Synthesis, antimicrobial and cytotoxicity evaluation of new cholesterol congeners

Article abstract of DOI:10.3762/bjoc.11.208

3β-Azidocholest-5-ene (3) and (3β)-3-(prop-2-yn-1-yloxy)cholest-5-ene (10) were prepared as substrates to synthesize a variety of three-motif pharmacophoric conjugates through CuAAC. Basically, these conjugates included cholesterol and 1,2,3-triazole moie

Full text of DOI:10.3762/bjoc.11.208

Synthesis, antimicrobial and cytotoxicity evaluation of new
cholesterol congeners
Mohamed Ramadan El Sayed Aly*1,2,§, Hosam Ali Saad1,3
and Shams Hashim Abdel-Hafez1,4
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2015, 11, 1922–1932.
1
Chemistry Department, Faculty of Science, Taif University,
1974-Hawyah-Taif, Kingdom of Saudi Arabia, 2Chemistry
Department, Faculty of Applied Science, Port Said University,
2522-Port Said, Egypt, 3Chemistry Department, Faculty of Science,
doi:10.3762/bjoc.11.208
2
Received: 12 June 2015
4
Accepted: 28 September 2015
Published: 16 October 2015
Zagazig University, Zagazig, 44511, Egypt and 4Chemistry
Department, Faculty of Science, Assuit University, 71516-Assuit,
Egypt
The article is dedicated to the Institute Curie team Prof. Monneret,
Dr. Florent and Dr. Johannes.
Email:
Mohamed Ramadan El Sayed Aly* - mrea34@hotmail.com
Associate Editor: K. N. Ganesh
*
Corresponding author
© 2015 Aly et al; licensee Beilstein-Institut.
License and terms: see end of document.
§
Tel.: +966 (0)540731939; +20 (0)100 507 3049
Keywords:
antimicrobial; chalcone; cholesterol; cytotoxicity; glycoside; triazole
Abstract
3
β-Azidocholest-5-ene (3) and (3β)-3-(prop-2-yn-1-yloxy)cholest-5-ene (10) were prepared as substrates to synthesize a variety of
three-motif pharmacophoric conjugates through CuAAC. Basically, these conjugates included cholesterol and 1,2,3-triazole
moieties, while the third, the pharmacophore, was either a chalcone, a lipophilic residue or a carbohydrate tag. These compounds
were successfully prepared in good yields and characterized by NMR, MS and IR spectroscopic techniques. Chalcone conjugate 6c
showed the best antimicrobial activity, while the lactoside conjugate 27 showed the best cytotoxic effect in vitro.
Introduction
Cholest-5-en-3β-ol (cholesterol, 1) is an amphiphilic-like Cholesterol gives eukaryotic membranes sufficient mechanical
steroidal constituent of eukaryotic cell membranes. It acts as stiffness against cationic selective antimicrobials (CSAs) such
fluidity buffer and it is essential for membrane integrity and as antimicrobial peptides (AMPs) [4] and ceragenins I
permeability. Besides, it is a substrate for the biosynthesis of (Figure 1) [5]. These CSAs selectively bind to the over
steroidal hormones, bile acids and vitamin D. Pathological expressed negatively charged peripheral phospholipids on the
accumulation of oxygenated cholesterol (oxysterol) metabolites internal bacterial cell membranes. Following membrane associ-
contributes to the prognosis of major chronic diseases. Choles- ation, deformation occurs causing bilayer destabilization and
terol is completely absent in prokaryotic organisms [1-3].
cell lysis [6]. According to this mechanism, synthetic polycar-
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Beilstein J. Org. Chem. 2015, 11, 1922–1932.
