Bistacrine derivatives as new potent antimalarials

Article abstract of DOI:10.1016/j.bmc.2016.06.003

Linking two tacrine molecules results in a tremendous increase of activity against Plasmodia in comparison to the monomer. This finding prompted the synthesis of a library of monomeric and dimeric tacrine derivatives in order to derive structure–activity relationships. The most active compounds towards chloroquine sensitive Plasmodium strain 3D7 and chloroquine resistant strain Dd2 show IC₅₀values in the nanomolar range of concentration, low cytotoxicity and target the cysteine protease falcipain-2, which is essential for parasite growth.

Full text of DOI:10.1016/j.bmc.2016.06.003

Bioorganic & Medicinal Chemistry 24 (2016) 3636–3642
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bistacrine derivatives as new potent antimalarials
Ines Schmidt a, Gabriele Pradel b, Ludmilla Sologub b, Alexandra Golzmann b, Che J. Ngwa b,
Anna Kucharski a, Tanja Schirmeister c, Ulrike Holzgrabe a,
⇑
a Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
b Institute of Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany
c Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55099 Mainz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Linking two tacrine molecules results in a tremendous increase of activity against Plasmodia in
comparison to the monomer. This finding prompted the synthesis of a library of monomeric and dimeric
tacrine derivatives in order to derive structure–activity relationships. The most active compounds
towards chloroquine sensitive Plasmodium strain 3D7 and chloroquine resistant strain Dd2 show IC50
values in the nanomolar range of concentration, low cytotoxicity and target the cysteine protease
falcipain-2, which is essential for parasite growth.
Received 15 February 2016
Revised 30 May 2016
Accepted 2 June 2016
Available online 4 June 2016
Keywords:
Bistacrines
Ó 2016 Elsevier Ltd. All rights reserved.
Antimalarials
Falcipain-2 inhibitors
1. Introduction
substitution pattern and tether length, and evaluation of the
antimalarial activity. Inhibition of b-hematin formation, which is
In 2013 the World Health Organization estimated 198 million
cases of malaria, whereof 584,000 were fatal.1 The causative
pathogens of this disease are four species of the genus Plasmodium,
i.e., P. falciparum, P. vivax, P. ovale and P. malariae, all of them being
sporozoic parasites. Malaria—still remaining one of the most
deadly diseases—occurs mainly in Africa, where 78% of the deaths
apply to children under the age of five.2 However, the disease also
appears in South-East Asia and South America. Nearly 50% of the
world population lives in an area of risk. Due to the small number
of efficient therapeutics and the increasing rate of resistance the
search for new drugs against malaria is urgently needed.
Tacrine 1a (1,2,3,4-tetrahydroacridine, THA, Fig. 1), which is
known as a reversible acetylcholinesterase inhibitor, was in clinical
use for the treatment of Alzheimer’s disease. It is a common
approach to expand the application of drugs that have already been
evaluated as drug leads for other diseases.3–5 Interestingly, in an
antiprotozoal screening tacrine was found to exhibit antimalarial
the assumed mechanism of action of chloroquine, was tested
because of the similarity of the aminoquinoline moiety of
chloroquine and tacrine.6 Additionally, the inhibition of the cysteine
protease falcipain-2 (FP2), which is essential for parasite survival by
playing a key role in host hemoglobin degradation, was investigated.
2. Results and discussion
2.1. Chemistry
Monomeric tacrine derivatives were prepared as reported for
the unsubstituted compound by means of condensation and
subsequent cyclization of 2-aminobenzonitriles and cyclohex-
anones in toluene with catalytic amounts of p-toluene sulfonic acid
(Scheme 1).7 All compounds 1a–1f were obtained in moderate
yields after recrystallisation from acetone.
The synthesis of dimers using tacrine derivatives and 1,x-dibro-
activity (IC50 = 12.5 lM against chloroquine sensitive strain 3D7).
moalkanes as alkylating reagents failed due to side reactions in
addition to low reproducibility. Thus, the dimerisation reaction
started off from 9-chloro-1,2,3,4-tetrahydroacridine derivatives,
which were obtained by condensation of equimolar amounts of
the appropriate 2-aminobenzoic acids with cyclohexanones using
an excess of POCl3 (Scheme 2).8 The excess of POCl3 and high tem-
peratures are necessary to prevent the formation of 4-quinolinones.
The achieved 9-chloro-intermediates (2a–2j) were heated
Furthermore, the linking of two tacrine moieties with an alkyl
chain of varying length resulted in a tremendous increase of the
activity.
These preliminary results prompted the synthesis of a library of
monomeric and dimeric tacrine derivatives with different
⇑
Corresponding author. Tel.: +49 (0) 931/31 85461; fax: +49 (0) 931/31 85494.
E-mail address: ulrike.holzgrabe@uni-wuerzburg.de (U. Holzgrabe).
under reflux with 0.5 equiv of 1,x-diaminoalkanes for several
http://dx.doi.org/10.1016/j.bmc.2016.06.003
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

I. Schmidt et al. / Bioorg. Med. Chem. 24 (2016) 3636–3642
3637
M]
Table 1
NH2
Increasing activities and cytotoxicities in relation to substitution
Compound R1
R2
Yield [%] IC50 (3D7) [
l
M] J774.1 [l
N
1a
1a
1b
H
H
H
68
40
12.50 4.65
1.52 0.28
>100
43.0
2-Propyl
Figure 1. Tacrine (1,2,3,4-tetrahydroacridine, THA).
1c
1d
H
7-Cl
50
24
4.02 1.87
0.21 0.06
43.2
8.40
2-Propyl 7-Cl
1e
1f
H
H
7-NO2 10
7-Me 31
3.23 2.73
2.04 0.03
44.6
44.3
hours in phenol under an argon atmosphere. Dimeric tacrine
compounds 3a–3i, 4a–4g, 5a and 5b were obtained in mostly good
yields.
Chloroquine: IC50 (3D7) = 20 nM, IC50 (J774.1) = 18.4 lM.
2.2. Biology
Table 2
2.2.1. In vitro activity against Plasmodium falciparum
In vitro activities against Plasmodium falciparum strains 3D7 and Dd2
The antimalarial activity and cytotoxicity of monomeric
compounds 1a–1f, unsubstituted dimeric compounds 3a–3i and
substituted dimeric compounds 4a–4g, 5a and 5b were deter-
mined in vitro against Plasmodium falciparum chloroquine sensitive
strain 3D7, and against J774.1 macrophages by means of the
Malstat9 and the AlamarBlueÒ assay,10 respectively. Additionally,
the unsubstituted dimeric compounds 3a–3i as well as 5a and b
were tested against the chloroquine resistant strain Dd2.
For the monomeric compounds 1a–1f, the following structure–
activity relationships could be derived: Introduction of a weak
electron-donating propyl group in position 2 leads to an increase
of activity to a low, single-digit micromolar IC50 value (Table 1).
