Laccase-catalyzed domino reaction between catechols and 6-substituted 1,2,3,4-tetrahydro-4-oxo-2-thioxo-5-pyrimidinecarbonitriles for the synthesis of pyrimidobenzothiazole derivatives

Article abstract of DOI:10.1021/jo401193e

The laccase-catalyzed domino reaction between catechols and 6-substituted 1,2,3,4-tetrahydro-4-oxo-2-thioxo-5-pyrimidinecarbonitriles using aerial O ₂ as the oxidant delivers new pyrimidobenzothiazole derivatives. The complete structure elucida

Full text of DOI:10.1021/jo401193e

Article
pubs.acs.org/joc
Laccase-Catalyzed Domino Reaction between Catechols
and 6‑Substituted 1,2,3,4-Tetrahydro-4-oxo-2-thioxo-
5-pyrimidinecarbonitriles for the Synthesis
of Pyrimidobenzothiazole Derivatives
Heba T. Abdel-Mohsen, Jurgen Conrad, and Uwe Beifuss*
̈
Bioorganische Chemie, Institut fur Chemie, Universitat Hohenheim, Garbenstrasse 30, D-70599 Stuttgart, Germany
̈
̈
S
* Supporting Information
ABSTRACT: The laccase-catalyzed domino reaction between
catechols and 6-substituted 1,2,3,4-tetrahydro-4-oxo-2-thioxo-5-
pyrimidinecarbonitriles using aerial O2 as the oxidant delivers
new pyrimidobenzothiazole derivatives. The complete structure
elucidation of the ring-proton deficient heterocyclic products
and the unambiguous determination of the regioselectivity of
the reactions have been achieved by extended NMR spectroscopic methods including HSQMBC, super long-range HMBC, and 15N
measurements.
the polymerization of numerous phenolics15 as well as the
INTRODUCTION
■
degradation of lignin.16
Since there are a large number of biologically active heterocycles,
their selective and efficient preparation is at the core of organic
synthesis.1 In view of the growing importance of enzyme-
catalyzed transformations in organic synthesis, it is surprising that
the number of methods for the synthesis of hetereocycles based
on enzyme-catalyzed transformations is rather limited.2 Among
the most well-known methods are the lipase-catalyzed double
bond epoxidation3 and the generation of lactones using Baeyer−
Villiger monooxygenases.4 More recently, we became interested
in the laccase-catalyzed transformations. Laccases are multi-
copper oxidases that are produced by fungi, plants, and pro-
karyotes, can be easily isolated and purified, and can be obtained
commercially.5 Laccases catalyze the oxidation of numerous
substrates using aerial oxygen as the oxidant.6 Employing laccase-
mediator systems, the substrate range of laccases can be extended
considerably.6d,e,7 The use of laccase-catalyzed transformations
is not restricted to simple oxidations such as the oxidation of
alcohols to aldehydes,8 the oxidative coupling of thiols to di-
sulfides9 or the aromatization of 1,4-dihyropyridines to the cor-
responding pyridines.10 They can also be combined with
chemical transformations like the Diels−Alder reaction11 or 1,4-
additions to new domino processes. Thus, the laccase-catalyzed
oxidation of catechols and hydroquinones can be combined
with the reaction of the resulting o- or p-benzoquinones with
C-,12 N-,6c,13 and S-nucleophiles.14 Using this approach, we have
been able to demonstrate that the laccase-catalyzed oxida-
tion of catechols in the presence of 1,3-dicarbonyls allows
the synthesis of a number of O-heterocycles12f−h,j,k such as
6-substituted 7,8-dihydroxybenzofuro[3,2-c]pyridin-1(2H)-
ones, 10-substituted 8,9-dihydroxybenzofuro[3,2-c]quinolin-
6(5H)-ones, and 8,9-dihydroxy-5-thiocoumestans.12g Apart
from applications in organic synthesis, laccases play also an
important role in polymer chemistry. They have been used for
Benzothiazoles exhibit a wide range of interesting pharmaco-
logical properties.17 Pyrimidobenzothiazole derivatives, for example,
are known for their antiallergic,18 antibacterial,19 and antifungal
activities.20 Some pyrimidobenzothiazoles have been demonstrated
to have a high affinity to the benzodiazepine receptor.21 This is
why a number of methods have been developed for the synthesis
of this heterocyclic skeleton. They include the condensation of
2-aminobenzothiazoles with acetylene carboxylic acids22 and
acetylene dicarboxylic acid derivatives,21a 2-aminofumarates,21a
and β-ketoesters.23 Pyrimidobenzothiazoles can also be
obtained by a three-component reaction of 2-aminobenzo-
thiazoles with aldehydes and β-ketoesters.24 The amidines of
2-aminobenzothiazoles have also been employed as substrates
for the synthesis of pyrimidobenzothiazoles.25 In summary,
the number of approaches to the pyrimidobenzothiazoles is
rather limited. This is why the development of new methods
for their preparation is highly desirable. In view of the fact that
so far no enzyme-catalyzed synthesis of the pyrimidobenzo-
thiazole skeleton has been developed, we wondered whether
it is possible to develop a laccase-catalyzed synthesis of this
heterocyclic system that is based on the combination of a
laccase-catalyzed oxidation of catechols to o-benzoquinones
and its reaction with thioxo-5-pyrimidinecarbonitriles.
RESULTS AND DISCUSSION
■
Here we report on the laccase-catalyzed synthesis of pyrimido-
benzothiazoles by reaction of catechol (1a) and 3-methylcatechol
(1b) with 6-substituted 1,2,3,4-tetrahydro-4-oxo-2-thioxo-5-
pyrimidinecarbonitriles 5a−f using aerial oxygen as the oxidant.
Received: June 1, 2013
© XXXX American Chemical Society
A
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Figure 1. Substrates for the laccase-catalyzed domino reactions.
Table 1. Laccase-Catalyzed Domino Reaction between Catechol (1a) and 1,2,3,4-Tetrahydro-4-oxo-6-phenyl-2-thioxo-
5-pyrimidinecarbonitrile (5a) under Different Conditions
a
b
c
entry
laccase (U)
1a:5a
buffer (mL)
T (°C)
time (h)
6a:7a
yield 6a + 7a (%)
d
g
1
2
3
4
5
6
7
8
120
120
60
1:1
1.25:1
1.25:1
1.25:1
1.25:1
1.25:1
1.25:1
1.25:1
80
80
80
80
30
60
30
30
22
22
22
22
22
22
50
22
24
12
18
24
15
15
10
24
75:25
72:28
74:26
84
95
93
e
e
e
e
f
30
traces
95
60
71:29
74:26
75:25
120
60
95
e
e
90
a
b
c
1
Phosphate buffer (0.2 M) was used. Ratio determined from the H NMR spectrum of the reaction mixture of 6a and 7a. Yields refer to crude
d
e
f
yields. 0.50 mmol of 1a and 0.50 mmol of 5a were reacted. 0.63 mmol of 1a and 0.50 mmol of 5a were reacted. 1.25 mmol of 1a and 1 mmol of
5a were reacted. Traces of 5a were present in the crude product.
g
In addition, we present a NMR method that allows the
unequivocal structure elucidation of the pyrimidobenzothia-
mixture (1H NMR) of the two regioisomers 6a and 7a was
obtained. Further modifications of the experimental procedure
with respect to the amount of catalyst and buffer, reaction
temperature, and reaction time revealed that the best result was
achieved when 0.63 mmol of 1a and 0.50 mmol of 5a were
reacted in the presence of 60 U laccase in 30 mL of phosphate
buffer pH 6 and 5.5 mL of MeOH at room temperature under air
for 15 h (Table 1, entry 5). Under these conditions 6a and 7a
were formed in 95%. The reaction could be also run on a 1 mmol
scale (Table 1, entry 6). A control experiment demonstrated that
without the laccase the transformation did not take place (Table 1,
entry 8).
Apart from the two cyclization products 6a and 7a, no
other products were obtained. It should be noted that none
of the two possible noncyclic 1,4-addition products I and II
were isolated (Figure 2).
It is assumed that the reaction sequence starts with the
laccase-catalyzed oxidation of catechol (1a) to o-benzoquinone
(8a) (Scheme 1). This is followed by 1,4-addition of the
nucleophilic S atom of the 4-oxo-2-thioxo-5-pyrimidinecarboni-
trile 5a to deliver the two tautomers 9A and 9B as
intermediates. Subsequent oxidation of 9A or 9B produces
the tautomeric benzoquinones 10A and 10B, respectively.
Depending on which of the N atoms acts as nucleophile, the
intramolecular 1,4-addition gives either the type I product 6a or
1
zoles obtained. It is based on H−15N HMBC NMR cor-
relations at natural abundance and experimental 1H−13C
long-range coupling constants in addition to super long-
1
range H−13C HMBC correlations, as well as calculated 13C
chemical shifts for the structure elucidation of aromatic ring-proton
deficient heterocyclic compounds whose chemical structures only
hardly could be assessed by standard NMR methods.
The 6-substituted 1,2,3,4-tetrahydro-4-oxo-2-thioxo-5-pyrimi-
dinecarbonitriles 5a−f required for this study were synthesized
by three-component reactions between aromatic and aliphatic
aldehydes 2a−f, methyl cyanoacetate (3), and thiourea (4) in the
presence of equimolar amounts of K2CO3 in methanol according
to the procedure of Kambe et al. (Figure 1).26
In a first experiment, 0.50 mmol of catechol (1a) and 0.50 mmol
of 1,2,3,4-tetrahydro-4-oxo-6-phenyl-2-thioxo-5-pyrimidinecarboni-
trile (5a) were reacted in a mixture of phosphate buffer (0.2 M,
pH 6) and MeOH (85:15 v/v) for 24 h at room temperature using
a commercially available laccase from Agaricus bisporus (120 U,
6 U/mg) as the catalyst and air as the oxidant (Table 1, entry 1).
After acidic workup, 84% of a 75:25 mixture (1H NMR) of the
regioisomers 6a and 7a was isolated. Since the crude product still
contained traces of 5a (TLC), the reaction was repeated using a
slight excess of 1a (Table 1, entry 2). After 12 h, 95% of a 72:28
B
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and the ratio of the two regioisomers ranged from 51:49 to 92:8.
Obviously, the ratio was strongly affected by the substituent at
C-6 of the 4-oxo-2-thioxo-5-pyrimidinecarbonitriles 5b−f.
With aromatic substituents, such as the 4-methylphenyl group
in 5b and the 4-methoxyphenyl group in 5c, the ratios of 6:7
were similar to the ratio observed in the reaction between 1a
and 5a (Table 2, entries 2 and 3). When the ethyl-substituted
4-oxo-2-thioxo-5-pyrimidinecarbonitrile 5d was reacted, nearly
equal amounts of the isomers 6d and 7d were formed (Table 2,
entry 4). A similar result was observed in the transformation
between the n-propyl-substituted 4-oxo-2-thioxo-5-pyrimidine-
carbonitrile 5e and 1a (Table 2, entry 5). However, when the
isopropyl-substituted 4-oxo-2-thioxo-5-pyrimidinecarbonitrile 5f
was used as a substrate, the regioisomer 6f was formed in excess
over the regioisomer 7f; the ratio amounted to 92:8 (Table 2,
entry 6). It is assumed that the ratio of the two isomers depends
on the steric demand of the C-6 substituent of the 4-oxo-2-
thioxo-5-pyrimidinecarbonitriles 5a−f. Considering 10A and
10B as intermediates of the domino reaction, it is expected that
sterically more demanding C-6 substituents favor the formation
of type I products 6. This is supported by the finding that with
sterically less demanding unbranched alkyl groups as in 5d and
5e, the regioisomers 6 and 7 are formed in comparable amounts.
As expected, the amount of 6 increases when sterically more
demanding aryl groups or the isopropyl group are attached to
C-6. The mixtures of the regioisomers 6b−f and 7b−f were
also difficult to separate. In order to separate the isomers, the
mixtures were transformed into the corresponding diacetates
with yields ranging from 68 to 85% (Table 3). After separation
by flash chromatography on silica gel, the individual isomers
11b−f and 12b,d,e were obtained in analytically pure form.
In order to study the influence of substituents on the
o-benzoquinone moiety on the yield and the selectivity of the
pyrimidobenzothiazole synthesis, 3-methylcatechol (1b) was
reacted with different 6-substituted 1,2,3,4-tetrahydro-4-oxo-
2-thioxo-5-pyrimidinecarbonitriles 5a−f under the conditions
developed for the laccase-initited domino reaction between
catechol (1a) and 5a−f (Table 1, entry 5 and Table 2). It is
remarkable that in most cases only two out of four possible products
could be detected (1H NMR), namely, the type IA products
13a−f and the type IB products 14a−f (Table 4, Scheme 3).
