Synthesis, structure and antiproliferative activity of chiral polyamines based on a 2-azanorbornane skeleton

Article abstract of DOI:10.1016/j.tetasy.2016.06.009

A series of enantiopure 2-azanorbornane-based amines were prepared via a stereoselective aza-Diels–Alder reaction and further modifications of the obtained cycloadduct. In particular, derivatives containing two bicyclic moieties linked by [Formula presented] fragments were synthesized. Two dimeric derivatives exhibited a significant antiproliferative activity against selected cell lines, comparable to cisplatin in certain cases.

Full text of DOI:10.1016/j.tetasy.2016.06.009

Tetrahedron: Asymmetry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Synthesis, structure and antiproliferative activity of chiral
polyamines based on a 2-azanorbornane skeleton
a
a,
b
b
b
⇑
_
_
´
´
Karolina Kaminska , Elzbieta Wojaczynska , Joanna Wietrzyk , Eliza Turlej , Agnieszka Błazejczyk ,
Robert Wieczorek c
a
_
´
Department of Organic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspianskiego 27, 50 370 Wrocław, Poland
b Department of Experimental Oncology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 R. Weigla St., 53 114 Wrocław, Poland
c Faculty of Chemistry, University of Wrocław, 14 F. Joliot-Curie St., 50 383 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of enantiopure 2-azanorbornane-based amines were prepared via a stereoselective aza-Diels–
Alder reaction and further modifications of the obtained cycloadduct. In particular, derivatives containing
two bicyclic moieties linked by NACACAN fragments were synthesized. Two dimeric derivatives exhib-
ited a significant antiproliferative activity against selected cell lines, comparable to cisplatin in certain
cases.
Received 30 April 2016
Accepted 15 June 2016
Available online xxxx
Ó 2016 Published by Elsevier Ltd.
1. Introduction
2-Azanorbornane derivatives have been recognized as valuable
rigid analogues of various piperidine and pyrrolidine alkaloids,
2-Azanorbornane (2-aza-bicyclo[2.2.1]heptane) and 2-azanor-
bornene are intrinsically chiral bicyclic compounds that have been
identified as versatile precursors for the synthesis of various enan-
tiopure derivatives and are useful in asymmetric synthesis and in
biomedical studies.1 These compounds are readily available via
stereoselective aza-Diels–Alder reaction betweens cyclopentadi-
ene and chiral aza-dienophiles, which are typically imines that
are formed from aldehydes and enantiopure amines (Fig. 1).2–5
Protocols have been developed for the preparation of cycloadduct
1 on the gram and even kilogram scale.6,7 The major isomer exo-
1 could be reduced to give alcohol 2, which could be further used
for the synthesis of various 2-azanorbornane8–10 and bridged aze-
pane derivatives,11 which are useful as chiral building blocks,
ligands and catalysts in asymmetric synthesis.
while 2-azabicyclo[2.2.1]heptane-3-carboxylic acid has been used
as a proline mimic in an approach to develop constrained oligopep-
tides of possible therapeutic utility.1,12–14 The bicyclic chiral
2-azanorbornane or 2-azanorbornene system has also been found
useful in the enantioselective preparation of cyclopentanoids. In
particular, 2-azabicyclo[2.2.1]hept-5-en-3-one (Vince lactam) has
been utilized in the synthesis of carbocyclic nucleoside analogues,
many of which exhibit significant antibiotic, antitumor and antiviral
activity (carbovir derivatives).15
Aliphatic polyamines, such as putrescine, spermidine and sper-
mine, are found in the cells of all living organisms.16 These com-
pounds are essential for cell growth and proliferation and
participate in diverse physiological processes, including the regula-
tion of gene expression and cell signaling. In their cationic (proto-
nated) form, polyamines are known to bind to DNA, RNA, proteins
and phospholipids. Since cellular division is associated with an
increased level of polyamines, these compounds are considered
biochemical markers of malignant tumors and other diseases.17,18
Synthetic analogues of these compounds have been shown to exhi-
bit antineoplastic activity against various tumor cell lines, both
in vitro and in vivo and are currently undergoing various clinical
trials for cancer treatment.19–21 The medical applications of
polyamines are more widespread;22 due to their coordination abil-
ities; polyamines are potent chelating agents that have been used,
for example, to treat iron overload.23 Various chiral diamines,
especially those bearing an NACACAN motif, have also been
successfully used in asymmetric synthesis.24–29
1. HIO4
CO2Et
OH
2.
Ph
1. H2, Pd(C),
K2CO3
NH2
H
H
Ph
N
COOEt
N
Ph
OH
OH
2. LiAlH4/THF
3.
2
1
exo-
major isomer
CO2Et
Figure 1. Synthesis of 2-azanorbornane derivatives.2–6,8
⇑
Corresponding author. Tel.: +48 71 3202410; fax: +48 71 3202427.
E-mail address: elzbieta.wojaczynska@pwr.edu.pl (E. Wojaczyn´ ska).
http://dx.doi.org/10.1016/j.tetasy.2016.06.009
0957-4166/Ó 2016 Published by Elsevier Ltd.
