One pot synthesis of thiazolodihydropyrimidinones and evaluation of their anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2004.06.001

2-(5-Arylfurfurylidene/5-nitrofurfurylidene)-5-aryl-7-(2, 4-dichloro-5-fluorophenyl)-5H-thiazolo[2,3-b]-pyrimidin-2(1H)-ones 5 and 6 are synthesized by a novel three component reaction of 4,6-diarylpyrimidino-2(1H)- thiones 4, monochloroacetic acid, arylf

Full text of DOI:10.1016/j.ejmech.2004.06.001

European Journal of Medicinal Chemistry 39 (2004) 777–783
www.elsevier.com/locate/ejmech
Original article
One pot synthesis of thiazolodihydropyrimidinones
and evaluation of their anticancer activity
B. Shivarama Holla a,*, B. Sooryanarayana Rao a,1, B.K. Sarojini a,2, P.M. Akberali b
a Department of Post Graduate Studies and Research in Chemistry, Mangalore University, Mangalagangotri 574199, Karnataka, India
b Strides Arco Laboratories, 120 A & B, Industrial Area, Baikampady, New Mangalore 575011, Karnataka, India
Received 5 January 2004; received in revised form 14 May 2004; accepted 1 June 2004
Available online 28 July 2004
Abstract
2-(5-Arylfurfurylidene/5-nitrofurfurylidene)-5-aryl-7-(2,4-dichloro-5-fluorophenyl)-5H-thiazolo[2,3-b]-pyrimidin-2(1H)-ones 5 and 6
are synthesized by a novel three component reaction of 4,6-diarylpyrimidino-2(1H)-thiones 4, monochloroacetic acid, arylfurfuraldehydes
and 5-nitro-2-furfuraldenediacetate, respectively. The newly synthesized compounds are characterized by elemental analysis, IR, 1H NMR
and mass spectral studies. These compounds exhibited in vitro antitumour activity with moderate to excellent growth inhibition against a panel
of 60 cell lines of leukemia, non-small cell lung cancer melanoma, ovarian cancer, prostate cancer and breast cancer.
© 2004 Elsevier SAS. All rights reserved.
Keywords: MCR; Three component reaction; Thiazolodihydropyrimidinones; Arylfurfural; 5-nitrofurfuraldiacetate; Anticancer activity
* Corresponding author. Fax: +91-824-2287-367.
E-mail address: hollabs@yahoo.com (B.S. Holla).
1 Present address: Department of Chemistry, Organosilicon and Synthesis Laboratory, National Tsing Hua University, Hsinchu, 60013, Taiwan, ROC.
2 Present address: Department of Chemistry, P. A. College of Engineering, Nadupadavu, Mangalore 574153, Karnataka, India.
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.06.001

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B.S. Holla et al. / European Journal of Medicinal Chemistry 39 (2004) 777–783
1. Introduction
Multi-component reactions (MCR) are special types of
theoretically useful organic reactions in which three or more
starting materials react to give a product [1]. Convergent
synthesis pathways generally show advantages over linear or
divergent approaches with respect to time, speed, yield and
reproducibility. Among organic reactions, MCR with more
than two starting materials are assembled to afford a complex
product. Therefore, they constitute a superior tool for diver-
sity oriented and complexity-generating synthesis for drug
discovery [2,3]. Well known examples of MCR, which form
heterocycles, are Bignilli reaction [4] and Hantzsch dihydro-
pyridine synthesis [5].
Pyrimidine derivatives are known for their varied biologi-
cal properties. Brugnatelli was the first scientist to isolate
“Alloxan”, a pyrimidine derivative in 1818, and later this
compound was found to possess antineoplastic properties
[6]. Nucleosides of pyrimidine bases have been used exten-
sively as antiviral and anticancer agents [7]. Recently,
fluoropyrimidines- and flurouracil-based combination
therapy is used in the treatment of gastrointestinal cancer and
solid tumors [8]. Fluropyrimidinones are found to be me-
tabolites of dihydropyrimidinones that are subtype-selective
antagonists of the ␣1a-adreginic receptor antagonists [9].
Thiazoles and their derivatives are found to be associated
with various biological activities such as antibacterial, anti-
fungal and anti-inflammatory activities [10–13]. Presence of
fluorine in a molecule enhances drug persistence and lipid
solubility [14] and also groups like 5-nitrofuran, arylfur-
furans contribute to the biological activities [15]. Based on
these reports and also in continuation of our search for
bioactive molecules, possessing anticancer activity [16], it
was contemplated to synthesize thiazolodihydropyrimidino-
nes in a one-pot reaction. Results of such studies are dis-
cussed in this paper.
Scheme 1. Synthesis of 4-aryl-6-(2,4-dichloro-5-fluorophenyl)-3,4-
dihydropyrimidin-2(1H)-thiones(4a-e).
