Economical and scalable synthesis of 6-amino-2-cyanobenzothiazole

Article abstract of DOI:10.3762/bjoc.12.189

2-Cyanobenzothiazoles (CBTs) are useful building blocks for: 1) luciferin derivatives for bioluminescent imaging; and 2) handles for bioorthogonal ligations. A particularly versatile CBT is 6-amino-2-cyanobenzothiazole (ACBT), which has an amine handle for straight-forward derivatisation. Here we present an economical and scalable synthesis of ACBT based on a cyanation catalysed by 1,4-diazabicyclo[2.2.2]octane (DABCO), and discuss its advantages for scale-up over previously reported routes.

Full text of DOI:10.3762/bjoc.12.189

Economical and scalable synthesis of
6-amino-2-cyanobenzothiazole
Jacob R. Hauser1,2, Hester A. Beard1,2, Mary E. Bayana1,3, Katherine E. Jolley1,3,
Stuart L. Warriner1,2 and Robin S. Bon*2,4
Letter
Open Access
Address:
Beilstein J. Org. Chem. 2016, 12, 2019–2025.
1School of Chemistry, University of Leeds, 2Astbury Centre for
Structural Molecular Biology, 3Institute of Process Research and
Development, and 4Leeds Institute of Cardiovascular and Metabolic
Medicine, LIGHT Laboratories, University of Leeds, Leeds LS2 9JT,
UK
doi:10.3762/bjoc.12.189
Received: 09 June 2016
Accepted: 24 August 2016
Published: 13 September 2016
Email:
Associate Editor: M. Rueping
Robin S. Bon* - r.bon@leeds.ac.uk
© 2016 Hauser et al.; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
ACBT; cyanation; 2-cyanobenzothiazoles; DABCO; luciferins;
organocatalysis
Abstract
2-Cyanobenzothiazoles (CBTs) are useful building blocks for: 1) luciferin derivatives for bioluminescent imaging; and 2) handles
for bioorthogonal ligations. A particularly versatile CBT is 6-amino-2-cyanobenzothiazole (ACBT), which has an amine handle for
straight-forward derivatisation. Here we present an economical and scalable synthesis of ACBT based on a cyanation catalysed by
1,4-diazabicyclo[2.2.2]octane (DABCO), and discuss its advantages for scale-up over previously reported routes.
Introduction
Functionalised 2-cyanobenzothiazoles (CBTs, 1) are key build- Even in the presence of other thiols and amines, CBTs react
ing blocks for the synthesis of luciferins 3 [1-3], substrates of rapidly and selectively with 1,2-aminothiols under physiologi-
natural and engineered firefly luciferases that are widely used cal conditions. This selective and bioorthogonal reactivity of
for bioluminescence imaging (BLI) [4,5]. The typical prepara- CBTs has been exploited in the development of the CBT liga-
tion of luciferins 3 involves straightforward condensation of tion as a useful and fast bioorthogonal reaction (k ≈ 10 M−1s−1)
CBTs 1 with D-cysteine (2) (Scheme 1). For BLI applications, [9] for site-specific labelling or immobilisation of proteins 4,
luciferins are often generated in vivo from CBTs [4-7], which either at an N-terminal cysteine residue or at a 1,2-aminothiol
are easier to modify and handle due to their higher stability, cell group incorporated into a non-natural amino acid (Scheme 1)
permeability, and reactivity than the full luciferin scaffolds [10,11]. In addition, CBT derivatives have been used for the
[2,5,8].
synthesis of polymeric nanostructures in cellulo [12,13].
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Scheme 1: Functionalised CBTs 1 can be used for the synthesis of luciferins 3 and for bioorthogonal ligations such as the site-specific labelling/im-
mobilisation of proteins 4.