Figure 2. Possible noncyclic 1,4-addition products of the reaction
between 1a and 5a.
the type II product 7a. The finding that the type I product 6a was
formed in excess indicates that N-3 (marked in blue) of tautomer
10A is more reactive than N-1 (marked in green) of 10B. This is
supported by intermolecular nucleophilic substitution reactions of
different S-alkylated/benzylated thioxopyrimidines with halides.27
Using the mixture of 6a and 7a, the structure elucidation
of 6a and 7a proved to be difficult. This is why we tried
to separate the two regioisomers. However, due to their high
polarity and their low solubility even in polar solvents,
they could not be separated. To increase their solubility,
the pyrimidobenzothiazoles 6a and 7a were transformed into
the corresponding diacetates 11a and 12a. For this purpose, the
mixture of 6a and 7a was reacted with an excess of acetic
anhydride in the presence of catalytic amounts of dimethylami-
nopyridine (DMAP) in pyridine28 to deliver 77% of a mixture of
11a and 12a (Scheme 2). The two regioisomeric diacetates 11a
and 12a could be separated by flash chromatography on silica gel
to deliver the analytically pure heterocycles 11a and 12a in 50%
and 8%, respectively.
Then, the protocol developed for the reaction between 1a
and 5a was applied to the transformations of catechol (1a) with
1,2,3,4-tetrahydro-4-oxo-2-thioxo-5-pyrimidinecarbonitriles
5b−f carrying different aryl or alkyl groups at the 6-position
(Table 2). First of all, in all cases the corresponding
pyrimidobenzothiazole derivatives 6 and 7 were formed
exclusively. Again, none of the 1,4-addition products I and II
could be detected. The yields were in the range of 83−96%,
Scheme 1. Possible Mechanism for the Reaction of Catechol (1a) with 1,2,3,4-Tetrahydro-4-oxo-6-phenyl-2-thioxo-
5-pyrimidinecarbonitrile (5a)
C
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Scheme 2. Acetylation of the Mixture of 6a and 7a
Table 2. Laccase-Catalyzed Domino Reaction between Catechol (1a) and 6-Substituted 1,2,3,4-Tetrahydro-4-oxo-2-thioxo-
a
5-pyrimidinecarbonitriles 5a−f
b
entry
5
R
6,7
6:7
yield 6 + 7 (%)
c
d
d
1
2
3
4
5
6
a
b
c
d
e
f
C6H5
a
b
c
d
e
f
71:29 , 77:23
95
96
90
83
84
c
4-CH3-C6H4
4-CH3O-C6H4
C2H5
76:24 , 68:32
c
,d
66:34
c
d
d
53:47 , 57:43
c
C3H7
51:49 , 55:45
c,d
CH(CH3)2
92:8
92
a
b
c
1
Experiments were carried out using 0.63 mmol of 1a and 0.50 mmol of 5a−f. Yields refer to crude yields. Ratio determined from H NMR
d
spectrum of the reaction mixture of 6 and 7. Ratio determined from 13C NMR spectrum of the reaction mixture of 6 and 7.
Table 3. Acetylation of 6,7
a
b
b
entry
6,7
R
11,12
yield 11 + 12 (%)
yield 11 (%)
yield 12 (%)
1
2
3
4
5
6
a
b
c
d
e
f
C6H5
a
b
c
d
e
f
77
81
77
76
85
68
50
46
45
35
38
8
23
c
4-CH3-C6H4
4-CH3O-C6H4
C2H5
16
36
c
C3H7
CH(CH3)2
45
a
b
c
Yields refer to crude yield of the acetylation step. Yields refer to isolated products after flash chromatography. 12c and 12f Could not be isolated
in pure form.
The yields were between 78% and 93%, and the ratio of the two
isomers ranged from 73:27 to 83:17.
regarding the nucleophilic 1,4-addition of C- and S-nucleophiles
to o-methyl benzoquinones,12f,g,j,29 it was no surprise that the 1,4-
addition of 5a−f at C-5 of 8b is preferred over the 1,4-addition at
C-4 of 8b. The observation that 15A/15B selectively delivers the
type IA products 13 may be attributed to (a) the higher reactivity
of N-3 (marked in blue) in comparison to N-1 (marked in green)
and (b) the steric interactions between the CH3 group of the
o-benzoquinone moiety and the R group in 16B. It should
be mentioned that the oxidation/intramolecular 1,4-addition
sequence starting from 18A/18B results in the formation of the
type IB products 14. Type IIB products 20 could not be detected.
The formation of type IB products 14 may be due to the fact
that N-3 (marked in blue) in 19A is more reactive than N-1
(marked in green) (Scheme 3).
It is assumed that the reaction starts with the laccase-catalyzed
oxidation of 3-methylcatechol (1b) to 8b followed by a nucleo-
philic 1,4-attack of the thiol group of 5a−f at either C-4 or C-5 of
o-methyl benzoquinone (8b) (Scheme 3). Attack at C-5 results in
the formation of a tautomeric mixture of 15A and 15B. After
oxidation to 16A and 16B, respectively, the intramolecular 1,4-
addition delivers the corresponding type IA products 13 and type
IIA products 17, respectively. In contrast, the nucleophilic addi-
tion of 5 at C-4 of 8b would give 18A and 18B, which after
oxidation to 19A and 19B, respectively, and final intramolecular
1,4-addition would produce type IB products 14 and type IIB
products 20, respectively. In accordance with previous results
D
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Table 4. Laccase-Catalyzed Domino Reaction between 3-Methylcatechol (1b) and 6-Substituted 1,2,3,4-Tetrahydro-4-oxo-
2-thioxo-5-pyrimidinecarbonitriles 5a−f
a
b
entry
5
R
13,14
13:14
yield 13 + 14 (%)
1
2
3
4
5
6
a
b
c
d
e
f
C6H5
a
b
c
d
e
f
overlapped peaks
82:18
91
93
89
82
78
85
4-CH3-C6H4
4-CH3O-C6H4
C2H5
overlapped peaks
c
80:20
c
C3H7
83:17
CH(CH3)2
73:27
a
b
c
Ratio determined from 1H NMR spectrum of the reaction mixture of 13 and 14. Yields refer to crude yields. The 1H NMR spectra of 13d/14d
and 13e/14e revealed the presence of traces of a third compound of unknown structure.
Scheme 3. Possible Mechanism for the Reaction of 3-Methylcatechol (1b) with 6-Substituted 1,2,3,4-Tetrahydro-4-oxo-
2-thioxo-5-pyrimidinecarbonitriles 5a−f
Since we did not succeed in separating the highly
polar isomeric pyrimidobenzothiazoles 13 and 14, they
were reacted with acetic anhydride and catalytic amounts of
DMAP to yield mixtures of the corresponding diacetates
21a−f and 22a−f with yields between 70% and 78% (Table 5).
Despite an increase in solubility, the diacetates 21a−f and
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Table 5. Acetylation of 13,14
a
b
entry
13,14
R
21,22
yield 21 + 22 (%)
21:22
yield 21 + 22 (%)
c
1
2
3
4
5
6
a
b
c
d
e
f
C6H5
a
b
c
d
e
f
78
76
70
72
76
90:10
53
43
53
41
57
58
c
c
c
c
4-CH3-C6H4
4-CH3O-C6H4
C2H5
84:16
87:13
85:15
,
,
d
d
d
C3H7
87:13 , 81:19
c,d
CH(CH3)2
75
77:23
a
b
c
Yields refer to crude yield. Yields refer to isolated yield of the mixtures of 21 and 22. Ratio determined from 13C NMR spectrum of the mixture of
d
1
21 and 22 after column chromatography. Ratio determined from H NMR spectrum of the mixture of 21 and 22 after column chromatography.
Figure 3. Structures of the two regioisomers 11e and 12e.
22a−f could not be separated by TLC or column chro-
were of the same order of magnitude, (ii) the 13C chemical
shifts could not easily be assigned by, e.g., standard HMBC, and
(iii) there were no 13C NMR data of similar structures in the
literature available. The latter holds true also for 15N chemical
shift considerations. Nevertheless, we found a fast and reliable
solution for the unequivocal determination of the regioselec-
matography.
Structure Elucidation by NMR. Preliminary analysis of
the 1H NMR spectra of 11,12 and 21,22, respectively, revealed
that the laccase-catalyzed reactions between catechols 1 and
6-substituted 1,2,3,4-tetrahydro-4-oxo-2-thioxo-5-pyrimidine-
carbonitriles 5a−f produces two regioisomeric pyrimidobenzo-
thiazoles 6,7 and 13,14, respectively (Tables 2 and 4). However,
their structural identification was challenging since single crystals
for X-ray analysis could not be obtained so far. The limited
solubility of the acetylated reaction products 11,12 and 21,22 in
almost all NMR solvents did not allow insensitive 13C−13C and/
or 13C−15N NMR correlation spectroscopy at natural abundance
to unambiguously assign the complete heterocyclic skeleton of
the compounds. Moreover, all possible tricyclic derivatives suffer
from a lack of aromatic protons in such a way that classical NMR
methods (1H, selective NOE/ROE, standard gHMBC) can
provide only partial information for assignment purposes but not
sufficient information for resolving regioselectivity issues. As
an example, the two regioisomers 11e and 12e (Figure 3) possess
only 2 aromatic protons in ring A and none in rings B and C
(Figure 3).
This raised the question whether there is any NMR-based
solution to differentiate between the two regioisomers without
tedious derivatization and/or degradation reactions. Unfortu-
nately, NOE experiments with the major isomer 11e displayed
no NOE enhancements at all between the propyl protons
and the aromatic protons of ring A. This is due to the fact that
their spatial distance is greater than 5 Å. A differentiation of the
isomers by comparison of the 13C chemical shifts of the C rings
of 11e (δ 172.6, 166.0, 158.1, 93.5 ppm) and 12e (δ 166.6,
162.3, 160.4, 98.6 ppm) was also impossible since (i) the values
1
tivity by indirect detection of H−15N long-range gHMBC
correlations, which could be recorded in 24−40 h without
cryo probe technology despite the limited solubility of the
compounds (ca. 10−50 mg/mL). The underlying idea is this: if
1
we detect H−15N long-range correlations with two different
15N chemical shifts (irrespective of the value itself), one of them
3
4
originating from a J and J correlation of the two aromatic
3
protons of ring A and the other from a J correlation of the
propyl protons, then the structure of the major regioisomer
1
has to be 11e (Figure 4 A). However, if the H−15N gHMBC
spectrum displays long-range correlations of the aromatic
protons of ring A and the propyl protons to only one (= the same)
15N chemical shift, then the structure of the minor regioisomer has
to be 12e (Figure 4 B). Using this approach, both the C-2/C-4
substitution issue and the regioselectivity of the right part of the
molecule (ring C) of all regioisomers 11 and 12 as well as 21
could be unequivocally determined.
The next question was how to prove the connectivity between
rings A and C of the heterocyclic isomers. In case of the
bisacetylated reaction products 21 and 22 this was a particularly
difficult problem since both of them have only a single aromatic
proton. A prerequisite for solving this problem was a fully
assigned carbon skeleton with the focus on the quaternary
1
carbons C-10a, C-4, C-5a, and C-9a. In the standard H−13C
gHMBC (optimized for JCH = 8 Hz) of, for example, 11e each of
the latter two at δ 122.2 and 132.8 (or vice versa) ppm showed
F
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1
Figure 4. Part of the H−15N gHMBC spectrum of 11e (A) and 12e (B) at natural abundance.
6J coupling (between 9-H and C-3) that can hardly be observed,
the proton at δ 8.77 ppm was assigned as 6-H and the proton at
δ 8.14 ppm as 9-H (Figures 5, 6B).
1
two H−13C long-range correlations to both aromatic ring A
protons at δ 8.77 and 8.14 ppm. This precludes any assignment
of the respective positions (Figures 5, 6A). However, a gHMBC
1
Evaluation of the experimental H−13C long-range coupling
constants (absolute values) between 9-H and C-5a (3J =
9.3 Hz) and C-9a (2J = 3.0 Hz) as well as between 6-H and
C-5a (2J = 4.2 Hz) and C-9a (3J = 8.8 Hz) allowed the assign-
ment of the carbons C-5a and C-9a at δ 132.8 and 122.2 ppm,
3
respectively, since JCH coupling constants (absolute values)
2
in aromatic ring systems are significantly greater than JCH and
4JCH coupling constants (Figure 7).12g,31
In order to support the experimental assignments, we computed
the 13C NMR chemical shifts by DFT quantum mechanical DFT
calculations at the DFT GIAO mPW1PW91/6-311+G(2d,p)//
mPW1PW91/6-31G(d) level of theory32 and found that the
calculated values are in good agreement with the experimental data
for C-5a (δ calcd 132.3, exptl 132.8 ppm) and C-9a (δ calcd 124.9,
exptl 122.2 ppm). The remaining quaternary carbons C-4 and
C-10a were assigned on the basis of computationally calculated
13C chemical shifts of C-4 (δ calcd 158.3, exptl 158.1 ppm) and of
C-10a (δ calcd 166.6, exptl 166.0 ppm), as the observed super
long-range 1H−13C HMBC correlations of δ 158.1 and 166.0 ppm
didn’t allow an unambiguous assignment of C-4 and C-10a.
The structures of the minor isomers 12a,b,d,e were elucidated
and fully assigned in the same way as described for compounds
11a−f. Fortunately, strong ROEs between the aromatic proton 6-H
and the protons of the side chain at C-4 in the minor compounds
12a,b,d,e confirmed our concept of the differentiation between the
Figure 5. Important 2J, 3J, 4J, and 5J HMBC correlations from 6-H and
9-H of 11e.
pulse sequence modified according to Seto et al.30 enabled the
1
detection of weak H−13C super long-range correlations (4JCH
,
5JCH, or even higher) in lieu of only JCH and JCH couplings.
