Fluorinated tetraketone derivatives of N-substituted carbazoles and their Eu(III) complexes for fluorescence immunoassay

Article abstract of DOI:10.1007/s10593-018-2300-4

[Figure not available: see fulltext.] New fluorinated tetraketone derivatives of N-substituted carbazoles were synthesized and tested as ligands for fluorescence immunoassay. The spectral properties of the obtained heterocyclic tetraketones and their Eu(III) complexes were studied. The complexes showed longwave absorption at 360–380 nm, high extinction coefficient values, long lifetime of excited states, and intense luminescence, allowing to consider the use of such lanthanide complexes in immunofluorescence analysis.

Full text of DOI:10.1007/s10593-018-2300-4

DOI 10.1007/s10593-018-2300-4
Chemistry of Heterocyclic Compounds 2018, 54(5), 528–534
Fluorinated tetraketone derivatives of N-substituted carbazoles
and their Eu(III) complexes for fluorescence immunoassay
Dmitriy E. Pugachov1,2, Tatiana S. Kostryukova2, Georgy V. Zatonsky2,
Sergey Z. Vatsadze3, Nikolai V. Vasil'ev1,2*
1 Moscow State Regional University,
10а Radio St., Moscow 105005, Russia; e-mail: nikolai-vasilev@mail.ru
2 State Research Institute of Biological Engineering,
75/1 Volokolamskoe Shosse, Moscow 125424, Russia; e-mail: pugachovdmitry@gmail.com
3 Lomonosov Moscow State University,
1-3 Leninskie Gory, Moscow 119991, Russia; e-mail: zurabych@gmail.com
Translated from Khimiya Geterotsiklicheskikh Soedinenii,
2018, 54(5), 528–534
Submitted November 7, 2017
Accepted March 28, 2018
New fluorinated tetraketone derivatives of N-substituted carbazoles were synthesized and tested as ligands for fluorescence
immunoassay. The spectral properties of the obtained heterocyclic tetraketones and their Eu(III) complexes were studied. The complexes
showed longwave absorption at 360–380 nm, high extinction coefficient values, long lifetime of excited states, and intense luminescence,
allowing to consider the use of such lanthanide complexes in immunofluorescence analysis.
Keywords: carbazole derivatives, europium, fluorinated β-diketones, tetraketones, immunofluorescence analysis, luminescence.
Europium complexes of fluorinated aromatic β-di-
ketones are some of the most successful reagents for
immunofluorescence analysis used in the medical
diagnostics of various pathologies.1–5 Suitable reagents
of this type must meet the following criteria: maximum
stability of the rare earth metal complex, high extinction
coefficients of the ligand, sufficient triplet state energy
of the ligand for sensibilization of the metal ion, as well
as long luminescence decay time of the obtained
complexes.6–9 A series of new heterocyclic compounds
with advanced properties have been obtained over the
recent decades, namely, such as complex longwave
absorption maxima at 360–380 nm, high values of the
extinction coefficient, and increased stability constants of
europium complexes.10–15 The stability of the complexes
was improved along with an increased number of chelating
groups, while the inclusion of a longer conjugated chain in
the ligand and the presence of a dibenzoheterocyclic
system led both to increased extinction coefficient as well
as to bathochromic shift of the absorption maximum for
these complexes.12
Compounds
belonging
to
the
series
of
dibenzoheterocyclic tetraketones (bis-β-diketones) include
various dibenzothiophenes 1а10,11 and 1b,12 dibenzofurans
2,12 2,7-disubstituted carbazole 3a,13 and 3,6-disubstituted
carbazoles 3b,c.12 Of particular interest are tetraketone
derivatives of carbazole 3, since aqueous solutions of their
complexes with europium ions show longwave absorption
bands with large extinction coefficient values (λmax 370–
400 nm, ε 3–4·104 mol–1·l·cm–1) and long lifetime of the
luminescent state (between 500 and 700 µs).12–14 Carbazole-
based tetraketone 3, which contains a ketone linker with
(CF2)4CO2Me functional groups, is a marker that can be
used as universal reagent for various schemes of biological
microanalysis.16
The preparation of fluorinated tetraketones on the basis
of N-ethyl-substituted carbazoles 3a,b and the formation of
their europium complexes have been described in a patent12
and a journal article.13 Other N-substituted fluorinated
tetraketones of carbazole series have not yet been obtained
and their complexation properties and sensibilization of
luminescence with europium ions have not been studied.