The precursor of D-aminoluciferin, 6-amino-2-cyanobenzothia- an alternative synthesis of ACBT that avoids the use of cyanide
zole (ACBT, 8), is an attractive building block for BLI probes (Scheme 2B) [7]. This route makes use of 4,5-dichloro-1,2,3-
and for handles for CBT ligations, because of the ease of dithiazolium chloride (Appel’s salt, 12), and variations of the
derivatisation of its amino group. Like other functionalised procedure have enabled the synthesis of different CBT deriva-
CBTs, ACBT is available from commercial suppliers, but tives, some of which on multi-gram scale [6,7,16,17]. However,
expensive. Thus, the development of an economical and scal- Appel’s salt is relatively expensive and available from a limited
able synthesis of ACBT would enable studies that require larger number of suppliers, and its synthesis requires the use of the
amounts of this building block.
highly toxic reagent sulfur monochloride [18].
The traditional synthesis of ACBT 8 involves the cyanation of Here, we report an alternative, practical and economical proce-
6-amino-2-chlorobenzothiazole (7) with an excess of potassium dure for the preparation of ACBT 8. Our route involves a mild,
cyanide (KCN) in hot DMSO under dilute conditions organocatalytic cyanation. In addition, the use of filtrations and
(Scheme 2A) [14,15]. However, this reaction is sluggish (partly crystallisations for purification, in combination with control of
because of the poor solubility of KCN in DMSO), requires elab- reaction rate and heat output in the cyanation step, makes this
orate extractive workups for the removal of DMSO (persistent procedure readily scalable.
traces of which are detrimental to purification) and gives low
and variable yields (typically 30–50% in our hands). In addi- Results and Discussion
tion, because DMSO can quickly penetrate the skin, working In order to install the cyano group of ACBT 8 under mild condi-
with KCN in DMSO requires substantial safety measures. tions, catalytic cyanation procedures were considered. Initial
Therefore, this route does not allow straightforward scale-up attempts to use the palladium or copper-catalysed cyanation of
under laboratory conditions. Prescher and co-workers reported 6-amino-2-halobenzothiazoles with potassium hexaferricyanide
Scheme 2: Reported synthetic routes to ACBT 8: A) Original route reported by Takakura et al. [14] and Wang et al. [15]. B) The improved route re-
ported by McCutcheon et al. [7].
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– a slow-releasing cyanide source – were unsuccessful. Instead, from ethanolysis of 13 and subsequent hydrolysis of the inter-
a report on the use of 1,4-diazabicyclo[2.2.2]octane (DABCO) mediate imidate) were formed as well (Table 1, entry 2; Sup-
as a catalyst for the cyanation of heteroaryl halides [19] inspired porting Information File, Figure S7). In contrast, the use of
us to explore the DABCO-catalysed cyanation of 2-chloroben- acetonitrile (with a small volume of water to facilitate dissolu-
zothiazoles.
tion/addition of NaCN) resulted in full conversion to 13, and
pure 13 which was isolated in 75% yield after quenching with
Indeed, the treatment of 2-chloro-6-nitrobenzothiazole (6) with iron(III) chloride solution, extraction and simple filtration
DABCO (15 mol %) and sodium cyanide (NaCN, 1.05 equiv) in through a silica plug (Table 1, entry 3). The straightforward
DMSO/water 1:1, at room temperature, resulted in full conver- work-up/purification procedure – in the absence of DMSO as a
sion to 2-cyano-6-nitrobenzothiazole (13, Table 1), a known co-solvent – outweighed the slightly reduced reaction yield on
precursor of ACBT 8 [7]. Any unreacted cyanide in the reac- this scale.
tion mixture was safely quenched by the addition of an iron(III)
chloride solution, and pure ACBT 8 was isolated in 90% yield For scale-up of the DABCO-catalysed cyanation, 6 was synthe-
after (extensive) extractive work-up and flash chromatography. sised by straight-forward nitration of the significantly cheaper
No conversion of 6 to 13 was observed in the absence of starting material 2-chlorobenzothiazole (16) [15], and isolated
DABCO, which demonstrates the essential role of DABCO as a in 82% yield after crystallisation from ethanol (Scheme 3).
cyanation catalyst under these conditions. In addition, when the Recrystallisation of 16 from acetonitrile required only 25% of
more electron rich 6-amino-2-chlorobenzothiazole (7) was sub- the solvent volume, but resulted in a slightly lower yield (73%).