Thus, the aromatic proton at δ 8.14 ppm exhibits two additional
1H−13C super long-range correlations to the quaternary carbons
at δ 158.1 and 166.0 ppm, whereas the aromatic proton at δ 8.77
ppm showed two additional 1H−13C super long-range correlations
to two quaternary carbons at δ 158.1 and C-3 at δ 93.5 ppm.
2
3
5
On the basis of a J coupling between 6-H and C-3 rather than a
1
Figure 6. Part of the normal (A) and the super long-range (B) H−13C gHMBC spectrum of 11e.
G
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Figure 7. Part of the HSQMBC spectrum of 11e.
Figure 8. Part of the ROESY spectrum of 12e.
regioisomers 11 and 12 based on the presence of one or two
indirectly detectable 15N chemical shifts (Figure 8).
Again, the calculated 13C chemical shifts are in a good agree-
ment with the experimental data: 12e C-10a (δ calcd 164.5,
exptl 166.6 ppm), C-2 (δ calcd 158.4, exptl 162.3 ppm), C-4 (δ
calcd 160.7, exptl 160.4 ppm), C-5a (δ calcd 130.7, exptl 132.8
ppm), C-9a (δ calcd 125.6, exptl 121.8 ppm). Interestingly,
in compounds 12a,b a strong shielding of the aromatic proton
6-H (e.g δ 5.54 ppm in 12a) induced by the ring current effect of
the aryl moiety indicate the spatial proximity between 6-H and the
phenyl substituent at C-4 (Figure 9). Moreover, the aryl groups
caused 13C chemical shift differences of ca. 7 ppm of C-2 in
compounds 11a−c compared to 11d−f and ca. 4 ppm of C-4 in
compounds 12a,b compared to 12d,e. The 13C chemical shift
calculations of C-2 (δ calcd 166.1, exptl 165.6 ppm) of, e.g., the
phenyl-substituted compounds 11a and C-4 (δ calcd 156.1, exptl
156.7 ppm) of, e.g., 12a also support this observation.
Figure 9. Selective 1D DPFG NOESY spectrum of 12a.
separation of the two isomers but caused a remarkable increase
in solubility. Let us consider the regioisomers obtained from the
reaction of 1b with 5e: here the whole molecule is represented
by a single aromatic proton (Figure 10).
In the reactions of the unsymmetrical catechol 1b with 5a−f,
theoretically four different regioisomers can be obtained
1
(Scheme 3). However, the H NMR spectra of the crude
products exhibited in most cases a mixture of two regioisomers.
Acetylation of the crude products 13,14 did not help in the
In the 1H−15N gHMBC spectrum of the mixture of 21e,22e
the major isomer 21e showed long-range correlations with two
H
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Figure 10. Structures of the four possible regioisomers obtained from the reaction of 1b with 5e.
1
Figure 11. Part of the H−15N gHMBC spectrum of the mixture of 21e and 22e.
different 15N chemical shifts indicating either a structure of
type IA or of type IB (Figure 11). Unfortunately, due to the low
concentration in the mixture no 1H−15N long-range correlations
could be observed for the minor isomer 22e.
2
3
21e. Similarly, JCH and JCH HMBC correlations of the propyl
protons 1′-H and 2′-H of 22e with a ring C carbon at δ 172.6 ppm
(C-2) establish structure type I in lieu of type II for which
a 13C chemical shift of δ ≈ 160 ppm (C-4) would be expected
(Figure 12).
The question of whether the minor isomer has the same
arrangement in ring C as the major one or the alternative
arrangement of type IIA or type IIB products could be answered
as follows: As previously observed for compounds 11 and 12, the
13C chemical shift of the quaternary ring C carbon directly
bonded to the side chain differ significantly in size (Δδ ≈ 10
ppm) depending on type I/II regioselectivity, e.g., δ 172.6 ppm
(C-2) in 11e and δ 160.4 ppm (C-4) in 12e. Thus, the
unambiguous identification (gHMBC) of the ring C carbon to
which the side chain is attached and comparison of its 13C
chemical shift with the above values provides another possibility
for a type I/II differentiation in the case of missing 1H−15N long-
The remaining problem of the differentiation between the
structures 21e and 22e was unequivocally solved by analysis
of the experimental 1H−13C long rang coupling constants
(absolute values) between the aromatic ring A proton and the
quaternary carbons C-5a and C-9a attached to N at δ ≈ 132 ppm
and S at δ ≈ 123 ppm, respectively. In the HSQMBC spectrum
the aromatic A ring proton of the major isomer 21e at δ 7.97 ppm
revealed a JCH coupling constant of 9.5 Hz to C-5a at δ 131.9 ppm
(= 3JCH coupling) as well as a JCH coupling constant of 3.3 Hz to
C-9a at δ 123.1 ppm (= 2JCH coupling); this is why it was assigned
as 9-H (type IA product). In contrast, JCH2 coupling constants
of 3.8 Hz to C-5a at δ 132.0 ppm (= JCH coupling) and
1
range correlations. In the H−13C gHMBC spectrum of the
2
3
3
mixture 21e,22e JCH and JCH long-range correlations between
the propyl protons 1′-H and 2′-H and a ring C carbon at δ 172.1
ppm (= C-2) confirmed structure type I for the major isomer
9.2 Hz to C-9a at δ 122.4 ppm (= JCH coupling) were
observed for the minor isomer 22e, establishing a type IB
structure as well as position 6-H for the aromatic A ring
I
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1
Figure 12. Part of H−13C gHMBC spectrum of the mixture of 21e and 22e.
Figure 13. Part of the HSQMBC spectrum of the mixture of 21e and 22e.
proton at δ 8.65 ppm (Figure 13). The remaining full NMR
assignment of the molecules was carried out as described for
compounds 11 and 12.
could hardly be assessed by standard NMR methods, was
1
achieved and was based on H−15N HMBC NMR correlations
at natural abundance and experimental 1H−13C long-range
1
coupling constants in addition to super long-range H−13C
HMBC correlations, as well as calculated 13C chemical shifts.
CONCLUSION
■
In summary, we have developed a method for the synthesis
of pyrimidobenzothiazole derivatives by a laccase-catalyzed
EXPERIMENTAL SECTION
General Remarks. All commercially available reagents were used
without further purification. Laccase from A. bisporus (6 U/mg, ASA
■
domino reaction between catechols and 6-substituted 1,2,3,4-
tetrahydro-4-oxo-2-thioxo-5-pyrimidinecarbonitriles. The method
makes use of aerial oxygen as an oxidant to initiate the domino
reaction, which can be interpreted as a domino oxidation/
intermolecular 1,4-addition/oxidation/intramolecular 1,4-addition.
The transformations can be performed under mild conditions
and deliver the corresponding pyrimidobenzothiazoles with
yields up to 96%. The regioselectivity of the process depends
both on the structure of the catechols and the structure of the
substituents of the 6-substituted 1,2,3,4-tetrahydro-4-oxo-2-thioxo-
5-pyrimidinecarbonitriles. Complete structure elucidation of the
regioisomeric ring-proton deficient pyrimidobenzothiazoles, which
Spezialenzyme GmbH, Wolfenbuttel) is commercially available.
̈
Solvents used for extraction and purification were distilled prior to
use. Thin-layer chromatography (TLC) was performed on TLC silica
gel 60 F254. Reaction temperatures are reported as bath temperatures.
Products were purified either by crystallization or by flash column
chromatography on silica gel. Melting points were recorded on a
melting point apparatus with open capillary tubes and are uncorrected.
IR spectra were measured using ATR insturment. UV−vis spectra
1
were recorded with a spectrophotometer. H and 13C NMR spectra
were recorded at (500/125 and 300/75 MHz) in DMSO-d6 at room
temperature or at 40 °C. The 1H and 13C chemical shifts were referenced
to residual solvent signals at δH = 2.49 and δC = 39.5 (DMSO-d6) relative
J
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to TMS. Splitting patterns are designated as s (singlet), d (doublet),
t (triplet), q (quartet), sex (sextet), sep (septet), m (multiplet), and br
(broad). 1D (1H, 13C) and 2D NMR (COSY, ROESY, gHSQCAD,
gHMBCAD) measurements were performed using standard pulse
sequences. HSQMBC parameters: sw = 4000−6000 Hz, sw1 = 8000−
12000 Hz, np = 8192, fn = 16384, jnxh = 8, ni = 128−512, nt = 16−64,
d1 = 1s, 40 °C. Linear prediction in f1. Super long-range band selective
gHMBC parameters: sw = 5000 Hz, sw1 = 11000−13000 Hz, np = 2048,
fn = 4096, jnxh = 1 Hz, ni = 64−128, d1 = 1s, nt = 24−96, 40 °C. Linear
prediction in f1. 15N chemical shifts were indirectly determined using
NH); 13C NMR (75 MHz, DMSO-d6) δ 85.1, 118.9, 127.9, 128.2,
130.0, 137.7, 162.5, 167.3, 183.0; MS (EI, 70 eV) m/z (%) 229 (100)
[M]+, 201 (12).
1,2,3,4-Tetrahydro-6-(4-methylphenyl)-4-oxo-2-thioxo-5-pyrimi-
dinecarbonitrile (5b).
1
1H−15N gc2HMBC (50.7 MHz). H−15N gc2HMBC (Chempack 4.1)
parameter: sw = 5000 Hz, sw1 = 10136 Hz, np = 4096, fn = 8192, jnxh =
3 Hz, ni = 32−64, d1 = 1s, nt = 600−1200, 40 °C. Nitromethane
(100 μL) in DMSO-d6 (25 μL) in a capillary was used as an external
standard (δ = 0 ppm). Copies of the NMR spectra were prepared
using SpinWorks.33 Mass spectra and high resolution mass spectra
were recorded using either EI method or ESI method. The intensities
are reported as percentages relative to the base peak (I = 100%).
Computational Studies. All calculations reported in this paper
were performed within density functional theory, using the Gaussian
03 package.32d 13C NMR chemical shifts of selected compounds 11a,
11e, 12a and 12e were calculated as follows: the rigid structures were
optimized with the MM2 force field implemented in Chem3D Pro.34
In the second step, the optimized structures were subsequently
reoptimized at the AM1 level followed by the RHF/3-21G level and
finally by the B3LYP/6-31G(d) level of theory within the Gaussian 03
package. In the final step, the gas-phase 13C NMR chemical shifts of
the reoptimized geometries were computed at the mPW1PW91/
6-311+G(2d,p)//mPW1PW91/6-31G(d) level of theory. The refer-
ences TMS and benzene for the MSTD approach according to Sarotti
and Pellegrinet32e were computed in the same manner as for the
aforementioned selected compounds. Theoretical 13C NMR chemical
shifts (δa) were derived by the following equation: δa = σref − σa + δref
where σref and σa are the calculated NMR isotropic magnetic shielding
tensors of the reference compound and carbon a of the compound of
interest: σTMS = 186.965 and σbenzene = 54.4127 at the mPW1PW91/
6-311+G(2d,p)// mPW1PW91/6-31G(d) level; δref represents the
According to the general procedure I, a mixture of 4-methylbenzalde-
hyde (2b) (120 mg, 1 mmol), thiourea (3) (76 mg, 1 mmol), methyl
cyanoacetate (4) (99 mg, 1 mmol), and K2CO3 (138 mg, 1 mmol) was
reacted for 7 h. Purification gave 1,2,3,4-tetrahydro-6-(4-methylphen-
yl)-4-oxo-2-thioxo-5-pyrimidinecarbonitrile (5b) as a white solid in
35% yield (86 mg, 0.35 mmol): mp 289−291 °C (lit.26 290−291 °C);
Rf = 0.28 (CH2Cl2/EtOAc = 4:1); 1H NMR (300 MHz, DMSO-d6) δ
2.39 (s, 3H, 1″-H), 7.36 (d, 3J (2′-H, 3′-H) = 8.1 Hz, 3J (5′-H, 6′-H) =
8.1 Hz, 2H, 3′-H and 5′-H), 7.57 (d, 3J (2′-H, 3′-H) = 8.1 Hz, 3J (5′-
H, 6′-H) = 8.1 Hz, 2H, 2′-H and 6′-H), 13.08 (s, 2H, NH); 13C NMR
(75 MHz, DMSO-d6) δ 21.1, 90.2, 115.0, 126.6, 128.7, 129.0, 142.4,
158.7, 161.1, 176.4; MS (EI, 70 eV) m/z (%) 243 (68) [M]+, 195
(100), 167 (24), 118 (28).
1,2,3,4-Tetrahydro-6-(4-methoxyphenyl)-4-oxo-2-thioxo-5-pyri-
midinecarbonitrile (5c).