Besides, exchanging the aromatic proton in position 7 by a halo-
gen substituent as chlorine results in a further significant increase of
activity. Of note, electron-withdrawing substituents such as a nitro
group as well as electron-donating groups, e.g., methyl groups, in
these positions show similar effects. Therefore, a single substitution
in position 7 increases activity independently of the electronical
structure of the introduced substituent, whereas the combination
of chlorine in position 7 and propyl in position 2 is superior. Thus,
7-chloro-2-propyl-1,2,3,4-tetrahydroacridine 1d exhibited the
H
N
H
N
n
N
N
Compound
n
IC50 (3D7)
IC50 (Dd2)
M]
J774.1
[lM]
SIa/b
113
(7.40)
86.9
(10.7)
48.8
(5.97)
2.86
(3.27)
29.7
27.1
(8.64)
17.3
(6.33)
36.0
[
lM]
[
l
3a
3b
3c
3d
2
3
4
5
0.40 0.07
0.49 0.16
0.17 0.02
0.63 0.18
6.11 2.90
3.99 3.30
1.39 0.38
0.55 0.09
45.2
42.6
8.30
1.80
3e
3f
6
7
0.10 0.03
0.07 0.01
n.d.
0.22 0.16
2.97
1.90
3g
3h
3i
8
9
0.11 0.01
0.05 0.02
0.30 0.02
0.15 0.03
0.14 0.2
1.90
1.80
1.80
(12.0)
16.4
(12.9)
10 0.11 0.01
highest antimalarial activity (IC50 = 0.21
lM) (Table 1).
n.d.: not determined.
NH2
a
8
1
4
9
IC50 (J774.1)/IC50(3D7).
CN
NH2
2
7
a
b
R2
R1
R2
R1
IC50 (J774.1)/IC50(Dd2).
6
3
O
N
5
: R1 = H, R2 = H
1
1a
1b
: R = 2-propyl, R2 = H
The dimerization leads to a substantial increase in activity,
depending on the length of the linker (compounds 3a–3i) (Table 2).
Interestingly, even a short linker of two methylene units displays a
30-fold enhancement of activity in comparison to THA, which
could be increased up to IC50 = 50 nM for a chain length of six to
nine methylene groups.
1c: R1 = H, R2 = 7-Cl
1d: R1 = 2-propyl, R2 = 7-Cl
1e: R1 = H, R2 = 7-NO2
: R1 = H, R2 = 7-Me
1f
Scheme 1. Reagents and conditions: (a) p-TsOH, toluene, 30–192 h, reflux. The
description of positions for R1 and R2 is outlined.
R2
R1
R2
Cl
H
N
H
N
n
COOH
NH2
N
N
a
b
R2
R1
R2
R1
O
R1
N
: R1 = H, R2 = H
2a
3a-i: R1 = H, R2 = H, n = 2 - 10
: R1 = 3,3-dimethyl, R2 = H
: R1 = 3-methyl, R2 = 6-Cl
:
:
R
1 = 3,3-dimethyl, R2 = H, n = 6
2b
4a
4b
4c:
4d:
R1 = 3-methyl, R2 = 6-Cl, n = 6
R
R
2c
2d: R1 = 3,3-dimethyl, R2 = 6-Cl
1 = 3,3-dimethyl, R2 = 6-Cl, n = 6
1 = H, R2 = 6-Cl, n = 6
2e: R1 = H, R2 = 6-Cl
: R1 = 2-methyl, R2 = 6-Cl
2f
4e: R1 = 2-methyl, R2 = 6-Cl, n = 6
: R1 = 3-methyl, R2 = 7-NO2
R1 = 3-methyl, R2 = 7-NO2, n = 6
2g
2h
4f
:
4g
: R1 = 3,3-dimethyl, R2 = 7-NO2
:
R1 = 3,3-dimethyl, R2 = 7-NO2, n = 6
2i: R1 = H, R2 = 7-OMe
2j: R1 = 2-OEt, R2 = 6-Cl
5a:
5b:
R
R
1 = H, R2 = 7-OMe, n = 9
1 = 2-OEt, R2 = 6-Cl, n = 9
Scheme 2. Reagents and conditions: (a) excess POCl3, 20 min–4 h, reflux; (b) 1,x-diaminoalkane, NaI, phenol, 2–4 h, reflux.

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I. Schmidt et al. / Bioorg. Med. Chem. 24 (2016) 3636–3642
Table 3
In vitro activities against Plasmodium falciparum strain 3D7 of dimeric, substituted compounds
R2
R2
R1
H
N
H
N
n
N
N
R1
Compound
n
R1
R2
IC50 (3D7) [
lM]
J774.1 [
lM]
SI
(IC50 (Dd2) [
lM])
4a
4b
4c
4d
4e
4f
6
6
6
6
6
6
6
9
3,3-Dimethyl
3-Methyl
3,3-Dimethyl
H
2-Methyl
3-Methyl
3,3-Dimethyl
H
H
0.03 0.002
0.05 0.003
0.03 0.007
0.02 0.007
0.03 0.007
0.02 0.014
0.04 0.014
0.11 0.004
1.80
1.50
1.50
1.80
7.80
25
60
30
50
90
260
1250
198
15
6-Cl
6-Cl
6-Cl
6-Cl
7-NO2
7-NO2
4g
5a
7.90
1.70
7-Methoxy
(0.16 0.035)*
0.20 0.047
5b
9
2-Ethoxy
—
6-Cl
—
1.74
18.4
9
(0.16 0.035)*
0.02 0.005
Chloroquine
920
*
Chloroquine resistant strain Dd2.
A ‘the longer the linker, the better the activity’-correlation was
chloroquine is able to form a complex with heme and thus hinders
the detoxification of heme. Ncokazi and Egan14 developed a simple
colorimetric high-throughput screening assay, which is based on
the formation of a low spin complex of pyridine with heme, but
not with hemozoin. The absorbance of this complex obeys
Lambert–Beer’s law, making it useful for quantitating the actual
heme concentration in heme/hemozoin mixtures. This tool allows
for investigating whether compounds inhibit the formation of
b-hematin.
In the experiments chloroquine was used as a positive and DMSO
as a negative control. We were able to obtain an IC50 value for
chloroquine of 3.01 equiv with respect to the inserted heme. This
is comparable to the values given in the literature (1.91 equiv).14
Tacrine derivatives did not inhibit hemozoin formation, so we assert
that it is definitely not addressed by the compounds reported herein
(data not shown). This is supported by the fact that the activities of
these compounds are similar in both chloroquine sensitive and
resistant strains.
found in the chloroquine resistant strain Dd2, too. Of note, whereas
unsubstituted dimers with short linkers are less active against
chloroquine resistant strain (factor 10), the dimers with longer
chains (>7 carbons) exhibit the same activity towards both
chloroquine sensitive and resistant strains. This is confirmed by
substituted compounds 5a and 5b showing almost the same IC50
values in both strains (Table 3). This hints to a mode of action
which might be different from chloroquine. The cytotoxicity
(J774.1) is affected in the same manner. Nevertheless, the most
active compound 3h was found to have an acceptable selectivity
index of 36 in case of 3D7 strain and 12 in Dd2.
In order to study the influence of the substituents of the tacrine
moiety on the antimalarial activity of the dimers, compounds hav-
ing a linker of six and nine methylene units were synthesized.
These linker lengths showed the highest activities of unsubstituted
dimeric tacrine derivatives.
Surprisingly the substituted dimers with a six-membered linker
showed
a significantly higher antiplasmodial activity in the
two-digit nanomolar range of concentration in comparison to com-
pounds 5a and 5b with a nine-membered linker, but the tacrine
substituents did not influence the activity (Table 3). However,
the dimers 4e, 4f and 4g are less toxic resulting in a selectivity
index of 1250 for 4f, which is sufficient for therapeutic use. The
cytotoxicity of the other compounds might be due to very high
lipophilicity (clogP ꢀ 10), which also has negative impact on oral
bioavailability and druggability.