jected to the DABCO-catalysed cyanation conditions, only In view of potential dangers involved in the use of NaCN on
starting material was isolated, even upon heating the reaction large scale, a calorimetric analysis was conducted to monitor
mixture to 100 °C (not shown). This suggests that, although the the heat output of the cyanation reaction. In this experiment,
cyanation may be adaptable to other 2-halobenzothiazole deriv- aqueous NaCN (49 mg in 2 mL) was slowly added to a solution
atives, electron-deficient substrates may be required for the of 6 (200 mg) and DABCO (15 mol %) in acetonitrile (20 mL;
reactions to proceed under mild conditions.
compound 6 dissolves in acetonitrile only upon addition of
DABCO). The calorimeter jacket temperature was set at 1 °C,
In view of scale-up of the DABCO-catalysed cyanation of 6, an and power compensation by an internal heater coil was used to
alternative solvent (mixture) was sought in order to eliminate maintain the reactor temperature at 21 °C. The reaction resulted
safety issues and work-up problems resulting from the use of in an endotherm (Figure 1A). The total power released or con-
DMSO. An initial attempt to run the reaction in water failed sumed by the reaction (QTotal) was calculated by subtracting
because of the low aqueous solubility of 6 – resulting in a the power generated due to the temperature and specific heat
biphasic reaction mixture – and the slow hydrolysis of 6 under capacity of the feed (QDose) from the compensatory power
the reaction conditions according to LC–MS (not shown). The supplied by the internal heater coil (QComp). Because QDose
conversion of 6 was complete in ethanol. However, in addition remained at zero throughout the reaction, the traces for QComp
to the expected cyano compound 13, significant amounts of side and QTotal overlap in Figure 1A. From QTotal, an energy
products 14 (resulting from ethanolysis of 6) and 15 (resulting consumption of 0.42 kJ was determined for the addition of
Table 1: DABCO-catalysed cyanation of 6: solvent studies.a
entry
solvent(s)
conversion of 6b
ratio 13:14:15c
yield 13d
1
2
3
DMSO/H2O 1:1
EtOH
100%
100%
100%
100:0:0
17:32:51
100:0:0
90%
ND
MeCN/H2O 10:1
75%
aReactions were performed using 200 mg 6 in 20 mL solvent. bConversion of 6 was determined by LC–ESIMS. cIdentities of 13–15 in the reaction
mixtures were confirmed by 1H NMR and LC–MS and product ratios were determined by 1H NMR after work-up. dYields were determined after purifi-
cation; ND = not determined.
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Figure 1: Calorimeter traces for the addition of aqueous NaCN (A) or water (B) to a solution of 6 and DABCO in acetonitrile. QComp: compensatory
power; QTotal: total power; QDose: power generated due to the temperature and heat capacity of the feed; energy: heat energy. Because QDose ≈ 0
over the course of the experiment, traces for QComp (orange) and QTotal (blue) overlap.
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Beilstein J. Org. Chem. 2016, 12, 2019–2025.
1 mmol of NaCN in 2 mL of water. A control experiment, in sole use of filtrations and crystallisations for purification of all
which only water was added to 6 and DABCO in acetonitrile, intermediates and products, in combination with the
showed an endotherm of 0.51 kJ, corresponding to a calculated endothermic nature of the controlled cyanation procedure, will
heat of mixing of water into the reaction mixture of enable straightforward further scale up, if required. A direct
ΔH = 4.57 kJ mol−1 (see Supporting Information File 1). These comparison of our route to ACBT 8 to those published by
observations complicated accurate quantification of the heat of others is shown in the Supporting Information File 1, clearly
reaction (because of different properties of the feeds in the two revealing our route as the most suitable for scale-up.
experiments). The difference in endotherms between the cyana-
Experimental
tion reaction (Figure 1A) and the control experiment
(Figure 1B) suggests the overall DABCO-catalysed reaction of General experimental
6 and NaCN is exothermic, but that the endothermic addition of All chemical reagents were purchased from commercial
water to the acetonitrile solution outweighs this exotherm under suppliers and used without further purification. The identity and
the experimental conditions. Our results suggest that a slow ad- purity of known compounds was confirmed through the com-
dition of a dilute aqueous NaCN solution is a safe method for parison of experimentally obtained data to values reported in
scale-up of the cyanation reaction, provided the concentration the literature. 1H and 13C NMR spectra were recorded in deuter-
of the solution allows an overall reaction endotherm to be main- ated solvents on a Bruker Avance 500 spectrometer. Chemical
tained to prevent potential thermal runaway upon scale-up.
shifts are referenced to residual solvent peaks and are quoted in
ppm. Coupling constants (J) are reported to the nearest 0.1 Hz.