´
Please cite this article in press as: Kaminska, K.; et al. Tetrahedron: Asymmetry (2016), http://dx.doi.org/10.1016/j.tetasy.2016.06.009

´
K. Kaminska et al. / Tetrahedron: Asymmetry xxx (2016) xxx–xxx
2
Taking into account the utility of 2-azanorbornane as a versatile
both diastereomers were quantitatively reduced by LiAlH4 to yield
the desired amine (1S,3R,4R)-7, which exhibits spectroscopic char-
acteristics that are different from its ring-expanded isomer.11 Its
reaction with aldehyde 3 and subsequent reduction formed a
C2-symmetrical triamine 8 in 50% yield (Fig. 3). The secondary
amine group was substituted with alkyl chlorides (benzyl chloride
and triphenylmethyl chloride) and benzoyl chloride to afford the
corresponding tertiary amines 9 and 10 and amide 11, respectively
(45–70% yield). All new enantiopure compounds were fully
characterized by HRMS, IR, 1H and 13C NMR spectroscopy, and their
structures were further confirmed by 2D NMR spectroscopy. For
molecules of amines 5, 8–11 containing two connected bicyclic
subunits, an effective C2 symmetry is expected. This was the case
for triamines 8 and 9 since a single set of 1H NMR resonances as
well as 13C NMR signals were observed for these compounds.
However, the substitution of the central nitrogen atom with a trityl
or benzoyl group, or elongation of the linker joining the two
chiral platform and the ability of binding both to biomolecules
and various metal ions exhibited by polyamines, we decided
to combine these structural motifs in the molecules of new
hybrid ligands. Herein, we describe the synthesis of enantiopure
2-azanorbornane amine derivatives bearing one, two or three
NACACAN fragments, including those containing two bicyclic
units. We have also reported studies of their biological (anticancer)
activity.
2. Results and discussion
Enantiopure aldehyde exo-3 (Fig. 2), which was obtained by the
Swern oxidation of alcohol 2,30,31 served as a convenient starting
material for all syntheses. This compound has been previously
used to prepare various 3-substituted 2-azanorbornanes, including
Schiff bases, sulfur and phosphorus derivatives.31–34 Here, the reac-
tion of aldehyde 3 with (1,1-diphenyl)methylamine led to imine 4
in 72% yield, whereas the use of ethylenediamine resulted in
diimine containing two 2-azanorbornane fragments, which was sub-
sequently reduced with sodium borohydride to yield tetramine 5
(Fig. 2).
2-azanorbornane fragments, resulted in
a decrease in the
symmetry. For compounds 5 and 11, this is manifested by the
doubling of the number of spectroscopic resonances in 1H and
13C NMR spectra. When the temperature was raised to 330 K, only
one set of signals was observed for benzyl derivative 11, some
of which were significantly broadened. However, this increase of
molecular symmetry was not observed for tetramine 5 up to
330 K (a temperature limit of chloroform-d solvent). In the case
of trityl derivative 10, very broad resonances were found in 1H
and 13C NMR spectra recorded at room temperature. After raising
the temperature to 330 K, they became narrower but an obscured
multiplet structure of 1H NMR peaks indicated that the fast
exchange limit was not achieved. These observations suggest that
at room temperature, the lowest energy is achieved for the
conformations in which the two subunits are not equivalent.
DFT calculations performed on compounds 5, 7–11 illustrated
the significant differences in geometries of triamine 8, and its
derivatives bearing a substituent on the central nitrogen atom
9–11. Increasing the size and rigidity of this group significantly
changed the conformation of the CACANACAC chain linking the
two subunits from fully relaxed in compound 8 to bent in
compound 10 (Fig. 4). This is reflected in the decrease of the
distance of the chain ends (3-C atoms of the two 2-azanorbornane
fragments) from 4.95 Å 8 to 4.21 and 4.13 Å (benzoyl and benzyl
derivatives 11 and 9, respectively) and 3.88 Å (trityl-substituted
amine 10). Thus, substitution of the central nitrogen atom
introduced steric hindrance, which placed the two bicyclic
subunits closer to each other, which may result in the observed
differentiation of the NMR shifts.
Ph
H2N
Ph
Ph
Ph
(COCl)2
DMSO
N
Ph
N
O
2
Na2SO4,
CH2Cl2
N
H
Ph
4
3
72%
1. H2NCH2CH2NH2 (0.5 eq.),
Na2SO4, CH2Cl2
2. NaBH4, MeOH
N
Ph
N
H
2
5
52%
Figure 2. Preparation of compounds 4 and 5.
Another approach to dimeric C2-symmetrical systems utilizes
amine 8 based on its 2-azanorbornane skeleton. Our previous
attempt to prepare this compound by the nucleophilic substitution
of alcohol 2 with an azide followed by reduction resulted in ring
expansion and the formation of bridged azepane derivatives.11
Thus, a different synthetic strategy was adopted (Fig. 3). Aldehyde
3 was converted into oxime 6. When hydroxylamine hydrochloride
was applied in the presence of pyridine, a mixture of cis–trans iso-
mers was obtained in a 66:34 ratio. However, when pyridine was
replaced with triethylamine, the reaction proceeded with high
stereoselectivity, and the (E):(Z) ratio increased to 96:4. Isomer
separation was not necessary before the next reaction step because
Our DFT calculations do not explain the observed spectroscopic
differences between the benzyl and benzoyl derivatives 9 and 11.
