Syntheses of 1,2,3-triazolyl salicylamides with inhibitory activity on lipopolysaccharide-induced nitric oxide production

Article abstract of DOI:10.1016/j.bmcl.2011.02.034

A 28-membered 1,2,3-triazolyl salicylamide library was synthesized via a Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition and evaluated for their abilities to inhibit NO production in LPS-activated RAW264.7 macrophage cells. Among 28 analogues, 29g showed a significant inhibitory activity (IC ₅₀ = 12.8 μM). The inhibitory effects of 29g on LPS-mediated NO production in macrophage cells appeared to be associated with the suppression of iNOS expression.

Full text of DOI:10.1016/j.bmcl.2011.02.034

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1953–1957
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Syntheses of 1,2,3-triazolyl salicylamides with inhibitory activity on
lipopolysaccharide-induced nitric oxide production
Jieun Yoon a, Lan Cho b, Sang Kook Lee b, , Jae-Sang Ryu a,
⇑
⇑
a Center for Cell Signaling and Drug Discovery Research, College of Pharmacy and Division of Life and Pharmaceutical Sciences, Ewha Womans University,
11-1 Daehyun-Dong, Seodaemun-Gu, Seoul 120-750, Republic of Korea
b College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A 28-membered 1,2,3-triazolyl salicylamide library was synthesized via a Cu(I)-catalyzed azide-alkyne
1,3-dipolar cycloaddition and evaluated for their abilities to inhibit NO production in LPS-activated
Received 19 January 2011
Revised 7 February 2011
Accepted 9 February 2011
Available online 13 February 2011
RAW264.7 macrophage cells. Among 28 analogues, 29g showed
(IC50 = 12.8 M). The inhibitory effects of 29g on LPS-mediated NO production in macrophage cells
appeared to be associated with the suppression of iNOS expression.
a significant inhibitory activity
l
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Salicylamide
Click chemistry
Library
Triazole
iNOS
Nitric oxide (NO) is an important signaling molecule that is
involved in a wide range of pathophysiological responses such as
vasodilation, nonspecific host defense, ischemia reperfusion injury,
chronic inflammation and carcinogenesis. In mammals, NO is pro-
The earliest inhibitors of NOS were direct analogs of the natural
substrate
N-nitroarginine (
3), and N-iminoethyllysine (
L
-arginine such as N-monomethyl arginine (
-NA, 2),4 N-nitroarginine methyl ester (
-NIL, 4)5,6 as shown in Figure 1. Unfor-
L
-NMMA, 1),3
L
L-NAME,
L
duced from the enzymatic oxidation of
L
-arginine by three distinct
tunately, most of these inhibitors exhibited moderate potency and
poor selectivity against NOS isoforms, which limited their applica-
tion in vivo. Only GW274150 (5)7 is currently under Phase II
clinical evaluation for the treatment of rheumatoid arthritis and
migraine. Recently, various non-peptidic small molecule inhibitors
including indazole, isothioureas, amidine, 2-aminopyridines,
imidazopyridine, pyrazoles and pyrroles have emerged as NOS
inhibitors.8 Such small molecules are structurally distinct from
isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS),
endothelial NOS (eNOS) and inducible NOS (iNOS). nNOS is located
in both the central and peripheral nervous systems, and controls
the production of NO, which acts as a neurotransmitter.1 eNOS is
found primarily in vascular endothelium, and regulates blood pres-
sure and the vascular tone.2 iNOS is ubiquitous, Ca2+/calmodulin-
independent, and implicated in the pathogenesis of numerous
diseases. Both nNOS and eNOS are constitutively expressed, and
produce NO at a low level. However, iNOS is frequently overex-
pressed by pro-inflammatory and/or carcinogenic stimuli such as
L-arginine and can thus avoid interference with physiological
processes that depend on arginine transport and metabolism. In
particular, AR-C102222 (6), one of the competitive inhibitors,
showed good selectivity for iNOS and good efficacy in a rat adju-
vant-induced arthritis model.9 More recently, an imidazole-based
inhibitor (BBS-4, 7)10 and a thiazole-based inhibitor (KLYP956,
8)11 were reported to be iNOS dimerization inhibitors. Mechanisti-
cally, these inhibitors disrupt the formation of the active iNOS
dimer by direct coordination of the imidazole or the thiazole to
the heme iron in the active site of the enzyme.12 Although progress
has been made in the development of iNOS inhibitors, there is still
a need for potent and selective inhibitors that would be more drug-
like and suitable for clinical use. In our ongoing efforts to develop
potent, selective, and orally available non-peptidic iNOS inhibitors,
we herein describe the synthesis and biological evaluation of a
interleukin-1b, tumor necrosis factor-
a and lipopolysaccharide
(LPS) in macrophages, endothelial cells and smooth muscle cells.
