A new application of the mild thiolation concept for an efficient three-step synthesis of 2-cyanobenzothiazoles: A new approach to Firefly-luciferin precursors

Article abstract of DOI:10.1080/17415993.2011.641558

An easy, low cost and upscalable three-step synthesis of Firefly luciferin precursors is presented. The 6-alkoxy-2-cyano-benzothiazoles (1) were synthesized starting from easily available O-alkylated 4-aminophenoles (2) via a mild thiolation, Jacobsen cyclization and dehydratization sequence.

Full text of DOI:10.1080/17415993.2011.641558

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A new application of the “mild
thiolation” concept for an
efficient three-step synthesis of 2-
cyanobenzothiazoles: a new approach
to Firefly-luciferin precursors
Hendryk Würfel a , Dieter Weiss a , Rainer Beckert a & Angelika
Güther a
a Institut für Organische Chemie und Makromolekulare Chemie,
Friedrich-Schiller Universität Jena, Humboldtstr. 10, Germany
Published online: 06 Jan 2012.
To cite this article: Hendryk Würfel , Dieter Weiss , Rainer Beckert & Angelika Güther (2012):
A new application of the “mild thiolation” concept for an efficient three-step synthesis of 2-
cyanobenzothiazoles: a new approach to Firefly-luciferin precursors, Journal of Sulfur Chemistry,
33:1, 9-16
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Journal of Sulfur Chemistry
Vol. 33, No. 1, February 2012, 9–16
A new application of the “mild thiolation” concept for an
efficient three-step synthesis of 2-cyanobenzothiazoles:
a new approach to Firefly-luciferin precursors
Hendryk Würfel, Dieter Weiss, Rainer Beckert* and Angelika Güther
Institut für Organische Chemie und Makromolekulare Chemie, Humboldtstr. 10, Friedrich-Schiller Univer-
sität Jena, Germany
(Received 26 October 2011; final version received 13 November 2011)
An easy, low cost and upscalable three-step synthesis of Firefly luciferin precursors is presented.
The 6-alkoxy-2-cyano-benzothiazoles (1) were synthesized starting from easily available O-alkylated
4-aminophenoles (2) via a mild thiolation, Jacobsen cyclization and dehydratization sequence.
NH2
N
CN
3 steps
R
S
O
1
O
2
18 - 25%
R
Keywords: sulfur; mild thiolation; luciferin; heterocycles; ethers
1. Introduction
2-Cyanobenzothiazoles (1) are important building blocks for natural products, functional dyes
and innovative materials. Firefly luciferin (3), a natural product, is of outstanding importance in
these areas. The substance was found in the beetle Photinus pyralis and is known to be a part of
the most efficient chemiluminescence systems nowadays. For that reason, it plays a major role
in applications used for medicinal diagnostics (1), biochemistry and forensic trace analysis (2)
(Figure 1).
6-Methoxy-2-cyano-benzothiazole (1a) plays a key role as a precursor for preparations of
3. The first synthesis starting from p-anisidine (2a) was published in 1961 (3). More than 30
years later, Toya et al. (4) presented a one-step access to 1a which employed the commercially
available 2-amino-6-methoxybenzothiazole (4) (Scheme 1). In subsequent years, a large number
*Corresponding author. Email: c6bera@uni-jena.de
ISSN 1741-5993 print/ISSN 1741-6000 online
© 2012 Taylor & Francis
http://dx.doi.org/10.1080/17415993.2011.641558
http://www.tandfonline.com

10 H. Würfel et al.
O
N
S
N
S
OH
HO
3
Figure 1. Firefly-luciferin.
NH2
White et. al (1961)
6 Steps
N
S
O
CN
2a
O
1a
Toya et. al (1992)
N
S
NH2
1 Step
O
4
Scheme 1. Classical synthesis of the precursor molecule (1a).
of modified protocols, mostly patents, were published (5a, 5b). However, a new approach for
the synthesis of the Firefly precursor (1a) remains desirable. This paper presents a substantially
improved protocol for the synthesis of (1a) with a far superior “scalability” and more favorable
cost basis. This new procedure is easily applicable not only in a research and teaching laboratory
setting, but also in an industrial environment.
