Laccase-catalyzed green synthesis and cytotoxic activity of novel pyrimidobenzothiazoles and catechol thioethers

Article abstract of DOI:10.1039/C6RA28102H

The laccase-catalyzed reaction between unsubstituted catechol and 2-thioxopyrimidin-4(1H)-ones using aerial O₂ as the oxidant delivers novel pyrimidobenzothiazoles with high yields in an aqueous solvent system under mild reaction conditions. With 4-substituted catechols, catechol thioethers are formed exclusively. The synthetic protocols developed provide a sustainable approach for these compound classes. In addition, the cytotoxicity of the products against HepG2 cell line is reported. Most compounds exhibit antiproliferative activities with IC₅₀ values at the micromolar level. A structure-activity relationship study will facilitate the further development of these compounds as cytotoxic agents.

Full text of DOI:10.1039/C6RA28102H

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PAPER
Laccase-catalyzed green synthesis and cytotoxic
activity of novel pyrimidobenzothiazoles and
catechol thioethers†‡
Cite this: RSC Adv., 2017, 7, 17427
a
b
c
d
b
H. T. Abdel-Mohsen, J. Conrad, K. Harms, D. Nohr and U. Beifuss*
The laccase-catalyzed reaction between unsubstituted catechol and 2-thioxopyrimidin-4(1H)-ones using
aerial O as the oxidant delivers novel pyrimidobenzothiazoles with high yields in an aqueous solvent
2
system under mild reaction conditions. With 4-substituted catechols, catechol thioethers are formed
exclusively. The synthetic protocols developed provide a sustainable approach for these compound
classes. In addition, the cytotoxicity of the products against HepG2 cell line is reported. Most
compounds exhibit antiproliferative activities with IC50 values at the micromolar level. A structure–
activity relationship study will facilitate the further development of these compounds as cytotoxic agents.
Received 12th December 2016
Accepted 15th February 2017
DOI: 10.1039/c6ra28102h
rsc.li/rsc-advances
natural origin (e.g. enzymes), the development of one-pot multi-
step reactions, which reduce the amount of waste formed
Introduction
Pyrimidines are the building blocks of pyrimidine nucleosides during reaction and work up as well as the development of
4,6
and thiamine (vitamin B1). Therefore, the pyrimidine skeleton environmentally benign reaction conditions.
is regarded as an interesting scaffold for the development of
Hepatocellular carcinoma (HCC) is regarded as one of the
1
molecules with potential applications in medicine. Over the leading causes of cancer related mortality in the world. Every
years, a wide range of pharmaceutical agents with a pyrimidine year more than half a million new cases with HCC are diag-
1
–3
moiety have been developed. Zidovudine, stavudine and other nosed worldwide. Hepatitis B, hepatitis C viruses and non-
2
,3a
potent anti-HIV agents are pyrimidines.
drugs with a pyrimidine ring are the antitumor agents uoro- development of chronic liver disease and subsequent develop-
Other important alcoholic fatty liver disease are the main risk factors for the
7
a,b
uracil (I), tegafur (II), nimustine (III), monastrol (IV) and pazo- ment of HCC. Despite the tremendous progress that has been
2,3b,c
panib (V) (Fig. 1).
achieved in cancer therapy over the last decades, the currently
Recently, the development of more environmentally friendly available drugs suffer from serious disadvantages, such as lack
approaches for the synthesis of pharmaceutical active ingredi-
ents has increasingly come into focus of the pharmaceutical
4
chemistry. Green chemistry is a valuable concept for the
development of new, more effective, less toxic and cost efficient
methods for the synthesis of bioactive molecules. This can be
achieved, for example, by developing highly atom economic
5
transformations, using nontoxic reagents and catalysts of
a
Chemistry of Natural and Microbial Products Department, Pharmaceutical Industries
Research Division, National Research Centre, Cairo, Egypt
b
Bioorganische Chemie, Institut f u¨ r Chemie, Universit a¨ t Hohenheim, Garbenstr. 30,
Stuttgart, D-70599, Germany. E-mail: ubeifuss@uni-hohenheim.de; Fax: +49 711
4
59 22951; Tel: +49 711 459 22171
c
Fachbereich Chemie, Universit a¨ t Marburg, Hans-Meerwein-Str. 4, D-35032 Marburg,
Germany
d
Institut f u¨ r Biologische Chemie und Ern a¨ hrungswissenscha, Universit a¨ t Hohenheim,
Garbenstr. 30, Stuttgart, D-70599, Germany
†
This paper is dedicated to Professor Dr Dr hc Wolfgang Haubold on the occasion
th
of his 80 birthday.
1
‡
Electronic supplementary information (ESI) available: Copies of the H NMR
13
and C NMR spectra. CCDC 1514742 and 1514743. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c6ra28102h
Fig. 1 Antitumor agents with a pyrimidine moiety.
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3b,7c
of selectivity, toxicity and resistance that limit their use.
For
these reasons, the development of novel anti-tumor agents
remains a challenge.
Against this background, the importance of enzyme-
catalyzed transformations in organic synthesis is steadily
8
growing. Laccases (benzenediol:
2
O oxidoreductase E.C.
1
.10.3.2.), which are multicopper oxidases, are among the most
9
attractive enzymes in this respect. Over the last few years, lac-
cases have proven their ability to catalyze a number of impor-
tant oxidative transformations in aqueous solvent systems
under mild reaction conditions (temperature, pH, pressure)
using aerial oxygen as an oxidant. The oxidation of the
substrates is linked to the reduction of oxygen to water which is
the only byproduct of laccase-catalyzed reactions. Moreover, the
application range of laccases can be broadened by employing
mediators. Using laccase-mediator systems, the oxidation of
substrates with higher oxidation potentials can also be ach-
10
ieved. Laccases have been successfully used to catalyze the
11
oxidation of a number of functional groups, the dimerization
12
of phenolic compounds by oxidative coupling and the oxida- Fig. 3 Substrates for the laccase-catalyzed transformations.
tion of catechols and hydroquinones to the corresponding
highly reactive quinoid systems, followed by reaction with
1
3–16
different nucleophiles.
simple 1,4-adducts
been made available.
With the latter approach, a range of
Results and discussion
as well as heterocyclic systems has
1
3–15
16
Fig. 3 depicts the structures of the catechols 1 and the 2,3-
dihydro-2-thioxopyrimidin-4(1H)-ones 2 that were chosen as
substrates for the enzyme-catalyzed oxidative transformations.
The catechols 1a–c are commercially available, while the 2,3-
dihydro-2-thioxopyrimidin-4(1H)-ones 2a–h were synthesized by
In contrast to the additions of C and N nucleophiles, only
little is known about laccase-initiated 1,4-additions of S
Recently, we have studied the laccase-
catalyzed generation of o-quinones and their reaction with
different S nucleophiles for the synthesis of thioethers as well
as pyrimidobenzothiazoles.
have shown the successful application of laccases for the
synthesis of 2,3-ethylenedithio-1,4-quinones.
Schauer et al. reported on the multiple C–S bond formation
between p-hydroquinones and aromatic thiols using laccase as
1
5,16b,d
nucleophiles.
reaction of different acyclic b-ketoesters 6a–h with thiourea
1
5b,16d
Ragauskas and coworkers
ꢀ
(
7) under basic conditions in ethanol at 80 C (general
17
procedure I). 2,3,6,7-Tetrahydro-2-thioxo-1H-cyclopenta[d]pyr-
imidin-4(5H)-one (2i) was obtained from cyclic b-ketoester 6i as
substrate (Scheme 1).
1
6b
In 2016,
First, the laccase-catalyzed reactions with the unsubstituted
catechol (1a) were studied. In an initial experiment, 0.58 mmol
1
5a
a catalyst.
Here, we report on the laccase-catalyzed reaction between
catechols 1 and 2,3-dihydro-2-thioxopyrimidin-4(1H)-ones 2 for
the green synthesis of novel biologically active compounds.
Depending on the substitution pattern of the catechols 1, either
pyrimidobenzothiazoles 3, 4 or catechol thioethers 5 are formed
(
1.16 equiv.) catechol (1a) were reacted with 0.50 mmol (1
equiv.) 2,3-dihydro-6-methyl-2-thioxopyrimidin-4(1H)-one (2a)
in the presence of 12 U laccase from Agaricus bisporus (1.2 U
mg
buffer (pH 6) and ethanol under air (general procedure II). The
reaction proceeded smoothly at room temperature and deliv-
ered 97% of a crude product (purity > 95%; H NMR) of a 37 : 63
ꢁ1 18
) as a catalyst in 20 mL of a 9 : 1 mixture of phosphate
(Fig. 2). Moreover, the cytotoxic activity of selected reaction
products against HepG2 cell line is reported.
1
mixture of the two regioisomers 7,8-dihydroxy-4H-2-methyl-
pyrimido[2,1-b]benzothiazol-4-one (3a) and 7,8-dihydroxy-2H-4-
methyl-pyrimido[2,1-b]benzothiazol-2-one (4a) aer 12 h (Table
1, entry 1). A control experiment was conducted to show that the
Fig. 2 Structures of the regioisomeric pyrimidobenzothiazoles 3, 4 Scheme
and the catechol thioethers 5. 2a–i.
1
Synthesis of 2,3-dihydro-2-thioxopyrimidin-4(1H)-ones
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Table 1 Laccase-catalyzed reaction between catechol (1a) and 2,3- Optimization experiments revealed that the reactions can be
dihydro-2-thioxopyrimidin-4(1H)-ones 2a–h
driven to completion by simply increasing the amount of lac-
case and solvent by a factor of 2. When 0.29 mmol catechol (1a)
were reacted with 0.25 mmol of the 2,3-dihydro-2-thioxopyr-
imidin-4(1H)-ones 2g, h in the presence of 12 U laccase from A.
bisporus in 20 mL of a 9 : 1 mixture of phosphate buffer (pH 6)
and ethanol under air (general procedure III) a mixture of
regioisomers 3g and 4g in 89% yield (purity of crude product
1
>
95%; H NMR) and the regioisomer 3h in 95% yield were
1
isolated (purity of crude product >95%; H NMR) (Table 1,
entries 7, 8).
It can be expected that the reactions proceed via the laccase-
catalyzed in situ oxidation of catechol (1a) to the highly reactive
o-quinone (8a) (Scheme 2). The latter one is then attacked at C-4 by
the S-atom of the 2,3-dihydro-2-thioxopyrimidin-4(1H)-ones 2a–h
to give the tautomers 9A and 10A. The intermolecular 1,4-addition
is followed by a second laccase-catalyzed oxidation, namely the
oxidation of the intermediates 9A and 10A to 9B and 10B,
respectively. The nal intramolecular 1,4-addition proceeds either
by nucleophilic attack of N-3 or N-1 and delivers the correspond-
ing regioisomeric cyclization products 3 and 4, respectively.
Yield of
3 + 4 (%)
1
2
d
e
Entry
2
R
R
t (h)
3 + 4
3 : 4
a
1
a
b
c
d
e
f
g
h
a
CH
3
H
H
H
H
H
H
CH
CH
H
12
12
18
13
16
16
28
17
72
a
b
c
d
e
f
g
h
—
37 : 63
54 : 46
44 : 56
76 : 24
100 : 0
100 : 0
36 : 64
100 : 0
—
97
95
94
95
97
78
89f
95
a
2
C
C
2
H
H
5
a
3
3
7
a
4
CH(CH
C(CH
CF
CH
CF
CH
3
)
2
a
5
3
)
3
a
1
6
3
It is interesting to note that the substituent R at C-6 of the
b
7
3
3
2
,3-dihydro-2-thioxopyrimidin-4(1H)-ones 2a–h exerts a tremen-
dous inuence on the ratio of the regioisomeric 7,8-dihydroxy-
H-pyrimido[2,1-b]benzothiazol-4-ones 3 and 7,8-dihydroxy-2H-
b
8
3
3
c
g
9
3
—
4
a
General procedure II: reactions were carried out using 0.58 mmol
catechol (1a), 0.50 mmol 2,3-dihydro-2-thioxopyrimidin-4(1H)-ones 2a–
f, laccase (A. bisporus, 10 mg, 12 U), phosphate buffer pH 6 (18 mL)
b
and EtOH (2 mL). General procedure III: reactions were carried out
using 0.29 mmol catechol (1a), 0.25 mmol 2,3-dihydro-2-
thioxopyrimidin-4(1H)-ones 2g, h, laccase (A. bisporus, 10 mg, 12 U),
c
phosphate buffer pH 6 (18 mL) and EtOH (2 mL). Reaction was
carried out using 0.58 mmol 1a, 0.50 mmol 2a, phosphate buffer pH 6
d
(
18 mL) and EtOH (2 mL). Ratios of regioisomers were determined
1
e
by H NMR of the crude products. Yields refer to crude products
purity > 95%, as determined by H NMR). Crude product contain
traces of unknown impurities. No reaction.
