One-pot synthesis of new (1,3-Thiazolo[5,4-b]pyridin-2-yl)benzenediols and their antiproliferative activities against human cancer cell lines

Article abstract of DOI:10.1002/cbdv.201100007

A one-pot synthesis of new 4-(1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3- diols has been described. The compounds were prepared by the reaction of sulfinylbis[(2,4-dihydroxyphenyl)methanethione] derivatives, with various substituents in the aryl rings, w

Full text of DOI:10.1002/cbdv.201100007

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
One-Pot Synthesis of New (1,3-Thiazolo[5,4-b]pyridin-2-yl)benzenediols and
Their Antiproliferative Activities against Human Cancer Cell Lines
by Joanna Matysiak*a), Monika M. Karpin´skab), Andrzej Niewiadomya)b), Joanna Wietrzykc), and
Dagmara Kłopotowskac)
a) Department of Chemistry, University of Life Sciences, Akademicka 15, PL-20-950 Lublin
(phone: þ48-81-4456816; fax: þ48-81-5333549; e-mail: joanna.matysiak@up.lublin.pl)
b) Institute of Industrial Organic Chemistry, Annopol 6, PL-03-236 Warszawa
c) Department of Experimental Onkology, Institute of Immunology and Experimental Therapy, Polish
Academy of Sciences, R. Weigla 12, PL-53-114 Wrocław
A one-pot synthesis of new 4-(1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diols has been described.
The compounds were prepared by the reaction of sulfinylbis[(2,4-dihydroxyphenyl)methanethione]
derivatives, with various substituents in the aryl rings, with 2-chloropyridin-3-amines. Their structures
were deduced from IR and, 1H- and 13C-NMR spectroscopic, mass spectrometric, and elemental analyses.
The antiproliferative properties of some of the products against human cancer cell lines were comparable
to those of cisplatin. Structureꢀactivity analysis showed that the presence of hydrophobic substituents in
both heterocyclic fused and phenyl rings of the compounds improves their biological effects. Further, an
additional OH group in the resorcinol moiety reduced the antiproliferative activity.
Introduction. – The thiazole and fused-thiazole heterocycles are important
structural components of biologically active molecules, and, as a result, they serve as
attractive targets for developing new, effective synthesis methods [1][2]. Because of
their pharmaceutical importance in the area of drug discovery, we have been interested
in developing an efficient protocol to prepare fused-thiazole scaffolds of 1,3-
thiazolo[5,4-b]pyridine that is relatively unexplored with regard to its biological
activity.
Several synthetic methods for the preparation of 1,3-thiazolo[5,4-b]pyridine
derivatives have been well-documented, but all approaches have some restraints. The
most common route involves the reaction of 2-chloropyridin-3-amine with alkyl,
aralkyl, or aryl isothiocyanate in absolute EtOH [3][4]. Krayushkin and co-workers
described the oxidation of compounds, in which a monothioxamide fragment is linked
to a heterocyclic ring leading to the formation of fused thiazole derivatives with a
carboxamide group. This approach was applied to the synthesis of previously unknown
derivatives, and its sensitivity to electronic factors was noticed [5–8]. 1,3-Thiazolo[5,4-
b]pyridine-2-carboxamides were also prepared by the reaction of 2-nitropyridin-3-
amine with dithioesters containing a carbamoyl group [9]. A one-step synthesis from
appropriately substituted chloro-nitro-pyridine, and thioamide or thiourea has been
recently described by Sahasrabudhe et al. In particular, the reaction was used to
prepare a large number of 6-nitrothiazolo[5,4-b]pyridine derivatives with various
substituents in 2-position [10]. Treatment of 4-chloro-N-(2-chloropyridin-3-yl)- or 4-
chloro-N-(4-chloropyridin-3-yl)-1,2,3-dithiazol-5H-imines with catalytic amounts of
tetraalkylammonium iodide gave 1,3-thiazolo[5,4-b]pyridine-2-carbonitriles [11].
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
49
The formation of 1,3-thiazolo[4,5-b]pyridine, via transformation of 1,2,4-triazine
rings [12] or 2-(tetrazolylsulfanyl)pyridin-3-amines containing an aryl substituent in the
tetrazole moiety under acidic conditions, has been also reported [13]. Couture and
Grandclaudon described the conversion of the C¼O group of N-(2-chloropyridin-3-
yl)benzamides to the C¼S group with Lawessonꢂs reagent, followed by ring closure
providing desired 1,3-thiazolo[5,4-b]pyridines [14]. This method was applied by Lee
et al. [15]. Other derivatives were prepared from ethyl 5-amino-2-(methylsulfanyl)-
thiazole-4-carboxylate and benzylidinemalononitrile [16].
