Synthetic approaches towards [18F]fluoro-DOG1, a potential radiotracer for the imaging of gastrointestinal stromal tumors

Article abstract of DOI:10.1016/j.tetlet.2018.07.050

Gastrointestinal stromal tumors (GIST) can currently only be identified by invasive biopsy sampling followed by immunohistochemical analysis. It would however be highly advantageous to have a radioligand able to bind the calcium-activated chloride channel

Full text of DOI:10.1016/j.tetlet.2018.07.050

Tetrahedron Letters 59 (2018) 3332–3335
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
1
8
Synthetic approaches towards [ F]fluoro-DOG1, a potential radiotracer
for the imaging of gastrointestinal stromal tumors
a
a
b
c
a,
⇑
Martin Prause , Sabrina Niedermoser , Ralf Schirrmacher , Carmen Wängler , Björn Wängler
a
Molecular Imaging and Radiochemistry, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, Theodor-Kutzer-Ufer
-3, 68167 Mannheim, Germany
Department of Oncology, Division Oncological Imaging, University of Alberta, 11560 University Avenue, Edmonton, AB T6G 1Z2 Canada
1
b
c
Biomedical Imaging, Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, Theodor-Kutzer-Ufer 1-3,
68167 Mannheim, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Gastrointestinal stromal tumors (GIST) can currently only be identified by invasive biopsy sampling fol-
lowed by immunohistochemical analysis. It would however be highly advantageous to have a radioligand
able to bind the calcium-activated chloride channel DOG1, which is specific for GIST, and thus enable the
sensitive, non-invasive and specific functional imaging of the disease by Positron Emission Tomography
Received 7 June 2018
Revised 17 July 2018
Accepted 20 July 2018
Available online 21 July 2018
(
PET). For this purpose, we developed different synthetic strategies towards a 4-phenylthiazole-2-amine-
1
8
based labeling precursor that can be directly reacted with F-fluoride to yield a radiotracer intended to
bind DOG1. Of these, a boronic acid pinacol ester radiolabeling precursor could be efficiently reacted with
Keywords:
DOG1
Gastrointestinal stromal tumors
Fluorine-18
Positron Emission Tomography (PET)
1
8
18
F in a one-step reaction, and the target radioligand [ F]fluoro-DOG1 be obtained in radiochemical
yields of 34.0 ± 11.1% within 85 min overall synthesis time.
Ó 2018 Elsevier Ltd. All rights reserved.
Introduction
as well as specificity for the identification of GIST and are therefore
of great interest for the sensitive and correct diagnosis of this dis-
ease, especially in those malignancies exhibiting no mutations in
KIT [13–15].
Gastrointestinal stromal tumors (GIST) are the most common
mesenchymal tumors [1], and most of them (85–95%) have pri-
mary mutations in the receptor tyrosine kinase KIT (also known
as c-Kit or CD117) and PDGFRA (platelet-derived growth factor
receptor alpha) leading to permanent activation of these kinases
DOG1 (also known as TMEM16A or ANO1) is expressed on
epithelial tissue, including the interstitial cells of Cajal (ICC), which
are supposed to be the origin of GIST [16–18]. It possesses
pacemaker activity in ICCs and is therefore involved in the regula-
tion of peristalsis in the gastrointestinal tract [19] as well as in
regulating transepithelial water transport. This mechanism is also
involved in the mucociliary clearance of the lungs, making DOG1
an interesting target for research on cystic fibrosis, asthma and
other diseases as well [20–22]. In oncology, the ion channel
DOG1 is used as a reliable biomarker in clinical routine for the dis-
tinction between GIST and other sarcoma due to its very high
expression rate in 98% of all GIST biopsies [2,15,23].
Although DOG1-specific antibodies for immunohistochemistry
exist and show high sensitivity and specificity for the target
protein [13–15], they bind to an intracellular domain of the ion
channel. This limits their usefulness as imaging agents in an
in vivo imaging setting and results in the demand for an ideally
small molecule radiotracer being able to address DOG1.