Once expressed at high levels, iNOS is essentially unregulated,
and can produce NO at cytotoxic levels, resulting in local tissue
damage and many inflammatory diseases including rheumatoid
arthritis, osteoarthritis, inflammatory bowel disease and multiple
sclerosis. Thus, the control of NO production through iNOS inhibi-
tion is very important in the treatment of numerous disease
mediated by the overproduction of NO.
⇑
Corresponding authors. Tel.: +82 2 3277 3008; fax: +82 2 3277 2851 (J.-S.R.);
tel.: +82 2 880 2475; fax: +82 2 762 8322 (S.K.L).
E-mail addresses: sklee61@snu.ac.kr (S.K. Lee), ryuj@ewha.ac.kr (J.-S. Ryu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.034

1954
J. Yoon et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1953–1957
O
NH
O
NH
O
F
F
H
N
N
S
R1
N
OR2
N
OH
n
H
H
NH2
N
CN
NH2
N
L-NMMA (1): R1 = MeNH; R2 = H; n = 1
L-NA (2): R1 = NO2NH; R2 = H; n = 1
L-NAME (3): R1 = NO2NH; R2 = Me; n = 1
L-NIL (4): R1 = Me; R2 = H; n = 2
GW274150 (5)
NH2
AR-C102222 (6)
N
O
S
H
N
N
Cl
O
O
Cl
N
N
HN
O
O
N
N
N
CO2Et
N
H
N
H
O
N
N
NH2
O
NH2
F
BBS-4 (7)
KLYP956 (8)
9
10
Figure 1. Structures of NOS inhibitors.
O
N3
NH2
N
H
1. c-HCl, NaNO2,
O
neat
rt, 1.5 h
O
O
HO
H2O, 0 oC, 10 min
CH3OH, H2SO4
NH2
NH2
N3
HO
HO
H3CO
HO
12 (87%)
H3CO
14 (92%)
80 oC, 18 h
2. NaN3, H2O
0 oC, 40 min
O
HO
N3
NH
N
11
13 (60%)
neat
rt, 6.5 h
HO
15 (90%)
O
NH2
N
H
HO
neat
rt, 6 h
1. c-HCl, NaNO2,
H2O, 0 oC, 10 min
N3
O
O
O
CH3OH, H2SO4
80 oC, 22 h
HO
HO
H3CO
HO
H3CO
19 (97%)
2. NaN3, H2O
NH2
NH2
HO
N3
oC, 60 min
O
N
0
NH
16
17 (92%)
18 (80%)
HO
N3
neat
rt, 30 h
20 (84%)
Scheme 1. Synthesis of azide building blocks.
Cl
1,2,3-triazolyl salicylamide library as
inhibitors.
a novel class of iNOS
Salicylamide is a non-prescription drug with anti-inflammatory,
analgesic and antipyretic properties. Furthermore, the salicylamide
scaffold is frequently found in several anti-inflammatory drugs and
molecules. Many drugs having a salicylamide scaffold or its deriv-
atives, are also orally available.13 Thus, a salicylamide scaffold is a
good starting point for new anti-inflammatory drug development.
In addition, using a 1,2,3-triazole scaffold in iNOS inhibitor devel-
opment would offer several advantages: (i) a 1,2,3-triazole can be
the bioisostere of the amide bond or the heteroaromatic ring,
which is observed in known inhibitors 6–10, (ii) small molecules
containing a 1,2,3-triazole moiety exhibit a wide range of biologi-
cal activity, (iii) substituents can be varied through the use of dif-
ferent azides or alkynes, (iv) 1,2,3-triazoles can be easily
assembled by click chemistry.
21
25
22
23
24
CF3
F
CF3
F
26
Figure 2. Structure of alkyne building blocks.