1
f
(
g
formation of the two regioisomers 3a and 4a does not proceed
in the absence of the laccase (Table 1, entry 9). Subsequently,
catechol (1a) was reacted with the other monosubstituted 2,3-
dihydro-2-thioxopyrimidin-4(1H)-ones 2b–f under similar
conditions (Table 1, entries 2–6). With 2b–d, mixtures of the
corresponding regioisomers 3b–d and 4b–d were obtained in
1
excellent yields (purity of crude products >95%; H NMR) (Table
1, entries 2–4). When the 6-tert-butyl- and the 6-triuoromethyl
substituted 2,3-dihydro-2-thioxopyrimidin-4(1H)-ones 2e, f were
used as substrates, the 7,8-dihydroxy-4H-pyrimido[2,1-b]
benzothiazol-4-ones 3e and 3f were isolated exclusively (Table
1
, entries 5, 6). It seems that with increasing space demand of
1
the R group in the 2,3-dihydro-2-thioxopyrimidin-4(1H)-ones 2,
the formation of regioisomers 3 is favoured over the formation
of 4.
When the disubstituted 2,3-dihydro-2-thioxopyrimidin-
4(1H)-ones 2g, h which carry an additional methyl group in 5-
position were employed as substrates, the reactions were not
complete even aer 72 h. In addition to the cyclized products
Scheme 2 Mechanistic proposal for the laccase-catalyzed synthesis
1
3g/4g and 3h, uncyclized material could be detected by H NMR. of pyrimidobenzothiazoles 3, 4.
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pyrimido[2,1-b]benzothiazol-2-ones 4. With unbranched alkyl
groups at C-6, the formation of 7,8-dihydroxy-2H-pyrimido[2,1-
b]benzothiazol-2-ones 4 is either favoured or roughly equal
amounts of the two isomers 3 and 4 are observed (Table 1,
entries 1–3, 7). With branched alkyl groups (i-propyl- or tert-
butyl group) or a triuoromethyl group at C-6, the 7,8-dihy-
droxy-4H-pyrimido[2,1-b]benzothiazol-4-ones
3 are strongly
favoured (Table 1, entries 4–6, 8). These ndings are in good
agreement with the assumption that the intramolecular 1,4-
addition via N-1 in 10B to regioisomers 4 (Scheme 2) is hindered
by sterically demanding as well as strongly electronegative
substituents.
The reactions between 1a and 2a–g delivered only either one
major product (3e, 3f) or two major products, consisting of Scheme 3 Mechanistic proposal for the laccase-catalyzed synthesis
of catechol thioethers 5a–i.
exclusively mixtures of the regioisomeric products 3a–d,g and
4
a–d,g in remarkably high yields. No other products could be
1
detected by H NMR. Only the reaction between 1a and 2h
delivered in addition to 3h traces of an additional product of
unknown structure (Table 1, entry 8).
1
7
6 and 92% (purity of crude products >95%; H NMR) (Table 2).
Obviously, only the S atoms of the 2,3-dihydro-2-thioxopyr-
imidin-4(1H)-ones 2 act as nucleophiles in the intermolecular
Subsequently, the laccase-catalyzed reactions of 4-
substituted catechols 1b, c with 2,3-dihydro-2-thioxopyrimidin-
1,4-additions. This can be attributed to the higher nucleophi-
licity of 2-S in comparison to N-1 and N-3 (Scheme 3).
Finally, the laccase-catalyzed reactions between the 4-
substituted catechols 1b, c and 2,3,6,7-tetrahydro-2-thioxo-1H-
cyclopenta[d]pyrimidin-4(5H)-one (2i) were studied (general
procedure II) to give the corresponding catechol thioethers 5j, k
in high yields (Table 3).
It can be taken for granted that the reactions start with the
laccase-catalyzed oxidation of the 4-substituted catechols 1b, c to
the corresponding o-benzoquinones 8b, c. This is followed by
intermolecular 1,4-addition of the 2,3-dihydro-2-thioxopyrimidin-
4
(1H)-ones 2a, c–e, g were studied. For this purpose, 4-methyl-
catechol (1b) and 4-ethylcatechol (1c) were reacted with selected
,3-dihydro-2-thioxopyrimidin-4(1H)-ones 2a, c–e, g under the
2
conditions developed for the reactions of unsubstituted cate-
chol (1a) (general procedure II). In all cases, the corresponding
thioethers were formed exclusively with yields ranging between
Table 2 Laccase-catalyzed synthesis of catechol thioethers 5
4(1H)-ones 2 via the S atom at C-5 to afford the corresponding
catechol thioethers 5a–i as sole products. Due to the presence of
a methyl or an ethyl substituent at C-4 of the catechol, the
intramolecular 1,4-addition occurs exclusively at C-5 (Scheme 3).
It should be highlighted that the reactions presented are
highly efficient and deliver the products 3, 4 and 5 in remarkably
Table
3 Synthesis of dihydroxyphenylthio-6,7-dihydro-3H-cyclo-
penta[d]pyrimidin-4(5H)-ones 5j,
k
by laccase-catalyzed trans-
formations under aerobic conditions
a
1
2
b
Entry
1
R
2
R
R
Time (h)
5
Yield (%)
1
2
3
4
5
6
7
8
9
b
b
b
b
b
c
c
c
c
CH
CH
CH
CH
CH
3
3
3
3
3
a
c
CH
3
H
H
H
H
CH
H
H
H
CH
12
13
16
17
20
14
12
16
16
a
b
c
d
e
f
g
h
i
82
88
92
90
86
90
76
81
91
C
3
H
7
d
e
g
a
d
e
g
CH(CH
C(CH
3
)
)
2
2
3
)
3
CH
CH
3
3
C
2
C
2
C
2
C
2
H
H
H
H
5
5
5
5
3
a
b
Entry
1
R
5
Yield (%)
CH(CH
C(CH
CH
3
3
)
3
1
2
b
c
CH
C
3
j
k
90
76
3
3
2
H
5
a
General procedure II: reactions were carried out using 0.58 mmol 4-
a
General procedure II: reactions were carried out using 0.58 mmol 4-
substituted catechols 1b, c, 0.50 mmol 2,3,6,7-tetrahydro-2-thioxo-1H-
cyclopenta[d]pyrimidin-4(5H)-one (2i), laccase (A. bisporus, 10 mg, 12
U), phosphate buffer pH 6 (18 mL) and EtOH (2 mL). Yields refer to
crude products (purity > 95%, as determined by H NMR).
substituted catechols 1b, c, 0.50 mmol 2,3-dihydro-2-thioxopyrimidin-
4
buffer pH 6 (18 mL) and EtOH (2 mL). Yields refer to crude products
(1H)-ones 2a, c–e, g, laccase (A. bisporus, 10 mg, 12 U), phosphate
b
b
1
(purity > 95%, as determined by H NMR).
1
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high yields and in a highly chemoselective manner. The reactions
proceed without the formation of any relevant amounts of
byproducts arising from competing reactions, such as (a) the
homocoupling of the catechols 1; i.e., the reaction of the o-
quinone intermediates 8 with the corresponding parent cate-
chols 1, which is followed by formation of the benzofurans or (b)
the formation of disuldes. We assume that this favourable
outcome is the result of a combination of several factors. Among
them are (a) the high nucleophilicity of the S-nucleophilic
substrates 2, (b) the use of a laccase which is particularly suit-
able for this type of reactions and (c) the careful choice of reac-
tion conditions, such as pH, reaction temperature and substrate
concentrations. This view is supported by previous work done in
2
3
4
5
Fig. 5 Important J, J, J and J HMBC correlations of 5d.
16k
our laboratory.
The reactions between catechols and 1,3-
dicarbonyls prove that the selection of laccase from A. bisporus as
the catalyst as well as the choice of suitable reaction conditions
exert a tremendous impact on product yields.
3
and C-7 ( J ¼ 7.6 Hz) allows the successful assignment of the
quaternary carbons C-5a at d 128.82 ppm and C-7 at dC
45.25 ppm. Similarly, two strong correlations can be seen from
C
1
3
3
H-6 (dH 7.52 ppm) to C-9a ( J ¼ 8.4 Hz) and C-8 ( J ¼ 6.2 Hz), to
be assigned at d 112.65 ppm and d 144.68 ppm, respectively.
The quaternary carbons C-2, C-4, C-10a of ring C were assigned
by standard gHMBC and super long range gHMBC at d 166.32,
48.31, 164.60 ppm, respectively (Fig. 4).
In the reactions between 4-substituted catechols 1b, c and 2-
Structure elucidation
C
C
Structures of all pyrimidobenzothiazole regioisomers 3, 4 as
well as catechol thioethers 5 were unambiguously elucidated by
mass spectrometry and NMR spectroscopy including 2D NMR
C
1
1
13
for the full assignment of the H and C chemical shis.
1
Analysis of the H NMR spectrum of the crude products ob-
thioxopyrimidin-4-ones 2a, c–e, g, i single products were formed
exclusively. The products 5a–i consist of the 2 ring systems A
and B. Taking 5d as an example, the complete assignment of
tained from the reaction between catechol (1a) and 2-
thioxopyrimidin-4-ones 2a–h showed the appearance of either
a mixture of 2 regioisomers 3 (type I) and 4 (type II) or a single
regioisomer 3. 2D ROESY as well as super long range gHMBC
experiments were carried out to differentiate between the 2
regioisomers. Taking the mixture of 3a and 4a as an example, in
1
13
ring A was carried out by PIP-HSQMBC H– C correlations. The
1
13
difficulty in assigning C-2, C-4, and C-6 was solved by H– C
super long gHMBC along with standard gHMBC correlations
0
4
between 6 -H at d
H
7.66 ppm and C-2 at d
C
165.32 ppm ( J) as
165.32 ppm
0
4
a a strong ROESY correlation between the methyl group at C-4
2.73 ppm) to both the aromatic proton 6-H (d 7.52 ppm)
and the aromatic proton 3-H (d 6.07 ppm) conrms the type II
regioisomer. However, in 3a only a single ROESY correlation
well as between 2 -CH
3
at d 2.42 ppm and C-2 at d
H
C
5
00
J), between 2 -H at d 1.14 and C-6 at d 176.73 ppm ( J) and
H C
3
(d
H
H
(
2
H

nally between 5-H at dH 6.43 ppm and C-4 at d 167.61 ( J) as
C
2
well as C-6 at d 176.73 ppm ( J) (Fig. 5).
C
between the methyl group at C-2 (d 2.26 ppm) and the aromatic
proton at 3-H (dH 6.17 ppm) can be observed (Fig. 4). J HMBC
H
Unequivocal evidence for the structures of 3f and 5a was
provided by X-ray crystal structure analysis. The molecular
4
19
correlations (as observed in the super long range gHMBC
structures of 3f and 5a are depicted in Fig. 6 and 7.
spectrum) between 6-H (d
H
8.44 ppm) and C-4 (d
C
159.97 ppm)
along with 9-H (d 7.30 ppm) and C-10a (d
H
C
161.71 ppm)
Greenness of the reactions
established a type I regioisomer. Evaluation of the experimental
1
13
The newly developed methods for the synthesis of pyr-
imidobenzothiazoles 3, 4 and catechol thioethers 5 address many
of the principles of green chemistry. The reactions are enzyme-
H– C long-range coupling constants (PIP-HSQMBC), for
3
example in 4a, between 9-H (d
H
7.25 ppm) to C-5a ( J ¼ 9.5 Hz)
6,20
Fig. 6 Molecular structure of 7,8-dihydroxy-4H-2-trifluoromethyl-
pyrimido[2,1-b]benzothiazol-4-one (3f), derived from X-ray crystal
Fig. 4 Important ROESY, J, J, and J HMBC correlations of 3a and 4a. structure analysis.