0009-3122/18/54(5)-0528©2018 Springer Science+Business Media, LLC
528

Chemistry of Heterocyclic Compounds 2018, 54(5), 528–534
ClO2S
S
O
O
F2 F2
C
C
C
C
F2
F2
O
O
S
1a
SO2Cl
O
O
O
O
R
R
F3C
CF3
O
O
N
O
O
Et
X
3a
1b
2
3b
3c
X = NEt, X = NH
X = S, X = O,
R = CF3, CF3(CF2)n, CF4CO2Me
Figure 1. Structural formulas of tetraketone derivatives of dibenzoheterocycles obtained in this study.
Thus, in the current work we considered it worthwhile to
Scheme 2
obtain fluorinated tetraketone derivatives of N-substituted
carbazoles and to study their spectral properties, as well as
to determine the spectral-luminescence characteristics of
their europium complexes, including experiments under
buffered conditions suitable for performing immunoassays.17
The introduction of aryl substituents at the nitrogen atom
was considered an advantage, since such substituents
exhibit auxochromic effects and can improve the spectral
characteristics of compounds.
The synthesis of the target compounds 3c–h was
accomplished by using various routes, which provided the
products in preparatively useful yields (Schemes 1–4).
Carbazole 4c was alkylated with methyl iodide in the
presence of sodium hydroxide, with the formation of
N-methylcarbazole 4d18 in 95% yield. In order to obtain the
previously known N-phenylcarbazole 4e, carbazole 4c was
arylated with phenyl iodide in the presence of lithium
chloride and copper iodide.19 4-Nitrophenylcarbazole 4f
was obtained through arylation of carbazole potassium salt
with nitrobenzene according to a published procedure,20
which involved an oxidative process.
in the presence of strong bases (Scheme 3). The alkylation
occurred exclusively at the nitrogen atom without affecting
the acetyl groups, as evidenced by NMR spectra.
Scheme 3
O
O
O
O
Me
Me
Me
Me
i for 5g
5h
ii for
N
N
H
R
5c
5g R = Ph (77%)
i: PhCH2Cl, t-BuOK, DMF, rt, 6 h
ii: 4-O2NC6H4CH2Br, LiH, DMF, rt, 14h
Scheme 1
h R = 4-NO2C6H4 (93%)
i for 4d
ii for 4e
iii for 4f
Synthesis of the target tetraketones 3c–h was performed
by Claisen reaction using lithium hydride as base. It is
known that the use of lithium hydride promotes
condensation reactions involving esters of fluorinated
acids,22 and compounds 3c–h were obtained in sufficiently
high yields (Scheme 4), including the previously known
compound 3с.12
N
R
N
H
4c
4d R = Me (95%)
e R = Ph (70%)
f R = 4-O2NC6H4 (68%)
i: MeI, NaOH, DMSO, rt, 5 h
ii: PhI, K2CO3, LiCl, CuI, DMF, 140°C, 48 h
iii: PhNO2, t-BuOK, Et2O, 50°C, 16 h
1H and 19F NMR spectra of the obtained compounds 3c–h
were in agreement with the proposed structures. An
unusual physicochemical property observed for the
majority of the synthesized carbazoles was their low
solubility in various types of solvents, which prevented the
acquisition of 13C NMR spectra for compounds 3f,h.
Similarly to other fluorinated 1,3-dicarbonyl compounds,23
compounds 3c–h existed in their enol forms in solutions.
The enol proton of these compounds, as a rule, was not
The acetylation of carbazoles 4c–e was performed under
Friedel–Crafts reaction conditions according to the
standard procedure21 in dichloromethane and the formation
of diacetylcarbazoles 5c–e occurred in satisfactory yields
(Scheme 2).
Diacetylcarbazole 5c was found to be a good starting
material for the preparation of N-benzyl- and N-4-nitro-
benzyl-substituted diacetylcarbazoles 5g,h, which were
obtained by treatment with the appropriate benzyl halides
1
manifested in H NMR spectra. IR spectra also showed
absorption bands in the region of 1583–1675 cm–1 that
were characteristic for the enol form СОС=СОН.24
529

Chemistry of Heterocyclic Compounds 2018, 54(5), 528–534
Scheme 4
The N-substituted tetraketones 3d–g could be
conveniently recrystallized from i-PrOH–EtOAc mixture,
compound 3f – from DMSO, while compound 3h was
recrystallized from EtOAc. No suitable solvent system
could be identified for the recrystallization of NH-
derivative 3c. The purification of compound 3c was
performed by sorption on alumina, followed by washing of
the adsorbed product from impurities and elution of the
target compound by CHCl3–MeOH mixture containing
10% of formic acid.