2.2.3. Inhibition of falcipain-2
Falcipain-2 (FP2) is localized in the acidic food vacuole and is
mainly responsible for the host hemoglobin degradation.15 Within
this pathway, the parasites obtain amino acids for their own
protein synthesis and thus for their survival.16 Many chemotypes
such as peptides, e.g., peptidyl aziridines,17 vinyl sulfones,18 pep-
tidomimetics19 or nonpeptidic compounds such as chalcones,20
isoquinolines,21 and thiosemicarbazones22 act as FP-2-inhibitors
and validate this target for antimalarials.
2.2.2. Inhibition of b-hematin formation
To examine the inhibitory effect of the compounds against
recombinant falcipain-2 Cbz-Phe-Arg-AMC was used as a sub-
strate. Falcipain-2 cleaves the fluorescent aminomethyl-coumarin
(AMC) from the substrate. This enables monitoring the enzyme
activity by measuring the fluorescence intensity which is directly
proportional to the concentration of AMC. The first step was an ini-
Hence inhibiting the formation of b-hematin (hemozoin) is the
assumed mode of action for chloroquine11 and the tacrine skeleton
resembles the quinoline moiety of chloroquine, the inhibition of
b-hematin formation was investigated.
In the intraerythrocytic stage of plasmodial infection more than
80% of the infected host red blood cells hemoglobin is degraded in
the digestive vacuole of the parasites.12 The resulting oligopeptides
and amino acids are transferred back into the cytoplasm, whereas
the non-proteinaceous component heme (ferroprotoporphyrin IX)
stays in the digestive vacuole. This is toxic for the parasites because
huge amounts of reactive oxygen species are being generated.
Therefore heme is detoxificated by conversion into nontoxic, insol-
uble hemozoin (malaria pigment). Chugh et al.13 assume that
tial screening using a 20 lM concentration of test compounds,
whereas pure DMSO was used as negative control. Only six
compounds with good activity in vitro passed this screening with
an inhibition >35% and were chosen for further kinetic experi-
ments in order to determine the IC50 values (Table 4).23 Therefore,
the substrate was added to a buffer solution containing enzyme
and different inhibitor concentrations (compounds 3g, 5b:
0–100 lM; 3h, 4d: 0–50 lM, 4f, 5a: 0–25 lM). Fluorescence was

I. Schmidt et al. / Bioorg. Med. Chem. 24 (2016) 3636–3642
3639
Table 4
Inhibition of falcipain-2
R2
R1
R2
R1
H
N
H
N
n
N
N
Compound
R1
R2
n
% Inhibition [I] = 20
lM
IC50 (FP-2) [
lM]
IC50 (3D7) [lM]
3g
3h
4d
4f
5a
5b
H
H
H
H
H
6-Cl
7-NO2
7-Methoxy
6-Cl
8
9
6
6
9
9
34.4
45.9
36.9
56.7
62.2
25
17.1
11.5
17.3
5.2
14.9
n.d.
0.11
0.05
0.02
0.02
0.11
0.20
3-Methyl
H
2-Ethoxy
n.d.: not determined.
observed over a period of 10 min. The assays were performed in
duplicate. The straight lines of derived progress curves of all tested
compounds indicate a non time-dependent, reversible mode of
inhibition. IC50 values were determined by plotting the inhibitor
7.4: MeOH 3:7, flow: 1.5 mL/min. LC/MS was conducted on an
Agilent 1100 analytical HPLC with DAD detection and an Agilent
LC/MSD Trap (Agilent Technologies, Böblingen, Germany). ESI-MS
data was conducted in positive mode. For LC/MS, following condi-
concentration against
regression analysis.
D
F/min and subsequent nonlinear
tions were used: Agilent Zorbax SB-CN (50 Â 4.6 mm, 3.5
lm), (A)
CH3CN + 0.1% acetic acid, (B) H2O + 0.1% acetic acid, gradient: 95%
A (0–5 min), 95% ? 10% A (5–10 min), 10% A (10–15 min), flow:
0.4 mL/min. MS-detection: ESI, nebulizer pressure: 40 psi, drying
gas: 9 l/min, drying gas temperature: 350 °C. All reagents and sol-
vents were obtained from commercial suppliers and were used
without further purification.
The derived low micromolar IC50 values indicate
a
good
inhibition of the compounds to the enzyme. The IC50 values for
the falcipain-2-inhibition are substantially higher than those
determined for Plasmodia, but they are in the similar range of
concentration. This might indicate that falcipain-2 represents at
least one target of the compounds studied.
Tacrine7 (1a) and compounds 1c,24 1e,25 1f,24 2a,8 2e,8 2f,26 2i,27
and 3a–i28,29 were synthesized as reported.
3. Conclusion
4.1.2. 2-Propyl-1,2,3,4-tetrahydroacridine-9-amine (1b)
2-Aminobenzonitrile (236 mg, 2.00 mmol) and 4-propyl-cyclo-
hexanone (337 mg, 2.40 mmol) were condensed with p-toluene-
sulfonic acid (69 mg, 0.34 mmol) in toluene (10 mL) for 96 h under
reflux. Isolation was performed as described for the compound 1a.7
Yield: 40% (193 mg) yellowish powder. Mp: 186–187 °C. Rf = 0.44
~
1636, 1424, 1011, 753. 1H NMR (CDCl3, d [ppm], J [Hz]): 7.89 (1H,
d, 3J = 8.4, C5-H), 7.69 (1H, dd, 3J = 8.3, 4J = 1.0, C8-H), 7.56 (1H,
ddd, 3J = 8.4, 6.8, 4J = 1.0, C6-H), 7.36 (1H, dd, 3J = 8.3, 6.8, 4J = 1.1,
C7-H), 4.69 (2H, br s, NH2), 3.11 (1H, ddd, 2J = 17.4, 3J = 5.0, 3.4,
C4-H2), 3.00 (1H, ddd, 2J = 17.4, 3J = 11.8, 5.5, C4-H2), 2.73 (1H,
ddd, 2J = 15.8, 3J = 5.5, 4J = 1.3, C1-H2), 2.17 (1H, dd, 2J = 15.8,
3J = 10.2, C1-H2), 2.03–2.10 (1H, m, C3-H2), 1.82–1.91 (1H, m,
C2-H), 1.41–1.55 (5H, m, C3-H2, C2-CH2-CH2-CH3), 0.97 (3H, t,
3J = 6.8 Hz, C2-CH2-CH2-CH3). 13C NMR (CDCl3, d [ppm]): 158.7
(C4a), 146.6 (C10a), 146.5 (C9), 128.9 (C5), 128.6 (C6), 124.0 (C7),
119.7 (C8), 117.2 (C8a), 110.3 (C9a), 39.1 (C2-CH2-CH2-CH3), 33.9
(C4), 31.0 (C2), 30.8 (C1), 29.1 (C3), 20.3 (C2-CH2-CH2-CH3), 14.5
(C2-CH2-CH2-CH3). ESI-MS: m/z 241.2 [M+H]+.
