Bipiperidine conjugates as soluble sugar surrogates in DNA-intercalating antiproliferative polyketides

Article abstract of DOI:10.1039/c6cc00890a

DNA-intercalating polyketide glycosides are important leads for cancer therapeutics, yet their use is often limited by their low solubility and challenging synthetic protocols. To overcome these limitations, we employed 1,4′-bipiperidine-1′-carbamate residues as sugar surrogates in daunorubicin and chartreusin, yielding water-soluble derivatives and prodrugs with dramatically improved antiproliferative activities.

Full text of DOI:10.1039/c6cc00890a

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To exclude unspecific effects we prepared the 1,4'-bipiperidine-1'- characterized (Scheme 2). HMBC-NMR data and C-NMR shift
carbamate adducts of polyphenols, resistomycin ( ) and benastatin ( calculations revealed that 11 is mono-substituted at position C-10 as
For all derivatives antiproliferative in the natural glycoside (see supplementary information Figure S13).
5
6,
13, 14
7
) from Streptomyces species.
activities were markedly decreased (see Table 1). Consequently,
installation of the 1,4'-bipiperidine-1'-carbamate would be
unfavorable for polyphenols
5-7, likely because of the dramatic
structural changes. A decrease in bioactivity of related carbamate
16
1
5
derivatives has also been reported for glyfoline and S16020-2.
Thus, we reasoned that the relatively large 1,4'-bipiperidine
substituent would be more suitable for the replacement of a
disaccharyl residue.
Scheme 2 Synthesis of carbamates starting from the aglycone using Steglich
conditions. Yields: 12 11 (19%)+13(73%) ∑92%; 16 18(14%)+20(84%) ∑98%;
19(31%)+21(69%) ∑>99%.
→
→
17→
We found that 11 exhibit an eight-fold lower GI50 value towards
HUVEC cells than the natural product chartreusin ( ). Its cytotoxicity
measured using HeLa cells was only slightly lower. The di-substituted
analogue 13 proved to be soluble at 10 mg/mL in aqueous buffer,
2
ꢀ
which allowed the omission of the co-solvent DMSO in cell-based
assays. Additionally, the antiproliferative activity of 13 is substantially
lower compared to
2 (1.56ꢀµM for 2 and 0.15ꢀꢁM for 13, for K-562
leukemia cells). Notably, the carbamic acid of the bipiperidine alone
proved to be completely inactive at all cell-based assays (Table 1: pip
-
OH).
To evaluate the scope of the method we also prepared analogues of
1
8
less potent chartreusin derivatives such as bromochartreusin (14
)
1
9
and norchartreusin (15), which were practically inactive in human
Scheme 1 Synthesis of carbamates starting from isolated natural products. a) cancer cell-line assays. By conjugation the antiproliferative potency
KOH, MeOH, H O, 2 h, 80 °C, >99% (gly for daunosamine); b) DMAP, pyridine,
,4'-bipiperidine-1'-carbonyl chloride, 72 h, 45 °C, 53%; c) DMAP, pyridine, 1,4'-
bipiperidine-1'-carbonyl chloride, 24 h, r.t. 75% for ; 47% for ; 76% for 10
2
was increased by up to two orders of magnitude. The presence of two
,4'-bipiperidine-1'-carbamate residues further improved solubility
1
1
8
9
.
and bioactivity, yielding compounds (13
, 20-21) that are active at
nanomolar concentrations (see Table 1).
This assumption was tested using chartreusin (
2), a potent DNA
Because of their outstanding antiproliferative activities we sought to
validate the mode of action of compounds 13 20 21 in comparison to
the natural DNA intercalator . We used a band-shift assay
evaluate DNA-intercalating properties. Buffered solutions of the test
compounds ( and 11 21) were individually titrated with a DNA
intercalator isolated from the soil-bacterium Streptomyces
4
,
5
,
-
chartreusis
.