There is little information available about biological activity of 1,3-thiazolo[5,4-
b]pyridines. For example, 3-cyclopentyl-N-(5-methoxythiazolo[5,4-b]pyridin-2-yl)-2-
[4-(4-methylpiperazine-1-sulfonyl)phenyl]propionamide activates the GK enzyme in
vitro at low nanomolar concentrations and significantly reduces glucose levels [17].
Thiazolo[5,4-b]pyridines with a MeO group at C(5) show potent inhibitory activities
for Ab42 fibrillization at the micromolar level for Alzheimerꢂs disease treatment [15],
whereas 5-chloro[1,3]thiazolo[5,4-b]pyridin-2-amines are H3 receptor antagonists [4].
2,6-Difluoro-3-methoxybenzamide derivatives are potent antistaphylococcal com-
pounds with suboptimal drug-like properties [18]. They act as inhibitors of the
bacterial cell-division protein, FtsZ. Other derivatives were described as useful
antimicrobial agents effective against a variety of human and veterinary pathogens
including, among others, Gram-positive and Gram-negative aerobic and anaerobic
bacteria, as well as mycobacteria [19][20]. 1,3-Thiazolo[5,4-b]pyridines act also as
amplifiers of phleomycin [21].
Additionally, our early studies showed that 2-(2,4-dihydroxyphenyl)benzo-1,3-
thiazoles possess promising biological properties [22]. Therefore, we decided to
prepare the isosteric fused ring with the heteroatom N as the potential atom interacting
with the molecular target containing a 2,4-dihydroxyphenyl moiety. The 2,4-dihydroxy-
phenyl substituent is mainly modified at C(5) by the hydrophobic substituents Cl, Me,
and Et according to the literature. It was pointed out that they interact with a
hydrophobic pocket of HSP90 protein as a molecular target of resorcyl derivatives [23].
These groups of compounds are commonly described as promising anticancer drugs of
non-standard mechanism of action. Some of them are in the stage of clinical trials [24].
Additionally, for comparison, we decided to prepare compounds with a third OH group
in a resorcinol moiety in two different positions. The Me substituents at C(5) or/and
C(6) of the fused heterocyclic ring might protect its metabolically sensitive positions
and change the lipophilicity of compounds [25]. The compounds were designed and
synthesized as potential anticancer substances.
Results and Discussion. – Chemistry. Based on similarities with the reaction of 2-
nitropyridin-3-amines with thioamides (thioureas) [9], and the transformation of N-(2-
chloropyridin-3-yl)benzamides by treatment with Lawessonꢂs reagent [15], which leads
to corresponding 1,3-thiazolo[5,4-b]pyridines, a synthesis of analogs making use of 2-
chloropyridin-3-amine, or the 5- or 6-Me derivatives is proposed (Scheme 1). This type
of reagents enables the reaction with some electrophilic reagents elaborated by us, i.e.,
sulfinylbis[(2,4-dihydroxyphenyl)methanethione] derivatives (STB, S3MTB, S5MTB,
SETB, SClTB, S3TTB, and S6TTB; Scheme 1) [26], towards thioamidation and then
cyclization by elimination of HCl (Scheme 2). The presence of the halogen atom at

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Scheme 1. Synthesis of 4-(1,3-Thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diols
Scheme 2. Reaction Mechanism of the Formation of 4-(1,3-Thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diols
C(2) of pyridin-3-amine is a necessary condition for the reaction. The yield and purity
of products depend on the kind of substituents (X, Y) in this ring, and also on the kind

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
51
of substituent R in the dihydroxyphenyl moiety of the electrophilic reagent. The
cyclization process depends on amideꢀthiolimine isomerization and ionization ability
of the SH group regulated by ring substituents. The reaction promotes a tendency of the
compounds with thioamide moiety toward transition into the thiolimine forms
compared with the analogous ones with an O-atom [27]. Electrophilic reagents were
obtained according to the method described previously, i.e., by treatment of the
corresponding dithioic acids with SOCl2 [26].
In the 1H-NMR spectrum, the unsubstituted 1,3-thiazolo[5,4-b]pyridine ring in 1–7
shows three doublets of doublets at ca. 8.56 (J¼4.6, 1.4 Hz), 8.32 (J¼8.2, 1.4 Hz), and
7.55 ppm (J¼8.2, 4.6 Hz), corresponding to HꢀC(5), HꢀC(7), and HꢀC(6), respec-
tively. The 5-Me derivatives exhibit the characteristic two doublets at 8.27 and 7.45 ppm
with J¼8.3 Hz, and the 6-Me derivatives most often show two doublets at 8.27 and
7.45 ppm with J¼1.3 Hz. The signals of the b-resorcinol moiety in the case of additional
substitution at C(5) appear as two characteristic singlets at ca. 7.9 and 6.6 ppm,
corresponding to HꢀC(3) and HꢀC(6), respectively. The OH groups are sometimes
invisible in the background on the base line.
