2,3-Diaryl-substituted indole based COX-2 inhibitors as leads for imaging tracer development

Article abstract of DOI:10.1039/c4ra05650g

A series of 2,3-diaryl-substituted indoles containing a fluorine or methoxy group was synthesized via Fischer indole synthesis, McMurry cyclization, or Bischler-Moehlau reaction to identify potential leads for positron emission tomography (PET) radiotracer development as well as for optical imaging. All 2,3-diaryl-substituted indoles possess autofluorescent properties with an emission maximum in a range of 443-492 nm, which is acceptable for biological studies in vitro and, in part, in vivo. The molecular structure of compounds 3a and 3j was confirmed by X-ray crystal structure analysis. COX inhibitory activity was evaluated by a fluorescence-based and enzyme immunoassay-based assay. Redox activity of all target compounds was also determined. All synthesized 2,3-diaryl-substituted indoles are inhibitors of COX-2 enzyme in the low micromolar range. Compounds 3e, 3f, 3g and 3m displayed a 30-40% inhibition of COX-2 at 0.1 μM concentration while compounds 3f and 3g also exhibited COX-1 inhibitory activity. Various compounds like 3g showed substantial antioxidative potential (R_DIENE = 2.85, R_HAVA = 1.98), an effect that was most measurable with methoxy-substituted compounds. With respect to PET radiotracer synthesis, OMe-containing compound 3j was selected as a promising candidate for carbon-11 labeling, and F-containing compound 3m as a lead for the development of a fluorine-18 labeled derivative. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra05650g

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2,3-Diaryl-substituted indole based COX-2
inhibitors as leads for imaging tracer
Cite this: RSC Adv., 2014, 4, 38726
development†‡
Markus Laube,ab Christoph Tondera,ab Sai Kiran Sharma,c Nicole Bechmann,ab
Franz-Jacob Pietzsch,ad Arne Pigorsch,e Martin Kockerling,e Frank Wuest,c
¨
ab
a
*
*
and Torsten Kniess
Jens Pietzsch
A series of 2,3-diaryl-substituted indoles containing a fluorine or methoxy group was synthesized via Fischer
indole synthesis, McMurry cyclization, or Bischler–Mohlau reaction to identify potential leads for positron
¨
emission tomography (PET) radiotracer development as well as for optical imaging. All 2,3-diaryl-
substituted indoles possess autofluorescent properties with an emission maximum in a range of 443–
492 nm, which is acceptable for biological studies in vitro and, in part, in vivo. The molecular structure of
compounds 3a and 3j was confirmed by X-ray crystal structure analysis. COX inhibitory activity was
evaluated by a fluorescence-based and enzyme immunoassay-based assay. Redox activity of all target
compounds was also determined. All synthesized 2,3-diaryl-substituted indoles are inhibitors of COX-2
enzyme in the low micromolar range. Compounds 3e, 3f, 3g and 3m displayed a 30–40% inhibition of
COX-2 at 0.1 mM concentration while compounds 3f and 3g also exhibited COX-1 inhibitory activity.
Various compounds like 3g showed substantial antioxidative potential (RDIENE ¼ 2.85, RHAVA ¼ 1.98), an
effect that was most measurable with methoxy-substituted compounds. With respect to PET radiotracer
synthesis, OMe-containing compound 3j was selected as a promising candidate for carbon-11 labeling,
and F-containing compound 3m as a lead for the development of a fluorine-18 labeled derivative.
Received 12th June 2014
Accepted 13th August 2014
DOI: 10.1039/c4ra05650g
www.rsc.org/advances
leads to the biosynthesis of prostaglandins and thromboxanes
as mediators of a variety of important physiological functions
Introduction
like vasodilation, regulation of inammatory response, and
platelet aggregation. While COX-1 is constitutively expressed,
COX-2 is nearly absent under normal physiological conditions.
However, various inammatory stimuli induce COX-2 expres-
sion and function. In a clinical picture, overexpression of COX-2
leads to high levels of eicosanoids resulting in acute inam-
mation, pain and fever. Hence, inammation is treated by
inhibition of COX-2. First generation of COX inhibitors are
termed as non-steroidal anti-inammatory drugs (NSAIDs),
which target both COX-1 and COX-2. Typical examples of
NSAIDs represent ibuprofen, indomethacin, and diclofenac.
COX-1 inhibition through NSAIDs can cause undesirable side
effects such as peptic ulcer or stomach bleeding. To overcome
these drawbacks, a second generation of compounds, termed as
COXIBs, was developed. COXIBs like celecoxib, valdecoxib, and
rofecoxib are characterized by high COX-2/COX-1 selectivity
prole. These selective COX-2 inhibitors bind competitively to
the cyclooxygenase active site of COX-2, but not COX-1, and
hence block COX-2-mediated prostaglandin synthesis. Later it
became clear that complete inhibition of COX-2 also blocks the
formation of vasodilators like prostaglandin I2 and, conse-
quently, resulted in a shi from the COX pathway to the lip-
oxygenase pathway. This shi was accompanied with increased
Since the discovery of the cyclooxygenase-2 (COX-2) isoenzyme
in the early 1990s it has been well accepted that this isoform of
cyclooxygenases plays an essential role in a number of various
(patho)physiological processes such as chronic inammatory
diseases, neurodegenerative disorders, and cancer. Both iso-
forms (COX-1 and COX-2) catalyze the conversion of arach-
idonic acid into prostaglandin H2 (PgH2), which subsequently
aDepartment Radiopharmaceutical and Chemical Biology, Institute of
Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf,
01328 Dresden, Germany. E-mail: t.kniess@hzdr.de; j.pietzsch@hzdr.de; Tel:
+493512602760; +493512602622
b
¨
Department of Chemistry and Food Chemistry, Technische Universitat Dresden,
01062 Dresden, Germany
cDepartment of Oncology, Cross Cancer Institute, University of Alberta, Edmonton,
Alberta, Canada T6G 1Z2
dCentre for Translational Bone, Joint, and So Tissue Research, Medical Faculty and
¨
University Hospital, Technische Universitat, 01307 Dresden, Germany
eDepartment of Inorganic Solid State Chemistry, Institute of Chemistry, University of
Rostock, Albert-Einstein-Str. 3a, 18059 Rostock, Germany
† This work is dedicated to the 75th birthday of Prof. Dr Bernd Johannsen.
‡ Electronic supplementary information (ESI) available. CCDC 963601, 963602,
963609 and 963572. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c4ra05650g
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cardiovascular risk. This matter of facts demonstrates that the assess efficacy of novel drugs based on their molecular mode of
search of new lead structures with high COX-2 selectivity but action. We are interested in radionuclide-guided functional
without cardiac or other side effects is an ongoing research molecular imaging of COX-2 by means of positron emission
eld.1
tomography (PET). The use of selective COX-2 inhibitors as PET
Most COXIBs are characterized by a central ve membered radiotracers for molecular imaging of COX-2 would provide
heterocyclic core structure (pyrrole, thiazole, oxazole, furan and valuable information on tumor prognosis, metastasis, or
imidazole) with two adjacent aromatic rings bearing a methyl- therapy response.23–25
sulfonyl or aminosulfonyl group as COX-2 pharmacophore. This
Up to now, a number of radiotracers basing on clinically
basic concept was extended for the design of new lead struc- used COXIBs like celecoxib and valdecoxib have been
tures possessing a six-membered or bicyclic heterocyclic core.2,3 described;26,27 as well as novel compounds with hitherto
The indole motif is a classical pharmacophore present in the unknown pharmacological prole were synthesized, radio-
non-selective COX inhibitor indomethacin which was as core- labeled and evaluated in vitro and in vivo.24 Some recent studies
structure for the design of various selective COX-2 inhibitors with 18F-labeled pyrazole28 or azulene29 based COX-2 inhibitors
reported by Black et al. (Scheme 1).4 Later, novel N-substituted have successfully demonstrated a specic uptake of radio-
indole carboxyclic acid esters,5 and various 3,6- and 2,6-disub- labeled COX-2 probes in inammatory lesions as well as in
stituted indole derivatives were described as potent and selec- xenograed tumors and gave the proof of principle of targeting
tive COX-2 inhibitors.6–8 Particularly high potent and selective COX-2 in vivo with radiotracers. However, despite of promising
COX-2 inhibitors as 2,3-diaryl substituted indoles were reported pre-clinical results a suitable COX-2 specic radiotracer for
by Hu and Guo et al.9–11 Moreover, quantitative structure– human use is still absent, a fact that is stimulating radiophar-
activity relationship (SAR) analysis on benzyl-substituted maceutical research in that eld.
indoles demonstrated a correlation of the C-2 and C-3 substi-
In our efforts to develop radiolabeled COX-2 inhibitors we
tution pattern with the found high COX-2 inhibitory and selected 2,3-diaryl-substituted indole as lead structure, and we
selectivity prole.12 More recently, Kaur et al.13 discussed recently reported on the radiosynthesis and radio-
various N-1 and C-3 substituted indole Schiff bases as selective pharmacological evaluation of 3-(4-[18F]uorophenyl)-2-(4-
COX-2 inhibitors. A selection of potent and selective COX-2 methylsulfonylphenyl)-1H-indole as novel 18F-radiotracer for
inhibitors based on indoles is depicted in Scheme 1.
PET imaging of COX-2. Although the radiotracer displayed a
Over the last years, COX-2 has also been associated with the promising in vitro and in vivo stability prole, no uptake into
development and progression of cancer. Elevated COX-2 levels COX-2-expressing human HT-29 tumor xenogras transplanted
are found in many human epithelium-derived malignancies. in NMRI nu/nu mice could be observed.30 We concluded that
This nding correlates with aggressiveness, metastatic and the COX-2 inhibitory activity and selectivity prole of the
invasive potential of tumors.14,15 Exemplarily, human malignant compound in vivo was not suitable for radiotracer applications.
melanoma, a non-epithelial tumor that is characterized by a However, potential of varying the C-2/C-3 substitution pattern in
marked inammatory response and high metastatic potential, the indole scaffold as well as the innovative and highly effective
has been shown to overexpress COX-2,16,17 and COX-2 was radiosynthesis via McMurry cyclization prompted us to
proposed as a prognostic marker in melanomata and also in synthesize a comprehensive series of 2,3-diaryl-substituted
various epithelial tumors.18–21 Consequently, tumor-promoting indoles containing uorine or methoxy groups as leads for the
inammation has been recognized as an emerging hallmark of development of respective 11C- and 18F-radiolabeled PET
cancer, and is a current therapeutic target for the development radiotracers. The motivation of the present work was to identify
of anticancer drugs.22
from a pool of 2,3-diaryl-substituted indoles one or two candi-
Current anticancer drug design and discovery increasingly dates with nanomolar affinity towards COX-2, most favorable
includes non-invasive molecular imaging methodologies to COX-2/COX-1 selectivity, having uorine or methoxy
Scheme 1 Potent COX-2 inhibitors containing an indole motif.
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substituents suitable to be replaced by uorine-18 or carbon-11 and p-uorobenzonitrile for the synthesis of the methoxy
respectively. derivative. Apparently under the used reaction conditions,
In this paper we describe the synthesis of thirteen 2,3-diaryl- boron trichloride caused a demethylation of p-anisidine so that
substituted indoles. We in detail assessed their chemical and the hydroxy derivative 1c was formed. Since similar but slightly
uorescent spectroscopic characteristics. All compounds were milder reaction conditions were used by Dinsmore and Berg-
screened for their COX-2 inhibitory activity and selectivity man35 for the synthesis of 2-amino-30-uoro-5-methoxy-benzo-
prole using a uorescence-based and an enzyme immuno- phenone, we also applied this procedure for the synthesis of the
assay-based COX assay. Many indoles possess antioxidative methoxy derivative but no product was formed. We assumed
properties which is an important parameter for their exerted that the methoxy-derivative was not directly accessible by this
¨
biological activity prole. Antioxidative potential of novel indole route and we applied the Bischler–Mohlau synthesis as shown
compounds was assessed and compared with prominent below to get access to a 5-methoxy-substituted indole. As a
indole-based antioxidant melatonin.31,32 Concluding SAR second note, 1c was not stable in alkaline solution during work-
studies assisted us in the selection of suitable candidates for up so that for isolation of 1c neutralization of the reaction
radiotracer development.
mixture with NaHCO3 and extraction before column chroma-
tography was applied. The amino substituted benzophenones
1a–1d were converted into the N-(2-benzoyl)phenyl-benzamides
2a–2g by reaction with the corresponding methylsulfonyl- or
aminosulfonyl-substituted benzoyl chloride and Et3N in THF in
62–96% yield. In the nal step, the benzamides 2a–2g were
cyclized under McMurry conditions to yield the 2,3-diaryl-
substituted indoles 3a–2g in 21–85%.
Results and discussion
Chemistry
The synthetic routes towards 2,3-diaryl-substituted indoles are
shown in Schemes 2 and 4, both follow in general literature
procedures which however have been improved and modied
by us for higher yields and products purity.