Notably, its poor solubility has hampered the
1
9, 20
6, 7
development of this compound for therapy. Initial logD and logS
2
to
calculations for a mono-substituted product (11) indicated a logD
2
-
value comparable to
2 but an increased (10-fold higher) solubility.
solution, monitoring their UV-Vis spectra. DNA intercalation is clearly
visible by a bathochromic band shift of the aglycone absorption bands
due to base-pair interaction with the DNA double strand (Figure 2).
Aglycone 12, which was prepared using an optimized synthetic
1
7-19
route,
obtained two products, the mono- and di-substituted chartreusin
analogues (11 13). Both compounds were purified and fully
was transformed according to the established protocol. We
+
2
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lead structure
daunorubicin
resistomycin benastatin A benastatin B
chartreusin
bromochartreusin
norchartreusin
reference compounds
iri cip pip-OH
compound no.
R*
1
4
3
5
8
6
9
7
10
2
12
11
13
14
16
18
20
15
17
19
21
1
gly
H
pip
H
pip
H
pip
H
pip
R
R
R
1
2
3
**
**
**
Me
H
gly
Me Me Me Br
Br
H
H
Br
H
Br
pip
H
H
H
H
H
H
H
pip
H
pip
pip
H
H
H
pip
H
2
2
2
pip pip gly
pip pip gly
M. vaccae
HUVEC
Kꢀ562
7.83 21.1 <0.13 21.9 0.40 18.0 0.40 17.9 1.22 >150 23.6 8.65 8.86 >125 21.1 15.9 2.5 >156 48.6 17.6 85.3 0.60
0.19 98.1 13.5 0.80 30.8 11.4 62.8 19.7 17.9 6.25 >150 0.76 0.26 66.3 >125 0.59 0.83 >80 >156 0.86 0.68 4.78 >150 715
0.03 115 7.26 0.53 31.5 12.2 >72 16.1 53.8 1.56 >150 3.59 0.15 8.79 >125 1.85 0.63 31.6 >156 0.78 0.16 2.05 117 >800
0.90 111 28.0 0.53 68.0 10.4 >72 14.5 >72 12.7 >150 17.0 11.8 48.2 117 9.94 2.79 70.7 >156 17.3 12.4 83.2 >150 >800
HeLa
logD (pH 7.4) 0.91 2.20 1.90 4.84 5.29 5.11 6.85 5.39 7.12 2.46 3.75 2.66 0.81 2.72
4
2.92 1.06 1.95 3.24 2.15 0.29 0.72 ꢀ0.81
logS (pH 7.4) ꢀ3.53 ꢀ3.82 ꢀ4.34 ꢀ5.13 ꢀ7.37 ꢀ4.18 ꢀ10.3 ꢀ3.71 ꢀ9.62 ꢀ7.27 ꢀ6.6 ꢀ6.6 ꢀ5.12 ꢀ7.6 ꢀ7.11 ꢀ7 ꢀ5.4 ꢀ6.82 ꢀ6.11 ꢀ6.13 ꢀ4.68 ꢀ3.93 ꢀ2.28
Table 1. Survey of antimicrobial (MIC value [µg/mL]), antiproliferative (GI50 [µg/mL] for HUVEC and Kꢀ562) and cytotoxic (CC50 [µg/mL] for HeLa)
1
activities. Residues marked with an asterisk (*) see Figure 1 and Scheme 1, with two asterisks (**) see Scheme 2; pip, 1,4'ꢀbipiperidineꢀ1'ꢀcarbamoyl; gly ,
2
daunosaminyl, gly , digitalosylꢀfucosyl (see Scheme 1), pipꢀOH, 1,4'ꢀbipiperidineꢀ1'ꢀcarboxylic acid. Test strain: Mycobacterium vaccae 10670. Reference
21
compounds: iri, irinotecan cip, ciprofloxacin. LogD and logS values were calculated using the ChemAxon prediction software.