bonates arising from organocatalytic ring-opening polymeriza- build new membranes [10]. This emerging propensity of
tion of cholesterol monomers were reported to create self- nascent cancer cells for cholesterol uptake is an attractive target
assemblies possessing high interior charge density and wide to use cholesterol as vehicle to increase the bioavailability of
spectrum antimicrobial activity [6]. Interestingly, the causative anticancer drugs. Thus, SuberAniloHydroxamic acid–choles-
vector of human gastritis and peptic ulcer Helicobacter pylori is terol conjugates (SAHA–cholesterol) [11], cholesterol-based
known to elevate serum cholesterol levels in infected patients. charged liposomes encaging doxorubicin [12] or curcumin [13]
This bacterial strain elevates the serum cholesterol levels and showed higher activity compared with the native drugs. Syn-
involves a specific enzyme known as cholesterol-α-glucosyl- thetic coumarin-caged cholesterol derivatives, for instance II,
transferase to glycosylate cholesterol via α-glycosidic linkage were triggered to release bioactive coumarines by photolysis at
and incorporates it into its cytoplasmic membrane. In this way it 350 nm [14]. Dendrogenin A (DDA, III) is a natural metabolite
boosts resistance to host immune defense and antibiotics as well in healthy mammals. It arises from conjugation of 5,6α-epoxy-
[
5,7].
cholesterol (5,6α-EC) with histamine. In vitro studies showed
that DDA induced tumor cell re-differentiation and death. This
In another case, eukaryotic cell membranes are supported by a explains why it is down-regulated during carcinogenesis and
membrane-associated cholesterol efflux regulatory protein opens the door for nucleophilic addition of amines to 5,6α-EC
(
CERP). This protein, also known as ABCA1, is a major regu- as a new lead for developing potential anticancer drugs [15].
lator of cellular cholesterol [8]. Synthetic BODIPY–cholesterol Apart from antimicrobial and antiproliferative activities of
conjugates were reported as probes for visualization of intracel- cholesterol derivatives, other pharmacologic activities were
lular cholesterol pools and for monitoring cholesterol efflux reported for them. Thus, cholesterol-conjugated C-peptides, for
from cells to extracellular receptors [9]. ABCA1 plays an example IV, are potent inhibitors of the Ebola virus glycopro-
inevitable role in the resistance against tumorgenesis through tein-mediated cell entry [16], while cholesterol-derived amines
depletion of cholesterol from cells under cancer threat, where exhibited a strong antiviral activity against the influenza A virus
cancer onset requires elevated intracellular cholesterol levels to (IFV). These amines were able to disrupt the cholesterol-rich
Figure 1: Structure of ceragenin (CSA-8) and selected cholesterol conjugates.
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Beilstein J. Org. Chem. 2015, 11, 1922–1932.
lipid envelope and inactivate viral invasion [17]. Cholesterol- bromine atom of compound 2 with the N3 group was ensued by
based hydrazones exhibited insecticidal activity against the refluxing with NaN3 in dry DMF to afford 3β-azidocholest-5-
larval stage of Mythimna separate (Walker) [18]. ene (3) after inversion of the configuration again at the C-3
Cholesterol–carbamate conjugates, for instance (3β)-cholest-5- carbon. The product could be isolated in good yield and the IR
en-3-yl (2-aminoethyl)carbamate (V), were used to prepare spectrum showed the N3 stretching as medium band at
nontoxic unilamellar vesicles as nanocarriers for gene delivery 2081 cm−1. Other methods for related syntheses were reported
into Neuro2A cells, which are involved in neurodegenerative in [33,34].
diseases [19]. Also, the involvement of cholesterol metal ion
complexes in Alzheimer’s disease was reviewed [20]. Choles- The target cholesterol–chalcone conjugates 6a–c and 7a,b were
terol glycosides are known for their immunostimulant activities prepared by reacting 3β-azidocholest-5-ene (3) with propargy-
[
21]. Finally, the ability of cholesterol derivatives to self- lated chalcones 4a–c and 5a,b [24] under CuAAC conditions
assembly and gelation as supramolecular gels was reviewed [35]. The reactions proceeded fairly in gently refluxing THF/
22]. They are beneficially applicable in materials science, reac- H2O mixture containing L-ascorbic acid as reducing agent and a
[
tion media, sensing and responsive materials, energy supply, catalytic amount of CuSO4·5H2O.
biomedicine, and tissue engineering [23].
