Synthesis of novel benzo[4,5]thiazolo[1,2-a]pyrimidine-3-carboxylate derivatives and biological evaluation as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2013.08.024

A novel series of building blocks consisting of benzo[4,5]thiazolo[1,2-a] pyrimidine-3-carboxylate have been synthesized as potential anticancer compounds. These compounds were prepared from 2-aminobenzothiazole, benzaldehyde and ethyl acetoacetate in eth

Full text of DOI:10.1016/j.ejmech.2013.08.024

Accepted Manuscript
Synthesis of Novel Benzo[4,5]thiazolo[1,2-a]pyrimidine-3-carboxylate Derivatives and
Biological Evaluation as Potential Anticancer Agents
Lingaiah Nagarapu, Satheeshvarma Vanaparthi, Rajashaker Bantu, C. Ganesh
Kumar
PII:
S0223-5234(13)00530-8
10.1016/j.ejmech.2013.08.024
EJMECH 6369
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 May 2013
Revised Date: 13 August 2013
Accepted Date: 14 August 2013
Please cite this article as: L. Nagarapu, S. Vanaparthi, R. Bantu, C Ganesh Kumar , Synthesis
of Novel Benzo[4,5]thiazolo[1,2-a]pyrimidine-3-carboxylate Derivatives and Biological Evaluation
as Potential Anticancer Agents, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.08.024.
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Graphical Abstract
Synthesis of Novel Benzo[4,5]thiazolo[1,2-a]pyrimidine-
Leave this area blank for abstract info.
3-carboxylate Derivatives and Biological Evaluation as
Potential Anticancer Agents
Lingaiah Nagarapua*, Satheeshvarma Vanaparthia, Rajashaker Bantua, Ganesh Kumar Cb
A novel series of building blocks consisting of benzo[4,5]thiazolo[1,2-a]pyrimidine-3-carboxylate as
potential tumor growth inhibitors were synthesized and screened for their cytotoxicity against the human
cancer cell lines in vitro.

ACCEPTED MANUSCRIPT
Synthesis of Novel Benzo[4,5]thiazolo[1,2-a]pyrimidine-3-
carboxylate Derivatives and Biological Evaluation as Potential
Anticancer Agents
Lingaiah Nagarapua*, Satheeshvarma Vanaparthia, Rajashaker Bantua, Ganesh
Kumar Cb
aOrganic Chemistry Division II, Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500007, India
(CSIR). Tel.: +91-40-27191509/10/11; Fax: +91-40-27193382; E-mail:lnagarapuiict@yahoo.com
bCentre for Medicinal Chemistry & Pharmacology, Indian Institute of Chemical Technology,Tarnanka,
Hyderabad-500007, India (CSIR).
ABSTRACT
A novel series of building blocks consisting of benzo[4,5]thiazolo[1,2-a]pyrimidine-3-
carboxylate have been synthesized as potential anticancer compounds. These compounds
were prepared from 2-aminobenzothiazole, benzaldehyde and ethyl acetoacetate in ethylene
glycol by catalyzing with TBAHS to give benzo[4,5]thiazo[1,2-a]pyrimidine derivative 4
followed by the formation of amide by reaction with several secondary amines in good
yields. The cytotoxicity of these compounds was evaluated against human cancer cell lines
in vitro (A549, HeLa, MDA-MB-231 and MCF-7). Compound 5b exhibited promising
cytotoxicity with IC50 values of 0.58 and 1.58 ꢀM specifically against human breast
adenocarcinoma cell lines, MCF-7 and MDA-MB-231, while compound 5a showed
promising cytotoxicity against MDA-MB-231 (IC50 value of 5.01 ꢀM).
Keywords: Pyrimidine-3-carboxylate, Cytotoxicity, Growth inhibition, Lung Carcinoma
A549 cell line, Human breast adenocarcinoma cell.
_________________________________________________________________________
1. Introduction
The treatment of tumour therapy remains an important and challenging therapeutic
problem; the majority of the solid tumours are currently not curable by chemotherapy. Half
of all cancer patients fail to respond to chemotherapy or relapse from the initial response
and ultimately die from their metastatic disease [1]. The discovery and development of new,
more active, more selective, and less toxic compounds for the treatment of malignancy are
one of the most important goals in medicinal chemistry. In recent years, interest in the
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development of new anticancer drugs increased mainly from emerging resistance against
drugs. The development of potential drugs has efficiently improved therapeutic index and
tumour growth inhibition. The aim of most cancer chemotherapeutic drugs currently in
clinical use is to kill malignant tumour cells by inhibiting some of the mechanisms implied
in cellular division. Early approaches of selectively inhibit tumour growth were generally
disappointing in clinical studies. The investigation of tumour growth inhibitors is a major
obstacle in the medical field [2]. For these reasons, the developments of novel antitumour
drugs and their derivatives is still necessary and have very much demand.
Pyrimidines have a long distinguished history extending from the days of their discovery
as important constituents of nucleic acids to their current use in the chemotherapy of AIDS.
The pyrimidine ring is found in vitamins like thiamine, riboflavin and folic acid. During the
last two decades, several pyrimidine derivatives have been developed as chemotherapeutic
agents and have found wide clinical applications. Benzothiazoles represent a class of
heterocyclic compounds of great importance in biological chemistry. They exist in many
condensed fused systems that were found to possess a wide range of activity [3-5]. Various
literature reports display numerous fused pyrimidine ring systems and their
chemotherapeutic activities as anticancer [6-8], antibacterial [9] and antiviral [10] agents.