UV absorption spectra recorded for aqueous solutions of
the substituted carbazoles 3c–h (Table 1) showed
pronounced maxima in the region of 354–364 nm and, as a
rule, also in the region of 321–325 nm. The latter
maximum was absent in UV spectrum of compound 3f,
apparently due to the influence of the conjugated nitro
group. The absorption spectra were in agreement with the
previously published data about longwave absorption
features observed for tetraketone derivatives of
carbazole.12,16
Figure 2. Absorption spectra of carbazoles 3с,е and their complexes
with Eu3+ ions in the presence of TOPO (ligand–Eu–TOPO ratio
3:2:6) (ligand concentration 10–5 M) in Tris buffer (0.05 М) and
Triton Х-100 (0.1%).
well as an increase of optical density was observed along
with the emergence of absorption maximum in the region
of 380 nm, which is characteristic for complex formation
with fluorinated bis-β-diketones.12,16 Besides that,
complexes of all compounds showed strong, prolonged
luminescence (Table 2).
It should be noted that in the case of N-substituted
carbazoles 3d–h the introduction of electron-donating
substituents (R = Me (compound 3d), Ph (compound 3e), or
CH2Ph (compound 3g)) increased the duration and intensity
of luminescence compared to the unsubstituted carbazolyl
tetraketone 3c in aqueous medium and micellar systems, as
well as relative to the nitro derivatives 3f,h. The weaker
luminescence characteristics of complexes 3f,h can be
linked to the known effect of luminescence quenching due
to the presence of nitro group.26 The luminescence intensity
was substantially higher in micellar solution in the presence
of Triton Х-100. There were no substantial differences of
The spectral-luminescence properties of 3:2 Eu3+
complexes of tetraketones 3c–h were studied in aqueous
solutions with an excess of a known luminescence-
enhancing agent – trioctylphosphine oxide (TOPO) in
buffer solutions (Tris buffer) in the presence of surfactant
(Triton X-100) or without it. The role of the hydrophobic
coligand TOPO is to saturate the coordination sphere of
europium ion and to exclude water molecules from it,
which would otherwise quench the luminescence.25
The absorption spectrum of N-phenyl-substituted carbazole
3е, compared to the spectrum of unsubstituted carbazole 3с,
as well as the spectra of their europium complexes in the
presence of TOPO are presented in Figure 2. The
luminescence characteristics of the complexes are given in
Table 2. The complex formation of compounds 3c–h with
europium ion was monitored from absorption spectra: the
absorption maxima of the complexes were shifted to the
long wavelength region compared to free molecules, as
Table 2. Luminescence of tetraketone 3c–h
complexes with Eu3+ ions and TOPO in 0.05 M aqueous Tris
buffer solution and Triton Х-100 (0.1%)*
Tris buffer, рН 7.2
λexc, I, τ,
Tris buffer, рН 7.2 + Triton Х-100
Table 1. The absorption spectra of carbazoles 3c–h in aqueous
solution of Tris buffer (0.05 M) and Triton Х-100 (0.1%)*
Ligand
λexcit
nm
,
I,
τ,
µs
nm arb units ×104 µs
arb units ×104
Com-
pound
λabs, nm Optical density, arb units
ε·104 mol·l–1·cm–1
378
378
374
368
376
374
35.6
48.3
55.4
19.1
56.2
11.8
560
575
565
520
575
434
372
374
368
362
370
334
125.4
142.5
161.8
68.9
686
701
711
692
710
568
3c
3d
3e
3f
321, 355
323, 364
325, 357
354
0.356; 0.371
0.379; 0.381
0.436; 0.393
0.428
3.7 (355 nm)
3.8 (364 nm)
3.9 (357 nm)
4.3
3c
3d
3e
3f
150.0
37.2
3g
3h
325, 362
325, 358
0.399; 0.376
0.369; 0.340
3.8 (362 nm)
3.4 (358 nm)
3g
3h
* The concentration of compounds 3c–h was 10–5 M.
* Ligand–Eu–TOPO = 3:2:6, concentration of ligand 10–5 M, λemiss 615 2 nm.
530

Chemistry of Heterocyclic Compounds 2018, 54(5), 528–534
Table 4. Luminescence of Eu3+ complexes of carbazoles 3c,e,g
luminescence parameters between compounds containing a
under buffered conditions*
phenyl ring conjugated to the carbazole system or a non-
conjugated benzyl analog.