The synthesis presented here is related to the method used by Seto et al. (6). However, the
advantage here lies in the formation of the thioamide (5) a simple, low cost, one-pot procedure.
Whereas the established protocol uses the costly trichloroacetamide in a low yielding procedure
with hydrogen sulfide to react with p-anisidine (2a), the new process takes advantage of the
reaction of low-cost chloroacetamide (6) with elemental sulfur and base to afford the thioamide
(5) in good yields (60–70 %). In larger-scale preparation, in the range of 100 g of 2a, the yield
of the Seto method is comparable (7) to ours. Nonetheless, with an increase in scale, there are
clearly disadvantages in the Seto method when compared with the new procedure presented here.
2. Results and discussion
O-Alkylated 4-aminophenols (2), which are commercially available or synthesized by simple
alkylation reactions (8) (Scheme 2: R = Me, i-Prop, cyclo-pentyl, benzyl), are the starting point
of the synthesis. They can easily be transformed with chloroacetamide (6) into thioamides (5).
This procedure, known as “mild thiolation” (9), requires a CH-acidic substrate (6), elemental
sulfur and a base. Working in a polar aprotic solvent (DMF, DMSO), the preactivated sulfur
is able to selectively oxidize CH2 groups. Extensive investigations about the “mild thiolation”
were the subject of papers in the 1980s (10). The authors showed that a mixture of sulfur and
triethylamine in DMF is best suited for the oxidative conversion of CH-acidic compounds to
their dithiocarboxylic acid derivatives under mild conditions. On the one hand, the triethylam-
monium salts of the corresponding dithiocarboxylic acid produced can be converted directly in

Journal of Sulfur Chemistry 11
a one pot procedure to their alkyl dithiocarboxylates (11) via alkylation. On the other hand, the
corresponding thioamides are formed if a primary or secondary amine is present (12) in the reac-
tion mixture (Scheme 2). In our approach, using the amines of type 2, the method allowed a direct
route to a series of substituted thioxoacetamides (5a–d). From numerous test reactions, the com-
bination of DMF and triethylamine were proven conditions for the planned transformations. Thus,
the aminophenol ethers (2a–d) were converted in a smooth reaction (6–8 h, room temperature)
to their corresponding thioamides (5a–d) (Scheme 3). According to the literature, changes in the
substitutions pattern of the aromatic amine lead to adjustments in the reaction conditions (10).
S
O
NH2
DMF/NEt3 RT
2/8S8
HN
O
NH2
6--8 h
Cl
NH2
60--70%
6
O
O
R
R
5
2
2a, 5a: R = Me
2b, 5b: R = iso-Propyl
2c, 5c: R = cyclo-Pentyl
2d, 5d: R = Benzyl
Scheme 2. The “mild thiolation” concept as one-pot reaction yielding thioamides (5).
S
S
O
X
R1
NH2
DMF/NEt3
2/8S8
R
S
S
O
O
S
NH2
Cl
NH2
HNEt3
O
6
NH2
NH2
NH
R2
R2
Scheme 3. The “mild thiolation” concept in general.
A major advantage is that the thioamides (5a–d) can be cyclized to the corresponding ben-
zothiazoles (7a–d) without further purification. The excess of sulfur that might still remain in
the products can be completely filtered off by dissolving (5a–d) in a basic aqueous solution. The
cyclization reaction was carried out with a basic aqueous solution of (5) and K3[Fe(CN)6] (13a,
13b). Test reactions showed that this reagents offers the best yields and, in addition, easy work
up of the resulting products (Scheme 4).
The final dehydratization reaction of the amide was carried out under mild conditions (14)
in dry DMF with POCl3 below room temperature which leads to better yields of (1) than the
previously used method (6). Attempts to introduce the nitril group prior to the “mild thiola-
tion” step failed completely. Most likely, the in situ formed nucleophilic sulfur species attack

12 H. Würfel et al.
H
N
H2O / NaOH
K3[Fe(CN)6]
S
O
N
S
50--65 %
NH2
O
R
H2N
O
O
R
7
5
DMF < 10 °C
POCl3 1--2 h
60--80 %
5a, 7a, 1a = Me
5b, 7b, 1b = iso-Propyl
5c, 7c, 1c = cyclo-Pentyl
5d, 7d, 1d = Benzyl
N
CN
S
O
R
1
Scheme 4. Cyclization to benzothiazoles (7) and their dehydratization to the final products (1).
the nitrile group forming dithioxalicacid diamides (15) which could be detected by thin-layer
chromatography (TLC).