According to the general procedure I, a mixture of 4-methoxybenzalde-
hyde (2c) (136 mg, 1 mmol), thiourea (3) (76 mg, 1 mmol), methyl
cyanoacetate (4) (99 mg, 1 mmol), and K2CO3 (138 mg, 1 mmol) was
reacted for 7 h. Purification gave 1,2,3,4-tetrahydro-6-(4-methoxyphen-
yl)-4-oxo-2-thioxo-5-pyrimidinecarbonitrile (5c) as a white solid in 26%
yield (67 mg, 0.26 mmol): mp 280−282 °C (lit.26 280−281 °C); Rf =
chemical shift of the reference compound δTMS = 0 ppm; δbenzene
=
128.5 ppm (benzene plus TMS at 125 MHz). An HP Compaq with a
2.39 GHz processor and 2 GB of RAM was used for the calculations.
General Procedure I for the Synthesis of 6-Substituted
1,2,3,4-Tetrahydro-4-oxo-2-thioxo-5-pyrimidinecarbonitrile
(5a−f).26 An oven-dried 10-mL vial with a magnetic stir bar was charged
with a mixture of an aldehyde 2 (1 mmol), thiourea (3) (1 mmol,
76 mg), methyl cyanoacetate (4) (1 mmol, 99 mg), and K2CO3 (1 mmol,
138 mg) in methanol (2.5 mL). The vial was sealed, and the reaction
mixture was left stirring at 80 °C for 7 h. After completion of the reaction,
the potassium salt of 5a−f that precipitates during the reaction was
collected and washed with methanol. The crude solid was stirred in water
at ∼80 °C until a clear solution was obtained. After cooling, the solution
was acidified with acetic acid, and stirring was continued for 30 min. The
deposited precipitate was collected to give solid crude product, which was
further purified by recrystallization from methanol to yield 5a−f.
1,2,3,4-Tetrahydro-4-oxo-6-phenyl-2-thioxo-5-pyrimidinecarbo-
nitrile (5a).
1
0.30 (CH2Cl2/EtOAc = 4:1); H NMR (300 MHz, DMSO-d6) δ 3.84
3
3
(s, 3H, 1″-H), 7.10 (d, J (2′-H, 3′-H) = 8.9 Hz, J (5′-H, 6′-H) = 8.9
3
3
Hz, 2H, 3′-H and 5′-H), 7.65 (d, J (2′-H, 3′-H) = 8.9 Hz, J (5′-H,
6′-H) = 8.9 Hz, 2H, 2′-H and 6′-H), 13.07 (s, 2H, NH); 13C NMR
(75 MHz, DMSO-d6) δ 55.3, 84.3, 113.3, 119.2, 129.6, 130.0, 160.9,
162.6, 166.3, 182.4.
1,2,3,4-Tetrahydro-6-ethyl-4-oxo-2-thioxo-5-pyrimidinecarboni-
trile (5d).
According to the general procedure I, a mixture of propionaldehyde
(2d) (58 mg, 1 mmol), thiourea (3) (76 mg, 1 mmol), methyl
cyanoacetate (4) (99 mg, 1 mmol), and K2CO3 (138 mg, 1 mmol)
was reacted for 7 h. Purification gave 1,2,3,4-tetrahydro-6-ethyl-4-
oxo-2-thioxo-5-pyrimidinecarbonitrile (5d) as a white solid in 25%
yield (45 mg, 0.25 mmol): mp 257−259 °C (lit.26 258−259 °C);
Rf = 0.30 (CH2Cl2/EtOAc = 4:1); 1H NMR (300 MHz, DMSO-d6)
δ 1.19 (t, J (1′-H, 2′-H) = 7.5 Hz, 3H, 2′-H), 2.56 (q, J (1′-H,
2′-H) = 7.5 Hz, 2H, 1′-H), 13.03 (s, 2H, NH); 13C NMR (75
MHz, DMSO-d6) δ 12.4, 25.5, 90.0, 114.0, 158.3, 166.8, 176.2; MS
(EI, 70 eV) m/z (%) 181 (100) [M]+, 123 (32), 56 (48).
According to the general procedure I, a mixture of benzaldehyde (2a)
(106 mg, 1 mmol), thiourea (3) (76 mg, 1 mmol), methyl cyanoacetate
(4) (99 mg, 1 mmol), and K2CO3 (138 mg, 1 mmol) was reacted
for 7 h. Purification gave 1,2,3,4-tetrahydro-4-oxo-6-phenyl-2-thioxo-5-
pyrimidinecarbonitrile (5a) as a white solid in 36% yield (83 mg, 0.36
mmol): mp 298−300 °C (lit.26 300−302 °C); Rf = 0.26 (CH2Cl2/
3
3
1
EtOAc = 4:1); H NMR (300 MHz, DMSO-d6) δ 7.53−7.61 (m, 3H,
3′-H, 4′-H and 5′-H), 7.63−7.68 (m, 2H, 2′-H and 6′-H), 13.16 (s, 2H,
K
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1,2,3,4-Tetrahydro-4-oxo-6-propyl-2-thioxo-5-pyrimidinecarbo-
nitrile (5e).
3-Cyano-7,8-diacetyloxy-4H-2-phenyl-pyrimido[2,1-b]-
benzothiazol-4-one (11a) and 3-Cyano-7,8-diacetyloxy-2H-4-phe-
nyl-pyrimido[2,1-b]benzothiazol-2-one (12a).
According to the general procedure I, a mixture of butyraldehyde (2e)
(72 mg, 1 mmol), thiourea (3) (76 mg, 1 mmol), methyl cyanoacetate
(4) (99 mg, 1 mmol), and K2CO3 (138 mg, 1 mmol) was reacted for
7 h. Purification gave 1,2,3,4-tetrahydro-4-oxo-6-propyl-2-thioxo-
5-pyrimidinecarbonitrile (5e) as a white solid in 25% yield (49 mg,
0.25 mmol): mp 276−278 °C (lit.26 260−261 °C); Rf = 0.50 (CH2Cl2/
EtOAc = 4:1); H NMR (300 MHz, DMSO-d6) δ 0.93 (t, J (2′-H,
3′-H) = 7.5 Hz, 3H, 3′-H), 1.64 (sex, 3J (1′-H, 2′-H) = 7.5 Hz, 3J (2′-H,
3′-H) = 7.5 Hz, 2H, 2′-H), 2.54 (t, 3J (1′-H, 2′-H) = 7.5 Hz, 2H, 1′-H),
13.02 (br, 2H, NH); 13C NMR (75 MHz, DMSO-d6) δ 13.3, 21.3, 33.5,
90.6, 114.2, 158.3, 165.3, 176.2; MS (EI, 70 eV) m/z (%) 195 (100)
[M]+, 180 (29) and 137 (12); HRMS calcd for C8H9N3OS (195.0466),
found 195.0444.
According to the general procedure II, a suspension of catechol (1a)
(69 mg, 0.63 mmol), 1,2,3,4-tetrahydro-4-oxo-6-phenyl-2-thioxo-5-
pyrimidinecarbonitrile (5a) (115 mg, 0.50 mmol), methanol (5.5 mL),
phosphate buffer (0.2 M, pH 6, 30 mL), and laccase from A. bisporus
(10 mg, 6 U/mg) was stirred for 15 h. Workup gave a mixture of 6a
and 7a in 95% yield (159 mg, 0.48 mmol). The obtained crude
product was reacted with acetic anhydride (255 mg, 2.50 mmol) and
DMAP (6 mg, 0.05 mmol) in pyridine (2 mL) under argon for 5 h.
Workup gave the acetylated mixture of 11a and 12a in 77% (153 mg,
0.37 mmol). The obtained mixture was separated by column
chromatography to give 3-cyano-7,8-diacetyloxy-4H-2-phenyl-
pyrimido[2,1-b]benzothiazol-4-one (11a) as a white solid in 50%
yield (100 mg, 0.24 mmol) and 3-cyano-7,8-diacetyloxy-2H-4-phenyl-
pyrimido[2,1-b]benzothiazol-2-one (12a) as a pale yellow solid in 8%
yield (15 mg, 0.04 mmol).
1
3
1,2,3,4-Tetrahydro-6-isopropyl-4-oxo-2-thioxo-5-pyrimidinecar-
bonitrile (5f).
3-Cyano-7,8-diacetyloxy-4H-2-phenyl-pyrimido[2,1-b]-
benzothiazol-4-one (11a). Mp 262−264 °C; Rf = 0.24 (CH2Cl2/
EtOAc = 20:1); UV (MeCN) λmax (log ε) 230 (4.39), 254 (4.31), 300
̃
(4.20), 369 nm (4.24); IR (ATR) ν 3113 (CH), 2935 (CH), 2217
According to the general procedure I, a mixture of isobutyraldehyde
(2f) (72 mg, 1 mmol), thiourea (3) (76 mg, 1 mmol), methyl
cyanoacetate (4) (99 mg, 1 mmol), and K2CO3 (138 mg, 1 mmol) was
reacted for 7 h. Purification gave 1,2,3,4-tetrahydro-6-isopropyl-4-oxo-
2-thioxo-5-pyrimidinecarbonitrile (5f) as a white solid in 32% yield
(62 mg, 0.32 mmol): mp 250−252 °C; Rf = 0.40 (CH2Cl2/EtOAc =
4:1); UV (MeCN) λmax (log ε) 204 (4.31), 215 (4.20), 271 (4.15),
(CN), 1780 (CO), 1763 (CO), 1672 (CO), 1536, 1503, 1460,
1
1174 cm−1; H NMR (500 MHz, DMSO-d6) δ 2.35 (s, 3H, 2″-H or
2‴-H), 2.36 (s, 3H, 2″-H or 2‴-H), 7.58−7.66 (m, 3H, 3′-H, 4′-H and
5′-H), 8.00−8.02 (m, 2H, 2′-H and 6′-H), 8.17 (s, 1H, 9-H), 8.82
(s, 1H, 6-H); 13C NMR (125 MHz, DMSO-d6) δ 20.15 (C-2″ or C-2‴),
20.20 (C-2″ or C-2‴), 91.5 (C-3), 114.3 (C-6), 115.6 (CN), 118.1
(C-9), 122.5 (C-9a), 128.6 (C-3′ and C-5′), 128.7 (C-2′ and C-6′),
131.8 (C-1′), 132.7 (C-5a), 134.7 (C-4′), 141.2 (C-7), 141.3 (C-8),
158.9 (C-4), 165.6 (C-2), 165.8 (C-10a), 167.9 (C-1‴), 168.1 (C-1″);
15N NMR (indirectly determined, 50.7 MHz, DMSO-d6) δ −191 (N-5);
̃
312 nm (4.28); IR (ATR) ν 3256 (NH), 3155 (NH), 2981 (CH),
1
2936 (CH), 2226 (CN), 1676 (CO), 1546, 1202 cm−1; H NMR
(300 MHz, DMSO-d6) δ 1.29 (d, 3J (1′-H, 2′-H) = 7.0 Hz, 6H, 2′-H),
3
MS (ESI) m/z (%) 442 (100) [M + Na]+, 437 (30), 420 (58) [M + 1]+;
HRMS calcd for C21H13N3O5SNa (442.0468), found 442.0474.
3.03 (sept, J (1′-H, 2′-H) = 7.0 Hz, 1H, 1′-H), 12.77 (s, 1H, NH),
13.05 (s, 1H, NH); 13C NMR (75 MHz, DMSO-d6) δ 18.8, 32.2, 89.2,
114.1, 158.5, 169.4, 176.4; MS (EI, 70 eV) m/z (%) 195 (100) [M]+,
180 (29), 137 (12); HRMS calcd for C8H9N3OS (195.0466), found
195.0439.
3-Cyano-7,8-diacetyloxy-2H-4-phenyl-pyrimido[2,1-b]-
benzothiazol-2-one (12a). Mp 157−159; Rf = 0.28 (CH2Cl2/EtOAc =
2:1); UV (MeCN) λmax (log ε) 208 (4.47), 234 (4.44), 267 (4.47),
306 nm (3.94); IR (ATR) ν
̃
3052 (CH), 2231 (CN), 1770 (CO),
General Procedure II for the Laccase-Catalyzed Domino
Reaction. A 100-mL round-bottomed flask with a magnetic stir
bar was charged with a solution or suspension of the catechol 1 (0.63
mmol) and the 6-substituted 1,2,3,4-tetrahydro-4-oxo-2-thioxo-
5-pyrimidinecarbonitrile 5 (0.50 mmol) in hot methanol (3−7 mL).
Phosphate buffer (0.2 M, pH 6, 30 mL) and laccase from A. bisporus
(10 mg, 6 U/mg) were added, and the mixture was left stirring under
air at room temperature overnight (12−20 h). The reaction mixture
was acidified with 2 M HCl to pH 4. The precipitated product was
filtered with suction using a Buchner funnel. The filter cake obtained
was washed with aq NaCl (15%, 20 mL) and dried to give the crude
products 6,7 or 13,14. The obtained mixture of isomers 6,7 or 13,14
was placed in an oven-dried 10-mL vial and charged with acetic
anhydride (255 mg, 2.50 mmol) and DMAP (6 mg, 0.05 mmol). The
vial was sealed, evacuated, and backfilled with argon, and then freshly
distilled pyridine (2 mL) was added. The reaction mixture was stirred
at room temperature for 5−7 h followed by neutralization with 2 M
HCl (12 mL). The precipitated product was filtered, washed with
water, and dried to give the acetylated solid crude products 11,12
or 21,22, which were further purified by flash chromatography over
silica gel using the eluent system (petroleum ether/CH2Cl2 = 1:1 →
CH2Cl2/EtOAc = 1:2).