1 h
3 days
The quantum yields were determined relative to the known
value for naphthoyltrifluoroacetone (NTA) in enhancing
solution (0.69)27 according to the following formula:28
Ligand
λexc
,
I,
τ,
λexc
,
I,
τ, µs
nm arb units ×104 µs
nm arb units ×104
372
370
374
116,0
153,4
138,4
657
628
614
376
372
376
64,4
110,0
105,1
601
617
594
3c
3e
3g
Φx = ((It,x·εref·сref)/(It,ref·εx·сx))·Φref,
where It,x and It,ref – integrated luminescence intensities for
unknown and reference compounds, ε (mol/(l·cm)) – the
values of extinction coefficients, с (mol/l) – concentrations
of the analyzed and known compounds, Φ – quantum yield.
The enhancing solution was prepared according to a
procedure described in patent.29
Despite the fact that the quantum yields of the
complexes of the obtained compounds were lower than for
the reference compound NTA, the so-called brightness
parameter – the product of extinction coefficient and
quantum yield (Table 3) was higher for the studied
complexes, which in turn allowed to obtain superior
luminescence intensity and, as a consequence, to improve
the sensitivity of the analysis.
Compounds 3e,g were selected for studying the
luminescence under the same conditions as used for
immunoassay (in buffer solution, Table 4). In order to
decrease the influence of protein present in the commercial
reagent on the complex formation process, the complexes
were obtained in dimethylformamide by maintaining the
ligand–Eu3+–TOPO (3:2:6) mixture for 2 h. These
experiments revealed good intensity and lifetime of
luminescence phenomena. When the ligand concentration
was at the level of 10–5 М, the complexes of compounds
3e,g showed superior stability over several days, compared
to unsubstituted carbazole 3c. The complex with NTA lost
90% of its luminescence over the first 4 h.
By studying the methods for preparation of fluorinated
tetraketones on the basis of N-substituted carbazoles, we
developed preparative procedures for the synthesis of the
target compounds, including new 1,1'-(9Н-carbazole-3,6-
diyl)bis(4,4,4-trifluorobutane-1,3-diones). The spectral-
luminescence characteristics of the obtained tetraketones as
complexes with europium ion allow to view them as
promising reagents for applications in time-resolved
immunoassays.
* Ligand–Eu–TOPO ratio = 3:2:6, ligand concentration 10–5 M, λemiss 615
2
Experimental
IR spectra over the range of 400–3600 cm–1 were
1
recorded on a Vector 22 spectrometer in KBr pellets. H,
19F, and 13С NMR spectra were acquired on JEOL JNM-
ECX400 (400, 376 and 100 MHz, respectively) and Bruker
Avance III 500 (500, 470 and 125 MHz, respectively)
instruments in DMSO-d6, using TMS as internal standard
for 1H and 13C NMR spectra or CFCl3 for 19F NMR spectra.
1Н and 13С NMR signals were assigned by using gradient
versions of two-dimensional 1H–1H COSY, 1H–1H
TOCSY, 1H–1H ROESY, 1H–13C HSQC, and 1H–13C
1
HMBC experiments. Mixing time was 80 ms for H–1H
TOCSY experiment, 200 ms for 1H–1H ROESY
experiment, while 1H–13C HMBC experiment was
optimized for spin-spin coupling constant JCH = 8.0 Hz.
Elemental analysis was performed on a PerkinElmer CHN
РЕ 2400 SII instrument. Melting points were determined in
glass capillaries on
a Gallenkamp MPD350.BM3.5
apparatus. Electronic absorption spectra were recorded on a
Shimadzu UV-1650PC spectrometer in 1-cm quartz
cuvette. The luminescence of europium complexes
obtained from carbazoles 3c–h was registered using a
Thermo Fisher Scientific Varioscan Flash microplate
reader with time delay of 100 µs (td) and integration time of
1000 µs (tg) in the presence of air oxygen.
Commercial reagents and solvents were used in this
work, which had been prepared according to published
procedures.30 The medium for buffered analysis procedure
was obtained from TTG-Neoscreen commercial reagent kit
(Immunoscreen, Russia).
The properties and spectral characteristics of compounds
3c, 4d–f, 5c,d were in agreement with the available
literature data.12,18–21,28,31
9-Methyl-9Н-carbazole (4d).18 A mixture of carbazole 4с
(1.0 g, 5.99 mmol) in DMSO (15 ml) and NaOH (0.48 g,
12.00 mmol) was cooled and treated by the addition of MeI
(1.28 g, 9.01 mmol) with stirring at room temperature for
5 h. The mixture was then poured into ice water (100 ml).