For chartreusin (
When the glycoside residue is replaced by the carbamate (11
) the band shift decreases to around 4 nm. A second pip substituent
as in 13 20 21) causes a markedly reduced band shift, which
correlates with decreased DNA intercalation. These results are in
2
) a maximum red shift of about 8 nm was detected.
,
18 and
1
9
(
,
-
2
0
contrast to the observed activities of the di-substituted analogues 13
21
,
2
0
-
.
Figure 2 UV-Vis band shift titration of chartreusin (
2) and two selected
carbamates 18 and 20 as a function of added DNA equivalents. *: The zero-value
Figure 3 Metabolic profiling using HPLC/HRMS of cell culture incubated for 48 h
with 20 including negative control and reference compounds. Overlaid LC-EIC
profiles of the medium incubated with 20 Overlaid HPLC-EIC profiles of the
A plausible explanation of this unexpected finding would be a prodrug medium including K-562 cells incubated with 18 HPLC-EIC profiles of the
within the x-axis is interpolated.
A
.
B
.
C
2
2
corresponding reference compounds 16, 18 and 20.
mechanism. Thus, we monitored the fate of bromo compound 20 in
cell assays, taking advantage of its unique isotopic pattern. In the
presence of K-562 leukemia cells, specific (degradation) products of
Conclusions
2
2
0
0
were detected by metabolic profiling (HPLC/HRMS). We found that
remains intact when incubated in medium alone (negative control)
In this study we have designed and evaluated a series of polyketide
glycoside analogues with the aim to use bipiperidine residues as sugar
surrogates. We found that it is feasible to replace the daunosamine
residue in daunorubicin with the bipiperidine carbamate. This novel
analogue proved to be a potent antiproliferative agent, albeit less
active than the parent compound. The best results were obtained for
chartreusin derivatives, where the disaccharide residue was replaced
with the bicyclic heterocycle. We obtained several water-soluble
chartreusin derivatives with substantially improved antiproliferative
activities and decreased cytotoxicity compared to the corresponding
glycosides. Water solubility was always higher for the di-substituted
chartreusin derivatives, and their antiproliferative potency was
(
Figure 3A), yet two hydrolysis products are formed in the presence of
K-562 cells (Figure 3B). High-resolution mass spectra and comparison
of the retention times with the aglycone 16 and the mono-substituted
product 18
(Figure 3C) revealed the identity of 16 (aglycone) and 18
(
mono-substituted analogue). These findings strongly support the
model according to which the di-carbamoylated compounds act as
well-soluble prodrugs, which are converted into the mono-substituted
congeners, which represent potent DNA intercalators.
tenfold higher than the natural product (2). Band-shift assays and in
vivo monitoring revealed that the di-substituted chartreusin
analogues serve as prodrugs that are cleaved in vivo. The resulting
mono-substituted derivatives have pronounced DNA-intercalating
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Representative studies using 1,4'ꢀbipiperidine moieties to
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glycosides, the antiproliferative activities of the chartreusin
derivatives are up to 200 times higher. These findings may encourage
related studies to replace glycoside residues in natural products. Thus
not only the pharmacological properties may be improved, but also
their synthesis may be greatly facilitated.
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Acknowlegements
We thank R. Hermenau and P. Wein for assistance in synthesis, A.
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Financial support by the BMBF (GenBioCom in the GenoMik program)
to C.H. is gratefully acknowledged.
Notes and references
#
These authors contributed equally to the manuscript.
a
Leibniz Institute for Natural Product Research and Infection Biology,
Hans Knöll Institute (HKI), Beutenbergstr. 11a, D-07745, Jena,
Germany. E-Mail: christian.hertweck@hki-jena.de
b
Friedrich Schiller University, Jena, Germany
†
Electronic Supplementary Information (ESI) available: Experimental
details, analytical data, NMR spectra and results of cytotoxicity testing.
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