The calculated structure of tetramine 5 did not show any deviation
of the expected C2 symmetry observed in the NMR spectra of this
compound. A possible explanation for this could be that not only
the structure of minimum energy but also the dynamic behaviour
of the molecules should be cosidered. The rigidity of C(O)-phenyl
fragment present in 11 may limit the ability of conformational
changes of this molecule.
NH2OH.HCl
Et3N
LiAlH4
THF
N
Ph
OH
N
Ph
3
N
NH2
7
6
90%, (E):(Z) = 96:4
75%
Three derivatives 5, 8, and 9 were sufficiently soluble in ethanol
for use in biological activity assays. These chiral polyamines were
tested for their antiproliferative activity toward cancer (colon, lung
and leukemia) and normal (human endothelial and mouse fibrob-
lastic) cell lines. The highest antiproliferative properties (compara-
ble to cisplatin in some cell lines) were exhibited by tetramine 5
and triamine 9 (Table 1). Compound 9 exhibited selectivity toward
cancer and endothelial cells; this compound also inhibited prolifer-
ation at concentrations 2–4 times lower than those that inhibit the
proliferation of the normal embryonic fibroblast BALB/3T3 cell
lines, which are recommended by various agencies that introduce
3
1. (1 eq)
2. NaBH4,
MeOH
RCl
N
Ph
N
Ph
N
N
H
Ph
K2CO3,
CH3CN
R
N
N
Ph
9: R = CH2Ph (70%)
8
50%
10:
R = CPh3 (64%)
11: R = COPh (45%)
Figure 3. Synthesis of compounds 7–11.
´
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´
3
K. Kaminska et al. / Tetrahedron: Asymmetry xxx (2016) xxx–xxx
Figure 4. Comparison of DFT-calculated structures of triamine 8 and its N-trityl derivative 10. Hydrogen atoms omitted for clarity. Difference of conformations of chains
linking the two subunits in both structures shown in red.
(1H, 500 MHz) spectrometers; in some cases, TMS was used as
the internal standard; otherwise, a residual solvent peak was used
Table 1
Antiproliferative activity of chiral polyamines 5, 8, and 9
a
as the reference. Optical rotations were measured using an Optical
Activity Ltd. Model AA-5 automatic polarimeter. High-resolution
mass spectra were recorded using a microTOF-Q and WATERS
LCT Premier XE instrument operating in the electrospray ionization
mode. Products were chromatographically separated on silica gel
60 (230–400 mesh; Merck). Thin layer chromatography was car-
ried out using silica gel 60 precoated plates (Merck).
Compound
Cell line/IC50
HLMEC
[l
g/ml]
A549
HT-29
MV4-11
BALB/3T3
5
8
9
31.8 1.2
NAb
4.1 0.05
2.2 0.5
3.5 0.4
50.6 3.5
2.5 0.2
2.9 0.6
3.2 0.06
30.9 3.6
3.1 0.3
23.8 1.6
NTc
2.9 0.4
0.2 0.1
34.0 5.6
NTc
10.5 5.4
0.7 0.3
Cisplatin
0.2 0.07
a
Data are presented as the IC50 [lM] mean standard deviation (SD). Ethanol did
not affect cell proliferation at the concentrations used.
b
4.2. Preparation of compounds
NA—not active at the concentrations used.
c
NT—not tested.
The aza-Diels–Alder cycloadduct exo-(1R,3R,4S)-1 was synthe-
sized by reacting cyclopentadiene with an iminium ion derived
from ethyl glyoxylate and (S)-1-phenylethylamine as previously
described.6,35 Compound 1 was reduced to alcohol 2 as described
previously.9,32,35 Compound 2 was oxidized under Swern condi-
tions to form (1S,3R,4R)-2-[(S)-1-phenylethyl]-2-azabicyclo[2.2.1]
heptan-3-al 3.30,31
new methods for testing toxicity. Therefore, we concluded that this
compound selectively inhibits the proliferation of cancer and
endothelial cells.
We also analyzed the cell cycle distribution of MV4-11 leuke-
mic cells after exposure to the two most active compounds. The
percentage of PI-positive (dead) cells in any of the analyzed sam-
ples did not exceed 4%. The tested compounds, even in concentra-
4.2.1. Synthesis of oxime 7
tions that exceeded the IC50 value (6 lg/ml for 9 and 50 lg/ml for
Aldehyde 3 was converted into oxime 6 according to a well-
established synthetic procedure. A mixture of 3 (1.10 g, 4.85 mmol),
hydroxylamine hydrochloride (0.84 g, 12 mmol, 2.5 eq.) and tri-
ethylamine (0.84 g, 5.4 mmol) was stirred in 15 mL of methylene
chloride for 24 h at room temperature. Saturated aqueous NaHCO3
(5 mL) and water (15 mL) were then added, and the aqueous phase
was extracted with dichloromethane (3 x 10 mL). The combined
organic extracts were evaporated to dryness, and the residue was
chromatographed on a silica gel column (n-hexane–ethyl acetate,
1:1 v/v), to yield 1.02 g of (E)-oxime 7 as a white solid.
