NMR method for simultaneous host-guest binding constant measurement

Article abstract of DOI:10.1021/jo4027963

An NMR-based relative binding affinity measurement method has been developed in which differences in the binding affinities of different hosts toward a particular guest (ΔlogK_ass values) are measured in the same solution. As an advancement, the

Full text of DOI:10.1021/jo4027963

Article
pubs.acs.org/joc
NMR Method for Simultaneous Host−Guest Binding Constant
Measurement
Sandip A. Kadam,† Kristjan Haav,† Lauri Toom,‡ Toiv Haljasorg,† and Ivo Leito*,†
̃
†Institute of Chemistry, University of Tartu, Ravila 14a, 50411 Tartu, Estonia
‡Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia
S
* Supporting Information
ABSTRACT: An NMR-based relative binding affinity measurement method has been developed in which differences in the
binding affinities of different hosts toward a particular guest (ΔlogKass values) are measured in the same solution. As an
advancement, the method allows the simultaneous determination of several ΔlogKass values in a single run. As a proof of
principle, the method was used to measure binding affinity differences of a number of indolocarbazole- and urea-based synthetic
receptors toward acetate ion in DMSO-d6/H2O (99.5%:0.5% m/m). As a result, a binding affinity scale containing 33 receptors
and spanning 2.32 log units with excellent self-consistency (consistency standard deviation = 0.01 log unit) was created. Together
with the very good agreement of the results with those obtained by UV−vis spectrophotometry, this demonstrates the high
accuracy of the method and the fact that the NMR and UV−vis techniques can be used interchangeably (in spite of the very
different concentrations used in these techniques). Additionally, it was found for symmetrical receptor molecules from the same
compound family that there is a correlation between the acetate binding affinity of a receptor and the 15N chemical shift of the
nitrogen atoms of its binding centers.
The binding constant Kass expressing the affinity of a host H
toward a guest G is in the case of 1:1 stoichiometry defined by
eqs 1 and 2:
INTRODUCTION
■
Molecular recognition, one of the core processes in supra-
molecular chemistry, involves binding of a complementary
guest to a host via non-covalent interactions. It is the
prerequisite for self-organization, self-assembly, transport, and
supramolecular catalysis.1,2 The binding affinity of a host
toward particular guest under equilibrium conditions can be
quantified by the binding (or association or stability) constant
(Kass) (eq 1). Binding constants are key characteristics of
supermolecules, and when determined for a series of super-
molecules, they can be useful for predicting the properties of
new molecular assemblies.3,4 Differences in binding strength
(Kass ratios) for binding of different guests by the same host
characterize the selectivity of the interaction, which is very
important in molecular recognition studies and is a key
parameter used in the design of synthetic molecular receptors.
Without question, the accurate and reliable determination of
binding constants plays an important role in supramolecular
studies.
Kass
(1)
H + G XoooY HG
aHG
aHaG
Kass
=
(2)
wherein aHG, aH, and aG are the activities of the species in the
solution. Binding constants are generally measured directly
according to eq 2.5 The consistency of the measurements is
mostly evaluated by repeatability (standard deviation of
measurements repeated within the same day).6 Possible
systematic effects, which often are the main source of
measurement error, are left out of consideration. Systematic
effects in the case of anion binding by synthetic receptors may
be caused, for example, by ion pairing7 and homoconjugation
Received: December 17, 2013
Published: February 18, 2014
© 2014 American Chemical Society
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Scheme 1. Structures of the Molecular Receptors
(association of an acid and its anion).8 Both of these can
significantly decrease the activity of the free anion, leading to
biased results. As another example, even low levels of water in
organic solvents (often used as media) decrease the effective
activity of both hydrogen-bond (HB) donors and acceptors by
selective solvation. Systematic effects such as those described
here introduce bias by shifting all of the results in a series in the
same direction, while at the same time the agreement between
the individual results can be good, leaving the wrong
impression of highly accurate data.
[H1G][H2]
[H2G][H1]
Δlog Kass = log
(5)
Because many sources of error cancel with relative binding
affinity measurements, it is possible to obtain highly accurate
results. The proposed method is analogous by nature to relative
acidity and basicity measurement methods, which have been
used for the determination of pKa values in nonaqueous
media6,8 and in competition experiments.10
In ref 9, the usability and high accuracy of the obtained data
were successfully demonstrated on a series of synthetic anion
receptors. Besides its numerous merits as a method for studying
host−guest binding, UV−vis spectroscopy also has important
limitations: the presence of a chromophore is needed in the
host molecule, there should be sufficient spectral change upon
host−guest binding, and the solvent should be transparent over
the spectral range used. Furthermore, the measurement and
(especially) calculation procedures are rather complex.9
The omnipresence and ease of use of NMR spectroscopy in
organic synthesis have contributed to making NMR analysis
one of the most common methods used to study binding
affinities.11−14 Besides enabling the measurement of binding
constants, it also gives important additional information on the
possible side processes (e.g., possible deprotonation, associa-
tion, etc.). If the complexation reaction is fast (which is usually
the case), then the association or dissociation degree can be
easily monitored from the change in chemical shift.
In a previous work,9 a UV−vis spectrophotometric relative
binding affinity measurement method was reported. It is based
on measuring the relative binding affinity of two hosts H1 and
H2 toward the same guest when all of the species are dissolved
in the same solvent, as described by eq 3:
Δlog Kass
H1 + H2G XooooooooY H1G + H2
(3)
The relative binding affinity is expressed by ΔlogKass, which is
defined in eq 4:
Δlog Kass = log Kass(H1G) − log Kass(H2G)
a
a
a
H1G H2
= log
a
H2G H1
(4)
From eqs 3 and 4 it can be seen that the need to determine the
activity of the guest is eliminated. This means that the possible
side processes involving the guest (e.g., ion pairing and
homoconjugation) influence binding to both hosts simulta-
neously, cancel out, and thus do not affect the measurement
result. The activities of the free and bound hosts enter eq 4 as
ratios. Thus, possible factors affecting the hosts also largely
cancel (e.g., the composition of the solvent is automatically
identical for both hosts). A reasonable assumption to make is
that the ratios of activity coefficients of γ(Hx)/γ(HxG) are
similar for the two host molecules.6,8 Consequently, the
activities in eq 4 can be replaced with the equilibrium
concentrations:
In this work, we have developed a relative binding affinity
measurement method based on NMR spectroscopy. As a proof
of concept, this method was applied to the measurement of
binding constants between a series of synthetic chelating anion-
binding receptor building blocks (different ureas and
indolocarbazoles; see Scheme 1) as HB donors and acetate
anions as HB acceptors. The obtained data were compared with
the results obtained by UV−vis spectrophotometry.
Anion recognition and detection is important in medicine,
chemical industry, and the environment. The design of
synthetic anion receptors has become a prominent research
field.15−21 Therefore, the development of sensitive and selective
anion receptors that are able to function in detection,
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a
Table 1. Scale of Relative Binding Affinities for Acetate in DMSO-d6/H2O (99.5%:0.5% m/m)
a
Solvent: DMSO-d6/H2O (99.5%:0.5% m/m). In all cases, the binding stoichiometry was 1:1. ΔlogKass values in rectangles were determined using
b
c
the UV−vis method. Standard uncertainties for comparison of logKass values on the scale. Standard uncertainties for comparison of logKass values
with those from other research groups.
extraction, or transport is of high interest. Derivatives of
urea22,23 and indolocarbazole24 are among the most frequently
used building blocks in anion receptor design.
bazole (30) (see Table 2). The consistency of the assigned
absolute logKass values with the measured ΔlogKass values can
be evaluated by the consistency standard deviation of the scale
(s),8 which is found according to the following equation:
RESULTS
■
SS
nm − nc
1H NMR-Based Relative Measurements Study. Alto-
gether 78 relative acetate binding measurements between 33
receptors were carried out in DMSO-d6/H2O (99.5%:0.5% m/
m) using the NMR-based relative binding measurement
method. For comparison and validation of the NMR method,
17 relative binding measurements between 11 receptors were
carried out in DMSO/H2O (99.5%:0.5% m/m) using the
previously reported UV−vis spectrophotometric method. The
resulting scale of relative binding affinities for acetate ranging
over 2.32 log units and incorporating measurements from both
methods is presented in Table 1. Each arrow in the scale
corresponds to the difference in absolute binding affinity
between two receptor molecules on the logarithmic scale
expressed as ΔlogKass values. Each additional measurement
contributes to circular validation25 of the whole scale. The
absolute logKass values of the receptors on the scale were found
by minimizing the sum of the squares of the differences
between the directly measured ΔlogKass values and the assigned
logKass values, which is denoted as SS in the following
equation:8
s =
(7)
where nm = 95 is the number of ΔlogKass measurements and nc
= 33 is the number of absolute logKass values that were
determined. For the current scale, which includes both the
NMR and UV−vis results, s = 0.01 log units. The separate s
values for the NMR and UV−vis results are 0.01 and 0.02,
respectively. These s values indicate high consistency of the
results and good agreement between the NMR and UV−vis
data, enabling us to put all of the results on the same scale. This
also suggests that there is no practical difference in the choice
of method, in spite of the very different concentrations used in
NMR and UV−vis measurements. The values in blue boxes on
the scale in Table 1 were measured via UV−vis spectropho-
tometry. The high consistency of the results enabled differ-
entiation between receptors with a binding strength difference
of less than 0.05 logKass units. This is significantly lower than is
possible in the case of absolute measurements, especially if they
are performed in different laboratories.
The logKass differences of the anchor compounds obtained
from the relative measurements are also well-consistent with
the differences of their absolute logKass values.
Absolute Binding Measurements. The absolute logKass
values were obtained for the receptors 4-nitroindolocarbazole
(30), indolocarbazole (22), and 1,10-dichloroindolocarbazole
(26). For each of them, the logKass value was measured in at
least two different days. The values of logKass for 22 and 26
were obtained using both UV−vis and NMR methods. Several
independent data sets were obtained on each day, and for each
nm
SS =
{Δlog Ki − [log Kass(HyG) − log Kass(HxG)]}2
∑
ass
i=1
(6)
Every ΔlogKiass value is the directly measured relative binding
strength of the hosts Hy and Hx. The absolute logKass values for
all of the compounds were found by anchoring the scale by the
least-squares procedure to the logKass values of indolocarbazole
(22), 1,10-dichloroindolocarbazole (26), and 4-nitroindolocar-
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Table 2. Results of Measurements of Absolute logKass Values
a
b
b
b
c
c
receptor
method
absolute logKass
s
n
CI (95%)
absolute logKass from scale
difference
30
22
26
22
26
UV−vis
UV−vis
UV−vis
NMR
3.75
3.15
2.09
3.17
2.19
0.04
0.01
0.05
0.25
0.06
5
0.05
0.01
0.09
0.41
0.14
3.76
3.1
0.005
−0.002
0.057
5
4
4
3
2.15
3.14
2.15
−0.020
−0.040
NMR
a
b
Values of logKass were obtained as averages of independent measurement runs. s is the standard deviation, n the number of measurement runs, and
CI (95%) the confidence interval of the mean value at 95% probability. Difference between the logKass value obtained for the same receptor
c
molecule from the scale by the least-squares procedure and the directly determined absolute logKass value.
Figure 1. 1H NMR spectra for measuring the relative binding affinity of receptors 1 and 27 for acetate in DMSO-d6/H2O (99.5%:0.5% m/m). The
titration proceeds from bottom to top. The bottom spectrum corresponds to solution without added titrant.
of the data sets three independent calculation procedures were
applied.9 The results of the measurements are presented in
Table 2 and display very good agreement between the UV−vis
and NMR results.
The binding scale for acetate was anchored to the three
independently measured logKass values for receptors 30, 22, and
26. The small absolute values of the differences between the
values of logKass from the scale and the directly determined
values (last column of Table 2) demonstrate the good
consistency between the absolute and the relative measurement
results and offer evidence for absence of artificial expansion or
contraction of the scale.