Since the two heterocyclic moieties, benzothiazoles and pyrimidines, constitute two active
pharmacophores that are highly active against antitumour and antimicrobial, combining the
two is expected to have a synergistic effect on their biological properties. Also substituted
thiazolopyrimidine ring systems were reported to possess antitumour activity [11]. The
reported significance of such synthons generated the interest to exploit this valuable
structure in the designing and the synthesis of new benzo[4,5]thiazolo[1,2-a]pyrimidine-3-
carboxylate derivatives.
Our on-going research programme [12-16], is to discover and develop tumour growth
inhibitors and apoptosis inducers as potential new anti cancer agents, we obtained designed
and synthesized novel benzo[4,5]thiazolo[1,2-a]pyrimidine-3-carboxylate derivatives which
has antitumour activity. Analysis of structure-activity relationships identified the
benzothiazole and pyrimidines nucleus as being essential for potent activity, and
substitution at the carboxylic acid 3’-position of the benzothiazopyrimidine ring when
condensed with methyl piperazine and piperidine moieties increases the cytotoxicity activity
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against human breast cancer cell lines MDA-MB-231 and MCF-7 more efficiently, when
compare to the secondary amines which shows moderate activity with the above cell line in
vitro. When chain length was increased with the condensation of β-alanine at the third
position of benzothiazopyrimidine ring, decreases the cytotoxicity compare to piperidine
and methylpiperazine moieties 5a and 5b. Significantly, (2-methyl-4-phenyl-4H-
benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)(piperidine-1-yl) methanone (5a), and (2-methyl-4-
phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)(4-methylpiperazin-1-yl)methanone
(5b) outperformed their best cytotoxicity activity against breast cancer cell lines in the IC50
range of 0.58-5.08 µM.
2. Results and discussion
2.1 Chemistry
2.1 Synthesis of [4,5]thiazo[1,2-a]pyrimidine
The key intermediate used for the synthesis of final compounds of both the series was 2-
methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylic acid 4, which in turn
was prepared by one-pot condensation of 2-aminobenzothiazole (1) with substituted
benzaldehyde (2) and ethyl acetoacetate (3) in the presence of ethylene glycol by catalyzing
with TBAHS to give crude compound which was hydrolyzed using KOH, ethanol refluxed
to get the targeted compound, 2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-
3-carboxylic acid (4), in quantitative yield (80%) (Scheme 1). Formation of compound 4
was confirmed by the presence of C=N stretching at 1542 and C-S-C stretching band at
1254 cm-1 in IR spectrum.
<<< Scheme 1. >>>
Treatment of pyrimidine-3-carboxylic acid 4 with 2o-amines in the presence of HOBt,
EDC.HCl and triethylamine at room temperature gave the (2-methyl-4-phenyl-4H-
benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)(piperidin-1-yl)methanone derivatives (5a-e) in
good to excellent yields (Scheme 2). Structure of 5a was elucidated on the basis of
stretching band at 1634 cm-1 of C=O.
In
another
reaction,
ethyl-3-(2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-
a]pyrimidine-3-carboxamido)propanoate (6) was obtained in good yield by the condensation
of pyrimidine-3-carboxylic acid (4) with β-alanine ethyl ester in the presence of HOBt,
EDC.HCl and triethylamine and the compound 6 was confirmed based on appearance of
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bands at 1642, 1623 cm-1 for C=O and ester carbonyl stretching in IR spectrum. The H
NMR spectra showed the characteristic ester signals at δ 1.22 as triplet representing three
hydrogens and δ 3.42-3.52 as a multiplet representing for two protons of ester –CH2. Upon
hydrolyzing the compound
6
was converted to 3-(2-methyl-4-phenyl-4H-
using KOH,
benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxamido)propanoic acid (7)
ethanol at reflux temperature. Treatment of compound 7 with piperidine in the presence of
HOBt, EDC.HCl and triethylamine at room temperature gave the targeted methyl-N-(3-
oxo-3-(piperidin-1-yl)propyl)-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-
carboxamide (8a) in good yield. The formation of the compound 8a was evident from mass
spectrum [ES+] by the appearance of molecular ion peak at m/z 461, IR spectrum shows
1
bands at 1613 and 1632 cm-1 which are attributed to C=O stretching, H NMR by the
appearance of piperidine protons at δ 1.41-1.71 as a multiplet representing for six protons
and δ 3.38-3.57 as multiplet representing for four protons. The synthetic strategy to prepare
the targeted compounds 5a-e and 8a-e is illustrated in Scheme 2 and 3.
<<< Scheme 2. >>>
<<< Scheme 3. >>>
In our studies we have planned to synthesize a new analogues of benzo[4,5]thiazo[1,2-
a]pyrimidine through a one pot-three component reaction of 2-aminobenzothiazole,
benzaldehyde and ethyl acetoacetate in ethylene glycol by catalyzing with TBAHS to give
benzo[4,5]thiazo[1,2-a]pyrimidine derivative which was hydrolyzed by using KOH, ethanol
at reflux conditions to give corresponding carboxylic acid 4. The required
benzothiazopyrimidine derivatives 5a-e was achieved by the reaction between carboxylic
acid 4 and a variety of 2o-amines. In the next step, acid 4 was also reacted with β-alanine
ethyl ester to give the corresponding ester 6. The ester was hydrolyzed with KOH, ethanol
reflux to give corresponding acid 7, which subsequently reacted with 2o-amine to give the
desired product 8. Here we have to study the structure-activity relationship between these
two types of compounds. The synthesized compounds were evaluated for anti-cancer
activity against different human cancer cell lines in vitro.