From 3-(uorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-indole
(3a) crystals suitable for X-ray structure analysis could be
obtained by slow evaporation of a solution of 3a in ethyl acetate
at room temperature. This unambiguously conrmed the
molecular structure of the compound (Fig. 1, for detailed results
including the X-ray structure analysis of the intermediate 2c see
the Experimental section and the ESI‡). Interestingly, the plane
of the methylsulfonyl-substituted phenyl ring in compound 3a
is only slightly twisted out of the plane of the indole core
(dihedral angle: 26.74ꢀ) in comparison to the uoro-substituted
phenyl ring (dihedral angle: 52.89ꢀ). This gives evidence for the
preferential interaction of the electron-rich indole system with
the electron-decient methylsulfonyl-substituted phenyl ring.
The synthesis of the sulfonyl acetamide derivative 3h was
accomplished by the reaction of compound 3g with acetyl
The synthesis of 2-sulfonylphenyl-3-phenyl-1H-indoles 3a–3g
(Scheme 2) followed the synthetic strategy of Hu et al.10
involving a McMurry cyclization as the nal step to build up the
indole core structure. For this approach the benzophenone
derivatives 1a–1d served as starting materials. Compounds 1a
and 1d were obtained in 25% and 21% yield, respectively, from
N-tosyl protected anthranilic acid by conversion into the cor-
responding acid chloride with PCl5 and subsequent reaction
with uorobenzene or anisole under Friedel–Cras conditions
followed by detosylation using a mixture of acetic acid and
perchloric acid.30,33 Compounds 1b and 1c were synthesized in
one step by Friedel–Cras acylation using BCl3 to achieve
selective ortho-benzoylation relative to the amino residue.34
Following this route by starting from p-toluidine and p-uoro-
benzonitrile, 1b was obtained in 21% yield. Comparable to that,
the yield for 1c was 23% but it should be noted that the hydroxy
derivative 1c was obtained unexpectedly by using p-anisidine
ꢀ
chloride in acetic acid at 90 C in 70% yield (Scheme 3).
The 3-sulfonylphenyl-2-phenyl indoles 3i–3l were synthe-
sized by application of a Fischer indole synthesis following the
Scheme 2 Synthesis of 2-sulfonylphenyl substituted indoles 3a–3g by McMurry cyclization. Reagents and conditions: (a) for 1a and 1d: (i) PCl5,
50 ꢀC, (ii) AlCl3, 80 ꢀC (1a) and 5–10 ꢀC (1d), (iii) HClO4/CH3COOH, 100 ꢀC; (b) for 1b–1c: (i) BCl3, AlCl3, toluene, reflux, (ii) 2 M HCl, reflux; (c) 4-
(R2O2S)C6H4COCl, NEt3, THF, rt; (d) TiCl4, Zn, THF, 65 ꢀC.
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pure chlorosulfuric acid at temperatures about ꢁ78 ꢀC rising
slowly to room temperature and subsequent reaction of the
crude product in ethyl acetate with aqueous ammonia solution
what gave the desired product 5d in 34% yield.38 Compounds
5a–5d were then converted into the appropriate 3-sulfonyl-
phenyl-2-phenyl-1H-indoles 3i–3l by Fischer indole synthesis
using phenylhydrazine and BF3$Et2O as Lewis acid. However,
the low yields about 20% prompted us to test the synthesis of 3j
exemplarily by the McMurry pathway. For that, 2-amino-40-
(methylsulfonyl)benzophenone was allowed to react with 4-
methoxybenzoyl chloride to form N-[2-(4-(methylsulfonyl)-
benzoyl)phenyl]-4-methoxybenzamide which was subsequently
and without isolation of the intermediate reacted under
Fig. 1 Molecular structure of compound 3a in the crystal (ORTEP plot:
displacement thermal ellipsoids are drawn at 50% probability level).
McMurry conditions in
a
two step/one-pot procedure
(Scheme 5) as described by us39 for the syntheses of 2-carba-
boranyl-substituted indoles. This approach gave 3j in slightly
better 32% yield over two steps. Crystals suitable for X-ray
structure analysis could be obtained from a solution of 3j in
ethyl acetate–petroleum ether 50 : 50 by slow evaporation at
room temperature which unambiguously revealed the molec-
ular structure of 3j (Fig. 2, for detailed results see the Experi-
mental section and the ESI‡). Interestingly, in this compound
the molecular geometry differs in comparison to 3a. The plane
of the phenyl ring in 2-position is substantially twisted out of
the plane of the indole-core (dihedral angle: 52.89ꢀ), likely due
to the electron-rich methoxy-substituent in para-position.
Consistently although not as stabilized as in compound 3a, the
methylsulfonyl-substituted phenyl ring is again less twisted out
of the plane of the indole core (dihedral angle: 39.62ꢀ)
compared to the neighboring phenyl ring.
Scheme 3 Synthesis of compound 3h by acetylation. Reagents and
conditions: (a) CH3COCl, CH3COOH, reflux.
synthetic strategy of Guo et al.11 (Scheme 4). For that purpose, 1-
phenyl-2-sulfonylphenyl ethanones 5a–5d served as key inter-
mediates. The methylsulfonyl-substituted compounds 5a–5c
were obtained starting from 4-(methylsulfonyl)phenylacetic
acid by conversion to its acid chloride with SOCl2 and subse-
quent reaction under Friedel–Cras conditions with ethox-
ybenzene, anisole, and uorobenzene, respectively (for detailed
results of X-ray crystal structure analysis of the intermediate 5c
see the Experimental section and the ESI‡).36 The sulfamoyl-
substituted ethanone 5d was synthesized in two steps: 1-(4-
uorophenyl)-2-phenylethanone (4) was prepared in a yield of
51% by Friedel–Cras reaction of phenylacetic acid chloride
and uorobenzene as described by Singh et al.37 followed by a
chlorosulfonylation and ammonolysis at the para-position of
the 2-phenyl ring. In detail this was performed by utilization of
As shown before, demethylation during the BCl3-mediated
Friedel–Cras acylation hampered the synthesis of
a
Scheme
5 Synthesis of compound 3j by McMurry cyclization.
Reagents and conditions: (i) NEt3, THF, rt, (ii) TiCl4, Zn, THF, 65 ꢀC.
Scheme 4 Synthesis of 3-sulfonyl-substituted indoles 3i–3l by Fischer indole synthesis. Reagents and conditions: (a) for 5a–5c: (i) SOCl2, (ii)
R2C6H5, AlCl3, 15–20 ꢀC; (b) AlCl3, fluorobenzene, DCM, rt; (c) for 5d: (i) ClSO3H, (ii) NH3(aq.), ethyl acetate, rt; (d) BF3$Et2O, CH3COOH, 130 ꢀC.
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Fig. 2 Molecular Structure of compound 3j in the crystal (ORTEP plot:
displacement thermal ellipsoids are drawn at 50% probability level).
Fig. 3 The chemical shifts of 3m in 1H-NMR (DMSO-d6, 400 MHz).
The arrows (grey) indicate NOESY cross peaks.
corresponding 5-methoxy-substituted indole 3m via the
McMurry-cyclization pathway. Thus, we decided to utilize
cyclooxygenase-2 by confocal laser induced cryouorescence
microscopy in human melanoma cells using the uorescent
indole 3g.17 With this in mind and because one of the COX
assays used by us is based on the detection of a uorescent dye
with an excitation wavelength of 530–540 nm, we initially eval-
uated the optical properties of the compounds 3a–3m. For that
purpose, the UV-vis absorbance and uorescence excitation as
well as emission spectra were recorded (Table 1). Fig. 4 shows
the normalized uorescence excitation and emission spectra of
compounds 3j and 3m as examples for the indole based COX-2
inhibitors. In summary, all indoles are characterized by an
excitation maxima at l > 300 nm, a large stokes shi of 106–160
nm and a uorescence emission maxima in the range of 444–
492 nm. In comparison to 3g which was already used to visu-
alize COX-2 in vitro, all tested indoles showed very similar
uorescence excitation and emission spectra so that they might
be used for similar applications. Of note, 2-(4-sulfonylphenyl)-
substituted indoles like 3m have an excitation maximum
between 338–354 nm whereas 3-(4-sulfonylphenyl)-substituted
indoles like 3j showed two bands with nearly similar intensity at
300–304 nm and 329–334 nm. As observed by X-ray crystallog-
raphy for 3a and 3j, this also indicates that the interaction of the
phenyl rings with the indole core differs substantially in
dependence of the position of the sulfonyl moiety. Finally, no
uorescence signal is observed by excitation at 530 nm allowing
¨
another type of ring closure reaction, the Bischler–Mohlau
cyclization for the synthesis of the methoxy-substituted deriva-
¨
tive 3m (Scheme 6). The Bischler–Mohlau cyclisation has been
used for the reaction of various methoxy-substituted anilines
with 2-bromo-ketone derivatives.40,41 In our case, 1-(4-uoro-
phenyl)-2-sulfonylphenyl ethanone (5c) was brominated to give
the precursor 5e in 52% yield.42 Then 5e was allowed to react
with p-anisidine at 170 ꢀC in ethanol under pressure to form 3m
¨
in 37% yield. The regioselectivity of the Bischler–Mohlau reac-
tion is hardly predictable, so the corresponding two isomers
with opposite substituent position could be formed (Scheme 6).
Actually, during the workup a set of by-products was observed.
However, we isolated one main product for its identication
NOESY experiments were performed. Finally by the help of this
technique the molecular structure of the 2-[4-(methylsulfonyl)-
phenyl]-3-(4-uorophenyl)-5-methoxy-1H-indole
(3m)
was
conrmed (Fig. 3, NOESY spectra are included in the ESI‡).
Optical properties
Optical properties have been previously reported for 2,3-
diphenyl-1H-indoles and some derivatives were found to be
highly uorescent with emission maxima in the range of 410–
475 nm.43–47 We recently presented the visualization of
Scheme 6 Synthesis of 3m by Bischler–Mohlau cyclization. Reagents and conditions: (a) Br2, benzoyl peroxide (cat.), CCl4/CHCl3, 78 ꢀC; (b)
¨
EtOH, 170 ꢀC.
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Table 1 Optical properties of 2,3-diaryl-substituted indoles
Absorption labs
Excitation maximum
lexc [nm]
Emission maximum
lem [nm]
[nm] (3 [104 Mꢁ1 cmꢁ1])
R1
F
F
F
F
F
OCH3
OCH3
OCH3
SO2CH3
SO2CH3
SO2CH3
SO2NH2
F
R2
R3
H
H
CH3
CH3
OH
H
H
H
H
H
3a
3b
3c
3d
3e
3f
3g
3h
3i
SO2CH3
SO2NH2
SO2CH3
SO2NH2
SO2NH2
SO2CH3
SO2NH2
SO2NHAc
OCH2CH3
OCH3
236 (2.24), 249 (2.03), 336 (1.61)
253 (2.08), 328 (1.70)
239 (5.62), 339 (2.03)
238 (4.06), 333 (2.10)
236 (4.33), 341 (2.05)
237 (2.87), 256 (2.54), 332 (1.60)
237 (3.71), 325 (1.61)
256 (2.98), 323 (1.80)
238 (1.97), 300 (1.73), 340sh (0.93)
238 (3.18), 300 (2.35), 344 (1.23)
238 (4.47), 296 (2.17), 337 (1.27)
237 (3.85), 296 (2.06), 329sh (1.36)
237 (4.52), 344 (2.54)
343
338
345
339
345
354
345
339
303
344
300
303
349
454
450
475
451
481
492
482
452
451
450
444
443
484
3j
3k
3l
F
F
H
H
OCH3
3m
SO2CH3
at the le part of Table 2. In all determinations celecoxib
was used as internal standard and for COX-1/COX-2
selectivity giving at least no difference between our results and
literature data.
Generally we found that all synthesized 2,3-diaryl-
substituted indoles are inhibitors of COX-2 in the low micro-
molar or sub-micromolar level. This is in accordance with
previous literature ndings from other groups describing 3a,
3b, 3f, 3g and 3k as selective COX-2-inhibitors.9,10,48 The results
from the assay used by us, showed that in 1 mM concentration
3a, 3b, 3e, 3g, 3i, 3j did inhibit more than 50% of COX-2 activity
and four derivatives (3e, 3f, 3g, 3m) inhibited even better,
between 30–40% in the 0.1 mM level. With view on regioisomers
it is difficult to predict any signicant impact from the position
of the methylsulfonyl or the aminosulfonyl group to COX-2
affinity. For the regioisomers 3a/3k a higher activity is observed
with the methylsulfone on the 2-phenyl ring, but for the pair 3b/
3l values in the same range were found. Furthermore, there is
no clear tendency if the methylsulfone substituent is superior to
the sulfonamide because by direct comparison of two deriva-
tives having the identical core but different sulfonyl substitu-
ents similar activities to COX-2 were found as demonstrated by
the pairs 3a/3b, 3c/3d and 3f/3g. Compounds with a uorine-
Fig. 4 Fluorescence excitation and emission spectra of compound 3j
and 3m. Emission spectra were acquired with an excitation wavelength
of 344 nm and 349 nm for 3j and 3m, respectively.
the determination of COX-inhibitory activity with the uores-
cence-based COX assay.