In the IR spectrum, there are strong bands in the region of ca. 3400–3100 and 1635–
1610 cmꢀ1, corresponding to n(OꢀH) and n(C¼N). The mass spectra of the compounds
showed molecular-ion peaks, however, with various intensities.
Antiproliferative Activity. The 1,3-thiazolo[5,4-b]pyridines compiled in Scheme 1
have been evaluated for their antiproliferative activities against human bladder cancer
HCV29T cells. The cytotoxic activity in vitro was expressed as IC50, the concentration of
the compound to inhibit proliferation rate of the tumor cells by 50% as compared to the
untreated cells. Cisplatin was used as a reference drug. The results of the screening are
collected in Table 1.
Table 1. Antiproliferative Activity of 4-(1,3-Thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diols against the
a
Human Cancer Cell Line HCV29T Expressed as IC50
)
Compound
IC50 [mg/ml]
Compound
IC50 [mg/ml]
1
2
3
4
5
6
7
8
9
8.34ꢁ1.27
3.39ꢁ0.24
6.70ꢁ0.54
2.37ꢁ0.37
7.61ꢁ0.44
22.14ꢁ2.83
39.82ꢁ2.36
5.61ꢁ1.20
4.09ꢁ0.26
10
11
12
13
14
15
16
17
4.24ꢁ1.80
4.45ꢁ1.12
3.30ꢁ1.11
10.64ꢁ0.58
6.29ꢁ1.03
6.75ꢁ2.14
3.29ꢁ1.27
18.40ꢁ3.16
2.66ꢁ1.15
Cisplatin
a) IC50 [mg/ml] indicates the compound concentration that inhibits the proliferation rate of tumor cells by
50% as compared to the untreated cells. The values are the meansꢁS.D. of nine independent
experiments.
Activity of compounds clearly depends on the substitution pattern of both rings.
Significantly, the highest effect against HCV29T cells was exhibited by compounds 2, 4,
12, and 16 (Table 1). The lowest antiproliferative effect was detected for the
compounds with a third OH group in the resorcinol moiety of 6, 7, and 17. Compounds

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2, 4, 9, 10, 12, and 16, as the most active derivatives of this set of tested 1,3-thiazolo[5,4-
b]pyridines, meet the cytotoxic-activity criterion against HCV29T cancer cell line for
potentially new antiproliferative drugs (IC50 ꢂ4 mg/ml) [28].
The selected compounds of the highest activity against HCV29T were also tested
against A549 (human non-small lung carcinoma), T47D (human breast cancer), and
SW707 (human rectal adenocarcinoma) cells (Table 2). Antiproliferative activities of
compounds 4, 12, and 16 against A549 cells were at the level of cisplatin. At the same
time, compound 4 met the cytotoxicity criterion against this cell line.
Table 2. Antiproliferative Activity of Some Compounds against the Human Cancer Cell Lines T47D,
A549, and SW707, Expressed as IC50
Compound
IC50 [mg/ml]
T47D
A 549
SW707
4
12
16
Cisplatin
4.29ꢁ0.84
4.81ꢁ2.05
6.28ꢁ3.19
2.03ꢁ1.61
3.32ꢁ1.19
6.14ꢁ0.24
4.45ꢁ0.52
5.29ꢁ1.85
10.88ꢁ3.39
10.78ꢁ1.54
11.64ꢁ2.31
3.13ꢁ1.49
Analyzing the structure of compounds and their activity, it was found that the
presence of a Me substituent at C(5) or C(6) of the 1,3-thiazolo[5,4-b]pyridine ring
increases the antiproliferative activity of compounds. The same effect is observed
where a Me (Et) substituent or a Cl-atom is present in the resorcinol moiety, compared
to the unsubstituted parent compound 1. The most beneficial is the presence of an Et
substituent at C(5) of the resorcinol moiety in 4. These effects are not additive when
two substituents are introduced into both rings; it is especially evident in the case of Et
derivatives (i.e., compounds 4 and 11 or 15). Introducing a Me group to the fused ring
of the compounds with three OH substituents in the aryl ring significantly increases the
activity (i.e., compounds 6 and 13 or 17). This analysis reveals that the hydrophobic
substituent (p>0) in the resorcinol ring increases antiproliferative activity. Similar
effects were observed for 2-(2,4-dihydroxyphenyl)-4H-3,1-benzothiazines [29]. It
confirms the earlier finding that the presence of a Cl-atom or Et substituent at C(5)
of the resorcinol moiety of resorcyl-azoles enhances their anticancer properties
[23][30][31].