The DABCO-catalysed cyanation reaction was scaled up in two Assignment of spectra was based on expected chemical shifts
steps: using 2 g and then 10 g of substrate 6 (Scheme 3). After and coupling constants, aided by COSY, HMQC, and HMBC,
quenching with iron(III) chloride solution, extractive work-up where appropriate. Calorimetry experiments were carried out
and filtration through a short silica plug, product 13 was isolat- using HEL AutoMATE parallel reactors with HEL WinISO
ed in 83–93% yield. A sample of 13 for melting point determi- 2225 and HEL IQ 1.2.16 software. Temperature was controlled
nation was obtained by recrystallisation from methanol. The using a Julabo refrigerated/heating circulator (Model FP50-HD)
synthesis of ACBT 8 was completed by reduction of the nitro and feed reactants introduced into the reaction using a Harvard
group in 13 (Scheme 3). In our hands, the use of iron powder in syringe pump (model pump 11) connected to a 20 mL dispos-
acetic acid proved more practical than the previously reported able syringe with an 8 inch, 16 gauge Luer fitting syringe
procedures with zinc powder and ammonium chloride [7]. It needle.
resulted in more consistent yields, an easier work-up, and no
persistent contamination of 8 with nitroso intermediates [6]. Reaction procedures
The reduction step was accomplished on a 1 g and 5 g scale 2-Chloro-6-nitro-1,3-benzothiazole (6) [15,20]
isolating ACBT 8 by filtration through a short silica plug to
give the desired product in 60–71% yield. Pure product 8 was
recrystallised from hot ethanol for melting point determination.
Conclusion
We have developed a straight-forward, practical and readily
scalable synthesis of ACBT 8, a valuable intermediate in the de- 2-Chloro-1,3-benzothiazole (16, 10.0 g, 58.95 mmol) was added
velopment of chemical probes for bioorthogonal ligation and portion wise to concentrated H2SO4 (60 mL) in a cooled round-
bioluminescent imaging. The procedure allowed the safe syn- bottomed flask (ice bath). Potassium nitrate (6.56 g,
thesis of 8 at multigram scale and in high purity. In addition, the 64.85 mmol) was added portion wise, and the resulting reaction
Scheme 3: Scale-up of ACBT synthesis.
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Beilstein J. Org. Chem. 2016, 12, 2019–2025.
mixture was stirred at 0 °C (ice bath) for 30 min, and then at stirred at room temperature for 24 h, diluted with water (1 L)
room temperature for 18 h. The solution was poured onto ice and unreacted iron was removed by filtration through celite.
and the formed precipitate collected by filtration. The collected The aqueous solution was extracted with EtOAc (4 × 500 mL).
solid was rinsed with ice cold water until acid free, and then The combined organic layers were washed with brine (2 ×
dried under reduced pressure. The crude product was further 300 mL), dried (Na2SO4) and concentrated in vacuo. The crude
purified by recrystallisation from MeCN (≈ 100 mL) or EtOH product was loaded onto a short plug of silica gel, flushed
(≈ 425 mL) to yield 6 as fine off-white needles. Yield from through with CHCl3 (ca. 1 L) and concentrated in vacuo to give
EtOH (10.37 g, 48.34 mmol, 82%), from MeCN (9.21 g, 42.89 8 as yellow microcrystals (3.02 g, 17.24 mmol, 71%). A sam-
mmol, 73%). Mp 193–194 °C (EtOH); 1H NMR (500 MHz, ple of 13 was crystallised from EtOH for melting point analysis.