The 13C NMR spectra of this series showed the C=O signal at δ
In light of this emerging propensity of cholesterol-based archi- values within the range of 187–188 ppm, while the 1H NMR
tectures to assimilate a plenty of pharmacological activities, spectra showed the trans configuration of the enone moiety due
cholesterol was propargylated, then reacted with azido-modi- to the high coupling constant of Jα,β 15.6 Hz, with the β-proton
fied quinoline and glucopyranosyl derivatives as part of a being more deshielded than the α-proton. The OCH2 signal was
previous study [24] to discover new antimicrobial and cyto- clearly observed in all derivatives at δ ≈ 5.30 ppm. On the other
toxic lead structures. Cholesterol conjugate VI (Figure 1) arose hand, the olefinic H-6 of cholesterol was observed at
from this consideration to be more active than ampicillin δ ≈ 5.40 ppm. The 1H NMR spectra of these compounds also
against the Gram-negative bacterial strain Escherichia coli showed the CH3-25 and CH3-26 signals of cholesterol as
(
ATCC 11775) and the Gram-positive bacterial strain Staphylo- doublets at δ ≈ 0.86 and 0.87 ppm with a coupling constant of
coccus aureus (ATTC 12600), while its antifungal activities J = 3.0 Hz. The CH3-21 was observed as doublet nearby the
against the filamentous fungal strain Aspergillus flavus (Link) previous signals, while the CH3-18 singlet was the most
and the yeast forming fungal strain Candida albicans (ATCC shielded at δ ≈ 0.85 ppm in all spectra. These five 1H NMR
7
102) were moderate compared with amphotericin B in vitro. In signals seemed to be a NMR identity fingerprint region of
the cytotoxicity study, this derivative was the most cytotoxic cholesterol. All these spectral data, besides the recorded mass
one against the prostate cancer PC3 cell line but it was 2.3 fold peaks at m/z values corresponding to the exact molecular weight
less active than doxorubicin in vitro. Therefore, this article of each derivative supported these azide–alkyne cycloadditions.
describes the synthesis of analogues of VI with different lipid,
glycon and chalcone [25,26] tags to assay and evaluate their in The second set of cholesterol conjugates (Scheme 2 and
vitro antimicrobial and cytotoxic activities against the above Scheme 3) was prepared by CuAAC of (3β)-3-(prop-2-yn-1-
mentioned microbial organisms and the prostate cancer PC3 cell yloxy)cholest-5-ene (10) with azidoalcanols 9a,b [24] and
line. It is worth mentioning that the bacterial [27,28] and fungal 3β-azidocholest-5-ene (3). These investigations aimed to
[
29,30] strains in this consideration were elected as they repre- address whether the terminal surface recognition glycon tag was
sent the main microbial classes for our in vitro antimicrobial necessary to stimulate the biological activity of triazolocholes-
evaluation. On the other hand, prostate cancer was considered terol [24] or just an alternative unique OH group, as in conju-
because it is highly prevalent malignancy and on the second gates 11a,b, or even without it as in derivatives 12 and 13, can
place in the list of cancer-related deaths due to its high retain its activity. Particularly, hydroxyalkyl-1,2,3-triazoles
metastatic potential [31].
were reported as valuable pharmacophores [36].
Results and Discussion
Chemistry
The products were isolated in good yields and the H-5 signal of
triazole (1H NMR) could be observed as a singlet at
Cholest-5-en-3β-ol (1) was activated as bromide in very good δ ≈ 7.5 ppm. Compound 11a was further converted into the
yield under Appel conditions [32], which means treatment with corresponding bromo derivative 12 in good yield under the
CBr4/PPh3 to afford 3α-bromocholest-5-ene 2 due to inversion same conditions used to prepare compound 2. This step aimed
of the configuration at the C-3 carbon (Scheme 1). The O–Hstr to have an alkylating probe that might target nucleic acids or
band of 1 disappeared upon this step. An SN2 substitution of the proteins in the tested biological systems.
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Beilstein J. Org. Chem. 2015, 11, 1922–1932.