Biological evaluation
Fluorescence-based COX-assay. The in vitro COX inhibitory substituent generally show a lower inhibition of COX-2 because
activity of the indoles 3a–3m and celecoxib as a reference was in all investigated compounds its replacement by a methoxy
evaluated with a uorescence-based COX assay (“COX Fluores- group lead to increased inhibition as has been demonstrated
cent Inhibitor Screening Assay Kit”, Item no. 700100, Cayman for the cases 3a/3f, 3b/3g, and 3k/3j. Acetylation of the sulfon-
Chemical, Ann Arbor, USA) that utilizes the COX-mediated amide resulted likewise in loss of activity as visible in 3g versus
reduction of PGG2 to PGH2 to oxidize 10-acetyl-3,7-dihydroxy- 3h, a similar reduction is observed by replacement of a methoxy
phenoxazine to resorun. This highly uorescent compound by an ethoxy group (3i versus 3j). Notable is the impact of the
can easily be analyzed with an excitation wavelength of 530– substituent in 5-position of the indole system (R3) on the COX-2
540 nm and emission wavelength of 585–595 nm. The results of inhibitory activity. By direct comparison of a hydrogen (3a), a
the COX inhibitory activity of the 2,3-diaryl-substituted indoles methyl- (3c) and a methoxy-group (3m) the latter gives the best
3a–3m from this uorescence-based COX-assay are summarized inhibition to COX-2 whereas the methyl substituent lead to
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Table 2 COX-inhibitory and antioxidant properties of 2,3-diphenyl-1H-indole derivatives 3a–3ma
Fluorescence-based COX-assay
% inhibition
EIA-based COX-assay
% inhibition
Redox-activity
COX-1
COX-2
COX-1
COX-2
R1
R2
R3
10 mM
1 mM
1 mM
0.1 mM
RDIENE
RHAVA
10 mM
1 mM
1 mM
0.1 mM
3a
3b
3c
3d
3e
3f
3g
3h
3i
F
F
F
F
SO2CH3
SO2NH2
SO2CH3
SO2NH2
SO2NH2
SO2CH3
SO2NH2
SO2NHAc
OCH2CH3
OCH3
H
H
CH3
CH3
OH
H
H
H
H
H
38
60
19
45
40
72
90
51
30
31
20
25
6
28
15
32
20
12
42
85
39
23
20
10
20
8
60
55
20
30
54
49
69
39
65
81
30
50
40
88
32
28
20
6
1.21
1.32
0.98
0.98
1.48
1.73
2.85
1.55
1.02
1.54
1.19
1.28
1.78
0.97
1.71
3.36
1.25
1.36
0.98
0.99
1.53
1.84
1.98
1.32
1.17
1.29
1.09
1.18
1.71
0.96
1.52
2.88
n.i.
51
n.d.
n.d.
50
41
98
86
n.d.
n.i.
n.d.
n.i.
37
10
n.i.
92
93
n.d.
n.d.
100
90
98
80
n.d.
61
n.d.
96
60
82
10
30
40
32
12
12
28
5
20
42
80
19
F
100
n.i.
70
100
n.i.
63
OCH3
OCH3
OCH3
SO2CH3
SO2CH3
SO2CH3
SO2NH2
F
80
n.d.
3j
3k
3l
n.i.
32
F
F
H
H
OCH3
n.i.
50
n.d.
17
72
100
65
3m
SO2CH3
100
100
20
Celecoxib
Melatonin
Quercetin
30
53
28
39
n.d.
30
15
a
n.i. no inhibition. n.d. not determined.
decreased affinity. Even a hydroxyl group at this position is well results are shown in the middle part of Table 2. The redox
tolerated (3e) and results in 40% inhibition at 0.1 mM level. activity is expressed as RDIENE and RHAVA ratios indicating effects
In summary in the series of thirteen 2,3-diaryl substituted of the compounds tested on either lipid oxidation or protein
indoles the COX-2 inhibitory activity of the known compounds oxidation. In both approaches R values (RDIENE or RHAVA) higher
was conrmed and, compared to 3a, three even more potent than 1 indicate antioxidant activity while a R value of about 1
derivatives, 3e, 3g and 3m could be identied.
stands for no effect. For comparison, celecoxib, the indole
Furthermore, we determined the inhibition prole of all hormone melatonin, and the polyphenol quercetin were
compounds to COX-1 and found that most of the derivatives studied. As expected, celecoxib did not exhibit any redox activity
lacked COX-1 inhibitory activity as expected. However the in the model systems used. In contrast, both quercetin and
methoxy-substituted compounds 3f–3g attracted our attention melatonin showed a substantial antioxidative activity both on
because 3f–3g appeared to be moderate inhibitors of COX-1, lipid and protein oxidation. Interestingly, all tested compounds
and especially 3g was even a more potent COX-1 than COX-2 with an unsubstituted 5-position in the indole heterocycle (R3 ¼
inhibitor. The last nding was in harsh contrast to the litera- H) showed a signicant antioxidative action that was mostly
ture, and we suspected false positive results since the uores- comparable to melatonin. Noteworthy, compound 3g showed
cence-based COX-assay is inuenced by antioxidants as noted the highest antioxidative potential which was signicantly
by the manufacturer. Particularly, the indole heterocycle is a well- higher than that of melatonin but less active than quercetin.
characterized antioxidant pharmacophore49,50 and it had to be This antioxidative action of indole compounds is hypothesized
considered that possibly antioxidative properties of 3a–3m to follow a mechanism discussed in detail for other indole
interfered with the assay and caused these deviations. This antioxidants like 2-phenylindoles.53 The proposed mechanism
hypothesis was supported by our rst ndings in parallel in vitro essentially would allow the presence of substituents like a
experiments that indole 3g was able to inhibit not only the methoxy group or a hydroxyl group at 5-position. Gozzo et al.
formation of PGE2 but also the formation of 8-iso-PGF2a gener- demonstrated that the presence of 5-methoxy or 5-hydroxyl
ated via non-enzymatic oxidation aer the exposure of endothe- groups in melatonin analogues induces benecial effects on
lial cells in both monolayer and organo-type aortic ring models to their antioxidative activity.54 In the present study the introduc-
ionizing radiation.51,52 Thus, we decided to evaluate the antioxi- tion of a methoxy or a hydroxyl group at position 5 also resulted
dant capacity of the indole based COX-2 inhibitors 3a–3m.
in higher antioxidant activity as is demonstrated for 3e/3b as
Redox activity–structure relationships. The antioxidant well as 3m/3a, respectively. In contrast, the introduction of a
potency of the COX-inhibitors was investigated using a low- methyl group at position 5 substantially attenuates antioxidant
density lipoprotein (LDL) copper-/iron-peroxidation model. The activity as demonstrated for 3c/3a and 3d/3b. These data further
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support the suggested mechanism. Furthermore, a benecial slightly more potent than 3j. A similar statement can be made
effect also could be observed when methoxy groups were for the position of the aminosulfonyl and methylsulfonyl
introduced in para-position of 2- or 3-phenyl rings (3f/3a; 3g/3b). substituents because 3a/3b were both highly selective inhibitors
Overall, the presence of methoxy groups favored antioxidant of COX-2 and within the pair 3f/3g only 3f was a selective COX-2
potential of indole derivatives with a slightly higher antioxidant inhibitor. In contrast to the uorescence-based assay, also no
activity for methoxy in para-position of the 3-phenyl ring clear tendency was observed for the comparison of uorine with
compared to para-position of the 2-phenyl ring (3f/3j). Of methoxy substituted compounds since in both groups very
interest, the sulfonamide moiety increases the antioxidant selective as well as unselective inhibitors were found.
activity when compared with the methylsulfone as demon-
strated for 3b/3a, 3g/3f, and 3l/3k. This tendency also was
observed in a study of Walter and colleagues who reported
Conclusions
prooxidative effects related to the sulfone agents rofecoxib and The objective of this work was to develop and characterize in
etoricoxib, respectively, compared to redox-neutral effects of detail a series of 2,3-diaryl-substituted indoles selectively
sulfonamides celecoxib and valdecoxib.55 Regarding protein inhibiting cyclooxygenase-2, and preferably bearing a uorine
oxidation the relationship between antioxidative capacity and or methoxy-substituent, in order to gain a pool of versatile
different substituents in indole compounds are consistent with compounds promising for 18F- or 11C-PET radiotracer develop-
those observed for lipid oxidation.
ment. The position of the uoro and methoxy group – on the 2-
EIA-based COX-assay. Hence, some of the indoles exhibited aryl respectively on the 3-aryl substituent – and of the appro-
substantial antioxidative properties and in order to avoid false priate methylsulfonyl or aminosulfonyl moiety should be
positive results issued from the uorescence-based COX-assay altered with view on the resulting affinity towards COX-1 and
we decided to subject selected compounds (3a, 3b, 3e–3h, 3m) COX-2.
regarding their COX inhibitory activity to a second assay. For
For that purpose thirteen derivatives of 2,3-diaryl-substituted
that purpose we chose an enzyme immunoassay (EIA) based indoles have been synthesized by applying different ring closure
COX assay (“COX Inhibitor Screening Assay Kit”, Item no. reactions, as Fischer-indole synthesis, McMurry cyclization or
¨
560131, Cayman Chemical, Ann Arbor, USA). Since this assay is Bischler–Mohlau cyclization. Among them the McMurry cycli-
determining the COX-activity via EIA based detection of pros- zation proofed to be an excellent approach due to its regiose-
taglandins and not via a redox-coupled mechanism, it seemed lectivity, modularity and the good yields that were obtained. The
promising to eliminate the false positive results from our spectroscopic and uorescent properties of all 2,3-diaryl-
experiments. The results are displayed in the right part of Table substituted indoles revealed that all the compounds are uo-
2. On a rst look at the compound with the highest antioxidative rescent with emission maxima acceptable for biological inves-
potential 3g, it turned out that this substance is, although not tigations in vitro and partly in vivo.
very selective, a slightly better COX-2 than COX-1 inhibitor with
To determine the COX inhibition activity two separate in
98% compared to 70% inhibition at the 1 mM level, respectively. vitro assays, one uorescence-based and one enzyme immuno-
A similar result was found for 3f which showed in the uores- assay-based, were used. This exceptional approach was essential
cence-based assay only moderate selectivity to COX-2 but in the because it is known that each individual assay can have its
enzyme immunoassay-based COX-assay no inhibition of COX-1 limitations regarding the chemical and physical properties of
and 90% inhibition of COX-2 at the 1 mM level. These ndings the subjected compounds. In our case we found that the indoles
support the hypothesis that the results of the uorescence- do not only show uorescence, some of them possess a
based assay for 2,3-diaryl-substituted indoles concerning COX-2 considerable redox activity which is known to interact with the
are affected by their redox activity. In contrast, for compounds uorescence-based assay, and as a consequence false results
3e, 3j and 3m that were already found to be COX-2 selective with may be occur. Indeed, for 3g that showed the highest redox
the uorescence-based assay, no such tendency was observed activity (RDIENE ¼ 2.85, RHAVA ¼ 1.98) of the tested indoles and
although these compounds had comparable R values with 3f. In appeared in the uorescence-based assay as a more potent
an additional experiment we subjected melatonin that is also a COX-1 than COX-2 inhibitor, the immunoassay-based determi-
5-methoxy-substituted indole derivative and an antioxidant to nation revealed that this compound was slightly more affine
both COX assays. As a result we found only a weak inhibition of towards COX-2.
both enzymes by melatonin with a slightly higher activity but
Summarizing the in vitro behavior of all 2,3-diaryl-
comparable selectivity in the uorescence-based assay. These substituted indoles it can be stated that all compounds are
nding let us conclude that the redox activity at this level inhibiting COX-2, but with a slight gradation. Some of them
(RDIENE and RHAVA value about 1.7) has only minor effect on the turned out to be unselective COX inhibitors in one (3e, 3f)
result of the assays. Consistently, for compounds having a respectively both (3g, 3h) of the assays and are hence unsuitable
RDIENE and RHAVA value of about 1.3, that means only slightly candidates for radiotracer development. Surprisingly the
redox active compounds (3a, 3b, 3l), the inhibitory activity to introduction of a methyl-substituent to the 5-position of the
COX-1 and COX-2 is more or less comparable for both assays.
indole lead to a remarkable loss of activity, hence the derivatives
As in the uorescence-based assay, no clear tendency for the 3c and 3d have to be excluded as well. Since a high selectivity for
regioisomers was found since the regioisomers 3a/3k as well as COX-2 is an important criterion for potential radiotracers, 3a,
3f/3j showed similar COX-2/COX-1 selectivity although 3f seems 3b, 3j, 3l and 3m emerged as most promising candidates in the
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following order of increasing inhibitory activity based on 0.1 mM
concentrations: 3b # 3l < 3a # 3j < 3m.
Spectroscopic and uorescent properties were determined at
the “Synergy 4 Hybrid Multi-Mode Microplate Reader” from
The radiolabeled COX-2 inhibitor [18F]3a corresponding to BioTek Company. The samples were dissolved in an appropriate
the non-radioactive compound 3a has been recently evaluated amount of DMSO to give a 10 mM stock solution. To 20 mL of
by ourselves and showed no substantial tumor accumulation, this stock solution was added 80 mL DMSO, 100 mL TWEEN 20
due to fast elimination and insufficient affinity to COX-2.30 The and 9800 mL PBS to yield a 20 mM test solution for the
COX-2 inhibitors 3b and 3l both have a uoro-substituent measurement of the extinction coefficient. Using BioCell 1 cm
making them to suitable candidates for radiotracer develop- Quartz Vessels from BioTec Company, the extinction coeffi-
ment with uorine-18. However, they possess an aminosulfonyl cients were determined from the absorption spectra. All
group that is known to interact in vivo with carbonic anhydrases extinction coefficients are given in 3/dm3 molꢁ1 cmꢁ1 at the
in the erythrocytes, resulting in a slow blood clearance. This was specied wavelength in nm. The uorescence excitation and
demonstrated for 125I-radiolabeled celecoxib derivatives56 and emission spectra were determined using a 100 mM solution of
should be kept in mind for the development of a 18F-radio- the test compound in a solution of 1% DMSO and 1% TWEEN
labeled analogue of 3b and 3l. The derivative 3j does not contain 20 in PBS. In both experiments, baseline correction was done
a uorine substituent. Hence, radiolabeling with carbon-11 using a solution of 1% DMSO and 1% TWEEN 20 in PBS.
could be a possible approach as demonstrated by Tanaka et al.57
but the low metabolic stability of the methoxy group in vivo is
always limiting the radiotracer application.