2.2 Effects of the compounds on the viability of human cancer cells
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The newly synthesized benzo[4,5]thiazolo[1,2-a]pyrimidine-3-carboxylate derivatives
(5a-e) were further evaluated for their in vitro cytotoxicity against breast adenocarcinoma
(MDA-MB-231, MCF-7), cervical cancer (HeLa), and alveolar adenocarcinoma (A549) cell
lines using the standard MTT assay[17]. From the data reported in Table 1, most of the
prepared compounds possessed good cytotoxicity effect on all the tested cell lines and
potencies of some compounds were comparable to the standard doxorubicin. The
compounds 5a, 5b and 5c showed promising inhibitory activity against human breast
adenocarcinoma cell lines, MCF-7 and MDA-MB-231, with IC50 values ranging from 0.58
µM-11.74 µM, while compounds 5d and 5e showed moderate inhibitory activities.
Compounds 8a and 8e exhibited good cytotoxicity against all the tested cell lines with IC50
values ranging from 11.18 µM-25.69 µM.
It has been observed from activity Table 1, that the compound with methyl piperazine
and piperidine moieties attached to 2-Methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-
a]pyrimidine-3-carboxylic acid are showed higher cytotoxicity as compared with other
2o amines. For example, compound 5b bearing methyl piperazine moiety showed most
potent cytotoxicity in human breast adenocarcinoma cell lines MDA-MB-231 and MCF-
7with the IC50 values 1.58 µM and 0.58µ M. The compound 5a bearing piperidine moiety
showed most potent cytotoxicity in human breast adenocarcinoma cell line MDA-MB-231
with the IC50 value 5.01µM. The compounds 5c-5e is showing moderate potent cytotoxicity
against human breast adenocarcinoma cell lines. The compounds 8a and 8e showed
moderate cytotoxicity against four cancer cell lines HeLa, MDA-MB-231, MCF-7, and
A549 which are Human cervical cancer cell line, Human breast adenocarcinoma cell line,
and Human alveolar adenocarcinoma cell line respectively. The results indicated that the
attachment of methyl piperazine and piperidine ring with the basic moiety of 2-methyl-4-
phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylic acid (4) facilitated the
increase of their antitumour activity.
<<<<<<Table 1>>>>>>
3. Conclusion
In conclusion, various substituted (2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-
a]pyrimidin-3-yl)(piperidin-1-yl)methanone derivatives (5a-e) and 2-methyl-N-(3-oxo-3-
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(piperidin-1-yl)propyl)-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxamide
(8a-e) have been prepared from 2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-
a]pyrimidine-3-carboxylic acid (4) and screened for in vitro cytotoxicity activity against
A549, HeLa, MDA-MB-231 and MCF-7 cell lines. In general, most compounds showed
significant cytotoxicity effect against human breast adenocarcinoma MDA-MB-231 and
MCF-7 tumour cell lines, altogether compounds 5a and 5b are effective in suppressing the
MDA-MB 231 and MCF-7 cell growth. The findings suggested that these compounds would
be very useful for further investigation of the mechanism of cell proliferation, morphology,
differentiation and apoptosis in our next research project and some of these may be lead
compounds for cancer research.
4. Experimental section
4.1. Chemistry
All commercial reagents and solvents were used as received without further purification
unless specified reaction solvents were used for distillation. The reactions were monitored
and Rf value were determined using analytical thin layer chromatography (TLC) with Merck
Silica gel 60 and F254 precoated plates (0.25 mm thickness). Spot on the TLC plates were
visualized using ultraviolet light (254 nm). Flash column chromatography was performed
with Merck silica gel 60 (100-200 mesh). Melting points were determined in capillaries and
1
are uncorrected. H NMR spectra were recorded on Bruker DRX-300, Varian 400 and
Varian-500 NMR spectrometers. 13C NMR spectra’s were recorded on Bruker DRX-300.
Proton chemical shifts are reported in ppm(δ) relative to internal tetramethylsilane (TMS, δ
0.00 or with the solvent reference relative to TMS employed as the internal standard
(CDCl3, δ 7.26 ppm; DMSO-d6 δ 2.54) and multiplicities of NMR signals are designated as
s (singlet), d (doublet), t (triplet), q (quartet), br (broad), m (multiplet, for unresolved lines),
etc. Infrared (IR) spectra were recorded on a Perkin Elmer FT-IR 400 spectrometer; data is
reported in wave numbers (cm-1). Mass spectra were recorded on Agilent Technologies
1100 Series (Agilent Chemistation Software). High-resolution mass spectra (HRMS) were
obtained by using ESI-QTOF mass spectrometry.
4.2 Synthesis of 2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylic
acid (4)
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2-Aminobenzothiazole (1 mmol), benzaldehyde (2 mmol), and ethyl acetoacetate (2
mmol) in ethylene glycol (5 mL) were mixed in a flask, and TBAHS (30 mol %) was added
at room temperature. The resulting mixture was heated at 120 oC for 1hr, and completion of
the reaction was monitored by TLC. After completion of the reaction, the reaction mixture
was allowed to cool, and poured into water (50 mL) and stirred for 15 min, the solid
separated was filtered through Buckner funnel and washed with water (2 x 10 mL) and dried
over reduced pressure to obtain the crude compound. The above crude compound was
dissolved in ethanol (10 mL), added 1N potassium hydroxide solution (10 mL) and the
reaction mixture was refluxed for 2 hrs, and completion of the reaction was monitored by
TLC. After completion of the reaction, the reaction mixture was allowed to cool, and
neutralized with diluted hydrochloric acid and stirred for 15 min, the solid separated was
filtered through Buckner funnel washed with water (2 x 5 mL), dried over reduced pressure.