2
3
4
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Table 4 IC50 of selected compounds 3, 5 against HepG2 cell line
a,b
Entry
Product
IC50 (mM)
1
2
3
4
5
6
7
8
9
3d
3e
3f
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
23.28 ꢂ 2.16
12.70 ꢂ 0.53
12.01 ꢂ 1.46
>40
21.25 ꢂ 0.46
7.77 ꢂ 0.30
14.30 ꢂ 0.11
30.57 ꢂ 1.05
27.82 ꢂ 2.36
2.74 ꢂ 0.29
14.92 ꢂ 1.16
31.98 ꢂ 1.43
40.48 ꢂ 3.52
31.34 ꢂ 3.57
0.28 ꢂ 0.04
10
11
12
13
14
15
Fig. 7 Molecular structure of 2-(4,5-dihydroxy-2-methylphenylthio)-
6
-methylpyrimidin-4(3H)-one (5a), derived from X-ray crystal struc-
ture analysis.
Doxorubicin
a
b
IC50 are the mean of 2–5 independent experiments ꢂ SE. DMSO
alone (2% nal concentration) had no effect on the cell viability.
catalyzed transformations using completely safe and non-toxic
aerial oxygen as the sole oxidant. The transformations deliver
the pyrimidobenzothiazoles 3, 4 and the catechol thioethers 5
exclusively and with high yields. The only byproduct formed is
water which stems from the reduction of oxygen. The laccase-
catalyzed domino reactions presented combine several indi-
vidual reactions in multistep processes. This allows the reduction
of the amount of waste formed during reaction and work up and
complies well with the rst principle of green chemistry. In
addition, environmentally benign reaction conditions have been
developed. Taking the preparation of 3e as a typical example for
the synthesis of pyrimidobenzothiazoles 3, the E-factor (kg waste
5
b (IC50 ¼ 21.25 mM, Table 4, entry 5) or introduction of
a methyl group in the 5-position in 5e (IC50 ¼ 30.57 mM, Table 4,
entry 8) increases the potency. Moreover, the presence of
a branched isopropyl group at C-6 in 5c results in a great
increase in potency (IC50 ¼ 7.77 mM, Table 4, entry 6) compared
to 5a (IC50 > 40 mM, Table 4, entry 4). However, the presence of
a bulky tert-butyl group in 5d results in a decrease of activity
(
IC50 ¼ 14.30 mM, Table 4, entry 7) in comparison to 5c. In the
0
21
ꢁ1
5f–i series, where an ethyl group is present at C-2 , the intro-
per kg product) of the process is 4.57 kg kg . The atom
5
duction of an isopropyl group in 5g increases the activity (IC50
¼
economy of this transformation amounts to 94%. The reaction
2.74 mM, Table 4, entry 10) and the presence of a tert-butyl group
was carried out in a mixture of phosphate buffer (pH 6.0) and 10
in 5h decreases the potency (IC50 ¼ 14.92 mM, Table 4, entry 11)
in comparison to 5g. Introduction of a methyl group at C-5 of 5i
results in a slight decrease of the cytotoxic activity (IC50 ¼ 31.98
mM, Table 4, entry 12) in comparison to 5f (IC50 ¼ 27.82 mM,
Table 4, entry 9). The presence of a bicyclic system in 5j and 5k
has no favourable effect on the cytotoxic activity compared to 5a
and 5f. Comparison of the IC50 values of the 5a–e, j series with
the 5f–i, k series reveals that the most potent compounds are 5c
and 5g which have an isopropyl group at C-6. The presence of an
vol% ethanol, which are completely safe and environmentally
22
acceptable solvents. The method highlights the use of enzymes
in catalytic rather than stoichiometric amounts. The turnover
number of the laccase in this process is high, for the synthesis of
3e TON amounts to 4656; the turnover frequency of this trans-
formation is good; in the example discussed TOF amounts to 291
ꢁ1
h
. Taking the preparation of 5j as a representative example for
the synthesis of thioethers 5, the E-factor of this process was
ꢁ1
calculated to be 5.00 kg kg . The atom economy of this process,
calculated as 94%, is also very good. The turnover number and
the turnover frequency of the laccase for the synthesis of 5j are
0
ethyl group at C-2 of 5g increases the cytotoxic potency in
comparison to 5c. In the pyrimidobenzothiazole derivatives 3d–
f, it was found that replacing the isopropyl group at C-2 of 3d
ꢁ
1
high. They amount to 4320 and 308.57 h , respectively.
(
(
IC50 ¼ 23.28 mM, Table 4, entry 1) with a tert-butyl group in 3e
IC50 ¼ 12.70 mM, Table 4, entry 2) or a triuoromethyl group in
In vitro cytotoxic activity
3f (IC50 ¼ 12.01 mM, Table 4, entry 3) results in an increase of the
antiproliferative activity. The presented study provides a novel
class of compounds, which will be further optimized to increase
their potency. The mechanism of their cytotoxic activity is still
under study.
Selected compounds and doxorubicin (positive control) were
evaluated for their in vitro cytotoxic activity against HepG2
23
cancer cell line using SRB assay. The IC50 values of the tested
compounds are summarized in Table 4. All compounds tested
exhibited cytotoxic activities, but all of them were less potent
than doxorubicin.
Experimental
Chemistry
In the catechol thioether series 5a–k, the substituents on C-5,
0
C-6 and C-2 have a great inuence on the cytotoxic activity. In
compounds 5a–e, it is assumed that increasing the chain length
General remarks. All chemicals were purchased from
from methyl in 5a (IC50 > 40 mM, Table 4, entry 4) to n-propyl in commercial suppliers. Laccase from Agaricus bisporus (1.2 U
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ꢁ
1
18
mg
) was purchased from ASA Spezialenzyme. Solvents used
in extraction and purication were distilled prior to use. The pH
of the buffer was adjusted using a pH 330/SET-1 pH-meter.
Analytical thin layer chromatography (TLC) was performed on
precoated silica gel 60 F245 aluminium plates (Merck) with
visualization under UV light. Flash chromatography was carried
out on silica gel MN 60, 0.04–0.053 mm (Macherey & Nagel).
Melting points were determined on a B u¨ chi melting point
apparatus B-545 with open capillary tubes and are uncorrected.
IR spectra were measured on a Perkin-Elmer Spectrum One (FT-
According to general procedure I, ethyl propionylacetate (6b)
1.44 g, 10 mmol), thiourea (7) (0.76 g, 10 mmol), KOH (0.56 g,
(
1
2
0 mmol), ethanol (20 mL) were reacted. Work up gave 6-ethyl-
,3-dihydro-2-thioxopyrimidin-4(1H)-one (2b) as a white powder
ꢀ
24a
ꢀ
(
0.78 g, 50%), mp 227–229 C (lit. 228.5–230.5 C); R
CH Cl (300 MHz; DMSO-d ) 1.08 (3H, t, J ¼
/EtOAc ¼ 2 : 1); d
.5 Hz, CH ), 5.66 (1H, s, 5-H) and
2.24 (2H, s, 1-H and 3-H); d (75 MHz; DMSO-d ) 11.60, 24.73,
02.04, 158.21, 161.19, 175.97.
Synthesis and analytical data of 2,3-dihydro-6-propyl-2-thio-
f
¼ 0.44
3
1
13
(
2
2
H
6
IR-spectrometer). H and C NMR spectra were recorded at 300
75) MHz and at 500 (125) MHz on Varian Unity Inova instru-
ments using DMSO-d and pyridine-d as solvents. The chem-
ical shis were referenced to the solvent signals at dH/C 2.49/
9.50 ppm (DMSO-d ) and 8.71/149.80 ppm (pyridine-d ) rela-
3
7
1
1
3
), 2.35 (2H, q, J ¼ 7.5 Hz, CH
2
(
C
6
6
5
3
6
5
xopyrimidin-4(1H)-one (2c).
tive to TMS as internal standards. 2-D ROESY, gHSQC,
ASAPHMQC, standard gHMBCAD, super long range gHMBCAD
as well as band selective gHMBC, PIP-HSQMBC and gCOSY
spectra were recorded on a Varian Unity Inova spectrometer
(500 MHz). Coupling constants J [Hz] were directly taken from
the spectra and are not averaged. Low resolution electron
impact mass spectra (EI-LRMS) and exact electron impact mass
spectra (HRMS) were recorded at 70 eV on a Finnigan MAT 95
instrument. Low resolution electron spray ionisation mass
spectra (ESI-LRMS) and exact electron spray ionisation mass
spectra (HRMS) were recorded on a Bruker Daltonics (micro
TOFQ) instrument. The intensities are reported as percentages
relative to the base peak (I ¼ 100%).
According to general procedure I, ethyl butyrylacetate (6c)
3.16 g, 20 mmol), thiourea (7) (1.52 g, 20 mmol), KOH (1.12 g, 20
mmol), ethanol (20 mL) were reacted. Work up gave 2,3-dihydro-6-
propyl-2-thioxopyrimidin-4(1H)-one (2c) as a white powder (1.40 g,
(
ꢀ
24a
ꢀ
4
1%), mp 216–218 C (lit. 218–220 C); R
f
¼ 0.52 (CH
2
Cl
2
/EtOAc
3
0
¼
2 : 1); d (300 MHz; DMSO-d ) 0.86 (3H, t, J ¼ 7.4 Hz, 3 -H), 1.53
H
6
3
0
3
0
(
2H, sex, J ¼ 7.4 Hz, 2 -H), 2.30 (2H, t, J ¼ 7.7 Hz, 1 -H), 5.65
General procedure I for the synthesis of 2,3-dihydro-2-thio-
xopyrimidin-4(1H)-ones 2a–h and 2,3,6,7-tetrahydro-2-thioxo-
(
1
1H, s, 5-H) and 12.20 (2H, s, 1-H and 3-H); d (75 MHz; DMSO-d )
C
6
3.20, 20.50, 33.35, 102.90, 156.97, 161.17, 176.08.
Synthesis and analytical data of 2,3-dihydro-6-isopropyl-2-thio-
xopyrimidin-4(1H)-one (2d).
17
1
H-cyclopenta[d]pyrimidin-4(5H)-one (2i). A 50 or 150 mL
round bottomed ask with a magnetic stir bar was charged with
a suspension of b-ketoester 6, thiourea (7) and KOH in ethanol.
ꢀ
The mixture was stirred at 80 C for 5 h. Ethanol was evaporated
under reduced pressure to one third of its original volume, the
reaction mixture was poured into water and neutralized with 2
N HCl. The resulting mixture was le stirring overnight at rt.
Filtration gave the crude product which was puried by crys-
tallization from methanol.
According to general procedure I, ethyl isobutyrylacetate (6d)
Synthesis and analytical data of 2,3-dihydro-6-methyl-2-thio-
xopyrimidin-4(1H)-one (2a).
(
1
1.58 g, 10 mmol), thiourea (7) (0.76 g, 10 mmol), KOH (0.56 g,
0 mmol), ethanol (20 mL) were reacted. Work up gave 2,3-
dihydro-6-isopropyl-2-thioxopyrimidin-4(1H)-one (2d) as a white
ꢀ
24a
ꢀ
powder (0.60 g, 35%), mp 176–178 C (lit. 179–180 C); R
.50 (CH Cl (300 MHz; DMSO-d ) 1.11 (6H, d,
/EtOAc ¼ 2 : 1); d
J ¼ 6.9 Hz, CH(CH ), 2.65 (1H, sep, J ¼ 6.9 Hz, CH(CH ),
.65 (1H, s, 5-H) and 12.20 (2H, br s, 1-H and 3-H); d (75 MHz;
f
¼
0
2
2
H
6
3
3
3
)
2
3 2
)
5
C
DMSO-d ) 20.39, 30.33, 100.33, 161.33, 162.22, 176.08.
According to general procedure I, ethyl acetoacetate (6a)
6
Synthesis and analytical data of 6-tert-butyl-2,3-dihydro-2-thio-
xopyrimidin-4(1H)-one (2e).
(
5
6.50 g, 50 mmol), thiourea (7) (3.81 g, 50 mmol), KOH (2.81 g,
0 mmol), ethanol (80 mL) were reacted. Work up gave 2,3-
dihydro-6-methyl-2-thioxopyrimidin-4(1H)-one (2a) as a white
ꢀ
24a
ꢀ
powder (5.00 g, 70%), mp > 300 C (lit. > 300 C); R ¼ 0.54
f
(
CH Cl /EtOAc ¼ 2 : 1); d (300 MHz; DMSO-d ) 2.05 (3H, s, 6-
2
2
H
6
3
CH ), 5.66 (1H, s, 5-H), 12.22 (2H, br, 1-H and 3-H).