The obtained precipitate was filtered off, washed wih water
(3×25 ml), and dried at reduced pressure over P2O5. The
compound was used for the further synthetic steps without
additional purification. Yield 1.03 g (95%), white powder,
mp 90–93°C (mp 90–92°C18).
Table 3. The relative quantum yields of Eu3+ complexes of
tetraketones 3c–h in the presence of enhancing solution* and their
brightness parameters
Ligand
Quantum yield
ε·Φ
0.24
0.27
0.28
0.11
0.27
0.07
0.69
26640
30780
34440
14190
31890
7770
3c
3d
3e
3f
9-Phenyl-9Н-carbazole (4e).19 A solution of carbazole
4с (0.50 g, 2.99 mmol) in DMF (25 ml) was treated by the
addition of K2CO3 (1.24 g, 8.98 mmol), CuI (0.57 g,
0.29 mmol), LiCl (0.13 g, 2.99 mmol), and iodobenzene
3g
3h
NTA
26320
* Eu3+ concentration of 10–6 М (λexcit 340 2, λemiss 615 2 nm).
531

Chemistry of Heterocyclic Compounds 2018, 54(5), 528–534
(0.67 g, 3.29 mmol). The mixture was stirred under argon
AcCl (0.87 ml, 12.12 mmol), and compound 4f (1.00 g,
3.47 mmol, added in the form of 3% solution in CH2Cl2)
analogously to the procedure for preparation of carbazole
derivative 5c. The yellow powder was washed during the
isolation procedure with EtOH (40 ml) and Me2CO (40 ml).
Yield 1.07 g (83%), yellow powder, stable to 320°С.
atmosphere at 140°С for 48 h. After cooling to room
temperature, the mixture was poured into 10% NH4Cl
solution (50 ml), extracted with EtOAc (3×20 ml), the organic
phase was washed with 10% NH4Cl solution (2×10 ml) and
H2O (2×10 ml), and dried over anhydrous Na2SO4. The
solvent was evaporated at reduced pressure, and the residue
was recrystallized from MeOH. Yield 0.51 g (70%), white
needles, mp 87–88°С (mp 86–87°C19).
1
IR spectrum, ν, cm−1: 1687 (С=О). H NMR spectrum,
δ, ppm (J, Hz): 2.70 (6H, s, CH3); 7.57 (2H, d, J = 7.8,
H carbazole); 8.00 (2Н, d, J = 8.0, Н Ar); 8.09 (2Н, d, J = 8.0,
Н Ar); 8.52 (2H, d, J = 7.8, H carbazole); 9.15 (2H, s,
H carbazole). Found, %: C 70.90; H 4.27; N 7.47.
C22H16N2O4. Calculated, %: C 70.96; H 4.33; N 7.52.
1,1'-(9-Benzyl-9Н-carbazole-3,6-diyl)diethanone (5g). A
suspension of carbazole 5c (0.31 g, 1.24 mmol) and
t-BuOK (0.55 g, 4.96 mmol) in DMF (10 ml) was treated
by the addition of benzyl chloride (0.31 g, 1.86 mmol) as a
solution in DMF (10 ml). After the addition was complete,
the mixture was stirred at room temperature for 6 h. The
reaction mixture was poured into 5% hydrochloric acid
(40 ml) with ice, the precipitate was filtered off, the
obtained solid was washed with H2O (3×30 ml) and then
with cold EtOH. The product was recrystallized from
EtOAc. Yield 0.33 g (77%), yellowish crystals, mp 236–
238°С. IR spectrum, ν, cm−1: 1698 (С=О). 1H NMR
spectrum, δ, ppm (J, Hz): 2.66 (6H, s, CH3); 3.75 (2H, s,
CH2); 7.15–7.27 (5Н, m, H Ph); 7.66 (2H, d, J = 7.8,
H carbazole); 8.06 (2H, dd, J = 7.8, J = 1.2, H carbazole);
9.05 (2H, d, J = 1.2, H carbazole). Found, %: C 80.96;
H 5.65; N 4.14. C23H19NO2. Calculated, %: C 80.92;
H 5.61; N 4.10.
9-(4-Nitrophenyl)-9Н-carbazole (4f).20 A suspension
of carbazole 4с (2.42 g, 14.49 mmol) in anhydrous Et2O
(50 ml) was treated with a single portion of t-BuOK
(1.95 g, 17.41 mmol) and stirred at room temperature for
1.5 h. The precipitate was filtered off, washed with Et2O
(2×15 ml), and dried at reduced pressure. The obtained
potassium salt of carbazole was added to nitrobenzene (30 ml)
and the dark-brown mixture was stirred at 50°С for 16 h.