The above crude product was recrystallized from ethanol to obtain the title compound 4 as a
o
light yellow solid (80 % yield). m.p: 168-172 C; IR (KBr, v): 3469, 3094, 2822, 1675,
1542, 1456, 1313, 1254, 753, 703 cm-1: 1H NMR (500 MHz, CDCl3+DMSO-d6): δ 2.48 (s,
3H, CH3), 6.54 (s, 1H, CH), 7.21-7.39 (m,4H, Ar-H), 7.41- 7.44 (d, 2H, Ar-H), 7.65 (m, 3H,
Ar-H). 13C NMR(75 MHz, CDCl3+ DMSO-d6 ): 17.1, 57.1, 103.9, 112.7, 122.4, 123.1,
125.1, 125.9, 126.8, 127.6, 127.8, 134.5, 137.3, 159.8, 164.5; MS (ESI): m/z 323 (M+H)+;
HRMS: cacld for C18H15O2N2S: 323.08487, Found 323.08423.
4.3 General procedure for the synthesis of compounds (5a-e)
Compound 4 (1mmol) was dissolved in freshly distilled dichloromethane under
nitrogen atmosphere .Then HOBt (1.1 mmol), EDC,HCl (1.1 mmol) were added to it. The
reaction mixture was stirred for 5 minutes, and piperidine (1.2 mmol) was added slowly
followed by the addition of triethylamine (2 mmol) and stirred for 2 hrs at room
temperature. The progress of the reaction was monitored by TLC. After completion of the
reaction, water (10 mL) was added to the reaction mixture and extracted with
dichloromethane. Organic layer was collected and dried over anhydrous Na2SO4. Solvent
was removed under reduced pressure and the resultant compound was recrystallized with
ether to afford 5a as light brown solid with 79 % yield.
4.3.1.(2-Methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)(piperidine-1-yl)
methanone (5a): Brown Solid(79% yield): m.p: 154-158 oC; IR (KBr, v): 3537, 3374, 3285,
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2934, 2850, 1665, 1634, 1584, 1543, 1467, 1274, 1262, 1207, 1023, 756, 747 cm-1: H
NMR (500 MHz, CDCl3): δ 1.08-1.57 (m, 6H, piperidine), 1.84 (s, 3H, CH3), 2.91-3.02 (m,
4H, piperidine), 6.35 (s, 1H, CH), 6.62 (d, J =7.41 Hz,1H, Ar-H), 7.01-7.07 (m, 2H, Ar-H),
7.21-7.24 (m, 2H, Ar-H), 7.29 (m, 3H, Ar-H), 7.37 (d, J =7.41 Hz,1H, Ar-H). 13C NMR (75
MHz, CDCl3): δ 20.5, 24.1, 25.2, 42.4, 47.2, 60.1, 107.4, 111.4, 121.6, 123.1, 126.1, 128.3,
129.1, 136.5, 138.3, 140.1, 160.4, 168.1(CO): MS (ESI): m/z 390 (M+H)+; HRMS: cacld for
C23H24ON3S: 390.16346, Found 390.16272.
4.3.2.(2-Methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)(4-methylpiperazin-
1-yl)methanone (5b): White solid (75 % yield ): m.p: 176-178 oC; IR (KBr, v): 3430, 2928,
2846, 2785, 1600, 1541, 1458, 1430, 1384, 1259, 1206, 1115, 746 cm-1: 1H NMR (300
MHz, CDCl3+DMSO-d6): δ 1.81 (s, 3H, CH3), 2.03 (s, 3H, CH3), 2.41 (m 2H, piperazine),
2.88-3.09 (m, 4H, piperazine), 3.95 (m, 2H, piperazine), 6.30 (s, 1H, CH), 6.74 (d, J =7.08
Hz, 1H, Ar-H), 7.10 (m, 2H,Ar-H), 7.26-7.35 (m, 5H, Ar-H), 7.51 (d, J =6.90 Hz, 1H, Ar-
H). 13C NMR (75 MHz, CDCl3+DMSO-d6 ): δ 20.0, 45.1, 53.4, 59.1, 77.7, 78.1, 78.3, 78.5,
106.7, 110.9, 121.5, 121.9, 122.8, 125.6, 125.8, 128.0, 128.7, 137.6, 139.5, 159.2, 167.0;
MS (ESI): m/z 405 (M+H)+; HRMS: cacld for C23H25ON4S: 405.17436, Found 405.17416.
4.3.3.(2-Methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)(morpholino)
methanone (5c): Light brown solid (83 % yield ): m.p: 218-222 oC; IR (KBr, v): 3435, 3045,
2970, 2910, 2853, 1636, 1611, 1550, 1454, 1423, 1319, 1276, 1204, 1109, 1001, 748, 740
cm-1: 1H NMR (300 MHz, CDCl3): δ 1.84 (s, 3H, CH3), 2.89-2.94 (m, 1H, morpholine), 3.0-
3.22 (m, 4H,morpholine), 3.59-3.64 (m, 1H, morpholine), 4.0-4.1(m, 2H, morpholine),
6.34(s, 1H, CH), 6.63 (d, J = 9.06 Hz, 1H, Ar-H), 7.03-7.07 (m, 2H, Ar-H), 7.29-7.40 (m,
4H, Ar-H), 7.51 (m, 1H, Ar-H), 7.69 (m, 1H, Ar-H). MS (ESI): m/z 392 (M+H)+; HRMS:
cacld for C22H22O2N3S: 392.14272, Found 351.14246
4.3.4.(2-Methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)(4-phenylpiperazin-
1-yl)methanone (5d): White solid (82 % yield ): m.p: 194-198 oC; IR (KBr, v): 3435, 3066,
1
2917, 1635, 1597, 1561, 1466, 1431, 1334, 1234, 1203, 1003, 740 cm-1: H NMR (300
MHz, CDCl3): δ δ 1.88 (s, 3H, CH3), 2.59-2.68 (m, 1H, piperazine), 2.74-2.80 (m, 1H,
piperazine), 3.10-3.31 (m, 5H, piperazine), 4.18 (m, 1H, piperazine), 6.37 (s, 1H, CH),
6.61-6.70 (m, 3H, Ar-H), 6.82 (t, J =7.55 Hz, 1H, Ar-H), 7.02-7.07 (m, 2H, Ar-H), 7.15-
7.24 (m, 3H, Ar-H), 7.29-7.31 (m, 4H, J = Ar-H), 7.36-7.40 (m, 1H, Ar-H). 13C NMR (75
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MHz, CDCl3): δ 20.6, 29.6, 48.7, 49.5, 60.0, 106.8, 111.4, 116.6, 120.4, 121.7, 122.8, 123.3,
126.0, 126.2, 128.6, 129.0, 129.2, 138.2, 140.1, 150.7, 160.8, 168.3; MS (ESI): m/z 467
(M+H)+; HRMS: cacld for C28H27ON4S: 467.19001, Found 467.18756.