The protection of the phenolic OH-group is of major importance in this synthetic approach.
Although the OH-group remained unchanged during the thiolation reaction, a subsequent selective
alkylation as well as an ortho-cyclization failed after the thioamide was formed. The commonly
used methylether moiety needs harsh reaction conditions to get cleaved (16) so it is far from being
an applicable protecting group. First results showed that other ways of cleaving the methyl ether
also affect the nitrile group. Therefore, different ethers were considered as protecting groups. The
literature shows (17a, 17b) that these derivatives are cleaved easier while keeping nitrile groups
in the molecule unchanged.
3. Conclusion
The “mild thiolation” of chloroacetamide (6) in the presence of an aminophenol ether (2) consti-
tutes an easy way of preparing thioxalicacid amides (5). They can easily be cyclized to yield the
corresponding alkoxy substituted benzothiazole (7) without further purification steps. The final
dehydratization led to 2-cyano-6-alkoxy-benzothiazoles (1) in good yields and high purities. The
synthetic approach shown here presents an easy and efficient way to produce Firefly-luciferin
precursor molecules. In our opinion, the synthesis can easily be upscaled to 100 g approaches.
4. Experimental
All solvents were of ACS reagent grade or better unless otherwise noted. All reactions were
carried out under atmospheric pressure without special need for an inert atmosphere. Unless
otherwise stated, there was no special need to exclude moisture. The reagents were purchased
from Sigma-Aldrich, Alfa Aesar or Roth. TLC was performed on glass plates coated with silica
gel 60. Visualization was observed by UV light at 254 nm or 366 nm.
1H and 13C NMR spectra for all compounds were acquired in DMSO-d6, CDCl3 and acetone-d6
on a Bruker AC 250 operating at (250 and 63 MHz, respectively). Chemical shifts (δ) are reported
in parts per million (ppm) and referenced with the residual undeuterated solvent. The mass spectra
were recorded on a MAZ 95 XL and an FINNIGAN MAT SSQ 710 with electron impact (EI) and
on a Waters Acquity ultra-performance liquid chromatography (UPLC) coupled to a quadrapole
time-of-flight micro-mass spectrometer (Waters) equipped with electrospray ionization (ESI) in
a positive ion mode. Column: BEH C18 UPLC column (2.1 mm, 1.7 μm; Waters). All melting
points were measured on a Galen III Boetius System and are uncorrected.

Journal of Sulfur Chemistry 13
4.1. General procedure for the preparation of thioamides (5a–d) starting from amines
(2a–d) using the “mild thiolation” concept
To a mixture of 0.5 mol of amine (2), 1 mol of sulfur (32.0 g) and 80 ml of triethylamine in 500 mL
DMF, 0.55 mol (51.4 g) of chloroacetamide (6) was added slowly in a way that the temperature
did not exceed 30◦C. The reaction mixture was stirred for 8 h and poured into 5 l of water. After
filtration, the product was obtained as a yellow powder with elemental sulfur impurities. The
powder can be used for the next synthesis step without further purification. For analysis, a sample
was taken up in a small portion of acetone filtered and evaporated in vacuo. Recrystallization gave
the pure compounds in yields of 73–65%.
4.1.1. 2-(4-Methoxyphenylamino)-2-thioxoacetamide (5a)
Yellow solid, m.p. 187–189◦C (water–ethanol); yield 70%; 1H NMR (DMSO-d6) δ (ppm): 3.76
(s, 3H, OCH3); 6.97 (d, 2H, ArH); 7.89 (d, 2H, ArH); 8.08 (s, 1H, NH); 8.13 (s, 1H, NH); 12.01
(s, 1H, NH); 13C NMR (DMSO-d6) δ (ppm): 55.8; 114.1; 125.2; 132.0; 158.0; 162.8; 185.3; MS
(m/z, ESI): 211.055 [M + H]; C9H10N2SO2 (210.25); Calcd. C 51.41, H 4.79, N 13.32, S 15.25;
found C 51.35, H 4.70, N 13.40, S 15.38.