1650 (CO), 1608, 1594, 1515 cm−1; 1H NMR (500 MHz, DMSO-d6)
δ 2.14 (s, 3H, 2″-H), 2.27 (s, 3H, 2‴-H), 5.51 (s, 1H, 6-H), 7.70−7.71
(m, 5H, phenyl H), 8.02 (s, 1H, 9-H); 13C NMR (125 MHz, DMSO-d6)
δ 19.8 (C-2″), 20.1 (C-2‴), 99.4 (C-3), 111.6 (C-6), 114.1 (CN), 118.7
(C-9), 121.8 (C-9a), 128.3 (C-2′ and C-6′), 129.2 (C-4′), 129.8 (C-3′
and C-5′), 131.9 (C-1′), 132.8 (C-5a), 140.2 (C-7 or C-8), 140.3 (C-7
or C-8), 156.6 (C-4), 162.3 (C-2), 166.2 (C-10a), 167.4 (C-1″), 167.9
(C-1‴); 15N NMR (indirectly determined, 50.7 MHz, DMSO-d6) δ
−210.53 (N-5); MS (ESI) m/z (%) 442 (100) [M + Na]+, 337 (11);
HRMS calcd for C21H13N3O5SNa (442.0468), found 442.0459.
3-Cyano-7,8-diacetyloxy-4H-2-(4-methyl)phenyl-pyrimido[2,1-b]-
benzothiazol-4-one (11b) and 3-Cyano-7,8-diacetyloxy-2H-4-
(4-methyl)phenyl-pyrimido[2,1-b]benzothiazol-2-one (12b).
L
dx.doi.org/10.1021/jo401193e | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
According to the general procedure II, a suspension of catechol (1a)
(69 mg, 0.63 mmol), 1,2,3,4-tetrahydro-6-(4-methylphenyl)-4-oxo-2-
thioxo-5-pyrimidinecarbonitrile (5b) (122 mg, 0.50 mmol), methanol
(7 mL), phosphate buffer (0.2 M, pH 6, 30 mL), and laccase from A.
bisporus (10 mg, 6 U/mg) was stirred for 12 h. Workup gave a mixture
of 6b and 7b in 96% (168 mg, 0.48 mmol). The obtained crude
product was reacted with acetic anhydride (255 mg, 2.50 mmol) and
DMAP (6 mg, 0.05 mmol) in pyridine (2 mL) under argon for 5 h.
After workup the acetylated mixture of 11b and 12b was isolated
in 81% (169 mg, 0.39 mmol). The obtained mixture was separated
by column chromatography to give 3-cyano-7,8-diacetyloxy-4H-2-
(4-methyl)phenyl-pyrimido[2,1-b]benzothiazol-4-one (11b) as a white
solid in 46% yield (96 mg, 0.22 mmol) and 3-cyano-7,8-diacetyloxy-
2H-4-(4-methyl)phenyl-pyrimido[2,1-b]benzothiazol-2-one (12b) as a
white solid in 23% yield (48 mg, 0.11 mmol).
column chromatography to give 3-cyano-7,8-diacetyloxy-4H-2-(4-
methoxy)phenyl-pyrimido[2,1-b]benzothiazol-4-one (11c) as a white
solid in 45% yield (91 mg, 0.20 mmol): mp 210−212 °C; Rf = 0.46
(CH2Cl2/EtOAc = 10:1); UV (MeCN) λmax (log ε) 240 (4.40), 340
̃
nm (4.51); IR (ATR) ν 3112 (CH), 2924 (CH), 2851 (CH), 2216
(CN), 1782 (CO), 1765 (CO), 1671 (CO), 1602, 1532, 1516
cm−1; 1H NMR (500 MHz, DMSO-d6) δ 2.34 (s, 3H, 2″-H or 2‴-H),
2.35 (s, 3H, 2″-H or 2‴-H), 3.86 (s, 3H, 4′-OCH3), 7.14 (d like,
3
3J (2′-H, 3′-H) = 9.0 Hz, J (5′-H, 6′-H) = 9.0 Hz, 2H, 3′-H and 5′-
3
3
H), 8.07 (d like, J (2′-H, 3′-H) = 8.5 Hz, J (5′-H, 6′-H) = 8.5 Hz,
2H, 2′-H and 6′-H), 8.14 (s, 1H, 9-H), 8.78 (s, 1H, 6-H); 13C NMR
(125 MHz, DMSO-d6) δ 20.17 (C-2″ or C-2‴), 20.22 (C-2″ or C-2‴),
55.5 (4′-OCH3), 89.9 (C-3), 114.1 (C-6), 114.2 (C-3′ and C-5′),
116.0 (CN), 118.1 (C-9), 122.4 (C-9a), 126.6 (C-1′), 130.9 (C-2′ and
C-6′), 132.7 (C-5a), 141.1 (C-7), 141.3 (C-8), 159.1 (C-4), 162.4
(C-4′), 164.6 (C-2), 165.4 (C-10a), 168.0 (C-1‴), 168.1 (C-1″); 15N
NMR (indirectly determined, 50.7 MHz, DMSO-d6) δ −191.24 (N-5),
−154.96 (N-1); MS (ESI) m/z (%) 472 (100) [M + Na]+, 450 (17)
[M + 1]+; HRMS calcd for C22H15N3O6SNa (472.0574), found
472.0565.
3-Cyano-7,8-diacetyloxy-4H-2-(4-methyl)phenyl-pyrimido[2,1-b]-
benzothiazol-4-one (11b). Mp 246−248 °C; Rf = 0.44 (CH2Cl2/
EtOAc = 20:1); UV (MeCN) λmax (log ε) 233 (4.40), 252 (4.33), 312
̃
(4.34), 368 nm (4.29); IR (ATR) ν 3112 (CH), 2217 (CN), 1779
1
(CO), 1764 (CO), 1672 (CO), 1532, 1480 cm−1; H NMR
(500 MHz, DMSO-d6) δ 2.34 (s, 3H, 2″-H or 2‴-H), 2.35 (s, 3H, 2″-
3-Cyano-7,8-diacetyloxy-4H-2-ethyl-pyrimido[2,1-b]-
benzothiazol-4-one (11d) and 3-Cyano-7,8-diacetyloxy-2H-4-ethyl-
pyrimido[2,1-b]benzothiazol-2-one (12d).
3
H or 2‴-H), 2.38 (s, 3H, 4′-CH3), 7.42 (d like, J (2′-H, 3′-H) =
3
8.1 Hz, J (5′-H, 6′-H) = 8.1 Hz, 2H, 3′-H and 5′-H), 7.94 (d like,
3
3J (2′-H, 3′-H) = 8.1 Hz, J (5′-H, 6′-H) = 8.1 Hz, 2H, 2′-H and 6′-
H), 8.15 (s, 1H, 9-H), 8.78 (s, 1H, 6-H); 13C NMR (125 MHz,
DMSO-d6) δ 20.2 (C-2″ or C-2‴), 20.3 (C-2″ or C-2‴), 21.0 (4′-CH3),
90.9 (C-3), 114.2 (C-6), 115.8 (CN), 118.1 (C-9), 122.5 (C-9a), 128.9
(C-2′ and C-6′), 129.3 (C-3′and C-5′), 131.8 (C-1′), 132.7 (C-5a),
141.2 (C-7), 141.3 (C-8), 142.4 (C-4′), 159.0 (C-4), 165.3 (C-2), 165.6
(C-10a), 168.0 (C-1‴), 168.1 (C-1″); 15N NMR (indirectly determined,
50.7 MHz, DMSO-d6) δ −190.63 (N-5), −156.72 (N-1); MS (ESI)
m/z (%) 456 (100) [M + Na]+, 434 (10) [M + 1]+; HRMS calcd for
C22H15N3O5SNa (456.0625), found 456.0616.
According to the general procedure II, a solution of catechol (1a)
(69 mg, 0.63 mmol), 1,2,3,4-tetrahydro-6-ethyl-4-oxo-2-thioxo-5-
pyrimidinecarbonitrile (5d) (91 mg, 0.50 mmol), methanol (3 mL),
phosphate buffer (0.2 M, pH 6, 30 mL), and laccase from A. bisporus
(10 mg, 6 U/mg) was stirred for 18 h. Workup gave a mixture of 6d
and 7d in 83% (120 mg, 0.42 mmol). The obtained crude product was
reacted with acetic anhydride (255 mg, 2.50 mmol) and DMAP (6 mg,
0.05 mmol) in pyridine (2 mL) under argon for 5 h. Workup gave
the acetylated mixture of 11d and 12d in 76% (118 mg, 0.32 mmol).
The obtained mixture was separated by column chromatography to
give 3-cyano-7,8-diacetyloxy-4H-2-ethyl-pyrimido[2,1-b]benzothiazol-
4-one (11d) as a white solid in 35% yield (54 mg, 0.15 mmol) and
3-cyano-7,8-diacetyloxy-2H-4-ethyl-pyrimido[2,1-b]benzothiazol-2-one
(12d) as a white solid in 16% yield (25 mg, 0.07 mmol).
3-Cyano-7,8-diacetyloxy-2H-4-(4-methyl)phenyl-pyrimido[2,1-b]-
benzothiazol-2-one (12b). Mp 236−238; Rf = 0.36 (CH2Cl2/EtOAc
= 2:1); UV (MeCN) λmax (log ε) 211 (4.51), 234 (4.46), 266 (4.49),
306 nm (3.98); IR (ATR) ν
̃
3112 (CH), 2217 (CN), 1779 (CO),
1763 (CO), 1672 (CO), 1531, 1516, 1479 cm−1; 1H NMR
(500 MHz, DMSO-d6) δ 2.15 (s, 3H, 2″-H), 2.27 (s, 3H, 2‴-H), 2.47
3
(s, 3H, 4′-CH3), 5.56 (s, 1H, 6-H), 7.53 (d like, J (2′-H, 3′-H) =
3
8.0 Hz, J (5′-H, 6′-H) = 8.0 Hz, 2H, 3′-H and 5′-H), 7.59 (d like,
3
3J (2′-H, 3′-H) = 8.0 Hz, J (5′-H, 6′-H) = 8.0 Hz, 2H, 2′-H and
6′-H), 8.02 (s, 1H, 9-H); 13C NMR (125 MHz, DMSO-d6) δ 19.8
(C-2″), 20.0 (C-2‴), 20.9 (4′-CH3), 99.2 (C-3), 111.7 (C-6), 114.1
(CN), 118.5 (C-9), 121.7 (C-9a), 126.2 (C-1′), 128.1 (C-2′ and
C-6′), 130.2 (C-3′and C-5′), 132.7 (C-5a), 140.1 (C-7 or C-8), 140.2
(C-7 or C-8), 142.1 (C-4′), 156.8 (C-4), 162.3 (C-2), 166.1 (C-10a),
167.4 (C-1″), 167.8 (C-1‴); 15N NMR (indirectly determined, 50.7
MHz, DMSO-d6) δ −211.14 (N-5); MS (ESI) m/z (%) 456 (100)
[M + Na]+, 434 (10) [M + 1]+; HRMS calcd for C22H15N3O5SNa
(456.0625), found 456.0614.
3-Cyano-7,8-diacetyloxy-4H-2-ethyl-pyrimido[2,1-b]-
benzothiazol-4-one (11d). Mp 258−260 °C; Rf = 0.30 (CH2Cl2/
EtOAc = 20:1); UV (MeCN) λmax (log ε) 206 (4.44), 222 (4.51), 342
̃
(4.28), 356 nm (4.34); IR (ATR) ν 3105 (CH), 2939 (CH), 2223
(CN), 1787 (CO), 1768 (CO), 1677 (CO), 1538, 1463, 1188
1
3
cm−1; H NMR (500 MHz, DMSO-d6) δ 1.28 (t, J (1′-H, 2′-H) =
7.5 Hz, 3H, 2′-H), 2.34 (s, 3H, 2″-H or 2‴-H), 2.35 (s, 3H, 2″-H or
2‴-H), 2.83 (q, 3J (1′-H, 2′-H) = 8.0 Hz, 2H, 1′-H), 8.14 (s, 1H, 9-H),
8.77 (s, 1H, 6-H); 13C NMR (125 MHz, DMSO-d6) δ 11.6 (C-2′),
20.15 (C-2″ or C-2‴), 20.19 (C-2″ or C-2‴), 29.5 (C-1′), 92.8 (C-3),
114.2 (C-6), 114.7 (CN), 118.1 (C-9), 122.2 (C-9a), 132.8 (C-5a),
141.1 (C-7), 141.2 (C-8), 158.1 (C-4), 166.2 (C-10a), 167.9 (C-1‴),
168.0 (C-1″), 173.8 (C-2); 15N NMR (indirectly determined, 50.7
MHz, DMSO-d6) δ −189.94 (N-5), −152.41 (N-1); MS (ESI) m/z
(%) 394 (100) [M + Na]+, 372 (50) [M + 1]+, 330 (20); HRMS calcd
for C17H13N3O5SNa (394.0468), found 394.0473.
3-Cyano-7,8-diacetyloxy-4H-2-(4-methoxy)phenyl-pyrimido-
[2,1-b]benzothiazol-4-one (11c).
According to the general procedure II, a suspension of catechol (1a)
(69 mg, 0.63 mmol), 1,2,3,4-tetrahydro-6-(4-methoxyphenyl)-4-oxo-2-
thioxo-5-pyrimidinecarbonitrile (5c) (130 mg, 0.50 mmol), methanol
(7 mL), phosphate buffer (0.2 M, pH 6, 30 mL), and laccase from A.
bisporus (10 mg, 6 U/mg) was stirred for 18 h. Workup gave a mixture
of 6c and 7c in 90% (165 mg, 0.45 mmol). The obtained crude
product was reacted with acetic anhydride (255 mg, 2.50 mmol) and
DMAP (6 mg, 0.05 mmol) in pyridine (2 mL) under argon for 5 h.