4.3.5.(2-Methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)(4-(4-nitrophenyl)
piperazin-1-yl)methanone (5e): Yellow solid (76.1 % yield): m.p: 222-226 oC; IR (KBr, v):
3434, 2918, 1634, 1597, 1542, 1398, 1320, 1305, 1278, 1114, 997, 822, 743 cm-1; 1H NMR
(500 MHz, CDCl3): δ 1.90 (s, 3H, CH3), 2.92 (m, 1H, piperazine), 3.09 (m, 1H, piperazine),
3.22-3.27 (m, 3H, piperazine), 3.46 (m, 1H, piperazine), 3.55 (m, 1H, piperazine), 4.25 (m,
1H, piperazine), 6.40 (s, 1H, CH), 6.66 (d, J = 8.99Hz, 3H, Ar-H), 7.07-7.12 (m, 2H, Ar-H),
7.21-7.26 (m, 1H, Ar-H), 7.29-7.34 (m, 4H, Ar-H), 7.41-7.43 (m, 1H, Ar-H), 8.09( d, J
13
=8.99,2H, Ar-H). C NMR (75 MHz, CDCl3): δ 20.7, 45.4, 46.5, 46.7, 60.0, 106.4, 111.5,
112.9, 121.8, 122.8, 123.5, 125.8, 126.0, 126.3, 128.7, 129.3, 138.0, 138.9, 140.3, 154.1,
161.1, 168.6; MS (ESI): m/z 512 (M+H)+; HRMS: cacld for C28H26O3N5S: 512.17509,
Found 512.17334.
4.4 Synthesis of ethyl 3-(2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-
carboxamido)propanoate (6)
Compound 4 (1mmol) was dissolved in freshly distilled dichloromethane (5 mL)
under nitrogen atmosphere. Then HOBt (1.1 mmol), EDC, HCl (1.1mmol) were added to it.
The reaction mixture was stirred for 5 minutes, and β-alanine ethyl ester hydrochloride (1.5
mmol) was added slowly, followed by the addition of triethylamine (2 mmol) and stirred for
2 h at room temperature. The progress of the reaction was monitored by TLC. After
completion of the reaction, water (10 mL) was added to the reaction mixture and extracted
with dichloromethane. Organic layer was collected and dried over anhydrous Na2SO4.
Solvent was removed under reduced pressure and the resultant compound was recrystallized
with ether to afford the title compound 6 as pale brown solid with 62% yield. m.p: 148-151
oC; IR (KBr, v): 3235, 3044, 2989, 2939, 1728, 1642, 1623, 1583, 1536, 1473, 1334, 1324,
1
1296, 1208, 1190, 1154, 1027, 753, 693 cm-1: H NMR (300 MHz, CDCl3): δ 1.22 (t, J
=7.55, 6.79 Hz,3H, CH3), 2.12 (s, 3H, CH3), 2.39-2.52 (m, 2H, COCH2), 3.42-3.52 (m, 2H,
CH2), 4.00-4.13 (m, 2H, NHCH2), 5.96 (bt, 1H, NH), 6.40 (s, 1H, CH), 6.83-6.86 (d, J
=7.55 Hz, 1H, Ar-H), 7.04-7.32 (m, 6H, Ar-H), 7.38-7.40 (d, J = 7.55 Hz, 2H, Ar-H). 13C
NMR (75 MHz, CDCl3): δ 14.0, 21.7, 33.7, 34.7, 58.4, 60.5, 109.5, 111.4, 121.7, 123.0,
9

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123.3, 126.2, 128.1, 128.8, 138.1, 140.5, 141.3, 168.0, 172.3; MS (ESI): m/z 422 (M+H)+;
HRMS: cacld for C23H24O3N3S: 422.15329, Found 422.15244.
4.5
Synthesis
of
3-(2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-
carboxamido)propanoic acid (7)
The compound 6 (1.2 mmol) was dissolved in ethanol (10 mL), added 1N potassium
hydroxide solution (10 mL) and the reaction mixture was refluxed for 1 hr, and completion
of the reaction was monitored by TLC. After completion of the reaction, the reaction
mixture was allowed to cool to room temperature, neutralized with diluted hydrochloric acid
and the solid separated was filtered through Buckner funnel and washed with water and
dried over reduced pressure to obtain the crude compound. The above crude product was
recrystallized from ethanol to obtain the title compound 7 as a light green solid (65 % yield).
o
m.p: 142-146 C; IR (KBr, v): 3318, 2924, 2852, 1624, 1526, 1456, 1259, 1206, 1122, 749,
1
699 cm-1: H NMR (300MHz, DMSO-d6): δ 2.08 (s, 3H, CH3), 2.37-2.42 (m, 2H, COCH2),
3.42-3.44 (m, 2H, NHCH2), 6.47 (s, 1H, CH), 6.92-6.95 (d, J =7.93 Hz, 1H, Ar-H), 7.07-
7.31 (m, 7H, Ar-H), 7.50 (d, J =7.17Hz, 1H, Ar-H), 7.56 (broad, NH). 13C NMR (75 MHz,
DMSO-d6): δ 21.3, 34.7, 35.7, 57.2, 109.5, 111.4, 122.1, 122.5, 123.3, 126.1, 126.4, 128,
128.7, 137.8, 140.6, 141.4,159.5,166.6; MS (ESI): m/z 394 (M+H)+; HRMS: cacld for
C21H20O3N3S: 394.12199, Found 394.12210.