4.1.2. 2-(4-iso-Propoxyphenylamino)-2-thioxoacetamide (5b)
Yellow solid, m.p. 127–128◦C (water–ethanol), yield 66%; 1H NMR (DMSO-d6) δ (ppm): 1.25
(d, 6H, CH3); 4.60 (hep, 1H, CH); 6.94 (d, 2H, ArH); 7.8–7.92 (m, 2H, ArH); 8.08 (s, 1H, NH);
8.13 (s, 1H, NH); 11.98 (s, 1H, NH); 13C NMR (DMSO-d6) δ (ppm): 22.2; 69.9; 115.6; 125.1;
131.6; 156.3; 162.7; 184.8; MS (m/z, EI): 238 [M]; 196; 152 [Basepeak]; 120; 108; 94; 81;
65; C11H13N2SO2 (237.30); Calcd. C 55.68, H 5.52, N 11.81, S 13.51; found C 55.56, H 5.48,
N 11.74, S 13.68.
4.1.3. 2-(4-Cyclopentyloxyphenylamino)-2-thioxoacetamide (5c)
Yellow solid, m.p. 149–150◦C (water–ethanol), yield 73%; 1H NMR (DMSO-d6) δ (ppm): 1.5–
1.75 (m, 6H, CH2); 1.8–2.0 (m, 2H, CH2); 4.75–4.85 (m, 1H, CH); 6.88–6.95 (m, 2H, ArH);
7.85–7.93 (m, 2H, ArH); 8.08 (s, 1H, NH); 8.13 (s, 1H, NH); 11.98 (s, 1H, NH);13C NMR
(DMSO-d6) δ (ppm): 24.1; 32.7; 79.3; 115.4; 125.0; 131.5; 156.4; 162.6; 184.9; MS (m/z, EI): 265
[M+1]; 264 [M/Basepeak]; 263 [M–1]; 196; 195; 188; 152; 151; 126; 108; 69; 45; C13H16N2SO2
(264.34); Calcd. C 59.07, H 6.10, N 10.60, S 12.13; found C 58.98, H 6.08, N 10.54, S 12.38.
4.1.4. 2-(4-Benzyloxyphenylamino)-2-thioxoacetamide (5d)
Yellow solid, m.p. 158–159◦C (ethanol–acetone), yield 65%; 1H NMR (DMSO-d6) δ (ppm): 5.12
(s, 2H, CH2); 7.05 (d, 2H, ArH); 7.2–7.5 (m, 5H, ArH); 7.90 (d, 2H, ArH); 8.1 (s, 1H, NH); 8.14
(s, 1H, NH); 12.02 (s, 1H, NH). 13C NMR (DMSO-d6) δ (ppm): 69.9; 115.0; 125.1; 128.2; 128.3;
128.9; 132.1; 137.3; 157.1; 162.7; 185.3; MS (m/z, EI): 286 [M]; 285 [M–1]; 210; 195; 150; 122;
108; 91 [Basepeak]; 65; 45; C15H14N2SO2 (286.35); Calcd. C 62.92, H 4.93, N 9.78, S 11.20;
found C 62.84, H 4.88, N 9.68, S 11.42.

14 H. Würfel et al.
4.2. General procedure for the Jacobsen cyclization reaction of the thioamides (5a–d)
yielding benzothiazoles (7a–d)
Thioamide (20 mmol) was dissolved in the necessary amount of 10% NaOH. The mixture was
filtered to obtain a sulfur-free yellow solution. This solution was added dropwise to 60 mmol
(19.7 g) of K3[Fe(CN)6] dissolved in 150 ml of water. After the addition, the reaction mixture was
stirred for one additional hour. In the case of thioamide 5d, the solution was heated to 60◦C for
3 h. The formed precipitate was filtered off and washed with water until the filtrate was colorless.
The pale yellow product was dried in vacuo and used for the next reaction step without further
purification. For analysis, samples were recrystallized from ethanol–DMF.
