Aminoalkoxyfluorenones and aminoalkoxybiphenyls: DNA binding modes

Article abstract of DOI:10.1016/j.bioorg.2019.01.024

Many evidences suggest that DNA-drug interaction in the minor groove and the intercalation of drugs into DNA may play critical roles in antiviral, antimicrobial, and antitumor activities. As a continuous effort to develop novel antiviral agents, the series of planar fluorenone (3a–7d) were synthesized and used along with nonplanar biphenyls (11a–d) for the comparative analysis of their interaction with DNA. The chemical structure of new compounds was confirmed by ¹H NMR, ¹³C NMR and mass spectra as well as elemental analysis. DNA affinity of 3a–7d and 11a–d was evaluated by ethidium bromide displacement assay. Affinity constant (lgKa) of 3a–7d was found to be approximately two orders of magnitude higher than constants of corresponding 11a–d. The molecular docking of aminoalkoxybiphenyls (11a–d) into minor grove of five different nucleotide sequences (d(CCIICICCII), d(CGCGTTAACGCG), d(CGCGATATCGCG), d(GGCCAATTGG), d(GGATATATCC)) demonstrated their binding capacity to the specific DNA site. The linear least squares fitting technique was successfully applied to derive an equation describing the relationship between lgKa and SF.

Full text of DOI:10.1016/j.bioorg.2019.01.024

Accepted Manuscript
Aminoalkoxyfluorenones and aminoalkoxybiphenyls: DNA binding modes
Svitlana O. Zanoza, Kyrylo O. Klimenko, George V. Maltzev, Tetiana I.
Bykova, Igor A. Levandovskiy, Sergiy A. Lyakhov, Sergiy A. Andronati,
Mikhail L. Bondarev
PII:
S0045-2068(18)30999-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.024
YBIOO 2728
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 September 2018
13 December 2018
14 January 2019
Please cite this article as: S.O. Zanoza, K.O. Klimenko, G.V. Maltzev, T.I. Bykova, I.A. Levandovskiy, S.A.
Lyakhov, S.A. Andronati, M.L. Bondarev, Aminoalkoxyfluorenones and aminoalkoxybiphenyls: DNA binding
modes, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.01.024
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Aminoalkoxyfluorenones and aminoalkoxybiphenyls: DNA binding modes
Svitlana O. Zanozaa , Kyrylo O. Klimenkoa,b, George V. Maltzeva, Tetiana I. Bykovac, Igor
A. Levandovskiyd, Sergiy A. Lyakhova, Sergiy A. Andronatia, Mikhail L. Bondareve,*
a
A. V. Bogatsky Physico-Chemical Institute of National Academy of Science of Ukraine, 86
Lyustdorfskaya doroga, Odessa 65080, Ukraine
b
Laboratoire de Chemoinformatique, (UMR 7140 CNRS/UniStra) Université de Strasbourg, 1,
rue B. Pascal, Strasbourg 67000, France
c I. I. Mechnikov National University, Department of Chemistry, 2 Dvoryanskaya, Odessa 65026,
Ukraine
d
Department of Organic Chemistry, National Technical University of Ukraine "Igor Sikorsky
Kyiv Polytechnic Institute”, 37 Pr. Pobedy, Kyiv, Ukraine
e
Department of Pharmaceutical Sciences, School of Pharmacy, Hampton University, Kittrell
Hall, Hampton, VA 23668, USA
*Corresponding author.
E-mail address: mikhail.bondarev@hamptonu.edu (M.L. Bondarev)
1

A B S T R A C T
Many evidences suggest that DNA-drug interaction in the minor groove and the intercalation of
drugs into DNA may play critical roles in antiviral, antimicrobial, and antitumor activities. As a
continuous effort to develop novel antiviral agents, the series of planar fluorenone (3a-7d) were
synthesized and used along with nonplanar biphenyls (11a-d) for the comparative analysis of
1
their interaction with DNA. The chemical structure of new compounds was confirmed by H
NMR, 13C NMR and mass spectra as well as elemental analysis. DNA affinity of 3a-7d and 11a-
d was evaluated by ethidium bromide displacement assay. Affinity constant (lgKa) of 3a-7d was
found to be approximately two orders of magnitude higher than constants of corresponding 11a-
d. The molecular docking of aminoalkoxybiphenyls (11a-d) into minor grove of five different
nucleotide sequences (d(CCIICICCII), d(CGCGTTAACGCG), d(CGCGATATCGCG),
d(GGCCAATTGG), d(GGATATATCC)) demonstrated their binding capacity to the specific
DNA site. The linear least squares fitting technique was successfully applied to derive an
equation describing the relationship between lgKa and SF.
Keywords: Aminoalkoxybiphenyls, Aminoalkoxyfluorenones, DNA affinity, DNA minor grove
Intercalation and molecular docking
1. Introduction
Essentially there are two chemotherapeutic approaches for treating viral diseases. The first
approach is the development of selective antiviral drugs [1-3] which, however, can become
redundant due to the drug resistance caused by the mutation of virus genes. Thus, establishing a
new drug using this methodology is a lengthy, expensive and sometimes inefficient process. The
2