5,6-disubstituted-5H-thiazolo[2,3-b]-pyrimidin-3-ones. This
compound was then converted into arylidene derivatives by
condensing the same with aromatic aldehydes in the presence
of piperidine. In the present investigation, it was contem-
plated to carry out the synthesis of the title compounds 5,6
in a one-pot synthesis. Thus, 2-(5-arylfurylidene/5-nitro-
furfurylidene)-5-(aryl)-7-(2,4-dichloro-5-fluorophenyl)-5H-
thiazolo[2,3-b]-pyrimidin-2(1H)-ones were prepared in
good yields by refluxing 4,6-diaryl-3,4-dihydropyrimidin-
2(1H)-thiones, monochloroacetic acid, anhydrous sodium
acetate with the corresponding arylfurfuraldehyde/5-nitro-2-
furfural diacetate in acetic acid–acetic anhydride medium
(Tables 2 and 3, Scheme 2). The structures of these com-
pounds were established on the basis of elemental analysis,
IR, 1H NMR and mass spectral studies.
2. Chemistry
1,3-Diaryl-2-propen-1-ones (chalcones) 3 were prepared
by condensing substituted benzaldehydes 2 with 2,4-
dichloro-5-fluoroacetophenones 1 in the presence of sodium
hydroxide under the Clasein–Schmidt reaction conditions.
These chalcones provided suitable building blocks for the
synthesis of 4,6-diaryl-3,4-dihydropyrimidin-2(1H)-thiones
4. These compounds were prepared in good yields by reflux-
ing 1,3-diaryl-2-propen-1-ones 3 and thiourea in the pres-
ence of ethanolic potassium hydroxide (Scheme 1). The
characterization data of these compounds are given in
Table 1. These pyrimidin-2-thiones 4 were further used for
the synthesis of N-bridged heterocycles. From the previous
study, it is clear that the preparation of 2-arylidene-5,6-
diaryl-5H-thiazolo[2,3-b]-pyrimidin-3-ones starting from
pyrimidine-2-thione involves two steps. In the first step,
pyrimidine-2-thione was condensed with monochloroacetic
acid in the presence of anhydrous sodium acetate to yield
3. Results and discussion
The IR spectrum of the compound 4a showed an absorp-
tion band at 3414 cm−1 corresponding to the –NH stretching
frequency of the pyrimidin-2-thione.An absorption band was
seen at 1182 cm−1 due to the stretching frequency of C~S
group. The absence of characteristic absorption bands due to
carbonyl and S–H groups of the starting materials clearly
confirmed the formation of pyrimidin-2-thiones. The absorp-

B.S. Holla et al. / European Journal of Medicinal Chemistry 39 (2004) 777–783
779
Table 1
Characterization data of compounds 4-aryl-6-(2,4-dichloro-5-fluorophenyl)-3,4-dihydropyrimidin-2(1H)-thiones (4a–e)
Compound
R
M.p.
(°C)
Yield
(%)
84
Molecular formula
N-Analysis found [Calc.]
C-Analysis found [Calc.]
no.
4a
4b
4c
4d
4e
H
98–100
86–88
112–14
95–97
103–105
C16H11Cl2FN2S
C17H13Cl2FN2OS
C18H15Cl2FN2O2S
C17H11Cl2FN2O2S
C16H10Cl3FN2S
7.90 [7.93]
7.34 [7.31]
6.79 [6.77]
7.02 [7.05]
7.26 [7.22]
54.50 [54.34]
53.28 [53.26]
52.38 [52.30]
51.30 [51.38]
49.50 [49.54]
4-OCH3
3,4-(OCH3)2
3,4-O-CH2O-
4-Cl
86
81
76