5), did not significantly affect the cell cycle distribution. Only com-
pound 9 arrested the cell cycle in the S phase (20%) compared to
the ethanol control (17% of cells), although only to a small degree.
Cisplatin decreased the percentage of cells in the S stage and
increased the percentage in the G2M stage (data not shown). This
finding suggests that the mechanism of the action of the tested
compounds is independent of the cell cycle phase.
3. Conclusions
Among the newly obtained enantiopure chiral amines based on
the 2-azanorbornane skeleton, compounds 5 and 9 (a benzyl
derivative of 8) containing two bicyclic fragments exhibited an
antiproliferative activity against selected cancer cell lines. These
results show the utility of the new hybrid molecules combining
chiral 2-azanorbornane skeletons and amine (NACACAN) linkers
in biological activity studies.
4.2.1.1. (1S,3R,4R)-2-[(S)-1-Phenylethyl]-2-azabicyclo[2.2.1]hep-
tan-3-al oxime 6. Yield 90%, (E:Z) 96:4. For the (E) isomer:
Mp = 132 °C. [a]
20 = +16.9 (c 0.26, CH2Cl2). 1H NMR (300 MHz,
D
CDCl3) d = 1.17–1.39 (m, 3H), 1.31 (d, 3H, J = 6.5 Hz), 1.60–1.66
(m, 2H), 1.78–1.80 (m, 1H), 1.99–2.03 (m, 1H), 2.23–2.24 (m,
1H), 2.63 (d, 1H, J = 6.5 Hz), 3.54 (q, 1H, J = 6.5 Hz), 3.69 (s, 1H),
6.72 (d, 1H, J = 6.5 Hz), 7.35–7.17 (m, 5H) ppm. 13C NMR
(75 MHz, CDCl3) d = 22.3, 22.6, 29.6, 35.5, 43.4, 58.6, 61.2, 67.4,
127.5, 128.0, 128.3, 145.0, 154.3 ppm. IR: 1451 (AC@NA), 3195
(AOH) cmꢀ1. HRMS (ESI-TOF): m/z calculated for (C15H21N2O)+
([M+H]+) 245.1654; found 245.1645.
4. Experimental
4.1. General
4.2.2. Reduction of oxime 6 to amine 7
IR spectra were recorded using a Perkin Elmer System 2000 FTIR
spectrophotometer. 1H and 13C NMR spectra were measured on
Bruker Avance DRX 300 (1H, 300 MHz) or Bruker Avance 500
A modified literature procedure was used for this reaction.36
A
solution of oxime 6 (0.58 g; 2.4 mmol) in anhydrous THF (10 mL)
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K. Kaminska et al. / Tetrahedron: Asymmetry xxx (2016) xxx–xxx
4
was added dropwise to a suspension of LiAlH4 (0.23 g, 6.0 mmol) in
the same solvent (10 mL). The resulting mixture was stirred for
24 h at room temperature. Aqueous NaOH was then added to the
reaction mixture, which was then filtered and evaporated. The
solid residue was dissolved in 15 mL of CH2Cl2 and acidified with
1 M aqueous HCl. The separated aqueous phase was rendered alka-
line with 20% aqueous NaOH and extracted with CH2Cl2
(3 ꢁ 10 mL). The organic phases were collected, dried over Na2SO4
and evaporated, to yield crude amine 7, which was then chro-
4.2.3.2. N,N0-Bis{[(1S,3R,4R)-2-((S)-1-phenylethyl)-2-azabicyclo
[2.2.1]heptan-3-yl]methyl}ethan-1,2-diamine 5. Yellow oil. Yield
0.51 g (75%), [a]
20 = +41.1 (c 0.32, CH2Cl2); d = 1.19–1.44 (m, 8H),
D
1.30 (d, 3H, J = 6.5 Hz), 1.31 (d, 3H, J = 6.5 Hz), 1.47 (dd, 1H,
J1 = 12.0 Hz, J2 = 2.6 Hz), 1.57–1.63 (m, 3H), 1.70–1.76 (m, 3H),
1.80–1.85 (m, 2H), 1.90–2.02 (m, 5H), 2.10–2.17 (m, 3H), 2.40–
2.44 (m, 1H), 3.41 (q, 1H, J = 6.5 Hz), 3.43 (q, 1H, J = 6.5 Hz), 3.57
(s, 2H), 7.17–7.35 (m, 10H) ppm. 13C NMR (75 MHz, CDCl3):
d = 22.4, 22.5, 29.3, 35.45, 35.50, 41.1, 41.4, 49.0, 52.4, 54.4, 54.5,
58.6, 61.16, 61.20, 69.1, 69.2, 127.1, 127.2, 128.0, 128.1, 128.2,
128.3, 146.26, 146.34 ppm. IR (film): 3299, 3063, 3027, 2968,
2869, 1454, 1306, 1166, 754, 701 cmꢀ1. HRMS (ESI-TOF): m/z cal-
culated for [C32H47N4]+ ([M+H]+) 487.3801; found 487.3810.
matographed on
a silica column using chloroform–methanol
(90/10, v/v) as the solvent.