An absolute binding constant measurement for receptor 3
was also attempted, but high scatter of the parallel measure-
ment results was observed. The reason probably is that NMR
spectroscopy is not suitable for accurate measurements of high
absolute binding constant values: because of the high
concentrations used in NMR measurements, almost all of the
anions added to the solution at any titration point are bound,
and finding the concentration of free anions is difficult. At the
same time, reproducible results were obtained with relative
measurements in the region of logKass = 3.6−4.0, implying that
in contrast to the absolute binding constant measurements by
NMR spectroscopy, the relative method described here enables
the measurement of high binding affinities.
DISCUSSION
■
Advantages of Relative Binding Measurement Using
NMR Spectroscopy. Measurement of binding constants via
NMR spectroscopy is performed by monitoring the changes in
1
the chemical shift of one or more proton signals in the H
NMR spectra of the receptor molecule at different guest
concentrations. For a 1:1 binding equilibrium under the
conditions of fast exchange, the chemical shift of the signal is
linearly dependent on the degree of host−guest association, α
(see eq 8),26 which can be found directly from the titration
spectra of a mixture containing both hosts. As outlined above,
applying the relative binding affinity measurement method
eliminates the need to determine the activity of the guest. This
becomes particularly useful if binding constants with high
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1
Figure 2. H NMR spectra for measuring the relative binding affinities between receptors 3, 23, and 36 toward acetate in DMSO-d6/H2O
(99.5%:0.5% m/m). The titration proceeds from bottom to top.
affinity are determined with the NMR approach. Because of the
high concentrations used in NMR analysis, it can be difficult to
determine the small content of the unbound ligand in solution
when absolute measurements are made, but with the relative
measurement this is not necessary. Therefore, if the ladder
approach described in this work is used, then high binding
affinities can be measured using NMR spectroscopy without
problems.
formation is faster) in comparison with receptor 27.
Calculation results confirmed that receptor 1 has a higher
binding affinity toward acetate than receptor 27.
UV−vis spectrophotometry is limited to measuring the
difference in the binding affinities of only two receptors toward
a selected anion. NMR spectroscopy does not have this
limitation, as differences in the binding affinities of more than
two receptors can be determined simultaneously within a single
measurement series. This capability enables the same amount
of data to be obtained with NMR spectroscopy around three
times faster than is possible with UV−vis spectrophotometry.
In UV−vis spectrophotometry measurements it is also
necessary to titrate solutions containing only one receptor
molecule. The degrees of association are found from the
titration spectra of the mixture via complex multiregression
analysis using also the spectral data from the titrations of the
two pure receptors. Furthermore, in the NMR method the
binding process for each host toward the selected guest can be
1
Figure 2 shows H NMR spectra for the measurement of the
affinities of receptors 3, 23, and 36 toward acetate. The
chemical shift of the NH protons of each receptor molecule is
observed, and the degrees of association for all of the receptors
are found. The relative binding constants (ΔlogKass values)
between receptors H1 and H2, H2 and H3, and H1 and H3 can
be calculated according to eq 9. During the titration, receptor 3
showed a larger change in the NH chemical shift (Δδ = 3.61
ppm) than receptor 36 (Δδ = 3.26 ppm) and receptor 23 (Δδ
= 3.36 ppm). The differences in binding affinity between
receptors 3 and 36, 36 and 23, and 3 and 23 are accordingly
0.45, 0.05, and 0.50 logKass units. The higher the binding
affinity of a receptor, the larger is the proportion of anion-
bound receptor molecules in solution and the faster its NH
proton signal shifts downfield (deshielding), which a general
feature in NMR spectra upon HB formation.26 The aromatic
protons show only negligible shielding shifts upon binding (Δδ
= 0.02 ppm) because they are distant from the binding center.
Substituent and Solvent Effects on the Binding
Affinity. As observed previously,9 electron-withdrawing groups
(EWGs) (e.g., −NO2, −COOBu, −Cl) increase the HB
1
followed directly from the H NMR spectra, which is not
possible in the case of the UV−vis method. This enables the
difference in binding affinities toward a particular guest to be
estimated already during the titration. Possible side processes,
such as deprotonation or additional unwanted association
processes, can be better observed with NMR than with UV−vis.
Under the conditions of fast exchange, the change in the
chemical shift is observed, not the change in signal intensity.5
Chemical shifts can be measured much more accurately than
signal intensities, and there is no necessity to correct for the
dilution during the NMR titration. Figure 1 demonstrates the
NMR measurement of the relative binding affinity of two
receptors toward acetate. The difference in the chemical shifts
of the NH protons of the free receptor and the receptor−anion
complex of compounds 1 and 27 are Δδ = 3.69 and 3.59 ppm,
respectively. During the addition of acetate, the NH proton
chemical shift of receptor 1 moves downfield faster (complex
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donicity of the receptor molecule, leading to an increase in the
binding affinities toward anions. In broad terms, one nitro
group is as powerful as two chloro or two ester groups, but the
effect of the substituents depends also on their positions. When
bulky groups (concrete data are available for −Cl and −CF3)
are located next to the binding site, as in the case of 1-
substitution or 1,10-disubstitution, the anion complexation is
hindered. This effect is further enhanced by the negative charge
of the substituent, leading to charge−charge repulsion.
It is of interest to compare the present results in DMSO-d6
containing 0.5% water with those obtained earlier by us in
acetonitrile (AN) containing 0.5% water.9 Acetonitrile has very
low HB-donating and -accepting abilities, and therefore,
binding of anions to receptor molecules in acetonitrile is
stronger than in more competitive solvents such as DMSO.
When the main solvent is changed from AN to DMSO, the
binding affinity decreases by more than 1 logKass unit (Table 3).
Figure 3. Relationship between the acetate binding constants (logKass)
of the symmetrical indolocarbazole receptors and the 15N chemical
shifts of the ring nitrogen atoms.
symmetrically substituted indolocarbazoles. There is a strong
correlation between these two parameters (R2 = 0.977) if the
1,10-disubstituted compounds (25 and 26) are not included.
These two compounds have significantly lower binding
affinities toward acetate than could be predicted from the 15N
chemical shifts. The obvious reason is that binding of acetate to
these compounds is sterically hindered, while the 15N chemical
shifts are not influenced by steric effects.
For the unsymmetrically substituted indolocarbazoles, there
are two different chemical shifts in the 15N NMR spectrum. We
attempted to correlate either of them individually or their
average with the value of logKass. Worse correlations were
observed in all of these cases (the highest was R2 = 0.938),
indicating in this case a more complex pattern of interrelation
among the charge distribution in the receptor, the shielding of
the nitrogen atoms, and the binding strength.
Table 3. Comparison of Binding Constant Values Measured
in DMSO and AN (Both Containing 0.5% Water)
logKass
receptor
1,3-diphenylurea
DMSO
AN
difference
3.20
4.01
3.14
3.14
3.15
2.76
3.76
3.72
3.55
3.57
3.39
1.69
2.15
4.28
5.20
4.46
4.46
4.50
4.24
5.24
5.20
5.05
5.09
4.95
3.36
3.84
1.08
1.19
1.32
1.32
1.35
1.48
1.48
1.49
1.51
1.52
1.56
1.67
1.69
3,4,4′-trichlorodiphenylurea
indolocarbazole
2,7-dimethoxyindolocarbazole
2-methoxyindolocarbazole
1-chloroindolocarbazole
4-nitroindolocarbazole
4,7-dichloroindolocarbazole
2,7-dichloroindolocarbazole
2-nitroindolocarbazole
2,9-dichloroindolocarbazole
1,10-bis(trifluoromethyl)indolocarbazole
1,10-dichloroindolocarbazole
CONCLUSION
■
The applicability of an NMR method for relative binding
affinity measurement has been demonstrated, and its
advantages have been outlined: it removes the necessity to
determine the activity of the guest, substantially simplifies the
calculation model for relative binding affinity measurement,
enables estimation of difference in binding affinity during the
titration experiment, and enables determination of more than
two relative binding affinities simultaneously in a single
experiment. The good agreement between the measurement
results obtained using NMR spectroscopy and UV−vis
spectrophotometry shows that it makes no difference which
experimental technique is used. This is a valuable conclusion
because the concentration ranges used in UV−vis and NMR
methods usually differ by around 2 orders of magnitude.
There are also changes in binding affinity order. It can be seen
that the binding affinities of diphenylureas decrease by slightly
more than 1 order of magnitude and that this is not strongly
dependent on their substitution. The decrease in binding
affinity in the case of indolocarbazoles is in the range of 1.3−1.7
logKass units, and the change is clearly dependent on the
substitution: indolocarbazole and methoxyindolocarbazoles
display changes of around 1.3 logKass units. Indolocarbazoles
with EWGs (chloro, nitro) display a decrease of around 1.5
logKass units in binding affinity. If the EWGs are at positions 1
and 10, then the change amounts to around 1.7 logKass units. In
the latter case, there seems to be a synergy between the action
of the EWGs and the steric effect. Three factors are at work.
First, the anion is repelled by the negative charge of the EWGs
and by the steric hindrance, preventing it from assuming a
suitable conformation. Second, the solvent molecule is neutral
and is not repelled by the negative charge. Third, the hydrogen
atoms of the NH groups carry strong positive partial charge and
are thus in principle strong HB donors, and DMSO is a much
stronger HB acceptor than AN. Therefore, in DMSO the
solvent molecules compete more efficiently with acetate anions
than in AN.
EXPERIMENTAL SECTION
■
Instruments. NMR measurements were carried out on a 200 MHz
NMR spectrometer, and UV−vis spectrophotometric measurements
were carried out on a double-beam spectrophotometer. The water
content of the DMSO solvent was checked with a coulometric Karl
Fischer titrator. Melting points were determined in open capillaries
and are uncorrected. All of the receptor molecules were characterized
on a 400 MHz NMR spectrometer. High-resolution mass spectra were
obtained on an FT-ICR mass spectrometer with a 7 T magnet using
negative ion electrospray ionization. The ionization chamber temper-
ature was set to 40 °C, the spray needle voltage to −3500 V, the shield
voltage to −300 V, the nebulizing gas (N2) pressure to 25 psi, and the
drying gas (N2) pressure to 10 psi at 200 °C. The ion capillary voltage
was optimized for every compound infused. The mass axis was
calibrated in negative mode daily prior to infusion experiments using
15N NMR Chemical Shifts. It is of interest to compare the
trends in the 15N chemical shifts with the trends in logKass
values when the substitution pattern changes. Figure 3 displays
the relationship between these two parameters in the case of
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Scheme 2. Synthesis of Different Substituted Indolocarbazole- and Urea-Based Receptors
an in-house calibration mixture containing perfluorinated Brønsted
superacids C12F10NO4S2H (anion m/z 475.91145) C8F18NO4S2H
(anion m/z 579.89868), C12F26NO4S2H (anion m/z 779.88591), and
C18F37O6S3H2 (anion m/z 1111.83499). In addition, two ions formed
during ionization, C8F17NO2H− (m/z 497.94620) and C4F9NO2S−
(m/z 296.95115), were used for calibration. Purification of the
compounds was performed by flash chromatography on silica gel
(pore size 60 Å, 230−400 mesh). Analytical thin-layer chromatography
(TLC) was conducted on TLC plates (silica gel 60 with fluorescent
UV254 marker on aluminum sheets). The samples for HRMS were
dissolved in DMSO so that the concentration of the stock solution was
1.0 mg/mL. A 1 μL aliquot was dissolved in 1 mL of methanol, so the
concentration of the infusion solution was roughly 1 μg/mL.
Solvents and Chemicals. The solvent for binding measurements
(DMSO with 0.5% water) was prepared using anhydrous 99.9%
DMSO (for UV−vis measurements) or 99.8% DMSO-d6 (for NMR
measurements) and water from a Milli-Q Advantage A10 system. The
final water content of the solvent was checked by Karl Fischer titration
and was always between 0.45% and 0.55%. Titrant solutions were
prepared from tetrabutylammonium acetate (TBAA) (99%). The
solvent for the synthesis, THF (Romil, 99.9%, according to Karl
Fischer titration, water content less than 5 ppm), was dried by means
of continuous circulation through a column filled with alumina and
was delivered inside a glovebox. DCM, DMF, and acetone were dried
as described in ref 27.