[
2,3]. In 2002, the FDA approved the tyrosine kinase inhibitor Ima-
tinib for the treatment of GIST, which prolonged the medium sur-
vival of patients suffering from metastatic GIST from 19 months
without treatment to over 50 months with Imatinib treatment
[
4–8]. The high specificity of Imatinib towards KIT causes only
minor side effects and results in good treatment results as long
as no mutations occur [9–12]. GIST is commonly identified via
biopsy sampling and subsequent immunohistochemical and/or
genetic analysis. For immunohistochemical analysis, antibodies
against KIT and CD34 are usually employed. However, antibodies
against the calcium-activated chloride channel DOG1 (DOG: dis-
covered on GIST) have proven to possess an even higher sensitivity
⇑
Corresponding author.
E-mail addresses: Martin.Prause@medma.uni-heidelberg.de (M. Prause), Sabrina.
Such a radioligand would make burdensome biopsy sampling of
GIST-suspect lesions dispensable as it would enable the correct
diagnosis of the disease via non-invasive in vivo imaging with PET.
Niedermoser@medma.uni-heidelberg.de (S. Niedermoser), schirrma@ualberta.ca
R. Schirrmacher), Carmen.Waengler@medma.uni-heidelberg.de (C. Wängler),
(
Bjoern.Waengler@medma.uni-heidelberg.de (B. Wängler).
https://doi.org/10.1016/j.tetlet.2018.07.050
0
040-4039/Ó 2018 Elsevier Ltd. All rights reserved.

M. Prause et al. / Tetrahedron Letters 59 (2018) 3332–3335
3333
Results and discussion
compounds was shown to be feasible at mild reaction tempera-
tures via triazenes [24–26], forming highly reactive diazonium
ion intermediates in situ upon the addition of trifluoromethansul-
fonic acid [27–29]. Although the labeling precursor could be
obtained in four steps in moderate overall yield (19%), this
approach also resulted in the formation of many by-products dur-
Thus, the aim of this work was to develop a 18F-labeled small
molecule PET radiotracer intended to specifically bind to DOG1
and to allow the efficient in vivo visualization of GIST via PET.
For this purpose, a labeling precursor had to be developed
1
8
18
which enables the efficient, ideally one-step introduction of
F
ing F-labeling and no product formation could be observed using
via nucleophilic substitution and thus provides an efficient synthe-
sis of the target radiotracer.
different labeling conditions (see ESI for details). Another difficulty
using this type of reaction is that the optimization of reaction
parameters is not easy since by-products can readily be formed
with every compound in the reaction mixture, e.g. solvents
[27,28], and considerable amounts of volatile [ F]HF are generated
under the acidic reaction conditions, further decreasing the achiev-
able radiochemical yields.
As a biologically active scaffold for the target radioligand, a
4
-phenylthiazole-2-amine core structure (Fig. 1) was chosen as
1
8
Namkung and co-workers were able to show by high-through-
put-screening of different compound classes that this substance
class is best suited to give highly potent activators, as well as inhi-
bitors, of DOG1 [21,22].
Thus, we finally decided to change the synthetic strategy to the
copper-catalyzed radiofluorination of aryl boronic acid esters. This
promising radiofluorination pathway is applicable to electron-poor
as well as electron-rich precursor compounds and is known for
its high selectivity and relatively mild reaction conditions for
A particular challenge in developing a 18F-labeled 4-phenylthi-
azole-2-amine derivative was the position that the radiolabel had
1
to be introduced into. The R position is – despite the unfavorably
high electron density – the best opportunity for the introduction of
1
8
18
[
F]fluoride. Previously it was shown that the introduction of a 4-
F-introduction [30,31].
1
fluorophenyl substituent in the R position is well tolerated with
regard to the binding characteristics compared with the lead struc-
ture, whereas F-introduction in the R position resulted in a con-
siderably decreased biological activity (Fig. 1A) [22].
The target boronic acid pinacol ester (BPin) precursor 3 was
obtained within three steps in good overall yield (37%, Scheme 1).