After workup the acetylated mixture of 11c and 12c was isolated in
77% (156 mg, 0.35 mmol). The obtained mixture was separated by
3-Cyano-7,8-diacetyloxy-2H-4-ethyl-pyrimido[2,1-b]-
benzothiazol-2-one (12d). Mp 224−226 °C; Rf = 0.22 (CH2Cl2/
EtOAc = 2:1); UV (MeCN) λmax (log ε) 213 (4.33), 235 (4.44), 262
̃
(4.52), 306 nm (4.05); IR (ATR) ν 3120 (CH), 2978 (CH), 2230
(CN), 1773 (CO), 1650 (CO), 1602, 1498, 1183 cm−1; 1H
NMR (500 MHz, DMSO-d6) δ 1.35 (t, 3J (1′-H, 2′-H) = 7.6 Hz, 3H,
2′-H), 2.33 (s, 3H, 2″-H or 2‴-H), 2.34 (s, 3H, 2″-H or 2‴-H), 3.34
(q, 3J (1′-H, 2′-H) = 7.5 Hz, 2H, 1′-H), 7.92 (s, 1H, 6-H), 8.05 (s, 1H,
9-H); 13C NMR (125 MHz, DMSO-d6) δ 11.3 (C-2′), 20.3 (C-2″ or
M
dx.doi.org/10.1021/jo401193e | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
C-2‴), 20.5 (C-2″ or C-2‴), 25.7 (C-1′), 97.8 (C-3), 113.3 (C-6),
114.7 (CN), 118.5 (C-9), 121.8 (C-9a), 132.8 (C-5a), 140.6 (C-7),
141.4 (C-8), 162.2 (C-4), 162.4 (C-2), 166.5 (C-10a), 168.1 (C-1‴),
168.2 (C-1″); 15N NMR (indirectly determined, 50.7 MHz, DMSO-
d6) δ −211.01 (N-5); MS (ESI) m/z (%) 394 (100) [M + Na]+,
372 (5) [M + 1]+; HRMS calcd for C17H13N3O5SNa (394.0468),
found 394.0458.
3-Cyano-7,8-diacetyloxy-4H-2-isopropyl-pyrimido[2,1-b]-
benzothiazol-4-one (11f).
3-Cyano-7,8-diacetyloxy-4H-2-propyl-pyrimido[2,1-b]-
benzothiazol-4-one (11e) and 3-Cyano-7,8-diacetyloxy-2H-4-prop-
yl-pyrimido[2,1-b]benzothiazol-2-one (12e).
According to the general procedure II, a solution of catechol (1a)
(69 mg, 0.63 mmol), 1,2,3,4-tetrahydro-6-isopropyl-4-oxo-2-thioxo-5-
pyrimidinecarbonitrile (5f) (98 mg, 0.50 mmol), methanol (3 mL),
phosphate buffer (0.2 M, pH 6, 30 mL), and laccase from A. bisporus
(10 mg, 6 U/mg) was stirred for 20 h. Workup gave a mixtrure of 6f
and 7f in 92% yield (139 mg, 0.46 mmol). The obtained crude product
was reacted with acetic anhydride (255 mg, 2.50 mmol) and DMAP
(6 mg, 0.05 mmol) in pyridine (2 mL) under argon for 5 h. Workup
gave a mixture of 11f and 12f in 68% (121 mg, 0.31 mmol), which was
separated by column chromatography to give 3-cyano-7,8-diacetyloxy-
4H-2-isopropyl-pyrimido[2,1-b]benzothiazol-4-one (11f) as a white
solid in 45% yield (80 mg, 0.21 mmol): mp 239−241 °C; Rf = 0.39
(CH2Cl2/EtOAc = 10:1); UV (MeCN) λmax (log ε) 223 (4.50), 239
According to the general procedure II, a solution of catechol (1a)
(69 mg, 0.63 mmol), 1,2,3,4-tetrahydro-4-oxo-6-propyl-2-
thioxo-pyrimidine-5-carbonitrile (5e) (98 mg, 0.50 mmol), methanol
(3 mL), phosphate buffer (0.2 M, pH 6, 30 mL), and laccase from A.
bisporus (10 mg, 6 U/mg) was stirred for 20 h. Workup gave a mixture
of 6e and 7e in 84% yield (127 mg, 0.42 mmol). The obtained crude
product was reacted with acetic anhydride (255 mg, 2.50 mmol) and
DMAP (6 mg, 0.05 mmol) in pyridine (2 mL) under argon for 7 h.
Workup gave the acetylated mixture of 11e and 12e in 85% yield
(138 mg, 0.36 mmol). The obtained mixture was separated by column
chromatography to give 3-cyano-7,8-diacetyloxy-4H-2-propyl-
pyrimido[2,1-b]benzothiazol-4-one (11e) as a white solid in 38%
yield (62 mg, 0.16 mmol) and 3-cyano-7,8-diacetyloxy-2H-4-propyl-
pyrimido[2,1-b]benzothiazol-2-one (12e) as a white solid in 36% yield
(58 mg, 0.15 mmol).
̃
(4.28), 341 (4.26), 355 nm (4.34); IR (ATR) ν 3114 (CH), 2972
(CH), 2221 (CN), 1771 (CO), 1685 (CO), 1541, 1491, 1465,
1
3
1188 cm−1; H NMR (500 MHz, DMSO-d6) δ 1.27 (d, J (1′-H,
2′-H) = 6.8 Hz, 6H, 2′-H), 2.35 (s, 6H, 2″-H and 2‴-H), 3.27
(sept, 3J (1′-H, 2′-H) = 6.8 Hz, 1H, 1′-H), 8.13 (s, 1H, 9-H), 8.76
(s, 1H, 6-H); 13C NMR (125 MHz, DMSO-d6) δ 20.29 (C-2″
or C-2‴), 20.33 (C-2″ or C-2‴), 20.5 (C-2′), 34.3 (C-1′), 92.2
(C-3), 114.3 (C-6), 114.8 (CN), 118.1 (C-9), 122.4 (C-9a), 132.9
(C-5a), 141.15 (C-7), 141.24 (C-8), 158.4 (C-4), 166.6 (C-10a),
168.1 (C-1‴), 168.2 (C-1″), 177.0 (C-2); 15N NMR (indirectly
determined, 50.7 MHz, DMSO-d6) δ −189.24 (N-5), −155.35
(N-1); MS (ESI) m/z (%) 408 (39) [M + Na]+, 403 (39)
[M + NH4]+, 386 (67) [M + 1]+; HRMS calcd for C18H19N4O5S
(403.1071), found 403.1069.
3-Cyano-7,8-diacetyloxy-4H-2-propyl-pyrimido[2,1-b]-
benzothiazol-4-one (11e). Mp 243−245 °C; Rf = 0.22 (CH2Cl2/
EtOAc = 20:1); UV (MeCN) λmax (log ε) 206 (4.34), 223 (4.41), 342
3-Cyano-7,8-diacetyloxy-6-methyl-4H-2-phenyl-pyrimido[2,1-b]-
benzothiazol-4-one (21a) and 3-Cyano-7,8-diacetyloxy-9-methyl-
4H-2-phenyl-pyrimido[2,1-b]benzothiazol-4-one (22a).
̃
(4.17), 356 nm (4.24); IR (ATR) ν 3123 (CH), 2965 (CH), 2937
(CH), 2878 (CH), 2224 (CN), 1786 (CO), 1768 (CO), 1676
1
(CO), 1537, 1486, 1462 cm−1; H NMR (500 MHz, DMSO-d6)
3
3
δ 0.97 (t, J (2′-H, 3′-H) = 8.0 Hz, 3H, 3′-H), 1.77 (sex, J (1′-H,
2′-H) = 7.5 Hz, 3J (2′-H, 3′-H) = 7.5 Hz, 2H, 2′-H), 2.34 (s, 3H, 2″-H
3
or 2‴-H), 2.35 (s, 3H, 2″-H or 2‴-H), 2.78 (t, J (1′-H, 2′-H) =
7.5 Hz, 2H, 1′-H), 8.14 (s, 1H, 9-H), 8.77 (s, 1H, 6-H); 13C NMR
(125 MHz, DMSO-d6) δ 13.2 (C-3′), 20.15 (C-2″ or C-2‴), 20.19
(C-2″ or C-2‴), 20.7 (C-2′), 37.9 (C-1′), 93.5 (C-3), 114.2 (C-6),
114.8 (CN), 118.0 (C-9), 122.2 (C-9a), 132.8 (C-5a), 141.1 (C-7),
141.2 (C-8), 158.1 (C-4), 166.0 (C-10a), 167.9 (C-1‴), 168.1 (C-1″),
172.6 (C-2); 15N NMR (indirectly determined, 50.7 MHz, DMSO-d6)
δ −189.56 (N-5), −151.61 (N-1); MS (ESI) m/z (%) 408 (100) [M +
Na]+, 386 (11) [M + 1]+; HRMS calcd for C18H15N3O5SNa
(408.0625), found 408.0637.
According to the general procedure II, a suspension of 3-methyl-
catechol (1b) (78 mg, 0.63 mmol), 1,2,3,4-tetrahydro-4-oxo-6-phenyl-
2-thioxo-5-pyrimidinecarbonitrile (5a) (115 mg, 0.50 mmol),
methanol (5.5 mL), phosphate buffer (0.2 M, pH 6, 30 mL), and
laccase from A. bisporus (10 mg, 6 U/mg) was stirred for 18 h. Workup
gave a mixture of 13a and 14a in 91% yield (160 mg, 0.46 mmol). The
obtained crude product of 13a and 14a was reacted with acetic
anhydride (255 mg, 2.50 mmol) and DMAP (6 mg, 0.05 mmol) in
pyridine (2 mL) under argon for 5 h. Workup gave the acetylated
mixture of 21a and 22a in 78% yield (155 mg, 0.36 mmol). The
obtained mixture was purified by column chromatography to give
a mixture of 3-cyano-7,8-diacetyloxy-6-methyl-4H-2-phenyl-pyrimido-
[2,1-b]benzothiazol-4-one (21a) and 3-cyano-7,8-diacetyloxy-9-meth-
yl-4H-2-phenyl-pyrimido[2,1-b]benzothiazol-4-one (22a) as a pale
yellow solid in 53% yield (105 mg, 0.24 mmol): mp 229−231 °C; Rf =
0.30 (CH2Cl2/EtOAc = 20:1); UV (MeCN) λmax (log ε) 204 (4.52),
232 (4.40), 259 (4.27), 274 (4.20), 304 (4.21), 375 nm (4.20); IR
3-Cyano-7,8-diacetyloxy-2H-4-propyl-pyrimido[2,1-b]-
benzothiazol-2-one (12e). Mp 258−260 °C; Rf = 0.24 (CH2Cl2/
EtOAc = 2:1); UV (MeCN) λmax (log ε) 213 (4.34), 235 (4.44), 262
̃
(4.53), 306 nm (4.05); IR (ATR) ν 3110 (CH), 2936 (CH), 2229
(CN), 1776 (CO), 1647 (CO), 1598, 1496, 1426, 1186 cm−1; 1H
NMR (500 MHz, DMSO-d6) δ 1.04 (t, 3J (2′-H, 3′-H) = 7.5 Hz, 3H,
3
3
3′-H), 1.76 (sex, J (1′-H, 2′-H) = 8.0 Hz, J (2′-H, 3′-H) = 8.0 Hz,
2H, 2′-H), 2.33 (s, 3H, 2″-H or 2‴-H), 2.34 (s, 3H, 2″-H or 2‴-H),
3
3.30 (t, J (1′-H, 2′-H) = 7.5 Hz, 2H, 1′-H), 7.86 (s, 1H, 6-H), 8.05
(s, 1H, 9-H); 13C NMR (125 MHz, DMSO-d6) δ 13.0 (C-3′), 19.9
(C-2′), 20.3 (C-2″ or C-2‴), 20.5 (C-2″ or C-2‴), 33.6 (C-1′), 98.6
(C-3), 113.2 (C-6), 115.0 (CN), 118.4 (C-9), 121.8 (C-9a), 132.8
(C-5a), 140.5 (C-7), 141.2 (C-8), 160.4 (C-4), 162.3 (C-2), 166.6
(C-10a), 168.12 (C-1‴), 168.14 (C-1″); 15N NMR (indirectly
determined, 50.7 MHz, DMSO-d6) δ −210 (N-5); MS (ESI) m/z
(%) 408 (100) [M + Na]+, 386 (5) [M + 1]+; HRMS calcd for
C18H15N3O5SNa (408.0625), found 408.0618.