second one is the development of multi-target therapeutics or polypharmacological drugs that are
needed to treat systematic diseases such as cancer or viral ones.
The multi-target therapy may enhance clinical efficacy, while the possibility of serious side
effects is also increased. The inductors (IFNI) or non-specific resistance stimulators [4] play an
important role for the clinical needs and therefore, tilorone remain a great challenge to rationally
design effective multi-target therapeutics.
Two main pharmacological significant types of the drug-DNA interactions are known, it
is a minor groove DNA binding and intercalation of planar polycyclic compounds between two
sequential DNA base pairs [5]. Both intercalators and minor groove binders reveal antiviral
activity. It was previously suggested [6, 7] that the interferon-induced inhibition of viral
reproduction by planar polycyclic compounds is based on their intercalation into DNA. Thus,
well known DNA intercalators acridines and their bis-analogs exhibited both antiviral [8-10] and
interferon-inducing properties [11]. The same relationship between intercalation and antiviral
properties revealed among fluorenone [12-15], isatin hydrazone [16], naphthylamide [17] and
indoloquinoxaline [18] derivatives. We can assume that any distortion of the planarity of tilorone
or tiloron-like structures should weaken or even abolish the intercalation to DNA, and
consequently reduce or eliminate the interferon-inducing and antiviral activity. In order to
estimate the influence of such distortion on DNA binding mode and its relationship to the
antiviral activity, we have prepared nonplanar 11a-15e structures.
However, in contrast to our expectation, we found that these compounds have shown the
antiviral activity in vitro on L929 (mouse fibroblast cell line), PST (primary swine testicle) , RF
(rat fibroblast cell line) against vesicular stomatitis virus (VSV) and herpes simples virus type I
(HSV) both in preventive and therapeutical modes [19, 20] and also induced interferon [21, 22].
3

These observations may suggest that the compounds are involved in an alternative DNA-binding
mode. In this paper we specifically focus on the ability of 4,4'-dihydroxybiphenyl derivatives
11a-15e to participate in DNA minor-groove recognition.
2. Results and Discussion
Chemistry 2.1.
The synthetic route for aminoalkoxyfluorenones 3a-7d derivatives is outlined in Scheme 1.
Scheme 1. Reagents and conditions: (a) (CH2)2Cl2, 20% NaOH/H2O, TBAB, 75 С; (b)
BrCHnBr, K2CO3/DMF, 80 °С; (c) NaI, TBAI, H2O/toluene, reflux; 10h; (d) appropriate
amines/DMF, rt.
4

The intermediates 2a-g, were generally obtained by O-alkylation of 2,7-dihydroxyfluoren-9-one
1 with a set of dihaloalkanes which have an straight alkyl chain from two to six methylene units.
The alkylation was carried out either under phase-transfer catalysis as for 2b or in DMF at 80 °C
in the presence of anhydrous K2CO3 as for compounds 2b-e. More reactive iodides 2f-g were
prepared from 2a-b by Finkelstein type of reactions. All halide derivatives 2a-g were then
converted to the corresponding amines 3a-7d at room temperature within 10 to 30 days.
The preparation and structural elucidation of compounds 3a, 3c, 4a-4c, 5a and as well as the
final aminoalkoxybiphenyls 11a-15e (Fig.1) were early reported [23, 24].
Fig. 1. Structures of aminoalkoxybiphenyls 11a-15e
As shown in Scheme 2, benzidine derivatives 10a-e were prepared by the acetylation of
commercially available benzidine 8 with chloroacetyl chloride to afford chloroacetamide
compound 9, followed by amination with appropriate amines.
5

Scheme 2. Reagents and conditions: (a) ClCH2COCl, toluene, reflux, 2 h; (b) appropriate
amines/DMF, rt.
The chemical structures of the target compounds were confirmed by elemental analysis, mass
spectrometry and NMR.
2.2.1. DNA binding studies
2.2.1.1 Analysis of DNA-ligand interaction
It is well known [25] that the compound can be considered a DNA intercalator if it meets
the following criteria: (i) it can increase the specific DNA viscosity; (ii) it should show a
bathochromic shift of its maximum absorption in the presentce of DNA; (iii) it is able to compete
with a known intercalator for DNA binding; (iv) it is able to unwind of supercoiled plasmid.
The specific DNA viscosity measurements were carried out with compounds 11a-15e
(concentration range: 30 - 300 μmol/L) in the presence of 2.5 times excess amount of DNA under the
conditions similar to those previously described [25]. None of 11a-15e led to increase of DNA
solution specific viscosity. Moreover, compound 15a even decreases viscosity (Table 1)
Table 1. Specific viscosity of solutions 3a-7a, 11a-15a
Compound
ŋ
σ
Compound
ŋ
σ
0.156
0.213*
0.216*
0.168*
0.002
0.003
0.003
0.001
DNA
3a
0.158**
0.155**
0.122*
0.003
0.002
0.002
11a
13a
15a
5a
7a
*P < 0.05; **P > 0.05
6

Any bathochromic shift of maximum absorption in the electronic spectra of 11a-15d
(concentration range: 0 - 30 μmol/L) with their DNA mixtures was not observed (Fig. 2a,
compound 11a) although such shifts were found in spectra of 3a-7d with their DNA mixtures
(Fig. 2b, compound 3a) [26].
7

a.
A
263
A
B
C
0.5
0.25
0
λ, nm
220
245
270
295
320
b.
A
270
A
0.3
0.2
0.1
0.0
274
B
C
D
E
λ, nm
220
245
270
295
320
Figure 2. (a) Electron specrum in UV-range – 11a compound at CDNA, M = 0 (A); 1 (B); 10
(C); (b) – 3a compound at CDNA, M = 0 (A); 5 (B); 10 (C); 20 (D); 30 (E)
8

In addition, all target compounds (concentration range: 0.1-500 μmol/L) compete with ethidium
bromide (EB) in binding to DNA. Based on the above experimental results, we concluded that
the 4,4-aminoalkoxybiphenyl ligands 11a-15d do not bind to DNA through the intercalation
mode.
2.2.1.2 DNA affinity
The affinity of ligands for DNA (Table 2) was evaluated by the association constants (lgKa)
which were calculated from their С50 values as early described [27, 28].
Table 2. Biphenyls and fluorenones affinity (lgKa) to DNA.*
Biphenyls (11a-15d), pKa for n = 2-6
Fluorenones (3a-7d), pKa for n = 2-6
R
2
3
4
5
6
2
3
4
5
6
a
4.74
5.07
5.23
5.51
5.93
6.89
7.23
7.50
7.58
7.78
b
5.00
4.82
5.11
5.26
5.47
5.45
5.63
5.81
5.47
5.59
5.71
5.59
5.49
5.57
5.26
7.26
7.44
7.33
7.68
7.74
7.61
7.69
7.77
7.76
7.78
7.87
7.73
7.68
7.80
7.61
c
d
*±ε < 0.1
The affinity of aminoalkoxybiphenyls was dependent on the side chain length. As chain length
grows, increases until it reaches four to five carbon length. At this point, degrees of freedom
contribution becomes dominant and Ka begins decreasing.
2.3.Structure-DNA affinity relationships
Simple orthogonal three factor dispersion analysis determined that all three independent
9