4.2.2.1. (1S,3R,4R)-2-[(S)-1-Phenylethyl]-3-aminemethyl-2-azabi-
cyclo[2.2.1]heptane 7. Yellow oil. Yield 0.41 g (75%), [a D
]
20 = ꢀ26.1 (c
0.23, CH2Cl2). 1H NMR (300 MHz, CDCl3) d = 1.22–1.41 (m, 3H), 1.35
(d, 3H, J = 6.6 Hz), 1.60–1.80 (m, 4H), 1.95–2.02 (m, 2H), 2.15–2.17
(m, 1H), 3.45 (q, 1H, J = 6.6 Hz), 3.61 (s, 1H), 7.22–7.37 (m, 5H)
ppm. 13C NMR (75 MHz, CDCl3) d = 22.4, 22.5, 29.5, 35.5, 39.9,
46.8, 58.8, 61.1, 72.0, 127.4, 128.1, 128.5, 145.5 ppm. IR: 3305,
3026, 2954, 2867, 1671, 1602, 1493, 1451, 1369, 1122, 761,
701 cmꢀ1. HRMS (ESI-TOF): m/z calculated for (C15H23N2)+ ([M
+H]+) 231.1861; found 231.1872.
4.2.3.3.
Bis-{[(1S,3R,4R)-2-((S)-1-phenylethyl)-2-azabicyclo
[2.2.1]heptan-3-yl]methyl}amine 8. Yellow oil. Yield 0.17 g
(50%), [
a]
D
20 = ꢀ13.9 (c 0.5, CH2Cl2); 1H NMR (500 MHz, CDCl3):
d = 1.13–1.24 (m, 6H), 1.27 (d, 6H, J = 6.5 Hz), 1.29–1.33 (m, 2H),
1.47–1.58 (m, 6H), 1.76 (dd, 2H, J1 = 9.1 Hz, J2 = 2.7 Hz), 1.87 (d,
2H, J = 4 Hz), 1.89–1.94 (m, 2H), 3.36 (q, 2H, J = 6.5 Hz), 3.52 (s,
2H), 7.20–7.27 (m, 10H) ppm. 13C NMR (125 MHz, CDCl3):
d = 22.3, 22.4, 28.9, 35.3, 40.6, 55.3, 58.6, 61.1, 69.3, 127.2, 128.1,
128.2, 145.9 ppm. IR (film) 3304; 2967, 2869, 1451, 1165,
700 cmꢀ1. HRMS (ESI-TOF): m/z calculated for [C30H42N3]+ ([M
+H]+) 444.3379; found 444.3380.
4.2.3. Preparation of imines and their reduction to amines
The aldehyde exo-(1S,3R,4R)-3 (0.34 g; 1.5 mmol) and (1,1-
diphenyl)methylamine (0.27 g; 1.5 mmol) were dissolved in dry
dichloromethane (25 mL). Anhydrous Na2SO4 was then added,
and the reaction mixture was stirred for 24 h at room temperature.
Sodium sulfate was removed by filtration under vacuum. After fil-
tration, water (10 mL) was added to the reaction mixture. The
organic phase was separated, and the aqueous phase was extracted
with dichloromethane (3 x 10 mL). The combined organic extracts
were washed with water and brine, dried (Na2SO4) and evaporated,
to yield imine 4, which was purified on a silica column using chlo-
roform–methanol (90/10 v/v) as the eluent.
A similar procedure was applied when reacting aldehyde 3 with
ethylenediamine (4.0 mmol of aldehyde and 2.0 mmol of diamine
were used), to yield a diimine, which was used without further
purification. A part of the crude product (0.37 g; 0.6 mmol) was dis-
solved in 10 mL of dry methanol; then, NaBH4 (0.037 g; 0.99 mol,
1.3 equiv) was added to the solution, which was then stirred for
24 h at room temperature. After evaporation of methanol, dichloro-
methane was added, and the organic layer was washed with water
and brine and dried over anhydrous Na2SO4. The solvent was evapo-
rated, and the resulting product 5 was purified by column chro-
matography using chloroform–methanol (70/30 v/v) as the eluent.
Aldehyde 3 was also reacted with amine 7 (4.4 mmol of both
reagents were used) in an analogous manner and the product
reduced in situ with sodium borohydride to yield tetramine 8
which was purified using column chromatography on silica.
4.2.4. Nucleophilic substitution of the secondary amine group
in exo-(1S,3R,4R)-8
Potassium carbonate (0.14 g, 1 mmol) was added to a solution
of exo-(1S,3R,4R)-8 (0.21 g, 0.48 mmol) and benzyl chloride
(0.07 mL, 0.6 mmol) in acetonitrile (15 mL), and the reaction mix-
ture was stirred for 48 h at room temperature. Chloroform
(15 mL) was then added, and the solution was washed with water
and brine. The organic layer was dried over anhydrous Na2SO4.