Receptor Molecules. The following commercially available
receptor molecules were used: 3,4,4′-trichlorodiphenylurea (35) and
1,3-diphenylurea (36). Receptors 22−34 were the same as in ref 9. All
of the remaining receptors were synthesized in this study.
Synthesis of Indolocarbazoles and Ureas. A series of mono- or
disubstituted indolocarbazole and urea-type anion receptor molecules
(compounds 1−6 and 8−21) were prepared as illustrated in Schemes
2 and 3. The general synthetic strategy was as follows. The reaction of
3-hydrazinobenzoic acid with cyclohexane-1,2-dione under reflux in
concentrated H2SO4 and subsequent dehydrogenation with 10% Pd/C
yielded compounds 1, 2, and 3 (Scheme 2).9 A similar reaction with 3-
methylphenylhydrazine yielded compounds 4, 5, and 6 (in this case,
dehydrogenation proceeded without the dehydrogenation catalyst).
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Scheme 3. Synthesis of Ester Derivatives of Indolocarbazoles
Intermediate 7 was prepared by refluxing phenylhydrazine and
cyclohexane-1,2-dione in 1:1 (v/v) aqueous acetic acid. Compound
8 was prepared by refluxing 7 and 2-(trifluoromethyl)phenylhydrazine
hydrochloride in concentrated H2SO4 and n-butanol and then
dehydrogenating with p-chloranil (Scheme 2). The synthesis of
indolocarbazoles substituted with a single ester group began from 3-
hydrazinobenzoic acid and intermediate 7, which were refluxed in n-
butanol and concentrated H2SO4; subsequent dehydrogenation using
10% Pd/C afforded compounds 12 and 13 (Scheme 3). Compound
12 was converted to compound 14 by hydrolysis, and a series of ester
derivatives of indolocarbazole were prepared from 14. Compound 14
reacted with different substituted benzyl halides and alkyl halides in
DMF in the presence of K2CO3 as described in Scheme 3, leading to
compounds 15−18, 20, and 21.28 Compound 19 was prepared from
14 using 1,2-diiodoethane in dry acetone (Scheme 3). Finally,
compounds 9−11 were prepared from the reactions of benzene-1,2-
diamine,29 1-aminonaphthalene,30 and 3-nitroaniline,31 respectively,
with phenyl isocyanide in dry DCM or THF.
Preparation of Compounds 1−3. Cyclohexane-1,2-dione (0.13
g, 1.18 mmol) and 3-hydrazinobenzoic acid (0.40 g, 2.63 mmol) were
suspended in n-butanol (24 mL), and then concentrated H2SO4 (0.23
mL) was added dropwise via syringe. The mixture was heated to reflux
for 60 h and then concentrated under reduced pressure. The thick oily
mass was then dissolved in dry DMF (10 mL), and 10% Pd/C (0.03 g,
10% by weight) was added. The mixture was heated to reflux under an
atmosphere of N2 for 24 h and then filtered to remove the 10% Pd/C,
which was washed with hot DMF (3 × 3 mL). H2O (50 mL) was
added to the combined filtrates to precipitate the crude product, which
was collected by filtration and washed with H2O (2 × 20 mL). The
mixture of structural isomers was a light-yellow solid, which was
washed in diethyl ether (30 mL). One isomer, dibutyl indolo[2,3-
a]carbazole-4,7-dicarboxylate (3) (light-green solid, 0.10 g, 0.21 mmol,
18.5% yield), was soluble in diethyl ether and remained in the filtrate.
The other two isomers were insoluble and remained in the solid
fraction, which was mixed with hot ethyl acetate (30 mL) and filtered.
Dibutyl indolo[2,3-a]carbazole-2,7-dicarboxylate (2) (light-yellow
solid, 0.060 g, 0.13 mmol, 11.1% yield), dissolved in ethyl acetate
and was obtained from the filtrate. Dibutyl indolo[2,3-a]carbazole-2,9-
dicarboxylate (1) (light-yellow solid, 0.070 g, 0.15 mmol, 12.9% yield)
did not dissolve and was obtained as the solid residue.
Data for 1: Mp: decomposed above 350 °C. Rf = 0.68 (50% THF in
hexane). 1H NMR (400.1 MHz, DMSO-d6, +25 °C): δ 11.56 (bs, 2H,
4
5
NH-11,12); 8.37 (dd, JHH = 1.5 Hz, JHH = 0.8 Hz, 2H, CH-1,10);
3
8.29 (dm, JHH = 8.2 Hz, 2H, CH-4,7); 8.04 (bs, 2H, CH-5,6); 7.84
(ddd, 3JHH = 8.2 Hz, 4JHH = 1.5 Hz, 6JHH = 0.4 Hz, 2H, CH-3,8); 4.35
(t, 3JHH = 6.6 Hz, 4H, 2 × OCH2); 1.76 (m, 4H, 2 × OCH2CH2); 1.49
3
(m, 4H, 2 × CH2CH3); 0.99 (t, JHH = 7.4 Hz, 6H, 2 × CH3). 13C
NMR (100.6 MHz, DMSO-d6, +25 °C): δ 166.5 (COO); 138.5 (C-
10′,12′); 127.2 (C-4′,7′ and C-11′,11″); 125.9 (C-2,9); 120.3 (C-
5′,6′); 119.7 (CH-3,8 and CH-4,7); 113.3 (CH-1,10); 112.8 (CH-5,6);
64.2 (OCH2); 30.4 (OCH2CH2); 18.8 (CH2CH3); 13.6 (CH3). 15N
NMR (40.6 MHz, DMSO-d6, +25 °C): δ 116.14 (NH-11,12). IR
(ATR-FT-IRS) ν
̃
: 3355, 2958, 1705, 1668 cm−1. ESI-ICR (m/z):
solvent ∼0.1% DMSO/MeOH, calcd for C28H27N2O4 [M − H]−
455.19763, found 455.19742.
Data for 2: Mp: decomposed above 350 °C. Rf = 0.55 (50% THF in
hexane). 1H NMR (400.1 MHz, DMSO-d6, +25 °C): δ 11.77 (bs, 1H,
3
5
NH-11); 11.34 (bs, 1H, NH-12); 8.53 (dd, JHH = 8.7 Hz, JHH = 0.8
4
5
Hz, 1H, CH-6); 8.39 (dd, JHH = 1.5 Hz, JHH = 0.6 Hz, 1H, CH-1);
3
5
5
8.27 (ddd, JHH = 8.2 Hz, JHH = 0.6 Hz, JHH = 0.5 Hz, 1H, CH-4);
8.00 (dd, 3JHH = 8.1 Hz, 4JHH = 1.1 Hz, 1H, CH-10); 7.96 (dd, 3JHH
8.7 Hz, 5JHH = 0.4 Hz, 1H, CH-5); 7.84 (dd, 3JHH = 8.2 Hz, 4JHH = 1.5
=
3
4
Hz, 1H, CH-3); 7.79 (dd, JHH = 7.5 Hz, JHH = 1.1 Hz, 1H, CH-8);
7.52 (dd, 3JHH = 8.1 Hz, 3JHH = 7.5 Hz, 1H, CH-9); 4.46 (t, 3JHH = 6.6
3
Hz, 2H, 7-COOCH2); 4.34 (t, JHH = 6.6 Hz, 2H, 2-COOCH2); 1.80
(m, 2H, 7-COOCH2CH2); 1.75 (m, 2H, 2-COOCH2CH2); 1.49 (m,
2H, 7-COO(CH2)2CH2); 1.48 (m, 2H, 2-COO(CH2)2CH2); 0.98 (m,
6H, 2 × CH3). 13C NMR (100.6 MHz, DMSO-d6, +25 °C): δ 167.5
(7-COO); 166.5 (2-COO); 140.0 (C-10′); 138.5 (C-12′); 127.16 (C-
4′ or C-11″); 127.11 (C-4′ or C-11″); 126.7 (C-11′); 125.8 (C-2);
124.6 (C-7); 123.9 (CH-9); 122.0 (CH-8); 121.4 (C-7′); 119.72 (CH-
3 or CH-4); 119.69 (CH-3 or CH-4); 119.6 (C-5′); 119.4 (C-6′);
116.6 (CH-6); 116.2 (CH-10); 113.3 (CH-1); 111.8 (CH-5); 64.4 (7-
COOCH2); 64.2 (2-COOCH2); 30.38 (2-COOCH2CH2 or 7-
COOCH2CH2); 30.32 (2-COOCH2CH2 or 7-COOCH2CH2); 18.8
(2-COO(CH2)2CH2 or 7-COO(CH2)2CH2); 13.6 (2-COO-
(CH2)3CH3 and 7-COO-(CH2)3CH3). 15N NMR (40.6 MHz,
DMSO-d6, +25 °C): δ 117.05, 115.23 (NH-11,12). IR (ATR-FT-
IRS) ν
̃
: 3334, 2957, 1714, 1657, 1256 cm−1. ESI-ICR (m/z): solvent
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The Journal of Organic Chemistry
Article
4
6
5
∼0.1% DMSO/MeOH, calcd for C28H27N2O4 [M − H]− 455.19763,
found 455.19750.
1H, CH-8); 2.86 (ddd, JHH = 0.9 Hz, JHH = 0.6 Hz, JHH = 0.3 Hz,
4
4
5
3H, CH3-7); 2.51 (ddd, JHH = 0.8 Hz, JHH = 0.6 Hz, JHH = 0.3 Hz,
3H, CH3-2). 13C NMR (100.6 MHz, DMSO-d6, +25 °C): δ 139.5 (C-
12′); 138.9 (C-10′); 134.0 (C-2); 131.6 (C-7); 125.6 (C-11′); 125.4
(C-11″); 124.3 (CH-9); 122.4 (C-7′); 121.5 (C-4′); 120.5 (CH-3);
120.4 (CH-8); 120.3 (C-6′); 119.7; (C-5′); 119.5 (CH-4); 113.6 (CH-
6); 111.6 (CH-1); 111.4 (CH-5); 109.2 (CH-10); 21.7 (CH3-2); 20.7
(CH3-7). 15N NMR (40.6 MHz, DMSO-d6, +25 °C): δ 113.47 (NH-
11); 111.85 (NH-12).
Data for 3: Mp: 155 °C. Rf = 0.29 (50% THF in hexane). 1H NMR
(400.1 MHz, DMSO-d6, +25 °C): δ 11.53 (bs, 2H, NH-11,12); 8.43
3
4
(bs, 2H, CH-5,6); 8.01 (dd, JHH = 8.1 Hz, JHH = 1.1 Hz, 2H, CH-
1,10); 7.78 (dd, 3JHH = 7.5 Hz, 4JHH = 1.1 Hz, 2H, CH-3,8); 7.52 (dd,
3JHH = 8.1 Hz, 3JHH = 7.5 Hz, 2H, CH-2,9); 4.47 (t, 3JHH = 6.6 Hz, 4H,
2 × OCH2); 1.81 (m, 4H, 2 × OCH2CH2); 1.50 (m, 4H, 2 ×
3
CH2CH3); 0.98 (t, JHH = 7.4 Hz, 6H, 2 × CH3). 13C NMR (100.6
Preparation of Compound 7. Cyclohexane-1,2-dione (3.00 g,
26.78 mmol) and phenylhydrazine (3.01 g, 26.78 mmol) were
dissolved in a 1:1 (v/v) mixture of acetic acid (40 mL) and water (40
mL), and the reaction mixture was stirred at reflux temperature for 5 h.
After disappearance of the starting material (as monitored by TLC),
the reaction mixture was quenched in aqueous NaHCO3 (1 M, 1 L).