Conclusions. – In conclusion, an efficient one-pot synthesis of new 4-(1,3-
thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diols has been developed. The studies indicat-
ed high level of antiproliferative activities of some products. Structureꢀactivity
relationship analyses indicated that the presence of hydrophobic substituents at C(5) or
C(6) of the fused ring, or at C(5) of the phenyl residue (especially the Et group)
improves the antiproliferative activities against human cancer cell lines. Taking into
account these findings, further studies including design, synthesis, and analysis of
subsequent derivatives, as well as new groups of compounds with the 2,4-dihydroxy-
phenyl substituent will be carried out.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
53
Experimental Part
General. M.p.: BꢀCHI B-540 (Switzerland) melting-point apparatus; uncorrected. IR Spectra:
PerkinꢀElmer FT-IR 1725X spectrophotometer (in KBr); ˜n in cmꢀ1; recorded in the range of 600–
4000 cmꢀ1. 1H-NMR Spectra: Bruker DRX 500 instrument in (D6)DMSO, CDCl3 or CD3OD solns.; d in
ppm rel. to Me4Si as internal standard, J in Hz. EI-MS (70 eV): AMD-604; in m/z (rel. %). Elemental
analysis: PerkinꢀElmer 2400; within ꢁ0.4% of the theoretical values.
General Procedure for the Synthesis of Compounds 1–17. A mixture of 2-chloropyridin-3-amine
(SigmaꢀAldrich; compounds 1–7) or 6-chloro-3-methylpyridin-5-amine (SigmaꢀAldrich; compounds 8,
10–13), or 6-chloro-2-methylpyridine-5-amine (Acros Organics; compounds 9, 14–17; 0.002 mol), and
the corresponding electrophile (0.0015 mol; Scheme 1) in MeOH (10 ml) was heated to reflux for 3 h.
The hot mixture was filtered. The filtrate was concentrated, and the formed solid was filtered and
crystallized from MeOH (5 ml).
4-(1,3-Thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (1). Yield 89%. M.p. 252–2538. IR: 3068 (OH),
3024 (arom. CꢀH), 1632 (C¼N), 1599 (C¼C), 1561 (C¼C), 1224 (CꢀOH). 1H-NMR ((D6)DMSO):
11.48 (s, HOꢀC(3)); 10.23 (s, HOꢀC(1)); 8.54 (dd, J¼4.7, 1.4, HꢀC(5’)); 8.35 (dd, J¼8.2, 1.4, HꢀC(7’));
7.89 (s, HꢀC(5)); 7.55 (dd, J¼8.2, 4.7, HꢀC(6’)); 6.49 (d, J¼1.7, HꢀC(2)); 6.45 (dd, J¼8.7, 1.8, HꢀC(6)).
EI-MS: 244 (100, Mþ ), 216 (10), 187 (37), 174 (3), 155 (4), 137 (2), 122 (2), 108 (11), 93 (3), 82 (5), 69
(4), 51(4), 39 (7). Anal. calc. for C12H8N2O2S (244.27): C 59.00, H 3.30, N 11.47; found: C 59.22, H 3.29, N
11.50.
2-Methyl-4-(1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (2). Yield 87%. M.p. 287–2898. IR:
3240 (OH), 3092 (OH), 3035 (arom. CꢀH), 2925 (CH), 2856 (CH), 1624 (C¼N), 1598 (C¼C), 1557
(C¼C), 1507 (C¼C), 1214 (CꢀOH). 1H-NMR (CD3OD): 12.25 (s, HOꢀC(3)); 10.43 (s, HOꢀC(1)); 8.55
(dd, J¼4.7, 1.4, HꢀC(5’)); 8.35 (dd, J¼8.2, 1.5, HꢀC(7’)); 7.58 (m, HꢀC(6’), HꢀC(5)); 6.53 (d, J¼8.7,
HꢀC(6)); 2.07 (s, Me). EI-MS: 258 (100, Mþ ), 229 (25), 201 (6), 175 (4), 129 (5), 106 (3), 83 (3), 65 (3),
39 (6). Anal. calc. for C13H10N2O2S (258.30): C 60.45, H 3.90, N 10.85; found: C 60.28, H 3.89, N 10.89.
4-Methyl-6-(1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (3). Yield 79%. M.p. 241–2438. IR:
3236 (OH), 3128 (OH), 2922 (CH), 2855 (CH), 1615 (C¼N), 1526 (C¼C), 1505 (C¼C), 1215 (CꢀOH).