structural factors (Factor A is the structure of terminal amino group, Factor B is polymethylene
fragment length and Factor C is the nature of polycyclic system) had a remarkable impact on
affinity. As we can see from Table 3, polycyclic fragment has the biggest impact, and the
contribution of polymethylene fragment length was approximately 30 times less than that of
Factor A. The structure impact of terminal amino group equals 0.64% only.
Table 3. Dispersion analysis (α = 0.05) of the contribution of structure factors to DNA
affinity
Variation
SS
DF MS
F
P-values
F-test Contrib., %
source
Factor A
Factor B
Factor C
Residual
Total
0.330
1.956
3
4
1
0.110
0.489
4.344
0.011
2.9113
2.6787
4.1596
0.64
3.80
4.6610-8
2.0110-29
19.295
94.04
1.53
48.444
0.786
48.444 1911.383
31 0.025
39
51.516
100.00
SS is the sum of squares due to the source; DF is degrees of freedom in the source; MS is the
mean sum of squares due to the source; F is F-statistic.
2.4 Molecular docking
There are three DNA sites where interactions with ligand can occur: (i) major grove (ii) minor
grove (iii) between stacked nucleic bases (DNA intercalation). It is unlikely for
aminoalkoxybiphenyls to bind to the major grove, since major grove ligands are proteins or
bulky molecules (e.g., Ditercalinium) of size that greatly exceeds the ones of
aminoalkoxybiphenyls [29, 30]. Theoretical basis for DNA intercalation is the presence of planar
polycyclic fragment of appropriate size (at least tricyclic one for a potent intercalation, in most
10

cases) in the ligand [31]. Aminoalkoxybiphenyls do not possess this feature, making DNA
intercalation improbable despite aminoalkoxybiphenyls similarity to the known intercalators,
such as actinomycins [32, 33]. Thus, we chose minor grove as the site for molecular docking.
Non-intercalative DNA binding ligands, which may show various degrees of sequence
specificity for binding to the minor groove of DNA, usually represent another class of important
therapeutic drugs with broad-spectrum antiviral, antibacterial, and antitumor activity. To
investigate the possible involvement of the aminoalkoxybiphenyls 11a-15d in binding to the
minor groove of DNA, we analyzed the ability of these agents to recognize specific nucleotide
sequence of DNA using a molecular modeling approach.
Docking of 11a-15d into oligonucleotide [34] showed that they can potentially bind to the DNA
minor groove. According to Gibbs–Helmholtz law, δG depends on . Scoring function ( )
reflects energy gain in the formation of complex ligand-receptor. In case of
aminoalkoxybiphenyls binding to minor groove d(CCIICICCII)2 sequence (Fig.3), the
correlation between and was defined by Spearman ρ rank correlation coefficient.
11

Figure 3. Binding of the aminoalkoxybiphenyls in the DNA minor groove d(CCIICICCII)2
sequence (in blue – native ligand netropsin; in yellow – aminoalkoxybiphenyl, in gray –
polynucleotide; on the left and on the right are two different projections of the same molecule)
and (Table 5) distribution normality was assessed by Q-Q plot and Shapiro-Wilk test.
Since most points are located near the trend line in Q-Q plot and Shapiro-Wilk W had p-value
above significance level (α=0.05), we assumed that both and data have distribution
similar to normal one (Fig.4).
12

Table 5. and values for biphenyls (sequence d(CCIICICCII)2)
Compounds ID
Compounds ID
4.74
4.95
4.82
5.11
4.94
5.07
4.85
5.47
5.45
5.03
5.23
5.01
5.81
5.47
5.12
5.51
5.59
5.71
5.59
5.05
5.31
5.93
5.57
5.26
5.05
-11.72
-12.5
-14.00
-14.80
-15.66
-14.41
-13.66
-15.32
-13.85
-13.97
-15.37
-14.89
-13.47
-13.48
11a
11b
11c
11d
11e
12a
12b
12c
12d
12e
13a
13b
13c
13d
13e
14a
14b
14c
14d
14e
15a
15b
15c
15d
15e
-13.26
-11.91
-12.34
-12.76
-13.33
-14.57
-13.42
-13.63
-14.09
-13.11
-13.65
13

lgKa
6
5.8
5.6
5.4
5.2
5
y = 0.2447x + 5.2983
R² = 0.9665
4.8
4.6
-3
-2
-1
0
1
2
3
Normal Theoretical Quantiles
SF
-11
-12
-13
-14
-15
-16
-17
y = 0.984x - 13.528
R² = 0.9842
-3
-2
-1
0
1
2
3
Normal Theoretical Quantiles
Figure 4. Quantile-quantile plots of (top plot) or (bottom plot) data distribution
vs normal distribution (solid line)
14

Therefore, usage of parametric statistics is justified in this case. Linear regression equation was
calculated using least squares method (eq.1).
(
)
1
Measurement of linear regression goodness-of-fit was crucial, as well as checking, if there are
any outlier prediction values for this regression (Table 6).
Table 6. Statistic assessment of linear regression goodness-of-fit (sequence
d(CCIICICCII)2)
MAE
MAPE
RMSE
R2
0.18
3.43%
0.24
0.467
0%
outliers
It is worth noticing that data has low scattering with
. Overall statistic
parameters show acceptable level of regression fit (Fig. 5). Outliers were defined using the
Grubbs test for outliers [35], i. e. compound is an outlier if difference between observed and
predicted values exceeds triple RMSE. Total number of outlier is given as percent of all data
points.
15