The formed precipitate was filtered and washed with water. The dried
crude solid was washed with pentane to obtain pure compound 7
(3.53 g, 71.4% yield) as a brown solid.9
MHz, DMSO-d6, +25 °C): δ: 167.5 (COO); 140.0 (C-10′,12′); 126.6
(C-11′,11″); 124.6 (C-4,7); 123.9 (CH-2,9); 121.9 (CH-3,8); 121.4
(C-4′,7′); 118.9 (C-5′,6′); 116.1 (CH-1,10); 115.6 (CH-5,6); 64.4
(OCH2); 30.3 (OCH2CH2); 18.8 (CH2CH3); 13.6 (CH3). 15N NMR
(40.6 MHz, DMSO-d6, +25 °C): δ 116.06 (NH-11,12). IR (ATR-FT-
IRS) ν
̃
: 3321, 2960, 1718, 1662 cm−1. ESI-ICR (m/z): solvent ∼0.1%
DMSO/MeOH, calcd for C28H27N2O4 [M − H]− 455.19763, found
455.19745.
Preparation of Compounds 4−6. Cyclohexane-1,2-dione (0.60
g, 5.35 mmol) and m-tolylhydrazine hydrochloride (2.1 g, 13.36
mmol) were suspended in n-butanol (24 mL), and then concentrated
H2SO4 (0.6 mL) was added dropwise via syringe. The mixture was
heated to reflux for 65 h. After disappearance of the starting material
(as monitored by TLC), the reaction mixture was cooled to room
temperature. The formed precipitate was filtered, and 2,9-
dimethylindolo[2,3-a]carbazole (4) (0.55 g, 1.93 mmol, 36.2% yield)
was isolated on the filter as a brown solid. The filtrate was
concentrated under reduced pressure, and the crude product obtained
from the filtrate was purified by column chromatography (silica 230−
400 mesh), eluting with 6−7% ethyl acetate in hexane, to afford a
3:84:13 mixture of structural isomers 4, 4,7-dimethylindolo[2,3-
a]carbazole (5), and 2,7-dimethylindolo[2,3-a]carbazole (6) (0.52 g,
1.83 mmol, 34.2% yield) as a brown solid.
Preparation of Compound 8. Compound 7 (0.10 g, 0.53 mmol)
and 2-(trifluoromethyl)phenylhydrazine hydrochloride (0.22 g, 1.07
mmol) were dissolved in n-butanol (10 mL), and the mixture was
stirred at room temperature for 15 min. Then concentrated H2SO4
(0.01 mL) was added dropwise. The mixture was stirred at reflux
temperature for 60 h. After disappearance of the starting material (as
monitored by TLC), the reaction mixture was cooled to room
temperature and concentrated under reduced pressure. The residue
was dissolved in ethyl acetate (25 mL), and the solution washed with
water (50 mL). The ethyl acetate layer was evaporated under reduced
pressure to obtain the 5,6-dihydro intermediate as a thick oil. It was
dissolved in toluene (10 mL), and p-chloranil (0.10 g, 1.05 mmol) was
added. The reaction mixture was stirred for 2 h at reflux temperature.
After disappearance of the intermediate (as monitored by TLC), the
reaction mixture was concentrated under reduced pressure. The
concentrated mass was dissolved in ethyl acetate (25 mL), and the
solution was washed with saturated NaHSO4 (50 mL) and water (2 ×
50 mL). The ethyl acetate solution was dried over anhydrous MgSO4
for 5 min and filtered. The filtrate was evaporated under reduced
pressure to obtain the crude product, which was purified by column
chromatography (silica 230−400 mesh), eluting with 1−2% ethyl
acetate in hexane, to afford 1-(trifluoromethyl)indolo[2,3-a]carbazole
(8) (0.090 g, 0.27 mmol, 51.7% yield) as a gray solid.
Data for 4: Mp: decomposed above 350 °C. Rf = 0.29 (30% ethyl
1
acetate in hexane). H NMR (400.1 MHz, DMSO-d6, +25 °C): δ
3
10.88 (bs, 2H, NH-11,12); 7.99 (dm, JHH = 7.9 Hz, 2H, CH-4,7);
7.81 (bs, 2H, CH-5,6); 7.45 (ddq, 4JHH = 1.5 Hz, 5JHH = 0.7 Hz, 4JHH
0.7 Hz, 2H, CH-1,10); 7.01 (ddq, 3JHH = 7.9 Hz, 4JHH = 1.5 Hz, 4JHH
=
=
0.6 Hz, 2H, CH-3,8); 2.49 (m, 6H, CH3-2,9). 13C NMR (100.6 MHz,
DMSO-d6, +25 °C): δ 139.4 (C-10′,12′); 133.7 (C-2,9); 125.5 (C-
11′,11″); 121.6 (C-4′,7′); 120.3 (CH-3,8); 119.8 (C-5′,6′); 119.3
(CH-4,7); 111.4 (CH-1,10); 111.2 (CH-5,6); 21.6 (CH3-2,9). 15N
NMR (40.6 MHz, DMSO-d6, +25 °C): δ 112.16 (NH-11,12). IR
Data for 8: Mp: 198.7 °C. Rf = 0.36 (10% ethyl acetate in hexane).
1H NMR (400.1 MHz, DMSO-d6, +25 °C): δ 11.44 (bs, 1H, NH-12);
(ATR-FT-IRS) ν
̃
: 3186, 3023, 1399, 1043 cm−1. ESI-ICR (m/z):
3
solvent ∼0.1% DMSO/MeOH, calcd for C20H15N2 [M − H]−
283.12407, found 283.12403.
11.09 (bs, 1H, NH-11); 8.45 (dm, JHH = 7.8 Hz, 1H, CH-4); 8.16
(dddd, 3JHH = 7.8 Hz, 4JHH = 1.2 Hz, 5JHH = 0.7 Hz, 5JHH = 0.6 Hz, 1H,
3
CH-7); 7.97 (dd, JHH = 8.3 Hz, 5JHH = 0.4 Hz, 1H, CH-5); 7.95 (dd,
Data for 4, 5, and 6 (3:84:13 mixture): Mp: decomposed above
5
3
3JHH = 8.3 Hz, JHH = 0.4 Hz, 1H, CH-6); 7.71 (ddd, JHH = 8.2 Hz,
314.6−316.0 °C. Rf = 0.29 (30% ethyl acetate in hexane). IR (ATR-
5
3
4JHH = 1.0 Hz, JHH = 0.7 Hz, 1H, CH-10); 7.70 (ddq, JHH = 7.6 Hz,
FT-IRS) ν
̃
: 3392, 3047, 1650, 1608 cm−1. ESI-ICR (m/z): solvent
∼0.1% DMSO/MeOH, calcd for C20H15N2 [M − H]− 283.12407,
4
3
4JHH = 1.1 Hz, JHF = 0.8 Hz, 1H, CH-2); 7.41 (ddd, JHH = 8.2 Hz,
4
3
3JHH = 7.1 Hz, JHH = 1.2 Hz, 1H, CH-9); 7.36 (ddq, JHH = 7.8 Hz,
found 283.12381.
1
5
3
3JHH = 7.6 Hz, JHF = 0.9 Hz, 1H, CH-3); 7.22 (ddd, JHH = 7.8 Hz,
Data for 5: H NMR (400.1 MHz, DMSO-d6, +25 °C): δ 11.05
(bs, 2H, NH-11,12); 7.97 (bs, 2H, CH-5,6); 7.54 (ddq, 3JHH = 8.1 Hz,
4JHH = 1.0 Hz, 6JHH = 0.6 Hz, 2H, CH-1,10); 7.29 (ddq, 3JHH = 8.1 Hz,
3JHH = 7.2 Hz, 5JHH = 0.4 Hz, 2H, CH-2,9); 7.00 (ddq, 3JHH = 7.2 Hz,
4JHH = 1.0 Hz, 4JHH = 0.9 Hz, 2H, CH-3,8); 2.88 (ddd, 4JHH = 0.9 Hz,
4
3JHH = 7.1 Hz, JHH = 1.0 Hz, 1H, CH-8). 13C NMR (100.6 MHz,
DMSO-d6, +25 °C): δ 138.7 (C-10′); 133.9 (q, 3JCF = 2.0 Hz, C-12′);
126.0 (C-11″); 125.7 (C-4′); 125.3 (C-11′); 125.0 (q, 1JCF = 271.5 Hz,
3
CF3-1); 124.9 (CH-9); 124.2 (CH-4); 123.4 (C-7′); 121.4 (q, JCF
=
5
6JHH = 0.6 Hz, JHH = 0.4 Hz, 6H, CH3-4,7). 13C NMR (100.6 MHz,
4.6 Hz, CH-2); 120.8 (C-6′); 119.8 (CH-7); 119.03 (CH-8); 119.01
(C-5′); 118.5 (CH-3); 112.7 (CH-5); 111.57 (q, 2JCF = 32.2 Hz, C-1);
111.55 (CH-10); 111.4 (CH-6). 15N NMR (40.6 MHz, DMSO-d6,
DMSO-d6, +25 °C): δ 139.0 (C-10′,12′); 131.6 (C-4,7); 125.4 (C-
11′,11″); 124.3 (CH-2,9); 122.2 (C-4′,7′); 120.3 (CH-3,8); 120.0 (C-
5′,6′); 113.6 (CH-5,6); 109.1 (CH-1,10); 20.6 (CH3-4,7). 15N NMR
(40.6 MHz, DMSO-d6, +25 °C): δ 113.39 (NH-11,12).
+25 °C): δ 112.96 (q, JNF = 0.8 Hz, NH-12); 113.77 (NH-11). 19F
4
NMR (188.3 MHz, DMSO-d6, +25 °C): δ −59.71 (CF3). IR (ATR-
Data for 6: 1H NMR (400.1 MHz, DMSO-d6, +25 °C): δ 11.05 (bs,
1H, NH-11); 10.86 (bs, 1H, NH-12); 8.01 (dddq, 3JHH = 7.9 Hz, 5JHH
FT-IRS) ν
̃
: 3436, 3384, 1308, 1105 cm−1. ESI-ICR (m/z): solvent
∼0.1% DMSO/MeOH, calcd for C19H10F3N2 [M − H]− 323.08015,
found 323.08015.
5
5
3
= 0.6 Hz, JHH = 0.6 Hz, JHH = 0.3 Hz, 1H, CH-4); 7.91 (dd, JHH
=
8.2 Hz, 5JHH = 0.6 Hz, 1H, CH-6); 7.86 (dd, 3JHH = 8.3 Hz, 5JHH = 0.5
Hz, 1H, CH-5); 7.51 (ddq, JHH = 8.0 Hz, JHH = 0.9 Hz, JHH = 0.6
Hz, 1H, CH-10); 7.49 (ddq, JHH = 1.5 Hz, JHH = 0.8 Hz, JHH = 0.6
Hz, 1H, CH-1); 7.27 (ddq, JHH = 8.0 Hz, JHH = 7.1 Hz, JHH = 0.3
Hz, 1H, CH-9); 7.03 (ddq, JHH = 7.9 Hz, JHH = 1.5 Hz, JHH = 0.6,
1H, CH-3); 6.98 (ddq, JHH = 7.1 Hz, JHH = 0.9 Hz, JHH = 0.9 Hz,
Preparation of Compound 9. Benzene-1,2-diamine (0.68 g, 6.28
mmol) was dissolved in dry DCM (75 mL), and then phenyl
isocyanate was added dropwise (1.64 g, 13.83 mmol). The reaction
mixture was heated to reflux under under an atmosphere of N2 for 17
h. After disappearance of the starting material (as monitored by TLC),
the formed precipitate was filtered and washed with diethyl ether to
3
4
6
4
4
5
3
3
5
3
4
4
3
4
4
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The Journal of Organic Chemistry
Article
obtain pure compound 9 (2.20 g, 11.89 mmol, 96.3% yield) as a white
filtrate was concentrated under reduced pressure to obtain a thick oily
liquid. The crude product was purified by column chromatography
(silica 230−400 mesh, 1% methanol in DCM) to get butyl indolo[2,3-
a]carbazole-4-carboxylate (13) (3.2 g, 8.98 mmol, 27.7% yield) as a
yellow solid. The insoluble precipitate was dissolved in dry DMF (50
mL), and Pd/C (0.3 g, ∼10 wt %) was added. The reaction mixture
was heated to reflux under an atmosphere of N2 for 24 h and then
filtered. The Pd/C remained on the filter and was washed with DMF
(3 × 10 mL). The filtrate was diluted with H2O (200 mL), and a
precipitate was formed. This was collected by filtration, washed with
H2O (200 mL), and dried under vacuum to yield butyl indolo[2,3-
a]carbazole-2-carboxylate (12) (4.20 g, 11.79 mmol, 36.4% yield) as
an off-white solid.
solid.