Mono- and dimeric tacrine derivatives with different substitu-
tion patterns of the tacrine moiety and linker lengths were synthe-
sized and tested for their antimalarial activity. Especially dimers
were found to have an antiplasmodial activity in the nanomolar
concentration range, but this was often associated with cytotoxic-
ity. Nevertheless one compound (4f) could be identified which
exhibited a very high selectivity index (>1000) and thus can be
regarded as a new lead compound. Since the cytotoxicity of the
other compounds might be due to their high lipophilicity, the
new lead compound will be optimized with regard to an even
higher antiplasmodial activity along with a decreased lipophilicity.
In addition, the inhibitory activity towards falcipain-2 has to be
considered.
(CHCl3/MeOH, 6:1). Purity: 99 %. IR
m
[cmÀ1]: 3493, 3301, 3050,
4. Experimental
4.1. Chemistry
4.1.1. Instruments and analyses
Melting points were determined with a Sanyo Gallenkamp
melting point apparatus MPD350 BM 3.5 and are reported uncor-
rected. Reactions were monitored by thin-layer chromatography
(TLC) using coated silica gel plates (Merck, Darmstadt, Germany),
detection under ultraviolet light (k = 254 nm). Column chromatog-
raphy was performed on Merck Silica Gel 60 (70–230 mesh; Merck,
Darmstadt). IR-Spectra were recorded on a Jasco FT-IR-6100 spec-
trometer (Jasco, Groß-Umstadt, Germany) using an ATR unit. 1H
(400.132 MHz) and 13C (100.613 MHz) NMR spectra were acquired
on a Bruker AV 400 instrument (Bruker Biospin, Ettlingen, Ger-
many). Chemical shifts are expressed in d (ppm) relative to TMS
as internal standard. The purities of the synthesized compounds
were determined via HPLC (Knauer Eurospher C18-column,
4.1.3. 7-Chloro-2-propyl-1,2,3,4-tetrahydroacridine-9-amine
(1d)
2-Amino-5-chlorobenzonitrile (305 mg, 2.00 mmol) and 4-pro-
pyl-cyclohexanone (337 mg, 2.40 mmol) were condensed with
p-toluene-sulfonic acid (69 mg, 0.34 mmol) in toluene (10 mL) for
72 h under reflux. Isolation was performed as described for com-
pound 1a. Yield: 24 % (132 mg) yellow solid. Mp: 208209 °C.
[cmÀ1]: 3493,
~
m
Rf = 0.53 (CHCl3/MeOH, 6:1). Purity: >99%. IR
3299, 3126, 1639, 1445, 1128, 678. 1H NMR (DMSO-d6, d [ppm], J
[Hz]): 8.27 (1H, d, 4J = 2.3, C8-H), 7.62 (1H, d, 3J = 9.0, C5-H), 7.46
(1H, dd, 3J = 9.0, 4J = 2.3, C6-H), 6.44 (2H, br s, NH2), 2.85 (2H, m,
C4-H2), 2.75 (1H, dd, 2J = 17.0, 3J = 5.0, C1-H2), 2.06 (1H, dd,
2J = 17.0, 3J = 10.5, C1-H2), 1.94–1.90 (1H, m, C3-H2), 1.79–1.68
150 Â 4.6 mm, 5
lm), mobile phase: 25 mM phosphate buffer, pH

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I. Schmidt et al. / Bioorg. Med. Chem. 24 (2016) 3636–3642
(1H, m, C2-H), 1.54–1.44 (2H, m, C2-CH2-CH2-CH3), 1.43–1.34 (3H,
m, C3-H2, C2-CH2-CH2-CH3), 0.93 (3H, t, 3J = 7.1, C2-CH2-CH2-CH3).
13C NMR (DMSO-d6, d [ppm]): 158.1 (C4a), 147.5 (C10a), 144.9
(C9), 130.1 (C5), 128.3 (C6), 127.0 (C7), 121.1 (C8), 117.7 (C8a),
109.5 (C9a), 38.6 (C2-CH2-CH2-CH3), 33.3 (C2), 33.2 (C4), 30.4 (C1),
28.8 (C3), 19.5 (C2-CH2-CH2-CH3), 14.2 (C2-CH2-CH2-CH3). ESI-MS:
m/z 275.2 [M+H]+.
1301, 848, 741. 1H NMR (CDCl3, d [ppm], J [Hz]): 9.13 (1H, d,
4J = 2.5, C8-H), 8.42 (1H, dd, 3J = 9.2, 4J = 2.5, C6-H), 8.08 (1H, d,
3J = 9.2, C5-H), 3.30–3.21 (2H, m, C1-H2, C4-H2), 2.93 (1H, ddd,
2J = 17.7, 3J = 11.1, 6.1, C1-H2), 2.75 (1H, dd, 2J = 17.9, 3J = 10.8,
C4-H2), 2.15–1.98 (2H, m, C2-H2, C3-H), 1.55 (1H, dtd, 2J = 13.8,
3J = 11.1, 6.3, C2-2), 1.17 (3H, d, 3J = 6.5, C3-CH3). 13C NMR (CDCl3,
d [ppm]): 164.0 (C4a), 148.8 (C10a), 145.8 (C7), 143.2 (C9), 131.9
(C9a), 130.7 (C5), 124.8 (C8a), 122.9 (C6), 121.2 (C8), 42.9 (C4), 30.4
(C2), 28.8 (C3), 27.3 (C1), 21.5 (C3-CH3).
4.1.4. 9-Chloro-3,3-dimethyl-1,2,3,4-tetrahydroacridine (2b)
Anthranilic acid (1.00 g, 7.29 mmol) and 3,3-dimethylcyclohex-
anone (1.01 g, 8.02 mmol) were refluxed in 8 mL POCl3 for 6 h.
The reaction was cooled to room temperature and kept in an ice
bath. The solution was carefully neutralized with saturated
NaHCO3-solution. The mixture was then extracted three times
with 40 mL chloroform and the combined organic phases were
dried over MgSO4 and concentrated in vacuo. The crude product
was recrystallized from acetone to afford the pure compound
2b.8 Yield: 50 % (896 mg) yellow solid. Mp: 63–64 °C. Rf = 0.76
~
ESI-MS: m/z 277.1 [M+H]+.
4.1.8. 9-Chloro-3,3-dimethyl-7-nitro-1,2,3,4-tetrahydro-
acridine (2h)
2-Amino-5-nitrobenzoic acid (728 mg, 4.00 mmol) and 3,3-
dimethylcyclohexanone (505 mg, 4.00 mmol) were refluxed in
10 mL POCl3 for 1 h. Isolation was performed as described for com-
pound 2b. Yield: 52% (607 mg) beige solid. Mp: 162–163 °C.
[cmÀ1]: 3083, 1617, 1519, 1473,
~
m
Rf = 0.83 (EtOAc/PE, 1:1). IR
m
[cmÀ1]: 3059, 1633, 1583, 1482, 1136, 1315,
1311, 899, 738. 1H NMR (CDCl3, d [ppm], J [Hz]): 9.12 (1H, d,
4J = 2.5, C8-H), 8.42 (1H, dd, 3J = 9.2, 4J = 2.5, C6-H), 8.09 (1H, d,
4J = 9.2, C5-H), 3.08 (2H, t, 3J = 6.9, C1-H2), 2.95 (2H, s, C4-H2), 1.78
(2H, t, 3J = 6.9, C2-H2), 1.08 (6H, s, C3-(CH3)2). 13C NMR (CDCl3, d
[ppm]): 163.8 (C4a), 149.0 (C10a), 145.8 (C7), 143.1 (C9), 130.7 (C5),
130.2 (C9a), 124.8 (C8a), 122.9 (C6), 121.2 (C8), 47.2 (C4), 35.0 (C2),
30.2 (C3), 27.9 (C3-(CH3)2), 25.1 (C1). ESI-MS: m/z 291.1 [M+H]+.