After removal of the solvent, the residue was chromatographed
on a silica column. Elution with chloroform/methanol (95/5 v/v)
afforded the tertiary amine 9. Compounds 10 and 11 were
prepared in a similar manner from 8 (0.30 g, 0.68 mmol) using
triphenylmethyl chloride (0.19 g, 0.68 mmol) and benzoyl chloride
(0.08 mL, 0.68 mmol), respectively.
4.2.4.1. N-Benzyl-bis-{[(1S,3R,4R)-2-((S)-1-phenylethyl)-2-azabi-
cyclo[2.2.1]heptan-3-yl]methyl}amine 9. Yellow oil. Yield 0.18 g
(70%), [a]
20 = +65.8 (c 0.38, CH2Cl2); 1H NMR (500 MHz, CDCl3):
D
d = 0.60 (d, 2H, J = 12.3 Hz), 1.07–1.13 (m, 4H), 1.27 (d, 6H,
J = 6.3 Hz), 1.30–1.40 (m, 4H), 1.57–1.61 (m, 2H), 1.77 (bs, 2H),
1.83 (d, 2H, J = 10.2 Hz), 1.91–1.93 (m, 2H), 2.01 (d, 1H,
J = 13.8 Hz), 2.16 (s, 2H), 2.66 (d, 1H, J = 13.8 Hz), 3.36 (q, 2H,
J = 6.2 Hz), 3.51 (s, 2H), 6.99 (d, 2H, J = 7.0 Hz), 7.13 (t, 1H,
J = 7.0 Hz), 7.18 (t, 2H, J = 7.0 Hz), 7.24–7.29 (m, 10H) ppm. 13C
NMR (75 MHz, CDCl3): d = 22.4, 22.6, 28.9, 34.9, 39.9, 58.7, 58.8,
59.5, 61.2, 67.4, 126.2, 127.1, 127.6, 128.0, 128.5, 128.8, 141.1,
146.0 ppm. IR (film): 3062, 3026, 2968, 2868, 1492, 1453, 1305,
700 cmꢀ1. HRMS (ESI-TOF): m/z calculated for [C37H48N3]+ ([M
+H]+) 534.3447; found 534.3558.
4.2.3.1. (E)-1,1-Diphenyl-N-{[(1S,3R,4R)-2-((S)-1-phenylethyl)-2-
azabicyclo[2.2.1]heptan-3-yl]methylene}methanamine 4. White
solid. Yield 0.43 g (72%), [
a
]
D
20 = ꢀ24.9 (c 0.42, CH2Cl2); 1H NMR
(500 MHz, CDCl3): d = 1.30 (d, 1H, J = 9.2 Hz), 1.35 (d, 3H,
J = 6.5 Hz), 1.41–1.52 (m, 2H), 1.64–1.70 (m, 1H), 1.74–1.80 (m,
1H), 2.01–2.07 (m, 1H), 2.38 (d, 1H, J = 3.9 Hz), 2.82 (d, 1H,
J = 5.0 Hz), 3.57 (q, 1H, J = 6.5 Hz), 3.73 (s, 1H), 4.99 (s, 1H), 6.81
(dd, 2H, J1 = 7.5 Hz, J2 = 2.0 Hz), 7.00 (tt, 1H J1 = 7.4 Hz,
J2 = 1.3 Hz), 7.10–7.31 (m, 10H), 7.34 (t, 2H, J = 7.6 Hz) ppm. 13C
NMR (125 MHz, CDCl3): d = 22.6, 23.0, 29.2, 36.2, 44.4, 58.7, 61.1,
71.6, 77.0, 126.6, 126.7, 127.0, 127.2, 127.6, 127.90, 127.95,
128.1, 128.2, 143.0, 143.5, 145.3, 170.2 ppm. IR (film): 3063,
3029, 2952, 2844, 1665, 1452, 1026, 745, 701 cmꢀ1. HRMS (ESI-
TOF): m/z calculated for [C28H31N2]+ ([M+H]+) 395.2487; found
395.2477.
4.2.4.2. N-1,1,1-Triphenylmethyl-bis-{[(1S,3R,4R)-2-((S)-1-pheny-
lethyl)-2-azabicyclo[2.2.1]heptan-3-yl]methyl}amine 10. White
solid. Yield 0.24 g (64%), [a]D
20 = +57.2 (c 0.6, CH2Cl2); 1H NMR
(500 MHz, CDCl3, 330 K): d = 0.85–1.15 (m, 2H), 1.16 (s, 6H), 1.32–
1.39 (m, 7H), 1.55 (s, 1H), 1.73–1.78 (m, 4H), 2.01 (s, 2H), 2.14 (s,
2H), 2.91 (s, 2H), 3.20 (s, 2H), 3.56 (s, 2H), 6.89–7.29 (m, 25H) ppm.