1H-NMR ((D6)DMSO): 10.30 (s, HOꢀC(3)); 8.54 (dd, J¼4.7, 1.4, HꢀC(5’)); 8.31 (dd, J¼8.2, 1.4,
HꢀC(7’)); 7.89 (s, HꢀC(5)); 7.55 (dd, J¼8.2, 4.7, HꢀC(6’)); 6.60 (s, HꢀC(2)); 2.13 (s, Me). EI-MS: 258
(100, Mþ ), 229 (12), 201 (10), 187 (13), 120 (5), 106 (4), 83 (4), 69 (4), 39 (6). Anal. calc. for
C13H10N2O2S (258.30): C 60.45, H 3.90, N 10.85; found: C 60.57, H 3.91, N 10.82.
4-Ethyl-6-(1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (4). Yield 89%. M.p. 264–2658. IR: 3427
(OH), 3106 (arom. CꢀH), 2964 (CꢀH), 2923 (CꢀH), 2856 (CꢀH), 1609 (C¼N), 1500 (C¼C), 1205
(CꢀOH). 1H-NMR ((D6)DMSO): 10.34 (s, HOꢀC(3)); 8.56 (dd, J¼4.7, 1.4, HꢀC(5’)); 8.36 (dd, J¼8.2,
1.4, HꢀC(7’)); 7.91 (s, HꢀC(5)); 7.58 (q, J¼8.2, 4.7, HꢀC(6’)); 6.64 (s, HꢀC(2)); 2.56 (m, MeCH2); 1.17 (t,
MeCH2). 13C-NMR ((D6)DMSO): 165.8; 160.5; 156.0; 155.0; 145.3; 143.8; 129.9; 128.4; 123.3; 122.0;
109.7; 102.4; 22.2; 14.3. EI-MS: 272 (55, Mþ ), 257 (100, [MꢀMe]þ ), 229 (3), 187 (7), 136 (3), 83 (3), 69
(3), 39 (3). Anal. calc. for C14H12N2O2S (272.31): C 61.74, H 4.44, N 10.28; found: C 61.80, H 4.43, N
10.31.
4-Chloro-6-(1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (5). Yield 86%. M.p. 234–2378. IR:
1
3022 (OH, arom. CꢀH), 1619 (C¼N), 1598 (C¼C), 1192 (CꢀOH). H-NMR ((D6)DMSO): 11.68 (s,
HOꢀC(1)); 10.73 (s, HOꢀC(3)); 8.56 (dd, J¼4.7, 1.4, HꢀC(5’)); 8.36 (dd, J¼8.2, 1.4, HꢀC(7’)); 7.91 (s,
HꢀC(5)); 7.58 (q, J¼8.2, 4.7, HꢀC(6’)); 6.72 (s, HꢀC(2)). EI-MS: 278 (100, Mþ ), 243 (11), 215 (8), 187
(23), 136 (7), 121 (5), 95 (4), 69 (6), 51 (5), 39 (4). Anal. calc. for C14H7ClN2O2S (278.71): C 51.71, H
2.53, N 10.05; found: C 51.90, H 2.52, N 10.02.
4-(1,3-Thiazolo[5,4-b]pyridin-2-yl)benzene-1,2,3-triol (6). Yield 76%. M.p. 337–3408. IR: 3042 (OH,
arom. CꢀH), 1629 (C¼N), 1606 (C¼C), 1192 (CꢀOH). 1H-NMR ((D6)DMSO): OH signals not visible in
the background on the base line; 8.56 (dd, J¼4.7, 1.4, HꢀC(5’)); 8.36 (dd, J¼8.2, 1.4, HꢀC(7’)); 7.57 (q,
J¼8.2, 4.7, HꢀC(6’)); 7.51 (d, J¼8.7, HꢀC(5)); 6.56 (d, J¼8.7, HꢀC(6)). 13C-NMR ((D6)DMSO): 166.7;
155.2; 150.3; 147.4; 145.3; 144.5; 133.1; 129.8; 122.0; 119.4; 110.9; 108.9. EI-MS: 260 (100, Mþ ), 231 (4),
203 (3), 187 (3), 176 (4), 161 (3), 67 (7), 39 (3). Anal. calc. for C12H8N2O3S (260.27): C 55.38, H 3.10, N
10.76; found: C 55.52, H 3.09, N 10.71.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2-(1,3-Thiazolo[5,4-b]pyridin-2-yl)benzene-1,3,5-triol (7). Yield 69%. M.p. 285–2878. IR: 3338
(OH), 2923 (CH), 2854 (CH), 1621 (C¼N), 1222 (CꢀOH). EI-MS: 260 (100, Mþ ), 231 (3), 203 (4), 190
1
(4), 176 (10), 163 (5), 116 (6), 69 (4), 39 (3). H-NMR ((D6)DMSO): 12.53 (s, HOꢀC(1), HOꢀC(3));
10.23 (s, HOꢀC(5)); 8.56 (dd, J¼4.6, 1.4, HꢀC(5’)); 8.32 (dd, J¼8.2, 1.4, HꢀC(7’)); 7.55 (dd, J¼8.2, 4.6,
HꢀC(6’)); 6.01 (s, HꢀC(4,6)). Anal. calc. for C12H8N2O3S (260.27): C 55.38, H 3.10, N 10.76; found: C
55.20, H 3.08, N 10.72.