Synthesis and analytical data of 6-ethyl-2,3-dihydro-2-thio-
xopyrimidin-4(1H)-one (2b).
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ꢁ
1
According to general procedure I, ethyl 4,4-dimethyl-3- (atr)/cm 3150 (NH), 2874 (CH), 1664 (C]O), 1570 and 1220;
5
oxovalerate (6e) (1.72 g, 10 mmol), thiourea (7) (0.76 g, 10
mmol), KOH (0.56 g, 10 mmol), ethanol (20 mL) were reacted. 12.85 (2H, br, 1-H and 3-H); d (75 MHz; DMSO-d ) 9.48 (q, J
C,F
d
(300 MHz; DMSO-d ) 1.93 (3H, q, J ¼ 3.3 Hz, 5-CH ) and
H
6
H,F
3
4
C
6
1
Work up gave 6-tert-butyl-2,3-dihydro-2-thioxopyrimidin-4(1H)- ¼ 2.5 Hz, 5-CH ), 115.57 (br, C-5), 119.89 (q, J ¼ 275.7 Hz,
3
2
C,F
ꢀ
24a
one (2e) as a white powder (0.50 g, 27%), mp 179–181 C (lit.
CF
Synthesis and analytical data of 2,3,6,7-tetrahydro-2-thioxo-1H-
), 5.63 (1H, s, 5-H), 11.79 and cyclopenta[d]pyrimidin-4(5H)-one (2i).
(75 MHz; DMSO-d ) 27.30, 34.49,
00.50, 161.16, 163.36, 176.49.
3
), 135.16 (q, JC,F ¼ 34.6 Hz, C-6), 161.18, 174.55.
ꢀ
178–180 C); R
f
¼ 0.63 (CH
2
Cl
2
/EtOAc ¼ 2 : 1); d
H
(300 MHz;
DMSO-d
6
) 1.21 (9H, s, C(CH
3 3
)
1
1
2.34 (2H, s, 1-H and 3-H); d
C
6
Synthesis and analytical data of 6-triuoromethyl-2,3-dihydro-
-thioxopyrimidin-4(1H)-one (2f).
2
According to general procedure I, ethyl 2-oxocyclopentane-
carboxylate (6i) (4.69 g, 30 mmol), thiourea (7) (2.28 g, 30 mmol),
KOH (1.68 g, 30 mmol), ethanol (30 mL) were reacted. Work up
gave
2,3,6,7-tetrahydro-2-thioxo-1H-cyclopenta[d]pyrimidin-
According
triuoroacetoacetate (6f) (1.84 g, 10 mmol), thiourea (7)
0.76 g, 10 mmol), KOH (0.56 g, 10 mmol), ethanol (20 mL) were
reacted. Work up gave 6-triuoromethyl-2,3-dihydro-2-thio-
xopyrimidin-4(1H)-one (2f) as a white powder (0.69 g, 35%), mp
to
general
procedure
I,
ethyl-4,4,4-
ꢀ
4
(5H)-one (2i) as a white powder (1.20 g, 24%), mp > 300 C
24a
ꢀ
(lit. 336–337 C); R
f
¼ 0.38 (CH
2
Cl
2
/EtOAc ¼ 2 : 1); d
H
(300
(
3
MHz; DMSO-d
6
) 1.94 (2H, quin, J ¼ 7.3 Hz, 6-H), 2.47 (2H, ov. t
3
3
like, J ¼ 7.3 Hz, 7-H), 2.67 (2H, t, J ¼ 7.6 Hz, 5-H), 12.17 and
1
2.18 (2H, br, 1-H and 3-H); d
1.00, 115.52, 156.51, 159.52, 175.57.
General procedure II for the laccase-catalyzed domino reac-
tion between catechols 1 and 2,3-dihydro-2-thioxopyrimidin-
(1H)-ones 2. A 100 mL round bottomed ask with a magnetic
stir bar was charged with a solution or suspension of a catechol
(0.58 mmol) and a 2,3-dihydro-2-thioxopyrimidin-4(1H)-ones 2
0.50 mmol) in ethanol (2 mL). Phosphate buffer (0.2 M, pH 6,
C 6
(75 MHz; DMSO-d ) 20.78, 26.60,
ꢀ
24b
ꢀ
2
46–248 C (lit. 247–249 C); R
(300 MHz; DMSO-d ) 6.40 (1H, s, 5-H), 12.83 (1H, s, 1-H or 3-
H) and 13.50 (1H, br, 1-H or 3-H); d (75 MHz; DMSO-d ) 105.14
), 140.56
f
¼ 0.50 (CH
2
Cl
2
/EtOAc ¼ 2 : 1);
3
d
H
6
C
6
3
1
(
(
q, JC,F ¼ 3.9 Hz, C-5), 118.83 (q, JC,F ¼ 273.7 Hz, CF
3
4
2
q, JC,F ¼ 36.1 Hz, C-6), 159.84, 176.82.
Synthesis and analytical data of 2,3-dihydro-5,6-dimethyl-2-
1
(
thioxopyrimidin-4(1H)-one (2g).
ꢁ
1
18 mL) and laccase from A. bisporus (10 mg, 12 U, 1.2 U mg )
were added and the mixture was stirred under air at rt for the
time given. The reaction mixture was acidied with 2 N HCl to
pH ꢃ 4. The precipitated product was ltered with suction using
a Buchner funnel. The lter cake was washed with water. The
crude products obtained aer drying exhibit a purity of >95%
According to general procedure I, ethyl 2-methylacetoacetate
6g) (7.21 g, 50 mmol), thiourea (7) (3.81 g, 50 mmol), KOH
2.81 g, 50 mmol), ethanol (60 mL) were reacted. Work up gave
1
(

H NMR). Analytically pure products were obtained by column
Cl Cl /EtOAc/MeOH ¼
/EtOAc ¼ 1 : 1 to CH
: 1 : 0.1) of the crude products.
General procedure III for the laccase-catalyzed domino
(
(
2
ltration (CH
2
2
2
2
1
,3-dihydro-5,6-dimethyl-2-thioxopyrimidin-4(1H)-one (2g) as
ꢀ
24a
ꢀ
a white powder (3.80 g, 49%), mp 283–285 C (lit. 283–285 C);
reaction between catechol (1a) and 2,3-dihydro-2-thioxopyr-
imidin-4(1H)-ones 2g, h. A 100 mL round bottomed ask with
a magnetic stir bar was charged with a solution of catechol (1a)
R ¼ 0.48 (CH Cl /EtOAc ¼ 2 : 1); d (300 MHz; DMSO-d ) 1.74
f
2
2
H
6
(
3 3
3H, s, CH ), 2.09 (3H, s, CH ) and 12.19 (2H, br, 1-H and 3-H).
Synthesis and analytical data of 6-triuoromethyl-2,3-dihydro-
-methyl-2-thioxopyrimidin-4(1H)-one (2h).
(0.29 mmol) and 2,3-dihydro-2-thioxopyrimidin-4(1H)-ones 2g,
5
h (0.25 mmol) in ethanol (2 mL). Phosphate buffer (0.2 M, pH 6,
ꢁ
1
1
8 mL) and laccase from A. bisporus (10 mg, 12 U, 1.2 U mg )
were added and the mixture was stirred under air at rt for the
time given. The reaction mixture was acidied with 2 N HCl to
pH ꢃ 4. The precipitated product was ltered with suction using
a Buchner funnel. The lter cake was washed with water. The
crude products obtained aer drying exhibit a purity of >95%
1
According to general procedure I, ethyl-4,4,4-triuoro-2-
(
H NMR). Analytically pure products were obtained either by
Cl
/EtOAc ¼ 1 : 1 to
methylacetoacetate (6h) (1.98 g, 10 mmol), thiourea (7)
0.76 g, 10 mmol), KOH (0.56 g, 10 mmol), ethanol (15 mL) were
reacted. Work up gave 6-triuoromethyl-2,3-dihydro-5-methyl-
acetylation or by column ltration (CH
CH Cl
2
2
(
2
2
/EtOAc/MeOH ¼ 1 : 1 : 0.1) of the crude products.
Synthesis and analytical data of 7,8-dihydroxy-4H-2-methyl-
pyrimido[2,1-b]benzothiazol-4-one (3a) and 7,8-dihydroxy-2H-4-
methyl-pyrimido[2,1-b]benzothiazol-2-one (4a).
2
2
-thioxopyrimidin-4(1H)-one (2h) as a white powder (0.60 g,
ꢀ
9%), mp 244–246 C; R
f
¼ 0.66 (CH
2
Cl
2
/EtOAc ¼ 2 : 1); n
max
̃
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12.73 (C-9a), 128.40 (C-5a), 144.63 (C-8), 145.33 (C-7), 153.26
+
(C-4), 164.91 (C-10a) and 166.35 (C-2); MS (EI-70 eV) m/z 262 (M ,
+
100%), 263 ([M + H] , 37) and 219 (19); HRMS calcd for
C H N O S (262.0412), found 262.0413.
1
2
10 2 3
Synthesis and analytical data of 7,8-dihydroxy-4H-2-propyl-
According to general procedure II, catechol (1a) (64 mg, 0.58 pyrimido[2,1-b]benzothiazol-4-one (3c) and 7,8-dihydroxy-2H-4-
mmol), 2,3-dihydro-6-methyl-2-thioxopyrimidin-4(1H)-one (2a) propyl-pyrimido[2,1-b]benzothiazol-2-one (4c).
(71 mg, 0.50 mmol), ethanol (2 mL), phosphate buffer (0.2 M,
pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bisporus) were
reacted for 12 h. Workup gave a mixture of 7,8-dihydroxy-4H-2-
methyl-pyrimido[2,1-b]benzothiazol-4-one (3a) and 7,8-dihy-
droxy-2H-4-methyl-pyrimido[2,1-b]benzothiazol-2-one (4a) as
ꢀ
a brown powder (120 mg, 97%), mp > 300 C; R
f
¼ 0.27 (CH
2 2
Cl /
ꢁ1
EtOAc/MeOH ¼ 2 : 2 : 0.1); n
̃
max (atr)/cm
CH), 1628 (C]O), 1510 (C]N) and 1185; d
3406 (OH), 3023
(500 MHz; DMSO-
(
H
According to general procedure II, catechol (1a) (64 mg, 0.58
d ) of 3a 2.26 (3H, s, 2-CH ), 6.17 (1H, s, 3-H), 7.30 (1H, s, 9-H), mmol), 2,3-dihydro-6-propyl-2-thioxopyrimidin-4(1H)-one (2c)
6
3
8
.44 (1H, s, 6-H) and 9.66 (2H, ov. br, 7,8-OH); dH (500 MHz; (85 mg, 0.50 mmol), ethanol (2 mL), phosphate buffer (0.2 M,
DMSO-d ) of 4a 2.73 (3H, s, 4-CH
), 6.07 (1H, s, 3-H), 7.25 (1H, s, pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bisporus) were
-H), 7.52 (1H, s, 6-H) and 9.66 (2H, ov. br, 7,8-OH); d
(125 reacted for 18 h. Workup gave a mixture of 7,8-dihydroxy-4H-2-
MHz; DMSO-d ) of 3a 23.10 (2-CH
), 105.68 (C-3), 106.38 (C-6), propyl-pyrimido[2,1-b]benzothiazol-4-one (3c) and 7,8-dihy-
6
3
9
C
6
3
1
(
08.29 (C-9), 113.21 (C-9a), 128.12 (C-5a), 144.96 (C-7), 145.65 droxy-2H-4-propyl-pyrimido[2,1-b]benzothiazol-2-one (4c) as
ꢀ
C-8), 159.97 (C-4), 161.71 (C-10a) and 161.99 (C-2); d
C
(125 MHz; a brown powder (130 mg, 94%), mp 258–260 C; R
f
¼ 0.27
ꢁ
1
DMSO-d
6
) of 4a 21.15 (4-CH
3
), 104.76 (C-6), 108.97 (C-9), 110.80 (CH
C-3), 112.65 (C-9a), 128.82 (C-5a), 144.68 (C-8), 145.25 (C-7), 1625 (C]O), 1453 and 1307; d
2
Cl
2
/EtOAc ¼ 2 : 1); n
m
̃ ax (atr)/cm 3310 (OH), 2960 (CH),
(
1
H
(500 MHz; DMSO-d
6
) of 3c 0.89
3
0
0
0
48.31 (C-4), 164.60 (C-10a) and 166.32 (C-2); MS (EI-70 eV) m/ (3H, t, J ¼ 7.3 Hz, 3 -H), 1.65 (2H, ov., 2 -H), 2.49 (2H, ov., 1 -H),
+
z 248 (M , 100%), 220 (35) and 181 (15); HRMS calcd for 6.15 (1H, br s, 3-H), 7.30 (1H, s, 9-H) and 8.45 (1H, s, 6-H); d
H
3
0
C
11
H
8
N
2
O
3
S (248.0256), found 248.0253.