Syntheses
Finally, 3m turned out as the most promising candidate for
2-Amino-40-uorobenzophenone (1a). Synthesis and charac-
radiotracer development since this compound was potent and terization of compound 1a was recently reported.39 The
COX-2 selective in both assays showing furthermore in the EIA- compound was used as starting material for the following steps.
based method a complete inhibition of COX-2 in the 0.1 mM
2-Amino-40-uoro-5-methylbenzophenone (1b). Synthesis
level. The radiolabeling of 3m with uorine-18 can be per- and characterization of compound 1b was recently reported.39
formed by using the McMurry cyclization30 as has been The compound was used as starting material for the following
demonstrated for [18F]3a because the Bischler–Mohlau reaction steps.
¨
as labeling strategy seems to be ineffective for the introduction
of the radioisotope.
2-Amino-40-uoro-5-hydroxybenzophenone (1c). Reaction
was carried out under Schlenk-conditions. Under nitrogen
atmosphere, p-anisidine (1.982 g, 16.1 mmol) and a solution of
4-uorobenzonitrile (2.918 g, 24.1 mmol) in toluene (18 mL) was
added at 0 ꢀC to 1 M boron trichloride in toluene (18 mL,
18 mmol). Then freshly sublimated anhydrous aluminum
Experimental protocols
All commercial reagents and solvents were used without further chloride (2.43 g, 18.2 mmol) was added and the mixture was
purication unless otherwise specied. Flash chromatography heated to reux for 12 h. Aerwards, the mixture was cooled to
was conducted using MERCK Silica Gel (mesh size 40–63 mm) 0 ꢀC, water (1 mL) and 10% HCl (18 mL) were slowly added and
and RP-18 silica gel (Lichroprep RP-18, 25–40 mm). Thin-layer the mixture was heated to reux for 2 h again. The raw product
chromatography (TLC) was performed on Merck silica gel F-254 was isolated by vacuum ltration aer cooling the solution to
aluminum plates and Alugram RP-18W/UV254. Visualization 0 ꢀC. To the raw material was added water (10 mL) and a small
was carried out using UV (254 nm/366 nm). Analytical HPLC amount of NaHCO3 and this mixture was extracted with ethyl
analysis were carried out with a Luna C18 column (250 ꢂ acetate (3 ꢂ 50 mL). The organic phases were combined,
4.6 mm, 5 mm, Phenomenex) using an isocratic eluent (aceto- washed with saturated NaHCO3 (10 mL) and water (2 ꢂ 10 mL)
nitrile/water + 0.1% TFA 70/30) by a gradient pump L2500 and dried over Na2SO4. The crude, dried product was puried by
(Merck, Hitachi) with a ow rate of 1 mL minꢁ1. The products dry column vacuum chromatography (DCVC, petroleum ether–
were monitored by an UV detector L4500 (Merck, Hitachi) at 254 ethyl acetate 90 : 10 / 50 : 50). Compound 1c was obtained as a
nm. All nal compounds displayed $95% purity as determined yellow-brown solid (854 mg, 23%). mp 150–153 ꢀC; Rf 0.51
by analytical HPLC. Mass spectra were obtained on a Quattro/ (petroleum ether–ethyl acetate 50 : 50); 1H NMR (400 MHz;
LC mass spectrometer (MICROMASS) by electrospray ioniza- DMSO-d6) d ppm: 6.50 (2H, br s, NH2), 6.68 (1H, d, 4J ¼ 2.6 Hz,
3
3
tion. Melting points were determined on a Galen III (Cambridge
H
phenyl-6), 6.74 (1H, d, J ¼ 8.8 Hz, Hphenyl-3), 6.86 (1H, dd, J ¼
Instruments) melting points apparatus (Leica, Vienna, Austria) 8.8 Hz, 4J ¼ 2.7 Hz, Hphenyl-4), 7.34 (2H, t, 3J ¼ 8.8 Hz, HF-phenyl-3 /5 ),
0
0
3
4
0
0
and are uncorrected. Nuclear magnetic resonance spectra 7.64 (2H, dd, J ¼ 8.8 Hz, J ¼ 5.7 Hz, HF-phenyl-2 /6 ), 8.65 (1H, s,
were recorded on a Varian Inova-400. NMR spectra were refer- OH); 13C NMR (101 MHz; DMSO-d6) d ppm: 115.2 (d, 2J ¼ 22 Hz),
enced to the residual solvent shis for 1H and 13C, and to CFCl3 116.5, 117.2, 118.2, 124.0, 131.2 (d, 3J ¼ 9 Hz), 136.5 (d, 4J ¼ 3 Hz),
1
for 19F spectra as internal standard. For some compounds, 145.3, 146.0, 163.5 (d, J ¼ 249 Hz), 196.1; 19F NMR (376 MHz;
isotope effects on C NMR chemical shis58 were observed in DMSO-d6) d ppm: ꢁ109.7; m/z ESI-MS (ES+) 232 [M + H]+ (100%).
13
acetone-d6 which were caused by hydrogen/deuterium exchange
2-Amino-40-methoxybenzophenone (1d). Synthesis of 1d was
while measurement. The signals with deuterium isotope carried out in analogy to a literature procedure.10 Briey, 2-(4-
shis are indicated and the range from minimal to maximal methyl-phenylsulphonylamino)benzoic acid (4.38 g, 15 mmol)
value is given.
was suspended in dry anisole (20 mL) and PCl5 (5.57 g, 17.1
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mmol) was added at room temperature to the suspension in Hz, 3J ¼ 6.8 Hz, 4J ¼ 1.8 Hz, Hphenyl), 7.47–7.54 (3H, m, Hphenyl
/
portions. The mixture was heated at 50 ꢀC for 30 min and cooled SO2NH2), 7.63–7.71 (2H, m, Hphenyl), 7.76 (2H, dd, 3J 8.8 Hz, 4J ¼
to 0 ꢀC by an ice–salt mixture. Anhydrous AlCl3 (8.7 g, 65.4 5.6 Hz, HF-phenyl-2/6), 7.81 (2H, d, 3J ¼ 8.7 Hz, Hphenyl), 7.88 (2H,
mmol) was added in portions, while the temperature was kept d, 3J ¼ 8.6 Hz, Hphenyl), 10.75 (1H, br s, NH); 13C NMR (101 MHz;
between ꢁ3 C and 5 C. The mixture was stirred for 6 h at 5– DMSO-d6) d ppm: 115.3 (d, 2J ¼ 22 Hz), 124.5, 125.0, 125.6,
ꢀ
ꢀ
3
4
10 ꢀC and at room temperature overnight. Aer cooling at 0 ꢀC, 128.0, 130.1, 131.4, 132.0, 132.4 (d, J ¼ 9 Hz), 133.8 (d, J ¼ 3
ice-cold 1 M HCl (80 mL) was added carefully and the suspen- Hz), 136.1, 136.9, 146.7, 164.4, 164.6 (d, 1J ¼ 251 Hz), 193.8; 19
F
sion was treated with diethyl ether (50 mL), the aqueous phase NMR (376 MHz; DMSO-d6) d ppm: ꢁ107.2; ESI-MS (ES+) m/z 421
was separated and extracted with diethyl ether once more (2 ꢂ [M + Na]+ (100%), 399 [M + H]+ (69), 462 [M + Na + CH3CN]+ (69).
25 mL). The white precipitate from the aqueous phase was
N-[2-(4-Fluorobenzoyl)-4-methylphenyl]-4-(methylsulfonyl)-
ltered, washed with diethyl ether and dried under vacuum to benzamide (2c). Purication of the raw product obtained by
give the intermediate product N-[2-(4-methoxybenzoyl)phenyl]- general procedure A was carried out by column chromatography
4-(methyl)benzenesulfonamide (1.82 g, 4.77 mmol) that was (petroleum ether–ethyl acetate–NEt3 65 : 30 : 5 / 47.5 : 47.5 : 5).
used as raw material for the next step. For deprotection, the Starting from 1b (315 mg, 1.37 mmol) and 4-(methylsulfonyl)
intermediate (1.32 g, 3.46 mmol) was dissolved in a mixture of benzoyl chloride (300 mg, 1.37 mmol), 2c was obtained as pale
ꢀ
perchloric acid (70%, 7 mL) and acetic acid (2.75 mL), heated at yellow crystalline solid (540 mg, 96%). mp 195–197 C; Rf 0.40
100 C and kept for 3 h at this temperature. Aer cooling, the (petroleum ether–ethyl acetate 50 : 50); 1H NMR (400 MHz;
ꢀ
solution was added to crushed ice (50 mL) and neutralized with acetone-d6) d ppm: 2.36 (3H, s, CH3), 3.19 (3H, s, SO2CH3), 7.31
aqueous NH3 (25%). The yellow precipitate was allowed to (2H, t, 3J ¼ 8.8 Hz, HF-phenyl-3/5), 7.48 (1H, d, 4J ¼ 1.7 Hz, Hphenyl),
crystallize and collected by ltration to give 1d as yellow crystals 7.54 (1H, dd, 3J ¼ 8.4 Hz, 4J ¼ 2.1 Hz, Hphenyl), 7.87 (2H, dd, 3J ¼
(0.52 g, 21%). mp 75–77 ꢀC (lit.,59 75–76 ꢀC); Rf 0.57 (petroleum 8.9 Hz, 4J ¼ 5.5 Hz, HF-phenyl-2/6), 8.12 (2H, d, 3J ¼ 8.4 Hz, Hphenyl),
ether–ethyl acetate 30 : 70); 1H NMR (400 MHz; DMSO-d6) d 8.16 (2H, d, 3J ¼ 8.5 Hz, Hphenyl), 8.44 (1H, dd, 3J ¼ 8.4 Hz, 4J ¼ 3.8
3
ppm: 3.83 (3H, s, OCH3), 6.51 (1H, t, J ¼ 7.4 Hz, Harom), 6.79– Hz, Hphenyl), 11.29 (1H, br s, NH); 13C NMR (101 MHz; acetone-d6)
3
2
6.86 (3H, m, NH2/Harom), 7.05 (2H, d, J 8.6 Hz, Hanisole), 7.22– d ppm: 20.8, 44.1, 116.2 (d, J ¼ 22 Hz), 123.0*, 126.7*, 128.7,
7.32 (2H, m, Harom), 7.58 (2H, d, 3J ¼ 8.6 Hz, Hanisole); 13C NMR 129.0, 133.6 (d, 3J ¼ 9 Hz), 133.8*, 134.1*, 135.2*, 135.8 (d, 4J ¼ 3
(101 MHz; DMSO-d6) d ppm: 55.4 (CH3O), 113.5, 114.1, 116.8, Hz)*, 137.9*, 140.3*, 145.1*, 164.6*, 166.1 (d, 1J ¼ 252 Hz),
117.2, 131.3, 131.9, 133.3, 133.6, 151.3, 161.8, 196.6 (CO).
198.1*, * deuterium isotope shis were observed in the range of 7
to 112 ppb; 19F NMR (376 MHz; CD3CN) d ppm: ꢁ108.5; ESI-MS
(ESꢁ) m/z 410 ([M ꢁ H]ꢁ, 100%). Crystals suitable for X-ray crys-
tallography were obtained by slow evaporation from ethyl acetate
at room temperature. Detailed results of the single-crystal X-ray
General procedure A for the synthesis of N-(2-benzoylphenyl)
benzamides (2a–2g)
Under nitrogen atmosphere, to a solution of the appropriate 2- structure determination are given below and in the ESI.‡
aminobenzophenone (0.87 mmol) and NEt3 (138 mL) in THF 4-(Aminosulfonyl)-N-[2-(4-uorobenzoyl)-4-methylphenyl]-
(1.8 mL) was added the appropriate 4-sulfonylbenzoyl chloride benzamide (2d). Purication of the raw product obtained by
(0.87 mmol) in THF (1.0 mL). The mixture was stirred at room general procedure A was carried out by column chromatography
temperature for 2 h, ltered and the solid was washed with (petroleum ether–ethyl acetate 50 : 50). Starting from 1b (204
tetrahydrofuran. The ltrate was concentrated to dryness under mg, 0.89 mmol) and 4-sulfamoylbenzoyl chloride (194 mg, 0.89
reduced pressure and further puried as described below.
mmol), 2d was obtained as colorless crystalline solid (252 mg,
N-[2-(4-Fluorobenzoyl)phenyl]-4-(methylsulfonyl)benzamide 69%). mp 230–231 ꢀC; Rf 0.40 (petroleum ether–ethyl acetate
1
(2a). Synthesis and characterization of compound 2a was 50 : 50); H NMR (400 MHz; CD3CN) d ppm: 2.35 (3H, s, CH3),
recently reported.30 Compound 2a was used for the following 5.77 (2H, br s, SO2NH2), 7.24 (2H, t, J ¼ 8.9 Hz, HF-phenyl-3/5),
3
steps.