4-(6-Methyl-1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (8). Yield 86%. M.p. 268–2708. IR:
3428 (OH), 3069 (OH), 2921 (CH), 2859 (CH), 1635 (C¼N), 1594 (C¼C), 1510 (C¼C), 1212 (CꢀOH).
1H-NMR ((D6)DMSO): OH signals not visible in the background on the base line; 8.44 (d, J¼1.3,
HꢀC(5’)); 8.19 (m, HꢀC(7’)); 8.00 (d, J¼8.7, HꢀC(5)); 6.54 (d, J¼2.3, HꢀC(2)); 6.47 (dd, J¼2.3, 8.7,
HꢀC(6)); 2.46 (s, Me). EI-MS: 258 (100, Mþ ), 230 (8), 201 (31), 169 (3), 137 (2), 115 (5), 69 (4), 38 (9).
Anal. calc. for C13H10N2O2S (258.30): C 60.45, H 3.90, N 10.85; found: C 60.31, H 3.93, N 10.81.
4-(5-Methyl-1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (9). Yield 82%. M.p. 261–2638. IR:
3430 (OH), 3334 (OH), 3153 (OH), 2859 (CH), 1636 (C¼N), 1600 (C¼C), 1580 (C¼C), 1540 (C¼C),
1183 (CꢀOH). 1H-NMR ((D6)DMSO): OH signals not visible in the background on the base line; 8.29
(d, J¼8.3, HꢀC(7’)); 7.99 (d, J¼8.7, HꢀC(5)); 7.47 (d, J¼8.3, HꢀC(6’)); 6.54 (d, J¼1.7, HꢀC(2)); 6.48
(dd, J¼8.7, 1.8, HꢀC(6)); 2.65 (s, Me). 13C-NMR ((D6)DMSO): 164.9; 162.3; 158.4; 153.9; 153.6; 143.7;
130.5; 129.8; 122.2; 110.2; 108.7; 102.6; 22.4. EI-MS: 258 (100, Mþ ), 246 (13), 230 (13), 213 (46), 201 (22),
169 (2), 53 (4), 36 (17). Anal. calc. for C13H10N2O2S (258.30): C 60.45, H 3.90, N 10.85; found: C 60.57, H
3.93, N 10.80.
4-Methyl-6-(6-methyl-1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (10). Yield 88%. M.p. 253–
2568. IR: 3142 (OH), 3009 (arom. CꢀH), 2922 (CꢀH), 1625 (C¼N), 1209 (CꢀOH). 1H-NMR
((D6)DMSO): 12.30 (s, HOꢀC(1)); 10.50 (s, HOꢀC(3); 8.40 (d, J¼1.3, HꢀC(5’)); 8.15 (m, HꢀC(7’)); 7.52
(d, J¼8.7, HꢀC(5)); 6.61 (d, J¼8.7, HꢀC(6)); 2.64 (s, Me); 2.06 (s, Me). 13C-NMR ((D6)DMSO): 172.9;
163.7; 160.5; 155.1; 150.0; 148.0; 135.3; 132.0; 130.3; 114.3; 112.0; 111.6; 21.1; 11.5. EI-MS: 272 (100, Mþ ),
243 (30), 215 (12), 201 (7), 189 (5), 160 (3), 151 (3), 136 (4), 97 (4), 78 (2), 36 (11). Anal. calc. for
C14H12N2O2S (272.32): C 61.75, H 4.44, N 10.29; found: C 61.93, H 4.42, N 10.17.
4-Ethyl-6-(6-methyl-1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (11). Yield 78%. M.p. 215–
2178. IR: 3220 (OH), 3150 (OH), 2857 (CH), 1612 (C¼N), 1527 (C¼C), 1511 (C¼C), 1183 (CꢀOH).
1H-NMR ((D6)DMSO): 10.27 (s, HOꢀC(3)); 8.40 (d, J¼1.3, HꢀC(5’)); 8.17 (m, HꢀC(7’)); 7.84 (s,
HꢀC(5)); 6.59 (s, HꢀC(2)); 2.55 (q, J¼7.5, MeCH2); 2.45 (s, Me); 1.16 (t, J¼7.5, MeCH2). EI-MS: 286
(Mþ, 55), 271, (100, [MꢀMe]þ ), 243 (6), 215 (4), 201 (8), 143 (3), 97 (3), 69 (4), 39 (3). Anal. calc. for
C15H14N2O2S (286.35): C 62.92, H 4.93, N 9.78; found: C 63.04, H 4.91, N 9.81.