4.2.1. 6-Methoxybenzothiazol-2-carboxamide (7a)
White solid, m.p. 258◦C (subl.), yield 65%; 1H NMR (DMSO-d6) δ (ppm): 3.86 (s, 3H, OCH3);
7.20 (dd, 1H, ArH, 3J = 9 Hz, 4J = 2.5 Hz); 7.76 (d, 1H, ArH, 3J = 2.5 Hz); 7.99 (d, 1H, ArH,
3J = 9 Hz); 8.36 (s, 2H, NH). 13C NMR (DMSO-d6) δ (ppm): 55.4; 104.3; 116.6; 124.2; 137.8;
146.8; 158.0; 161.0; 161.7; MS (m/z, ESI): 209.029 [M + H]. C9H8N2O2S (208.24); Calcd. C
51.91; H 3.87; N 13.45; S 15.40; found C 51.88, H 3.77, N 13.50, S 15.25.
4.2.2. 6-iso-Propoxybenzothiazol-2-carboxamide (7b)
White solid, m.p. >360◦C (subl.), yield 63%; 1H NMR (DMSO-d6) δ (ppm): 1.39 (d, 6H, CH3,
3J = 6.0 Hz); 4.63 (m, 1H, CH, 3J = 6.0 Hz); 7.13 (dd, 1H,ArH, 4J = 2.0 Hz, 3J = 9.0 Hz); 7.37
(d, 1H, ArH, 4J = 2.0 Hz); 7.94 (d, 1H, ArH, 3J = 9.0 Hz). 13C NMR (DMSO-d6) δ (ppm): 21.9;
70.8; 106.0; 118.5; 125.1; 139.1; 147.3; 157.4; 160.0; 162.3; MS (m/z, ESI): 237.071 [M + H].
C11H12N2O2S (236.29); Calcd. C 55.91; H 5.12; N 11.86; S 13.57; found C 55.88, H 5.07, N
13.60, S 13.29.
4.2.3. 6-cyclo-Pentyloxybenzothiazol-2-carboxamide (7c)
White solid, m.p. >360◦C (subl.), yield 63%; H NMR (DMSO-d6) δ (ppm): 1.5–2.0 (m, 8H,
1
CH2); 4.89 (m, 1H, CH); 7.1–7.2 (m, 1H, ArH); 7.70 (s, 1H, ArH); 7.9–8.0 (m, 1H, ArH); 8.34
(s, 1H; NH). 13C NMR (DMSO-d6) δ (ppm): 24.1; 32.7; 80.0; 106.8; 118.4; 125.1; 138.6; 147.4;
157.3; 161.9; 162.3; MS (m/z, ESI): 263.77 [M + H]. C13H14N2O2S (262.33); Calcd. C 59.52;
H 5.38; N 10.68; S 12.22; found C 59.46; H 5.20; N 10.50; S 12.30.
4.2.4. 6-Benzyloxybenzothiazol-2-carboxamide (7d)
White solid, m.p. >360◦C (subl.), yield 60%; 1H NMR (DMSO-d6) δ (ppm): 5.19 (s, 2H, CH2);
4
7.24–7.5 (m, 6H, ArH); 7.84 (d, 1H, ArH, J = 2.5 Hz); 7.96 (s, 1H, NH); 7.99 (d, 1H, ArH,
3J = 9 Hz); 8.36 (s, 1H, NH). 13C NMR (DMSO-d6) δ (ppm): 70.4; 106.4; 117.9; 125.2; 128.3;
128.5; 129.0; 137.0; 138.6; 147.8; 158.0; 161.9; 162.7; MS (m/z, EI): 285 [M + 1]; 284 [M];
165; 122; 91 [Basepeak]; 65. C15H12N2O2S (284.33); Calcd. C 63.36; H 4.25; N 9.85; S 11.28;
found C 63.30; H 4.20; N 9.78; S 11.15.