1
Data for 9: Mp: 240.5 °C. Rf = 0.37 (5% methanol in DCM). H
NMR (400.1 MHz, DMSO-d6, +25 °C): δ 9.05 (bs, 2H, C6H5NH);
8.05 (bs, 2H, C6H4NH); 7.60 (AA′ of AA′XX′, 2H, CH-3,6); 7.47 (m,
4H, CH-2′,6′); 7.27 (m, 4H, CH-3′,5′); 7.09 (XX′ of AA′XX′, 2H,
CH-4,5); 6.96 (m, 2H, CH-4′). 13C NMR (100.6 MHz, DMSO-d6,
+25 °C): δ 153.2 (NHCONH); 139.8 (C-1′); 131.3 (C-1,2); 128.7
(CH-3′,5′); 124.0 (CH-3,6); 123.9 (CH-4,5); 121.7 (CH-4′); 118.1
(CH-2′,6′). 15N NMR (40.6 MHz, DMSO-d6, +25 °C): δ 108.58
̃
(C6H5NH); 99.81 (C6H4NH). IR (ATR-FT-IRS) ν: 3277, 3056, 1600,
1305 cm−1. ESI-ICR (m/z): solvent ∼0.1% DMSO/MeOH, calcd for
C20H17O2N4 [M − H]− 345.13570, found 345.13542.
1
Data for 12: Mp: 364.4 °C. Rf = 0.64 (50% THF in hexane). H
NMR (400.1 MHz, DMSO-d6, +25 °C): δ 11.32 (bs, 2H, NH-11,12);
Preparation of Compound 10. 1-Aminonaphthalene (0.20 g,
1.39 mmol) was dissolved in dry THF (20 mL), and then phenyl
isocyanate was added dropwise via syringe (0.21 g, 1.81 mmol). The
reaction mixture was stirred at room temperature under an atmosphere
of N2 for 17 h. A precipitate was formed, and the progress of the
reaction was monitored by TLC. After disappearance of the starting
material, the reaction mixture was cooled to 0 °C and filtered. The
crude solid product was washed with diethyl ether to obtain pure
compound 10 (0.30 g, 1.14 mmol, 82.0% yield) as a white solid.
8.36 (dd, 4JHH = 1.5 Hz, 5JHH = 0.7 Hz, 1H, CH-1); 8.25 (ddd, 3JHH
=
=
5
3
8.2 Hz, JHH = 0.7 Hz, 5JHH = 0.6 Hz, 1H, CH-4); 8.19 (dddd, JHH
7.8 Hz, 4JHH = 1.2 Hz, 5JHH = 0.8 Hz, 5JHH = 0.6 Hz, 1H, CH-7); 7.97
(bs, 2H, CH-5,6); 7.83 (dd, 3JHH = 8.2 Hz, 4JHH = 1.5 Hz, 1H, CH-3);
3
5
7.70 (ddd, JHH = 8.1 Hz, 4JHH = 1.0 Hz, JHH = 0.8 Hz, 1H, CH-10);
3
3
4
7.41 (ddd, JHH = 8.1 Hz, JHH = 7.1 Hz, JHH = 1.2 Hz, 1H, CH-9);
3
3
4
7.22 (ddd, JHH = 7.8 Hz, JHH = 7.1 Hz, JHH = 1.0 Hz, 1H, CH-8);
3
1
4.34 (t, JHH = 6.5 Hz, 2H, OCH2); 1.76 (m, 2H, OCH2CH2); 1.49
Data for 10: Mp: 211.0 °C. Rf = 0.42 (30% THF in hexane). H
(m, 2H, CH2CH3); 0.98 (t, 3JHH = 7.3 Hz, 3H, CH3). 13C NMR (100.6
MHz, DMSO-d6, +25 °C): δ 166.6 (COO); 139.2 (C-10′); 138.4 (C-
12′); 127.5 (C-4′); 127.5 (C-11″); 125.5 (C-2); 125.4 (C-11′); 124.9
(CH-9); 123.6 (C-7′); 121.0 (C-6′); 119.9 (CH-7); 119.7 (CH-3);
119.5 (CH-4); 119.3 (C-5′); 119.1 (CH-8); 113.1 (CH-1); 112.4 (CH-
5 or CH-6); 112.1 (CH-5 or CH-6); 111.7 (CH-10); 64.2 (OCH2);
30.4 (OCH2CH2); 18.8 (CH2CH3); 13.7 (CH3). 15N NMR (40.6
MHz, DMSO-d6, +25 °C): δ 114.97 (NH-12); 114.25 (NH-11). IR
NMR (400.1 MHz, DMSO-d6, +25 °C), δ 9.04 (bs, 1H, Ph-NH); 8.76
3
(bs, 1H, Naph-NH); 8.13 (dm, JHH = 8.3 Hz, 1H, CH-8); 8.02 (dd,
3JHH = 7.6 Hz, 4JHH = 1.2 Hz, 1H, CH-2); 7.93 (dm, 3JHH = 8.0 Hz, 1H,
CH-5); 7.64 (dm, 3JHH = 8.3 Hz, 1H, CH-4); 7.60 (ddd, 3JHH = 8.3 Hz,
4
3
3JHH = 6.8 Hz, JHH = 1.6 Hz, 1H, CH-7); 7.55 (ddd, JHH = 8.0 Hz,
4
3JHH = 6.8 Hz, JHH = 1.4 Hz, 1H, CH-6); 7.51 (m, 2H, CH-2′,6′);
7.48 (dd, 3JHH = 8.3 Hz, 3JHH = 7.6 Hz, 1H, CH-3); 7.31 (m, 2H, CH-
3′,5′); 6.99 (m, 1H, CH-4′). 13C NMR (100.6 MHz, DMSO-d6, +25
°C): δ 152.9 (NHCONH); 139.8 (C-1′); 134.3 (C-8a); 133.7 (C-4a);
128.8 (CH-3′,5′); 128.4 (CH-5); 125.9 (C-1); 125.84 (CH-6); 125.83
(CH-3); 125.7 (CH-7); 122.9 (CH-4); 121.8 (C-4′); 121.3 (CH-8);
118.1 (CH-2′,6′); 117.4 (CH-2). 15N NMR (40.6 MHz, DMSO-d6,
+25 °C): δ 108.89 (Ph-NH); 101.83 (Naph-NH). IR (ATR-FT-IRS)
(ATR-FT-IRS) ν
̃
: 3351, 2957, 1666, 1613 cm−1. ESI-ICR (m/z):
solvent ∼0.1% DMSO/MeOH, calcd for C23H19O2N2 [M − H]−
355.14520, found 355.14511.
Data for 13: Mp: 169.6 °C. Rf = 0.42 (50% THF in hexane). H
NMR (400.1 MHz, DMSO-d6, +25 °C): δ 11.49 (bs, 1H, NH-12);
1
3
5
11.08 (bs, 1H, NH-11); 8.48 (dd, JHH = 8.7 Hz, JHH = 0.5 Hz, 1H,
CH-5); 8.16 (dddd, 3JHH = 7.8 Hz, 4JHH = 1.2 Hz, 5JHH = 0.8 Hz, 5JHH
= 0.6 Hz, 1H, CH-7); 7.98 (dd, 3JHH = 8.1 Hz, 4JHH = 1.1 Hz, 1H, CH-
1); 7.90 (dd, 3JHH = 8.7 Hz, 5JHH = 0.5 Hz, 1H, CH-6); 7.77 (dd, 3JHH
ν
̃
: 3277, 3046, 1639, 1550 cm−1. ESI-ICR (m/z): solvent ∼0.1%
DMSO/MeOH, calcd for C17H13N2O [M − H]− 261.10333, found
261.10347.
Preparation of Compound 11. 3-Nitroaniline (0.20 g, 1.44
mmol) was dissolved in dry THF (20 mL), and then phenyl isocyanate
was added dropwise (0.26 g, 2.17 mmol). The reaction mixture was
stirred at room temperature under an atmosphere of N2 for 17 h. After
disappearance of the starting material (as monitored by TLC), the
formed precipitate was filtered and washed with diethyl ether to obtain
pure compound 11 (0.35 g, 1.36 mmol, 94.1% yield) as a white solid.
= 7.5 Hz, 4JHH = 1.1 Hz, 1H, CH-3); 7.71 (ddd, 3JHH = 8.1 Hz, 4JHH
=
1.0 Hz, 5JHH = 0.8 Hz, 1H, CH-10); 7.49 (dd, 3JHH = 8.1 Hz, 3JHH = 7.5
3
3
4
Hz, 1H, CH-2); 7.41 (ddd, JHH = 8.1 Hz, JHH = 7.1 Hz, JHH = 1.2
3
3
4
Hz, 1H, CH-9); 7.22 (ddd, JHH = 7.8 Hz, JHH = 7.1 Hz, JHH = 1.0
3
Hz, 1H, CH-8); 4.46 (t, JHH = 6.6 Hz, 2H, OCH2); 1.81 (m, 2H,
3
OCH2CH2); 1.50 (m, 2H, CH2CH3); 0.98 (t, JHH = 7.4 Hz, 3H,
CH3). 13C NMR (100.6 MHz, DMSO-d6, +25 °C): δ 166.6 (COO);
139.8 (C-12′); 139.2 (C-10′); 127.0 (C-11″); 125.2 (C-11′); 124.8
(CH-9); 124.4 (C-4); 123.53 (CH-2); 123.49 (C-7′); 121.71 (CH-3);
121.67 (C-4′); 120.4 (C-6′); 119.9 (CH-7); 119.0 (CH-8); 118.5 (C-
5′); 116.0 (CH-1); 115.9 (CH-5); 111.7 (CH-10); 111.4 (CH-6); 64.4
(OCH2); 30.3 (OCH2CH2); 18.8 (CH2CH3); 13.6 (CH3). 15N NMR
(40.6 MHz, DMSO-d6, +25 °C): δ 115.79 (NH-12); 113.28 (NH-11).
1
Data for 11: Mp: 209.3 °C. Rf = 0.74 (5% methanol in DCM). H
NMR (400.1 MHz, DMSO-d6, +25 °C): δ 9.20 (bs, 1H, 3-
4
NO2C6H4NH); 8.83 (bs, 1H, Ph-NH); 8.56 (ddd, JHH = 2.3 Hz,
5
3
4JHH = 2.2 Hz, JHH = 0.4 Hz, 1H, CH-2); 7.82 (ddd, JHH = 8.2 Hz,
4
3
4JHH = 2.3 Hz, JHH = 1.0 Hz, 1H, CH-4); 7.71 (ddd, JHH = 8.2 Hz,
4
3
4JHH = 2.2 Hz, JHH = 1.0 Hz, 1H, CH-6); 7.56 (ddd, JHH = 8.2 Hz,
5
3JHH = 8.2 Hz, JHH = 0.4 Hz, 1H, CH-5); 7.48 (m, 2H, CH-2′,6′);
IR (ATR-FT-IRS) ν
̃
: 3344, 2928, 1662, 1609 cm−1. ESI-ICR (m/z):
7.30 (m, 2H, CH-3′,5′); 7.00 (m, 1H, CH-4′). 13C NMR (100.6 MHz,
DMSO-d6, +25 °C): δ 152.4 (NHCONH); 148.1 (C-3); 141.0 (C-1);
139.2 (C-1′); 130.0 (CH-5); 128.8 (CH-3′,5′); 124.3 (CH-6); 122.3
(C-4′); 118.6 (CH-2′,6′); 116.2 (CH-4); 112.1 (CH-2). 15N NMR
(40.6 MHz, DMSO-d6, +25 °C): δ 371.8 (NO2); 109.66 (2 × NH). IR
solvent ∼0.1% DMSO/MeOH, calcd for C23H19O2N2 [M − H]−
355.14520, found 355.14509.