4-Chloro-6-(6-methyl-1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (12). Yield 86%. M.p. 227–
1
2298. IR: 3013 (OH, arom. CꢀH), 1627 (C¼N), 1609 (C¼C), 1188 (CꢀOH). H-NMR ((D6)DMSO):
11.60 (s, HOꢀC(1)); 10.90 (s, HOꢀC(3); 8.43 (d, HꢀC(5’)); 8.18 (m, HꢀC(7’)); 8.15 (s, HꢀC(5)); 6.86 (s,
HꢀC(2)); 2.44 (s, Me). EI-MS: 292 (100, Mþ ), 264 (6), 258 (4), 201 (21), 187 (2), 132 (3), 122 (3), 95 (3),
69 (5), 51 (4), 39 (5). Anal. calc. for C13H9ClN2O2S (292.74): C 53.34, H 3.10, N 9.57; found: C 53.57, H
3.09, N 9.62.
4-(6-Methyl-1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,2,3-triol (13). Yield 72%. M.p. 310–3138. IR:
3435 (OH), 3045 (arom. CꢀH), 2922 (CꢀH), 1631 (C¼N), 1598 (C¼C), 1548 (C¼C), 1183 (CꢀOH).
1H-NMR ((D6)DMSO): 11.95 (s, HOꢀC(3)); 9.13 (s, HOꢀC(1)); 8.73 (s, HOꢀC(2)); 8.40 (d, J¼1.5,
HꢀC(5’)); 8.15 (m, HꢀC(7’)); 7.41 (d, J¼8.7, HꢀC(5)); 6.53 (d, J¼8.7, HꢀC(6)); 2.45 (s, Me). 13C-NMR
((D6)DMSO): 167.2; 153.6; 149.8; 147.3; 147.1; 144.4; 133.0; 131.4; 128.3; 119.2; 110.7; 108.5; 17.8. EI-MS:
274 (100, Mþ ), 245 (5), 209 (7), 201 (6), 190 (8), 175 (7), 164 (4), 137 (3), 123 (4), 95 (2), 81 (2), 65 (1),
53 (2), 39 (2). Anal. calc. for C13H10N2O3S (274.30): C 56.92, H 3.67, N 10.21; found: C 57.13, H 3.66, N
10.18.
4-Methyl-6-(5-methyl-1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (14). Yield 81%. M.p. 251–
2548. IR: 3116 (OH), 2922 (CꢀH), 1624 (C¼N), 1504 (C¼C), 1236 (CꢀOH). 1H-NMR (CDCl3): 12.30 (s,
HOꢀC(1)); 10.31 (s, HOꢀC(3)); 8.23 (d, J¼8.3, HꢀC(7’)); 7.53 (d, J¼8.7, HꢀC(5)); 7.44 (d, J¼8.4,
HꢀC(6’)); 7.60 (d, J¼8.7, HꢀC(2)); 2.61 (s, Me); 2.07 (s, Me). EI-MS: 272 (100, Mþ ), 243 (22), 227 (7),
215 (10), 201 (6), 188 (3), 65 (4), 51 (3), 39 (7). Anal. calc. for C14H12N2O2S (272.32): C 61.75, H 4.44, N
10.29; found: C 61.92, H 4.42, N 10.32.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
55
4-Ethyl-6-(5-methyl-1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (15). Yield 77%. M.p. 270–
2738. IR: 3385 (OH), 3078 (arom. CꢀH), 2969 (CH), 1619 (C¼N), 1503 (C¼C), 1250 (CꢀOH). 1H-NMR
((D6)DMSO): 10.32 (s, HOꢀC(3)); 8.33 (d, J¼8.3, HꢀC(7’)); 7.87 (s, HꢀC(5)); 7.49 (d, J¼8.4, HꢀC(6’));
6.63 (s, HꢀC(2)); 2.67 (s, Me); 2.52 (m, MeCH2); 1.16 (t, MeCH2). 13C-NMR ((D6)DMSO): 165.1; 160.5;
156.7; 152.5; 152.4; 144.4; 131.4; 128.2; 123.3; 122.6; 109.5; 102.3; 22.1; 21.5; 14.3. EI-MS: 286 (54, Mþ ),
271 (100, [MꢀMe]þ ), 243 (7), 215 (4), 201 (7), 143 (7), 69 (3), 36 (29). Anal. calc. for C15H14N2O2S
(286.35): C 62.92, H 4.93, N 9.78; found: C 63.18, H 5.00, N 9.69.