푆퐹
-11
-12
-13
-14
-15
-16
푙푔퐾푎
5.90
4.50
4.70
4.90
5.10
5.30
5.50
5.70
Figure 5. ???????? vs ???? plot. Solid line is a trend line which corresponds to regression
equation (sequence d(CCIICICCII)2)
This regression equation was used for prediction of of benzidine derivatives (10a-10e).
10a-10e were synthesized, tested for DNA affinity and docked into the same oligonucleotide as
previously described compounds (Table 7), exhibiting the same minor groove binding mode.
Comparison of experimental value and calculated using regression equation showed
that calculated value is close to modelled one with MAE = 0.15 and RMSE = 0.11.
16

Table 7. and values for benzidines (sequence d(CCIICICCII)2)
Compounds ID
(theoretical)
4.83 ± 1.40
4.74 ± 0.02
4.95 ± 0.09
4.82 ± 0.02
5.11 ± 0.06
4.94 ± 0.02
-11.72
-12.5
10a
10b
10c
10d
10e
5.00 ± 1.15
5.16 ± 1.12
-13.26
-11.91
-12.34
4.87 ± 1.36
4.96 ± 1.23
Such good prediction encourages us to presume that applicability domain of above-mentioned
equation can comprise compounds of classes other than biphenyl as long as they tend to have
similar binding mode.
Aminoalkoxybiphenyls 11a-15e were also docked in another oligonucleotide (Table 8) [36],
where N,N'-bis[3-(4,5-dihidro-1H-imidazol-2-yl)phenyl]biphenyl-4,4'-dicarboxamide was bound
to d(CGCGAATTCGCG)2 sequence (Fig.6).
17

Figure 6. Binding of the aminoalkoxybiphenyls in the DNA minor groove sequence
d(CGCGAATTCGCG)2 (in red – native ligand netropsin; in yellow – aminoalkoxybiphenyl, in
gray – polynucleotide)
18

Table 8. and minimum values for biphenyls (sequence d(CGCGAATTCGCG)2)
Compounds ID
Compounds ID
Compounds ID
-11.77
-12.34
-12.30
-9.73
-11.54
-11.63
-12.57
-13.65
-12.93
-12.57
-14.15
-13.19
-12.09
-12.60
-14.46
-13.50
-12.85
-12.60
-12.23
-14.31
11a
11b
11c
11d
11e
12a
12b
12c
12d
12e
13a
13b
13c
13d
13e
14a
14b
14c
14d
14e
15a
15b
15c
15d
15e
-14.10
-12.07
-12.55
-13.00
-12.20
In this case correlation between and is absent (R2 = 0.014). The correlation between
and
in case of d(CCIICICCII)2 sequence and formation of complexes
aminoalkoxybiphenyls with DNA d(CGCGAATTCGCG)2 sequence in the rich GC DNA
regions in favor of selective binding aminoalkoxybiphenyls with GC DNA sequences.
3. Conclusion
The series of planar fluorenones (3a-7d) and nonplanar biphenyls (11a-d) were prepared for the
comparative analysis of interaction with DNA. Initial evaluation of the affinity for these two
series of compounds to DNA revealed the deference between the classic intercalation of
19

fluorenones and non-intercalating mechanism of action for biphenyls. Molecular docking studies
allowed us to suggest that aminoalkoxybiphenyls can potentially bind to a minor grove of DNA,
particularly to d(CCIICICCII)2 sequence. The negative correlation between lgKa and docking SF
was defined and a regression equation, which was capable of lgKa prediction for the structures
similar to biphenyls was derived. Despite the fact that the two series of compounds demonstrated
substantially different in affinity and binding mode to DNA, their pharmacological properties
were similar. Further studies on these DNA minor groove binders and their analogues, along
with extended structural investigation are required to better understand regulatory factors guiding
the ability to develop antiviral activity.
4 Experimental
4.1. Chemistry
All reagents and solvents were purchased from commercial sources and were used as
received, unless noted otherwise. Melting points were determined with a Kofler bench melting
1
point apparatus, and are uncorrected. H (500 MHz) and 13C NMR (125 MHz) spectra were
recorded on Bruker Avance 500 MHz spectrometer with TMS as an internal standard. Mass-
spectra (MS) were obtained with VG 70 70 EQ spectrometer (ionization was carried out by Ar
atomic beam with 10 kV energy; compounds were dissolved in 3-nitrobenzyl alcohol. All
reactions and the purity of the synthesized compounds were controlled by TLC on Merck
Aluminum Foil Silica gel 60 F254. UV-Vis spectroscopy was performed on a Shimadzu UV-
2401PC spectrophotometer. Microanalyses were performed for the indicated elements, and the
results are within 0.4% of calculated values. All buffers, intermediates and working solutions
were prepared using chemically pure grade reagents and distilled water.
20

4.1.1. 2,7-Bis(2-chloroethoxy)fluoren-9-one (2a)
A mixture of 2,7-dihydroxyfluorenone (1) (23.34 g, 0.11 mol), 1,2-dichloroethane (250 mL),
20% NaOH aqueous solution (50 mL) and tetrabutylammonium chloride (7.50 g, 0.027 mol)
were vigorously stirred at 75 °C for one day. After the reaction mixture was cooled, organic layer
was separated and washed with 5% NaOH (3  20 mL) and H2O (3  20 mL), dried (CaCl2) and
filtered. The filtrate was evaporated under reduced pressure to afford the pure 2b as a red solid
after recrystallization from i-PrOH (32.27 g, yield: 87%); mp 145 – 146 °С (Lit. mp 142 – 144
°С, [23]).
4.1.2. 2,7-Bis(2-chloropropoxy)fluoren-9-one (2b).
A mixture of 2,7-dihydroxyfluorenone (1) (23.34 g, 0.11 mol), 1,3-bromochloropropane (157.44
g, 1.0 mol) and anhydrous K2CO3 (64 g, 0.46 mol) in DMF (150 mL) was stirred at 80°С for one
day. Upon completion of reaction, the mixture was diluted with H2O (600 mL). The precipitate
was filtered and recrystallized from EtOH to give a solid (33.33 g, yield: 83%); mp: 124 – 125
°С.
4.1.3. 2,7-Bis(4-bromobutoxy)fluoren-9-one (2c).
A mixture of 2,7-dihydroxyfluorenone (1) (23.34 g, 0.11 mol), 1,4-dibromobutane (216 g, 1.00
mol) and anhydrous K2CO3 (64 g, 0.46 mol) in DMF (150 mL) was stirred at 80º С for one day.
Upon completion of reaction, the mixture was diluted with H2O (600 mL). The precipitate was
filtered and recrystallized from EtOH to give a solid; (39.25 g, yield: 74%); mp 109 – 110° С.
Intermediates 2d – 2e were papered in a similar manner to 2b – 2c
21