79
Solvent of crystallization: benzene and methanol
IR (KBr, cmax cm−1): 4a: 3414 (NH), 1563 (C~C), 1083 (C–F), 726 (C–Cl); 4b: 3419 (N–H), 2933(C=H), 1610 (C=N), 732 (C–Cl); 4c: 3420 (N–H), 2935
(C–H), 1565 (C=C), 1084 (C–F), 726 (C–Cl);
1H NMR (CDCl3 + DMSO-d6): 4a: d 5.06 (d, 1H, methine H J = 4.6 Hz), 5.26 (d, 1H, olefinic H, J = 4.6 Hz), 7.01–7.53 (m, 6H,Ar–H); 7.72 (s, 1H, NH); 7.81(s,
1H, NH); 8.05 (d, Ar–H, J = 9.3 Hz). 4b: d 3.9(s, 3H, OCH3), 5.07(d, 1H, methine H J = 4.8 Hz), 5.23 (d, 1H, olefinic H, J = 4.8 Hz), 7.02–7.6 (m, 6H, Ar–H)
8.05(d, 1H, Ar–H, J = 9.2 Hz); 7.7 (bs, 1H, N–H); 7.8 (s, 1H, N–H); 4c: d 3.86 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 5.07 (d, 1H, methine H J = 4.5 Hz), 5.22 (d,
1H, olefinic H, J = 4.5 Hz), 6.91(m, 2H, Ar–H) 7.21(d, 1H, Ar–H, J = 8.6 Hz); 7.52 (m, 2H, Ar–H), 7.62 (bs, 1H, N–H); 7.83 (s, 1H, N–H); 4d: d 5.04 (d, 1H,
methine H, J = 4.8 Hz), 5.12 (d, 1H, olefinic H, J = 4.8 Hz), 6.01(s, 2H, -OCH2O-), 6.78–7.43 (m, 5H, Ar–H), 7.83 (bs, 1H, N–H). 4e: d 4.9 (d, 1H, methine H
J = 5 Hz), 5.2 (d, 1H, olefinic H, J = 5 Hz), 7.2–7.8(m, H, Ar–H), 7.8 (bs, 1H, N–H); 8.03 (s, 1H, N–H);
MS: m/z (% abundance): 4a: 352 (96%, M+), 318 (24%, M+-H2S), 275 (38% M+-C6H5), 189 (100%, M+-2,4-dichlorophenyl radical), 77 (51%, phenyl cation)
4b: 382 (100%, M+), 348 (13%, M+-H2S), 276 (15%, M+-4-MeOC6H4), 219 (34%, M+-2,4-dichlorophenyl radical), 4c: 412 (80%, M+), 378 (15% M+-H2S), 275
(36% M+-163), 138 (15% 3,4-(OCH3)2C6H3); 4d: 396 (49%, M+), 362 (29%, M+-H2S), 275 (35%, 3,4-methylenedioxy phenyl radical), 234 (15%, M+-163); 4e:
386 (88%, M+), 352 (52%, M+-H2S), 275 (28% M+-111), 223 (100%, M+-163), 163(17%, 2,4-dichloro-5-fluorophenyl cation), 111(33%, 4-chlorophenyl
cation).
Table 2
Characterization data of compounds 2-(5-arylfurfurylidene)-5-(aryl)-(2,4-dichloro-5-fluorophenyl)-5H-thiazolo[2,3-b]pyrimidin-3-ones (5a–j)
Compound
R
R1
M.p.
(°C)
Yield
(%)
78
Molecular formula
N-Analysis found
[cald.]
C-Analysis found
[cald.]
No
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
4-NO2-2-CH3
4-NO2-2-CH3
4-NO2-2-CH3
4-NO2-2-CH3
4-NO2-2-CH3
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
2,4-dichloro
145–47
116–18
126–28
119–21
138–40
132–34
128–30
142–44
126–28
133–35
C30H18Cl2FN3O2S
C31H20Cl2FN3O5S
C32H22Cl2FN3O4S
C31H18Cl2FN3O6S
C30H12Cl3FN3O4S
C29H15Cl4FN2O2S
C30H17Cl4FN2O3S
C31H19Cl4FN2O2S
C30H15Cl4FN2O2S
C29H14Cl5FN2O2S
6.95 [6.93]
6.64 [6.60]
6.31 [6.30]
6.48 [6.46]
6.60 [6.55]
4.56 [4.54]
4.30 [4.33]
4.10 [4.14]
4.21 [4.24]
4.27 [4.30]
59.44 [59.40]
58.52 [58.49]
57.68 [57.65]
57.28 [57.23]
56.26 [56.20]
56.47 [56.49]
55.70 [55.72]
55.10 [55.02]
54.52 [54.54]
53.53 [53.49]
4-OCH3
3,4-(OCH3)2
3,4-O- CH2-O-
4-Cl
81
76
79
83
H
71
4-OCH3
3,4-(OCH3)2
3,4- O- CH2-O-
4-Cl
74
78
68
5j
73
Solvent of crystallization: glacial acetic acid