Preparation of Compound 14. Compound 12 (4.20 g, 11.79
mmol) was suspended in 2-propanol (100 mL), and then a solution of
KOH (32.0 g, 570.4 mmol) in H2O (120 mL) was added. The mixture
was heated under reflux temperature for 40 h until disappearance of
the starting material (as monitored by TLC). The reaction mixture
was then cooled to room temperature, and the aqueous layer pH was
adjusted to 7 using aqueous HCl (1 M). A precipitate was formed and
was extracted into ethyl acetate (200 mL). The ethyl acetate solution
was dried over anhydrous MgSO4 for 5 min and filtered. The filtrate
was evaporated under reduced pressure to obtain pure indolo[2,3-
a]carbazole-2-carboxylic acid (14) (3.20 g, 10.66 mmol, 90.4% yield)
as an off-white solid.
(ATR-FT-IRS) ν
̃
: 3309, 3269, 1638, 1523 cm−1. ESI-ICR (m/z):
solvent ∼0.1% DMSO/MeOH, calcd for C13H10N3O3 [M − H]−
256.07276, found 256.07289.
Preparation of Compounds 12 and 13. Compound 7 (6.00 g,
32.43 mmol) and 3-hydrazinobenzoic acid (5.40 g, 35.52 mmol) were
dissolved in n-butanol (250 mL). The mixture was stirred at room
temperature for 15 min, and then concentrated H2SO4 (3.00 mL) was
added dropwise. The mixture was stirred at reflux temperature for 40 h
until disappearance of the starting material (as monitored by TLC).
The mixture was then cooled to room temperature and kept there for
15 h. A yellow precipitate formed. The precipitate was filtered. The
Data for 14: Mp: decomposed above 350 °C. Rf = 0.13 (50% THF
1
in hexane). H NMR (400.1 MHz, DMSO-d6, +25 °C): δ 12.72 (bs,
2510
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The Journal of Organic Chemistry
Article
1H, COOH); 11.27 (bs, 1H, NH-12); 11.26 (bs, 1H, NH-11); 8.34
(dd, JHH = 1.5 Hz, JHH = 0.7 Hz, 1H, CH-1); 8.24 (ddd, JHH = 8.2
(CH-5 or CH-6); 111.7 (CH-10); 64.8 (CH2). 15N NMR (40.6 MHz,
DMSO-d6, +25 °C): δ 115.14 (NH-12); 114.45 (NH-11). IR (ATR-
4
5
3
5
3
Hz, 5JHH = 0.7 Hz, JHH = 0.5 Hz, 1H, CH-4); 8.18 (dddd, JHH = 7.8
Hz, 4JHH = 1.2 Hz, 5JHH = 0.9 Hz, 5JHH = 0.6 Hz, 1H, CH-7); 7.97 (bs,
2H, CH-5,6); 7.82 (dd, 3JHH = 8.2 Hz, 4JHH = 1.5 Hz, 1H, CH-3); 7.70
FT-IRS) ν
̃
: 3384, 3349, 1678, 1277 cm−1. ESI-ICR (m/z): solvent
∼0.1% DMSO/MeOH, calcd for C26H16N3O4 [M − H]− 434.11463,
found 434.11457.
3
4
5
(ddd, JHH = 8.1 Hz, JHH = 1.0 Hz, JHH = 0.9 Hz, 1H, CH-10); 7.41
(ddd, JHH = 8.1 Hz, JHH = 7.1 Hz, JHH = 1.2 Hz, 1H, CH-9); 7.22
Data for 17: Yield: 120.0 mg, 0.28 mmol, 85.7%. Light-yellow solid.
3
3
4
1
Mp: decomposed above 350 °C. Rf = 0.53 (50% THF in hexane). H
(ddd, JHH = 7.8 Hz, JHH = 7.1 Hz, JHH = 1.0 Hz, 1H, CH-8). 13C
NMR (100.6 MHz, DMSO-d6, +25 °C): δ 168.1 (COO); 139.1 (C-
10′); 138.3 (C-12′); 127.3 (C-11″); 127.2 (C-4′); 126.5 (C-2); 125.4
(C-11′); 124.8 (CH-9); 123.6 (C-7′); 120.9 (C-6′); 119.9 (CH-3);
119.8 (CH-7); 119.4 (C-5′); 119.3 (CH-4); 119.0 (CH-8); 113.3 (CH-
1); 112.2 (CH-5 or CH-6); 112.0 (CH-5 or CH-6); 111.7 (CH-10).
15N NMR (40.6 MHz, DMSO-d6, +25 °C): δ: 114.78 (NH-12);
3
3
4
NMR (400.1 MHz, DMSO-d6, +25 °C): δ 11.34 (bs, 1H, NH-11);
4
5
11.28 (bs, 1H, NH-12); 8.37 (dd, JHH = 1.5 Hz, JHH = 0.6 Hz, 1H,
3
5
5
CH-1); 8.26 (ddd, JHH = 8.2 Hz, JHH = 0.7 Hz, JHH = 0.6 Hz, 1H,
CH-4); 8.18 (dddd, 3JHH = 7.9 Hz, 4JHH = 1.2 Hz, 5JHH = 0.7 Hz, 5JHH
= 0.6 Hz, 1H, CH-7); 7.97 (bs, 2H, CH-5,6); 7.83 (dd, 3JHH = 8.2 Hz,
4JHH = 1.5 Hz, 1H, CH-3); 7.70 (ddd, JHH = 8.1 Hz, JHH = 1.0 Hz,
3
4
5JHH = 0.7 Hz, 1H, CH-10); 7.48 (AA′ of AA′XX′, 2H, CH-2,6Ph);
114.14 (NH-11). IR (ATR-FT-IRS) ν
̃
: 3410, 2951, 2831, 1679 cm−1.
3
3
4
7.41 (ddd, JHH = 8.1 Hz, JHH = 7.1 Hz, JHH = 1.2 Hz, 1H, CH-9);
3
3
4
ESI-ICR (m/z): solvent ∼0.1% DMSO/MeOH, calcd for C19H11O2N2
7.22 (ddd, JHH = 7.9 Hz, JHH = 7.1 Hz, JHH = 1.0 Hz, 1H, CH-8);
6.99 (XX′ of AA′XX′, 2H, CH-3,5Ph); 5.34 (s, 2H, CH2); 3.78 (s, 3H,
OCH3). 13C NMR (100.6 MHz, DMSO-d6, +25 °C): δ 166.4 (COO);
159.2 (CH-4Ph); 139.1 (C-10′); 138.3 (C-12′); 130.0 (CH-2,6Ph);
128.3 (C-1Ph); 127.5 (C-4′); 127.5 (C-11″); 125.3 (C-2); 125.3 (C-
11′); 124.9 (CH-9); 123.5 (C-7′); 121.0 (C-6′); 119.9 (CH-7); 119.7
(CH-3); 119.5 (CH-4); 119.3 (C-5′); 119.1 (CH-8); 113.9 (C-3,5Ph);
113.3 (CH-1); 112.4 (CH-5 or CH-6); 112.1 (CH-5 or CH-6); 111.7
(CH-10); 65.9 (CH2); 55.1 (OCH3). 15N NMR (40.6 MHz, DMSO-
d6, +25 °C): δ 114.98 (NH-12); 114.37 (NH-11). IR (ATR-FT-IRS)
[M − H]− 299.08260, found 299.08281.
General Procedure for Compounds 15−18, 20, and 21.
Compound 14 (0.16 or 0.32 mmol) and K2CO3 (2 equiv) were
dissolved in dry DMF (2−3 mL), and then the respective reactant (1.3
equiv) was added dropwise. The reactants were the following:
bromomethylbenzene, 1-bromomethyl-4-nitrobenzene, 1-chlorometh-
yl-4-methoxybenzene, 1-chloromethyl-3-methylbenzene, iodoethane,
and 2-iodopropane. The reaction mixture was stirred for 3 h at 80 °C
under an atmosphere of N2. After disappearance of the starting
material (as monitored by TLC), the reaction mixture was cooled to
room temperature and quenched in water, and the product was
extracted with ethyl acetate. The ethyl acetate solution was washed
with water and saturated aqueous NaCl solution, dried over MaSO4,
filtered, and concentrated under reduced pressure. The crude product
was washed with diethyl ether to get the pure compound 15−18, 20,
or 21 as a solid.
ν
̃
: 3372, 3336, 1669, 1245 cm−1. ESI-ICR (m/z): solvent ∼0.1%
DMSO/MeOH, calcd for C27H19N2O3 [M − H]− 419.14012, found
419.14024.
Data for 18: Yield: 100.0 mg, 0.24 mmol, 74.6%. Light-yellow solid.
Mp: decomposed above 324.5−326.5 °C. Rf = 0.55 (50% THF in
hexane). 1H NMR (400.1 MHz, DMSO-d6, +25 °C): δ 11.38 (bs, 1H,
4
5
NH-11); 11.34 (bs, 1H, NH-12); 8.40 (dd, JHH = 1.5 Hz, JHH = 0.6
Data for 15: Yield: 60.0 mg, 0.15 mmol, 92.3%. Light-yellow solid.
3
5
5
Hz, 1H, CH-1); 8.27 (ddd, JHH = 8.2 Hz, JHH = 0.6 Hz, JHH = 0.6
1
Mp: decomposed above 350 °C. Rf = 0.59 (50% THF in hexane). H
3
4
Hz, 1H, CH-4); 8.18 (dddd, JHH = 7.8 Hz, JHH = 1.2 Hz, 5JHH = 0.7
NMR (400.1 MHz, DMSO-d6, +25 °C): δ 11.36 (bs, 2H, NH-11,12);
5
3
Hz, JHH = 0.6 Hz, 1H, CH-7); 7.97 (bs, 2H, CH-5,6); 7.86 (dd, JHH
4
5
3
8.41 (dd, JHH = 1.5 Hz, JHH = 0.6 Hz, 1H, CH-1); 8.27 (dd, JHH
=
= 8.2 Hz, 4JHH = 1.5 Hz, 1H, CH-3); 7.70 (ddd, 3JHH = 8.1 Hz, 4JHH
=
=
8.2 Hz, 5JHH = 0.6 Hz, 1H, CH-4); 8.18 (ddd, 3JHH = 7.8 Hz, 4JHH = 1.2
Hz, JHH = 0.7 Hz, 1H, CH-7); 7.98 (bs, 2H, CH-5 or CH-6); 7.87
(dd, JHH = 8.2 Hz, JHH = 1.5 Hz, 1H, CH-3); 7.70 (ddd, JHH = 8.1
Hz, JHH = 1.0 Hz, JHH = 0.7 Hz, 1H, CH-10); 7.54 (m, 2H, CH-
5
3
3
1.0 Hz, JHH = 0.7 Hz, 1H, CH-10); 7.41 (ddd, JHH = 8.1 Hz, JHH
5
4
7.1 Hz, JHH = 1.2 Hz, 1H, CH-9); 7.34 (m, 1H, CH-2Ph); 7.33 (m,
1H, CH-5Ph); 7.32 (m, 1H, CH-6Ph); 7.22 (ddd, 3JHH = 7.8 Hz, 3JHH
7.1 Hz, 4JHH = 1.0 Hz, 1H, CH-8); 7.19 (m, 1H, CH-4Ph); 5.38 (s, 2H,
3
4
3
=
4
5
3
3
2,6Ph); 7.45 (m, 2H, CH-3,5Ph); 7.41 (ddd, JHH = 8.1 Hz, JHH = 7.1
Hz, 4JHH = 1.2 Hz, 1H, CH-9); 7.38 (m, 1H, CH-4Ph); 7.22 (ddd, 3JHH
= 7.8 Hz, 3JHH = 7.1 Hz, 4JHH = 1.0 Hz, 1H, CH-8); 5.43 (s, 2H, CH2).