4.1.4. 2,7-Bis(4-bromopentoxy)fluoren-9-one (2d)
Yield: 78%; mp 96–97 °С.
4.1.5. 2,7-Bis(4-bromohexoxy)fluoren-9-one (2e)
Yield: 71%; mp 83–84 °С.
4.1.6. 2,7-Bis(2-iodoethoxy)fluoren-9-one (2f).
A solution of NaI (24.00 g, 0.16 mol) and tetrabutylammonium iodide (29.55 g, 0.08 mol) in
H2O (70 mL) was added to a solution of 2b (26.98 g 0.08 mol) in toluene (200 mL). The mixture
was refluxed with vigorous stirring for 10 h and then cooled to the room temperature. The
organic layer was separated, washed with 5% Na2S2O3, H2O, dried (Na2SO4), and filtered. The
filtrate was evaporated under reduced pressure to provide a solid material; (39.95 g, Yield: 96
%); mp 154 – 155 °С.
Compound 2g was papered in a similar manner to 2f.
4.1.7. 2,7-Bis(3-iodopropoxy)fluoren-9-one (2g)
Yeild: 95%; mp 120 – 121 °С.
4.1.8. 2,7-Bis[2-(diethylamino)ethoxy]fluoren-9-one Dihydrochloride (3a)
Diethylamine (0.73 g, 0.01 mol) was added to a solution of 2f (1.10 g 0.002 mol) in DMF (5
mL). The reaction mixture was kept at room temperature for 5 days, diluted with H2O (200 mL),
and then acidified with HCl to achieve a pH of 2-3. The acidic solution was washed with CHCl3
(3  50 mL), and then basified with aqueous NaOH until pH was 12 – 13. The product was
22

extracted with CHCl3 (3  50 mL). The combined CHCl3 layers were washed with H2O until pH
of aqueous layer was about 7, dried (Na2SO4) and evaporated under reduced pressure. The
resultant residue was dissolved in anhydrous dioxane (20 mL) and then saturated anhydrous
solution of HCl in dioxane was added to the resulting solution while stirring. The solvent was
evaporated under reduced pressure to give a crude gummy residue that was suspended in
acetone. A stirred suspension was refluxed for 15 min, followed by filtration to give a solid
material. The procedure was repeated until a pure reddish solid product (3a) was afforded; (0.80
g, Yield: 83%); mp 236 – 237 °С (Lit. mp 235 – 236 °С [23]; FAB-MS m/z: M+ = 411.
Compounds 3b – 7d were papered in a similar manner to 3a.
4.1.9. 2,7-Bis[2-(dimethylamino)ethoxy]fluoren-9-one Dihydrochloride (3b)
Yield: 78%; mp 272 – 273 °С; FAB-MS m/z: M+ = 355; Lit. mp 278 – 280 °С [23].
4.1.10. 2,7-Bis(2-piperidin-1ylethoxy)fluoren-9-one Dihydrochloride (3c)
Yield: 87%; mp 304 – 305 °С; FAB-MS m/z: M+ = 435; Lit. mp 304 – 306 °С [23].
4.1.11. 2,7-Bis(2-azepan-1-ylethoxy)fluoren-9-one Dihydrochloride (3d)
1
Yield: 85%; mp 291 – 292 °С; H NMR (CDCl3) δ: 1.6 (m, 8 H, 2CH2+2CH2), 1.6 (m, 8 H,
2CH2+2CH2), 2.7 (t, 8 H, 2CH2+2CH2), 2.9 (t, J=5.8 Hz, 4 H, 2CH2), 4.0 (t, J=5.8 Hz, 4 H,
2CH2), 6.9 (dd, J=7.8, 2.3 Hz, 2 H, 2CH), 7.1 (d, J=2.0 Hz, 2 H, 2CH), 7.2 (d, J=7.8 Hz, 2 H,
2CH); 13C NMR (CDCl3) δ: 27.0, 28.0, 55.8, 56.3, 67.1, 110.4, 120.4, 120.9, 135.9, 137.5, 159.3,
193.7; FAB-MS m/z: M+ = 463; Anal. calc. for C29H38N2O3∙2HCl: C 65.04, H 5.23, N 7.53;
found C 64.70, H 5.57, N 7.87.
23