Spectral data: IR (KBr, cmax cm−1): 5a: 1684 (C=O), 1610 (C=N), 1516 (NO2), 1347 (NO2), 1075 (C–F), 736 (C–Cl); 5f: 1684 (C=O), 1586 (C=N), 732 (C–Cl);
1H NMR (CDCl3 + DMSO-d6) : 5a: d 2.68 (s, 3H, CH3), 5.92 (d, 1H, methine H, J = 4.4 Hz), 5.81 (d, 1H, olefinic H, J = 4.4 Hz), 6.91–7.53 (m, 9H, Ar–H and
furan H), 8.01 (d, 1H, Ar–H, J = 8.6Hz), 8.15 (m, 2H Ar–H), 9.75 (s, 1H, exocyclic vinylic H). 5b: d 2.68 (s, 3H, CH3), 3.9 (s, 3H, –OCH3) 5.8 (d, 1H, methine
H, J = 4.4 Hz), 5.91(d, 1H, olefinic H, J = 4.4 Hz), 6.91–7.63 (m, 9H,Ar–H and furan H), 8.0 (d, 1H,Ar–H, J = 8.6 Hz), 8.15 (m, 2HAr–H), 9.65 (s, 1H, exocyclic
vinylic H). 5c: d 2.91 (s, 3H, CH3), 3.6 (s, 3H, –OCH3), 3.9 (s, 3H, OCH3), 5.7 (d, 1H, methine H, J = 4.5 Hz), 5.85 (d, 1H, olefinic H, J = 4.5 Hz), 7.09–7.50
(m, 8H, Ar–H), 7.9 (d, 1H, J = 9 Hz) 8.1 (d, 1H, J = 9 Hz), 9.8 (s, 1H, exocyclic vinylic H); 5d: d 2.7 (s, 3H, CH3), 3.9 (s,3H, –OCH3 ), 6.05(s, 2H, methylene
dioxy), 5.6 (d, 1H, methine H, J = 5 Hz), 5.7 (d, 1H, olefinic H, J = 5 Hz), 7.05–7.65 (m, 8H, Ar–H and furan H), 7.95 (d, 1H, Ar–H, J = 8.5 Hz), 8.0(d, 1H
J = 8.5 Hz), 9.65 (s, 1H, exocyclic vinylic H). 5e: d 2.6 (s, 3H, CH3), 5.8 (d, 1H, methine H, J = 4.4 Hz), 5.9 (d, 1H, olefinic H, J = 4.4 Hz), 6.98–7.71 (m, 8H,
Ar–H and furan H), 7.95 (d, 1H, Ar–H, J = 8.7 Hz), 8.0 (d, 1H J = 8.7 Hz), 9.68 (s, 1H, exocyclic vinylic H). 5f: d, 5.85 (d, 1H, methine H, J = 4.6 Hz), 5.91 (d,
1H, olefinic H, J = 4.6 Hz), 6.66–7.40 (m, 10H, Ar–H and furan H), 7.9 (d, 1H, Ar–H, J = 8.9 Hz), 8.1 (d, 1H J = 8.9 Hz), 9.67(s, 1H, exocyclic vinylic H). 5g:
3.9 (s, 3H, –OCH3 ) 5.85 (d, 1H, methine H, J = 4.4 Hz), 5.91 (d, 1H, olefinic H, J = 4.4 Hz), 6.7–7.43 (m, 9H, Ar–H and furan H), 7.9 (d, 1H, Ar–H, J = 9 Hz),
8.0 (d, 1H, Ar–H, J = 9 Hz), 9.60 (s, 1H, exocyclic vinylic H). 5h: d 3.85 (s, 3H, –OCH3), 3.86 (s, 3H, OCH3), 5.9 (d, 1H, methine H, J = 4.8 Hz), 5.76 (d, 1H,
olefinic H, J = 4.8 Hz), 6.82 (d, 1H, furan-H, J = 3.7 Hz), 6.68–6.71 (m, 7H, Ar–H and furan H), 7.79–7.96 (m, 2H, Ar–H), 9.69 (s, 1H, exocyclic vinylic H); 5i:
3.9 (s, 3H, –OCH3), 6.05 (s, 2H, methylene dioxy), 5.8 (d, 1H, methine H, J = 5 Hz), 5.9 (d, 1H, olefinic H, J = 5 Hz), 7.05–7.65 (m, 8H, Ar–H and furan H),
7.95 (d, 1H, Ar–H, J = 8.5 Hz), 8.1 (d, 1H J = 8.5 Hz), 9.70 (s, 1H, exocyclic vinylic H). 5j: d 6.27 (d, 1H, methine H J = 4.5 Hz), 5.93 (d, 1H, olefinic H,
J = 4.5 Hz), 6.91–7.93 (m, 10H, Ar–H and furan H), 7.96(d, 1H, Ar–H J = 8.8 Hz), d, 1H,
Ar–H J = 8.6 Hz). MS: m/z (% abundance): 5b[Fab Mass]: 636 (100%, MH+), 635 (13%, MH+-CH3), 527 (10%, MH+-107), 163 (10% ,2,4-dichloro-5-
fluorophenyl cation), 107 (30%, 4-methoxyphenyl cation). 5d[Fab Mass]: 650 (100%, MH+), 528 (18%, M+-121), 487,(MH+-163), 163 (11%, 2,4-dichloro-5-
fluorophenyl cation), 121 (20%, 3,4-methoxyphenyl cation); 5f[FabMass]: 616 (22%, M++2), 538 (10%, M+-C6H5), 163 (8%, 2,4-dichloro-5-fluorophenyl
cation); 5j[FabMass]: 649 (100%, MH+), 651(100%, MH++2).