Then water (~50 ml) was added and nitrobenzene was
removed by steam distillation, the procedure was repeated
until the removal of nitrobenzene was complete. The
reaction mixture was then cooled, the yellowish-brown
precipitate was filtered off, washed with water (3×40 ml),
air-dried, and recrystallized from PhMe. Yield 2.84 g
(68%), yellow crystals, mp 208–210°C (mp 205–208°C20).
1,1'-(9Н-Carbazole-3,6-diyl)diethanone (5c).21 A suspen-
sion of AlCl3 (4.27 g, 32.10 mmol) in CH2Cl2 (35 ml) was
cooled and treated with AcCl (1.72 ml, 24.07 mmol) in
CH2Cl2 (8 ml). After a light-yellow mixture was formed, it
was cooled and treated by portionwise addition of
carbazole 4с (1.34 g, 8.08 mmol), the mixture was stirred
for 1 h at room temperature and then refluxed for 1.5 h.
The reaction mixture was poured into 15% hydrochloric
acid (200 ml) with ice. The obtained gray-brown
precipitate was filtered off, washed with 5% hydrochloric
acid, then with water to neutral pH, and recrystallized from
a mixture of EtOH–CHCl3 with activated carbon. Yield
1.52 g (75%), white crystals, mp 233–235°C (mp 233°C21).
1,1'-(9-Methyl-9Н-carbazole-3,6-diyl)diethanone (5d)
was obtained from AlCl3 (3.10 g, 23.48 mmol), AcCl (1.18
ml, 16.44 mmol), and carbazole 4d (0.85 g, 4.69 mmol)
analogously to the procedure for the preparation of
carbazole derivative 5c. The product was recrystallized
from Me2CO. Yield 0.94 g (76%), white crystals, mp 191–
193°С (mp 195°С31).
1,1'-[9-(4-Nitrobenzyl)-9Н-carbazole-3,6-diyl]dietha-
none (5h). A solution of compound 5c (0.51 g, 2.03 mmol)
and LiH (0.08 g, 10.13 mmol) in DMF (20 ml) was cooled
and treated by the addition of 4-nitrobenzyl bromide
(0.57 g, 2.64 mmol) as a solution in DMF (10 ml). A beige
precipitate formed after the addition, the suspension was
stirred for 14 h at room temperature, then poured into ice-
cold 5% hydrochloric acid solution (40 ml), the precipitate
was filtered off and washed with H2O, EtOH, and Me2CO.
Yield 0.73 g (93%), yellow powder. Mp 283–286°С
1
(decomp.). IR spectrum, ν, cm−1: 1694 (С=О). H NMR
spectrum, δ, ppm (J, Hz): 2.68 (6H, s, CH3); 5.95 (2H, s,
CH2); 7.32 (2H, d, J = 7.8, H carbazole); 7.74 (2Н, d,
J = 7.9, Н Ar); 8.08 (2H, dd, J = 7.8, J = 1.2, H carbazole);
8.13 (2H, d, J = 7.9, H Ar); 9.09 (2H, d, J = 1.2, H carbazole).
Found, %: C 71.57; H 4.75; N 7.33. C23H18N2O4.
Calculated, %: C 71.49; H 4.70; N 7.25.
1,1'-(9-Phenyl-9Н-carbazole-3,6-diyl)diethanone (5e)
was obtained at room temperature from AlCl3 (0.685 g,
5.14 mmol), AcCl (0.22 ml, 3.08 mmol), and compound 4e
(0.25 g, 1.03 mmol, added in the form of 10% solution in
CH2Cl2) analogously to the procedure for the preparation of
carbazole derivative 5c. The product was recrystallized
from EtOH. Yield 0.28 g (81%), white powder, mp 163–
165°С. IR spectrum, ν, cm−1: 1690 (С=О). 1H NMR
spectrum, δ, ppm (J, Hz): 2.68 (6H, s, CH3); 7.39 (2H, d,
J = 7.8, H carbazole); 7.57–7.72 (5Н, m, Н Ph); 8.05 (2H,
dd, J = 7.8, J = 1.2, H carbazole); 9.11 (2H, d, J = 1.2,
H carbazole). Found, %: C 80.65; H 5.19; N 4.11.
C22H17NO2. Calculated, %: C 80.71; H 5.23; N 4.28.