The synthesis started with the bromination of 4-acetylphenyl-
boronic acid pinacol ester at the alpha position with N-bromosuc-
1
8
3
In a first attempt, we synthesized a nitro precursor (see ESI for
details) of the target substance (Fig. 1C) over two steps in good
overall yield (64%). In the following radiosynthesis, no reaction of
the precursor molecule with [18F]fluoride could be observed at
lower temperatures of 80–100 °C. A possible explanation for this
is the high electron density of the aromatic system the nitro leav-
ing group is attached to which is caused by the 2-aminothiazole
ring. Thus, harsher reactions conditions were necessary which
resulted - despite the optimization of several reaction parameters
such as solvent, temperature, base and precursor concentrations
cinimide. The
a-bromoketone 1 was obtained after column
chromatography and subsequent recrystallization in good yield
1
13
(67%). The H and C NMR spectra of the product showed keto-
enol tautomerism and fast deuterium-hydrogen exchange and con-
firmed the identity of 1.
a-Bromoketone 1 was then reacted with
N-benzylthiourea in a Hantzsch’ thiazole synthesis, giving 2-
aminothiazole 2 in high yield (79%). The last step of the boronic
acid pinacol ester precursor synthesis was the formation of the
amide bond by condensation of 2-aminothiazole 2 and 3,4,5-
trimethoxybenzoyl chloride, giving the radiolabeling precursor
BPin-DOG1 (3) in good yield (70%) and excellent purity (>97%).
Noteworthy is that the boronic acid ester formed a reversible,
pH-dependent equilibrium with the respective borate complex in
aqueous solution which was observable by analytical HPLC and is
in accordance with earlier observations [32]. The non-radioactive
(
see ESI for details) - in significant decomposition of the precursor
18
before a considerable incorporation of [ F]fluoride could be
achieved. As the precursor decomposition occurred faster than
1
8
the incorporation of [ F]fluoride, no product could be isolated.
In a second attempt, we synthesized a triazene precursor (see
ESI for details) since the 18F-radiolabeling of electron rich aromatic
19
reference compound [ F]fluoro-DOG1 (5) was obtained in an
Figure 1. General structure of DOG1 activators based on the 4-phenylthiazole-2-amine scaffold (A), structural lead showing high potency in terms of DOG1 binding (B) and
the target 18F-labeled DOG1 radioligand (C).
Scheme 1. Schematic depiction of the reaction pathway towards the radiolabeling precursor boronic acid pinacol ester (DOG1-BPin, 3). Reagents and conditions:
(
(
a) N-bromosuccinimide (1.1 eq., 5.5 mmol), p-toluene sulfonic acid (1.1 eq., 5.5 mmol), MeCN (50 mL), 50 °C, 3 h (67%); (b) N-benzylthiourea (1.0 eq., 2.3 mmol), MeCN
6 mL + 0.017% TFA), 80 °C, 2 h (79%); (c) 3,4,5-trimethoxybenzoyl chloride (2.0 eq., 2.8 mmol), pyridine (8.0 eq., 11.3 mmol), toluene (10 mL), 110 °C, 8 h (70%).

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334
M. Prause et al. / Tetrahedron Letters 59 (2018) 3332–3335
Scheme 2. Schematic depiction of the reaction pathway towards the cold reference compound F-DOG1, 5). Reagents and conditions: (a) 2-Bromo-1-(4-fluorophenyl)ethan-1-
one (2 mmol), N-benzylthiourea (1.0 eq., 2 mmol), EtOH (8 mL), 80 °C, 30 min (quant.); (b) 3,4,5-trimethoxybenzoyl chloride (4 eq., 1 mmol), pyridine (8 eq., 2 mmol), toluene
(
5 mL), 110 °C, 5 h (89%).
Noteworthy, higher amounts of potassium carbonate resulted in
degradation of the copper catalyst during the reaction.
1
8
For the F-introduction, equimolar amounts of the catalyst
tetrakis(pyridine)copper(II) triflate (20 mmol) and the BPin precur-
sor 3 (20 mmol) were found to be ideal since the catalyst is subject
to decomposition processes under the radiolabeling conditions
applied. As described by Preshlock and co-workers [27], the solvent
plays an important role for this reaction type, an observation
which we were able to confirm. For example, using DMF, a [18F]
fluoride incorporation of only 36% could be achieved, whereas in
DMA, an incorporation rate of 74 ± 8% (n = 10) was found under
otherwise identical reaction conditions.
Scheme 3. Schematic depiction of the radiosynthesis of 18F-DOG1 ([18F]5).