tion bands due to (C–F) and (C–Cl) were seen at 1083 cm−1
and 726 cm−1, respectively. The 300 MHz 1H NMR spectrum
of compound 4a showed two doublets at d 5.06 (J = 4.6 Hz)
and 5.26 (J = 4.6 Hz), respectively, due to the methine and
olefinic protons of dihydropyrimidine ring. The two –NH
protons of the pyrimidine ring were seen as two broad sin-
glets at d 7.72 and 7.81, respectively. The aromatic protons
resonated as a complex multiplet in the region d 6.85–

780
B.S. Holla et al. / European Journal of Medicinal Chemistry 39 (2004) 777–783
Scheme 2. Synthesis of 2-(5-aryfufurylidene/nitrofufurylidene)-5-(aryl)-7-(2,4-dichloro-5-fluorophenyl)-5H-thiazolo[2,3-b]pyrimidin-3-ones(5a-j, 6a-e).
7.57 integrating for seven protons. The mass spectrum of the
compound 4d showed a molecular ion peak at m/z 396, which
is consistent with its molecular formula, C17H11Cl2FN2O2S.
A peak observed at m/z 362 is attributed to an ion obtained by
the loss of H2S from the molecular ion. A peak at m/z
121 corresponds to the formation of 3,4-methylene-
dioxyphenyl cation during the fragmentation process.A peak
at m/z is attributable to the radical cation of 2,4-dichloro-5-
fluoro-benzonitrile obtained during fragmentation. A peak
seen at m/z 275 was attributed to the cation obtained by the
loss of 3,4-dimethoxyphenyl radical from the molecular ion.
The base peak seen at m/z 44 is due to the formation of radical
cation of carbon monosulphide during the fragmentation
process. The spectroscopic data of some other compounds 4
are given in experimental section.
two doublets at d 5.90 (J = 4.8 Hz) and 5.76 (J = 4.8 Hz),
which are assigned to the methine protons. The shift of the
signals to higher d values is due to the deshielding effect of
the carbonyl group of thiazolidinone ring. The two methoxy
protons resonated as two closely packed singlets at d 3.85 and
3.86, respectively. The signal due to the exocyclic vinyl
protons was seen as a singlet at d 9.69. The signals of the
protons of the furan ring appeared as two sharp doublets at d
6.82 (J = 3.7 Hz) and 7.43 (J = 3.7 Hz), respectively. The
aromatic protons resonated as a complex multiplet in the
region d 6.81–7.98 integrating for eight protons. The mass
spectral data of these compounds further confirmed the for-
mation of condensed products 5 and 6. The FAB mass spec-
trum of the compound showed a molecular ion peak at 636,
which is consistent with the assigned molecular formula,
C31H20Cl2FN3O5S. A peak seen at m/z 107 was due to
4-methoxyphenyl cation. The mass spectrum of compound
6b showed a peak due to the protonated molecular ion at m/z
547, which is in agreement with the molecular formula
C24H14Cl2FN3O5S assigned for this compound. A peak seen
at m/z 189 is due to the formation of molecular ion of
2,4-dichloro-5-fluorobenzonitrile. Loss of CN radical from
this ion produced 2,4-dichloro-5-fluorophenyl cation as evi-
denced by the presence of peak at m/z 163. The spectral data
of the other newly synthesized compounds are given in the
experimental section.
Similarly the structure of the compounds 5 and 6 is estab-
lished on the basis of spectral data. IR spectrum of 5f showed
an absorption band at 1684 cm−1 indicating the presence of
amide carbonyl group in the molecule. Absence of the char-
acteristic absorption bands due to NH stretch clearly showed
that –NH protons were involved in the cyclization. An ab-
sorption band appearing at 1586 cm−1 is attributed to C~C
stretching frequency. The absorption bands due to (C–F) and
(C–Cl) were seen at 1083 cm−1 and 726 cm−1, respectively.
Further support for the formation of condensed products was
obtained by recording 1H NMR spectra of a few compounds.
1
The 300 MHz H NMR spectrum of compound 5h showed

B.S. Holla et al. / European Journal of Medicinal Chemistry 39 (2004) 777–783
781
4. Pharmacology
5. Conclusion
All the compounds tested showed moderate to good anti-
cancer activity. Among them compound 5j, 2-[5-(2,4-
dichlorophenylfurfurylidene)]-5-(4-chlorophenyl)-7-(2,4-
dichloro-5-fluorophenyl)-5H-thiazolo[2,3-b]-pyrimidin-
2(1H)-one showed highest activity against leukemia HL-
60(TB) cell line[GI50 = <0.001 µM, TGI = <0.001 µM,
LC50 = <0.001 µM]. The compounds 2-(5-nitrofurfury-
lidene)-5-[4-(3,4-methylenedioxyphenyl)]-7-(2.4-dichloro-
5-fluorophenyl)-5H-thiazolo[2,3-b]-pyrimidin-2(1H)-one
(6d) and 2-(5-nitrofurfurylidene)-5-[(4-chlorophenyl)]-7-
(2,4-dichloro-5-fluorophenyl)-5H-thiazolo[2,3-b]-pyrimidin-
2(1H)-one (6e) showed moderate activity against all the
tested cell lines and highest against leukemia SR cell line
[GI50 = 3.34 µM], CCRP-CEM Cell Line [GI50 = 3.24 µM]
and coloncancer HCT-116 cell line [GI50 = 3.56 µM]. Hence,
the presence of 5-nitro-2-furfuryl moiety and 4-chlorphenyl
groups as substituents tends to increase the anticancer prop-
erty in these molecules, although they did not prove cytotoxic
or cytostatic at the maximum tested concentration (100 µM).