(ATR) ν
̃
3101 (CH), 2970 (CH), 2226 (CN), 1761 (CO), 1681
(CO), 1539, 1504, 1370 cm−1; 1H NMR (500 MHz, DMSO-d6) of
21a δ 2.34 (s, 3H, 2‴-H), 2.35 (overlapped, 3H, 6-CH3), 2.41 (s, 3H,
2″-H), 7.59−7.64 (m, 2H, 3′-H and 5′-H), 7.65−7.67 (m, 1H, 4′-H),
7.99 (s, 1H, 9-H), 8.00−8.05 (m, 2H, 2′-H and 6′-H); 13C NMR (125
MHz, DMSO-d6) of 21a δ 16.5 (6-CH3), 19.8 (C-2″), 20.18 (C-2‴),
91.0 (C-3), 115.4 (C-9), 115.9 (CN), 123.4 (C-9a), 125.4 (C-6),
N
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The Journal of Organic Chemistry
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128.63 (C-3′ and C-5′), 128.8 (C-2′ and C-6′), 131.9 (C-5a), 132.0
(C-4′), 134.2 (C-1′), 141.2 (C-7), 141.6 (C-8), 158.9 (C-4), 165.2
(C-2), 166.6 (C-10a), 167.8 (C-1″), 167.9 (C-1‴); 15N NMR
(indirectly determined, 50.7 MHz, DMSO-d6) of 21a δ −187.44
3-Cyano-7,8-diacetyloxy-6-methyl-4H-2-(4-methoxy)phenyl-
pyrimido[2,1-b]benzothiazol-4-one (21c) and 3-Cyano-7,8-diacety-
loxy-9-methyl-4H-2-(4-methoxy)phenyl-pyrimido[2,1-b]-
benzothiazol-4-one (22c).
1
(N-5); H NMR (500 MHz, DMSO-d6) of 22a δ 2.35 (overlapped,
3H, 2″-H), 2.37 (s, 3H, 9-CH3), 2.40 (s, 3H, 2‴-H), 7.59−7.64 (m,
2H, 3′-H and 5′-H), 7.65−7.67 (m, 1H, 4′-H), 8.00−8.05 (m, 2H,
2′-H and 6′-H), 8.69 (s, 1H, 6-H); 13C NMR (125 MHz, DMSO-d6)
of 22a δ 14.6 (9-CH3), 19.8 (C-2‴), 20.21 (C-2″), 91.8 (C-3), 112.2
(C-6), 115.5 (CN), 122.7 (C-9a), 126.0 (C-9), 128.59 (C-3′ and
C-5′), 128.8 (C-2′ and C-6′), 131.8 (C-5a), 132.0 (C-4′), 134.6
(C-1′), 140.0 (C-8), 141.9 (C-7), 158.9 (C-4), 164.8 (C-2), 165.5
(C-10a), 167.76 (C-1‴), 168.1 (C-1″); MS (ESI) m/z (%) 456 (100)
[M + Na]+, 434 (11) [M + 1]+; HRMS calcd for C22H15N3O5SNa
(456.0625), found 456.0616.
According to the general procedure II, a suspension of 3-methyl-
catechol (1b) (78 mg, 0.63 mmol), 1,2,3,4-tetrahydro-6-(4-methox-
yphenyl)-4-oxo-2-thioxo-5-pyrimidinecarbonitrile (5c) (130 mg, 0.50
mmol), methanol (7 mL), phosphate buffer (0.2 M, pH 6, 30 mL),
and laccase from A. bisporus (10 mg, 6 U/mg) was stirred for 18 h.
Workup gave a mixture of 13c and 14c in 89% (170 mg, 0.45 mmol).
The obtained crude product of 13c and 14c was reacted with acetic
anhydride (255 mg, 2.50 mmol) and DMAP (6 mg, 0.05 mmol)
in pyridine (2 mL) under argon for 7 h. Workup gave the acetylated
mixture of 21c and 22c in 70% yield (146 mg, 0.32 mmol). The
obtained mixture was purified by column chromatography to give a
mixture of 3-cyano-7,8-diacetyloxy-6-methyl-4H-2-(4-methoxy)-
phenyl-pyrimido[2,1-b]benzothiazol-4-one (21c) and 3-cyano-7,8-
diacetyloxy-9-methyl-4H-2-(4-methoxy)phenyl-pyrimido[2,1-b]-
benzothiazol-4-one (22c) as a yellow solid in 53% (110 mg, 0.24
mmol): mp 214−216 °C; Rf = 0.50 (CH2Cl2/EtOAc = 20:1); UV
3-Cyano-7,8-diacetyloxy-6-methyl-4H-2-(4-methyl)phenyl-
pyrimido[2,1-b]benzothiazol-4-one (21b) and 3-Cyano-7,8-diace-
tyloxy-9-methyl-4H-2-(4-methyl)phenyl-pyrimido[2,1-b]-
benzothiazol-4-one (22b)
.
̃
(MeCN): λmax (log ε) 238 (4.30), 342 nm (4.39); IR (ATR) ν 2937
According to the general procedure II, a suspension of 3-
methylcatechol (1b) (78 mg, 0.63 mmol), 1,2,3,4-tetrahydro-6-(4-
methylphenyl)-4-oxo-2-thioxo-5-pyrimidinecarbonitrile (5b) (122 mg,
0.50 mmol), methanol (7 mL), phosphate buffer (0.2 M, pH 6,
30 mL), and laccase from A. bisporus (10 mg, 6 U/mg) was stirred for
15 h. Workup gave a mixture of 13b and 14b in 93% yield (170 mg,
0.47 mmol). The obtained crude product of 13b and 14b was reacted
with acetic anhydride (255 mg, 2.50 mmol) and DMAP (6 mg,
0.05 mmol) in pyridine (2 mL) under argon for 5 h. Workup gave
the acetylated mixture of 21b and 22b in 76% (159 mg, 0.36 mmol).
The obtained mixture was purified by column chromatography to
give a mixture of 3-cyano-7,8-diacetyloxy-6-methyl-4H-2-(4-methyl)-
phenyl-pyrimido[2,1-b]benzothiazol-4-one (21b) and 3-cyano-7,8-
diacetyloxy-9-methyl-4H-2-(4-methyl)phenyl-pyrimido[2,1-b]-
benzothiazol-4-one (22b) as a pale yellow solid in 43% yield (90 mg,
0.20 mmol): mp 257−259 °C; Rf = 0.40 (CH2Cl2/EtOAc = 20:1); UV
(MeCN) λmax (log ε) 234 (4.38), 259 (4.23), 315 (4.26), 376 nm
(CH), 2114 (CN), 1773 (CO), 1687 (CO), 1602, 1537, 1480
cm−1; 1H NMR (500 MHz, DMSO-d6) of 21c δ 2.30 (s, 3H, 6-CH3),
2.33 (s, 3H, 2‴-H), 2.40 (s, 3H, 2″-H), 3.81 (s, 3H, 4′-OCH3), 7.13
3
3
(d like, J (2′-H, 3′-H) = 8.5 Hz, J (5′-H, 6′-H) = 8.5 Hz, 2H, 3′-H
and 5′-H), 7.96 (s, 1H, 9-H), 8.08 (d like, 3J (2′-H, 3′-H) = 8.5 Hz, 3J
(5′-H, 6′-H) = 8.5 Hz, 2H, 2′-H and 6′-H); 13C NMR (125 MHz,
DMSO-d6) of 21c δ 16.5 (6-CH3), 19.8 (C-2″), 20.18 (C-2‴), 55.5
(4′-OCH3), 89.2 (C-3), 114.12 (C-3′ and C-5′), 115.1 (C-9), 116.3
̀
(CN), 123.3 (C-9a), 125.2 (C-6), 126.0 (C-1′), 130.9 (C-2′ and C-6),
131.9 (C-5a), 141.2 (C-7), 141.5 (C-8), 158.8 (C-4), 162.5 (C-4′),
164.2 (C-2), 166.1 (C-10a), 167.8 (C-1″), 167.9 (C-1‴); 15N NMR
(indirectly determined, 50.7 MHz, DMSO-d6) of 21c δ −189.58
(N-5), −155.30 (N-1); 1H NMR (500 MHz, DMSO-d6) of 22c δ 2.34
(s, 3H, 2″-H), 2.35 (s, 3H, 9-CH3), 2.39 (s, 3H, 2‴-H), 3.81 (s, 3H,
3
3
4′-OCH3), 7.13 (d like, J (2′-H, 3′-H) = 8.5 Hz, J (5′-H, 6′-H) =
3
8.5 Hz, 2H, 3′-H and 5′-H), 8.08 (d like, J (2′-H, 3′-H) = 8.5 Hz,
3J (5′-H, 6′-H) = 8.5 Hz, 2H, 2′-H and 6′-H), 8.64 (s, 1H, 6-H); 13C
NMR (125 MHz, DMSO-d6) of 22c δ 14.6 (9-CH3), 19.8 (C-2‴),
20.20 (C-2″), 55.5 (4′-OCH3), 90.1 (C-3), 112.1 (C-6), 114.07 (C-3′
and C-5′), 115.9 (CN), 122.5 (C-9a), 125.96 (C-9), 126.4 (C-1′),
130.9 (C-2′ and C-6′), 131.8 (C-5a), 139.8 (C-8), 141.9 (C-7), 159.0
(C-4), 162.4 (C-4′), 164.4 (C-2), 166.1 (C-10a), 167.75 (C-1‴),
168.0 (C-1″); MS (ESI) m/z (%) 486 (100) [M + Na]+; HRMS calcd
for C23H17N3O6SNa (486.0730), found 486.0736.
(4.12); IR (ATR) ν
̃
3115 (CH), 2940 (CH), 2159 (CN), 1770 (C
O), 1688 (CO), 1539, 1485 cm−1; 1H NMR (500 MHz, DMSO-d6)
of 21b δ 2.33 (s, 3H, 6-CH3), 2.34 (s, 3H, 2‴-H), 2.40 (overlapped,
3H, 2″-H), 2.41 (s, 3H, 4′-CH3), 7.41 (d like, 3J (2′-H, 3′-H) = 8.0 Hz,
3J (5′-H, 6′-H) = 8.0 Hz, 2H, 3′-H and 5′-H), 7.96 (d like, 3J (2′-H, 3′-
3
H) = 8.5 Hz, J (5′-H, 6′-H) = 8.5 Hz, 2H, 2′-H and 6′-H), 7.98 (s,
1H, 9-H); 13C NMR (125 MHz, DMSO-d6) of 21b δ 16.5 (6-CH3),
19.9 (C-2″), 20.19 (C-2‴), 20.97 (4′-CH3), 90.4 (C-3), 115.1 (C-9),
116.0 (CN), 123.4 (C-9a), 125.3 (C-6), 128.8 (C-2′ and C-6′), 129.2
(C-3′ and C-5′), 131.3 (C-1′), 131.9 (C-5a), 141.2 (C-7), 141.6
(C-8), 142.5 (C-4′), 158.7 (C-4), 165.0 (C-2), 166.4 (C-10a), 167.8
(C-1″), 167.9 (C-1‴); 15N NMR (indirectly determined, 50.7 MHz,
3-Cyano-7,8-diacetyloxy-2-ethyl-6-methyl-4H-pyrimido[2,1-b]-
benzothiazol-4-one (21d) and 3-Cyano-7,8-diacetyloxy-2-ethyl-9-
methyl-4H-pyrimido[2,1-b]benzothiazol-4-one (22d).
1
DMSO-d6) of 21b δ −187.50 (N-5), −154.41 (N-1); H NMR (500
MHz, DMSO-d6) of 22b δ 2.35 (s, 3H, 2″-H), 2.37 (s, 3H, 9-CH3),
2.40 (overlapped, 3H, 2‴-H), 2.41 (s, 3H, 4′-CH3), 7.41 (d like, 3J (2′-
H, 3′-H) = 8.0 Hz, 3J (5′-H, 6′-H) = 8.0 Hz, 2H, 3′-H and 5′-H), 7.94
3
3
(d like, J (2′-H, 3′-H) = 8.5 Hz, J (5′-H, 6′-H) = 8.5 Hz, 2H, 2′-H
and 6′-H), 8.67 (s, 1H, 9-H); 13C NMR (125 MHz, DMSO-d6) of 22b
δ 14.6 (9-CH3), 19.9 (C-2‴), 20.22 (C-2″), 20.95 (4′-CH3), 91.2
(C-3), 112.2 (C-6), 115.7 (CN), 122.6 (C-9a), 126.0 (C-9), 128.8
(C-2′ and C-6′), 129.2 (C-3′ and C-5′), 131.2 (C-1′), 131.7 (C-5a),
139.9 (C-8), 141.9 (C-7), 142.4 (C-4′), 159.0 (C-4), 164.6 (C-2),
165.3 (C-10a), 167.78 (C-1‴), 168.1 (C-1″); MS (ESI) m/z (%) 470
(100) [M + Na]+, 448 (11) [M + 1]+; HRMS calcd for C23H17N3-
O5SNa (470.0781), found 470.0777.
According to the general procedure II, a solution of 3-methylcatechol
(1b) (78 mg, 0.63 mmol), 1,2,3,4-tetrahydro-6-ethyl-4-oxo-2-thioxo-
5-pyrimidinecarbonitrile (5d) (91 mg, 0.50 mmol), methanol (3 mL),
phosphate buffer (0.2 M, pH 6, 30 mL), and laccase from A. bisporus
(10 mg, 6 U/mg) was stirred for 18 h. Workup gave a mixture of 13d
and 14d in 82% (124 mg, 0.41 mmol). The obtained crude product of
13d and 14d was reacted with acetic anhydride (255 mg, 2.50 mmol)
and DMAP (6 mg, 0.05 mmol) in pyridine (2 mL) under argon for 7 h.