4.3. General method for converting amides (7a–d) into nitriles (1a–d)
A solution of 21 mmol of amide (7) in 150 ml of DMF was cooled to 5◦C. To this solution, 4 ml of
POCl3 were added dropwise keeping the temperature below 10◦C. After all POCl3 was added, the

Journal of Sulfur Chemistry 15
mixture was stirred for 2 h while reaching room temperature. After the reaction was completed
(monitored by TLC), the reaction mixture was poured into 500 ml of ice water. The precipitate
formed was filtered off and washed with NaHCO3 solution and water. The product was dried
in vacuo and recrystallized to give white solids.
4.3.1. 6-Methoxybenzothiazol-2-carbonitrile (1a)
White solid, m.p. 125◦C (heptane), yield 65%; 1H NMR (acetone-d6) δ (ppm): 3.95 (s, 3H, OCH3),
7.32 (dd, 1H, ArH, 3J = 9.1 Hz, 4J = 2.5 Hz); 7.78 (d, 1H, ArH, 4J = 2.5 Hz); 8.1 (d, 1H, ArH,
3J = 9.1 Hz). 13C NMR (acetone-d6) δ (ppm): 55.6; 103.7; 113.2; 118.6; 125.4; 133.5; 137.7;
146.8; 160.6; MS (m/z, ESI): 191.026 [M + 1]. C9H6N2OS (190.22); Calcd. C 56.83; H 3.18; N
14.73; S 16.86; found C 56.77; H 3.15; N 14.66; S 16.75.
4.3.2. 6-iso-Propoxybenzothiazol-2-carbonitrile (1b)
White solid, m.p. 215◦C subl., yield 60%; H NMR (acetone-d6) δ (ppm): 1.37 (d, 6H, CH3,
1
4
3
J = 6 Hz); 4.79 (m, 1H, CH, J = 6 Hz); 7.29 (dd, 1H, ArH, J = 2.5 Hz, J = 9 Hz); 7.78 (d,
1H, ArH, 4J = 2.5 Hz); 8.06 (d, 1H, ArH, 3J = 9 Hz). 13C NMR (acetone d6) δ (ppm): 21.1; 70.7;
105.5; 113.2; 119.6; 125.5; 133.3; 137.8; 146.6; 158.9; MS (m/z, EI): 218 [M]; 176 [Basepeak];
147; 124; 96; 69; 43. C11H10N2OS (218.27); Calcd. C 60.53; H 4.62; N 12.83; S 14.69; found C
60.44; H 4.58; N 12.60; S 14.49.
4.3.3. 6-cyclo-Pentyloxybenzothiazol-2-carbonitrile (1c)
White solid, m.p. 78–79◦C (heptane), yield 60%; 1H NMR (DMSO-d6) δ (ppm): 1.62–2.10 (m,
4
3
8H, CH2); 4.80–4.90 (m, 1H, CH); 7.19 (dd, 1H, ArH, J = 2.5 Hz, J = 9 Hz); 7.31 (d, 1H,
4
3
ArH, J = 2.5 Hz); 8.05 (d, 1H, ArH, J = 9 Hz). 13C NMR (DMSO-d6) δ (ppm): 24.0; 32.8;
80.4; 104.6; 113.3; 119.5; 125.76; 132.9; 137.39; 146.5; 159.1; MS (m/z, ESI): 245.07 [M + H].
C13H12N2OS (244.31); Calcd. C 63.91; H 4.95; N 11.47; S 13.12; found C 63.85; H 4.90; N
11.28; S 12.99.
4.3.4. 6-Benzyloxybenzothiazol-2-carbonitrile (1d)
White solid, m.p. 139–140◦C (heptane), yield 80%; 1H NMR (DMSO-d6) δ (ppm): 5.18 (s, 2H,
CH2); 7.2–7.5 (m, 7H, ArH); 8.1 (d, 1H, ArH, 3J = 9 Hz). 13C NMR (DMSO-d6) δ (ppm): 70.8;
104.3; 113.2; 119.0; 125.9; 127.5; 128.4; 128.8; 133.5; 135.8; 137.4; 147.0; 159.5; MS (m/z, EI):
267 [M + 1]; 266 [M]; 92; 91; 65. C15H10N2OS (266.32); Calcd. C 67.65; H 3.78; N 10.52; S
12.04; found C 67.70; H 3.81; N 10.37; S 11.91.
Acknowledgement
The authors thank Roche Diagnostics GmbH, Penzberg, for financial support.
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