13C NMR (125 MHz, CDCl3, 330 K): d = 23.0, 23.2, 28.6, 35.6, 39.6,
56.3, 59.0, 61.2, 68.2, 78.1, 125.2, 126.3, 126.6, 127.86, 127.91,
130.4, 146.2 ppm. IR (KBr): 3469, 3056, 2965, 2937, 1490, 1447,
´
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K. Kaminska et al. / Tetrahedron: Asymmetry xxx (2016) xxx–xxx
760, 700 cmꢀ1. HRMS (ESI-TOF): m/z calculated for [C49H56N3]+ ([M
72 h of exposure to various concentrations of the tested agents.
For adherent cells, a sulforhodamine B (SRB) assay was performed,
and for leukemia cells, an MTT assay was performed, as previously
described.37
The results are presented as IC50 (inhibitory concentration 50)
values—the concentrations of the tested agents that inhibit the
proliferation of 50% of the cancer cell population.38 IC values were
calculated for each experiment separately, and mean values SD
are presented in the tables. Each compound was tested in triplicate
at each concentration in a single experiment, which was repeated 3
times.
+H]+) 686.4474; found 686.4451.
4.2.4.3. N-Benzoyl-bis-{[(1S,3R,4R)-2-((S)-1-phenylethyl)-2-azabi-
cyclo[2.2.1]heptan-3-yl]methyl} 11. White solid. Yield 0.21 g
(45%), [a]
20=+69.6 (c 0.66, CH2Cl2); 1H NMR (500 MHz, CDCl3):
D
d = 0.67–0.74 (m, 1H), 0.78–0.83 (m, 1H), 0.87–0.91 (m, 1H), 1.11
(d, 3H, J = 6.3 Hz), 1.15–1.42 (m, 6H), 1.23 (d, 3H, J = 6.1 Hz),
1.55–1.58 (m, 1H), 1.67–1.75 (m, 5H), 1.80–1.87 (m, 2H), 1.93
(dd, 1H, J1 = 13.9 Hz, J2 = 3.8 Hz), 2.60 (t, 1H, J = 12.4 Hz), 3.23 (q,
1H, J = 6.0 Hz), 3.34 (s, 1H), 3.40 (q, 1H, J = 6.0 Hz), 3.58 (s, 1H),
3.70 (t, 1H, J = 11.6 Hz), 7.09–7.11 (m, 2H), 7.27–7.32 (m, 7H),
7.37 (t, 4H, J = 7.2 Hz), 7.45 (d, 2H, J = 7.1 Hz) ppm. 13C NMR
(75 MHz, CDCl3): d = 22.3, 22.5, 23.8, 24.3, 27.8, 28.1, 34.7, 35.3,
37.5, 38.1, 45.0, 50.4, 59.2, 61.1, 61.2, 64.9, 65.2, 126.7, 127.3,
127.5, 128.17, 128.24, 128.3, 128.8, 137.3, 146.9, 147.4,
172.0 ppm. IR (film): 3436, 3028, 2969, 2872, 1632, 1425,
701 cmꢀ1. HRMS (ESI-TOF): m/z calculated for [C37H46N3O]+ ([M
+H]+) 548.3641; found 548.3641.
4.3.3. SRB assay
Cells were attached to the bottom of plastic wells by adding
cold 50% TCA (trichloroacetic acid, Sigma–Aldrich Chemie GmbH,
Steinheim, Germany) on top of the culture medium in each well.
The plates were incubated at 4 °C for 1 h and then washed five
times with tap water. Cellular material fixed with TCA was stained
with 0.1% sulforhodamine B (SRB, Sigma–Aldrich Chemie GmbH,
Steinheim, Germany) in 1% acetic acid (POCh, Gliwice, Poland) for
30 min. Unbound dye was removed by rinsing (4ꢁ) in 1% acetic
acid. Protein-bound dye was extracted with 10 mM unbuffered Tris
base (POCh, Gliwice, Poland), and the optical density at 540 nm
was determined using a computer-interfaced Synergy H4 pho-
tometer (BioTek Instruments, USA).
4.3. Biological activity tests
4.3.1. Cells
The HT-29 human colon cancer cell line was obtained from the
German Cancer Research Center (Deutsches Krebsforschungszen-
trum, DKFZ), Heidelberg, Germany. The cell line originated at the
Leibniz Institute DSMZ-German Collection of Microorganisms and
Cell Cultures, Braunschweig, Germany. The BALB/3T3 mouse
embryonic fibroblast, A549 human lung cancer, MV4-11 bipheno-
4.3.4. MTT assay
Twenty microliters of MTT solution (MTT: 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma–Aldrich Che-
mie GmbH, Steinheim, Germany; stock solution: 5 mg/ml)) was
added to each well, and the plates were then incubated for 4 h.
typic
B myelomonocytic leukemia and HLMEC human lung
microvascular endothelial cell lines were purchased from the
American Type Culture Collection (ATCC Rockville, Maryland,
USA). All cell lines are maintained at the Institute of Immunology
and Experimental Therapy (IIET), Wrocław, Poland.