4.6 General procedure for the synthesis of compounds (8a-e)
Compound 7 (1 mmol) was dissolved in freshly distilled dichloromethane (5 mL)
under nitrogen atmosphere. Then HOBt (1.1 mmol), EDC, HCl (1.1 mmol) were added to it.
The reaction mixture was stirred for 5 minutes, piperidine (1.2 mmol) was added in a slow
stream and followed by the addition of triethylamine (2 mmol) and the reaction mixture was
stirred for 1 hr at room temperature. The progress of the reaction was monitored by TLC.
After completion of the reaction, water (10 mL) was added to the reaction mixture and
extracted with dichloromethane. Organic layer was collected and dried over anhydrous
Na2SO4. Solvent was removed under reduced pressure and the resultant compound was
recrystallized with ether to afford 8a as brick red solid with 81% yield.
4.6.1.2-Methyl-N-(3-oxo-3-(piperidin-1-yl)propyl)-4-phenyl-4H-benzo[4,5]thiazolo[3,2-
o
a]pyrimidine-3-carboxamide(8a): Brick red solid (81% yield): m.p: 152-155 C; IR (KBr,
1
v): 3276, 2936, 2856, 1632, 1613, 1541, 1471, 1326, 1208, 1026, 829, 743, 699 cm-1: H
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NMR (500 MHz, CDCl3): δ 1.41-1.71(m, 6H, piperidine), 2.14 (s, 3H, CH3), 2.25-2.30 (m,
1H, COCH2), 2.42-2.47 (m, 1H, COCH2), 3.26-3.31(m, 2H, CH2), 3.38-3.57 (m, 4H,
piperidine), 6.34 (bt, 1H, NH), 6.40 (s, 1H, CH), 6.84-6.87 (d,J =7.34 Hz, 1H, Ar-H), 7.04-
7.07 (t, J =7.34 Hz, 1H, Ar-H), 7.10-7.14 (t, J =8.39, 7.34 Hz, 1H, Ar-H), 7.17-7.20 (m,
1H, Ar-H), 7.23-7.25 (m, 2H, Ar-H), 7.32-7.34 ( d, J=6.29 Hz, 2H, Ar-H), 7.37-7.39 (d, J
=7.34 Hz,1H,Ar-H). 13C NMR (75 MHz, CDCl3): δ 21.8, 24.4, 25.4, 26.2, 32.7, 35.0, 42.6,
46.2, 58.6, 109.6, 111.4, 121.8, 123.3, 126.2, 126.5, 128.1, 128.7, 138.3, 140.7, 161.1,
167.9(CO), 169.4(CO); MS (ESI): m/z 461 (M+H)+; HRMS: cacld for C26H29O2N4S:
461.20057, Found 461.19897.
4.6.2.2-Methyl-N-(3-(4-methylpiperazin-1-yl)-3-oxopropyl)-4-phenyl-4H-benzo[4,5]thiazolo
[3,2-a]pyrimidine-3-carboxamide (8b): White solid (71% yield): m.p: 162-166 oC; IR (KBr,
1
v): 3435, 2932, 1646, 1540, 1444, 1208, 746, 702 cm-1: H NMR (300 MHz, DMSO-d6): δ
2.11(s, 3H,CH3), 2.29(s, 3H, CH3), 2.30-2.48(m,4H, COCH2 & piperazine), 3.38-3.63 (m,
8H, NHCH2 & piperazine), 6.42 (s, 1H, CH), 6.89 (d, J = 7.36 Hz, 1H, Ar-H), 7.06-7.35 (m,
7H, Ar-H), 7.44 (d, J = 7.55 Hz, 1H, Ar-H), 7.60 (bt, 1H, NH). MS (ESI): m/z 476 (M+H)+;
HRMS: cacld for C26H30O2N5S: 476.21147, Found 476.21155, cacld for C26H29O2N5NaS:
498.19342, Found 498.19351:
4.6.3.2-Methyl-N-(3-morpholino-3-oxopropyl)-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyri
o
midine-3-carboxamide (8c): Pale brown solid (75 % yield): m.p: 198-202 C; IR (KBr, v):
1
3435, 3263, 3062, 1646, 1542, 1470, 1332, 1209, 1111, 745 cm-1: H NMR (300 MHz,
CDCl3): δ 2.14 (s, 3H, CH3), 2.28 (m, 1H, COCH2), 2.43 (m, 1H, COCH2), 3.31-3.39 (m,
2H, morpholine), 3.44-3.55 (m, 2H, CH2), 3.56-3.67 (m, 6H, morpholine), 6.27 (t,1H, NH),
6.41 (s, 1H, CH), 6.87 (d, J =7.55Hz,1H, Ar-H), 7.04-7.17 (m, 2H, Ar-H), 7.20-7.24 (m,
2H, Ar-H), 7.28-7.36 (m, 3H, Ar-H), 7.40 (d,J =7.55Hz,1H, Ar-H). 13C NMR (75 MHz,
CDCl3): δ 21.9, 32.5, 34.8, 41.7, 45.4, 58.5, 66.3, 66.7, 111.4, 121.8, 123.3, 126.2, 126.5,
128.1, 128.6, 163.3, 168.0; MS (ESI): m/z 463 (M+H)+; HRMS: cacld for C25H27O3N4S:
463.17984, Found 463.17868.