(EtOAc/PE, 1:2). IR
762. 1H NMR (CDCl3, d [ppm], J [Hz]): 8.18 (1H, dd, 3J = 8.3,
4J = 1.2, C8-H), 8.00 (1H, d, 3J = 8.4, C5-H), 7.67 (1H, ddd, 3J = 8.4,
7.0, 4J = 1.2, C6-H), 7.55 (1H, ddd, 3J = 8.3, 7.0, 4J = 1.1, C7-H), 3.05
(2H, t, 3J = 6.9, C1-H2), 2.92 (2H, br s, C4-H2), 1.75 (2H, t, 3J = 6.9,
C2-H2), 1.07 (6H, br s, C3-(CH3)2). 13C NMR (CDCl3, d [ppm]):
159.3 (C4a), 147.5 (C10a), 141.9 (C9), 129.4 (C8a), 128.8 (C5), 127.8
(C6), 126.7 (C7), 125.6 (C9a), 123.8 (C8), 48.0 (C4), 35.3 (C3), 30.2
(C2), 28.0 (C3-(CH3)2), 25.0 (C1). ESI-MS: m/z 246.1 [M+H]+.
4.1.9. 6,9-Dichloro-2-ethoxy-1,2,3,4-tetrahydroacridine (2j)
2-Amino-4-chlorobenzoic acid (1.20 g, 7.00 mmol) and
p-ethoxycyclohexanone30 (1.00 g, 7.93 mmol) were refluxed in
20 mL POCl3 for 2 h. Isolation was performed as described for com-
pound 2b. Yield: 80% (1.66 g) greyish solid. Mp: 59–60 °C. Rf = 0.34
~
4.1.5. 6,9-Dichloro-3-methyl-1,2,3,4-tetrahydroacridine (2c)
2-Amino-4-chlorobenzoic acid (686 mg, 4.00 mmol) and
3-methylcyclohexanone (494 mg, 4.40 mmol) were refluxed in
10 mL POCl3 for 2 h. Isolation was performed as described for com-
pound 2b. Yield: 54% (574 mg) yellowish solid. Mp: 92–93 °C.
[cmÀ1]: 2929, 1653, 1542, 1474,
~
m
(EtOAc/PE, 1:5). IR
m
[cmÀ1]: 3057, 1603, 1545, 1474, 1095, 936,
735. 1H NMR (CDCl3, d [ppm], J [Hz]): 8.05 (1H, d, 3J = 9.0, C8-H),
7.96 (1H, d, 4J = 2.1, C5-H), 7.45 (1H, dd, 3J = 9.0, 4J = 2.1, C7-H),
3.95–3.90 (1H, m, C2-H), 3.69–3.55 (2H, m, C2-O-CH2-CH3), 3.32–
3.19 (2H, m, C1-H2, C4-H2), 3.10–3.02 (2H, m, C1-H2, C4-H2), 2.13–
2.08 (2H, m, C3-H2), 1.23 (3H, t, 3J = 7.0, C2-O-CH2-CH3). 13C NMR
(CDCl3, d [ppm]): 160.1 (C4a), 147.1 (C10a), 142.1 (C9), 135.6 (C6),
127.7 (C7), 127.6 (C5), 127.1 (C9a), 125.3 (C8), 123.9 (C8a), 72.4 (C2),
63.8 (O-CH2-CH3), 33.8 (C4), 30.5 (C1), 27.1 (C3), 15.7 (O-CH2-CH3).
ESI-MS: m/z 296.1 [M+H]+.
Rf = 0.89 (EtOAc/PE, 1:2). IR
1315, 875, 812. 1H NMR (CDCl3, d [ppm], J [Hz]): 8.10 (1H, d,
3J = 9.0, C8-H), 8.00 (1H, s, C5-H), 7.48 (1H, dd, 3J = 9.0, 4J = 2.0,
C7-H), 3.24–3.16 (2H, m, C1-H2, C4-H2), 2.88 (1H, ddd, 2J = 17.7,
3J = 11.1, 6.1, C1-H2), 2.71 (1H, dd, 2J = 17.6, 3J = 10.8, C4-H2), 2.10–
1.96 (2H, m, C2-H2, C3-H), 1.51 (1H, dtd, 2J = 13.7, 3J = 11.1, 6.3,
C2-H2), 1.15 (3H, d, 3J = 6.5 Hz, C3-CH3). 13C NMR (CDCl3,
d
[ppm]): 160.8 (C4a), 146.7 (C10a), 141.7 (C9), 135.2 (C6), 128.9
(C9a), 127.7 (C7), 127.6 (C5), 125.3 (C8), 124.1 (C8a), 42.5 (C4), 30.7
(C2), 28.9 (C3), 27.2 (C1), 21.6 (C3-CH3). ESI-MS: m/z 266.1 [M+H]+.
4.1.10. N1,N6-Bis(3,3-dimethyl-1,2,3,4-tetrahydroacridine-9-yl)-
hexane-1,6-diamine (4a)
4.1.6. 6,9-Dichloro-3,3-dimethyl-1,2,3,4-tetrahydroacridine (2d)
2-Amino-4-chlorobenzoic acid (1.03 g, 6.00 mmol) and 3,3-
dimethylcyclohexanone (833 mg, 6.60 mmol) were refluxed in
10 mL POCl3 for 2 h. Isolation was performed as described for com-
pound 2b. Yield: 63% (1.06 g) yellowish solid. Mp: 102–103 °C.
[cmÀ1]: 3064, 1634, 1544, 1473,
~
m
Compound 2b (300 mg, 1.22 mmol) was heated with hexane-
1,6-diamine (71 mg, 0.61 mmol), NaI (10 mg) and phenol
(500 mg) at 180 °C for 2 h under argon atmosphere. After cooling
to room temperature, 20 mL of a 3 N KOH solution were added
and the crude product was extracted three times with 30 mL
EtOAc. The organic phases were combined, dried over Na2SO4,
and concentrated under reduced pressure. The resulting residue
was purified by flash column chromatography to afford pure com-
pound 4a.28 Yield: 59% (194 mg) yellowish solid. Mp: 170–172 °C.
~
Rf = 0.92 (EtOAc/PE, 1:1). IR
1362, 902, 812. 1H NMR (CDCl3, d [ppm], J [Hz]): 8.10 (1H, d,
3J = 9.0, C8-H), 7.99 (1H, s, C5-H), 7.49 (1H, dd, 3J = 9.0, 4J = 2.0,
C7-H), 3.02 (2H, t, 3J = 6.9, C1-H2), 2.89 (2H, br s, C4-H2), 1.74 (2H,
t, 3J = 6.9, C2-H2), 1.06 (6H, br s, C3-(CH3)2). 13C NMR (CDCl3, d
[ppm]): 160.6 (C4a), 147.3 (C10a), 141.5 (C9), 135.4 (C6), 128.1
(C9a), 127.7 (C7), 127.7 (C5), 125.3 (C8), 124.1 (C8a), 47.9 (C4), 35.2
(C2), 30.1 (C3), 27.9 (C3-(CH3)2), 24.95 (C1). ESI-MS: m/z 280.1
[M+H]+.