Eight newly synthesized compounds were screened for
their antitumour activities at NIH, Bethesda, Maryland, USA
under the Drug Discovery Programme of NCI according to
the procedure suggested by Boyd and Paull [17] in a primary
three cell line—one dose antitumour assay against NCI-H
(lung), MCF-7 (breast) and SF 268 (CNS). In the current
protocol, each cell line is inoculated on a preincubated mi-
crotiter plate. Test agents are added at a single concentration
and the culture is incubated for 48 h. End point determina-
tions are made with sulpharhodamine B, a protein binding
dye. Compounds which reduce the growth of any one of the
cell lines to 32% or less (negative numbers indicate cell kill)
are passed on for evaluation in a panel of 60 cell lines over a
5-log dose range (Table 3). In the present screening pro-
gramme, (Table 4) almost all the compounds showed moder-
ate to good antiproliferative activity on the whole panel of
60 cell lines (Table 5) derived from seven cancer types
namely lung, colon, melonoma, renal, ovarian, CNS and
leukamia. Their GI50, TGI and LC50 values were determined.
Table 3
Characterization data of compounds 2-(5-nitrofurfurylidene)-5-(aryl)-7-(2,4-dichloro-5-fluorophenyl)-5H-thiazolo[2,3-b]pyrimidin-3-ones (6a–e)
Compound
R
M.p.
(°C)
Yield
(%)
68
Molecular formula
N-Analysis found
[cald.]
C- Analysis found
[cald.]
No.
6a
6b
6c
6d
6e
H
78–80
81–83
109–11
85–87
106–108
C23H12Cl2FN3O4S
C24H14Cl2FN3O5S
C25H16Cl2FN3O6S
C24H12Cl2FN3O6S
C23H11Cl3FN3O4S
8.10 [8.14]
7.74 [7.69]
7.26 [7.29]
7.54 [7.50]
7.60 [7.62]
53.46 [52.74]
52.70 [52.74]
52.12 [52.08]
57.40 [57.42]
50.16 [50.13]
4-OCH3
3,4-(OCH3)2
3,4-O-CH2-O-
4-Cl
79
71
74
76
Solvent of crystallization: glacial acetic acid and DMF
IR (KBr, cmax cm–1): 6a: 3034(C–H), 1759 (C=O), 1598 (C=N), 1541 (NO2), 1354 (NO2), 1077 (C–F), 728 (C–Cl); 6c: 2935(C–H), 1713 (C=O), 1594 (C=N),
1514 (NO2), 1348 (NO2), 1079 (C–F), 729 (C–Cl).
1H NMR (CDCl3 + DMSO-d6): 6a: 6.01 (d, 1H, methine H J = 4.7 Hz), 5.75 (d, 1H, olefinic H, J = 4.7 Hz), 6.725–7.81 (m, 7H, Ar–H and furan H), 7.95 (d, 1H,
J = 9 Hz) 8.1(d, 1H, J = 9 Hz), 9.83 (s, 1H, exocyclic vinylic H); 6b: d 3.9 (s, 3H, –OCH3), 6.05 (d, 1H,methine H J = 4.8 Hz), 5.80 (d, 1H, olefinic H, J = 4.8 Hz),
6.70–7.6 (m, 6H, Ar–H and furan H), 7.92 (d, 1H, J = 9 Hz) 8.05 (d, 1H, J = 9 Hz), 9.82 (s, 1H, exocyclic vinylic H); 6c: d 3.83 (s, 3H, –OCH3), 3.84 (s, 3H,
OCH3), 6.18 (d, 1H,methine H J = 5.1 Hz), 5.89 (d, 1H, olefinic H, J = 5.1 Hz), 6.85–7.85 (m, 7H, Ar–H and furan H), 9.84 (s,1H, exocyclic vinylic H);6d: d
6.01(s, 2H, –OCH2O–), 6.04 (d, 1H, methine H J = 4.6 Hz), 5.71 (d, 1H, olefinic H, J = 4.6 Hz), 6.72–7.71 (m, 7H, Ar–H and furan H), 9.89 (s, 1H, exocyclic
vinylic H); 6e: 6.02 (d, 1H,methine H J = 4.5 Hz), 5.9 (d, 1H, olefinic H, J = 4.5 Hz), 6.85–7.78 (m, 8H, Ar–H and furan H), 9.84 (s, 1H, exocyclic vinylic H);
MS: m/z (% abundance): 6b: 547(16%, M++2), 502 (96%, M+-NO2), 275 (38%, M+–C6H5), 163 (8%, 2,4-dichloro-5-fluorophenyl cation), 83 (33%,
1-nitrocyclopropenyl cation). 6c: (M+is not observed), 340 (50% M+-2,4-dicloro-5-fluorobenzonitrile & NO2), 163 (8%, 2,4-dichloro-5-fluorophenyl cation),
223 (100%, M+-163), 163(17%, 2,4-dichloro-5-fluorophenyl cation), 111 (33%, 4-chlorophenyl cation).