CDCl3) δ 8.75 (d, J = 2.3 Hz, 1H, CH-7), 8.38 (dd, J = 9.0, 2.3 Rf 0.2 (SiO2, DCM); mp 219–220 °C (EtOH); 1H NMR
Hz, 1H, CH-5), 8.07 (d, J = 9.0 Hz, 1H, CH-4); 13C NMR (125 (500 MHz, CDCl3) δ 7.95 (d, J = 8.9 Hz, 1H, CH-4), 7.08 (d, J
MHz, CDCl3) δ 158.9 (C-2), 154.9 (C-6), 145.6 (C-7a), 136.6 = 2.2 Hz, 1H, CH-7), 6.95 (dd, J = 8.8, 2.2 Hz, 1H, CH-5), 4.14
(C-3a), 123.5 (CH-4), 122.4 (CH-5), 117.8 (CH-7).
(s, 2H, NH2); 13C NMR (125 MHz, CDCl3) δ 147.8 (C-6),
145.7 (C-7a), 138.3 (C-3a), 131.2(C-2), 126.1 (CH-4), 117.8
(CH-5), 113.7 (C≡N), 104.0 (CH-7).
6-Nitro-1,3-benzothiazole-2-carbonitrile (13) [19]
Supporting Information
Supporting Information File 1
Copies of 1H and 13C NMR spectra, details of calorimetry
experiments and comparison of routes to ACBT 8.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-12-189-S1.pdf]
A solution of NaCN (2.40 g, 48.97 mmol) in H2O (100 mL)
was added slowly to a stirred solution of 2-chloro-6-nitro-1,3-
benzothiazole (6, 10.0 g, 46.59 mmol) and DABCO (748 mg,
6.99 mmol) in MeCN (1000 mL). The reaction mixture was
stirred at room temperature for 24 h. Excess cyanide was
quenched by the addition of an aqueous FeCl3 solution (0.3 M,
30 mL). The reaction mixture was diluted with H2O (470 mL)
and extracted with EtOAc (3 × 400 mL). The organic layers
were combined, washed with brine (100 mL), dried with
Na2SO4, filtered, and concentrated in vacuo to give a yellow
solid. The crude product was loaded onto a short plug of silica
gel, flushed through with CHCl3 (ca. 1 L) and concentrated in
vacuo to give 13 as a white solid (7.89 g, 38.44 mmol, 83%). A
sample of 13 was crystallised from MeOH for melting point
analysis. Rf 0.37 (SiO2, CHCl3); mp 164–165 °C (MeOH); 1H
NMR (500 MHz, CDCl3) δ 8.96 (d, J = 2.1 Hz, 1H, CH-7), 8.52
(dd, J = 9.1, 2.1 Hz, 1H, CH-5), 8.38 (d, J = 9.1 Hz, 1H, CH-4);
13C NMR (125 MHz, CDCl3) δ 155.5 (C-6), 147.4 (C-7a),
141.9 (C-2), 135.7 (C-3a), 126.2 (CH-4), 123.2 (CH-5), 118.7
(CH-7), 112.1 (C≡N).
Acknowledgements
We thank EPSRC for funding (DTA studentship to HAB), Prof.
John Blacker for help with interpretation of calorimetry results
and Dr Martin Fisher and Dr James Murray for initial ACBT
scale up studies.
References
1. White, E. H.; McCapra, F.; Field, G. F. J. Am. Chem. Soc. 1963, 85,
337–343. doi:10.1021/ja00886a019
2. White, E.; Wörther, H. J. Am. Chem. Soc. 1966, 88, 2015–2019.
doi:10.1021/ja00961a030
3. Meroni, G.; Rajabi, M.; Santaniello, E. ARKIVOC 2009, 2009, 265–288.
doi:10.3998/ark.5550190.0010.109
4. Godinat, A.; Budin, G.; Morales, A. R.; Park, H. M.; Sanman, L. E.;
Bogyo, M.; Yu, A.; Stahl, A.; Dubikovskaya, E. A. A Biocompatible
“Split Luciferin” Reaction and Its Application for Non-Invasive
Bioluminescent Imaging of Protease Activity in Living Animals. Current
Protocols in Chemical Biology; Wiley, 2014; Vol. 6, pp 169–189.