7.43 (1H, d, 4J ¼ 1.6 Hz, Hphenyl), 7.51 (1H, dd, 3J ¼ 8.4 Hz, 4J ¼
4-(Aminosulfonyl)-N-[2-(4-uorobenzoyl)phenyl]benzamide 1.7 Hz, Hphenyl), 7.80 (2H, dd, 3J ¼ 8.9 Hz, 4J ¼ 5.5 Hz, HF-phenyl-2/6),
(2b). Purication of the raw product obtained by general 7.96–8.04 (4H, m, 3J ¼ 8.7 Hz, Hphenyl), 8.32 (1H, d, J ¼ 8.4 Hz,
3
procedure A was carried out by column chromatography
H
phenyl), 10.91 (1H, br s, NH); 13C NMR (101 MHz; CD3CN) d ppm:
(petroleum ether–ethyl acetate 50 : 50). Starting from 1a 21.4, 116.9 (d, 2J ¼ 22 Hz), 123.8, 127.7, 128.1, 129.5, 134.3 (d, 3J ¼
(258 mg, 1.20 mmol) and 4-sulfamoylbenzoyl chloride (262 mg, 9 Hz), 134.6, 135.2, 135.9, 136.4 (d, 4J ¼ 3 Hz), 138.2, 139.8, 147.7,
1.20 mmol), 2b was obtained as pale yellow solid (300 mg, 62%). 165.6, 166.9 (d, 1J ¼ 237 Hz), 199.1; 19F NMR (376 MHz; CD3CN) d
mp 202–203 ꢀC (lit.,11 204–206 ꢀC); Rf 0.36 (petroleum ether– ppm: ꢁ108.6; ESI-MS (ESꢁ) m/z 411 [M ꢁ H]ꢁ (100%).
1
ethyl acetate 50/50); H NMR (400 MHz; CD3CN) d ppm: 5.78
4-(Aminosulfonyl)-N-[2-(4-uorobenzoyl)-4-hydroxyphenyl]-
3
3
(2H, br s, SO2NH2), 7.20–7.30 (3H, m, J ¼ 8.9 Hz, J ¼ 7.9 Hz, benzamide (2e). Purication of the raw product obtained by
H
phenyl), 7.63 (1H, dd, 3J ¼ 7.9 Hz, 4J ¼ 1.5 Hz, Hphenyl), 7.70 (1H, general procedure A was carried out by column chromatography
t, 3J ¼ 7.9 Hz, 4J ¼ 1.7 Hz, Hphenyl), 7.80 (2H, dd, 3J ¼ 8.9 Hz, 4J ¼ (petroleum ether–ethyl acetate 50 : 50 / 0 : 100). Starting from
5.5 Hz, HF-phenyl-2/6), 8.00 (2H, d, 3J ¼ 8.6 Hz, Hphenyl), 8.05 (2H, 1c (250 mg, 1.08 mmol) and 4-sulfamoylbenzoyl chloride
3
3
4
d, J ¼ 8.8 Hz, Hphenyl), 8.50 (1H, dd, J ¼ 8.3 Hz, J ¼ 0.9 Hz, (224 mg, 1.02 mmol), 2e was obtained as yellow solid (275 mg,
1
H
phenyl), 11.15 (1H, br s, NH); H NMR (400 MHz; DMSO-d6) d 65%). mp 248–250 ꢀC; Rf 0.56 (ethyl acetate); 1H NMR (400 MHz;
ppm: 7.30 (2H, t, J ¼ 8.9 Hz, HF-phenyl-3/5), 7.37 (1H, t, J ¼ 7.8 acetone-d6) d ppm: 6.75 (2H, br s, SO2NH2), 7.07 (1H, 4J ¼ 2.9 Hz,
3
3
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Hphenyl), 7.18 (1H, dd, 3J ¼ 8.9 Hz, 4J ¼ 2.9 Hz, Hphenyl), 7.30 (2H, results of the single-crystal X-ray structure determination are
t, 3J ¼ 8.8 Hz, HF-phenyl-3/5), 7.87 (2H, dd, 3J ¼ 8.9 Hz, 4J ¼ 5.5 Hz, given below and in the ESI.‡
H
F-phenyl-2/6), 7.99–8.05 (4H, m), 8.25 (1H, d, 3J ¼ 8.9 Hz, Hphenyl),
2-[4-(Aminosulfonyl)phenyl]-3-(4-uorophenyl)-1H-indole (3b).
8.68 (1H, s, OH), 10.86 (1H, br s, NH); 13C NMR (101 MHz; Purication of the crude product obtained by general procedure
2
acetone-d6) d ppm: 116.1 (d, J ¼ 22 Hz), 119.2, 121.1, 125.3*, B was carried out by column chromatography (petroleum ether–
127.3*, 128.6, 129.1, 132.0*, 133.5 (d, 3J ¼ 9 Hz), 135.6 (d, 4J ¼ 3 ethyl acetate 50 : 50). Starting from 2b (220 mg, 0.54 mmol), 3b
Hz), 138.9*, 147.8*, 154.2, 164.6, 166.1 (d, 1J ¼ 252 Hz), 197.3, * was obtained as colorless solid (168 mg, 85%). mp 238–239 C
ꢀ
deuterium isotope shis were observed in the range of 11 to 101 (lit.,11 228–230 ꢀC); Rf 0.26 (petroleum ether–ethyl acetate
ppb; 19F NMR (376 MHz; acetone-d6) d ppm: ꢁ108.5; ESI-MS 50 : 50); UV/vis: lmax/nm 253, 328 (3/dm3 molꢁ1 cmꢁ1 20 800,
(ES+) m/z 437 [M + Na]+ (100%), 478 [M + Na + CH3CN]+, (72).
17 000); uorescence: lexc ¼ 338, lem ¼ 450 nm; H NMR (400
1
3
N-[2-(4-Methoxybenzoyl)phenyl]-4-(methylsulfonyl)benzamide MHz; CD3CN) d ppm: 5.68 (2H, br s, SO2NH2), 7.13 (1H, t, J ¼
(2f). Purication of the raw product obtained by general 8.0 Hz, 3J ¼ 7.1 Hz, Hindol), 7.18 (2H, t, 3J ¼ 9.0 Hz, HF-phenyl-3/5),
procedure A was carried out by washing the raw precipitate in 7.26 (1H, t, 3J ¼ 8.3 Hz, 3J ¼ 7.1 Hz, 4J 1.1 Hz, Hindol), 7.39 (2H,
methanol, ltration and drying under vacuum. Starting from 1d dd, 3J ¼ 8.9 Hz, 4J ¼ 5.5 Hz, HF-phenyl-2/6), 7.49–7.54 (2H, m,
(454 mg, 2.0 mmol) and 4-(methylsulfonyl)benzoyl chloride (484
mg, 2.2 mmol), 2f was obtained as pale yellow solid (689 mg,
H
H
indol), 7.59 (2H, d, 3J ¼ 8.7 Hz, Hphenyl), 7.81 (2H, d, 3J ¼ 8.7 Hz,
phenyl), 9.78 (1H, br s, NH); 13C NMR (101 MHz; CD3CN) d ppm:
2
84%). mp 170–171 ꢀC (lit.,11 160–161 ꢀC); Rf 0.25 (petroleum 113.1, 116.4, 117.1 (d, J ¼ 22 Hz), 120.7, 122.1, 124.8, 127.9,
ether–ethyl acetate 50 : 50); 1H NMR (400 MHz; DMSO-d6) d 130.0, 130.1, 132.7 (d, J ¼ 3 Hz), 133.5 (d, J ¼ 8 Hz), 134.1,
4
3
ppm: 3.26 (3H, s, SO2CH3), 3.81 (3H, s, OCH3), 7.01 (2H, d, 3J ¼ 138.0, 138.1, 143.5, 163.4 (d, J ¼ 243 Hz); 19F NMR (376 MHz;
1
8.8 Hz, Hanisole), 7.36 (1H, t, 3J ¼ 7.4 Hz, Harom), 7.48 (1H, d, 3J ¼ CD3CN) d ppm: ꢁ118.0; ESI-MS (ESꢁ) m/z 365 [M ꢁ H]ꢁ (100%).
7.6 Hz, 4J ¼ 1.2 Hz, Harom), 7.64 (1H, t, 3J ¼ 8.0 Hz, 4J ¼ 1.3 Hz,
3-(4-Fluorophenyl)-5-methyl-2-[4-(methylsulfonyl)phenyl]-1H-
H
H
H
arom), 7.69 (2H, d, 3J ¼ 8.8 Hz, Hanisole), 7.73 (1H, d, 3J ¼ 7.9 Hz, indole (3c). Purication of the crude product obtained by
arom), 7.90 (2H, d, 3J ¼ 8.4 Hz, Harom), 8.01 (2H, d, 3J ¼ 8.4 Hz, general procedure B was carried out by column chromatography
arom), 10.77 (1H, s, NH); ESI-MS (ES+) m/z: 432 [M + Na]+ (chloroform–methanol 90 : 10). Starting from 2c (400 mg, 0.97
(100%).
mmol), 3c was obtained as beige solid (221 mg, 60%). mp 239–
4-(Aminosulfonyl)-N-[2-(4-methoxybenzoyl)phenyl]benzamide 242 ꢀC; Rf 0.72 (chloroform–methanol 90 : 10); UV/vis: lmax/nm
(2g). Purication of the raw product obtained by general 239, 339 (3/dm3 molꢁ1 cmꢁ1 56 200, 20 300); uorescence: lexc
¼
procedure A was carried out by column chromatography 345, lem ¼ 476 nm; 1H NMR (400 MHz; acetone-d6) d ppm: 2.40
(petroleum ether–ethyl acetate 30 : 70). Starting from 1d (600 (3H, s, CH3), 3.13 (3H, s, SO2CH3), 7.08 (1H, dd, 3J ¼ 8.4 Hz, 4J ¼
mg, 2.64 mmol) and 4-sulfamoylbenzoyl chloride (640 mg, 2.92 1.4 Hz, Hindol), 7.21 (2H, t, 3J ¼ 8.9 Hz, HF-phenyl-3/5), 7.32 (1H, d,
mmol), 2g was obtained as pale yellow solid (853 mg, 78%). mp 4J ¼ 0.6 Hz, Hindol), 7.38–7.45 (3H, m, J ¼ 8.4 Hz, J ¼ 8.8 Hz,
162–166 ꢀC (lit.,11 173–174 ꢀC); 1H NMR (400 MHz; DMSO-d6) d 4J ¼ 5.5 Hz, 2HF-phenyl/1Hindol), 7.71 (2H, d, 3J ¼ 8.6 Hz, Hphenyl),
ppm: 3.81 (3H, s, OCH3), 7.01 (2H, d, 3J ¼ 8.8 Hz, Hanisole), 7.35 7.89 (2H, d, 3J ¼ 8.6 Hz, Hphenyl), 10.72 (1H, br s, NH); 13C NMR
(1H, t, 3J ¼ 7.6 Hz, 4J ¼ 1.0 Hz, Harom), 7.48 (1H, d, 3J ¼ 7.7 Hz, (101 MHz; acetone-d6) d ppm: 21.6, 44.2, 112.2*, 115.8*, 116.5
4J ¼ 1.5 Hz, Harom), 7.51 (2H, s, NH2), 7.64 (1H, t, 3J ¼ 7.8 Hz, 4J ¼ (d, 2J ¼ 21 Hz), 119.6, 125.8, 128.4, 129.3*, 129.8, 130.3, 132.3 (d,
3
3
1.5 Hz, Harom), 7.69 (2H, d, 3J ¼ 8.8 Hz, Hanisole), 7.74 (1H, d, 3J ¼ 4J ¼ 3 Hz), 132.8 (d, J ¼ 8 Hz), 133.2*, 136.2*, 138.8*, 140.6,
3
7.6 Hz, Harom), 7.82 (2H, d, 3J ¼ 8.5 Hz, Harom), 7.88 (2H, d, 3J ¼ 162.6 (d, 1J ¼ 246 Hz), * deuterium isotope shis were observed
8.4 Hz, Harom), 10.71 (1H, s, NH); 13C NMR (101 MHz; DMSO-d6) in the range of 36 to 143 ppb; 19F NMR (376 MHz; acetone-d6) d
d ppm: 55.6 (OCH3), 113.6, 124.6, 124.9, 125.7, 128.0, 129.8, ppm: ꢁ117.9; ESI-MS (ESꢁ) m/z 378 [M ꢁ H]ꢁ (100%).
130.2, 131.7, 131.7, 132.0, 136.2, 137.2, 146.6, 162.9, 164.3 (CO),
2-[4-(Aminosulfonyl)phenyl]-3-(4-uorophenyl)-5-methyl-1H-
indole (3d). Purication of the crude product obtained by
general procedure B was carried out by column chromatography
((1) chloroform–methanol 90 : 10; (2) petroleum ether–ethyl
acetate 50 : 50) and preparative HPLC (RP-18, CH3CN–H2O
(with 0.1% TFA) 50 : 50 / 70 : 30). Starting from 2d (220 mg,
194.1 (CO); ESI-MS (ES+) m/z: 433 [M + Na]+ (100%).
