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receptors (e.g., in activation of human liver X receptors) [6], and in
their capability to be modified by synthetic methods [7–9].
Aminomethylpyridines and related heteroaromatic amines,
including numbers of their derivatives, and the derived N-oxides,
had already been investigated for their pharmacological activity
[10–12], as markers of solid tumors [13] or agents in supramolec-
ular self-assembly [14]. Aromatic and tertiary amine N-oxides
were subjects of a comparative study for their potential action as
DNA intercalators as bioreductive prodrugs [15]. Potential of sev-
eral estrone and estradiol conjugates to form cis-dichloroplati-
num(II) complexes have recently been reported [16].
Lipophilic derivation of aminomethylpyridines is expected to
result in obtaining conjugates, bearing ester and amide bond in
each molecule. Their importance consists in enabling transporta-
tion of potentially biologically active compounds through biomem-
brane and they often form cationic immune-stimulating
complexes [17].
regular commercial sources in analytical grade and the solvents
were purified by general methods before use.
2.2. Synthetic protocol I: 4-[(3b)-lanosta-8,24-dien-3-yloxy]-4-
oxobutanoic acid (2) and 4-[(3b)-cholest-5-en-3-yloxy]-4-
oxobutanoic acid (12)
Succinic anhydride (14.7 mmol; 1.57 equiv) and DMAP
(1.584 mmol, 0.17 equiv) was added to a solution of (3b)-lanosta-
8,24-dien-3-ol (1) or (3b)-cholest-5-en-3-ol (11) (9.37 mmol) in
pyridine (28 mL). The reaction mixture was stirred over 7 days at
r.t. After stopping the reaction, the resulting mixture was poured
onto ice, and hydrochloric acid was added to adjust pH = 7, ex-
tracted with chloroform, and dried over sodium sulfate. Evapora-
tion of the solvent gave a solid which was purified by column
chromatography, affording the products (2) or (12) in 79% or 96%
yield, respectively, and with >99.9% analytical purity (Schemes
1–2). The analytical data of the products 2 and 12 are presented
in the Supplementary material.
Based on the above referenced facts, a clear connection exists
between the selected sterols, cholesterol and lanosterol, which
have been involved in the investigation focused on designing novel
potential cytotoxic agents. The objectives of this investigation have
consisted in (a) designing and synthesizing a series amides derived
2.3. Synthetic protocol II: preparation of 3–10 and 13–20
from lanosterol hemiesters and cholesterol hemiesters with
a
-, b-
(a) 1-(Pyridin-2-yl)-, 1-(pyridin-3-yl)- and 1-(pyridin-4-
yl)methanamine (21–23), their N-oxides (33–35) or 1-[4-(1-oxido-
pyridin-2-yl)phenyl]methanamine (36) (0.28 mmol; 1 equiv), DCC
(0.34 mmol; 1.2 equiv) and DMAP (0.085 mmol; 0.3 equiv) were
added to a solution of 2 or 12 (0.28 mmol) in dichloromethane
(3 mL), the reaction mixture was stirred for 1–7 days at r.t., and
then evaporated. The obtained residue was purified by chromatog-
raphy, affording the products 3–5, 7–10, 13–15 and 17–20 in the
yields 42–85%, and with >99.9% analytical purity (Schemes 1–2).
(b) T3P (0.16 mL; 0.27 mmol; 2 equiv) was added to a solution
of 2 or 12 (0.13 mmol) and 1-[4-(1-pyridin-2-yl)phenyl)methan-
amine hydrochloride (24) (0.16 mmol; 1.2 equiv) in pyridine
(2 mL). The reaction mixture was stirred for 3 days at r.t., then an
additional amount of T3P (0.39 mmol; 3 equiv) was added, and
stirring continued for an additional 3 h. The reaction mixture was
washed with a saturated solution of sodium bicarbonate, extracted
with chloroform and dried over sodium sulfate. Evaporation of the
solvent afforded a solid which was purified by column chromatog-
raphy, and gave the products 6 or 16 in the yields 87% or 63%,
respectively, and with >99.9% analytical purity (Schemes 1–2).
The analytical data of the products 3–10 and 13–20 are presented
in the Supplementary material.
and -aminomethylpyridines and their N-oxides, and (b) perform-
c
ing introductory cytotoxicity tests of the target compounds.
2. Experimental
2.1. General
The 1H NMR and the 13C NMR spectra were recorded on a Bru-
ker AVANCE 600 MHz spectrometer at 600.13 and 150.90 MHz in
CDCl3 using tetramethylsilane (d = 0.0) as internal reference. 1H
NMR data are presented in the following order: chemical shift (d)
expressed in ppm, number of protons, multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; b, broad), coupling con-
stants in Hertz, proton position in the molecule. For unambiguous
assignment of both 1H and 13C signals 2D NMR H,C-gHSQC and
gHMBC spectra were measured using standard parameters sets
and pulse programs delivered by producer of the spectrometer.
Infrared spectra were measured with a Nicolet 205 FT-IR spectrom-
eter. Mass spectra were measured with a waters ZMD mass spec-
trometer in a positive ESI mode and for some cases in negative
ESI mode. Analytical HPLC was carried out on a TSP (Thermosepa-
ration Products, USA) instrument equipped with a ConstaMetric
4100 Bio pump and a SpectroMonitor 5000 UV DAD. The analyses
of the products were performed on a reverse phase Nucleosil 120-5
C18 column (250 ꢀ 4 mm; Watrex, Czech Republic) using a meth-
2.4. Synthetic protocol III: FMOC-protected aminomethylaromates 25–
28
A solution of 9H-fluoren-9-ylmethyl carbonochloridate (FMOC-
Cl; 9.24 mmol; 1 equiv) in 1,4-dioxane (23.9 mL) was slowly added
to a stirred and ice cooled solution of 21–24 (9.25 mmol) in 1,4-
dioxane (12 mL) and 10% solution of sodium carbonate (25 mL),
and stirred overnight. The reaction mixture was washed with
water, extracted with chloroform, dried over sodium sulfate and
the solvent was evaporated. The obtained residue was purified
by chromatography, affording the products 25–28 in the yields
79%, 76%, 58% and 78%, respectively (Scheme 3). The analytical data
of the products 25–28 are presented in the Supplementary
material.
anol/water mixture (9:1, v/v) as mobile phase at 0.5–1.0 mL minꢁ1
.
The eluate was monitored at 220, 254, and 275 nm, and the UV
spectra were run from 200 to 300 nm. ACD/Labs software, ACD/
logD DB, version 12.01, was used for calculation of solubility, par-
tition coefficient (logP) and distribution coefficient (logD) of the
prepared compounds. Chemical names of the compounds used or
prepared in this work were generated according to the rules given
in the ACD/Labs software, and are based on general nomenclature
rules. TLC was carried out on silica gel plates (Merck 60F254) and
the visualization was performed by both, the UV detection, spray-
ing with the methanolic solution of phosphomolybdic acid (5%) fol-
lowed by heating. For column chromatography, silica gel 60
(0.063–0.200 mm) from Merck was used, and chloroform/metha-
nol mixtures (80:1–40:1) were employed as mobile phases. The
purity of the prepared compounds was checked by crystallization,
if possible (melting points are presented), and by HPLC, NMR and
MS analysis. All chemicals and solvents were purchased from
2.5. Synthetic protocol IV: FMOC-protected aminomethylaromate N-
oxides 29–32
A solution of peracetic acid (39%, 0.808 mmol, 3 equiv) in acetic
acid was added to a solution of 25–28 (0.272 mmol) in acetic acid
(2 mL). The mixture was heated to 80 °C for 2.5–4 h, and then