doi:10.1002/9780470559277.ch140047
6-Amino-1,3-benzothiazole-2-carbonitrile (8) [21]
5. Godinat, A.; Park, H. M.; Miller, S. C.; Cheng, K.; Hanahan, D.;
Sanman, L. E.; Bogyo, M.; Yu, A.; Nikitin, G. F.; Stahl, A.;
Dubikovskaya, E. A. ACS Chem. Biol. 2013, 8, 987–999.
doi:10.1021/cb3007314
6. Porterfield, W. B.; Jones, K.; McCutcheon, D. C.; Prescher, J.
J. Am. Chem. Soc. 2015, 137, 8656–8659. doi:10.1021/jacs.5b02774
7. McCutcheon, D. C.; Porterfield, W. B.; Prescher, J. Org. Biomol. Chem.
2015, 13, 2117–2121. doi:10.1039/C4OB02529F
Iron powder (68.05 g, 1218.55 mmol) was added to a suspen-
sion of 6-nitro-1,3-benzothiazole-2-carbonitrile (13, 5.00 g,
24.37 mmol) in acetic acid (500 mL). The reaction mixture was
8. White, E. H.; Steinmetz, M. G.; Miano, J. D.; Wildes, P. D.; Morland, R.
J. Am. Chem. Soc. 1980, 102, 3199–3208. doi:10.1021/ja00529a051
2024

Beilstein J. Org. Chem. 2016, 12, 2019–2025.
9. Lang, K.; Chin, J. W. ACS Chem. Biol. 2014, 9, 16–20.
doi:10.1021/cb4009292
10.Ren, H.; Xiao, F.; Zhan, K.; Kim, Y.-P.; Xie, H.; Xia, Z.; Rao, J.
Angew. Chem., Int. Ed. 2009, 48, 9658–9662.
doi:10.1002/anie.200903627
11.Nguyen, D. P.; Elliott, T.; Holt, M.; Muir, T. W.; Chin, J. W.
J. Am. Chem. Soc. 2011, 133, 11418–11421. doi:10.1021/ja203111c
12.Liang, G.; Ren, H.; Rao, J. Nat. Chem. 2010, 2, 54–60.
doi:10.1038/nchem.480
13.Ye, D.; Pandit, P.; Kempen, P.; Lin, J.; Xiong, L.; Sinclair, R.; Rutt, B.;
Rao, J. Bioconjugate Chem. 2014, 25, 1526–1536.
doi:10.1021/bc500254g
14.Takakura, H.; Kojima, R.; Urano, Y.; Terai, T.; Hanaoka, K.; Nagano, T.
Chem. – Asian J. 2011, 6, 1800–1810. doi:10.1002/asia.201000873
15.Wang, P.; Zhang, C.-J.; Chen, G.; Na, Z.; Yao, S. Q.; Sun, H.
Chem. Commun. 2013, 49, 8644–8646. doi:10.1039/c3cc43566k
16.McCutcheon, D. C.; Paley, M. A.; Steinhardt, R. C.; Prescher, J. A.
J. Am. Chem. Soc. 2012, 134, 7607. doi:10.1021/ja301493d
17.Steinhardt, R. C.; O’Neill, J. M.; Rathbun, C. M.; McCutcheon, D. C.;
Paley, M. A.; Prescher, J. A. Chem. – Eur. J. 2016, 22, 3671–3675.
doi:10.1002/chem.201503944
18.The National Institute for Occupational Safety and Health (NIOSH):
‘sulfur monochloride‘, December 4, 2014.
http://www.cdc.gov/niosh/idlh/10025679.html (accessed May 27, 2016).
19.Sviridov, S.; Kodumuru, V.; Liu, S.; Abreo, M.; Winther, M. D.;
Gschwend, H. W.; Kamboj, R.; Sun, S.; Holladay, M. W.; Li, W.; Tu, C.
Piperazine derivatives and their use as therapeutic agents. WO
2005011657 A2, Feb 10, 2005.
20.Katz, L. J. Am. Chem. Soc. 1951, 73, 4007–4010.
doi:10.1021/ja01152a132
21.Brinkman, J. A.; Donnell, A. F.; Kester, R. F.; Qian, Y.; Sarabu, R.;
So, S. Antiviral compounds. WO 2012175581 A1, Dec 27, 2012.
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