General procedure B for the synthesis of 2-(4-sulfonylphenyl)-
3-phenyl-1H-indoles (3a–3g)
Under nitrogen atmosphere, TiCl4 (146.2 mL, 253 mg, 1.3 mmol) 0.54 mmol), 3d was obtained as colorless solid (44 mg, 21%).
was added to a suspension of the appropriate benzamide (0.62 mp 242–244 ꢀC; Rf 0.38 (petroleum ether–ethyl acetate 50 : 50);
mmol) and Zn (170 mg, 2.5 mmol) in THF (5.5 mL). The UV/vis: lmax/nm 238, 333 (3/dm3 molꢁ1 cmꢁ1 40 600, 21 000);
resulting mixture was heated to 65 ꢀC for 1.5 h. The solvent was uorescence: lexc ¼ 339, lem ¼ 451 nm; 1H NMR (400 MHz;
removed and the mixture was puried as described below.
acetone-d6) d ppm: 2.39 (3H, s, CH3), 6.60 (2H, br s, SO2NH2),
3-(Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-indole (3a). 7.07 (1H, dd, 3J ¼ 8.3 Hz, 4J ¼ 1.4 Hz, Hindol), 7.21 (2H, t, 3J ¼ 8.9
Synthesis and characterization of compound 3a was recently Hz, HF-phenyl-3/5), 7.33 (1H, d, 4J ¼ 0.6 Hz, Hindol), 7.37–7.45 (3H,
reported.30 Compound 3a was used for the discussed experi- m, 3J ¼ 8.9 Hz, 3J ¼ 8.3 Hz, 4J ¼ 5.5 Hz, 2HF-phenyl/1Hindol), 7.63
3
3
ments. UV/vis: lmax/nm 236, 249, 336 (3/dm3 molꢁ1 cmꢁ1 (2H, d, J ¼ 8.6 Hz, Hphenyl), 7.84 (2H, d, J ¼ 8.6 Hz, Hphenyl),
22 400, 20 300, 16 100); uorescence: lexc ¼ 343, lem ¼ 454 nm. 10.66 (1H, br s, NH); 13C NMR (101 MHz; acetone-d6) d ppm:
2
Crystals suitable for X-ray crystallography were obtained by slow 21.6, 112.1*, 115.2*, 116.4 (d, J ¼ 21 Hz), 119.5, 125.6, 127.2,
4
3
evaporation from ethyl acetate at room temperature. Detailed 129.1, 129.8*, 130.1, 132.5 (d, J ¼ 3 Hz), 132.8 (d, J ¼ 8 Hz),
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133.6, 136.1*, 137.2*, 143.6*, 162.6 (d, 1J ¼ 244 Hz), * deuterium 125.9, 126.8, 128.1*, 130.9, 132.0, 135.9, 136.3, 142.4, 158.0,
isotope shis were observed in the range of 33 to 144 ppb; 19F *two carbon species with equivalent chemical shi; ESI-MS
NMR (376 MHz; acetone-d6) d ppm: ꢁ118.1; ESI-MS (MSꢁ) m/z (ES+) m/z 401 [M + Na]+ (80%).
379 [M ꢁ H]ꢁ (100%).
N-({4-[3-(4-Methoxyphenyl)-1H-indol-2-yl]phenyl}sulfonyl)-
2-[4-(Aminosulfonyl)phenyl]-3-(4-uorophenyl)-5-hydroxy-1H- acetamide (3h). The sulfonamide 3g (240 mg, 0.6 mmol) was
indole (3e). Purication of the crude product obtained by suspended in acetic acid (5 mL). Acetyl chloride (200 mL,
general procedure B was carried out by column chromatography 2.8 mmol) was added and the mixture was stirred under reux
(chloroform–methanol 90 : 10). Starting from 2e (240 mg, for 90 min. Aer cooling, the acetic acid was removed under
0.58 mmol), 3e was obtained as colorless solid (119 mg, 54%). reduced pressure and the residue was puried by column
mp 244–247 ꢀC; Rf 0.19 (petroleum ether–ethyl acetate 50 : 50); chromatography (petroleum ether–ethyl acetate 20 : 80). 3h was
UV/vis: lmax/nm 236, 341 (3/dm3 molꢁ1 cmꢁ1 43 300, 20 500); obtained as pale green powder (176 mg, 70%). mp 96–100 ꢀC; Rf
uorescence: lexc ¼ 345, lem ¼ 481 nm; 1H NMR (400 MHz; 0.32 (petroleum ether–ethyl acetate 20 : 80); UV/vis: lmax/nm
acetone-d6) d ppm: 6.60 (2H, br s, SO2NH2), 6.83 (1H, dd, 3J ¼ 8.7 256, 323 (3/dm3 molꢁ1 cmꢁ1 29 800, 18 000); uorescence: lexc
¼
1
Hz, 4J ¼ 2.3 Hz, Hindole-6), 6.95 (1H, d, 4J ¼ 2.3 Hz, Hindole-4), 7.20 339, lem ¼ 452 nm; H NMR (400 MHz; DMSO-d6) d ppm: 1.94
3
(2H, t, 3J ¼ 8.9 Hz, HF-phenyl-3/5), 7.34 (1H, d, 3J ¼ 8.6 Hz, Hindole-7), (3H, s, COCH3), 3.80 (3H, s, OCH3), 7.02 (2H, d, J ¼ 8.8 Hz,
7.40 (2H, dd, 3J ¼ 8.8 Hz, 4J ¼ 5.5 Hz, HF-phenyl-2/6), 7.61 (2H, d,
H
anisole), 7.06 (1H, t, 3J ¼ 8.0 Hz, 3J ¼ 7.1 Hz, 4J ¼ 0.8 Hz, Harom),
3J ¼ 8.7 Hz, Hphenyl), 7.78 (1H, br s, OH); 7.83 (2H, d, 3J ¼ 8.7 Hz, 7.20 (1H, t, 3J ¼ 8.2 Hz, 3J ¼ 7.1 Hz, 4J ¼ 1.1 Hz, Harom), 7.27 (2H,
3
3
H
phenyl), 10.54 (1H, br s, NH); 13C NMR (101 MHz; CD3CN) d d, J ¼ 8.7 Hz, Hanisole), 7.42–7.48 (2H, m, J ¼ 8.2 Hz, Harom),
ppm: 103.6*, 113.0*, 114.3*, 114.9*, 116.3 (d, 2J ¼ 21 Hz), 127.2, 7.66 (2H, d, 3J ¼ 8.6 Hz, Harom), 7.86 (2H, d, 3J ¼ 8.6 Hz, Harom),
129.0, 130.3*, 132.4*, 132.6, 132.7 (d, J ¼ 8 Hz), 134.1*, 137.3 11.68 (1H, s, NH), 12.13 (1H, s, NH); 13C NMR (101 MHz; DMSO-
3
(d, 4J ¼ 4 Hz)*, 143.5*, 152.8*, 162.5 (d, 1J ¼ 244 Hz), d6) d ppm: 23.3, 55.1, 111.7, 114.5, 115.3, 119.2, 120.0, 122.9,
*deuterium isotope shis were observed in the range of 12 to 126.6, 127.8, 128.1, 128.2, 130.9, 131.6, 136.5, 137.4, 137.6,
140 ppb; 19F NMR (376 MHz; acetone-d6) d ppm: 118.2; ESI-MS 158.1, 168.9; ESI-MS (ES+) m/z 443 [M + Na]+ (100%).
(MS+) m/z 383 [M + H]+ (100%).
1-(4-Fluorophenyl)-2-phenylethanone (4). 4 was synthesized
3-(4-Methoxyphenyl)-2-[4-(methylsulfonyl)phenyl]-1H-indole as described by Singh et al.37 Starting from phenylacetyl chloride
(3f). Purication of the crude product obtained by general (3.3 mL, 25 mmol), 4 was obtained following this protocol and
procedure B was carried out by column chromatography additional purication by column chromatography (petroleum
(petroleum ether–ethyl acetate 30 : 70). Starting from 2f (360 ether–ethyl acetate 85 : 15) as a colorless solid (2.47 g, 51%). mp
mg, 0.88 mmol), 3f was obtained as a pale yellow solid (182 mg, 80–82 ꢀC (lit.,38 83 ꢀC); Rf 0.36 (petroleum ether–ethyl acetate
1
55%). mp 222–226 ꢀC (lit.,11 218.5–220.5 ꢀC); Rf 0.47 (petroleum 85 : 15); H NMR (400 MHz; CD3CN) d ppm: 4.32 (2H, s, CH2),
ether–ethyl acetate 30 : 70); UV/vis: lmax/nm 237, 256, 332 (3/ 7.18–7.36 (7H, m, Hphenyl), 8.08 (2H, dd, 3J ¼ 9.0 Hz, 4J ¼ 5.5 Hz,
dm3 molꢁ1 cmꢁ1 28 700, 25 400, 16 000); uorescence: lexc
¼
H
F-phenyl-2/6); 13C NMR (101 MHz; CD3CN) d ppm: 45.9, 116.6 (d,
1
3
354, lem ¼ 492 nm; H NMR (400 MHz; DMSO-d6) d ppm: 3.25 2J ¼ 22 Hz), 127.7, 129.4, 130.7, 132.2 (d, J ¼ 10 Hz), 134.4 (d,
3
1
(3H, s, SO2CH3), 3.80 (3H, s, OCH3), 7.02 (2H, d, J ¼ 8.8 Hz, 4J ¼ 3 Hz), 136.1, 166.6 (d, J ¼ 252 Hz), 197.3; 19F NMR (376
H
anisole), 7.06 (1H, t, 3J ¼ 8.0 Hz, 3J ¼ 7.0 Hz, 4J ¼ 0.9 Hz, Harom), MHz; CD3CN) d ppm: ꢁ108.0; ESI-MS (ES+) m/z 215 [M + H]+
7.21 (1H, t, 3J ¼ 8.1 Hz, 3J ¼ 7.1 Hz, 4J ¼ 1.1 Hz, Harom), 7.28 (2H, (100%).
d, 3J ¼ 8.7 Hz, Hanisole), 7.45 (1H, d, 3J ¼ 8.0 Hz, Harom), 7.48 (1H,
d, 3J ¼ 8.1 Hz, Harom), 7.69 (2H, d, 3J ¼ 8.7 Hz, Harom), 7.90 (2H,
d, J ¼ 8.6 Hz, Harom), 11.70 (1H, s, NH); 13C NMR (101 MHz;
3
General procedure C for the synthesis of 2-[4-(methylsulfonyl)
phenyl]-1-phenyl-ethanones 5a and 5b
DMSO-d6) d ppm: 43.4, 55.0, 111.7, 114.4, 115.3, 119.1, 119.9,
122.8, 126.6, 127.1, 128.2*, 130.9, 131.5, 136.4, 137.5, 139.0, The reaction was carried out under Schlenk-conditions. A
158.0, *two carbon species with equivalent chemical shi; ESI- suspension of 4-(methylsulfonyl)phenylacetic acid (1.714 g,
MS (ES+) m/z 400 [M + Na]+ (100%).
8 mmol) in dichloromethane (1.6 mL) and 4 drops of DMF was
2-[4-(Aminosulfonyl)phenyl]-3-(4-methoxyphenyl)-1H-indole heated to 30 ꢀC. Freshly distilled thionyl chloride (1.00 g,
(3g). Purication of the crude product obtained by general 0.60 mL, 8.48 mmol) was added and the mixture was stirred for
procedure B was carried out by column chromatography 1.5 h at constant temperature. The solution was cooled to 15 ꢀC
(petroleum ether–ethyl acetate 20 : 80). Starting from 2g (300 and freshly sublimated AlCl3 (2.02 g, 15.15 mmol) was added in
mg, 0.73 mmol), 3g was obtained as pale yellow solid (184 mg, portions. Aer 15 min the appropriate benzene (9.66 mmol) was
48%). mp 282–285 ꢀC (lit.,11 280–282 ꢀC), Rf 0.51 (petroleum dropped to the mixture and it was stirred for 2 h. Then ethanol
ether–ethyl acetate 20 : 80); UV/vis: lmax/nm 237, 325 (3/dm3 (2.8 mL) was added dropwise at 10 ꢀC, followed by dichloro-
molꢁ1 cmꢁ1 37 100, 16 100); uorescence: lexc ¼ 345, lem ¼ 482 methane (20.6 mL) and water (10.2 mL) over a period of 20 min.
1
nm; H NMR (400 MHz; DMSO-d6) d ppm: 3.80 (3H, s, OCH3), The mixture was heated to 30 ꢀC and the organic phase was
3
3
6.79–7.08 (3H, m, J ¼ 7.7 Hz, Hanisole/Harom.), 7.19 (1H, t, J ¼ separated, washed with 5 M HCl (6 mL) and saturated Na2CO3
3
7.9 Hz, Harom), 7.27 (2H, d, J ¼ 7.9 Hz, Hanisole), 7.39 (2H, s, solution (6 mL), and concentrated by distillation to a volume of
NH2), 7.43–7.49 (2H, m, Harom), 7.61 (2H, d, 3J ¼ 8.2 Hz, Harom), 5 mL. Further purication was carried out as described below.
7.79 (2H, d, 3J 8.0, Harom), 11.64 (1H, s, NH); 13C NMR (101 MHz;
1-(4-Ethoxyphenyl)-2-[4-(methysulfonyl)phenyl]ethanone (5a).