Table 4
Anticancer activity screening data of compound 4, 5 and 6
Compound
Growth percentage a
NCI code
NCI-H (lung)
MCF-7 (breast)
SF268 (CNS)
Activity b
Active
Active
Active
Active
Active
Active
Active
Active
4b
4d
4e
5g
5i
5j
6d
6e
NSC 716879
NSC 716880
NSC 716878
NSC 716882
NSC 716883
NSC 716881
NSC 716885
NSC 716884
–5
09
26
37
24
30
–6
–24
07
27
14
34
11
43
14
27
12
17
19
07
–55
–54
–67
–38
Fixed concentration assay (100 µM; standard NCI protocol).
a Percentage cell growth reduction following 48-h incubation with test compounds (optical density, sulphorhodamine proceure) [17].
b ‘Active’ when growth percentage is <32% for any one of the three cell lines. Negative numbers indicate the cell kill.

782
B.S. Holla et al. / European Journal of Medicinal Chemistry 39 (2004) 777–783
Table 5
Sixty cell line in vitro antitumour screening data (GI50 in µM)
Panel cell line
Leukemia
CCRF-CEM
HL-60[TB]
K-562
4b
4d
4e
28.9
5g
5i
5j
6d
6e
30.2
11.3
15.2
17.7
21.7
52.4
21.8
–
38.8
27.2
35.2
28.2
26.1
–
–
3.24
15.6
5.10
8.92
9.32
3.34
4.83
12.3
13.1
12.8
5.16
–
0.003
32.1
23.4
22.8
31.9
20.6
56.2
15.6
14.9
40.6
–
<0.001
38.8
22.4
31.1
–
38.8
31.9
32.5
–
MOLT-4
RPMI-8226
SR
Non-small lung
A549/ATCC
EKVX
35.3
28.0
38.7
24.0
56.1
37.5
51.3
20.1
31.8
35.6
22.2
39.5
25.9
59.1
60.8
42.2
25.5
31.6
47.3
31.6
48.2
27.1
88.7
63.9
45.7
34.0
34.0
>100
–
46.1
–
>100
–
16.5
16.6
22.6
22.6
46.7
20.6
38.3
17.8
25.8
30.3
–
HOP-62
18.8
31.6
24.6
38.5
>100
48.5
52.2
58.7
31.1
20.7
38.6
19.3
36.9
49.1
10.6
26.1
22.4
36.5
47.9
60.2
84.4
11.5
12.9
15.8
15.3
14.3
10.0
18.3
HOP-92
NCIH-226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO-205
HCC-2998
HCT-116
HCT-15
19.1
23.9
27.0
28.7
26.2
29.2
30.9
20.9
23.2
27.5
37.4
27.9
38.0
36.0
41.0
23.3
35.8
46.8
36.5
49.4
70.6
79.9
20.1
35.1
33.8
37.3
60.1
51.1
38.7
–
78.6
12.8
38.0
35.6
39.8
87.5
33.6
18.7
12.9
5.10
9.6
3.94
–
35.4
34.1
25.7
57.5
35.8
3.56
8.19
6.63
24.5
4.91
HT-29
17.1
16.6
5.42
KM-12
SW-620
CNS cancer
SF-268
48.6
24.3
42.3
61.0
18.2
26.7
45.7
31.9
46.6
57.4
20.8
22.9
65.7
28.2
>100
76.7
37.6
29.6
35.6
43.1
34.6
31.6
38.4
34.7
46.3
40.7
35.3
28.7
>100
29.5
36.0
53.2
34.7
35.6
63.3
31.5
28.7
31.2
23.8
20.9
17.3
19.9
13.9
21.3
22.4
15.9
29.2
8.02
SF-295
SF-539
SNB-19
SNB-75
U-251
Melanoma
LOXIMVI
MALME-3M
M-14
37.3
40.6
32.5
25.0
46.9
11.8
24.6
28.3
37.1
30.7
40.4
20.9
40.2
10.3
26.3
35.9
48.7
>100
54.3
27.1
>100
11.6
31.6
57.0
38.0
99.1
55.4
20.1
>100
36.6
22.6
25.5
34.8
>100
28.0
26.0
91.8
28.5
30.0
16.0
57.9
>100
>100
40.4
>100
46.5
34.6
25.0
17.2
19.7
13.9
25.1
22.3
10.3
22.6
15.9
16.0
17.9
14.5
50.4
24.2
13.9
15.4
14.7
SKMEL-2
SKMEL-28
SKMEL-5
UACC-257
UACC-62
Ovarian cancer
IGROVI
31.4
33.7
2.83
43.0
40.3
44.1
27.4
30.2
38.1
4.1
41.2
33.1
35.8
>100
39.9
>100
30.3
–
34.2
–
33.0
–
16.3
16.8
15.8
21.7
11.9
46.5
15.8
–
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-O
37.3
39.4
37.8
21.5
33.2
58.8
42.0
21.5
41.9
>100
45.8
192
16.6
16.3
41.6
14.8
41.2
57.0