Scheme 1: Reagents and conditions: (a) CBr4, PPh3, DCM (74%); (b) NaN3, DMF, 100 °C (63%); (c) CuSO4·5H2O, L-ascorbic acid (L-AsAc), THF/
H2O [6a, R = H (40%); 6b, R = OMe (41%); 6c, R = NMe2 (68%); 7a, X = O (47%); 7b, X = S (60%)].
The 1,2,3-triazole-bridged bicholesterol 13 (Scheme 3) was enzymes involved in processing this valuable metabolite. There-
prepared in excellent yield. The H-5 signal of triazole fore, triazolocholesterol–glucopyranosylamine conjugates 16,
(
1H NMR) also was observed as singlet at δ = 7.78 ppm 17 and 20 (Scheme 4) were prepared to compare the pharmaco-
confirming the cycloaddition of derivatives 3 and 10.
logical effects of the modification of the D-glucopyranose
moiety in VI (Figure 1) as glucosamine in different forms and
D-Glucosamine is an essential constituent of many naturally with a hexyl spacer. Retaining the dimethylmaleoyl (DMM)
occurring oligosaccharides such as bacterial and fungal cell group in targets 16 and 20 was based on the finding that
walls. Mainly, it is available as N-acetylglucosamine in β-glyco- NDMM-protected phosphatidylcholine showed better antiprolif-
sidic linkages (β-D-GlcNAc) [37]. Chitinases are special erative activity than its natural hydrochloride congener [38].
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Beilstein J. Org. Chem. 2015, 11, 1922–1932.
Scheme 2: Reagents and conditions: (a) NaN3, DMF, 100 °C (9b, 47%); (b) CuSO4·5H2O, L-AsAc, THF/H2O [11a, n = 4 (90%); 11b, n = 9 (67%)];
(c) CBr4, PPh3, DCM (59%).
Scheme 3: Reagents and conditions: CuSO4·5H2O, L-AsAc, THF/H2O (96%).
Thus, to prepare these targets glucosyl donor 14 [39] was conjugate 16 in 84% yield without affecting the DMM group.
coupled with cholest-5-en-3β-ol (1) as glycosyl acceptor in the Despite, the two C=O groups could not be seen with certain at
presence of catalytic TMSOTf as promoter to afford 15 in 74% δ ≈ 174.00 ppm (13C NMR), the two maleimide CH3 groups
yield. The large anomeric coupling constant (J1,2 = 8.4 Hz) of were observed at δ = 8.80 ppm as a proof of structure.
the pyranoside moiety at δ = 5.30 ppm ensured the β-configur-
ation of this glycoside.
Substitution of the DMM group with an acetyl group was
performed under standard conditions, i.e., treatment with NaOH
Deacetylation of intermediate 15 under Zémplen conditions, for ring opening [39], HCl at pH 5 for amide cleavage,
i.e., catalytic NaOMe in MeOH [40], safely afforded the target peracetylation and then O-deacetylation. Under these condi-
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Beilstein J. Org. Chem. 2015, 11, 1922–1932.
Scheme 4: Reagents and conditions: (a) 1, TMSOTF, CH3CN, rt (74%); (b) NaOMe, MeOH (84%); (c) NaOH; HCl (pH 5); Ac2O/Pyr; NaOMe/MeOH
(37%); (d) 9a, TMSOTf, DCM (71%); (e) 10, CuSO4·5H2O, L-AsAc, THF/H2O (67%); (f) NaOMe, MeOH (75%).
tions, compound 17 was prepared in 37% yield. The C=O signal tag compared with glucose as previously investigated in the
13C NMR) was observed at δ = 183.10 ppm with concurrent case of VI [24]. Thus, coupling of glycosyl donor 21 [41] with
(
disappearance of the maleimide CH3 signal. A peak was acceptor 9a afforded maltoside 22 in low yield (Scheme 5).
observed at m/z value corresponding to the exact molecular CuAAC of substrate 22 with 10 yielded derivative 23 in 62%
mass.