MV4-11 leukemia and HLMEC endothelial cell lines were cul-
tured in RPMI 1640 medium (IIET, Wrocław, Poland), supple-
After the incubation was complete, 80 ll of lysing mixture was
added to each well (lysing mixture: 225 ml of dimethylformamide,
67.5 g of sodium dodecyl sulfate (Sigma–Aldrich Chemie GmbH,
Steinheim, Germany) and 275 ml of distilled water). The optical
densities of the samples were measured at 570 nm after 24 h using
a computer-interfaced Synergy H4 photometer (BioTek Instru-
ments, USA).
mented with 2 mM
L-glutamine, 1.5 g/L sodium bicarbonate,
4.5 g/L glucose (all were obtained from Sigma–Aldrich Chemie
GmbH, Steinheim, Germany) and 10% fetal bovine serum
(Thermo-Fisher Scientific Oy, Vataa, Finland). HT-29 and A549 cells
were cultured in RPMI 1640+Opti-MEM (1:1) (both were obtained
from IIET, Wrocław, Poland), and BALB/3T3 cells were cultured in
Dulbecco medium (IIET, Wrocław, Poland), supplemented with
4.3.5. Cell cycle assessment based on DNA content
Based on the IC50 results, two concentrations of chemicals were
chosen to study the effect of the chemicals on the cell cycle. The
tests were performed using the MV4-11 cell line. Approximately
0.2 ꢁ 106 cells/well were plated in 24-well plates; after 24 h of
2 mM L-glutamine (Sigma–Aldrich Chemie GmbH, Steinheim, Ger-
many) and 5% fetal bovine serum (Thermo-Fisher Scientific Oy,
Vataa, Finland). The HT-29 cell culture was supplemented with
1.0 mM sodium pyruvate (Sigma–Aldrich Chemie GmbH, Stein-
heim, Germany). All culture media were supplemented with
incubation, the cells were treated with compounds 9 (6
l
g/ml;
3 lg/ml) and 5 (50 lg/ml; 25 lg/ml) for 72 h. After incubation,
the cells were washed twice with phosphate-buffered saline
(PBS, IIET PAN, Wrocław) and then adjusted to a density of
1 ꢁ 106/ml and then fixed for 24 h in 70% ethanol (POCh, Gliwice)
at ꢀ20 °C. The cells were then washed twice in PBS and incubated
100 units/ml penicillin and 100 lg/ml streptomycin (both were
obtained from Polfa Tarchomin S.A., Warsaw, Poland). All cell lines
were grown at 37 °C under a humidified atmosphere containing 5%
CO2.
with RNAse (50 lg/ml, Fermentas, Germany) at 37 °C for 1 h. The
cells were stained for 30 min with propidium iodide (50 lg/ml,
Sigma–Aldrich Chemie GmbH, Steinheim, Germany) at 4 °C, and
the cellular DNA contents were measured using BD LSR Fortessa
II, BD, San Jose, USA. The percentage of stained cells in each cell
cycle phase was determined using ModFit LT (version 3.2, devel-
oped by Verity Software House, USA) and Flowing Software (ver-
sion 2.5.1 (freeware), developed by Perttu Terho, Turkey).
4.3.2. In vitro anti-proliferative assay
The compounds used in this experiment were dissolved in
99.8% ethanol (POCh, Gliwice, Poland), which also served as the
solvent control. Stock solutions of the test compounds were pre-
pared in RPMI 1640+Opti-MEM (1:1) (both were obtained from
IIET, Wrocław, Poland) at a concentration of 10 mg/ml and then
diluted in the same culture medium to obtain 100, 10, 1 and
4.4. DFT calculations
0.1 lg/ml final concentrations. Cisplatin (Accord Healthcare Lim-
ited, North Harrow, Middlesex, United Kingdom) was used as a
positive control.
Twenty-four hours before the tested compounds were added,
the cells were plated in 96-well plates (Sarstedt, Germany) at a
density of 1 ꢁ 104 cells per well. The results were measured after
Full geometry optimizations were performed by DFT with CAM-
B3LYP hybrid functional39 using 6-311G(d) basis set, the NMR
shielding tensors were calculated with Gauge-Independent Atomic
Orbital (GIAO) method.40 All calculations were done with Gaussian
09 C.01 suite of programs.41
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Please cite this article in press as: Kaminska, K.; et al. Tetrahedron: Asymmetry (2016), http://dx.doi.org/10.1016/j.tetasy.2016.06.009

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6
K. Kaminska et al. / Tetrahedron: Asymmetry xxx (2016) xxx–xxx
19. Bergeron, R. J.; McManis, J. S.; Liu, C. Z.; Feng, Y.; Weimar, W. R.; Luchetta, G. R.;
Acknowledgments
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37, 3464–3476.
Financial support from the Foundation of Polish Science (VEN-
TURES/2013–11/4) and from Wrocław Centre of Biotechnology
(the Leading National Research Centre programme (KNOW) for
years 2014–2018) is gratefully acknowledged.
20. Hahm, H. A.; Dunn, V. R.; Butash, K. A.; Deveraux, W. L.; Woster, P. M.; Casero,
R. A., Jr.; Davidson, N. E. Clin. Cancer Res. 2001, 7, 391–399.
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compounds 4–11) associated with this article can be found, in the
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