33.6
28.0
36.9
29.3
18.8
21.3
32.7
36.5
39.9
20.8
28.8
34.8
23.0
27.4
28.8
39.2
47.9
27.8
37.7
42.9
31.3
29.3
45.1
42.2
42.3
24.8
>100
80.6
39.6
47.7
12.1
32.9
42.9
22.6
69.2
>100
39.5
57.3
77.8
31.7
45.3
20.5
>100
>100
31.3
76.3
13.4
41.5
95.1
56.0
16.1
13.8
16.1
18.0
23.3
16.8
47.1
30.7
16.3
28.7
13.1
54.9
16.1
14.7
A498
ACHN
CAKI-1
RXP 393
SN12C
TK10
UO-31
(continued on next page)

B.S. Holla et al. / European Journal of Medicinal Chemistry 39 (2004) 777–783
783
Table 5
(continued)
Panel cell line
Prostate cancer
PC-3
4b
4d
4e
28.3
5g
5i
5j
6d
6e
19.9
39.9
21.4
49.0
99.3
70.9
69.1
63.6
>100
65.1
22.0
21.6
11.5
17.8
DU-145
62.8
Breast cancer
MCF7
32.6
25.7
27.3
79.1
12.8
11.1
32.7
18.7
32.2
51.7
23.8
70.0
34.5
35.6
57.0
20.2
31.2
64.1
23.6
>100
43.9
47.4
>100
19.2
26.9
44.4
25.5
34.8
30.8
28.1
47.2
19.6
26.0
45.9
22.6
14.0
32.2
37.9
37.8
21.1
33.5
50.8
23.6
29.6
54.3
53.4
45.8
19.2
10.0
34.4
23.0
61.0
13.4
55.0
16.8
17.3
13.3
30.8
50.7
56.4
16.1
46.4
15.1
19.0
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
MDA-N
BT-549
T-47D
The other two standard parameters TGI and LC50 were above 100 µM except for 5j, which is <0.001 µM.
Hence, it is concluded that there is ample scope for further
study in developing these compounds as anticancer agents.
ization data of the title compounds are given in Tables 2
and 3.
6. Experimental
Acknowledgements
Melting points were taken in open capillary tubes and are
uncorrected. IR spectra in KBr pellets were recorded on
JASCO FT-IR 5300 Infrared spectrophotometer.1H-NMR
spectra were recorded (CDCl3/CDCl3-DMSO-d6 mixture)
on a Brucker AC 300 F (300 MHz) NMR spectrometer using
TMS as an internal standard and the mass spectra were
recorded on a JEOL JMS 300 mass spectrometer operating at
70 eV.
The authors are grateful to Dr. V.L. Narayanan, National
Institute of Health (NIH), Bethesda, MA, USA, for arranging
the anticancer activity screening studies reported in this pa-
per. The authors are also thankful to the Director, RSIC,
Punjab University, Chandigarh and the Head RSIC, CDRI,
Lucknow for elemental and spectral analysis. B.S.N.R. is
grateful to Mangalore University for the award of a Senior
Research Fellowship.
6.1. General procedure for the preparation of 4-(aryl)-6-
(2,4-dichloro-5-fluorophenyl)-3,4-dihydropyrimidin-2(1H)-
thiones 4
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6.2. General procedure for the preparation of 2-(5-
arylfurfurylidene/5-nitrofurfurylidene)-5-(aryl)-7-(2,4-
dichloro-5-fluorophenyl)-5H-thiazolo[2,3-b]pyrimidin-3-
ones 5 and 6
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carboxaldehyde/5-nitro-2-furfural diacetate (10 mmol) was
heated under reflux for 3 h. The reaction mixture was cooled
and poured onto crushed ice with vigorous stirring. The
separated solid was filtered and washed with water, recrys-
tallized from suitable solvents to give 5 and 6. The character-
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