13C NMR (100.6 MHz, DMSO-d6, +25 °C): δ 166.4 (COO); 139.1
(C-10′); 138.3 (C-12′); 136.5 (C-1Ph); 128.5 (C-3,5Ph); 128.1 (CH-
4Ph); 128.0 (CH-2,6Ph); 127.6 (C-4′); 127.5 (C-11″); 125.3 (C-11′);
125.1 (C-2); 124.9 (CH-9); 123.5 (C-7′); 121.0 (C-6′); 119.9 (CH-7);
119.7 (CH-3); 119.5 (CH-4); 119.3 (C-5′); 119.0 (CH-8); 113.3 (CH-
1); 112.4 (CH-5 or CH-6); 112.1 (CH-5 or CH-6); 111.7 (CH-10);
66.0 (CH2). 15N NMR (40.6 MHz, DMSO-d6, +25 °C): δ 115.14
CH2); 2.35 (dd, JHH = 1.0 Hz, JHH = 0.6 Hz, 3H, CH3). 13C NMR
(100.6 MHz, DMSO-d6, +25 °C): δ 166.4 (COO); 139.2 (C-10′);
138.3 (C-12′); 137.7 (C-3Ph); 136.3 (C-1Ph); 128.7 (CH-4Ph); 128.6
(CH-2Ph); 128.4 (CH-5Ph); 127.6 (C-4′); 127.5 (C-11″); 125.4 (C-
11′); 125.15 (C-2); 125.12 (CH-6Ph); 124.9 (CH-9); 123.5 (C-7′);
121.0 (C-6′); 119.9 (CH-7); 119.7 (CH-3); 119.5 (CH-4); 119.3 (C-
5′); 119.0 (CH-8); 113.3 (CH-1); 112.4 (CH-5 or CH-6); 112.1 (CH-
5 or CH-6); 111.7 (CH-10); 66.0 (CH2); 21.0 (CH3). 15N NMR (40.6
MHz, DMSO-d6, +25 °C): δ 115.17 (NH-12); 114.49 (NH-11). IR
4
4
(ATR-FT-IRS) ν
̃
: 3377, 3335, 1671, 1282 cm−1. ESI-ICR (m/z):
̃
(NH-12); 114.45 (NH-11). IR (ATR-FT-IRS) ν: 3387, 3343, 1675,
solvent ∼0.1% DMSO/MeOH, calcd for C27H19N2O2 [M − H]−
403.14520, found 403.14556.
1282 cm−1. ESI-ICR (m/z): solvent ∼0.1% DMSO/MeOH, calcd for
C26H17N2O2 [M − H]− 389.12955, found 389.12955.
Data for 20: Yield: 40.0 mg, 0.12 mmol, 73.2%. Light-yellow solid.
Data for 16: Yield: 59.7 mg, 0.13 mmol, 59.7%. Yellow solid. Mp:
decomposed above 350 °C. Rf = 0.46 (50% THF in hexane). 1H NMR
(400.1 MHz, DMSO-d6, +25 °C): δ 11.34 (bs, 1H, NH-11); 11.32 (bs,
1H, NH-12); 8.44 (dd, 4JHH = 1.5 Hz, 5JHH = 0.6 Hz, 1H, CH-1); 8.30
(XX′ of AA′XX′, 2H, CH-3,5Ph); 8.29 (ddd, 3JHH = 8.2 Hz, 5JHH = 0.6
1
Mp: decomposed above 350 °C. Rf = 0.58 (50% THF in hexane). H
NMR (400.1 MHz, DMSO-d6, +25 °C): δ 11.33 (bs, 1H, NH-11);
4
5
11.29 (bs, 1H, NH-12); 8.37 (dd, JHH = 1.5 Hz, JHH = 0.7 Hz, 1H,
3
5
5
CH-1); 8.26 (ddd, JHH = 8.2 Hz, JHH = 0.7 Hz, JHH = 0.6 Hz, 1H,
CH-4); 8.18 (dddd, 3JHH = 7.8 Hz, 4JHH = 1.2 Hz, 5JHH = 0.8 Hz, 5JHH
= 0.6 Hz, 1H, CH-7); 7.97 (bs, 2H, CH-5,6); 7.83 (dd, 3JHH = 8.2 Hz,
3
4
Hz, 5JHH = 0.6 Hz, 1H, CH-4); 8.18 (dddd, JHH = 7.8 Hz, JHH = 1.2
Hz, 5JHH = 0.8 Hz, 5JHH = 0.6 Hz, 1H, CH-7); 7.98 (bs, 2H, CH-5,6);
4JHH = 1.5 Hz, 1H, CH-3); 7.70 (ddd, JHH = 8.1 Hz, JHH = 1.0 Hz,
3
4
3
4
7.90 (dd, JHH = 8.2 Hz, JHH = 1.5 Hz, 1H, CH-3); 7.81 (AA′ of
AA′XX′, 2H, CH-2,6Ph); 7.71 (ddd, 3JHH = 8.1 Hz, 4JHH = 1.0 Hz, 5JHH
5JHH = 0.8 Hz, 1H, CH-10); 7.42 (ddd, JHH = 8.1 Hz, JHH = 7.1 Hz,
3
3
4JHH = 1.2 Hz, 1H, CH-9); 7.22 (ddd, JHH = 7.8 Hz, JHH = 7.1 Hz,
4JHH = 1.0 Hz, 1H, CH-8); 4.38 (q, 3JHH = 7.1 Hz, 2H, CH2); 1.39 (t,
3JHH = 7.1 Hz, 3H, CH3). 13C NMR (100.6 MHz, DMSO-d6, +25 °C):
δ 166.5 (COO); 139.1 (C-10′); 138.3 (C-12′); 127.4 (C-4′,11″); 125.5
(C-2); 125.4 (C-11′); 124.8 (CH-9); 123.6 (C-7′); 121.0 (C-6′); 119.9
(CH-7); 119.7 (CH-3); 119.4 (CH-4); 119.3 (C-5′); 119.0 (CH-8);
113.1 (CH-1); 112.3 (CH-5 or CH-6); 112.0 (CH-5 or CH-6); 111.7
(CH-10); 60.5 (CH2); 14.3 (CH3). 15N NMR (40.6 MHz, DMSO-d6,
3
3
= 0.8 Hz, 1H, CH-10); 7.42 (ddd, 3JHH = 8.1 Hz, 3JHH = 7.1 Hz, 4JHH
=
1.2 Hz, 1H, CH-9); 7.23 (ddd, 3JHH = 7.8 Hz, 3JHH = 7.1 Hz, 4JHH = 1.0
Hz, 1H, CH-8); 5.57 (s, 2H, CH2). 13C NMR (100.6 MHz, DMSO-d6,
+25 °C): δ 166.2 (COO); 147.1 (C-4Ph); 144.3 (C-1Ph); 139.1 (C-
10′); 138.3 (C-12′); 128.6 (CH-2,6Ph); 127.8 (C-4′); 127.6 (C-11″);
125.3 (C-11′); 124.9 (CH-9); 124.7 (C-2); 123.7 (C-3,5Ph); 123.5 (C-
7′); 121.1 (C-6′); 119.9 (CH-7); 119.8 (CH-3); 119.6 (CH-4); 119.3
(C-5′); 119.1 (CH-8); 113.4 (CH-1); 112.4 (CH-5 or CH-6); 112.1
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The Journal of Organic Chemistry
Article
+25 °C): δ 114.96 (NH-12); 114.38 (NH-11). IR (ATR-FT-IRS) ν
̃
:
solutions of the anion titrant (TBAA) were in the range of 0.17−0.44
and 0.65−1.41 M, respectively. In the course of titration, the dilute
titrant was used to obtain degrees of association at different levels of
anion concentration. Near the end point of the titration, the
concentrated titrant was added, so that virtually all of the receptor
molecules in solution were converted to receptor−anion complexes
(8−10 equiv of titrant was eventually added). The medium used was
not buffered because the introduction of additional anionic species
would interfere with the binding process. In each measurement series,
approximately 14−18 spectra were recorded. Most of the spectra from
the titration experiments are provided in the Supporting Information.
From the spectra, the degrees of complexation for both receptor−
anion complexes at a particular titration step were found using the
following equation:
3357, 2995, 1674, 1282 cm−1. ESI-ICR (m/z): solvent ∼0.1% DMSO/
MeOH, calcd for C21H15N2O2 [M − H]− 327.11390, found
327.11377.
Data for 21: Yield: 80.0 mg, 0.23 mmol, 70.1%. Off-white solid. Mp:
decomposed above 350 °C. Rf = 0.64 (50% THF in hexane). 1H NMR
(400.1 MHz, DMSO-d6, +25 °C): δ 11.35 (bs, 1H, NH-11); 11.29 (bs,
1H, NH-12); 8.35 (dd, 4JHH = 1.5 Hz, 5JHH = 0.7 Hz, 1H, CH-1); 8.25
3
5
5
(ddd, JHH = 8.2 Hz, JHH = 0.7 Hz, JHH = 0.6 Hz, 1H, CH-4); 8.18
(dddd, 3JHH = 7.8 Hz, 4JHH = 1.2 Hz, 5JHH = 0.8 Hz, 5JHH = 0.6 Hz, 1H,
CH-7); 7.97 (bs, 2H, CH-5,6); 7.82 (dd, 3JHH = 8.2 Hz, 4JHH = 1.5 Hz,
3
4
5
1H, CH-3); 7.70 (ddd, JHH = 8.1 Hz, JHH = 1.0 Hz, JHH = 0.8 Hz,
1H, CH-10); 7.41 (ddd, JHH = 8.1 Hz, JHH = 7.1 Hz, 4JHH = 1.2 Hz,
3
3
3
3
4
1H, CH-9); 7.22 (ddd, JHH = 7.8 Hz, JHH = 7.1 Hz, JHH = 1.0 Hz,
1H, CH-8); 5.21 (sept, 3JHH = 6.2 Hz, 1H, OCH); 1.39 (d, 3JHH = 6.2
Hz, 6H, 2 × CH3). 13C NMR (100.6 MHz, DMSO-d6, +25 °C): δ
166.0 (COO); 139.2 (C-10′); 138.3 (C-12′); 127.4 (C-4′); 127.4 (C-
11″); 125.9 (C-2); 125.4 (C-11′); 124.9 (CH-9); 123.6 (C-7′); 121.0
(C-6′); 119.9 (CH-7); 119.7 (CH-3); 119.39 (CH-4); 119.35 (C-5′);
119.0 (CH-8); 113.1 (CH-1); 112.3 (CH-5 or CH-6); 112.0 (CH-5 or
CH-6); 111.7 (CH-10); 67.7 (OCH); 21.8 (CH3). 15N NMR (40.6
MHz, DMSO-d6, +25 °C): δ 114.88 (NH-12); 114.38 (NH-11). IR
δ − δH
[HxG]
[Hx] + [HxG]
x
α =
=
δ
− δ
HxG
Hx
(8)
The δ values refer to the chemical shifts of the NH protons. δ denotes
the chemical shift at the titration step, and δH and δH G are the
x
x
chemical shifts of the free receptor molecule and the receptor−anion
complex, respectively. Replacing the equilibrium concentrations in eq
5 with the degrees of association for H1 and H2 (α1 and α2,
respectively) gives the following equation for ΔlogKass:
(ATR-FT-IRS) ν
̃
: 3367, 3068, 1672, 1653 cm−1. ESI-ICR (m/z):
solvent ∼0.1% DMSO/MeOH, calcd for C22H17N2O2 [M − H]−
341.12955, found 341.12948.