(500 MHz; DMSO-d
6
) of 4c 1.04 (3H, t, J ¼ 7.1 Hz, 3 -H), 1.65
3
0
3
0
Synthesis and analytical data of 7,8-dihydroxy-4H-2-ethyl-pyr- (2H, ov. sex, J ¼ 7.7 Hz, 2 -H), 3.00 (2H, t, J ¼ 7.5 Hz, 1 -H), 6.03
imido[2,1-b]benzothiazol-4-one (3b) and 7,8-dihydroxy-2H-4-ethyl- (1H, br s, 3-H), 7.25 (1H, s, 9-H) and 7.41 (1H, s, 6-H); d
(125
0
C
0
0
pyrimido[2,1-b]benzothiazol-2-one (4b).
MHz; DMSO-d
6
) of 3c 13.50 (C-3 ), 20.87 (C-2 ), 38.26 (C-1 ),
1
5
1
05.26 (C-3), 106.39 (C-6), 108.29 (C-9), 113.24 (C-9a), 128.09 (C-
a), 144.95 (C-7), 145.66 (C-8), 160.10 (C-4), 161.84 (C-10a),
0
65.30 (C-2); d
C
(125 MHz; DMSO-d
0
6
) of 4c 13.14 (C-3 ), 20.09
0
(C-2 ), 34.20 (C-1 ), 104.63 (C-6), 109.00 (C-9), 109.73 (C-3),
1
12.73 (C-9a), 128.28 (C-5a), 144.72 (C-8), 145.38 (C-7), 151.48
+
(C-4), 164.98 (C-10a), 166.24 (C-2); MS (ESI) m/z 299 ([M + Na] ,
+
100%) and 277 ([M + H] , 35); HRMS calcd for C H N O S + Na
13 12 2 3
(
299.0461), found 299.0457.
Synthesis and analytical data of 7,8-dihydroxy-4H-2-isopropyl-
According to general procedure II, catechol (1a) (64 mg, 0.58
mmol), 6-ethyl-2,3-dihydro-2-thioxopyrimidin-4(1H)-one (2b)
pyrimido[2,1-b]benzothiazol-4-one (3d) and 7,8-dihydroxy-2H-4-
isopropyl-pyrimido[2,1-b]benzothiazol-2-one (4d).
(
78 mg, 0.50 mmol), ethanol (2 mL), phosphate buffer (0.2 M,
pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bisporus) were
reacted for 12 h. Workup gave a mixture of 7,8-dihydroxy-4H-2-
ethyl-pyrimido[2,1-b]benzothiazol-4-one (3b) and 7,8-dihydroxy-
2
H-4-ethyl-pyrimido[2,1-b]benzothiazol-2-one (4b) as a brown
ꢀ
powder (124 mg, 95%), mp 298–300 C; R
f
¼ 0.27 (CH
2 2
Cl /EtOAc
ꢁ
1
¼
2 : 1); n
̃
max (atr)/cm 3400 (OH), 2979 (CH), 1625 (C]O),
3
1
7
7
512 and 1299; d
H
(500 MHz; DMSO-d
6
) of 3b 1.17 (3H, t, J ¼
0
3
0
.5 Hz, 2 -H), 2.54 (2H, q, J ¼ 7.5 Hz, 1 -H), 6.16 (1H, s, 3-H),
According to general procedure II, catechol (1a) (64 mg, 0.58
.30 (1H, s, 9-H), 8.45 (1H, s, 6-H) and 9.65 (2H, ov. br, 7,8-OH);
mmol),
2,3-dihydro-6-isopropyl-2-thioxopyrimidin-4(1H)-one
2d) (85 mg, 0.50 mmol), ethanol (2 mL), phosphate buffer
0.2 M, pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bisporus)
3
0
d (500 MHz; DMSO-d ) of 4b 1.27 (3H, t, J ¼ 7.1 Hz, 2 -H), 3.09
H
6
(
(
3
0
(
(
2H, q, J ¼ 7.1 Hz, 1 -H), 6.02 (1H, s, 3-H), 7.25 (1H, s, 9-H), 7.49
1H, s, 6-H) and 9.65 (2H, br, 7,8-OH); d (125 MHz; DMSO-d ) of
C
6
were reacted for 13 h. Workup gave a mixture of 7,8-dihydroxy-
0
0
3b 12.25 (C-2 ), 29.56 (C-1 ), 104.36 (C-3), 106.37 (C-6), 108.30 (C-
4
7
H-2-isopropyl-pyrimido[2,1-b]benzothiazol-4-one (3d) and
9
), 113.24 (C-9a), 128.09 (C-5a), 144.94 (C-7), 145.65 (C-8), 160.25
,8-dihydroxy-2H-4-isopropyl-pyrimido[2,1-b]benzothiazol-2-one
(C-4), 161.82 (C-10a), 166.68 (C-2); d
C
(125 MHz; DMSO-d
6
) of 4b
ꢀ
(4d) as a brown powder (131 mg, 95%); mp 250–252 C; R
f
¼ 0.36
0
0
11.57 (C-2 ), 25.84 (C-1 ), 104.93 (C-6), 108.52 (C-3), 108.96 (C-9),
ꢁ1
(CH Cl /EtOAc ¼ 0.1 : 1); n
̃ (atr)/cm 3350 (OH), 2962 (CH),
2
2
max
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Paper
1
H 6 C
636 (C]O), 1517 and 1234; d (500 MHz; DMSO-d ) of 3d 1.17 (1H, s, 9-H), 8.47 (1H, s, 6-H), 9.82 and 9.90 (2H, 2s, 7,8-OH); d
3
0
3
0
3
(
(
6H, d, J ¼ 6.9 Hz, 2 -H), 2.77 (1H, sep, J ¼ 6.8 Hz, 1 -H), 6.15 (125 MHz; DMSO-d ) 105.31 (q, J ¼ 3.1 Hz, C-3), 106.33 (C-6),
6
C,F
1
1H, s, 3-H), 7.35 (1H, s, 9-H), 8.44 (1H, s, 6-H) and 9.77 (2H, ov. 108.35 (C-9), 114.19 (C-9a), 120.78 (q, J
¼ 273.0 Hz, CF ),
C,F
3
3
2
br, 7,8-OH); d (500 MHz; DMSO-d ) of 4d 1.32 (6H, d, J ¼ 127.82 (C-5a), 145.42 (C-7), 146.41 (C-8), 148.25 (q,
J
C,F
¼
H
6
0
0
6.3 Hz, 2 -H), 3.64 (1H, ov., 1 -H), 6.08 (1H, s, 3-H), 7.30 (1H, s, 9- 34.4 Hz, C-2), 159.47 (C-4), 164.29 (C-10a); MS (ESI) m/z 341 ([M
+
+
H), 7.68 (1H, s, 6-H) and 9.77 (2H, ov. br, 7,8-OH); d
C
(125 MHz; + K] , 49%), 303 ([M + H] , 100) and 283 (73); HRMS calcd for
S + H (303.0046), found 303.0046.
Synthesis and analytical data of 7,8-dihydroxy-4H-2,3-dimethyl-
(125 MHz; pyrimido[2,1-b]benzothiazol-4-one (3g) and 7,8-dihydroxy-2H-3,4-
DMSO-d ) of 4d 21.48 (C-2 ), 28.59 (C-1 ), 105.33 (C-6), 106.43 (C- dimethyl-pyrimido[2,1-b]benzothiazol-2-one (4g).
0
0
DMSO-d
), 108.34 (C-9), 113.20 (C-9a), 128.02 (C-5a), 144.98 (C-7), 145.77
C-8), 160.42 (C-4), 161.90 (C-10a) and 170.33 (C-2); d
6
) of 3d 21.19 (C-2 ), 34.65 (C-1 ), 103.22 (C-3), 106.53 (C-
11 5 3 2 3
C H F N O
6
(
C
0
0
6
3), 109.02 (C-9), 112.65 (C-9a), 128.00 (C-5a), 144.81 (C-8), 145.64
(
(
C-7), 158.10 (C-4), 165.25 (C-10a), 166.53 (C-2); MS (ESI) m/z 315
[M + K] , 20%), 277 ([M + H] , 100), 249 (8); HRMS calcd for
S + H (277.0641), found 277.0615. Column ltration
+
+
13 12 2 3
C H N O
using the eluent CH
2
2
Cl /EtOAc ¼ 1 : 1 gave 3d in pure form; mp
ꢀ
259–261 C.
Synthesis and analytical data of 7,8-dihydroxy-4H-2-tert-butyl-
pyrimido[2,1-b]benzothiazol-4-one (3e).
According to general procedure III, catechol (1a) (32 mg, 0.29
mmol), 2,3-dihydro-5,6-dimethyl-2-thioxopyrimidin-4(1H)-one
(
(
2g) (39 mg, 0.25 mmol), ethanol (2 mL), phosphate buffer
0.2 M, pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bisporus)
were reacted for 28 h. Workup gave a mixture of 7,8-dihydroxy-
4H-2,3-dimethyl-pyrimido[2,1-b]benzothiazol-4-one (3g) and
7
,8-dihydroxy-2H-3,4-dimethyl-pyrimido[2,1-b]benzothiazol-2-
ꢀ
one (4g) as a brown powder (58 mg, 89%), mp > 300 C; R
f
¼ 0.46
ꢁ
1
(
1
(
6
2
CH
631 (C]O), 1488 and 1185; d
3H, s, 3-CH ), 2.28 (3H, s, 2-CH
-H) and 9.61 (2H, ov. br, 7,8-OH); d
.00 (3H, s, 3-CH ), 2.73 (3H, s, 4-CH ), 7.25 (1H, s, 9-H), 7.58
2
Cl
2
/EtOAc ¼ 1 : 1); n
m
ax (atr)/cm 3352 (OH), 3023 (CH),
(500 MHz; DMSO-d ) of 3g 2.03
), 7.28 (1H, s, 9-H), 8.48 (1H, s,
(500 MHz; DMSO-d ) of 4g
According to general procedure II, catechol (1a) (64 mg, 0.58
mmol),
6-tert-butyl-2,3-dihydro-2-thioxopyrimidin-4(1H)-one
2e) (92 mg, 0.50 mmol), ethanol (2 mL), phosphate buffer
0.2 M, pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bisporus)
H
6
(
(
3
3
H
6
were reacted for 16 h. Workup gave 7,8-dihydroxy-4H-2-tert-
butyl-pyrimido[2,1-b]benzothiazol-4-one (3e) as a brown powder
3
3
(
1H, s, 6-H) and 9.61 (2H, ov. br, 7,8-OH); d (125 MHz; DMSO-
C
ꢀ
d ) of 3g 10.98 (3-CH ), 21.71 (2-CH ), 106.46 (C-6), 108.29 (C-9),
6 3 3
1
(C-8), 157.37 (C-2), 157.83 (C-10a) and 160.46 (C-4); d (125 MHz;
(
n
140 mg, 97%), mp > 300 C; R
f
¼ 0.38 (CH
2
Cl
2
/EtOAc ¼ 0.1 : 1);
ꢁ
1
12.82 (C-3), 113.33 (C-9a), 128.12 (C-5a), 144.77 (C-7), 145.52
̃max (atr)/cm 3295 (OH), 2954 (CH), 1638 (C]O), 1546 and
0
1
185; d
H
(500 MHz; DMSO-d
6
) 1.23 (9H, s, 2 -H), 6.21 (1H, s, 3-
C
DMSO-d
108.99 (C-9), 112.76 (C-9a), 116.70 (C-3), 129.21 (C-5a), 144.00
C-4), 144.49 (C-8), 145.02 (C-7), 162.86 (C-10a) and 166.21 (C-2);
analytically pure product was obtained by acetylation; MS (ESI)
6 3 3
) of 4g 12.01 (3-CH ), 17.97 (4-CH ), 105.06 (C-6),
H), 7.31 (1H, s, 9-H), 8.44 (1H, s, 6-H) and 9.75 (2H, br, 7,8-OH);
0
0
d
C
(125 MHz; DMSO-d ) 28.67 (C-2 ), 36.78 (C-1 ), 102.08 (C-3),
6
(
106.42 (C-6), 108.32 (C-9), 113.22 (C-9a), 127.90 (C-5a), 144.94
(
C-7), 145.73 (C-8), 160.52 (C-4), 161.36 (C-10a) and 172.33 (C-2);
+
+
+
+
m/z 369 ([M + Na] , 93%), 347 ([M + H] , 49), 305 (53) and 263
MS (ESI) m/z 329 ([M + K] , 31%) and 291 ([M + H] , 100); HRMS
calcd for C14 S + H (291.0798), found 291.0802.