4-Chloro-6-(5-methyl-1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diol (16). Yield 85%. M.p. 278–
1
2808. IR: 2995 (CꢀH), 1633 (C¼N), 1584 (C¼C), 1525 (C¼C), 1185 (CꢀOH), 1100 (CꢀCl). H-NMR
((D6)DMSO): 11.80 (s, HOꢀC(1)); 11.10 (s, HOꢀC(3)); 8.27 (d, J¼8.3, HꢀC(7’)); 8.14 (s, HꢀC(5)); 7.45
(d, J¼8.3, HꢀC(6’)); 6.87 (d, HꢀC(2)); 2.64 (s, Me). 13C-NMR ((D6)DMSO): 162.5; 157.1; 156.6; 153.5;
153.7; 143.7; 130.9; 128.4; 122.3; 112.2; 111.5; 103.8; 22.4. EI-MS: 292 (100, Mþ ), 265 (8), 259 (5), 201
(24), 132 (2), 121 (4), 94 (6), 69 (7), 51 (3), 39 (6). Anal. calc. for C13H9ClN2O2S (292.74): C 53.34, H
3.10, N 9.57; found: C 53.48, H 3.09, N 9.54.
4-(5-Methyl-1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,2,3-triol (17). Yield 82%. M.p. 319–3218. IR:
3450 (OH), 3127 (OH), 1630 (C¼N), 1227 (CꢀOH). 1H-NMR ((D6)DMSO): 9.42 (s, HOꢀC(2)); 8.31 (d,
J¼8.3, HꢀC(7’)); 7.46 (m, HꢀC(6’), HꢀC(5)); 6.54 (d, J¼8.7, HꢀC(6)); 2.65 (s, Me). EI-MS: 274 (100,
Mþ ), 245 (4), 217 (4), 201 (6), 190 (7), 175 (6), 137 (3), 123 (4), 81 (2), 39 (2). Anal. calc. for
C13H10N2O3S (274.30): C 56.92, H 3.67, N 10.21; found: C 57.13, H 3.65, N 10.24.
Bioassays. The following human cell lines were used in vitro: T47D (breast cancer), SW707 (rectal
adenocarcinoma), and A549 (nonsmall cell lung carcinoma) from the American Type Culture Collection
(Rockville, Maryland, USA), and HCV29T (bladder cancer) from the Fibiger Institute, DK-
Copenhagen. Twenty-four hours before the addition of the agents to be tested, the cells were plated in
96-well plates (Sarstedt, USA) at a density of 104 cells/well. All cell lines were maintained in the opti-
MEM medium supplement with 2 mm glutamine (Gibco; PL-Warsaw), streptomycin (50 mg/ml),
penicillin (50 U/ml; Polfa; PL-Tarchomin), and 5% fetal calf serum (Gibco, Grand Island, USA). The
cells were incubated at 378 in a humid atmosphere saturated with 5% CO2. The solns. of compounds
(1 mg/ml) were prepared ex tempore by dissolving the substance in 100 ml of DMSO completed with
900 ml of tissue culture medium. Then, the compounds were diluted in the culture medium to reach the
final concentrations ranging from 0.1 to 100 mg/ml. The solvent (DMSO) at the highest concentration
used in the test did not reveal any cytotoxic activity. Cisplatin was applied as a test reference agent. The
cytotoxicity assay was performed after 72 h exposure of the cultured cells at the concentration ranging
from 0.1 to 100 mg/ml of the tested agents. The sulforhodamine B (SRB) test to determine the cell
proliferation inhibition in in vitro culture was applied [32]. The cells attached to the plastic were fixed
with cold 50% CCl3CO2H (Aldrich-Chemie, Germany) added on the top of the culture medium in each
well. The plates were incubated at 48 for 1 h and then washed five times with tap water. The background
optical density was measured in the wells filled with culture medium without the cells. The cellular
material fixed with CCl3CO2H was stained with 0.4% SRB (Sigma; Germany) dissolved in 1% AcOH
(POCh, PL-Gliwice) for 30 min. The unbound dye was removed by rinsing (four times) with 1% AcOH,
and the protein-bound dye was extracted with 10 mm unbuffered Tris base (tris(hydroxymethyl)amino-
methane; POCh, PL-Gliwice) for determination of the optical density (at 540 nm) in a computer-
interfaced, 96-well microtiter plate reader Uniskan II (Labsystems, FI-Helsinki). The compounds were
tested in triplicates per experiment. The experiments were repeated at least three times.
Cisplatin was used for comparison. It is not characterized by great stability, but it has been used as the
reference drug in biological studies of new potential anticancer agents [33]. It was reported that it is
stable for 24 h in the admixtures containing NaCl concentrations of 0.3% or larger [34]. Our studies
showed that its biological effect starting with the 24-h incubation was stable and did not depend on
incubation time.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Received January 9, 2011