Preparation of Compound 19. Compound 14 (0.050 g, 0.16
mmol) and K2CO3 (0.060 g, 0.43 mmol) were dissolved in dry
acetone (3 mL) under nitrogen, and then 1,2-diiodoethane (0.032 g,
0.11 mmol) was added to the solution. The mixture was heated to
reflux for 15 h until disappearance of the starting material (as
monitored by TLC). Then the reaction mixture was quenched in water
and extracted with ethyl acetate. The ethyl acetate solution was washed
with water and a saturated aqueous solution of NaCl, dried over
MgSO4, and filtered. The ethyl acetate layer was concentrated under
reduced pressure. The crude product was purified by column
chromatography, eluting with 1−2% methanol in DCM, to afford
compound 19 (29 mg, 0.08 mmol, 48.9% yield) as a yellow solid.
Data for 19: Mp: decomposed above 300 °C. Rf = 0.5 (5%
α1(1 − α2)
(1 − α1)α2
Δlog Kass = log
(9)
The 1:1 binding ratios for the indolocarbazoles and substituted ureas
were confirmed in ref 9, and that of compound 9 was confirmed in ref
29.
Compound 5 was obtained as a mixture that also contained 4 and 6
(altogether 16% yield). Nevertheless, the ΔlogKass measurements
involving 5 were not disturbed by the impurities because the signals in
the NMR spectra were sufficiently separated (see pp 305 and 310 in
the SI). As further evidence that these measurements were reliable, the
within-series standard deviations s(ΔlogKass) were in the range of
0.001−0.008 log units and the circular validation showed good
consistency with other overlapping measurements.
1
methanol in DCM). H NMR (400.1 MHz, DMSO-d6, +25 °C): δ
4
11.32 (bs, 1H, NH-11); 11.31 (bs, 1H, NH-12); 8.41 (dd, JHH = 1.5
The experimental setup and UV−vis spectrophotometric relative
binding affinity measurement method were the same as used in the
previous work.9
5
3
5
Hz, JHH = 0.7 Hz, 1H, CH-1); 8.29 (ddd, JHH = 8.2 Hz, JHH = 0.7
Hz, 5JHH = 0.6 Hz, 1H, CH-4); 8.19 (dddd, JHH = 7.8 Hz, JHH = 1.2
3
4
Hz, 5JHH = 0.7 Hz, 5JHH = 0.6 Hz, 1H, CH-7); 7.99 (bs, 2H, CH-5,6);
Measurements of Absolute Binding Constants. The working
conditions and solvents used the NMR measurements of absolute
binding constants were the same as in measurements of relative
binding constants. The concentrations of the receptor molecules also
were similar to the ones used in the measurements of relative binding
constants. The concentrations of TBAA in the dilute and concentrated
solutions were in the ranges of 0.15−0.5 and 0.6−1.4 M, respectively.
Over the course of the titration, 12−17 spectra were recorded. The
spectrum of the free receptor was obtained before the first addition of
titrant. The spectrum of the receptor−anion complex was obtained at
the end of titration by adding a large excess of titrant. From the
weighing data the exact amounts of added titrant were found.
The UV measurements of absolute logKass values were performed as
in ref 9, except that the concentrations used were different: 0.185−
0.220 and 0.01−0.18 M for the concentrated and dilute titrant
solutions, respectively.
7.86 (dd, 3JHH = 8.2 Hz, 4JHH = 1.5 Hz, 1H, CH-3); 7.71 (ddd, 3JHH
=
=
8.1 Hz, 4JHH = 1.0 Hz, JHH = 0.7 Hz, 1H, CH-10); 7.42 (ddd, JHH
5
3
8.1 Hz, 3JHH = 7.1 Hz, 4JHH = 1.2 Hz, 1H, CH-9); 7.23 (ddd, 3JHH = 7.8
Hz, 3JHH = 7.1 Hz, 4JHH = 1.0 Hz, 1H, CH-8); 5.07 (s, 2H, CH2); 2.21
(s, 3H, CH3). 13C NMR (100.6 MHz, DMSO-d6, +25 °C): δ 202.1
(CO); 165.9 (COO); 139.1 (C-10′); 138.3 (C-12′); 127.7 (C-4′);
127.6 (C-11″); 125.4 (C-11′); 124.9 (CH-9); 124.5 (C-2); 123.5 (C-
7′); 121.1 (C-6′); 119.9 (CH-7); 119.8 (CH-3); 119.6 (CH-4); 119.3
(C-5′); 119.1 (CH-8); 113.4 (CH-1); 112.4 (CH-5 or CH-6); 112.1
(CH-5 or CH-6); 111.7 (CH-10); 68.7 (CH2); 26.0 (CH3). 15N NMR
(40.6 MHz, DMSO-d6, +25 °C): δ 115.11 (NH-12); 114.36 (NH-11).
IR (ATR-FT-IRS) ν
̃
: 3344, 2919, 2850, 1675 cm−1. ESI-ICR (m/z):
solvent 80% ACN/20% H2O (0.1% formic acid), calcd for
C22H15N2O3 [M − H]− 355.10882, found 355.10898.
15N NMR Measurements. The 15N NMR spectra were recorded
at 40.6 MHz (15N) on a 400 MHz NMR instrument. 15N NMR
chemical shifts were directly obtained from 15N INEPT or indirectly
1
1
1
from H-detected H−15N gs-HMBC or H−15N gs-HSQC spectra.
The 15N chemical shifts were referenced externally to the signal of neat
nitromethane (381.7 ppm).32
ASSOCIATED CONTENT
■
S
* Supporting Information
Relative binding measurement spectra (1H NMR and UV−vis
spectrophotometric), additional compound characterization
data (NMR, IR, and HRMS) of the synthesized compounds,
and an example calculation file of relative binding measure-
ments and uncertainty estimation procedures (in .xlsx format).
This material is available free of charge via the Internet at
http://pubs.acs.org.
Measurements of Relative Binding Constants. NMR measure-
ments were carried out with the 200 MHz instrument. Some
measurements were also repeated on the 400 MHz instrument. The
results did not differ. All of the solutions were prepared in 95.5%
DMSO-d6/0.5% water (m/m). The concentrations of receptor
molecules during the titration experiments were in the range of
0.006−0.009 M. The concentrations of the concentrated and dilute
2512
dx.doi.org/10.1021/jo4027963 | J. Org. Chem. 2014, 79, 2501−2513

The Journal of Organic Chemistry
Article
(24) Gale, P. A. Chem. Commun. 2008, 4525−4540.
AUTHOR INFORMATION
Corresponding Author
*E-mail: ivo.leito@ut.ee. Phone: +372 51 84 176.
Notes
■
(25) Leito, I.; Herodes, K.; Huopolainen, M.; Virro, K.; Kunnapas,
̈
A.; Kruve, A.; Tanner, R. Rapid Commun. Mass Spectrom. 2008, 22,
379−384.
(26) Eblinger, F.; Schneider, H.-J. J. Phys. Chem. 1996, 100, 5533−
5537.
The authors declare no competing financial interest.
(27) Armarego, W. L. F.; Chai, C. L. L. Purification of Laboratory
Chemicals, 6th ed.; Elsevier Butterworth-Heinemann: Burlington, MA,
2009.
ACKNOWLEDGMENTS
■
This work was supported by Grant 9105 from the Estonian
Science Foundation, the Estonian National Research and
Development Infrastructure Development Programme of
Measure 2.3 “Promotion of Development Activities and
Innovation” (Regulation 34) funded by the Enterprise Estonia
Foundation, and by Institutional Funding Project IUT20-14
from the Estonian Ministry of Education and Science. L.T. was
supported by Mobilitas Top Researcher Grant MTT2, and
S.A.K. was supported by DoRa Grant 30.1.6/886 from the
Archimedes Foundation. The authors thank Dr. Thomas
Netscher and Dr. Werner Bonrath from DSM Nutritional
Products for supplying the compounds for the MS mass axis
calibration.
(28) Huang, L.; Shi, A.; He, F.; Li, X. Bioorg. Med. Chem. 2010, 18,
1244−1251.
(29) Brooks, S. J.; Gale, P. A.; Light, M. E. Chem. Commun. 2005,
4696−4698.
(30) Lewis, F. D.; Delos Santos, G. B.; Liu, W. J. Org. Chem. 2005,
70, 2974−2979.
(31) Mayumi, K.; Kosuke, K.; Isao, A.; Hiroyuki, K.; Aya, T.
Tetrahedron 2012, 68, 4455−4463.
̌
(32) Marek, R.; Lycka, A. Curr. Org. Chem. 2002, 6, 35−66.
REFERENCES
■
(1) Lehn, J.-M. Science 2002, 295, 2400−2403.
(2) Lehn, J.-M. Angew. Chem., Int. Ed. Engl. 1988, 27, 89−112.
(3) Zhang, S. Nat. Biotechnol. 2003, 21, 1171−1178.
(4) Zeng, F.; Zimmerman, S. C. Chem. Rev. 1997, 97, 1681−1712.
(5) Hirose, K. Quantitative Analysis of Binding Properties. In
Analytical Methods in Supramolecular Chemistry; Schalley, C. A., Ed.;
Wiley-VCH: Weinheim, Germany, 2012; pp 27−66.
(6) Soovali, L.; Kaljurand, I.; Kutt, A.; Leito, I. Anal. Chim. Acta 2006,
̈
̈
566, 290−303.
(7) Sessler, J. L.; Gross, D. E.; Cho, W.-S.; Lynch, V. M.;
Schmidtchen, F. P.; Bates, G. W.; Light, M. E.; Gale, P. A. J. Am.
Chem. Soc. 2006, 128, 12281−12288.
(8) Kutt, A.; Leito, I.; Kaljurand, I.; Soovali, L.; Vlasov, V. M.;
̈
̈
Yagupolskii, L. M.; Koppel, I. A. J. Org. Chem. 2006, 71, 2829−2838.
(9) Haav, K.; Kadam, S. A.; Toom, L.; Gale, P. A.; Busschaert, N.;
Wenzel, M.; Hiscock, J.; Kirby, I.; Haljasorg, T.; Lokov, M.; Leito, I. J.
̃
Org. Chem. 2013, 78, 7796−7808.
(10) You, L.; Anslyn, E. V. In Supramolecular Chemistry: From
Molecules to Nanomaterials; Gale, P. A., Steed, J. W., Eds.; Wiley:
Chichester, U.K., 2012; Vol. 1, pp 135−160.
(11) Bates, G. W.; Light, M. E.; Albrecht, M.; Gale, P. A. J. Org.
Chem. 2007, 72, 8921−8927.
(12) Makuc, D.; Hiscock, J. R.; Light, M. E.; Gale, P. A.; Plavec, J.
Beilstein J. Org. Chem. 2011, 7, 1205−1214.
(13) Brooks, S. J.; Gale, P. A.; Light, M. E. Chem. Commun. 2006,
4344−4346.
(14) Caltagirone, C.; Gale, P. A. Chem. Soc. Rev. 2009, 38, 520−563.
(15) Gale, P. A. Coord. Chem. Rev. 2001, 213, 79−128.
(16) Sessler, J. L.; Davis, J. M. Acc. Chem. Res. 2001, 34, 989−997.
(17) De Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley,
A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997,
97, 1515−1566.
(18) Beer, P. D.; Gale, P. A. Angew. Chem., Int. Ed. 2001, 40, 486−
516.
(19) Amendola, V.; Esteban-Gomez, D.; Fabbrizzi, L.; Licchelli, M.
Acc. Chem. Res. 2006, 39, 343−353.
(20) Gunnlaugsson, T.; Glynn, M.; Tocci, G. M.; Kruger, P. E.;
Pfeffer, F. M. Coord. Chem. Rev. 2006, 250, 3094−3117.
(21) Bhosale, S. V.; Bhosale, S. V.; Kalyankar, M. B.; Langford, S. J.
Org. Lett. 2009, 11, 5418−5421.
(22) Li, A.-F.; Wang, J.-H.; Wang, F.; Jiang, Y.-B. Chem. Soc. Rev.
2010, 39, 3729−3745.
(23) Amendola, V.; Fabbrizzi, L.; Mosca, L. Chem. Soc. Rev. 2010, 39,
3889−3915.
2513
dx.doi.org/10.1021/jo4027963 | J. Org. Chem. 2014, 79, 2501−2513