1
8
Reagents and conditions: 3 (11.7 mg, 20 mmol), [ F]KF/K2.2.2 (16.7 mmol), K
6.02 mmol), K CO (0.76 mmol), [Cu(OTf) (py) ] (13.6 mg, 20 mmol), DMA (400 mL),
20 °C, 20 min, RCY: 34.0 ± 11.1%.
2 2 4
C O
(
2
3
2
4
1
The product [18F]5 was purified via semipreparative radio-
HPLC, trapped on a C18 cartridge and eluted with 90% ethanol
(containing 0.125 mg/mL ascorbic acid as stabilizing agent). The
ethanolic product solution was diluted with saline to a final EtOH
concentration of <10% (v/v) and the product was obtained in
excellent radiochemical purity (Fig. 2), radiochemical yields of
34.0 ± 11.1% and molar activities of 8.2 ± 3.0 GBq/mmol at the end
of synthesis within 85 min (n = 7).
excellent overall yield (89%) over two steps via Hantzsch’ thiazole
synthesis, first reacting 2-bromo-1-(4-fluorophenyl)ethan-1-one
and N-benzylthiourea followed by the condensation of intermedi-
ate (4) with 3,4,5-trimethoxybenzoyl chloride analogously to the
reaction pathway to the BPin precursor 3 (see ESI for details)
(Scheme 2).
The radiofluorination of 3 (Scheme 3) to product [18F]5 was
Thus, although all three reaction pathways towards [18F]5
should be able to generate the product by nucleophilic substitution,
performed according to the protocol described by Preshlock and
co-workers for electron-rich compounds [30].
1
8
[
F]5 could only by obtained using the BPin precursor approach.
Although the radiosynthesis of [ F]5 proceeded efficiently
1
8
18
[
F]fluoride was eluted from the QMA cartridge using an opti-
1
8
mized F-elution cocktail consisting of K2.2.2, potassium carbon-
ate and potassium oxalate in MeCN : H O (8 : 2, v/v, 1 mL).
under the optimized conditions using the BPin precursor, giving
the product in good RCYs, the achieved molar activity (determined
2
8
7
6
5
4
3
2
1
00
00
00
00
00
00
00
00
0
0
1
2
3
4
5
6
time [min]
Figure 2. Analytical radio-HPLC chromatograms of the purified radiotracer [18F]5 and of the reference compound 5. Black: radioactive signal of [18F]5, red: corresponding UV
signal of [18F]5 at 210 nm, blue: UV signal of reference compound 5 at 210 nm. Gradient: 0–1 min: 0–30% MeCN, 1–4 min: 30–70% MeCN, 4–5 min: 70–100% MeCN, 5–6 min:
1
00% MeCN.

M. Prause et al. / Tetrahedron Letters 59 (2018) 3332–3335
3335
by analytical radio-HPLC by the area of the obtained UV signal of
References
1
8
the carrier of [ F]5 at 214 nm wavelength and a standard curve
of 5) was rather low. As the semipreparative radio-HPLC purifica-
tion of the product proceeded well, we have no explanation for this
phenomenon.
[
[
[
1] A.W. Beham, I.-M. Schaefer, P. Schüler, S. Cameron, Ghadimi B. Michael, Int. J.
Colorectal Dis. 27 (2011) 689–700.
2] Z.A. Kamar, A.A.F. Sedky, O.H. Nada, N.M. Abd Raboh, N.M. El-Mishad, Egypt. J.
Pathol. 34 (2014) 89–95.
3] M. Linch, J. Claus, C. Benson, OncoTargets Ther. 6 (2013) 1011–1023.
Next, the stability and lipophilicity of [18F]5 were evaluated.
F]5 was demonstrated to be stable in a phosphate buffered solu-
[18
[4] C.D. Blanke, G.D. Demetri, M. von Mehren, M.C. Heinrich, B. Eisenberg, J.A.
Fletcher, C.L. Corless, C.D.M. Fletcher, P.J. Roberts, D. Heinz, E. Wehre, Z.
Nikolova, H. Joensuu, J. Clin. Oncol. 26 (2008) 620–625.