4.1.12. 2,7-Bis[3-(diethylamino)propoxy]fluoren-9-one Dihydrochloride (4a)
Yield: 90%; mp 252–253 °С; FAB-MS m/z: M+ = 439; Lit. mp 256–257 °С [23].
4.1.13. 2,7-Bis[3-(dimethylamino)propoxy]fluoren-9-one Dihydrochloride (4b).
Yield: 80%; mp 280 – 281 °С; FAB-MS m/z: M+ = 383; Lit. mp 282 – 283 °С [23].
4.1.13. 2,7-Bis(3-piperidin-1-ylpropoxy)fluoren-9-one Dihydrochloride (4c)
Yield: 90%; mp 278 – 279 °С; FAB-MS m/z: M+ = 463; Lit. mp 279 – 280 °С [23].
4.1.14. 2,7-Bis(3-azepan-1-ylpropoxy)fluoren-9-one Dihydrochloride (4d)
1
Yield: 76%; mp 266 – 267 °С; FAB-MS m/z: M+ = 491; H NMR (CDCl3) δ: 1.6 (m, 8 H,
2CH2+2CH2), 1.7 (m, 8 H, 2CH2+2CH2), 2.0 (t, 4 H, 2CH2), 2.7 (t, J=4.9 Hz, 8H, 2CH2+2CH2),
2.8 (t, J=4.9 Hz, 4H, 2CH2), 4.0 (t, J=5.9 Hz, 4 H, 2CH2), 6.9 (d, J=8.0 Hz, 2 H, 2CH), 7.1 (s, 2
13
H, 2CH), 7.2 (d, J=8.0 Hz, 2 H, 2CH); C NMR (CDCl3) δ: 26.6, 27.3, 46.4, 54.2, 55.1, 66.4,
110.0, 120.1, 120.3, 135.5, 137.0, 159.0, 193.4; Anal. calc. for C31H42N2O3∙2HCl: C 66.06, H
4.97, N 7.87; found C 66.38, H 4.79, N 7.91.
4.1.15. 2,7-Bis[4-(diethylamino)butoxy]fluoren-9-one Dihydrochloride (5a)
Yield: 77%; mp 230 – 231 °С; FAB-MS m/z: M+ = 467; Lit. mp 233–234 °С; [23]
4.1.16. 2,7-Bis[4-(dimethylamino)butoxy]fluoren-9-one Dihydrochloride (5b)
24

Yeild: 75%; mp 299–300 °С; FAB-MS m/z: M+ = 411; 1H NMR (CDCl3) δ: 1.6 (m, J=6.0 Hz, 4
H, 2CH2), 1.8 (m, J=6.0 Hz, 4 H, 2CH2), 2.2 (s, 12 H, 4CH3), 2.3 (t, 4 H, 2CH2), 4.0 (t, 4 H,
2CH2), 6.9 (d, J=8.0 Hz, 2 H, 2CH), 7.1 (s, 2 H, 2CH), 7.2 (d, J=8.0 Hz, 2 H, 2CH); 13C NMR
(CDCl3) δ: 23.8, 26.6, 45.1, 58.9, 67.8, 109.8, 120.0, 120.4, 135.5, 137.0, 159.0, 193.4; Anal.
calc. for C25H34N2O3∙2HCl: C 62.11, H 5.79, N 7.51; found C 62.08, H 5.59, N 7.28.
4.1.17. 2,7-Bis(4-piperidin-1-ylbutoxy)fluoren-9-one Dihydrochloride (5c)
1
Yeild: 80%; mp 253 – 254 °С; FAB-MS m/z: M+ = 491; H NMR (CDCl3) δ: 1.4 (m, 4 H,
2CH2), 1.6 (m, J=4.9 Hz, 9 H, 2CH2+2CH2), 1.6 (m, 4 H, 2CH2), 1.8 (m, J=6.6 Hz, 4 H, 2CH2),
2.3 (t, 4 H, 2CH2), 2.3 (t, J=7.7 Hz, 8 H, 2CH2+2CH2), 4.0 (t, J=6.0 Hz, 4 H, 2CH2), 6.9 (d,
J=7.8 Hz, 2 H, 2CH), 7.1 (s, 2 H, 2CH), 7.2 (d, J=8.0 Hz, 2 H, 2CH); 13C NMR (CDCl3) δ: 23.0,
24.1, 25.6, 26.9, 54.2, 58.6, 67.9, 109.8, 120.0, 120.4, 135.5, 137.0, 159.0, 193.4; Anal. calc. for
C31H42N2O3∙2HCl: C 66.06, H 4.97, N 7.87; found C 66.14, H 4.80, N 7.59.
4.1.18. 2,7-Bis(4-azepan-1-ylbutoxy)fluoren-9-one Dihydrochloride (5d)
1
Yield: 74%; mp 248 – 249 °С; FAB-MS m/z: M+ = 519; H NMR (CDCl3) δ: 1.6 (m, 12 H,
2CH2+2CH2, 2CH2), 1.6 (m, 8 H, 2CH2+2CH2), 1.8 (m, 4 H, 2CH2), 2.5 (t, J=7.4 Hz, 4 H,
2CH2), 2.6 (t J=5.8 Hz, 8 H, 2CH2+2CH2), 4.0 (t, J=6.4 Hz, 4 H, 2CH2), 6.9 (dd, J=8.2, 2.5 Hz, 2
13
H, 2CH), 7.1 7.1 (d, J=2.5 Hz, 2 H, 2CH), 7.2 (d, J=8.0 Hz, 2 H, 2CH); C NMR (CDCl3) δ:
23.6, 26.6, 26.8, 27.6, 55.1, 57.4, 68.0, 109.8, 120.0, 120.4, 135.5, 137.0, 159.0, 193.4; Anal.
calc. for C33H46N2O3∙2HCl: C 66.99, H 4.73, N 8.18; found C 66.74, H 4.93, N 8.26.
4.1.19. 2,7-Bis[5-(diethylamino)pentoxy]fluoren-9-one Dihydrochloride (6a)
25

