent anti-inflammatory activity.