[5] C.D. Blanke, C. Rankin, G.D. Demetri, C.W. Ryan, M. von Mehren, R.S. Benjamin,
A.K. Raymond, V.H.C. Bramwell, L.H. Baker, R.G. Maki, M. Tanaka, J.R. Hecht, M.
C. Heinrich, C.D.M. Fletcher, J.J. Crowley, E.C. Borden, J. Clin. Oncol. 26 (2008)
tion (1 mM, pH 7.0–7.4) for 2 h at ambient temperature. In terms of
lipophilicity/hydrophilicity, the distribution coefficient log
evaluated in 1-octanol/phosphate buffer (0.05 M, pH 7.4)) was
D
(
determined to be 2.51 ± 0.13 (n = 6). In addition to the stability test
performed in the phosphate buffered solution, we also evaluated
the stability of the radioligand in blood sera of different origin. In
human serum, the radiotracer showed excellent stability (>99%)
after 2 h at 37 °C. In contrast, [ F]5 decomposed rather quickly
in murine and rat serum, resulting in only 16.5% and 3.4% intact
tracer after 2 h at 37 °C, respectively (Fig. S1). Due to the high sta-
bility of the radiotracer in human serum, [18F]5 could nevertheless
be interesting for human GIST imaging. The next steps for in vitro
evaluation of the potential of the newly developed radiotracer for
human application in PET imaging could involve molecular model-
ing of the interaction of [18F]5 with DOG1 as well as evaluation of
the binding specificity of the radioligand to the target protein.
6
26–632.
[6] J. Call, C.D. Walentas, J.C. Eickhoff, N. Scherzer, BMC Cancer (2012) 12.
[
[
7] U. De Giorgi, J. Verweij, Mol. Cancer Ther. 4 (2005) 495–501.
8] J.S. Gold, S.M. van der Zwan, M. Gonen, R.G. Maki, S. Singer, M.F. Brennan, C.R.
Antonescu, R.P. De Matteo, Ann. Surg. Oncol. 14 (2007) 134–142.
[9] C.R. Antonescu, P. Besmer, T.H. Guo, K. Arkun, G. Hom, B. Koryotowski, M.A.
Leversha, P.D. Jeffrey, D. Desantis, S. Singer, M.F. Brennan, R.G. Maki, R.P.
DeMatteo, Clin. Cancer Res. 11 (2005) 4182–4190.
10] M. Debiec-Rychter, J. Cools, H. Dumez, R. Sciot, M. Stul, N. Mentens, H. Vranckx,
B. Wasag, H. Prenen, J. Roesel, A. Hagemeijer, A. Van Oosterom, P. Marynen,
Gastroenterology 128 (2005) 270–279.
18
[
[
11] A.W. Gramza, C.L. Corless, M.C. Heinrich, Clin. Cancer Res. 15 (2009) 7510–
7
518.
[12] J. Verweij, P.G. Casali, J. Zalcberg, A. LeCesne, P. Reichardt, J.Y. Blay, R. Issels, A.
van Oosterom, P.C.W. Hogendoorn, M. Van Glabbeke, R. Bertulli, I. Judson, Grp
ESTBS, Grp IS, A.G. Tria, Lancet 364 (2004) 1127–1134.
13] I. Espinosa, C.-H. Lee, M.K. Kim, B.-T. Rouse, S. Subramanian, K. Montgomery, S.
Varma, C.L. Corless, M.C. Heinrich, K.S. Smith, Z. Wang, B. Rubin, T.O. Nielsen, R.
S. Seitz, D.T. Ross, R.B. West, M.L. Cleary, M. van de Rijn, Am. J. Surg. Pathol. 32
[
Conclusion
(
2008) 210–218.
[
14] B. Liegl, J.L. Hornick, C.L. Corless, C.D.M. Fletcher, Am. J. Surg. Pathol. 33 (2009)
437–446.
15] R.B. West, C.L. Corless, X. Chen, B.P. Rubin, S. Subramanian, K. Montgomery, S.
Zhu, C.A. Ball, T.O. Nielsen, R. Patel, J.R. Goldblum, P.O. Brown, M.C. Heinrich,
M. van de Rijn, Am. J. Pathol. 165 (2004) 107–113.
Herein, we report the first example of a 18F-labeled small mole-
cule radiotracer intended for the non-invasive PET imaging of
DOG1. This included the organic syntheses of different radiolabel-
ing precursors as well as the development and optimization of the
[
[16] S.J. Hwang, P.J.A. Blair, F.C. Britton, K.E. O’Driscoll, G. Hennig, Y.R. Bayguinov, J.