DMSO-d6) d ppm: 55.1, 111.6, 114.4, 114.6, 119.0, 119.9, 122.6, For purication of the raw product obtained by general
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procedure C, the solution was ltered to yield 5a. Furthermore, boiling heat which was slowly cooled down to room temperature
the ltrate was concentrated to dryness, washed with a small overnight. This formed a small amount of tiny crystals. Then,
amount of petroleum ether–DCM 1 : 1 to obtain a further the solution was allowed to evaporate slowly what generated
amount of 5a. Starting from 4-(methylsulfonyl)phenylacetic acid crystals suitable for X-ray crystallography. Detailed results of the
(428 mg, 2 mmol) and 4-ethoxybenzene (0.304 mL, 295 mg, 2.41 single-crystal X-ray structure determination are given below and
mmol), 5a was obtained as colorless solid (192 mg, 30%). mp in the ESI.‡
188–190 ꢀC from ethanol (lit.,60 156–159 ꢀC); Rf 0.41 (petroleum
2-[4-(Aminosulfonyl)phenyl]-1-(4-uorophenyl)ethanone (5d).
1
ether–ethyl acetate 50 : 50); H NMR (400 MHz; CDCl3) d ppm: Chlorosulfuric acid (1.63 mL, 2.81 g, 23.9 mmol) was slowly
3
1.45 (3H, t, J ¼ 7.0 Hz, OCH2CH3), 3.04 (3H, s, SO2CH3), 4.11 added to compound 4 (650 mg, 3.03 mmol) in a ask at ꢁ78 ꢀC
(2H, q, 3J ¼ 7.0 Hz, OCH2CH3), 4.34 (2H, s, CH2), 6.94 (2H, d, 3J ¼ equipped with a drying tube lled with CaCl2. The mixture was
8.9 Hz, Hphenyl), 7.46 (2H, d, 3J ¼ 8.4 Hz, Hphenyl), 7.90 (2H, d, 3J allowed to warm up over a period of 60 min and then allowed to
3
¼ 8.4 Hz, Hphenyl), 7.98 (2H, d, J ¼ 8.9 Hz, Hphenyl); 13C NMR stir for 2 h at room temperature, poured over ice and extracted
(101 MHz; CDCl3) d ppm: 14.8, 44.7, 44.9, 64.0, 114.6, 127.8, with ethyl acetate (3 ꢂ 25 mL). The organic phase was washed
129.2, 130.8, 131.0, 139.2, 141.5, 163.5, 194.9; ESI-MS (MS+) m/z with water (10 mL), the organic phase was separated and then
341 [M + Na]+ (100%), 319 [M + H]+ (53), 659 (24).
25% aq. NH3 (5 mL) solution were added. The mixture was
1-(4-Methoxyphenyl)-2-[4-(methysulfonyl)phenyl]ethanone stirred for 1 h at room temperature. Then, the organic phase
(5b). For purication of the raw product obtained by general was separated, washed with 1 M HCl (21 mL) and brine (21 mL),
procedure C, the solution was ltered and the resulting solid concentrated to 5 mL and stored in the fridge. The resulting
(1.14 g) was further puried by sublimation under reduced solid was ltered off and petroleum ether was added to the
pressure to yield 836 mg of compound 5b. The residue from ltrate to yield further product. The solids were combined and
sublimation and the ltrate were combined, dried and further dried. 5d was obtained as colorless solid (303 mg, 34%). mp
puried by column chromatography (DCM–acetone 98 : 2) to 201–204 ꢀC (lit.,38 198–204 ꢀC); Rf 0.34 (petroleum ether–ethyl
yield 402 mg of compound 5b. Starting from 4-(methylsulfonyl)- acetate 50 : 50); 1H NMR (400 MHz; DMSO-d6)
d ppm:
3
phenylacetic acid (1.714 g, 8 mmol) and 4-methoxybenzene 4.53 (2H, s, CH2), 7.32 (2H, br s, SO2NH2), 7.38 (2H, t, J ¼ 8.9
3
(1.055 mL, 1.044 g, 9.66 mmol), 5b was obtained as colorless solid Hz, HF-phenyl-3/5), 7.44 (2H, d, J ¼ 8.3 Hz, Hphenyl), 7.78 (2H, d,
1
(1.24 g, 51%). mp 172–174 C; Rf 0.34 (DCM–acetone 98 : 2); H 3J ¼ 8.3 Hz, Hphenyl), 8.14 (2H, dd, 3J ¼ 8.9 Hz, 4J ¼ 5.5 Hz, HF-
ꢀ
NMR (400 MHz; CDCl3) d ppm: 3.04 (3H, s, SO2CH3), 3.88 (3H, s, phenyl-2/6); 13C NMR (101 MHz; DMSO-d6) d ppm: 44.3, 115.8 (d,
OCH3), 4.34 (2H, s, CH2), 6.96 (2H, d, 3J ¼ 8.9 Hz, Hphenyl), 7.46 3J ¼ 22 Hz), 125.6, 130.4, 131.3 (d, J ¼ 10 Hz), 133.0 (d, J ¼ 3
3
3
(2H, d, 3J ¼ 8.4 Hz, Hphenyl), 7.90 (2H, d, 3J ¼ 8.4 Hz, Hphenyl), 7.99 Hz), 139.2, 142.4, 165.1 (d, J ¼ 252 Hz), 195.7; 19F NMR (376
3
(2H, d, 3J ¼ 8.9 Hz, Hphenyl); 13C NMR (101 MHz; CDCl3) d ppm: MHz; DMSO-d6) d ppm: ꢁ106.1; ESI-MS (MS+) m/z 316 [M + Na]+
44.7, 44.9, 55.7, 114.2, 127.8, 129.4, 130.8, 131.0, 139.2, 141.5, (100%).
164.1, 194.9; ESI-MS (MS+) m/z 631 [2M + Na]+ (73%), 368 [M +
2-Bromo-1-(4-uorophenyl)-2-[4-(methylsulfonyl)phenyl]-
ethanone 5e. To a solution of 5c (1.528 g, 5.23 mmol) in CHCl3
Na + CH3CN]+ (100), 327 (56), 305 [M + H]+ (72).
1-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]ethanone (5c). (22 mL) and CCl4 (85 mL) was added dropwise a solution of Br2
To 4-(methylsulfonyl)phenylacetic acid (2.0 g, 9.2 mmol) was (0.270 mL, 5.13 mmol) in CCl4 (1.5 mL). Then, benzoyl peroxide
added thionyl chloride (4.0 mL, 6.6 g, 56 mmol) and the mixture (65 mg, 0.27 mmol) was added and the reaction mixture was
was heated to reux until the solution became clear. The excess of heated to 78 ꢀC until the brown color of the mixture originating
thionyl chloride was removed under reduced pressure. The from Br2 disappeared. The reaction mixture was cooled to room
resulting solid was suspended in uorobenzene (12 mL) and then temperature, washed with 5% Na2CO3 (20 mL) and brine
aluminum chloride (1.88 g, 13 mmol) was added in small (30 mL). Purication was carried out by column chromatog-
ꢀ
portions at 20–25 C. The mixture was heated to reux for 2 h, raphy (ethyl acetate–petroleum ether 5 : 5). In this way, 5e was
cooled to room temperature and poured onto a mixture of ice and obtained as a yellow solid (1.009 g, 52%). mp 134–141 ꢀC (lit.,47
1 M HCl (4 mL). The uorobenzene was removed by distillation 140–141 ꢀC); Rf 0.44 (ethyl acetate–petroleum ether 5 : 5); 1H
under reduced pressure and the raw product was ltered off, NMR (400 MHz; acetone-d6) d ppm: 3.15 (3H, s, SO2CH3), 7.03
washed with 1 M Na2CO3 (10 mL) and water (20 mL). Recrystal- (1H, s, CHBr), 7.33 (2H, t, 3J ¼ 8.8 Hz, HF-phenyl-3/5), 7.91 (2H, d,
3
lization from 95% ethanol yielded 5c as pale beige solid (1.13 g, 3J ¼ 8.5 Hz, HSO2-phenyl), 7.99 (2H, d, J ¼ 8.6 Hz, HSO2-phenyl),
42%). mp 182–185 ꢀC (lit.,38 182–183 ꢀC); Rf 0.29 (petroleum 8.25 (2H, dd, 3J ¼ 9.0 Hz, 4J ¼ 5.4 Hz, HF-phenyl-2/6); 13C NMR (101
ether–ethyl acetate 50 : 50); 1H NMR (400 MHz; acetone-d6) d MHz; acetone-d6) d ppm: 44.2, 48.9, 116.9 (d, 2J ¼ 22 Hz), 128.5,
4
3
ppm: 3.12 (3H, s, CH3), 4.59 (2H, s, CH2), 7.31 (2H, t, 3J ¼ 8.8 Hz, 131.4, 131.6 (d, J ¼ 3 Hz), 133.1 (d, J ¼ 10 Hz), 142.7, 142.8,
HF-phenyl-3/5), 7.59 (2H, d, 3J ¼ 8.3 Hz, Hphenyl), 7.91 (2H, d, 3J ¼ 8.3 166.9 (d, J ¼ 254 Hz), 190.3; 19F NMR (376 MHz; acetone-d6) d
1
3
4
Hz, Hphenyl), 8.20 (2H, dd, J ¼ 8.9 Hz, J ¼ 5.5 Hz, HF-phenyl-2/6); ppm: ꢁ106.1.
13C NMR (101 MHz; acetone-d6) d ppm: 44.4, 45.3, 116.5 (d, 2J ¼
3
4
22 Hz), 128.1, 131.7, 132.2 (d, J ¼ 9 Hz), 134.3 (d, J ¼ 3 Hz),
1
General procedure D for the synthesis of 3-(4-sulfonylphenyl)-
2-phenyl-1H-indoles (3i–3l)
140.8, 142.3, 166.6 (d, J ¼ 252 Hz), 195.7; 19F NMR (376 MHz;
acetone-d6) d ppm: ꢁ107.7; ESI-MS (MS+) m/z 315 [M + Na]+
(100%). Single crystals were obtained as follows: 5c was crystal- A mixture of phenylhydrazine (66 mL, 73 mg, 0.68 mmol) and the
lized from a mixture of acetone and acetonitrile saturated at the appropriate 1,2-diphenyl ethanone (0.68 mmol) was heated to
38738 | RSC Adv., 2014, 4, 38726–38742
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130 ꢀC for 30 min. Aer cooling to room temperature, acetic
acid (4.13 mL) and BF3$Et2O (36 mL, 40 mg, 0.29 mmol) were
added and the mixture was heated to reux for 1 h. Then, acetic
acid was removed by distillation under reduced pressure. The
resulting solid was suspended in water (3 mL), ltered, washed
with a small amount of water and dried. The mixture was
further puried as described below.
2-(4-Fluorophenyl)-3-[4-(methylsulfonyl)phenyl]-1H-indole
(3k). Purication of the raw product obtained by general
procedure D was carried out by column chromatography
(petroleum ether–ethyl acetate 50 : 50) and preparative HPLC
(RP-18, CH3CN–H2O (with 0.1% TFA) 50 : 50 / 70 : 30). Start-
ing from 5c (300 mg, 1.03 mmol), 3k was obtained as pale yellow
ꢀ
solid (73 mg, 19%). mp 234–235 C; Rf 0.57 (petroleum ether–
ethyl acetate 50 : 50); UV/vis: lmax/nm 238, 296, 337 (3/dm3
molꢁ1 cmꢁ1 44 700, 21 700, 12 700); uorescence: lexc ¼ 300,
lem ¼ 444 nm; 1H NMR (400 MHz; acetone-d6) d ppm: 3.15 (3H,
2-(4-Ethoxyphenyl)-3-[4-(methylsulfonyl)phenyl]-1H-indole (3i).
Purication of the raw product obtained by general procedure D
was carried out by column chromatography (petroleum ether–
ethyl acetate 60 : 40). Starting from 5a (216 mg, 0.68 mmol), 3i
was obtained as pale yellow solid (28 mg, 10%). mp 212–214 ꢀC;
Rf 0.56 (petroleum ether–ethyl acetate 50 : 50); UV/vis: lmax/nm
238, 300, 340 (3/dm3 molꢁ1 cmꢁ1 19 700, 17 300, 9300); uo-
rescence: lexc ¼ 303, lem ¼ 450 nm; 1H NMR (400 MHz; acetone-
d6) d ppm: 1.38 (3H, t, 3J ¼ 7.0 Hz, CH2CH3), 3.15 (3H, s,
SO2CH3), 4.08 (2H, q, 3J ¼ 7.0 Hz, CH2CH3), 6.94 (2H, d, 3J ¼ 8.8
3
3
4
s, SO2CH3), 7.09–7.20 (3H, m, J ¼ 8.9 Hz, J ¼ 7.5 Hz, J ¼ 1,
3
4
2Hphenyl/1Hindol), 7.23 (1H, t, J ¼ 7.6 Hz, J ¼ 1.0 Hz, Hindol),
7.46–7.58 (3H, m, 3J ¼ 8.9 Hz, 3J ¼ 7.9 Hz, 4J ¼ 5.5 Hz, 2Hphenyl
/
/
3
3
1Hindol), 7.63–7.68 (3H, m, J ¼ 8.4 Hz, J ¼ 7.8 Hz, 2Hphenyl
1Hindol), 7.95 (2H, d, 3J ¼ 8.4 Hz, Hphenyl), 10.83 (1H, br s, NH);
13C NMR (101 MHz; acetone-d6) d ppm: 44.4, 112.5, 113.3, 116.5
(d, 2J ¼ 22 Hz), 119.5, 121.4, 123.5, 128.5, 128.7, 129.7 (d, 4J ¼ 3
Hz), 131.7 (d, 3J ¼ 8 Hz), 131.2, 135.7, 137.5, 139.5, 142.1, 163.4
(d, 1J ¼ 246 Hz); 19F NMR (376 MHz; acetone-d6) d ppm: ꢁ115.3;
ESI-MS (ESꢁ) m/z 364 [M ꢁ H]ꢁ (100%).