Preparation of carbazoles 3c–h (General method). A
mixture of carbazole 5с–h (0.68 mmol) and THF (15 ml)
was stirred and treated by the addition of LiH (0.03 g,
3.40 mmol) and methyl trifluoroacetate (0.26 g,
2.03 mmol). The reaction mixture was stirred for 1.5–5 h at
63°С, evaporated, and treated with cold 5% hydrochloric
acid solution (20 ml). The precipitate was filtered off,
washed with water, and then dried over P2O5.
1,1'-(9Н-Carbazole-3,6-diyl)bis(4,4,4-trifluorobutane-
1,3-dione) (3c).12 The precipitate was dissolved in CHCl3,
adsorbed on alumina that was prepared by heating
according to Brockmann method, and the impurities were
1,1'-[9-(4-Nitrophenyl)-9Н-carbazole-3,6-diyl]dietha-
none (5f) was obtained from AlCl3 (2.31 g, 17.37 mmol),
532

Chemistry of Heterocyclic Compounds 2018, 54(5), 528–534
removed by elution with 3:1 CHCl3–MeOH system (200 ml).
123.1 (C-4a,b carbazole); 123.2 (C-4,5 carbazole); 125.0
(C-3',5' Ar); 127.2 (C-2,7 carbazole); 127.3 (C-2',6' Ar); 128.1
(C-4' Ar); 129.2 (C-3,6 carbazole); 136.9 (C-1' Ar); 145.0
(C-8a,9a carbazole); 173.4 (q, JCF = 36.7, 2=COH); 187.5
(2C=O). Found, %: C 60.84; H 3.30; N 2.71. C27H17F6NO4.
Calculated, %: C 60.80; H 3.21; N 2.63.
Carbazole (3c) was eluted from the sorbent with the same
solvent system, but additionally containing formic acid
(10%). The eluate was evaporated at reduced pressure.
Yield 0.26 g (87%), light-yellow powder, mp 263–264°С
(decomp.) (mp 260°C (decomp.)12).
1,1'-(9-Methyl-9Н-carbazole-3,6-diyl)bis(4,4,4-trifluo-
robutane-1,3-dione) (3d). The precipitate was recrystalli-
zed from 1:1 mixture of i-PrOH–EtOAc. Yield 0.26 g (83%),
yellow crystals, mp 229–232°C. IR spectrum, ν, cm−1: 1611,
1583 (С(O)=COH). 1H NMR spectrum, δ, ppm (J, Hz): 3.95
(3Н, s, CH3); 7.15 (2H, s, =CH); 7.78 (2H, d, J = 7.8,
H carbazole); 8.27 (2H, dd, J = 7.8, J = 1.2, H carbazole);
9.17 (2H, d, J = 1.2, H carbazole). 19F NMR spectrum, δ, ppm:
–75.12 (6F, s, 2CF3). 13C NMR spectrum, δ, ppm (J, Hz):
30.3 (CH3); 93.1 (2CH=); 111.0 (C-1,8 carbazole); 118.0
(q, JCF = 293.6, 2CF3); 123.6 (C-4а,b carbazole); 123.7
(C-4,5 carbazole); 128.2 (C-2,7 carbazole); 130.1 (C-3,6
carbazole); 144.9 (C-8а,9а carbazole); 173.3 (q, JCF = 36.7,
2=COH); 187.8 (2C=O). Found, %: C 55.23; H 2.94;
N 3.11. C21H13F6NO4. Calculated, %: C 55.15; H 2.87; N 3.06.
1,1'-(9-Phenyl-9Н-carbazole-3,6-diyl)bis(4,4,4-trifluoro-
butane-1,3-dione) (3e). The precipitate was recrystallized
analogously to compound 3d. Yield 0.28 g (79%), yellow
plates, mp 222–225°C. IR spectrum, ν, cm−1: 1607, 1592
(С(O)=COH). 1H NMR spectrum, δ, ppm (J, Hz): 7.15 (2H,
s, =CH); 7.45 (2Н, d, J = 7.8, H carbazole); 7.60–7.74 (5Н,
m, H Ph); 8.24 (2H, dd, J = 7.8, J = 1.2, H carbazole); 9.32
(2H, d, J = 1.2, H carbazole). 19F NMR spectrum, δ, ppm:
–76.57 (6F, s, 2CF3). 13C NMR spectrum, δ, ppm (J, Hz):
93.1 (2CH=); 111.4 (C-1,8 carbazole); 118.0 (q,
JCF = 282.4, 2CF3); 123.5 (C-4a,b carbazole); 123.6 (C-4,5
carbazole); 125.9 (C-4' Ph); 127.4 (C-2,7 carbazole); 127.8
(C-2',6' Ph); 129.6 (C-3',5' Ph); 131.0 (C-3,6 carbazole); 135.6
(C-1' Ph); 145.1 (C-8a,9a carbazole); 173.4 (q, JCF = 36.5,
2=COH); 187.3 (2C=O). Found, %: C 60.21; H 2.96; N 2.77.