yield. The 1H NMR showed that the B ring of the maltose
moiety was α-configurated at the glycosidic center (H-1B at
Conjugate 20 was prepared under similar conditions as δ = 5.40 ppm, J1,2 = 4.2 Hz (see Scheme 5 compound 21 for the
employed for the synthesis of compound 17. Thus, coupling of assignment of rings A and B of the maltose moiety) and the A
azidohexanol 9a with trichloroacetimidate 14 afforded the inter- ring β-configurated (H-1A at δ = 4.49 ppm, J1,2 = 7.8 Hz). The
mediate β-glycoside 18 (J1,2 = 8.4 Hz at δ = 5.18 ppm) in 71% traizole H-5 was observed as singlet at δ = 7.52 ppm, while the
yield. CuAAC of derivative 18 with compound 10 afforded cholesterol CH3 groups fingerprint signals were observed in the
compound 19 in 67% yield. The H-5 proton of the triazole upfield region of the spectrum.
moeity was observed at δ = 7.49 ppm which confirms a
successful cycloaddition step. Deacetylation of 19 afforded Deacetylation of compound 23 smoothly afforded the target
target spacer linked conjugate 20 in 75% yield. Unlike com- conjugate 24 in 78% yield. Finally, compound 28 (Scheme 6)
pound 16, the two C=O 13C NMR signals of conjugate 20 were was attempted to be prepared to investigate the cytotoxicity of a
clearly observed at δ = 174.28 ppm.
lactose scaffold with a cholesterol moiety at the C-3 carbon of
the B ring of the lactose. This is because chemically modified
Then, attention was given to prepare conjugate 24 (Scheme 5). 3β-lactosides were emerged as potential galectin-3 inhibitors.
This is to investigate the pharmacological effects of the maltose Galectin-3 is a member of the protein family known as galectins
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Beilstein J. Org. Chem. 2015, 11, 1922–1932.
Scheme 5: Reagents and conditions: (a) 9a, TMSOTF, DCM, rt (19%); (b) 10, CuSO4·5H2O, L-AsAc, THF/H2O (62%); (c) NaOMe/MeOH (78%).
A: Ring A of the maltose moiety, B: Ring B of the maltose moiety.
Scheme 6: Reagents & conditions: (a) Propargyl bromide, NaH, Et2O/DMF (quant. for both 26 and 30); (b) 3, CuSO4·5H2O, L-AsAc, THF/H2O (89%
for 27 and 74% for 31); (c) H2, Pd/C 10%, MeOH. A: Ring A of the lactose moiety, B: Ring B of the lactose moiety.
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Beilstein J. Org. Chem. 2015, 11, 1922–1932.
and this subfamily is believed to be involved in tumorigenesis Both conjugates were prepared in very good yields. Probe 38
[
42,43]. To this endeavor, 3β-O-propargylated lactose deriva- was acetylated to confirm the regioselectivity at the C-4 pos-
tive 26 was reacted with compound 3 to afford intermediate ition of the lactose B ring [46].
triazole bridged conjugate 27 in 89% yield. This intermediate
was attempted to be debenzylated by treatment with H2 and The 1H NMR of compound 39, thus, showed the H-4 proton of
Pd/C 10% but the benzyl groups could not be removed under the lactose B ring discriminated at δ = 5.46 ppm with a very
these conditions.
small coupling constant due to a weak equatorial–diaxial
coupling. Compounds 36 and 38 were subjected to reductive
For comparison reasons, derivative 31, that has the cholesterol debenzylation. While, compound 38 could be debenzylated in
moiety at the C-4 carbon of the B ring of the lactose moiety, excellent yield, quinoline derivative 36 could not be debenzy-
was prepared by CuAAC of substrate 30 with 3 to afford inter- lated even in the presence of Pd(OH)2.
mediate 31 in 74% yield. This intermediate resisted reductive
debenzylation under the same conditions and compound 32 also Consequently, it might be concluded that bulky substituents
could not be obtained [44,45].
such as cholesterol and quinoline hindered the complete reduc-
tive debenzylation, at least under the described conditions.