(
100); HRMS calcd for C H N O S + Na (369.0516), found
H
14
N
2
O
3
16 14 2 5
369.0522.
Synthesis and analytical data of 7,8-dihydroxy-4H-2-tri-
Synthesis and analytical data of 7,8-dihydroxy-4H-3-methyl-2-

uoromethyl-pyrimido[2,1-b]benzothiazol-4-one (3f).
triuoromethyl-pyrimido[2,1-b]benzothiazol-4-one (3h).
According to general procedure II, catechol (1a) (64 mg, 0.58
mmol), 6-triuoromethyl-2,3-dihydro-2-thioxopyrimidin-4(1H)-
one (2g) (98 mg, 0.50 mmol), ethanol (2 mL), phosphate buffer
0.2 M, pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bisporus)
were reacted for 16 h. Workup gave 7,8-dihydroxy-4H-2-tri-
According to general procedure III, catechol (1a) (32 mg, 0.29
mmol),
6-triuoromethyl-2,3-dihydro-5-methyl-2-thioxopyr-
imidin-4(1H)-one (2h) (52.5 mg, 0.25 mmol), ethanol (2 mL),
phosphate buffer (0.2 M, pH 6.0, 18 mL) and laccase (12 U,
0 mg, A. bisporus) were reacted for 17 h. Workup gave 7,8-
(
1

uoromethyl-pyrimido[2,1-b]benzothiazol-4-one (3f) as a brown
ꢀ
dihydroxy-4H-2-triuoromethyl-pyrimido[2,1-b]benzothiazol-4-
powder (118 mg, 78%), mp > 300 C; R
f
¼ 0.31 (cyclohexane/
ꢀ
ꢁ1
one (3h) as a brown powder (75 mg, 95%), mp > 300 C; R
f
¼ 0.49
EtOAc ¼ 1 : 1); n
m
ax (atr)/cm 3408 (OH), 1654 (C]O), 1526
(500 MHz; DMSO-d ) 6.79 (1H, s, 3-H), 7.42
ꢁ
1
(
1
CH Cl /EtOAc ¼ 2 : 1); n
̃
(atr)/cm 3533 (OH), 3120 (CH),
632 (C]O), 1531 (C]N) and 1224; d (500 MHz; DMSO-d )
H 6
2
2
max
(
C]N) and 1151; d
H
6
17436 | RSC Adv., 2017, 7, 17427–17441
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5
0
0
0
0
2
.20 (3H, q, JH,F ¼ 2.2 Hz, 3-CH
3
), 7.39 (1H, s, 9-H), 8.49 (1H, s, 118.85 (C-3 ), 124.96 (C-6 ), 135.39 (C-2 ), 145.57 (C-5 ), 149.60
0
6
-H) and 9.85 (2H, br, 7,8-OH); d (125 MHz; DMSO-d ) 10.18 (q, (C-4 ), 166.79 (C-2), 167.68 (C-4) and 170.09 (C-6); MS (ESI) m/z
C
6
4
+
+
+
JC,F ¼ 2.2 Hz, 3-CH ), 106.40 (C-6), 108.36 (C-9), 114.29 (C-9a), 331 ([M + K] , 47%), 315 ([M + Na] , 100), 293 ([M + H] , 83) and
3
1
1
16.29 (br s, C-3), 121.78 (q, J ¼ 274.6 Hz, CF ), 127.59 (C- 171 (53); HRMS calcd for C H N O S + Na (315.0774), found
C,F
3
14 16 2 3
2
5
1
7
a), 143.73 (q, JC,F ¼ 33.0 Hz, C-2), 145.22 (C-7), 146.27 (C-8), 315.0767.
+
59.79 (C-10a) and 160.82 (C-4); MS (ESI) m/z 355 ([M + K] ,
Synthesis and analytical data of 2-(4,5-dihydroxy-2-methyl-
2%), 317 ([M + H] , 74), 297 (100) and 249 (60); HRMS calcd for phenylthio)-6-isopropylpyrimidin-4(3H)-one (5c).
S + H (317.0202), found 317.0193.
Synthesis and analytical data of 2-(4,5-dihydroxy-2-methyl-
+
12 7 3 2 3
C H F N O
phenylthio)-6-methylpyrimidin-4(3H)-one (5a).
According to general procedure II, 4-methylcatechol (1b)
(72 mg, 0.58 mmol), 2,3-dihydro-6-isopropyl-2-thioxopyrimidin-
4
(1H)-one (2d) (85 mg, 0.50 mmol), ethanol (2 mL), phosphate
According to general procedure II, 4-methylcatechol (1b)
72 mg, 0.58 mmol), 2,3-dihydro-6-methyl-2-thioxopyrimidin-
(1H)-one (2a) (71 mg, 0.50 mmol), ethanol (2 mL), phosphate
buffer (0.2 M, pH 6.0, 18 mL) and laccase (12 U, 10 mg, A.
bisporus) were reacted for 12 h. Workup gave 2-(4,5-dihydroxy-
-methylphenylthio)-6-methylpyrimidin-4(3H)-one (5a) as
buffer (0.2 M, pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bis-
porus) were reacted for 16 h. Workup gave 2-(4,5-dihydroxy-2-
methylphenylthio)-6-isopropylpyrimidin-4(3H)-one
(
4
(5c)
as
ꢀ
a pale yellow powder (134 mg, 92%), mp 204–206 C; R
f
¼ 0.27
ꢁ
1
(CH
2
Cl
2
/EtOAc/MeOH ¼ 3 : 3 : 0.1); n
m
̃ ax (atr)/cm 3240 (OH),
2
ꢀ
2965 (CH), 1644 (C]O), 1509 and 1281; d
d
H
(300 MHz; pyridine-
a pale yellow powder (108 mg, 82%), mp 213–215 C; R
f
¼ 0.27
3
00
0
ꢁ
1
5
) 1.09 (6H, d, J ¼ 6.9 Hz, 2 -H), 2.42 (3H, s, 2 -CH
3
), 2.65 (1H,
(
2
CH
2
Cl
2
/EtOAc/MeOH ¼ 3 : 3 : 0.1); n
̃
max (atr)/cm 3406 (OH),
(500 MHz; pyri-
3
00
0
sep, J ¼ 6.9 Hz, 1 -H), 6.32 (1H, s, 5-H), 7.26 (1H, s, 3 -H) and
916 (CH), 1644 (C]O), 1508 and 1264; d
H
0
0
0
7.67 (1H, s, 6 -H); d
C
(75 MHz; pyridine-d
5
) 20.32 (2 -CH
3
), 21.20
dine-d ) 2.13 (3H, s, 6-CH ), 2.43 (3H, s, 2 -CH ), 6.29 (1H, s, 5-
5
3
3
0
0
00
0
0
0
0
(C-2 ), 35.60 (C-1 ), 103.23 (C-5), 116.82 (C-1 ), 118.77 (C-3 ),
124.94 (C-6 ), 135.40 (ov., C-2 ), 145.51 (C-5 ), 149.56 (C-4 ),
166.51 (C-2), 167.84 (C-4) and 174.91 (C-6); MS (ESI) m/z 315 ([M
H), 7.23 (1H, s, 3 -H) and 7.69 (1H, s, 6 -H); d (125 MHz;
C
0
0
0
0
0
pyridine-d ) 20.40 (2 -CH ), 23.67 (6-CH ), 105.59 (C-5), 116.82
5
3
3
0 0 0 0
(
5
C-1 ), 118.95 (C-3 ), 125.04 (C-6 ), 135.40 (ov., C-2 ), 145.63 (C-
+
+
0
0
), 149.64 (C-4 ), 166.69 (C-6), 167.13 (C-2) and 167.87 (C-4);
+ Na] , 100%), 293 ([M + H] , 44) and 171 (27); HRMS calcd for
S + H (293.0954), found 293.0950.
Synthesis and analytical data of 2-(4,5-dihydroxy-2-methyl-
+
+
14 16 2 3
C H N O
MS (ESI) m/z 287 ([M + Na] , 100%), 265 ([M + H] , 75) and
1
2
43 (60); HRMS calcd for C12
65.0643.
12 2 3
H N O S + H (265.0641), found
phenylthio)-6-tert-butylpyrimidin-4(3H)-one (5d).
Synthesis and analytical data of 2-(4,5-dihydroxy-2-methyl-
phenylthio)-6-propylpyrimidin-4(3H)-one (5b).
According to general procedure II, 4-methylcatechol (1b)
(72 mg, 0.58 mmol), 6-tert-butyl-2,3-dihydro-2-thioxopyrimidin-
4(1H)-one (2e) (92 mg, 0.50 mmol), ethanol (2 mL), phosphate
buffer (0.2 M, pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bis-
porus) were reacted for 17 h. Workup gave 2-(4,5-dihydroxy-2-
According to general procedure II, 4-methylcatechol (1b)
72 mg, 0.58 mmol), 2,3-dihydro-6-propyl-2-thioxopyrimidin-
(1H)-one (2c) (85 mg, 0.50 mmol), ethanol (2 mL), phosphate
buffer (0.2 M, pH 6.0, 18 mL) and laccase (12 U, 10 mg, A.
bisporus) were reacted for 13 h. Workup gave 2-(4,5-dihydroxy-
-methylphenylthio)-6-propylpyrimidin-4(3H)-one (5b) as
(
4
methylphenylthio)-6-tert-butylpyrimidin-4(3H)-one
a pale yellow powder (138 mg, 90%), mp 205–207 C; R
(5d)
as
ꢀ
1
f
¼ 0.45
ꢁ
(
2
CH
2
Cl
2
/EtOAc/MeOH ¼ 3 : 3 : 0.1); n
m
̃ ax (atr)/cm 3450 (OH),
957 (CH), 1638 (C]O), 1577 (C]N) and 1279; d (500 MHz;
H
2
0
0
0
ꢀ
pyridine-d
5
) 1.14 (9H, s, 2 -H), 2.42 (3H, s, 2 -CH
3
), 6.43 (1H, s, 5-
a pale yellow powder (128 mg, 88%), mp 189–191 C; R
f
¼ 0.40
0
0
ꢁ
1
H), 7.28 (1H, s, 3 -H) and 7.66 (1H, s, 6 -H); d (125 MHz; pyri-
), 28.73 (C-2 ), 37.20 (C-1 ), 102.37 (C-5),
116.75 (C-1 ), 118.72 (C-3 ), 124.98 (C-6 ), 135.72 (C-2 ), 145.49
(C-5 ), 149.58 (ov., C-4 ), 165.32 (C-2), 167.61 (C-4) and 176.73
(C-6); MS (ESI) m/z 345 ([M + K] , 40%), 329 ([M + Na] , 100), 307
([M + H] , 77), 229 (24) and 185 (54); HRMS calcd for
C
(
3
(
CH
2
Cl
2
/EtOAc/MeOH ¼ 3 : 3 : 0.1); n
̃max (atr)/cm 3439 (OH),
0
00
00
dine-d
5
) 20.34 (2 -CH
3
179 (NH), 2957 (CH), 1634 (C]O), 1508 (C]N) and 1227; d
H
0
0
0
0
3
00
300 MHz; pyridine-d ) 0.75 (3H, t, J ¼ 7.2 Hz, 3 -H), 1.58 (2H,
5
0
0
3
00
3
00
sex like, J ¼ 7.5 Hz, 2 -H), 2.39 (2H, t, J ¼ 7.6 Hz, 1 -H), 2.43
+
+
0
0
0
(
(
3H, s, 2 -CH
3
), 6.31 (1H, s, 5-H), 7.25 (1H, s, 3 -H) and 7.67
+
00
0
1H, s, 6 -H); d
C
(75 MHz; pyridine-d
5
) 13.64 (C-3 ), 20.34 (2 -
00
00
0
C H N O S + H (307.1111), found 307.1111.