27
minimal purification, and are versatile in joining diverse structures
without the prerequisite of protection steps. In particular, the
Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC),14
a representative process in click chemistry, provides a straightfor-
Click chemistry encompasses a group of powerful linking reac-
tions that are simple to perform, have high yields, require no or

J. Yoon et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1953–1957
1955
CuSO4 (2 mol%)
Na. Ascorbate
(10 mol%)
diazonium intermediate with sodium azide afforded 13 in 60%
overall yield. Following aminolysis with n-propylamine and piper-
idine yielded azidosalicylamides 14 and 15, respectively. Two
other compounds, the 4-azidosalicylamides 19 and 20 were
synthesized in an analogous manner. In our previous study, sali-
cylamides that obtained from arylamines and salicylic acid showed
a poor aqueous solubility. Thus, in this study, only alkylamines
were used to achieve better physical properties.
O
O
R2
R2
N
N
N
R1
N
R3
+
N
N3
R1
t-BuOH-H2O
rt, 3 d
HO
R3
HO
21 − 27
14, 15, 19, 20
28a − g
29a − g
30a − g
31a − g
Alkynes 21–27 were obtained from commercial sources (Fig. 2).
Synthesis of the triazole library was performed in the presence of
2 mol % of CuSO4 and 10 mol % of sodium ascorbate in 28 conical
tubes (Scheme 2). After three days, the resulting insoluble triazoles
were precipitated by centrifugation, and the supernatant was
removed by decantation. The solid residue was then washed sev-
eral times with H2O and ether to remove residual reagents. Where
a solid was not formed during the reaction, solvent was removed
by GeneVacTM. Then, the residue was washed in the same manner.
All synthesized compounds were submitted to HPLC and 1H NMR
analysis to verify their purity and authenticity. Of 28 compounds
Scheme 2. Click assembly of azides and alkynes.
ward route to 1,2,3-triazoles. To access diverse 1,2,3-triazole struc-
tures, we used CuAAC. Salicylamide-based azides were prepared
and coupled with various alkynes. The preparation of azide precur-
sors 14–15 began from 5-aminosalicylic acid (11), which, upon
treatment with methanol and H2SO4, was converted to the corre-
sponding methyl ester 12 in good yield (87%, Scheme 1). Installa-
tion of an azido group in the aromatic ring was accomplished by
diazotization of the amino group and treatment of the resulting
Table 1
iNOS inhibition of 1,2,3-triazolyl salicylamides synthesized by ‘click chemistry’
N
N
N
O
R3
N
H
HO
28
N
N
N
O
R3
N
HO
29
30
31
N
N
N
R3
HO
H
N
O
N
N
N
R3
HO
N
O
Compound
R3
% inhibitiona
IC50
(l
M)
Compound
R3
% inhibitiona
IC50 (lM)
28a
28b
28c
28d
28e
28f
28g
29a
29b
29c
29d
29e
29f
Phenyl
Isopentyl
n-Hexyl
m-Chlorophenyl
3,5-Difluorophenyl
4-n-Pentylphenyl
3,5-Bis(trifluoromethyl)phenyl
Phenyl
Isopentyl
n-Hexyl
m-Chlorophenyl
3,5-Difluorophenyl
4-n-Pentylphenyl
3,5-Bis(trifluoromethyl)phenyl
28.6
22.6
57.9
33.8
0
0
1.8
11.8
0.9
4.2
>20
>20
19.7
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
12.8
30a
30b
30c
30d
30e
30f
30g
31a
31b
31c
31d
31e
31f
Phenyl
Isopentyl
n-Hexyl
m-Chlorophenyl
3,5-Difluorophenyl
4-n-Pentylphenyl
3,5-Bis(trifluoromethyl)phenyl
Phenyl
Isopentyl
n-Hexyl
m-Chlorophenyl
3,5-Difluorophenyl
4-n-Pentylphenyl
3,5-Bis(trifluoromethyl)phenyl
1.1
7.3
11
0
0
0
0
0
1.9
8.3
0
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
35.4
9.1
18.4
94.1
0
0
0
29g
31g
a
Inhibition at 20 lM.