1
Yield: 72%; mp 212 – 213 °С; FAB-MS m/z: M+ = 495; H NMR (CDCl3) δ: 1.0 (t, J=7.1 Hz,
12 H, 4CH3), 1.4 (m, J=13.7, 7.1, 6.9 Hz, 4 H, 2CH2), 1.5 (m, 4 H, 2CH2), 1.8 (m, J=14.0, 6.9,
6.6 Hz, 4 H, 2CH2), 2.4 (m, 4 H, 2CH2), 2.5 (q, J=7.1 Hz, 8 H, 4CH2), 4.0 (t, J=6.4 Hz, 4 H,
2CH2), 6.9 (dd, J=8.1, 2.1 Hz, 2 H, 2CH), 7.1 (d, J=2.2 Hz, 2 H, 2CH), 7.2 (d, J=8.0 Hz, 2 H,
13
2CH); C NMR (CDCl3) δ: 11.2, 23.7, 26.4, 28.7, 46.5, 52.4, 68.0, 109.8, 120.0, 120.4, 135.5,
137.0, 159.0, 193.4; Anal. calc. for C31H46N2O3∙2HCl: C 65.59, H 4.94, N 8.52; found C 65.90,
H 5.25, N 8.52.
4.1.20 2,7-Bis[5-(dimethylamino)pentoxy]fluoren-9-one Dihydrochloride (6b)
Yield: 68 %; mp 214 – 215 °С; FAB-MS m/z: M+ = 439; 1H NMR (CDCl3) δ: 1.4 (m, J=7.4, 7.1,
6.9, 6.9 Hz, 4 H, 2CH2), 1.5 (m, 4 H, 2CH2), 1.8 (m, 4 H, 2CH2), 2.3 (s, 12 H, 4CH2), 2.3 (m, 4
H, 2CH2), 3.9 (t, J=6.4 Hz, 4 H, 2CH2), 6.9 (dd, J=8.2, 2.4 Hz, 2 H, 2CH), 7.1 (d, J=2.3 Hz, 2 H,
2CH), 7.2 (d, J=8.3 Hz, 2 H, 2CH); 13C NMR (CDCl3) δ: 23.8, 27.0, 29.0, 45.1, 59.4, 68.3,
110.2, 120.5, 120.7, 135.9, 137.4, 159.4, 193.8; Anal. calc. for C27H38N2O3∙2HCl: C 63.40, H
5.48, N 7.88; found C 63.28, H 5.29, N 7.91.
4.1.21. 2,7-Bis(5-piperidin-1ylpentoxy)fluoren-9-one Dihydrochloride (6c)
Yield: 73%; mp 231 – 232 °С; FAB-MS m/z: M+ = 519; 1H NMR (CDCl3) δ:1.4 (m, 8 H, 2CH2;
2CH2), 1.6 (m, 12 H, 2CH2+2CH2; 2CH2), 1.8 (m, 4 H, 2CH2), 2.3 (t, 4 H, 2CH2), 2.4 (t, 8 H,
2CH2+2CH2), 4.0 (t, J=6.4 Hz, 4 H, 2CH2), 6.9 (dd, J=8.2, 2.5 Hz, 2 H, 2CH), 7.1 (d, J=2.2 Hz,
2 H, 2CH), 7.2 (d, J=8.0 Hz, 2 H, 2CH); 13C NMR (CDCl3) δ: 23.8, 24.1, 25.6, 26.3, 28.7, 54.3,
59.0, 68.0, 109.8, 120.0, 120.4, 135.5, 137.0, 159.0, 193.4; Anal. calc. for C33H46N2O3∙2HCl: C
66.99, H 4.73, N 8.18; found C 67.04, H 4.60, N 8.49.
26

4.1.22. 2,7-Bis(5-azepan-1-ylpentoxy)fluoren-9-one Dihydrochloride (6d)
1
Yield: 69%; mp 223 – 224 °С; FAB-MS m/z: M+ = 547; H NMR (CDCl3) δ: 1.4 (m, 4 H,
2CH2), 1.5 (m, 12 H, 2CH2+2CH2, 2CH2), 1.6 (m, J=4.7 Hz, 4 H, 2CH2), 1.8 (m, 8 H,
2CH2+2CH2), 2.5 (t, J=7.7 Hz, 4 H, 2CH2), 2.6 (t, J=5.8 Hz, 8 H, 2CH2+2CH2), 4.0 (t, J=6.4 Hz,
4 H, 2CH2), 6.9 (dd, J=8.1, 2.3 Hz, 2 H, 2CH), 7.1 (d, J=2.5 Hz, 2 H, 2CH), 7.2 (d, J=8.2 Hz, 2
H, 2CH); 13C NMR (CDCl3) δ: 23.6, 26.6, 26.9, 27.5, 28.7, 55.2, 57.7, 68.0, 109.8, 120.0, 120.4,
135.5, 137.0, 159.0, 193.4; Anal. calc. for C35H50N2O3∙2HCl: C 67.83, H 4.52, N 8.46; found C
68.03, H 4.43, N 8.15.
4.1.23. 2,7-Bis[6-(diethylamino)hexoxy]fluoren-9-one Dihydrochloride (7a)
1
Yield: 72%; mp 177 – 178 °С; FAB-MS m/z: M+ = 523; H NMR (CDCl3) δ: 1.1 (t, J=7.0 Hz,
12 H, 4CH3), 1.3 (m, 4 H, 2CH2), 1.5 (m, 4 H, 2CH2), 1.5 (m, 4 H, 2CH2), 1.7 (m, J=14.1, 6.8
Hz, 4 H, 2CH2), 2.5 (t, 4 H, 2CH2), 2.6 (q, J=6.9 Hz, 8 H, 4CH2), 3.9 (t, J=6.1 Hz, 4 H, 2CH2),
6.9 (d, J=7.8 Hz, 2 H, 2CH), 7.1 (s, 2 H, 2CH), 7.2 (d, J=8.0 Hz, 2 H, 2CH); 13C NMR (CDCl3)
δ: 11.0, 25.9, 26.1, 27.2, 29.1, 46.8, 52.5, 68.4, 110.2, 120.5, 120.8, 135.9, 137.4, 159.4, 193.8;
Anal. calc. for C33H50N2O3∙2HCl: C 66.54, H 4.70, N 8.80; found C 66.61, H 4.98, N 9.05.
4.1.24. 2,7-Bis[6-(dimethylamino)hexoxy]fluoren-9-one Dihydrochloride (7b)
Yield: 65 %; mp 197 – 198 °С; FAB-MS m/z: M+ = 467; 1H NMR (CDCl3) δ: 1.3 (m, J=6.8, 6.8,
6.8, 6.8 Hz, 4 H, 2CH2), 1.4 (m, 4 H, 2CH2), 1.5 (dt, J=14.9, 7.5 Hz, 4 H, 2CH2), 1.7 (m, 4 H,
2CH2), 2.3 (s, 12 H, 4CH2), 2.4 (m, 4 H, 2CH2), 3.9 (t, J=6.4 Hz, 4 H, 2CH2), 6.9 (dd, J=8.2, 2.4
Hz, 2 H, 2CH), 7.1 (d, J=2.0 Hz, 2 H, 2CH), 7.2 (d, J=8.3 Hz, 2 H, 2CH); 13C NMR (CDCl3) δ:
27