15 18 2 3
CH
3
), 21.44 (C-2 ), 39.32 (C-1 ), 105.31 (C-5), 116.79 (C-1 ),
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Synthesis and analytical data of 2-(4,5-dihydroxy-2-methyl-
According to general procedure II, 4-ethylcatechol (1c)
phenylthio)-5,6-dimethylpyrimidin-4(3H)-one (5e).
(80 mg, 0.58 mmol), 2,3-dihydro-6-isopropyl-2-thioxopyrimidin-
4(1H)-one (2d) (85 mg, 0.50 mmol), ethanol (2 mL), phosphate
buffer (0.2 M, pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bis-
porus) were reacted for 12 h. Workup gave 2-(2-ethyl-4,5-dihy-
droxyphenylthio)-6-isopropylpyrimidin-4(3H)-one (5g) as a pale
ꢀ
yellow powder (116 mg, 76%); mp 158–160 C; R
f
¼ 0.50
ꢁ
1
(
2
CH
2
Cl
2
/EtOAc/MeOH ¼ 3 : 3 : 0.1); n
m
ax (atr)/cm 3323 (OH),
(300 MHz; pyridine-
According to general procedure II, 4-methylcatechol (1b)
962 (CH), 1630 (C]O), 1514 and 1252; d
H
(72 mg, 0.58 mmol), 2,3-dihydro-5,6-dimethyl-2-thioxopyrimidin-
3
00
3
0
d ) 1.09 (6H, d, J ¼ 7.0 Hz, 2 -H), 1.16 (3H, t, J ¼ 7.5 Hz, 2 -
5
4(1H)-one (2g) (78 mg, 0.50 mmol), ethanol (2 mL), phosphate
3
00
3
CH CH ), 2.66 (1H, sep, J ¼ 7.0 Hz, 1 -H), 2.86 (2H, q, J ¼
2
3
buffer (0.2 M, pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bisporus)
were reacted for 20 h. Workup gave 2-(4,5-dihydroxy-2-methyl-
phenylthio)-5,6-dimethylpyrimidin-4(3H)-one (5e) as a pale yellow
0
0
0
7
(
.5 Hz, 2 -CH CH ), 6.32 (1H, s, 5-H), 7.28 (1H, s, 3 -H) and 7.67
2
3
0
1H, s, 6 -H); d
C
0
(75 MHz; pyridine-d ) 15.80 (2 -CH CH ), 21.25
5 2 3
), 35.63 (C-1 ), 103.06 (C-5), 116.20 (C-
), 117.29 (C-3 ), 125.44 (C-6 ), 141.26 (C-2 ), 145.57 (C-5 ),
50.20 (ov., C-4 ), 167.33 (C-2), 168.02 (C-4) and 175.01 (C-6); MS
ESI) m/z 345 ([M + K] , 23%), 329 ([M + Na] , 100), 307 ([M + H] ,
00
00
(
1
1
C-2 ), 27.24 (2 -CH
2
CH
3
ꢀ
powder (120 mg, 86%), mp 229–331 C; R ¼ 0.45 (CH Cl /EtOAc/
0
0
0
0
0
f
2
2
ꢁ1
MeOH ¼ 3 : 3 : 0.1); n
m
̃
ax (atr)/cm 3420 (OH), 2926 (CH), 1625
(500 MHz; pyridine-d
), 2.46 (3H, s, 2 -CH
0
(
C]O), 1512 and 1264; d
H
5
) 2.06 (3H, s, 5-
), 7.24 (1H, s, 3 -H)
+
+
+
(
0
0
CH
3
), 2.13 (3H, s, 6-CH
3
3
2
1) and 215 (14); HRMS calcd for C H N O S + H (307.1111),
15 18 2 3
0
and 7.69 (1H, s, 6 -H); d
2
3
(
C
(125 MHz; pyridine-d
), 113.56 (C-5), 116.85 (C-1 ), 118.92 (C-
), 125.00 (C-6 ), 135.31 (C-2 ), 145.61 (C-5 ), 149.60 (C-4 ), 161.78
C-2), 162.20 (C-6) and 166.32 (C-4); MS (ESI) m/z 301 ([M + Na] ,
5%), 279 ([M + H] , 100), 263 (25) and 157 (62); HRMS calcd for
S + H (279.0798), found 279.0797.
Synthesis and analytical data of 2-(2-ethyl-4,5-dihydrox-
5
) 10.87 (5-CH
3
),
found 307.1103.
Synthesis and analytical data of 2-(2-ethyl-4,5-dihydrox-
yphenylthio)-6-tert-butylpyrimidin-4(3H)-one (5h).
0
0
0.41 (2 -CH
3
), 21.61 (6-CH
3
0
0
0
0
0
+
+
2
13 14 2 3
C H N O
yphenylthio)-6-methylpyrimidin-4(3H)-one (5f).
According to general procedure II, 4-ethylcatechol (1c) (80 mg,
0.58 mmol), 6-tert-butyl-2,3-dihydro-2-thioxopyrimidin-4(1H)-one
(2e) (92 mg, 0.50 mmol), ethanol (2 mL), phosphate buffer (0.2 M,
pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bisporus) were reacted
for 16 h. Workup gave 2-(2-ethyl-4,5-dihydroxyphenylthio)-6-tert-
butylpyrimidin-4(3H)-one (5h) as a pale yellow powder (130 mg,
According to general procedure II, 4-ethylcatechol (1c) (80 mg,
.58 mmol), 2,3-dihydro-6-methyl-2-thioxopyrimidin-4(1H)-one
2a) (71 mg, 0.50 mmol), ethanol (2 mL), phosphate buffer (0.2 M,
pH 6.0, 18 mL) and laccase (12 U, 10 mg, A. bisporus) were reacted
for 14 h. Workup gave 2-(2-ethyl-4,5-dihydroxyphenylthio)-6-
methylpyrimidin-4(3H)-one (5f) as a pale yellow powder (125 mg,
0
(
ꢀ
8
3
1
1%), mp 153–155 C; R
: 3 : 0.1); n
f
¼ 0.49 (CH
2
Cl
2
/EtOAc/MeOH ¼
ꢁ
1
m
̃ ax (atr)/cm 3410 (OH), 2962 (CH), 1632 (C]O),
512 (C]N) and 1275; d
0
0
0
H
(300 MHz; pyridine-d
0
5
) 1.15 (9H, s, 2 -
3
3
ꢀ
ꢁ
H), 1.17 (3H, t, J ¼ 7.5 Hz, 2 -CH
2
CH
3
), 2.84 (2H, q, J ¼ 7.4 Hz, 2 -
9
3
0%), mp 180–182 C; Rf ¼ 0.31 (CH Cl /EtOAc/MeOH ¼
2
2
0 0
CH CH ), 6.44 (1H, s, 5-H), 7.30 (1H, s, 3 -H) and 7.67 (1H, s, 6 -H);
2 3
1
m
: 3 : 0.1); ñ ax (atr)/cm 3400 (OH), 2964 (CH), 1645 (C]O), 1503
0
0
3
d
C
(75 MHz; pyridine-d ) 15.79 (2 -CH CH ), 27.24 (2 -CH CH ),
5
2
3
2
3
(
2
C]N) and 1278; d
H
(500 MHz; pyridine-d
), 2.13 (3H, s, 6-CH
), 6.31 (1H, s, 5-H), 7.27 (1H, s, 3 -H) and 7.69 (1H, s, 6 -H);
(125 MHz; pyridine-d
5
) 1.17 (3H, t, J ¼ 7.5 Hz,
0
0
00
0
0
0
3
0
28.75 (C-2 ), 37.19 (C-1 ), 102.12 (C-5), 116.14 (C-1 ), 117.20 (C-3 ),
2
-CH CH
3
3
), 2.88 (2H, q, J ¼ 7.5 Hz, 2 -
0
0
0
0
0
0
125.47 (C-6 ), 141.28 (C-2 ), 145.52 (C-5 ), 149.8 (ov., C-4 ), 166.20
CH CH
2
3
0
(C-2 or C-4), 167.80 (C-2 or C-4) and 176.82 (C-6); MS (ESI) m/z 343
d
C
5
) 15.88 (2 -CH
2
CH
3
), 23.64 (6-CH
3
), 27.30
+
+
0
0
0
0
([M + Na] , 100%), 321 ([M + H] , 38) and 185 (25); HRMS calcd for
S + H (321.1267), found 321.1260.
Synthesis and analytical data of 2-(2-ethyl-4,5-dihydrox-
(
1
2 -CH CH ), 105.54 (C-5), 116.11 (C-1 ), 117.44 (C-3 ), 125.45 (C-6 ),
41.24 (C-2 ), 145.68 (C-5 ), 149.92 (C-4 ), 166.67 (C-6), 167.74 (C-2)
and 167.93 (C-4); MS (ESI) m/z 301 ([M + Na] , 100%), 279 ([M + H] ,
1) and 143 (18); HRMS calcd for C13 S + H (279.0798),
found 279.0790.
2
3
0
0
0
16 20 2 3
C H N O
+
+
yphenylthio)-5,6-dimethylpyrimidin-4(3H)-one (5i).
2
14 2 3
H N O
Synthesis and analytical data of 2-(2-ethyl-4,5-dihydrox-
yphenylthio)-6-isopropylpyrimidin-4(3H)-one (5g).
According to general procedure II, 4-ethylcatechol (1c)
(80 mg, 0.58 mmol), 2,3-dihydro-5,6-dimethyl-2-thioxopyr-
imidin-4(1H)-one (2g) (78 mg, 0.50 mmol), ethanol (2 mL),
phosphate buffer (0.2 M, pH 6.0, 18 mL) and laccase (12 U,
17438 | RSC Adv., 2017, 7, 17427–17441
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1
0 mg, A. bisporus) were reacted for 16 h. Workup gave 2-(2- 3500 (OH), 2962 (CH), 1639 (C]O), 1534 (C]N) and 1285; d
H
3
0
ethyl-4,5-dihydroxyphenylthio)-5,6-dimethylpyrimidin-4(3H)-one (500 MHz; pyridine-d ) 1.20 (3H, t, J ¼ 7.6 Hz, 2 -CH CH ), 1.72
5
2
3
ꢀ
ꢁ
3
3
(
0
5i) as a pale yellow powder (133 mg, 91%), mp 185–187 C; R ¼ (2H, quin, J ¼ 7.5 Hz, 6-H), 2.56 (2H, t, J ¼ 7.5 Hz, 7-H), 2.75
f
1
3
3
0
.43 (CH Cl /EtOAc/MeOH ¼ 3 : 3 : 0.1); n
̃
(atr)/cm 3403 (2H, t, J ¼ 7.5 Hz, 5-H), 2.88 (2H, q, J ¼ 7.6 Hz, 2 -CH CH ),
2
2
max
2
3
0
0
(OH), 2927 (CH), 1629 (C]O), 1523 (C]N) and 1252; d
H
(500 7.24 (1H, s, 3 -H) and 7.72 (1H, s, 6 -H); d
C
(125 MHz; pyridine-
3
0
0
0
MHz; pyridine-d
5
) 1.19 (3H, t, J ¼ 7.5 Hz, 2 -CH
2
CH
3
), 2.06
d
5
) 15.89 (2 -CH
2
CH
3
), 21.36 (C-6), 27.30 (2 -CH
2
CH
3
), 27.40 (C-
3
0
0
0
(3H, s, 5-CH
3
), 2.13 (3H, s, 6-CH
3
), 2.88 (2H, q, J ¼ 7.5 Hz, 2 - 5), 34.85 (C-7), 115.21 (C-1 ), 117.41 (C-3 ), 119.48 (C-4a), 125.41
0
0
0
0
0
0
CH
pyridine-d
2
CH
3
), 7.27 (1H, s, 3 -H) and 7.70 (1H, s, 6 -H); d
C
(125 MHz; (C-6 ), 141.20 (C-2 ), 145.76 (C-5 ), 150.11 (C-4 ), 163.06 (C-4),
0
+
5
) 10.82 (5-CH
3
), 15.86 (2 -CH
2
CH
3
), 21.55 (6-CH
3
0
), 163.85 (C-2) and 171.04 (C-7a); MS (ESI) m/z 327 ([M + Na] ,
0
0
+
2
1
7.27 (2 -CH CH ), 113.42 (C-5), 116.19 (C-1 ), 117.38 (C-3 ), 100%), 305 ([M + H] , 41) and 169 (14); HRMS calcd for
25.40 (C-6 ), 141.15 (C-2 ), 145.64 (C-5 ), 149.80 (ov., C-4 ), 162.20 C H N O S + H (305.0954), found 305.0946.