1956
J. Yoon et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1953–1957
synthesized, 18 were obtained in very high purity (>95%), while the
others were obtained as a substrate-product mixtures, with their
purities estimated to be lower than 90%. The latter compounds
were resynthesized in the presence of 20 mol % of CuSO4, which
led to synthesis of batches with high purity.
LPS (1 g/mL)
A
+
+
+
+
+
29g ( M)
2.5
5
10 15
The 28 triazole compounds were next evaluated for their ability
to inhibit LPS-activated NO production by RAW264.7 macro-
phages. RAW 264.7 cells (5 Â 105 cells/mL) were incubated in
iNOS
-actin
24-well plates for 24 h. Then, LPS (1 lg/mL) and the triazole com-
pounds were added and incubated for another 20 h. As a measure
of NO produced, the accumulation of nitrite, a stable metabolite of
NO in the culture supernatant was assessed using the Griess reac-
tion15 according to a previously documented procedure.16 iNOS
inhibition of the tested triazole compounds was expressed as %
LPS (1 g/mL)
B
+
+
+
+
+
29g ( M)
inhibition at 20 l
M of inhibitor as shown in Table 1.17 Most of
2.5
5
10 15
the 5-(1H-1,2,3-triazol-1-yl)-salicylamides (28a–d, 28g, 29a,
29d–g) inhibited LPS-induced NO production more effectively than
4-(1H-1,2,3-triazol-1-yl)-salicylamides (30a–d, 30g, 31a, 31d–g).
In particular, 28c and 29g18 were the most potent; they exhibited
58% and 94% inhibition, respectively. Their IC50 values are listed in
iNOS
-actin
Table 1 (28c, 19.7
NO production in a dose-dependent manner (Fig. 3A). Under the
same assay condition, the IC50 for -NMMA (a positive control,
non-selective inhibitor of NOS) was 19.9 M. To further ensure
that 29g did not interfere with the survival of the macrophages,
Raw 264.7 cells were treated with 29g for 24 h. Cell viability was
then determined by MTT assay; it was not cytotoxic at the concen-
lM; 29g, 12.8 lM). 29g suppressed LPS-induced
Figure 4. Modulation of iNOS protein (A) and mRNA (B) expression by 29g in LPS-
stimulated macrophages. Cells were stimulated with LPS (1 g/mL) with or without
various concentrations of 29g for 4 h (mRNA) and 16 h (protein). Total RNA and
protein were isolated and further analyzed by RT-PCR and western blots, respec-
tively, as described in Supplementary data.
L
l
l
trations up to15 lM (Fig. 3B).
To clarify the possible mechanism of action involved in the ef-
fects of 29g, we examined the suppressive effects of 29g on iNOS
protein and iNOS mRNA expression in Raw 264.7 cells that were
treated with LPS (1 lg/mL) in the absence or presence of various
concentrations of 29g. As illustrated in Figure 4A, treatment with
LPS (1 lg/mL) for 16 h drastically increased the expression of iNOS
protein; co-treatment with 29g suppressed the expression of iNOS
protein in a concentration-dependent manner. A further study
revealed that 29g significantly inhibits, in
dependent manner, the LPS-induced increase of iNOS mRNA levels.
In particular, at a concentration of 15 M, expression of iNOS
a concentration-
l
protein and RNA was significantly inhibited by 29g. These data
suggest that inhibition of NO production by 29g is correlated with
suppression of iNOS gene and iNOS protein expression.
50
A
40
30
20
10
0
In conclusion, a 28-membered triazole library was synthesized
using a ‘click chemistry’ strategy. The minimum work-up using
centrifugation and decantation afforded pure compounds that
were suitable for the biological evaluation against iNOS. Several
of the synthesized compounds inhibited LPS-activated NO produc-
tion by RAW264.7 macrophages. The inhibitory effects of 29g were
associated with the suppression of iNOS expression. We believe
that this compound is a good candidate for further design of
non-peptidic iNOS inhibitors.
LPS− LPS+
2.5
5
10
15
LPS + 29g (µM)
Acknowledgements
120
100
80
B
This work was supported by the grant from the National Core
Research Center program (No. R15-2006-020) of the Ministry of
Science & Technology and the Korea Science and Engineering Foun-
dation and the grant from Basic Science Research Program (2009-
0075425) through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and Technol-
ogy. J. Yoon was supported by the Brain Korea 21 project.