Rf = 0.42 (CHCl3/MeOH, 10:2). Purity: >99%. IR
m
[cmÀ1]: 3057,
1603, 1545, 1474, 1095, 936, 735. 1H NMR (CDCl3, d [ppm], J
[Hz]): 7.95 (2H, d, 3J = 8.3, 4J = 1.1, C8-H), 7.90 (2H, dd, 3J = 8.4,
4J = 1.0, C5-H), 7.54 (2H, ddd, 3J = 8.4, 6.8, 4J = 1.1, C6-H), 7.33 (2H,
ddd, 3J = 8.3, 6.8, 4J = 1.0, C7-H), 3.89 (2H, br s, NH), 3.50 (4H, t,
3J = 7.1, Ca-H2), 2.84 (4H, s, C4-H2), 2.71 (4H, t, 3J = 6.8, C1-H2),
1.71 (4H, t, 3J = 6.8, C2-H2), 1.68–1.62 (4H, m, Cb-H2), 1.44–1.41
4.1.7. 9-Chloro-3-methyl-7-nitro-1,2,3,4-tetrahydroacridine
(2g)
(4H, m, Cc-H2), 1.04 (12H, s, C3-(CH3)2). 13C NMR (CDCl3,
d
2-Amino-5-nitrobenzoic acid (911 mg, 5.00 mmol) and
3-methylcyclohexanone (561 mg, 5.00 mmol) were refluxed in
10 mL POCl3 for 1 h. Isolation was performed as described for com-
pound 2b. Yield: 53% (735 mg) beige solid. Mp: 122–123 °C.
~
[ppm]): 158.3 (C4a), 150.6 (C9), 148.1 (C10a), 129.1 (C5), 128.3 (C6),
123.7 (C7), 123.1 (C8), 120.4 (C8a), 114.6 (C9a), 49.5 (Ca), 48.0 (C4),
35.6 (C2), 31.9 (Cb), 30.0 (C3), 28.0 (C3-(CH3)2), 26.9 (Cc), 22.2 (C1).
ESI-MS: m/z 268.2 [M+2H]2+
.
Rf = 0.63 (EtOAc/PE, 1:1). IR
m
[cmÀ1]: 3031, 1617, 1517, 1476,

I. Schmidt et al. / Bioorg. Med. Chem. 24 (2016) 3636–3642
3641
4.1.11. N1,N6-Bis(6-chloro-3-methyl-1,2,3,4-tetrahydro-acridine-
9-yl)hexane-1,6-diamine (4b)
3J = 5.1, 4J = 1.3, C1-H2), 2.16 (2H, dd, 2J = 15.6, 3J = 10.4, C1-H2),
2.00–1.86 (2H, m, C2-H, C3-H2), 1.66–1.63 (4H, m, Cb-H2), 1.48
(2H, dtd, 2J = 12.7, 3J = 11.5, 5.5, C3-H2), 1.42–1.38 (4H, m, Cc-H2),
1.13 (6H, d, 3J = 6.5, C2-CH3). 13C NMR (CDCl3, d [ppm]): 159.3
(C4a), 151.0 (C9), 148.0 (C10a), 134.4 (C6), 127.2 (C5), 124.6 (C8),
124.5 (C7), 118.3 (C8a), 115.6 (C9a), 49.4 (Ca), 33.4 (C4), 33.3 (C1),
31.7 (Cb), 30.7 (C3), 29.3 (C2), 26.8 (Cc), 22.1 (C2-CH3). ESI-MS: m/z
Compound 2c (200 mg, 0.76 mmol) was heated with hexane-1,6-
diamine (44 mg, 0.38 mmol), NaI (10 mg) and phenol (800 mg)
under reflux for 3 h. Isolation was performed as described for com-
pound 4a. Yield: 63% (136 mg) brownish solid. Mp: 156–157 °C.
~
Rf = 0.49 (CHCl3/MeOH, 10:2). Purity: 92%. IR
m
[cmÀ1]: 3052, 1603,
1552, 1489, 1154, 895, 734. 1H NMR (CDCl3, d [ppm], J [Hz]): 7.89
(2H, d, 4J = 1.8, C5-H), 7.80 (2H, d, 3J = 9.0, C8-H), 7.19 (2H, dd,
3J = 9.0, 1.8, C7-H), 3.84 (2H, br s, NH), 3.40 (4H, t, 3J = 6.6, Ca-H2),
3.04 (2H, ddd, 2J = 17.0, 3J = 4.2, 4J = 1.4, C4-H2), 2.69 (2H, ddd,
2J = 15.9, 3J = 5.7, 3.2, C1-H2), 2.59–2.51 (4H, m, C1-H2, C4-H2), 1.99–
1.86 (4H, m, C2-H2, C3-H), 1.60–1.55 (4H, Cb-H2), 1.39 (2H, dtd,
2J = 12.9, 3J = 10.9, 5.7, C2-H2), 1.37–1.33 (4H, Cc-H2), 1.12 (6H, d,
3J = 6.5, C3-CH3). 13C NMR (CDCl3, d [ppm]): 159.6 (C4a), 150.7 (C9),
148.0 (C10a), 134.2 (C6), 127.9 (C5), 124.6 (C8), 124.5 (C7), 118.6
(C8a), 115.5 (C9a), 49.6 (Ca), 42.2 (C4), 31.8 (Cb), 31.0 (C2), 28.9 (C3),
289.1 [M+2H]2+
.
4.1.15. N1,N6-Bis(3-methyl-7-nitro-1,2,3,4-tetrahydro-acridine-
9-yl)hexane-1,6-diamine (4f)
Compound 2g (300 mg, 1.08 mmol) was heated with hexane-
1,6-diamine (63 mg, 0.54 mmol), NaI (10 mg) and phenol
(500 mg) under reflux for 2 h. Isolation was performed as described
for compound 4a. Yield: 35% (116 mg) brownish solid. Mp: 195–
[cmÀ1]:
~
m
196 °C. Rf = 0.65 (CHCl3/MeOH, 10:2). Purity: >99 %. IR
3101, 1609, 1558, 1490, 1254, 1142, 834. 1H NMR (CDCl3, d
[ppm], J [Hz]): 9.03 (2H, d, 4J = 2.3, C8-H), 8.26 (2H, dd, 3J = 9.3,
4J = 2.3, C6-H), 7.91 (2H, d, 3J = 2.3, C5-H), 4.30 (2H, br s, NH),
3.70–3.65 (4H, m, Ca-H2), 3.14 (2H, dd, 2J = 17.5, 3J = 2.9, C4-H2),
2.75 (2H, ddd, 2J = 15.9, 3J = 5.8, 3.2, C1-H2), 2.69–2.58 (4H, m, C1-
H2, C4-H2), 2.11–2.06 (2H, m, C2-H2), 2.03–1.92 (2H, m, C3-H),
1.86–1.76 (4H, m, Cb-H2), 1.57 - 1.45 (6H, m, C2-H2, Cc-H2), 1.13
(6H, d, 3J = 6.5, C3-CH3). 13C NMR (CDCl3, d [ppm]): 178.8 (C4a),
152.1 (C9), 150.5 (C10a), 142.8 (C7), 130.3 (C5), 121.9 (C6), 121.6
(C8), 117.7 (C8a), 115.4 (C9), 49.5 (Ca), 42.7 (C4), 31.7 (Cb), 30.7 (C2),
28.7 (C3), 26.7 (Cc), 24.1 (C1), 21.5 (C3-CH3). ESI-MS: m/z 299.2
26.8 (Cc), 24.3 (C1), 21.6 (C3-CH3). ESI-MS: m/z 288.2 [M+2H]2+
.