4.6.4.2-Methyl-N-(3-oxo-3-(4-phenylpiperazin-1-yl)propyl)-4-phenyl-4H-benzo[4,5]thiazolo
o
[3,2-a]pyrimidine-3-carboxamide (8d): White solid (82 % yield): m.p:173-176 C; IR
1
(KBr, v): 3401, 2922, 1637, 1599, 1502, 1467, 1332, 1231, 1026, 753 cm-1: H NMR (300
MHz, CDCl3): δ 2.15 (s, 3H,CH3), 2.29-2.38 (m, 1H, COCH2), 2.47-2.56 (m,1H, CH2),
11

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3.08-3.18 (m, 4H, piperazine), 3.47-3.60 (m, 4H, piperazine), 3.62-3.66 (m, 1H, CH2),
3.72-3.78 (m, 1H, CH2), 6.31(bt, 1H, NH), 6.41(s, 1H, CH), 6.85-6.94 (m, 4H, Ar-H), 7.04-
7.41 (m, 10H, Ar-H). MS (ESI): m/z 538 (M+H)+; HRMS: cacld for C31H32O2N5S:
538.22712, Found 538.22583.
4.6.5.2-Methyl-N-(3-(4-(4-nitrophenyl)piperazin-1-yl)-3-oxopropyl)-4-phenyl-4H-benzo
[4,5]thiazolo[3,2-a]pyrimidine-3-carboxamide (8e): Yellow solid (89 % yield): m.p: 166-
o
168 C; IR (KBr, v): 3435, 2919, 1657, 1631, 1596, 1542, 1327, 1238, 1208, 1116, 1026,
830, 753, 699 cm-1: 1H NMR (500 MHz, CDCl3+DMSO-d6): δ 2.13 (s, 3H, CH3), 2.41-2.56
(m, 2H, COCH2), 3.45-3.52 (m, 6H, piperazine), 3.62 (m, 2H,CH2), 3.73 (m, 2H,
piperazine), 6.44 (s, 1H, CH), 6.87-6.93 (m, 3H, Ar-H), 7.05 (bt, 1H, NH), 7.09 (t, 1H, Ar-
H), 7.15-7.19 (q, 2H, Ar-H), 7.24-7.27 (t, 2H, Ar-H), 7.31-7.33 (d, J =7.41 Hz, 2H, Ar-H),
7.44 (d, J =8.47 Hz,1H, ArH), 7.63 (s, 2H, Ar-H). 13C NMR (75 MHz, CDCl3+ DMSO-d6):
δ 21.3, 32.1, 34.6, 43.8, 46.0, 57.8, 108.8, 110.8, 112.2, 121.4, 122.4, 122.9, 125.3, 125.9,
127.6, 128.1, 137.6, 138.1, 140.1, 141.2, 153.7, 160.4, 167.4(CO), 169.4(CO); MS (ESI):
m/z 583 (M+H)+; HRMS: cacld for C31H31O4N6S: 583.21220, Found 583.21167.
In vitro cytotoxicity testing
Cytotoxicity of the synthesized compounds was assessed on the basis of the
measurement of the in vitro growth in the 96 well plates by cell-mediated reduction of
tetrazolium salt to water insoluble formazan crystals by a previously described method [17].
Cell lines for testing in vitro cytotoxicity included HeLa derived from human cervical
cancer cells (ATCC No. CCL-2), A549 derived from human alveolar adenocarcinoma
epithelial cells (ATCC No. CCL-185), MDA-MB-231 derived from human breast
adenocarcinoma cells (ATCC No. HTB-26) and MCF7 derived from human breast
adenocarcinoma cells (ATCC No. HTB-22) were obtained from American Type Culture
Collection, Manassas, VA, USA. Doxorubicin was used as positive control for comparison
purpose. Dose-response curves were plotted for the test compounds and controls after
correction by subtracting the background absorbance from that of the blanks. The antitumor
potency of the compounds indicated by IC50 values (50% inhibitory concentration) were
calculated from the plotted absorbance data for the dose-response curves. IC50 values (in
µM) are expressed as the average of two independent experiments.
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References and notes
[1] M. R. Grever, B. A. Chabner, M, Cancer Principles and Practise of Oncology (1997)
385.
[2] R. H. Bradury (Ed.), Cancer in “Topics in Medicinal Chemistry”, Vol. 1, Springer-
Verlag, berlin Heidelberg, 2007.
[3] T. D. Badshaw, S. Wrigley, D. F. Shi, R. J. Schultz, K. D. Paull, M. F. G. Stevens, Br.
J. Cancer (1998) 745.
[4] S.Salamone, C.Colin, I. G. Vuissoz, S.Kuntz, S.Mazerbourg, S. Flament, H.Martin, L.
Richert, Y.Chapleur, M.Boisbrun. Eur. J. Med. Chem.51 (2012) 206.
[5] L.T.Pierce, M. M. Cahill, H. J. Winfield, F. O. McCarthy. Eur. J. Med. Chem.56
(2012) 292.
[6] C. O. Kappe, W. M. F. Fabian, M. A. Semones, Tetrahedron. 53 (2010) 2803.
[7] R.Determann. J.Dreher, K.Baumann, L.Preu, P.G.Jones, F.Totzke, C.Schachtele,
M.H.G. Kubbutat, C.Kunick. Eur. J. Med. Chem. 53 (2012) 254.