60
40
20
0
LPS+
2.5
5
10
15
Supplementary data
LPS + 29g (µM)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.034.
Figure 3. Effect of 29g on LPS-induced NO production. (A) RAW 264.7 cells
(5 Â 105 cells/mL) were incubated in 24 well plates for 24 h, and then treated with
LPS (1 lg/mL) in the presence or absence of 29g. After 20 h, supernatants were
References and notes
collected, and nitrite accumulation was measured by the Griess reaction. Nitrite
concentrations were determined by comparison with a sodium nitrite standard
curve. (B) Cell viability was measured by MTT assays as described in Supplementary
data.
1. Bredt, D. S.; Snyder, S. H. Annu. Rev. Biochem. 1994, 63, 175.
2. Palmer, R. M. J.; Ferrige, A. G.; Moncada, S. Nature 1987, 327, 524.

J. Yoon et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1953–1957
1957
3. Olken, N. M.; Rusche, K. M.; Richards, M. K.; Marletta, M. A. Biochem. Biophys.
Res. Commun. 1991, 177, 828.
Czarniecki, M. F.; Rokosz, L. L.; Stauffer, T. M.; Rindgen, D.; Taveras, A. G. Bioorg.
Med. Chem. Lett. 2008, 18, 1864.
4. Furfine, E. S.; Harmon, M. F.; Paith, J. E.; Garvey, E. P. Biochemistry 1993, 32,
8512.
5. Moore, W. M.; Webber, R. K.; Jerome, G. M.; Tjoeng, F. S.; Misko, T. P.; Currie, M.
G. J. Med. Chem. 1994, 37, 3886.
6. Connor, J. R.; Manning, P. T.; Settle, S. L.; Moore, W. M.; Jerome, G. M.; Webber,
R. K.; Tjoeng, F. S.; Currie, M. G. Eur. J. Pharmacol. 1995, 273, 15.
7. Young, R. J.; Beams, R. M.; Carter, K.; Clark, H. A. R.; Coe, D. M.; Chambers, C. L.;
Davies, P. I.; Dawson, J.; Drysdale, M. J.; Franzman, K. W.; French, C.; Hodgson,
S. T.; Hodson, H. F.; Kleanthous, S.; Rider, P.; Sanders, D.; Sawyer, D. A.; Scott, K.
J.; Shearer, B. G.; Stocker, R.; Smith, S.; Tackley, M. C.; Knowles, G. Bioorg. Med.
Chem. Lett. 2000, 10, 597.
14. For recent reviews on the Cu(I)-catalyzed azide-alkyne 1, 3-dipolar
cycloaddition reaction see: Meldal, M.; Tornøe, C. W. Chem. Rev. 2008, 108,
2952.
15. Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J. S.;
Tannenbaum, S. R. Anal. Biochem. 1982, 126, 131.
16. Hong, C. H.; Noh, M. S.; Lee, W. Y.; Lee, S. K. Planta Med. 2002, 68, 545.
17. Nitrite assay. For measuring nitrite accumulation in the media, RAW 264.7
cells (5 Â 105 cells/mL) were incubated in 24-well plates for 24 h, and then LPS
(1 lg/mL) and test samples were added and incubated for 20 h. Samples were
dissolved in DMSO, and final DMSO concentration in cell culture medium did
not exceed 0.1%. As a parameter of NO synthesis nitrite concentration in the
8. Maddaford, S.; Annedi, S. C.; Ramnauth, J.; Rakhit, S. Annu. Rep. Med. Chem.
2009, 44, 27.
9. (a) LaBuda, C. J.; Koblish, M.; Tuthill, P.; Dolle, R. E.; Little, P. J. Eur. J. Pain 2005,
10, 505; (b) Tinker, A. C.; Beaton, H. G.; Boughton-Smith, N.; Cook, T. R.; Cooper,
S. L.; Fraser-Rae, L.; Hallam, K.; Hamley, P.; McInally, T.; Nicholls, D. J.; Pimm, A.
D.; Wallace, A. V. J. Med. Chem. 2003, 46, 913.
supernatants of culture was assessed using the Griess reaction. Briefly, 100
of cell culture supernatants were collected and combined with 180 l of Griess
reagent (1% sulfanilamide in 5% H3PO4 and 0.1% N-(1-
naphthyl)ethylenediamine dihydrochloride solution) in a 96-well plate, and
then absorbance was measured at 540 nm. Nitrite concentrations were
ll
l
determined by comparison with
a
sodium nitrite standard curve.