25.9, 27.0, 27.0, 29.1, 45.0, 59.4, 68.4, 110.2, 120.5, 120.8, 135.9, 137.4, 159.4, 193.8; Anal.
calc. C29H42N2O3 ·2HCl: for C 64.55, H 5.19, N 8.22; found C 64.21, H 5.20, N 8.12.
4.1.25. 2,7-Bis(6-piperidin-1ylhexoxy)fluoren-9-one Dihydrochloride (7c)
1
Yield: 79%; mp 224 – 225 °С; FAB-MS m/z: M+ = 547. H NMR (CDCl3) δ: 1.3 (m, 4 H,
2CH2), 1.4 (m, J=4.5 Hz, 8 H, 2CH2+2CH2), 1.5 (m, J=7.3, 3.0 Hz, 4 H, 2CH2), 1.6 (d, J=5.0 Hz,
8 H, 2CH2; 2CH2), 1.8 (m, 4 H, 2CH2), 2.3 (t, 4 H, 2CH2), 2.4 (s, 8 H, 2CH2+2CH2), 3.9 (t, J=6.1
Hz, 4 H, 2CH2), 6.9 (dd, J=7.8, 2.3 Hz, 2 H, 2CH), 7.1 (d, J=2.0 Hz, 2 H, 2CH), 7.2 (d, J=7.8
13
Hz, 2 H, 2CH); C NMR (CDCl3) δ: 24.3, 25.7, 25.9, 26.6, 27.4, 29.1, 54.5, 59.3, 68.4, 110.2,
120.4, 120.8, 136.0, 137.4, 159.5, 193.9; Anal. calc. for C35H50N2O3·2HCl: C 67.83, H 4.52, N
8.46; found C 67.95, H 4.19, N 8.57.
4.1.26. 2,7-Bis(6-azepan-1-ylhexoxy)fluoren-9-one Dihydrochloride (7d)
Yield: 71%; mp 212–214 °С; FAB-MS m/z: M+ = 575; 1H NMR (CDCl3) δ: 1.4 (m, 4 H, 2CH2),
1.5 (m, 4 H, 2CH2), 1.6 (m, 12 H, 2CH2+2CH2, 2CH2), 1.8 (m, 12 H, 2CH2+2CH2, 2CH2), 2.7
(m, 4 H, 2CH2), 2.9 (m, 8 H, 2CH2+2CH2), 3.9 (t, J=6.1 Hz, 4 H, 2CH2), 6.9 (dd, J=8.0, 2.3 Hz,
2 H, 2CH), 7.1 (d, J=2.0 Hz, 2 H, 2CH), 7.2 (d, J=8.0 Hz, 2 H, 2CH); 13C NMR (CDCl3) δ: 25.5,
25.8, 25.9, 25.9, 27.0, 29.0, 55.0, 57.8, 68.3, 110.3, 120.5, 120.8, 135.9, 137.4, 159.4, 193.8;
Anal. calc. for C37H54N2O3Cl2 2HCl: C 68.61, H 4.32, N 8.71; found C 68.46, H 4.31, N 8.85.
4.1.27. N,N'-Biphenyl-4,4'-diylbis(2-chloroacetamide) (9)
Chloroacetyl chloride (35.24 g, 0.312 mol) was added dropwise to a stirred solution of benzidine
(8) (22.11 g, 0.12 mol) in toluene (500 mL) at room temperature. The resulting mixture was
28

refluxed for 2 h, cooled to room temperature, and concentrated under reduced pressure. The
resultant residue was suspended in CHCl3 (500 mL) and refluxed for an additional 0.5 h. The
suspension was cooled to room temperature, and then the precipitate was filtered off and washed
with CHCl3 (3  50 mL) yielded 32.38 g, 80%; mp. 275–276 °С. The product 9 was used in the
next step without further purification.
4.1.28. N,N'-Biphenyl-4,4'-diylbis[2-(diethylamino)acetamide] Dihydrochloride (10a)
Diethylamine (1.10 g, 0.015 mol) was added to a solution of 9 (1.01 g 0.003 mol) in DMF (5
mL). After the reaction was completed, the reaction mixture was diluted with H2O (10 mL) and
product was extracted with CHCl3 (3  20 mL). The combined CHCl3 layers were washed with
H2O until pH of aqueous layer was 7, dried (Na2SO4) and evaporated under reduced pressure to
dryness. The residue was dissolved in benzene (20 mL) and added to a stirred saturated
anhydrous solution of HCl in dioxane. The resultant precipitate was filtered off and suspended in
acetone. A stirred suspension was refluxed for 15 min, followed by filtration to give a solid
material. The procedure was repeated until a pure solid product (10a) was afforded; (0.99 g,
1
Yield: 68 %); mp 274 – 275 °С; FAB-MS m/z: MH+ = 411; H NMR (CDCl3) δ: 1.1 (t, J=7.3
Hz, 12 H, 4CH3), 2.6 (q, 8 H, 2CH2+2CH2), 3.2 (s, 4 H, 2CH2), 7.5 (d, J=8.2 Hz, 4 H, 4CH), 7.6
(d, J=8.2 Hz, 4 H, 4CH), 9.4 (s, 2 H, NH). Anal. calc. for C24H34N4O2∙2HCl: C 59.62, H 11.59,
N 7.51; found C 59.46, H 11.76, N 7.21.
Compounds 10b-e were papered in a similar manner to 10a
4.1.29. N,N'-Biphenyl-4,4'-diylbis[2-(dimethylamino)acetamide] Dihydro-chloride (10b)
1
Yield: 65%; mp 265 – 266 °С; FAB-MS m/z: MH+ = 355; H NMR (CDCl3) δ: 2.4 (s, 12 H,
29