[8] P. M. Lukasik, S. Elabar, F. Lam, H. Shao, X. Liu, A. Y. Abbas, S. Wang, Eur. J.
Med. Chem. 57 (2012) 311.
[9] K. S. Alwal, B. N. Swanson, S. E. Unger, D. M. Floyd, S. Moreland, A. Hedberg, B.
C. O’Reilly, J. Med. Chem. 34 (1991) 806.
[10] A. Ozair, A. K. Suroor, S. Nadeem, A. Waquar, Med. Chem. Res. 19 (2010) 1245.
[11] F. M. Salwa, M. F. Eman, E. A. Abd El-Galil, D. N. Abd El Shafy, Eur. J. Med.
Chem.45 (2010) 1494.
[12] L. Nagarapu, H. K. Gaikwad, K. Sarikonda, J. Mateti, R. Bantu, P.S. Raghu, K.M.
Mahduri, S.V. Kalivendi, Eur. J. Med. Chem. 46 (2010) 4720-4725.
[13] L. Nagarapu, J. Mateti, H. K. Gaikwad, R. Bantu, M. Sheebarani, N. J. P. Subhashini.
Bioorg. Med. Chem Lett. 21 (2011) 4138.
[14] L. Nagarapu, H. K. Gaikwad, R. Bantu, S. R. Manikonda, Eur. J. Med. Chem. 46
(2001) 2152.
[15] L. Nagarapu, V. Paparaju, A. Satyender, Bioorg. Med. Chem. Lett. 18 (2008) 2351.
[16] L. Nagarapu, A .Satyender, B. Rajashaker, K. Srinivas, P. Ruparani; K. Radhika, G.
Subhashini, Bioorg. Med. Chem. Lett. 18 (2008) 1167.
[17] T. Mosmann, J. Immunol. Methods, 65 (1983) 55.
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Supplementary data
Copies of 1H and 13C NMR spectra for all new compounds.
Figure/Scheme Captions:
Scheme 1: Synthesis of 2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-
carboxylic acid.
Scheme 2: Synthesis of (2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidin-3-
yl)(piperidine/4-methylpiperazin/morpholino/4-phenylpiperazin/4-
nitrophenyl) piperazin -1-yl) methanone derivatives.
Scheme 3: Synthesis of 2-methyl-N-(3-oxo-3-(piperidine/4-methylpiperazin/morpholino/4-
phenylpiperazin/4-nitrophenyl)-1-yl)propyl)-4-phenyl-4H-benzo[4,5]thiazolo
[3,2-a]pyrimidine-3-carboxamides.
Table 1: IC50 of the tested compounds against human tumor cell lines
14

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Table
Table 1: Cytotoxicity of the tested compounds against human tumor cell lines
Compounds
IC50a (µM)
HeLa
MDA-MB-231
MCF-7
11.74
0.58
10.084
77.58
29.01
16.79
-
A549
-
-
-
-
5.01
1.58
11.09
31.58
47.25
21.73
-
-
-
-
-
5a
5b
5c
5d
5e
8a
8b
8c
-
-
11.39
18.09
-
-
-
-
-
-
-
-
-
-
-
8d
8e
25.69
0.451
13.79
1.05
11.18
1.21
Doxb
0.501
a Data presented is the mean value of two independent determinations.
b Doxorubicin is positive control
Schemes
Scheme 1: Synthesis of 2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-
carboxylic acid.
15

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O
O
o
HOBt
2 -amine,
N
N
N
OH
EDC.HCl,Et3N,
CH2Cl2, r.t
X
S
S
N
CH3
N
CH3
5
4
5a
X,
= CH2;
5b = N-CH3
5c = O
5d
= N-Ph
5e = N-Ph-NO2
Scheme 2: Synthesis of (2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidin-3-
yl)(piperidine/4-methylpiperazin/morpholino/4-phenylpiperazin/4-
nitrophenyl) piperazin -1-yl) methanone derivatives.
O
O
HOBt
COOEt
HCl.NH2-CH2-CH2-COOEt
+
N
N
N
OH
CH3
EDC.HCl,Et3N,
CH2Cl2, r.t
H
S
S
N
CH3
N
6
4
O
O
2o-amine; HOBt
EDC.HCl,Et3N,
O
1N KOH;
Ethanol;
COOH
N
N
N
N
N
H
H
reflux
X
CH2Cl2, r.t
S
N
CH3
S
N
CH3
8
7
X, 8a = CH2;
8b = N-CH3
8c = O
8d
= N-Ph
8e = N-Ph-NO2
Scheme 3: Synthesis of 2-methyl-N-(3-oxo-3-(piperidine/4-methylpiperazin/morpholino/4-
phenylpiperazin/4-nitrophenyl)-1-yl)propyl)-4-phenyl-4H-benzo[4,5]
thiazolo[3,2-a]pyrimidine-3-carboxamides.
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Research Highlights
ꢀ To discover and develop tumor growth inhibitors.
ꢀ Synthesis of several new benzo[4,5]thiazolo[1,2-a]pyrimidine-3-carboxylate
analogues
ꢀ
Co
mpound 5a and 5b exhibited potent cytotoxicity against MDA-MB-231 and MCF-7
cells.

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Synthesis
of
Novel
Benzo[4,5]thiazolo[1,2-a]pyrimidine-3-
carboxylate Derivatives and Biological Evaluation as Potential
Anticancer Agents
Lingaiah Nagarapua,*, Satheeshvarma Vanaparthia, Rajashaker Bantua, Ganesh
Kumar Cb
1H NMR Spectra
1-7
13C NMR Spectra
8-12
Mass (HRMS) spectra
IR Spectra
13-19
20-20

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