2
3
0
0
0
0
1
5
16 2 3
+
(C-6), 162.43 (C-2) and 166.34 (C-4); MS (ESI) m/z 315 ([M + Na] ,
+
1
00%), 293 ([M + H] , 69) and 157 (41); HRMS calcd for
S + H (293.0954), found 293.0949.
Synthesis and analytical data of 2-(4,5-dihydroxy-2-methyl-
phenylthio)-6,7-dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-one (5j).
Biology
14 16 2 3
C H N O
Sulforhodamine assay. The human HepG2 cell line (kindly
provided by Prof. Dr Lutz Graeve; Institut f u¨ r Biologische Chemie
und Ern ¨a hrungswissenscha, Universit ¨a t Hohenheim) was grown
in DMEM/Hams's medium (Merck) containing 10% fetal bovine
serum (Merck) and 1% penicillin/streptomycin. Cells were inocu-
lated into 96 well microtiter plates (Sarstedt) in 100 mL at plating
densities ꢃ 5000 cells per well. Aer cell inoculation, the micro-
ꢀ
titer plates were incubated at 37 C, 5% CO
2
, 95% air and 100%
relative humidity for 24 h prior to addition of compounds under
According to general procedure II, 4-methylcatechol (1b) test or doxorubicin (Sigma, cat. no. D2975000). Experimental
(
72 mg, 0.58 mmol), 2,3,6,7-tetrahydro-2-thioxo-1H-cyclopenta[d] drugs were prepared as 4 mM solution in DMSO and stored frozen
pyrimidin-4(5H)-one (2i) (84 mg, 0.50 mmol), ethanol (2 mL), prior to use. At the time of drug addition, an aliquot of frozen
phosphate buffer (0.2 M, pH 6.0, 18 mL) and laccase (12 U, 10 mg, concentrate and pure DMSO were thawed and diluted with
A. bisporus) were reacted for 14 h. Workup gave 2-(4,5-dihydroxy-2- complete DMEM/Hams's medium containing 10% fetal bovine
methylphenylthio)-6,7-dihydro-3H-cyclopenta[d]pyrimidin-4(5H)- serum and 1% penicillin/streptomycin. An aliquot of 100 mL of 4%
ꢀ
one (5j) as a pale yellow powder (130 mg, 90%), mp 240–242 C; R
f
DMSO was added to the microtiter wells containing 100 mL of
̃
(atr)/cm 3480 medium. An aliquot of 100 mL of the drug dilution was added to
max
ꢁ1
¼
0.30 (CH Cl /EtOAc/MeOH ¼ 3 : 3 : 0.1); n
2
2
(
OH), 2924 (CH), 1639 (C]O), 1512 and 1278; d (500 MHz; the appropriate microtiter wells, resulting in the required nal
H
0
pyridine-d
5
) 1.72 (2H, quin, 6-H), 2.47 (3H, s, 2 -CH
3
), 2.57 (2H, t, drug concentrations (2.5–80 mM). Six wells were prepared for each
3
3
0
J ¼ 7.7 Hz, 7-H), 2.75 (2H, t, J ¼ 7.7 Hz, 5-H), 7.21 (1H, s, 3 -H) individual dose. Following drug addition, the plates were incu-
0
0
ꢀ
and 7.70 (1H, s, 6 -H); d
C
(125 MHz; pyridine-d
5
) 20.42 (2 -CH
3
0
), bated for an additional 48 h at 37 C, 5% CO
2
, 95% air, and 100%
1.37 (C-6), 27.41 (C-5), 34.89 (C-7), 115.94 (C-1 ), 118.93 (C-3 ), relative humidity. The assay was terminated by the addition of cold
0
2
1
(
0
0
0
19.48 (C-4a), 125.03 (C-6 ), 135.35 (ov., C-2 ), 145.71 (C-5 ), 149.80 trichloroacetic acid (TCA). Cells were xed in situ by gentle addi-
ov., C-4 ), 163.05 (C-4), 163.32 (C-2) and 171.02 (C-7a); MS (ESI) tion of 50 mL of cold 50% (w/v) TCA (nal concentration, 10% TCA)
m/z 313 ([M + Na] , 93%), 291 ([M + H] , 100) and 169 (67); HRMS and incubated for 60 min at 4 C. The supernatant was discarded,
calcd for C H N O S + H (291.0798), found 291.0788.
and the plates were washed ve times with tap water and air dried.
Synthesis and analytical data of 2-(2-ethyl-4,5-dihydrox- Sulforhodamine B (SRB) (Sigma-Aldrich, cat. no. S1402) solution
0
+
+
ꢀ
1
4
14 2 3
yphenylthio)-6,7-dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-one (5k).
(100 mL) at 0.4% (w/v) in 1% acetic acid was added to each well,
and plates were incubated for 10 min at room temperature. Aer
staining, unbound dye was removed by washing at least ve times
with 1% acetic acid and the plates were dried. Bound stain was
subsequently solubilized with 10 mM Trizma base pH 10.5, and
the absorbance was read on ELISA microplate reader at a wave-
length of 515 nm. IC50 was calculated for each experiment using
four parameter logistic equation (Graph Pad, Prism Version 5).
According to general procedure II, 4-ethylcatechol (1c) (80 mg, IC50 results represent the mean of 2–5 experiments with the
.58 mmol), 2,3,6,7-tetrahydro-2-thioxo-1H-cyclopenta[d]pyr- standard error of the mean indicating the variation.
0
imidin-4(5H)-one (2i) (84 mg, 0.50 mmol), ethanol (2 mL), phos-
phate buffer (0.2 M, pH 6.0, 18 mL) and laccase (12 U, 10 mg, A.
bisporus) were reacted for 14 h. Workup gave 2-(2-ethyl-4,5-dihy-
droxyphenylthio)-6,7-dihydro-3H-cyclopenta[d]pyrimidin-4(5H)-
Conclusions
To summarize, simple-to-perform, efficient and sustainable
methods for the synthesis of regioisomeric pyrimidobenzo-
thiazoles 3, 4 as well as catechol thioethers 5 by laccase-catalyzed
ꢀ
one (5k) as a pale yellow powder (115 mg, 76%), mp 178–180 C;
ꢁ1
R ¼ 0.36 (CH Cl /EtOAc/MeOH ¼ 3 : 3 : 0.1); n
̃
(atr)/cm
f
2
2
max
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domino reactions between catechols 1 and 2,3-dihydro-2-thio-
xopyrimidin-4-(1H)-ones 2 have been developed. The trans-
formations rely on the laccase-catalyzed oxidation of a catechol 1
to the corresponding o-benzoquinone 8 which in turn undergoes
Dev., 2016, 20, 1118; (b) F. Roschangar, R. A. Sheldon and
C. H. Senanayake, Green Chem., 2015, 17, 752; (c)
E. A. Peterson and J. B. Manley, Green Chemistry Strategies
for Drug Discovery, in RSC Drug Discovery Series, Royal
Society of Chemistry, Thomas Graham House, Cambridge,
2015; (d) P. Gupta and A. Mahajan, RSC Adv., 2015, 5,
26686; (e) H.-J. Federsel, Green Chem., 2013, 15, 3105.
5 B. M. Trost, Angew. Chem., Int. Ed., 1995, 34, 259.
6 (a) R. A. Sheldon, I. W. C. E. Arends and U. Hanefeld, Green
Chemistry and Catalysis, WILEY-VCH, Weinheim, 2007; (b)
P. T. Anastas and J. C. Warner, Green Chemistry Theory and
Practice, Oxford University Press, New York, 2000; (c)
K. Alfonsi, J. Colberg, P. J. Dunn, T. Fevig, S. Jennings,
T. A. Johnson, H. P. Kleine, C. Knight, M. A. Nagy,
D. A. Perry and M. Stefaniak, Green Chem., 2008, 10, 31; (d)
D. J. C. Constable, P. J. Dunn, J. D. Hayler,
G. R. Humphrey, J. L. Leazer Jr, R. J. Linderman, K. Lorenz,
J. Manley, B. A. Pearlman, A. S. Wells, A. Zaks and
T. Y. Zhang, Green Chem., 2007, 9, 411.
reaction with
a 2,3-dihydro-2-thioxopyrimidin-4(1H)-one 2.
Depending on the substitution pattern of the 2,3-dihydro-2-thi-
oxopyrimidin-4(1H)-one 2, the laccase-catalyzed reactions with
unsubstituted catechol (1a) deliver either 7,8-dihydroxy-4H-pyr-
imido[2,1-b]benzothiazol-4-ones 3 and/or 7,8-dihydroxy-2H-pyr-
imido[2,1-b]benzothiazol-2-ones 4. With 4-substituted catechols
1b, c, catechol thioethers 5 are formed exclusively. All reactions
can be performed under mild reaction conditions with aerial O2
as the oxidant, and the products are formed with yields ranging
between 76 and 97%. In addition, the cytotoxicity of selected
pyrimidobenzothiazoles 3 and catechol thioethers 5 against
HepG2 cell line is reported. A structure–activity relationship
study reveals that the most potent compounds are 5c (IC50 ¼ 7.77
mM) and 5g (IC50 ¼ 2.74 mM) which carry an isopropyl group at C-
0
6
. The presence of an additional ethyl group at C-2 of 5g
increases the cytotoxic potency in comparison to 5c.
7 (a) R. Dhanasekaran, A. Limaye and R. Cabrera, Hepatic
Med., 2012, 4, 19; (b) S. Mittal and H. B. El-Serag, J. Clin.
Gastroenterol., 2013, 47, S2; (c) E. Carr and J. A. Killman,
Basics of Cancer Chemotherapy (Clinical Nursing Series),
Western Schools Press, Canada, 3rd edn, 1995.
8 (a) S.-I. Shoda, H. Uyama, J.-I. Kadokawa, S. Kimura and
S. Kobayashi, Chem. Rev., 2016, 116, 2307; (b) D. Monti,
G. Ottolina, G. Carrea and S. Riva, Chem. Rev., 2011, 111,
4111; (c) F. Hollmann, I. W. C. E. Arends, K. Buehler,
A. Schallmey and B. B u¨ hler, Green Chem., 2011, 13, 226.
9 (a) O. V. Morozova, G. P. Shumakovich, M. A. Gorbacheva,
S. V. Shleev and A. I. Yaropolov, Biochemistry (Moscow),
2007, 72, 1136; (b) H. Claus, Micron, 2004, 35, 93; (c)
A. M. Mayer and R. C. Staples, Phytochemistry, 2002, 60,
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Acknowledgements
We thank Mr M. Wolf (Institut f u¨ r Chemie, Universit ¨a t
Hohenheim) for recording NMR spectra as well as Dipl.-Ing.
(
FH) J. Trinkner and D. Garnier (Institut f u¨ r Organische
Chemie, Universit ¨a t Stuttgart) for recording mass spectra.
Special thanks to Prof. Dr Lutz Graeve; (Institut f u¨ r Biologische
Chemie und Ern ¨a hrungswissenscha, Universit ¨a t Hohenheim)
for providing the HepG2 cell line. We acknowledge the help of
A. Flaccus and D. Mvondo (Institut f u¨ r Biologische Chemie und
Ern ¨a hrungswissenscha, Universit ¨a t Hohenheim) in carrying
out the SRB assay. H. T. A.-M. is grateful to Science Technology
Development Fund (STDF) for nancial support (Project ID
11978).
10 (a) S. Witayakran and A. J. Ragauskas, Adv. Synth. Catal.,
2009, 351, 1187; (b) A. Mikolasch and F. Schauer, Appl.
Microbiol. Biotechnol., 2009, 82, 605; (c) A. Kunamneni,
S. Camarero, C. Garc ´ı a-Burgos, F. J. Plou, A. Ballesteros
and M. Alcalde, Microb. Cell Fact., 2008, 7, 32.
Notes and references
1
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(a) E. Sherman, in Lippincott Illustrated Reviews:
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