10. Davey, D. D.; Adler, M.; Arnaiz, D.; Eagen, K.; Erickson, S.; Guilford, W.; Kenrick,
M.; Morrissey, M. M.; Ohlmeyer, M.; Pan, G.; Paradkar, V. M.; Parkinson, J.;
Polokoff, M.; Saionz, K.; Santos, C.; Subramanyam, B.; Vergona, R.; Wei, R. G.;
Whitlow, M.; Ye, B.; Zhao, Z. S.; Devlin, J. J.; Phillips, G. J. Med. Chem. 2007, 50,
1146.
11. Bonnefous, C.; Payne, J. E.; Roppe, J.; Zhuang, H.; Chen, X.; Symons, K. T.;
Nguyen, P. M.; Sablad, M.; Rozenkrants, N.; Zhang, Y.; Wang, L.; Severance, D.;
Walsh, J. P.; Yazdani, N.; Shiau, A. K.; Noble, S. A.; Rix, P.; Rao, T. S.; Hassig, C. S.;
Smith, N. D. .J. Med. Chem. 2009, 52, 3047.
12. Blasko, E.; Glaser, C. B.; Devlin, J. J.; Xia, W.; Feldman, R. I.; Polokoff, M. A.;
Phillips, G. B.; Whitlow, M.; Auld, D. S.; McMillan, K.; Ghosh, S.; Stuehr, D. J.;
Parkinson, J. F. J. Biol. Chem. 2002, 277, 295.
13. (a) Kung, P.-P.; Huang, B.; Zhang, G.; Zhou, J. Z.; Wang, J.; Digits, J. A.;
Skaptason, J.; Yamazaki, S.; Neul, D.; Zientek, M.; Elleraas, J.; Mehta, P.; Yin, M.-
J.; Hickey, M. J.; Gajiwala, K. S.; Rodgers, C.; Davies, J. F.; Gehring, M. R. J. Med.
Chem. 2010, 53, 499; (b) Lai, G.; Merritt, J. R.; He, Z.; Feng, D.; Chao, J.;
Experiments were performed at least three times with triplicate tests. In
these assays, 100% activity was defined as the difference between NO
production in the absence and presence of LPS for 20 h.% of Inhibition was
expressed as [1
– (NO level of sample/NO level of vehicle-treated
control)] Â 100. IC50 value was calculated using nonlinear regression
analyses (percent inhibition versus concentration).
18. Yield: 99%. TLC: Rf 0.24 (1:1 hexane/EtOAc). 1H NMR (400 MHz, DMSO-d6): d
10.48 (br s, 1H), 9.59 (s, 1H), 8.57 (s, 2H), 8.12 (s, 1H), 7.82 (dd, 1H, J = 8.8 Hz,
2.8 Hz), 7.66 (d, 1H, J = 2.8 Hz), 7.09 (d, 1H, J = 8.8 Hz), 3.60–3.22 (m, 4H), 1.65–
1.60 (m, 2H), 1.58–1.50 (m, 4H). 1H NMR (400 MHz, benzene-d6): d 11.04 (br s,
1H), 8.27 (s, 2H), 7.72 (s, 1H), 7.70 (d, 1H, J = 2.4 Hz), 7.03 (d, 1H, J = 8.8 Hz),
6.93 (s, 1H), 6.83 (dd, 1H, J = 8.8 Hz, 2.4 Hz), 3.25–3.20 (m, 4H), 1.11–1.05 (m,
6H). 13C NMR (100 MHz, benzene-d6): d 169.5, 160.7, 145.4, 133.4, 132.5 (q, JC-
F = 33.6 Hz), 127.0, 125.8 (m), 124.3, 123.8 (q, JC-F = 270.9 Hz), 121.6 (m), 121.5,
119.1, 118.6, 118.5, 46.7, 25.9, 24.2. LRMS (FAB) m/z (rel int): (pos) 485
([M+H]+, 100). HRMS m/z calcd C22H19F6N4O2 485.1412; found 485.1420.