4.1.12. N1,N6-Bis(6-chloro-3,3-dimethyl-1,2,3,4-tetrahydro-acri-
dine-9-yl)hexane-1,6-diamine (4c)
Compound 2d (280 mg, 1.00 mmol) was heated with hexane-
1,6-diamine (58 mg, 0.50 mmol), NaI (10 mg) and phenol
(500 mg) under reflux for 2 h. Isolation was performed as described
for compound 4a. Yield: 38% (117 mg) yellowish solid. Mp:
~
102–103 °C. Rf = 0.53 (CHCl3/MeOH, 10:2). Purity: 98%. IR
m
[cmÀ1]: 3052, 1605, 1555, 1488, 1076, 876, 737. 1H NMR (CDCl3,
d [ppm], J [Hz]): 7.92 (2H, br s, C5-H), 7.90 (2H, d, 3J = 9.1, C8-H),
7.27 (2H, ddd, 3J = 9.1, 4J = 2.2, C7-H), 3.96 (2H, br s, NH),
3.54–3.49 (4H, m, Ca-H2), 2.82 (4H, s, C4-H2), 2.66 (4H, t, 3J = 6.8,
C1-H2), 1.71 (4H, t, 3J = 6.8, C2-H2), 1.69–1.63 (4H, m, Cb-H2),
1.44–1.41 (4H, m, Cc-H2), 1.04 (12H, s, C3-(CH3)2). 13C NMR (CDCl3,
d [ppm]): 159.2 (C4a), 150.5 (C9), 149.2 (C10a), 134.7 (C6), 127.5 (C5),
124.8 (C8), 124.5 (C7), 116.3 (C8a), 114.3 (C9a), 49.6 (Ca), 47.8 (C4),
35.4 (C2), 31.9 (Cb), 29.9 (C3), 27.9 (C3-(CH3)2), 26.8 (Cc), 22.0 (C1).
[M+2H]2+
.
4.1.16. N1,N6-Bis(3,3-dimethyl-7-nitro-1,2,3,4-tetrahydro-acrid-
ine-9-yl)hexane-1,6-diamine (4g)
Compound 2h (300 mg, 1.03 mmol) was heated with hexane-
1,6-diamine (60 mg, 0.52 mmol), NaI (50 mg) and phenol (1.00 g)
under reflux for 2 h. Isolation was performed as described for com-
pound 4a. Yield: 52% (169 mg) orange solid. Mp: 165–166 °C.
~
ESI-MS: m/z 302.2 [M+2H]2+
.
Rf = 0.76 (CHCl3/MeOH, 10:2). Purity: 92%. IR
m
[cmÀ1]: 3041,
1613, 1583, 1493, 1260, 1151, 832. 1H NMR (CDCl3, d [ppm], J
[Hz]): 9.06 (2H, d, 4J = 2.3, C8-H), 8.28 (2H, dd, 3J = 9.3, 4J = 2.3,
C7-H), 7.92 (2H, d, 3J = 9.3, C5-H), 4.31 (2H, br s, NH), 3.74–3.68
(4H, m, Ca-H2), 2.84 (4H, s, C4-H2), 2.67 (4H, t, 3J = 6.7, C2-H2),
1.83 (4H, m, Cb-H2), 1.75 (4H, t, 3J = 6.7, C1-H2), 1.56 (4H, m,
Cc-H2), 1.05 (6H, s, C3-(CH3)2). 13C NMR (CDCl3, d [ppm]): 161.8
(C4a), 152.0 (C9), 150.9 (C10a), 142.8 (C7), 130.4 (C5), 121.9 (C6),
121.8 (C8), 117.8 (C8a), 114.4 (C9a), 49.5 (Ca), 47.7 (C4), 35.2 (C1),
31.8 (Cb), 29.9 (C3), 27.9 (C3-(CH3)2), 26.7 (Cc), 21.9 (C2). ESI-MS:
4.1.13. N1,N6-Bis(6-chloro-1,2,3,4-tetrahydroacridine-9-yl)-hexan-
1,6-diamine (4d)
Compound 2e (300 mg, 1.19 mmol) was heated with hexane-
1,6-diamine (70 mg, 0.60 mmol), NaI (10 mg) and phenol
(500 mg) under reflux for 2 h. Isolation was performed as described
for compound 4a. Yield: 35% (116 mg) yellow solid. Mp:
~
125–126 °C. Rf = 0.31 (CHCl3/MeOH, 10:2). Purity: 98 %. IR
m
[cmÀ1]: 3122, 1604, 1556, 1505, 1077, 803, 672. 1H NMR (CDCl3,
d [ppm], J [Hz]): 7.89 (2H, d, 4J = 2.2, C5-H), 7.86 (2H, d, 3J = 9.0,
C8-H), 7.26 (2H, dd, 3J = 9.0, 4J = 2.2, C7-H), 3.48–3.44 (4H, m,
Ca-H2), 3.03–3.01 (4H, m, C4-H2), 2.66–2.64 (4H, m, C1-H2),
1.92–1.89 (8H, m, C2-H2, C3-H2), 1.67–1.62 (4H, m, Cb-H2),
1.43–1.40 (4H, m, Cc-H2). 13C NMR (CDCl3, d [ppm]): 159. 8 (C4a),
150.8 (C9), 148.3 (C10a), 134.2 (C6), 127.8 (C5), 124.6 (C8), 124.5
(C7), 118.6 (C8a), 116.1 (C9a), 49.6 (Ca), 34.2 (C4), 31.8 (Cb), 26.9
m/z 313.3 [M+2H]2+
.
4.1.17. N1,N9-Bis(7-methoxy-1,2,3,4-tetrahydroacridine-9-yl)-
nonane-1,9-diamine (5a)
Compound 2i (1.00 g, 4.04 mmol) was heated with nonane-1,9-
diamine (320 mg, 2.02 mmol), NaI (100 mg) and phenol (2.00 g)
under reflux for 5 h. Isolation was performed as described for com-
pound 4a. Yield: 28% (330 mg) brownish foam. Mp: 61–62 °C.