O
dx.doi.org/10.1021/jo401193e | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Workup gave the acetylated mixture of 21d and 22d in 72% yield
(114 mg, 0.30 mmol). The obtained mixture was purified by column
chromatography to give a mixture of 3-cyano-7,8-diacetyloxy-2-ethyl-6-
methyl-4H-pyrimido[2,1-b]benzothiazol-4-one (21d) and 3-cyano-7,8-
diacetyloxy-2-ethyl-9-methyl-4H-pyrimido[2,1-b]benzothiazol-4-one
(22d) as a pale yellow solid in 41% yield (65 mg, 0.17 mmol): mp 212−
214 °C; Rf = 0.26 (CH2Cl2/EtOAc = 10:1); UV (MeCN) λmax (log ε)
6-H); 13C NMR (125 MHz, DMSO-d6) of 22e δ 13.2 (C-3′), 14.6
(9-CH3), 19.8 (C-2‴), 20.19 (C-2″), 20.6 (C-2′), 37.8 (C-1′), 93.8
(C-3), 112.1 (C-6), 114.7 (CN), 122.4 (C-9a), 126.0 (C-9), 132.0
(C-5a), 139.8 (C-8), 141.8 (C-7), 158.1 (C-4), 165.0 (C-10a), 167.8
(C-1‴), 168.0 (C-1″), 172.6 (C-2); MS (ESI) m/z (%) 422 (100)
[M + Na]+, 400 (11) [M + 1]+; HRMS calcd for C19H17N3O5SNa
(422.0781), found 422.0784.
3-Cyano-7,8-diacetyloxy-2-isopropyl-6-methyl-4H-pyrimido[2,1-
b]benzothiazol-4-one (21f) and 3-Cyano-7,8-diacetyloxy-2-iso-
propyl-9-methyl-4H-pyrimido[2,1-b]benzothiazol-4-one (22f).
̃
208 (4.31), 226 (4.30), 362 nm (4.12); IR (ATR) ν 2943 (CH), 2223
(CN), 1764 (CO), 1701 (CO), 1541, 1451, 1169 cm−1; 1H NMR
(500 MHz, DMSO-d6) of 21d δ 1.27 (t, 3J (1′-H, 2′-H) = 7.5 Hz, 3H,
2′-H), 2.29 (s, 3H, 6-CH3), 2.33 (s, 3H, 2‴-H), 2.39 (overlapped, 3H,
2″-H), 2.79 (q, 3J (1′-H, 2′-H) = 7.5 Hz, 2H, 1′-H), 7.97 (s, 1H, 9-H);
13C NMR (125 MHz, DMSO-d6) of 21d δ 11.7 (C-2′), 16.7 (6-CH3),
20.0 (C-2″), 20.32 (C-2‴), 29.2 (C-1′), 92.6 (C-3), 115.1 (CN), 115.3
(C-9), 123.3 (C-9a), 125.4 (C-6), 132.06 (C-5a), 141.2 (C-7), 141.6
(C-8), 157.9 (C-4), 167.3 (C-10a), 168.0 (C-1″), 168.1 (C-1‴), 173.4
(C-2); 15N NMR (indirectly determined, 50.7 MHz, DMSO-d6) of 21d
δ −186.65 (N-5), −152.66 (N-1); 1H NMR (500 MHz, DMSO-d6) of
22d δ 1.28 (t, 3J (1′-H, 2′-H) = 7.5 Hz, 3H, 2′-H), 2.34 (s, 3H, 2″-H),
2.35 (s, 3H, 9-CH3), 2.39 (overlapped, 3H, 2‴-H), 2.82 (q, 3J (1′-H, 2′-
H) = 7.5 Hz, 2H, 1′-H), 8.63 (s, 1H, 6-H); 13C NMR (125 MHz,
DMSO-d6) of 22d δ 11.8 (C-2′), 14.7 (9-CH3), 20.0 (C-2‴), 20.35
(C-2″), 29.6 (C-1′), 93.2 (C-3), 112.3 (C-6), 114.8 (CN), 122.6
(C-9a), 126.1 (C-9), 132.08 (C-5a), 139.9 (C-8), 141.8 (C-7), 158.3
(C-4), 165.3 (C-10a), 167.97 (C-1‴), 168.3 (C-1″), 173.9 (C-2); MS
(ESI) m/z (%) 408 (100) [M + Na]+, 386 (11) [M + 1]+; HRMS calcd
for C18H15N3O5SNa (408.0625), found 408.0631.
According to the general procedure II, a suspension of 3-methyl-
catechol (1b) (78 mg, 0.63 mmol), 1,2,3,4-tetrahydro-6-isopropyl-4-
oxo-2-thioxo-5-pyrimidinecarbonitrile (5f) (98 mg, 0.50 mmol), methanol
(3 mL), phosphate buffer (0.2 M, pH 6, 30 mL), and laccase from
A. bisporus (10 mg, 6 U/mg) was stirred for 20 h. Workup gave a mixture
of 13f and 14f in 85% yield (135 mg, 0.43 mmol). The obtained crude
product of 13f and 14f was reacted with acetic anhydride (255 mg,
2.50 mmol) and DMAP (6 mg, 0.05 mmol) in pyridine (2 mL) under
argon for 5 h. Workup gave the acetylated mixture of 21f and 22f in 75%
yield (128 mg, 0.32 mmol). The obtained mixture was purified by column
chromatography to give a mixture of 3-cyano-7,8-diacetyloxy-2-isopropyl-
6-methyl-4H-pyrimido[2,1-b]benzothiazol-4-one (21f) and 3-cyano-7,8-
diacetyloxy-2-isopropyl-9-methyl-4H-pyrimido[2,1-b]benzothiazol-4-one
(22f) as a white solid in 58% yield (99 mg, 0.25 mmol): mp 227−229 °C;
Rf = 0.27 (CH2Cl2/EtOAc = 20:1); UV (MeCN) λmax (log ε) 209 (4.43),
3-Cyano-7,8-diacetyloxy-6-methyl-4H-2-propyl-pyrimido[2,1-b]-
benzothiazol-4-one (21e) and 3-Cyano-7,8-diacetyloxy-9-methyl-
4H-2-propyl-pyrimido[2,1-b]benzothiazol-4-one (22e).
̃
224 (4.41), 359 nm (4.23); IR (ATR) ν 2978 (CH), 2932 (CH), 2221
1
(CN), 1778 (CO), 1698 (CO), 1540, 1496, 1188 cm−1; H NMR
3
(500 MHz, DMSO-d6) of 21f δ 1.27 (d like, J (1′-H, 2′-H) = 6.5 Hz,
6H, 2′-H), 2.30 (s, 3H, 6-CH3), 2.33 (s, 3H, 2‴-H), 2.39 (overlapped,
3H, 2″-H), 3.25 (sept, 3J (1′-H, 2′-H) = 6.0 Hz, 1H, 1′-H), 8.00 (s, 1H, 9-
H); 13C NMR (125 MHz, DMSO-d6) of 21f δ 16.7 (6-CH3), 20.0
(C-2″), 20.35 (C-2‴), 20.39 (C-2′), 34.0 (C-1′), 92.0 (C-3), 115.0 (CN),
115.2 (C-9), 123.3 (C-9a), 125.4 (C-6), 132.1 (C-5a), 141.2 (C-7), 141.6
(C-8), 158.0 (C-4), 167.6 (C-10a), 167.99 (C-1″), 168.1 (C-1‴), 176.5
(C-2); 15N NMR (indirectly determined, 50.7 MHz, DMSO-d6) of 21f δ
−186.67 (N-5), −156.01 (N-1); 1H NMR (500 MHz, DMSO-d6) of 22f
According to the general procedure II, a solution of 3-methylcatechol
(1b) (78 mg, 0.63 mmol), 1,2,3,4-tetrahydro-4-oxo-6-propyl-2-thioxo-
5-pyrimidinecarbonitrile (5e) (98 mg, 0.50 mmol), methanol (3 mL),
phosphate buffer (0.2 M, pH 6, 30 mL), and laccase from A. bisporus
(10 mg, 6 U/mg) was stirred for 20 h. Workup gave a mixture of
13e and 14e in 78% yield (124 mg, 0.39 mmol). The obtained crude
product of 13e and 14e was reacted with acetic anhydride (255 mg,
2.50 mmol) and DMAP (6 mg, 0.05 mmol) in pyridine (2 mL) under
argon for 7 h. Workup gave the acetylated mixture of 21e and 22e in
76% yield (120 mg, 0.30 mmol). The obtained mixture was purified by
column chromatography to give a mixture of 3-cyano-7,8-diacetyloxy-
6-methyl-4H-2-propyl-pyrimido[2,1-b]benzothiazol-4-one (21e) and
3-cyano-7,8-diacetyloxy-9-methyl-4H-2-propyl-pyrimido[2,1-b]-
benzothiazol-4-one (22e) as a pale yellow solid in 57% yield (90 mg,
0.23 mmol): mp 194−196 °C; Rf = 0.47 (CH2Cl2/EtOAc = 10:1);
UV (MeCN) λmax (log ε) 209 (4.44), 226 (4.43), 362 nm (4.25);
3
δ 1.27 (d like, J (1′-H, 2′-H) = 6.5 Hz, 6H, 2′-H), 2.34 (s, 3H, 2″-H),
3
2.35 (s, 3H, 9-CH3), 2.39 (overlapped, 3H, 2‴-H), 3.25 (sept, J (1′-H,
2′-H) = 6.0 Hz, 1H, 1′-H), 8.63 (s, 1H, 6-H); 13C NMR (125 MHz,
DMSO-d6) of 22f δ 14.7 (9-CH3), 20.0 (C-2‴), 20.35 (C-2″), 20.5
(C-2′), 34.3 (C-1′), 92.6 (C-3), 112.3 (C-6), 114.7 (CN), 122.6 (C-9a),
126.1 (C-9), 132.1 (C-5a), 139.9 (C-8), 141.9 (C-7), 158.5 (C-4), 165.6
(C-10a), 167.97 (C-1‴), 168.3 (C-1″), 176.9 (C-2); MS (ESI) m/z (%)
422 (100) [M + Na]+, 400 (13) [M + 1]+; HRMS calcd for C19H17N3-
O5SNa (422.0781), found 422.0784.
ASSOCIATED CONTENT
* Supporting Information
Full characterization of all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
̃
IR (ATR) ν 3092 (CH), 2974 (CH), 2878 (CH), 2225 (CN), 1773
■
(CO), 1705 (CO), 1548, 1171 cm−1; 1H NMR (500 MHz,
DMSO-d6) of 21e δ 0.99 (t, 3J (2′-H, 3′-H) = 7.5 Hz, 3H, 3′-H), 1.77
S
3
3
(sex, J (1′-H, 2′-H) = 7.5 Hz, J (2′-H, 3′-H) = 7.5 Hz, 2H, 2′-H),
2.30 (s, 3H, 6-CH3), 2.33 (s, 3H, 2‴-H), 2.39 (overlapped, 3H, 2″-H),
3
2.76 (t, J (1′-H, 2′-H) = 7.6 Hz, 2H, 1′-H), 7.97 (s, 1H, 9-H);
AUTHOR INFORMATION
Corresponding Author
*E-mail: ubeifuss@uni-hohenheim.de.
13C NMR (125 MHz, DMSO-d6) of 21e δ 13.3 (C-3′), 16.6 (6-CH3),
19.8 (C-2″), 20.17 (C-2‴), 20.5 (C-2′), 37.4 (C-1′), 93.3 (C-3),
115.0 (CN), 115.1 (C-9), 123.1 (C-9a), 125.3 (C-6), 131.9 (C-5a),
141.2 (C-7), 141.5 (C-8), 157.7 (C-4), 167.0 (C-10a), 167.78 (C-1″),
167.8 (C-1‴), 172.1 (C-2); 15N NMR (indirectly determined, 50.7
■
Notes
The authors declare no competing financial interest.
1
MHz, DMSO-d6) of 21e δ −187.63 (N-5), −152.45 (N-1); H NMR
(500 MHz, DMSO-d6) of 22e δ 0.98 (t, 3J (2′-H, 3′-H) = 7.6 Hz, 3H,
ACKNOWLEDGMENTS
We thank Ms. S. Mika for recording the NMR spectra and Dr.
A. Baskakova as well as Ms. K. Wohlbold (Institut fur Organische
3
3
■
3′-H), 1.77 (sex, J (1′-H, 2′-H) = 7.5 Hz, J (2′-H, 3′-H) = 7.5 Hz,
2H, 2′-H), 2.34 (s, 3H, 2″-H), 2.35 (s, 3H, 9-CH3), 2.39 (overlapped,
3
3H, 2‴-H), 2.79 (t, J (1′-H, 2′-H) = 7.2 Hz, 2H, 1′-H), 8.65 (s, 1H,
̈
P
dx.doi.org/10.1021/jo401193e | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
2007, 48, 5073. (k) Leutbecher, H.; Conrad, J.; Klaiber, I.; Beifuss, U.
Synlett 2005, 3126.
Chemie der Universitat Stuttgart) for recording the mass spectra.
H.T A.-M. is grateful to Deutscher Akademischer Austausch-
dienst (DAAD) for financial support.
̈
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