To investigate, whether the triazole or the cholesterol entities
are not compatible with these debenzylation conditions, probes Biology
3
6 and 38 (Scheme 7) were prepared by CuAAC of derivative The antibacterial activity of selected newly synthesized choles-
3
4 with compounds 35 and 9b, respectively [24]. terol conjugates was evaluated in vitro against E.coli (ATCC
Scheme 7: Reagents and conditions: (a) Bu2SnO, MeOH; propargyl bromide, TBAI, Tol (92%); (b) CuSO4·5H2O, L-AsAc, THF/H2O (76%); (c) H2,
Pd/C 10%, MeOH (0% for 37 and 62% for 40); (d) 9b, CuSO4·5H2O, L-AsAc, THF/H2O (71%); (e) Ac2O/Pyr (90%). A: Ring A of the lactose moiety,
B: Ring B of the lactose moiety.
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Beilstein J. Org. Chem. 2015, 11, 1922–1932.
1
1775) and S. aureus (ATTC 12600) according to the as active as ampicillin. However, conjugate VI from the
Kirby–Bauer disc diffusion method (Figure 2) [47].
previous investigation was still more active than these conju-
gates [24].
The results shown in Figure 2 revealed that the chalcone modi-
fied cholesterol derivatives 6a,c and 7b were the most potent The antifungal activity of selected newly synthesized chalcone
derivatives against E. coli. They were as active as ampicillin conjugates was evaluated in vitro against A. flavus (Link) and
and insignificantly varied with each other. Thus, the chalcones Candida albicans (ATCC 7102) similarly according to the
possessing unsubstituted phenyl, and p-dimethylaminophenyl as Kirby–Bauer disc diffusion method (Figure 2).
well as 2-thienyl alternatives were more active than other
congeners against E. coli. Despite, derivatives 6b, 7a, 11b, 12, Although, the series was inactive against A. flavus, they showed
1
3, 17, 20 and 24 varied significantly with the control, they some promising antifungal results against C. albicans. As
insignificantly varied with each other and they were 37–64% shown in Figure 2, only chalcone 6c was as active as the control
less active than ampicillin. Compound 11a, that is modified (amphotericin B). Cholesterols 6a,b, 7a,b, 11b, 13 and 20
with a C11 lipid tail, was the least active cholesterol derivative, significantly varied with the control and they were by 50–71%
thus, it was 73% less active than the control.
less active, while the cholesterol derivatives 11a, 12, 17 and 24
were entirely inactive.
On the other hand, derivatives 6c, 7b and the bicholesterol 13
were the most active cholesterols against S. aureus. All Therefore, clicked cholesterol–chalcone conjugates could
these derivatives insignificantly varied with the control afford, at least, one derivative of promising anticandidal activity
(
ampicillin). All other derivatives varied significantly with the which was even better than VI.
control without significant variation among each other. They
were 28–56% less active than the control. Therefore, choles- A group of target cholesterols were screened in vitro as cyto-
terol–chalcone conjugation could afford derivatives that were toxic agents against the human prostate cancer cell line PC3
Figure 2: In vitro antimicrobial activity of some new cholesterol derivatives against E.coli, S. aureus. A. flavus and C. albicans. Ampicillin was used as
positive control (A) in the case of E. coil and S. aureus, while, amphotericin B was used in the case of A. flavus and C. albicans. Different letters on
the column for each parameter varied significantly at p ≤ 0.05. These characters (a, b, etc) above the columns denote to statistical variances. Two or
more columns with different vertical values but specified with the same character mean that their variation is not significant, i.e., they are equally
active.
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Beilstein J. Org. Chem. 2015, 11, 1922–1932.
using the sulforhodamine B colorimetric (SRB) assay and
Supporting Information
doxorubicin as positive control (IC50 = 8.8 μM) (Figure 3) [48].
Supporting Information File 1
Experimental section.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-11-208-S1.pdf]
Acknowledgements
The financial support of Port Said and Taif Universities, Grant
Number 1–435–3248 are gratefully acknowledged. The valu-
able discussions with Prof. R. R. Schmidt at Konstanz Univer-
sity, Germany are kindly acknowledged too.
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