~
(Cc), 24.8 (C1), 23.1, 22.8 (C2, C3). ESI-MS: m/z 274.2 [M+2H]2+
.
m
[cmÀ1]: 3002,
4.1.14. N1,N6-Bis(6-chloro-2-methyl-1,2,3,4-tetrahydro-acridine-
9-yl)hexane-1,6-diamine (4e)
Rf = 0.42 (CHCl3/MeOH, 10:2). Purity: 99%. IR
1623, 1580, 1500, 1226, 1107, 1031, 828. 1H NMR (DMSO-d6, d
[ppm], J [Hz]): 7.65 (2H, d, 3J = 9.1, C5-H), 7.44 (2H, d, 4J = 2.7,
C8-H), 7.23 (2H, dd, 3J = 9.1, 4J = 2.7, C6-H), 5.61 (2H, br s, NH),
3.85 (6H, s, C7-O-CH3), 3.37–3.34 (4H, m, Ca-H2), 2.88 (4H, t,
3J = 6.2, C4-H2), 2.70 (4H, t, 3J = 6.0, C1-H2), 1.84–1.73 (8H, C2-H2,
C3-H2), 1.54–1.47 (4H, m, Cb-H2), 1.26–1.20 (4H, m, Cc-H2), 1.20–
1.13 (6H, m, Cd-H2, Ce-H2). 13C NMR (DMSO-d6, d [ppm]): 155.5
(C7), 154.7 (C4a), 150.3 (C9), 141.1 (C10a), 128.5 (C5), 120.6 (C8a),
120.4 (C6), 116.1 (C9a), 101.9 (C8), 55.4 (C7-O-CH3), 47.3 (Ca), 32.4
(C4), 30.5 (Cb), 28.8 (Cd), 28.6 (Ce), 26.2 (Cc), 25.2 (C1), 22.6, 22.2
Compound 2f (266 mg, 1.00 mmol) was heated with hexane-
1,6-diamine (58 mg, 0.50 mmol), NaI (15 mg) and phenol (1.00 g)
under reflux for 3 h. Isolation was performed as described for com-
pound 4a. Yield: 60% (175 mg) yellowish solid. Mp: 188–189 °C.
[cmÀ1]: 3063,
~
m
Rf = 0.46 (CHCl3/MeOH, 10:2). Purity: >99%. IR
1605, 1555, 1502, 1137, 923, 871. 1H NMR (CDCl3, d [ppm], J
[Hz]): 7.85 (2H, d, 3J = 9.0, C8-H), 7.84 (2H, d, 4J = 2.2, C5-H), 7.24
(2H, dd, 3J = 9.0, 4J = 2.2, C7-H), 3.98 (2H, br s, NH), 3.50–3.39 (4H,
m, Ca-H2), 3.07 (2H, ddd, 2J = 17.6, 3J = 5.5, 3.2, C4-H2), 2.98 (2H,
ddd, 2J = 17.6, 3J = 11.5, 5.7, C4-H2), 2.73 (2H, ddd, 2J = 15.6,
(C2, C3). ESI-MS: m/z 291.3 [M+2H]2+
.

3642
I. Schmidt et al. / Bioorg. Med. Chem. 24 (2016) 3636–3642
4.1.18. N1,N9-Bis(6-chloro-2-ethoxy-1,2,3,4-tetrahydro-acridine-
9-yl)nonane-1,9-diamine (5b)
test according to Ahmed et al.10. Macrophages were cultured in
RPMI medium containing 10% FCS. The compounds were dissolved
Compound 2j (1.00 g, 3.37 mmol) was heated with nonane-1,9-
diamine (268 mg, 1.69 mmol), NaI (100 mg) and phenol (3.00 g)
under reflux for 2 h. Isolation was performed as described for com-
pound 4a. Yield: 53% (603 mg) brownish foam. Mp: 152–153 °C
(as dihydrochloride). Rf = 0.45 (CHCl3/MeOH, 10:2). Purity: 97%.
~
in DMSO and serial dilutions ranging from 100
prepared. Incubation was done at 37 °C with 5% CO2 for 24 h. After
adding 20
L AlamarBlueÒ solution, the plates were incubated for
further 24 and 48 h. Optical densities were measured with a
Multiscan Ascent enzyme-linked immunosorbent assay (ELISA)
reader (Thermo Electron Corporation, Dreieich, Germany) applying
lM to 1.28 nM were
l
IR
m
[cmÀ1]: 3071, 1606, 1577, 1556, 1345, 1090, 802. 1H NMR
(CDCl3, d [ppm], J [Hz]): 7.89 (4H, m, C5-H, C8-H), 7.27 (2H,
dd, 3J = 9.0, 4J = 2.1, C7-H), 3.98 (2H, br s, NH), 3.87–3.81 (2H, m,
C2-H), 3.74–3.67 (2H, m, C2-O-CH2-CH3), 3.61–3.54 (2H, m,
C2-O-CH2-CH3), 3.50–3.46 (4H, m, Ca-H2), 3.20 (2H, dt, 2J = 17.9,
3J = 5.5, C3-H2), 3.05–2.96 (4H, m, C1-H2, C3-H2), 2.64 (2H,
dd, 2J = 15.4, 3J = 7.6, C1-H2), 2.26–2.19 (2H, m, C4-H2), 1.97–1.87
(2H, m, C4-H2), 1.68–1.61 (4H, m, Cb-H2), 1.38–1.33 (4H, m,
Cc-H2), 1.30–1.26 (6H, m, Cd-H2, Ce-H2), 1.25 (6H, t, 3J = 7.0,
C2-O-CH2-CH3). 13C NMR (CDCl3, d [ppm]): 158.4 (C4a), 151.4 (C9),
148.2 (C10a), 134.4 (C6), 127.8 (C5), 124.6 (C8), 124.5 (C7), 118.1
(C8a), 113.3 (C9a), 74.0 (C2), 63.9 (C2-O-CH2-CH3), 49.7 (Ca), 31.9
(Cb), 31.6 (C1), 31.4 (C3), 29.5 (Ce), 29.4 (Cd), 27.6 (C4), 27.0 (Cc),
a wavelength of k = 540 nm and a reference wavelength of
k = 630 nm.
Acknowledgements
This work was funded by the Deutsche Forschungsgemein-
schaft, Bonn, Germany (SFB 630, project A1). Thanks are due to
Elena Katzowitsch and Tobias Ölschläger for performing the
cytotoxicity assays and Heike Bruhn for worthwhile discussions.
References and notes
15.8 (C2-O-CH2-CH3). ESI-MS: m/z 339.2 [M+2H]2+
.
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4.2. Biological assays
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Compound concentrations between 10 nM and 100 lM were
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200
sodium acetate buffer (50 mM, pH 5.5, containing 5 mM EDTA
and 200 mM NaCl) 5 L enzyme solution, 10 L inhibitor stock
solution (400 M in DMSO) and 5 L substrate stock solution
ll. Cbz-Phe-Arg-AMC was used as substrate. To 180 lL of
l
l
l
l
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the slopes of the curves obtained (
an inhibitory effect greater than 35 % at 20
detailed assays. Therefore 185 L buffer, 5
L inhibitor solution and 5 L substrate solution were mixed
and the fluorescence intensity was recorded over 10 min. Inhibitor
concentrations were in the range of 0–100 in case of
compounds 3g and 5b, 0–50 M in case of compounds 3h und
D
F vs time). Compounds having
M were chosen for
L enzyme solution,
l
l
l
26. Skotnicki, J. S.; Gilman, S.C., 1990.
5
l
l
27. Spilovska, K.; Korabecny, J.; Kral, J.; Horova, A.; Musilek, K.; Soukup, O.;
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lM
l
4d, and 0–25 lM in case of compounds 4f and 5a. IC50 values were
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4.2.3. Cytotoxicity assay
The cytotoxicity of all compounds was determined in the macro-
phage cell line J774.1 using the AlamarBlueÒ-based cytotoxicity