R. Rock, B.D. Harfe, K.M. Sanders, S.M. Ward, J. Physiol. 587 (2009) 4887–4904.
[
[
1
8
corresponding F-radiosyntheses.
17] B. Manoury, A. Tamuleviciute, P. Tammaro, J. Physiol. 588 (2010) 2305–2314.
18] Y.D. Yang, H. Cho, J.Y. Koo, M.H. Tak, Y. Cho, W.-S. Shim, S.P. Park, J. Lee, B. Lee,
B.-M. Kim, R. Raouf, Y.K. Shin, U. Oh, Nature 455 (2008) 1210–1215.
Among the developed and tested approaches (using a nitro, a
triazene and an aryl boronic acid ester as precursors for subse-
1
8
quent F-radiolabeling), only the aryl boronic acid ester precursor
[19] K.M. Sanders, M.H. Zhu, F. Britton, S.D. Koh, S.M. Ward, Exp. Physiol. 97 (2012)
200–206.
[
18
enabled the efficient one-step incorporation of
F, thus
20] R. Tarran, B. Button, R.C. Boucher, Ann. Rev. Physiol. 68 (2006) 543–561.
21] W. Namkung, P.W. Phuan, A.S. Verkman, J. Biol. Chem. 286 (2011) 2365–2374.
demonstrating the superiority of this pathway for nucleophilic
[
1
8
F-radiolabeling of electron-rich precursor molecules over other,
[22] W. Namkung, Z. Yao, W.E. Finkbeiner, A.S. Verkman, FASEB J. 25 (2011) 4048–
062.
[
4
more common approaches.
23] S. Simon, F. Grabellus, L. Ferrera, L. Galietta, B. Schwindenhammer, T.
Muhlenberg, G. Taeger, G. Eilers, J. Treckmann, F. Breitenbuecher, M. Schuler,
T. Taguchi, J.A. Fletcher, S. Bauer, Cancer Res. 73 (2013) 3661–3670.
24] T.J. Tewson, M.J. Welch, ChemComm (1979) 1149–1150.
Acknowledgements
[
[
25] J.S. Ng, J.A. Katzenellenbogen, M.R. Kilbourn, J. Org. Chem. 46 (1981) 2520–
This work was supported by the European Community’s 7th
Framework Programme [FP7/2007-2013; grant agreement number
2528.
[26] M.N. Rosenfeld, D.A. Widdowson, ChemComm (1979) 914–916.
[
[
27] T. Pages, B.R. Langlois, J. Fluorine Chem. 107 (2001) 321–327.
28] T. Pages, B.R. Langlois, D. Le Bars, P. Landais, J. Fluorine Chem. 107 (2001) 329–
6
02306] and the German Federal Ministry of Education and
Research (BMBF) [funding code number 13GW0091E]. Further,
the authors thank Dr. W. Spahl (LMU, Munich) for ESI mass spec-
trometry measurements.
335.
[29] P.J. Riss, S. Kuschel, F.I. Aigbirhio, Tet. Lett. 53 (2012) 1717–1719.
[
30] S. Preshlock, S. Calderwood, S. Verhoog, M. Tredwell, M. Huiban, A. Hienzsch, S.
Gruber, T.C. Wilson, N.J. Taylor, T. Cailly, M. Schedler, T.L. Collier, J. Passchier, R.
Smits, J. Mollitor, A. Hoepping, M. Mueller, C. Genicot, J. Mercier, V.
Gouverneur, ChemComm 52 (2016) 8361–8364.
31] M. Tredwell, S.M. Preshlock, N.J. Taylor, S. Gruber, M. Huiban, J. Passchier, J.
Mercier, C. Genicot, V. Gouverneur, Angew. Chem. Int. Edit. 53 (2014) 7751–
A. Supplementary data
[
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.tetlet.2018.07.050.
7755.
[32] B.P. Carrow, J.F. Hartwig, J. Am. Chem. Soc. 133 (2011) 2116–2119.