3
3
4
Hz, Hphenyl), 7.12 (1H, t, J ¼ 8.0 Hz, J ¼ 7.1 Hz, J ¼ 1.0 Hz,
H
indol), 7.20 (1H, t, 3J ¼ 8.1 Hz, 3J ¼ 7.1 Hz, 4J ¼ 1.1 Hz, Hindol),
7.42 (2H, d, 3J ¼ 8.9 Hz, Hphenyl), 7.50 (1H, d, 3J ¼ 8.1 Hz, Hindol),
7.63–7.69 (3H, m, 3J ¼ 8.5 Hz, 2Hphenyl/1Hindol), 7.94 (2H, d, 3J ¼
8.5 Hz, Hphenyl), 10.71 (1H, br s, NH); 13C NMR (101 MHz;
acetone-d6) d ppm: 15.1, 44.5, 64.1, 112.3*, 112.3*, 115.6, 119.2,
121.2, 123.1, 125.3*, 128.4, 128.9*, 130.8, 131.2, 136.9*, 137.4*,
139.2, 142.7, 160.1, *deuterium isotope shis were observed in
the range of 37 to 146 ppb; ESI-MS (APcIꢁ) m/z 390 [M ꢁ H]ꢁ
(100%).
3-[4-(Aminosulfonyl)phenyl]-2-(4-uorophenyl)-1H-indole (3l).
Purication of the raw product obtained by general procedure D
was carried out by column chromatography (petroleum ether–
ethyl acetate 70 : 30) and preparative HPLC (RP-18, CH3CN–H2O
with 0.1% TFA, 50 : 50 / 70 : 30). Starting from 5d (200 mg,
0.68 mmol), 3l was obtained as colorless solid (70 mg, 28%). mp
162–163 ꢀC; Rf 0.23 (petroleum ether–ethyl acetate 70 : 30); UV/
vis: lmax/nm 237, 296, 329 (3/dm3 molꢁ1 cmꢁ1 38 500, 20 600,
2-(4-Methoxyphenyl)-3-[4-(methylsulfonyl)phenyl]-1H-indole
(3j). Purication of the raw product obtained by general proce-
dure D was carried out by column chromatography (petroleum
ether–ethyl acetate 60 : 40). Starting from 5b (206 mg, 0.68
mmol), 3j was obtained as pale yellow solid (60 mg, 23%). mp
1
13 600); uorescence: lexc ¼ 303, lem ¼ 443 nm; H NMR (400
MHz; acetone-d6) d ppm: 6.59 (2H, br s, SO2NH2), 7.10–7.20 (3H,
m, 3J ¼ 8.9 Hz, 3J ¼ 7.5 Hz, 2Hphenyl/1Hindol), 7.22 (1H, dd, 3J ¼
7.6 Hz, 3J ¼ 8.0 Hz, Hindol), 7.49–7.59 (5H, m, 3J ¼ 8.9 Hz, 3J ¼ 8.5
3
3
Hz, J ¼ 8.2 Hz, 4J ¼ 5.5 Hz, 4Hphenyl/1Hindol), 7.64 (1H, d, J ¼
8.0 Hz, Hindol), 7.91 (2H, d, 3J ¼ 8.6 Hz, Hphenyl), 10.78 (1H, br s,
NH); 13C NMR (101 MHz; acetone-d6) d ppm: 112.4, 113.5, 116.5
(d, 2J ¼ 22 Hz), 119.6, 121.3, 123.5, 127.3, 128.9, 129.8 (d, 4J ¼ 3
Hz), 130.9, 131.6 (d, 3J ¼ 8 Hz), 135.3, 137.5, 140.4, 142.5, 163.3
(d, 1J ¼ 246 Hz); 19F NMR (376 MHz; acetone-d6) d ppm: ꢁ115.5;
ESI-MS (ESꢁ) m/z: 365 [M ꢁ H]ꢁ (100%).
ꢀ
241–243 C; Rf 0.40 (petroleum ether–ethyl acetate 50 : 50); UV/
vis: lmax/nm 238, 300, 344 (3/dm3 molꢁ1 cmꢁ1 31 800, 23 500,
1
12 300); uorescence: lexc ¼ 344, lem ¼ 450 nm; H NMR (400
MHz; acetone-d6) d ppm: 3.15 (3H, s, SO2CH3), 3.83 (3H, s, OCH3),
6.96 (2H, d, 3J ¼ 8.7 Hz, Hphenyl), 7.12 (1H, t, 3J ¼ 7.5 Hz, Hindol),
7.20 (1H, t, 3J ¼ 7.6 Hz, 4J ¼ 0.9 Hz, Hindol), 7.43 (2H, d, 3J ¼ 8.7
3
Hz, Hphenyl), 7.50 (1H, d, J ¼ 8.1 Hz, Hindol), 7.59–7.69 (3H, m,
3J ¼ 8.3 Hz, 2Hphenyl/1Hindole), 7.94 (2H, d, 3J ¼ 8.3 Hz, Hphenyl),
10.71 (1H, br s, NH); 13C NMR (101 MHz; acetone-d6) d ppm: 44.5,
55.6, 112.3*, 112.3*, 115.1, 119.2, 121.2, 123.1, 125.5*, 128.4,
128.9*, 130.8, 131.2, 136.8*, 137.4*, 139.2, 142.6, 160.7,
*deuterium isotope shis were observed in the range of 38 to 145
ppb; m/z (ESI+) 378 [M + H]+ (100%). 5d was also synthesized in a
two-step/one-pot procedure including aminolysis and McMurry
reaction and puried by column chromatography ((1) petroleum
ether–ethyl acetate 50 : 50 / 0 : 100, (2) petroleum ether–ethyl
acetate 50 : 50) as previously described.39 Starting from 2-amino-
40-(methylsulfonyl)benzophenone (500 mg, 1.82 mmol) and p-
methoxybenzoyl chloride, 5d was obtained as colorless solid
having the same spectroscopic properties as described above (218
mg, 32%). From this batch, crystals suitable for X-ray crystallog-
raphy were obtained from a solution of 3j in ethyl acetate–
petroleum ether 50 : 50 by slow evaporation at room tempera-
ture. Detailed results of the single-crystal X-ray structure deter-
mination are given below and in the ESI.‡
3-(4-Fluorophenyl)-5-methoxy-2-[4-(methylsulfonyl)phenyl]-1H-
indole (3m). A solution of 5e (0.524 g, 1.41 mmol) and freshly
distilled 4-methoxyaniline (0.745 g, 6.05 mmol) in ethanol (20
mL) was heated to 170 ꢀC for 34 h in a pressure stable glass vial.
Aer that, the mixture was cooled to room temperature and the
solvent was removed under reduced pressure. The resulting
solid was suspended in ethyl acetate (20 mL), ltered, washed
with ethanol (3 mL) and dried. In this way, compound 3m was
obtained as a yellow solid (197 mg, 37%). mp 240–242 ꢀC; Rf 0.29
(petroleum ether–ethyl acetate 5 : 5); UV/vis: lmax/nm 237, 344
(3/dm3 molꢁ1 cmꢁ1 45 200, 25 400); uorescence: lexc ¼ 349,
lem ¼ 484 nm; 1H NMR (400 MHz; DMSO-d6) d ppm: 3.24 (3H, s,
SO2CH3), 3.73 (3H, s, OCH3), 6.82–6.94 (2H, m, Hindole), 7.28
3
(2H, t, J ¼ 8.9 Hz, HF-phenyl-3/5), 7.33–7.44 (3H, m, HF-phenyl-2/6
/
Hindole), 7.63 (2H, d, 3J ¼ 8.4 Hz, HSO2-phenyl), 7.89 (2H, d, 3J ¼ 8.5
Hz, HSO2-phenyl), 11.65 (1H, s, NH); 13C NMR (101 MHz; DMSO-
d6) d ppm: 43.4, 55.3, 99.8, 112.6, 113.6, 114.1, 115.9 (d, 2J ¼ 21
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Hz), 127.2, 128.2, 128.3, 131.1 (d, 4J ¼ 3 Hz), 131.7 (d, 3J ¼ 8 Hz), spectrometric determination of formation of g-glutamyl semi-
131.7, 132.6, 137.3, 139.1, 154.2, 161.0 (d, 1J ¼ 243 Hz); 19F aldehyde, a specic product of protein oxidation, which by
NMR (376 MHz; DMSO-d6) d ppm: ꢁ116.4; ESI-MS (ESꢁ) m/z 394 reduction forms 5-hydroxy-2-aminovaleric acid (HAVA) as
[M ꢁ H]ꢁ (100%).
described elsewhere in detail.65,66
In this study the results are expressed as ratios between the
duration of lag phase in the presence and in the absence of the
compound (RDIENE) and between the apoB-100 HAVA content in
the absence and in the presence of the compound (RHAVA).
Thus, in both approaches R values (RDIENE or RHAVA) higher than
1 indicate antioxidant activity, R values of about 1 mean that the
compound has no effect and R values lower than 1 suggests
prooxidant activity.
X-ray crystallography
The crystallographic data were collected with a Bruker-Nonius
Apex-X8 and Bruker-Nonius APEX-II CCD diffractometer,
˚
respectively, with Mo-Ka radiation (l ¼ 0.71073 A). The struc-
tures were solved using SHELXS-97 and rened against F2 on all
data by full-matrix least squares with SHELXL-97.61 All non-
hydrogen atoms were rened anisotropically; all hydrogen
atoms bonded to carbon atoms were placed on geometrically
calculated positions and rened using a riding model.‡
COX inhibitory assay
The COX inhibition activity against ovine COX-1 and human
COX-2 presented in Table 2 was determined at the given
concentrations using the uorescence based COX assay “COX
Fluorescent Inhibitor Screening Assay Kit” (catalog number
700100; Cayman Chemical, Ann Arbor, MI, USA) and the EIA
based COX assay “COX inhibitor Screening Assay Kit” (catalog
number 560131; Cayman Chemical, Ann Arbor, MI, USA)
according to the manufacturer's instructions.
LDL assay
In order to determine redox activity–structure relationships of
COX-2 inhibitors low-density lipoprotein (LDL) copper-/iron-
peroxidation models were used. Therefore, native albumin-free
LDL (density 1.006–1.063 g mLꢁ1) were isolated from the blood
plasma of healthy, normolipidemic, normoglycemic male
volunteers by sequential very fast ultracentrifugation (VFU) and
prepared for oxidation experiments as previously described by
us in detail.62 Blood plasma and LDL were all processed in
subdued light to prevent the photooxidation of LDL. All buffers
and solutions were made oxygen-free by degassing and purging
with argon. LDL apoB-100 was measured by immunoelectro-
phoresis using ‘ready-to-use’ agarose gels (Sebia, Issy-les-Mou-
lineaux, France). Immediately before oxidation of LDL, EDTA,
and salt from the density gradient were removed using a size
exclusion column (Econo-Pac 10DG, Bio-Rad) and phosphate-
buffered saline (PBS, 10 mM sodium phosphate, 150 mM
sodium chloride, pH 7.4) as the eluent.62
For lipid oxidation, to 200 mL-aliquots of native LDL (125 mg
apoB-100/mL, equal to 0.25 mM LDL) in 96 well plates (UV-Star
plates, UV transparent to 200 nm, Greiner, Germany) were
added 25 mL of an aqueous solution of CuSO4 (16 mM) and 25 mL
of the solution of the compound to be tested. All compounds
(novel COX-2 inhibitors, celecoxib, quercetin, and melatonin)
were tested at 1 mM concentration. The same preparation
without CuSO4 was used as control. The oxidative process was
monitored using a Biotek Instruments Synergy 4 thermostatic
UV/vis micro plate reader by following the formation of conju-
Acknowledgements
The authors thank the Helmholtz Association for funding a part
of this work through Helmholtz-Portfolio Topic “Technologie
¨
und Medizin – Multimodale Bildgebung zur Aularung des In vivo-
Verhaltens von polymeren Biomaterialien”. This work is also part
of the research initiative “Radiation-Induced Vascular Dysfunc-
tion (RIVAD)”. The excellent technical assistance of Peggy
Wecke, Mareike Barth, Catharina Heinig, and Sebastian Meister
is greatly acknowledged. Franz-Jacob Pietzsch is graduate
student member of the Integrated Research Training Group
“Matrixengineering” (within the Transregional Collaborative
Research Centre 67 “Functional biomaterials for controlling
healing processes in bone and skin – from material science to
clinical application” funded by German Research Foundation)
at Medical Faculty and University Hospital Carl Gustav Carus,
¨
Technische Universitat, Dresden.
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