C26H15F6NO4. Calculated, %: C 60.12; H 2.91; N 2.70.
1,1'-[9-(4-Nitrobenzyl)-9Н-carbazole-3,6-diyl]bis(4,4,4-
trifluorobutane-1,3-dione) (3h). The precipitated product
was recrystallized from EtOAc. Yield 0.27 g (69%), yellow
powder, mp 246–250°C (decomp.). IR spectrum, ν, cm−1:
1675, 1667 (С(O)=COH). 1H NMR spectrum, δ, ppm (J, Hz):
5.90 (2Н, s, СН2); 7.17 (2H, s, =CH); 7.35 (2Н, d, J = 7.7,
H Ar); 7.87 (2H, d, J = 7.8, H carbazole); 8.13 (2Н, d,
J = 7.7, Н Ar); 8.25 (2H, dd, J = 7.8, J = 1.2, H carbazole);
9.30 (2H, d, J = 1.2, H carbazole). 19F NMR spectrum,
δ, ppm: –75.37 (6F, s, 2CF3). Found, %: C 56.15; H 2.89;
N 4.95. C27H16F6N2O6. Calculated, %: C 56.07; H 2.79;
N 4.84.
Preparing solutions of Eu3+ complexes of carbazoles 3c–h
and TOPO in Tris buffer (General method). The
calculated amount of ligand 3c–h was dissolved in DMF
(2 ml) in order to obtain a solution with concentration equal
to 5 × 10–3 М. An aliquot of the solution was diluted with
0.05 М Tris buffer (рН 7.2) to obtain ligand concentration of
10–5 М. A freshly prepared 10–4 М solution of Eu3+ ions in
10–5 М НСl was added, followed by 2 × 10–4 М solution of
TOPO in EtOH and the necessary amount of Tris buffer in
order to obtain the desired solution of ligand–Eu–TOPO in
3:2:6 ratio containing 10–5 M of the ligand. This solution
was left in darkness for 2 h to allow for complex formation.
In order to obtain the micellar solution, 10% of Triton
Х-100 in EtOH was added to obtain the final concentration
of surfactant equal to 0.1%. The spectral-luminescence
characteristics were studied with 100 μl portions of the
complex solutions.
References
1,1'-[9-(4-Nitrophenyl)-9Н-carbazole-3,6-diyl]bis(4,4,4-
trifluorobutane-1,3-dione) (3f). The precipitated product
was recrystallized from DMSO (120°С). Yield 0.30 g
(77%), yellow needles, mp 276°C (decomp.). IR spectrum,
ν, cm−1: 1610, 1593 (С(O)=COH). 1H NMR spectrum, δ, ppm
(J, Hz): 7.17 (2H, s, =CH); 7.62 (2H, d, J = 7.8,
H carbazole); 8.03 (2Н, d, J = 7.7, Н Ar); 8.25 (2H, dd,
J = 7.8, J = 1.2, H carbazole); 8.54 (2Н, d, J = 7.7, H Ar);
9.30 (2H, d, J = 1.2, H carbazole). 19F NMR spectrum, δ, ppm:
–73.77 (6F, s, 2CF3). Found, %: C 55.42; H 2.61; N 5.03.
C26H14F6N2O6. Calculated, %: C 55.33; H 2.50; N 4.96.
1,1'-(9-Benzyl-9Н-carbazole-3,6-diyl)bis(4,4,4-trifluoro-
butane-1,3-dione) (3g). The precipitate was recrystallized
analogously to compound 3d. Yield 0.32 g (88%), light-
yellow needles, mp 232–234°C. IR spectrum, ν, cm−1: 1609,
1584 (С(O)=COH). 1H NMR spectrum, δ, ppm (J, Hz): 5.80
(1Н, s, СН2); 7.16 (2H, s, =CH); 7.17–7.29 (5Н, m, H Ph);
7.86 (2H, d, J = 7.8, H carbazole); 8.23–8.27 (2H, dd, J = 7.8,
J = 1.2, H carbazole); 9.24–9.26 (2H, d, J = 1.2,
H carbazole). 19F NMR spectrum, δ, ppm: –74.57 (6F, s,
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